EA041220B1 - CRYSTAL FORMS - Google Patents

Description

Cross-reference to related applications

This patent application claims priority to U.S. Provisional Patent Application Serial Numbers 62/570,573, filed October 10, 2017; 62/643,950, filed March 16, 2018; 62/656,668, filed April 12, 2018; 62/669,288, filed May 9, 2018; 62/676,417, filed May 25, 2018; and 62/712,707, filed July 31, 2018; the entire contents of each of which are incorporated herein by reference.

State of the art

RET is a single-pass transmembrane receptor belonging to the tyrosine kinase superfamily that is required for normal development, maturation, and maintenance of several tissues and cell types (Mulligan, L. M., Nature Reviews Cancer (2014) 14:173–186). The extracellular portion of RET contains four calcium-dependent cadherin-like repeats involved in ligand binding and a cysteine-rich juxtamembrane region required for proper folding of the RET extracellular domain, while the cytoplasmic portion of the receptor contains two tyrosine kinase subdomains.

RET signaling is mediated by binding of a group of soluble protein ligands (GFLs) of the glial cell line-derived neurotrophic factor (GDNF) family, which also includes neuroturin (NTRN), artemin (ARTN), and persephin (PSPN) (Arighi et al., Cytokine Growth Factor Rev. (2005) 16:441–67). Unlike other receptor tyrosine kinases, RET does not directly bind GFLs and requires an additional coreceptor: one of four members of the GDNF receptor α (GFRa) family, which are linked to the cell surface by a glycosylphosphatidylinositol linkage. GFLs and GFRa family members form binary complexes that in turn bind RET and recruit it to cholesterol-rich membrane subdomains, which are known lipid rafts where RET signaling occurs.

Upon binding to the ligand-coreceptor complex, RET dimerization and autophosphorylation on intracellular tyrosine residues recruits adaptor and signaling proteins to activate several downstream cascades. Binding of an adaptor protein to these docking sites results in activation of the Ras-MAPK and PI3K-Akt/mTOR signaling pathways or recruitment of the CBL family of ubiquitin ligases, which act to down-regulate RET in RET-mediated functions.

Aberrant expression and/or activity of RET has been demonstrated in various types of cancer and gastrointestinal disorders such as irritable bowel syndrome (IBS).

The essence of the invention

Compounds of formula I-IV, 4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (formula I); 6-(2-hydroxy-2methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (formula II); 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (formula III); and 6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (formula IV) are RET kinase inhibitors and are useful for the treatment of diseases such as proliferative diseases, including cancer.

Accordingly, compounds of formulas I-IV are proposed in this document.

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ιν and their pharmaceutically acceptable salts, amorphous and polymorphic forms.

This document also provides a crystalline form of the compound of formula I, said crystalline form being form A and being characterized by an X-ray powder diffraction (XRPD) pattern comprising peaks at °2θ values of 4.4±0.2, 14.6±0.2, and 18.3±0.2.

This document also provides a crystalline form of the compound of formula II, said crystalline form being form 1 and characterized by an X-ray powder diffraction (XRPD) pattern comprising peaks at °2θ values of 16.5±0.2, 18.9±0.2, and 26.0±0.2.

This document also provides a crystalline form of the compound of formula III, said crystalline form being form A and characterized by an X-ray powder diffraction (XRPD) pattern comprising peaks at °2θ values of 17.3±0.2, 19.2±0.2, and 23.9±0.2.

This document also provides a crystalline form of the compound of formula IV, said crystalline form being form A and characterized by an X-ray powder diffraction (XRPD) pattern comprising peaks at °2θ values of 8.3±0.2, 16.3±0.2, and 21.9±0.2.

This document also provides a solid oral pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of formulas I-IV, including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

This document also provides a liquid pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of formulas I-IV, including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The present invention also provides a method for treating cancer in a subject in need thereof, comprising administering a pharmaceutical composition containing a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

The present invention also provides a method for inhibiting cell proliferation, in vitro or in vivo, comprising contacting a cell with an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or with a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or with a pharmaceutical composition described herein.

This document also provides a method of treating a RET-associated disease or disorder in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition described herein.

The present invention provides a method for treating cancer and/or suppressing metastasis associated with a certain type of cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition described herein.

The present document also provides a method of treating cancer and/or suppressing metastasis associated with a certain type of cancer in a subject in need thereof, comprising (a) determining whether the cancer is associated with deregulation of the RET gene, RET kinase, or their expression or activity or level; and (b) if it is determined that the cancer is associated with deregulation of the RET gene, RET kinase, or their expression or activity or level, administering to said subject a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or its

- 2 041220 pharmaceutically acceptable salt, amorphous or polymorphic form.

The document also provides a method of treating RET-associated cancer in a subject, comprising administering to a subject identified or diagnosed with RET-associated cancer a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

The document also provides a method for treating a RET-associated cancer in a subject, comprising: determining whether the cancer in the subject is a RET-associated cancer; and administering to a subject confirmed to have a RET-associated cancer a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

This document also provides a method of treating a subject comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, to a subject having a medical history indicating that the subject suffers from deregulation of the RET gene, RET kinase, or their expression or activity or level.

The document also provides a method for selecting a treatment for a subject, comprising selecting a treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, to a subject identified or diagnosed with RET-associated cancer.

The document also provides a method for selecting a treatment for a subject suffering from cancer, comprising: determining whether the subject's cancer is a RET-associated cancer; and selecting a treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, to a subject confirmed to have a RET-associated cancer.

The document also provides a method for selecting a subject for treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, and said method comprises: identifying a subject suffering from a RET-associated cancer; and selecting a subject for treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form.

The document also provides a method for selecting a subject suffering from cancer for treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, and said method comprises: determining whether the subject's cancer is a RET-associated cancer; and selecting a subject confirmed to have a RET-associated cancer for treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form.

This document also provides the use of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof for the manufacture of a medicament for the treatment of RET-associated cancer in a subject.

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The document also provides a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, for use in the treatment of a subject identified or diagnosed with RET-associated cancer.

This document also provides a method for inhibiting RET kinase activity in a mammalian cell, comprising contacting the mammalian cell with a compound of formula I-VI or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form.

The document also provides a method for treating irritable bowel syndrome in a subject, comprising administering to a subject identified or diagnosed with irritable bowel syndrome a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

The document also provides a method for alleviating pain associated with irritable bowel syndrome in a subject in need thereof, comprising administering to a subject identified or diagnosed with irritable bowel syndrome a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

The present invention also provides a method for providing supportive care to a cancer patient, including preventing or minimizing gastrointestinal disorders, such as diarrhea, associated with treatment, including chemotherapeutic treatment, and said method comprises administering to the patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition described herein.

Also provided herein is a method for inhibiting metastasis (e.g., brain metastasis) of cancer in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of Formula I-IV, e.g., a pharmaceutical composition prepared using a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of Formula I-IV, e.g., a pharmaceutical composition prepared using a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is used in combination with another chemotherapeutic agent.

The document also provides a method of treating a subject suffering from cancer, comprising: (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with the first RET inhibitor used in step (a); and (c) administering a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent to said subject, if the subject has a cancer cell that has at least one RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with the first RET inhibitor used in step (a); or (d) administering to the subject additional doses of the first RET inhibitor used in step (a), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with the first RET inhibitor used in step (a).

The document also provides a method of treating a subject suffering from cancer, comprising: (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation that confers on the cancer cell

- 4 041220 or a tumor with increased resistance to treatment with the first RET inhibitor used in step (a); (c) administering to the subject a second RET inhibitor as monotherapy or in combination with another anticancer agent, if said subject has a cancer that has at least one RET inhibitor resistance mutation that confers increased resistance to treatment with the first RET inhibitor used in step (a); or (d) administering to the subject additional doses of the first RET inhibitor used in step (a), if the subject has a cancer cell that does not contain a RET inhibitor resistance mutation that confers increased resistance to treatment with the first RET inhibitor used in step (a); wherein said mutation is a substitution at amino acid position 804, such as V804M, V804L, or V804E.

The document also provides a method of treating a subject suffering from cancer, comprising: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously treated with one or more doses of a first RET inhibitor has one or more RET inhibitor resistance mutations that render the cancer cell or tumor increased resistant to treatment with a first RET inhibitor previously administered to the subject; and (b) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if the subject has a cancer cell that has at least one RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with a first RET inhibitor that has been previously administered to the subject; or (c) administering to the subject additional doses of a first RET inhibitor, if the subject has a cancer cell that does not contain a RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with a first RET inhibitor that has been previously administered to the subject.

The invention also provides a method of treating a subject suffering from a cancer, comprising: (a) determining whether a cancer cell in a sample obtained from a subject suffering from a cancer and previously receiving one or more doses of a first RET inhibitor has one or more RET inhibitor resistance mutations that render the cancer cell or tumor increased resistance to treatment with a first RET inhibitor that was previously administered to the subject; and (b) administering to the subject a second RET inhibitor as monotherapy or in combination with another anti-cancer agent, if the subject has a cancer cell that has at least one RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with a first RET inhibitor that was previously administered to the subject; or (c) administering additional doses of a first RET inhibitor that was previously administered to the subject if the subject has a cancer cell that does not have a RET inhibitor resistance mutation that renders the cancer cell or tumor increasingly resistant to treatment with the first RET inhibitor that was previously administered to the subject.

The document also provides a method of treating a subject suffering from cancer, comprising: (a) administering one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, over a period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations that render the cancer cell or tumor increased resistance to treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, used in step (a); and (c) administering a second RET inhibitor or a second compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, e.g., a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent to a subject having a cancer cell that has one or more RET inhibitor resistance mutations that render the cancer cell or tumor increased resistance to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising a compound of formula I-IV, e.g., a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, used in step (a); or (d) administering further doses of the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition comprising

- 5 041220 a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof used in step (a), to a subject having a cancer cell that does not have a RET inhibitor resistance mutation that confers on the cancer cell or tumor increased resistance to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof used in step (a).

Also provided herein is a method of treating a subject suffering from cancer, comprising: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously receiving one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, has one or more RET inhibitor resistance mutations that render the cancer cell or tumor increased resistance to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof that has previously been administered to the subject; (b) administering a second RET inhibitor or a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anticancer agent to a subject having a cancer cell that has one or more RET inhibitor resistance mutations that render the cancer cell or tumor increased resistance to treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, that has been previously administered to the subject; or (c) administering additional doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, that has been previously administered to a subject having a cancer cell that does not have a RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, that has been previously administered to the subject.

Also provided herein is a pharmaceutical combination for the treatment of cancer (e.g., a RET-associated cancer, such as a RET-associated cancer having one or more RET inhibitor resistance mutations) in a patient in need thereof, comprising (a) a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, (b) an additional therapeutic agent, and (c) optionally at least one pharmaceutically acceptable carrier, wherein the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and the additional therapeutic agent are formulated in the form of separate compositions or dosage forms for simultaneous, separate or sequential use for the treatment of cancer, wherein the amounts of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, forms, and an additional therapeutic agent together are effective for treating cancer. Also provided herein is a pharmaceutical composition comprising such a combination. Also provided herein is the use of such a combination for producing a medicament for treating cancer. Also provided herein is an industrial package or product comprising such a combination as a combination preparation for simultaneous, separate or sequential use; and a method for treating cancer in a patient in need thereof.

The present invention also provides a method for reversing or preventing acquired resistance to an anticancer drug, comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, to a patient at risk of developing or having acquired resistance to an anticancer drug. In some embodiments, the patient is administered a dose of an anticancer drug (e.g., administered to the patient at substantially the same time as a dose of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof).

The present invention also provides a method for delaying and/or preventing the development of cancer resistance to an anticancer drug in an individual, comprising administering to said individual an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, before, during or after the administration of an effective amount of an anticancer drug.

The document also provides a method of treating an individual suffering from cancer that is at increased risk of developing resistance to an anticancer drug, which comprises administering to said individual (a) an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, before, during or after administration of (b) an effective amount of an anticancer drug.

The present invention also provides methods of treating an individual suffering from a RET-associated cancer that has one or more RET inhibitor resistance mutations that enhance the resistance of the cancer to a first RET inhibitor (e.g., one or more amino acid substitutions in the kinase domain (e.g., amino acid positions 700-1012 in the wild-type RET protein), in the gatekeeper amino acid (e.g., amino acid position 804 in the wild-type RET protein), in the P-loop (e.g., amino acid positions 730-737 in the wild-type RET protein), in the XDFG residue (e.g., amino acid position 891 in the wild-type RET protein), in the solvent front amino acids of the ATP cleft (e.g., amino acid positions 806-811 in the wild-type RET protein), in the activation loop (e.g., amino acid positions 891-916 in the wild-type RET), in the C-helix and in the loop preceding the C-helix (e.g., amino acid positions 768-788 in the wild-type RET protein), and/or in the ATP-binding site (e.g., amino acid positions 730-733, 738, 756, 758, 804, 805, 807, 811, 881, and 892 in the wild-type RET protein) (e.g., a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D, and/or one or more of the RET inhibitor resistance mutations listed in Table 3 and 4), which comprise administering a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, before, during, or after administration of another anticancer drug (e.g., a second RET kinase inhibitor). See also J. Kooistra, G. K. Kanev, O. P. J. Van Linden, R. Leurs, I. J. P. De Esch, and C. De Graaf, KLIFS: A structural kinase-ligand interaction database, Nucleic Acids Res., vol. 44, no. D1, pp. D365-D371, 2016; and O. P. J. Van Linden, A. J. Kooistra, R. Leurs, I. J. P. De Esch, and C. De Graaf, KLIFS: A knowledge-based structural database to navigate kinase-ligand interaction space, J. Med. Chem., vol. 57, no. 2, pp. 249-277, 2014, which are both incorporated herein by reference in their entireties. In some embodiments, the wild-type RET protein is an illustrative wild-type RET protein described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials described herein are in the context of use according to this invention; other suitable methods and materials known in the art may also be used. The materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database accession numbers and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this description, including definitions, shall control.

Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims.

Description of graphic materials

Figs. 1A-1E are solubility plots of the compound of formula I (free base) in various solvent systems. Fig. 1A is a solubility plot in DCM/EtOH. Fig. 1B is a solubility plot in DMSO/EtOH. Fig. 1C is a solubility plot in DMSO/ _H2O . Fig. 1D is a solubility plot in THF/EtOH. Fig. 1E is a solubility plot in THF/H2O.

Fig. 2A-2G are X-ray powder diffraction patterns of the free base of the compound of formula I. Fig. 2A is an X-ray powder diffraction pattern of Form A of the compound of formula I (free base). Fig. 2B is an overlay of X-ray powder diffraction patterns of the free base from different lots before and after DSP analysis. Fig. 2C is a differential calorimetry trace of the free base. Fig. 2D is an isothermal (25°C) dynamic vapor sorption trace of the free base. Fig. 2E is a thermogravimetric analysis trace of the free base. Fig. 2F is a FTIR spectrum of the free base. Fig. 2G is the 1H NMR spectrum of the free base in DMSO-d ⁶ .

Fig. 3 shows a differential scanning calorimetry curve of the maleic acid salt of the compound of formula I identified in the initial salt screening.

Fig. 4 shows a differential scanning calorimetry curve of the acetic acid salt of the compound of formula I identified in the initial salt screening.

Fig. 5 shows a differential scanning calorimetry curve of the D-malic acid salt of the compound of formula I identified in the initial salt screening.

Fig. 6 shows a differential scanning calorimetry curve of the benzoic acid salt of the compound of formula I identified in the initial screening of salts.

Fig. 7 shows a differential scanning calorimetry curve of the L-tartaric acid salt of the compound of formula I identified in the initial salt screening.

Fig. 8 shows a differential scanning calorimetry curve of the citric acid salt of the compound of formula I identified in the initial salt screening.

Fig. 9 shows a differential scanning calorimetry curve of the propionic acid salt of the compound of formula I identified in the initial salt screening.

Fig. 10 shows a differential scanning calorimetry curve of the D-tartaric acid salt of the compound of formula I identified in the initial salt screening.

Fig. 11 shows a differential scanning calorimetry curve of the L-malic acid salt of the compound of formula I identified in the initial salt screening.

Fig. 12 shows a differential scanning calorimetry curve of the sulfuric acid salt of the compound of formula I identified in the initial salt screening.

Fig. 13 shows a differential scanning calorimetry curve of the hydrochloric acid salt of the compound of formula I, obtained on a scale of about 0.2 g.

Fig. 14 shows a differential scanning calorimetry curve of the hydrobromic acid salt of the compound of formula I, obtained on a scale of about 0.2 g.

Fig. 15 are differential scanning calorimetry curves of the hydrochloric acid salt of the compound of formula I obtained during the optimization process. Fig. 15A is a differential scanning calorimetry curve of the hydrochloric acid salt (prepared using DMA as a solvent). Fig. 15B is a differential scanning calorimetry curve of the hydrochloric acid salt (prepared using a DCM/EtOH mixture as a solvent).

Fig. 16A-16F are the hydrochloric acid salt curve of the compound of formula I prepared at the 2 g scale. Fig. 16A is the hydrochloric acid salt curve (prepared using DMA as a solvent). Fig. 16B is the isothermal (25°C) dynamic vapor sorption curve of the hydrochloric acid salt (prepared using DMA as a solvent). Fig. 16C is the thermogravimetric analysis curve of the hydrochloric acid salt (prepared using DMA as a solvent). Fig. 16D is a superimposition of the differential scanning calorimetry chart and the thermogravimetric analysis chart of the hydrochloric acid salt (prepared using DCM/EtOH as a solvent). Fig. 16E is a superposition of the X-ray powder diffraction patterns of the hydrochloric acid salt prepared using DMA or DCM/EtOH as solvent at different scales, before and after DSP. Fig. 16F is the 1H NMR spectrum of the hydrochloric acid salt in DMSO-d ⁶ .

Figs. 17A-17C are diagrams of the hydrobromide salt of the compound of formula I prepared on a 2 g scale. Fig. 17A is a superimposition of the differential scanning calorimetry diagram and the thermogravimetric analysis diagram of the hydrobromide salt. Fig. 17B is an X-ray powder diffraction pattern of the hydrobromide salt. Fig. 17C is a Fourier transform IR spectrum of the hydrobromide salt. Fig. 17D is a 1H NMR spectrum of the hydrobromide salt in DMSO-d ⁶ .

Fig. 18A-18H are diagrams of the L- and D-malic acid salts of the compound of formula I. Fig. 18A is a superposition of the differential scanning calorimetry curve and the thermogravimetric analysis curve of the L-malic acid salt. Fig. 18B is a superposition of the differential scanning calorimetry curve and the thermogravimetric analysis curve of the D-malic acid salt. Fig. 18C is a diagram of the isothermal (25°C) dynamic vapor sorption of the L-malic acid salt. Fig. 18D is a diagram of the isothermal (25°C) dynamic vapor sorption of the D-malic acid salt. Fig. 18E is a superposition of the X-ray powder diffraction diagrams of the L- and D-malic acid salts. Fig. 18F is a superposition of the FTIR spectra of the L- and D-malic acid salts. Fig. 18G is the 1H NMR spectrum of the malate salt in DMSO-d ⁶ . Fig. 18H is the 1H NMR spectrum of the malate salt in DMSO-d ⁶ . Figs. 18I-M are traces of two batches of the L-malate salt of the compound of formula I. Fig. 18I is the differential scanning calorimetry trace of the L-malate salt (Batch A). Fig. 18J is the thermogravimetric analysis trace of the L-malate salt (Batch A). Fig. 18K is the differential scanning calorimetry trace of the L-malate salt (Batch B). Fig. 18L is the thermogravimetric analysis trace of the L-malate salt (Batch B). Fig. 18M is a superposition of the X-ray powder diffraction patterns of the salts of L-malic acid (batch A and batch B) and the free base of the compound of formula I.

Figs. 19A-19F are diffraction patterns of polymorphic Form 1 of the compound of formula II.

- 8 041220

Fig. 19A is an X-ray powder diffraction pattern of fully dried Form 1. Fig. 19B is a differential scanning calorimetry curve of Form 1. Fig. 19C is a thermogravimetric/differential thermal analysis curve of Form 1. Fig. 19D is a gravimetric vapor sorption isotherm of Form 1. Fig. 19E is a kinetic gravimetric vapor sorption curve of Form 1. Fig. 19F is a 1H NMR spectrum of Form 1 in d ₆ -DMSO.

Figs. 20A-20E are diagrams of polymorph Form 2 of the compound of Formula II. Fig. 20A illustrates X-ray powder diffraction patterns of Form 2 (small scale suspension, large scale suspension, and fully dried). Fig. 20B is a differential scanning calorimetry curve of Form 2. Fig. 20C is a thermogravimetric/differential thermal analysis curve of Form 2. Fig. 20D is a gravimetric vapor sorption isotherm of Form 2. Fig. 20E is a kinetic gravimetric vapor sorption curve of Form 2.

Figs. 21A-21F are diagrams of polymorph Form 7 of the compound of Formula II. Fig. 21A illustrates X-ray powder diffraction patterns of Form 7 (small scale suspension, large scale suspension, and fully dried). Fig. 21B is a differential scanning calorimetry curve of Form 7. Fig. 21C is a thermogravimetric/differential thermal analysis curve of Form 7.

Fig. 21D is a gravimetric vapor sorption isotherm of Form 7. Fig. 21E is a kinetic gravimetric vapor sorption curve of Form 7. Fig. 21F is a 1H NMR spectrum of Form 7 in d ₆ -DMSO.

Fig. 22A-22F are diagrams of polymorph Form 8 of the compound of Formula II. Fig. 22A illustrates X-ray powder diffraction patterns of Form 8 (small scale suspension, large scale suspension, and fully dried). Fig. 22B is a differential scanning calorimetry curve of Form 8. Fig. 22C is a thermogravimetric/differential thermal analysis curve of Form 8. Fig. 22D is a gravimetric vapor sorption isotherm of Form 8. Fig. 22E is a kinetic gravimetric vapor sorption curve of Form 8. Fig. 22F is a 1H NMR spectrum of Form 8 in d ₆ -DMSO.

Figs. 23A-23F are diagrams of the phosphate salt of the compound of formula II. Fig. 23A is an X-ray powder diffraction pattern of the fully dried phosphate salt. Fig. 23B is a differential scanning calorimetry curve of the phosphate salt. Fig. 23C is a thermogravimetric/differential thermal analysis curve of the phosphate salt. Fig. 23D is a gravimetric vapor sorption isotherm of the phosphate salt. Fig. 23E is a kinetic gravimetric vapor sorption curve of the phosphate salt. Fig. 23F is a 1H NMR spectrum of Form 1 in d ₆ -DMSO.

Figs. 24A-24B are X-ray powder diffraction patterns after screening of hydrochloric acid salts of a compound of formula II. Fig. 24A illustrates patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4 hour cycles for 24 hours (after repeating the cycles). Fig. 24B shows patterns of compound of formula II in cas with at least peaks in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 25A-25B are X-ray powder diffraction patterns after screening of sulfuric acid salts of compound of formula II. Fig. 25A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 25B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 26A-26B are X-ray powder diffraction patterns after screening salts of compound of formula II with p-toluenesulfonic acid. Fig. 26A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 26B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 27A-27B are X-ray powder diffraction patterns after screening the methanesulfonic acid salts of compound of formula II. Fig. 27A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 27B illustrates the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and a relative humidity of

75% (after achieving stability).

Figs. 28A-28B are X-ray powder diffraction patterns after screening of salts of compound of formula II with naphthalene-2-sulfonic acid. Fig. 28A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 28B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 29A-29B are X-ray powder diffraction patterns after screening of salts of compound of formula II with benzenesulfonic acid. Fig. 29A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 29B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 30A-30B are X-ray powder diffraction patterns after screening of oxalic acid salts of compound of formula II. Fig. 30A illustrates patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 30B shows patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 31A-31B are X-ray powder diffraction patterns after screening salts of compound of formula II with 2-hydroxyethanesulfonic acid. Fig. 31A illustrates patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 31B shows patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 32A-32B are X-ray powder diffraction patterns after screening of L-aspartic acid salts of compound of formula II. Fig. 32A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 32B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 33A-33B are X-ray powder diffraction patterns after screening of maleic acid salts of compound of formula II. Fig. 33A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 33B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 34A-34B are X-ray powder diffraction patterns after screening of phosphoric acid salts of compound of formula II. Fig. 34A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 34B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 35A-35B are X-ray powder diffraction patterns after screening of salts of compound of formula II with ethanesulfonic acid. Fig. 35A illustrates the patterns of compound of formula II in each tested solvent after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 35B shows the patterns of compound of formula II in each tested solvent after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Fig. 36A-36B are X-ray powder diffraction patterns after screening of salts of the compound of formula II with L-glutamic acid. Fig. 36A illustrates the diagrams

- 10 041220 compounds of formula II in each tested solvent after subjecting the samples to temperature cycles from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 36B shows diagrams of the compound of formula II in each tested solvent after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 37A-37B are X-ray powder diffraction patterns after screening of L-tartaric acid salts of compound of formula II. Fig. 37A illustrates patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 37B illustrates patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 38A-38B are X-ray powder diffraction patterns after screening of fumaric acid salts of the compound of formula II. Fig. 38A illustrates the patterns of the compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 38B shows the patterns of the compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 39A-39B are X-ray powder diffraction patterns after screening of citric acid salts of compound of formula II. Fig. 39A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 39B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 40A-40B are X-ray powder diffraction patterns after screening of D-glucuronic acid salts of compound of formula II. Fig. 40A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 40B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 41A-41B are X-ray powder diffraction patterns after screening of salts of compound of formula II with L-malic acid. Fig. 41A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 41B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 42A-42B are X-ray powder diffraction patterns after screening of hippuric acid salts of compound of formula II. Fig. 42A illustrates patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 42B illustrates patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 43A-43B are X-ray powder diffraction patterns after screening of D-gluconic acid salts of compound of formula II. Fig. 43A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 43B illustrates the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 44A-44B are X-ray powder diffraction patterns after screening of L-lactic acid salts of compound of formula II. Fig. 44A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 44B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after

- 11 041220 achieving stability).

Figs. 45A-45B are X-ray powder diffraction patterns after screening of L-ascorbic acid salts of compound of formula II. Fig. 45A illustrates the patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 45B shows the patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 46A-46B are X-ray powder diffraction patterns after screening of benzoic acid salts of the compound of formula II. Fig. 46A illustrates the patterns of the compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 46B shows the patterns of the compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Figs. 47A-47B are X-ray powder diffraction patterns after screening of succinic acid salts of compound of formula II. Fig. 47A illustrates patterns of compound of formula II in each solvent tested after subjecting the samples to temperature cycling from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). Fig. 47B shows patterns of compound of formula II in each solvent tested after keeping the samples overnight in an oven at 40°C and 75% relative humidity (after stability was achieved).

Fig. 48 shows thermogravimetric/differential thermal analysis curves of the sulfate salt of the compound of formula II identified in the initial salt screening.

Fig. 49 shows thermogravimetric/differential thermal analysis curves of the tosylate salt of the compound of formula II identified in the initial salt screening.

Fig. 50 shows thermogravimetric/differential thermal analysis curves of the naphthalene-2-sulfonate salt of the compound of formula II identified in the initial salt screening.

Fig. 51 shows thermogravimetric/differential thermal analysis curves of the oxalate salt (1,4-dioxane/10% water) of the compound of formula II identified in the initial salt screening.

Fig. 52 shows thermogravimetric/differential thermal analysis curves of the oxalate salt (evaporation) of the compound of formula II identified in the initial salt screening.

Fig. 53 shows the thermogravimetric/differential thermal analysis curve of the phosphate salt (acetone/10% water) of the compound of formula II identified in the initial salt screening.

Fig. 54 shows the thermogravimetric/differential thermal analysis curve of the phosphate salt (IPA/10% water) of the compound of formula II identified in the initial salt screening.

Fig. 55 shows a thermogravimetric/differential thermal analysis curve of the tartrate salt of the compound of formula II identified in the initial salt screening.

Fig. 56 shows a thermogravimetric/differential thermal analysis curve of the fumarate salt of the compound of formula II identified in the initial salt screening.

Fig. 57 shows X-ray powder diffraction patterns of solids prepared from solvents tested in the solvent solubility screening of Form I of Formula II.

Figs. 58A-58D show X-ray powder diffraction patterns of the compound of formula II after temperature cycling experiments in various solvents and after overnight storage at 40°C and 75% RH.

Fig. 59 shows the X-ray powder diffraction patterns of the compound of formula II after evaporation experiments using different solvents.

Fig. 60 shows the X-ray powder diffraction patterns of the compound of formula II after quenching experiments using different solvents.

Fig. 61 shows the X-ray powder diffraction patterns of the compound of formula II after antisolvent experiments using different solvents.

Figs. 62A-62F are diagrams of the compound of formula II. Fig. 62A is an X-ray powder diffraction pattern of the compound of formula II. Fig. 62B is a differential scanning calorimetry curve of the compound of formula II. Fig. 62C is a thermogravimetric/differential thermal analysis curve of the compound of formula II. Fig. 62D is a dynamic vapor sorption isotherm of the compound of formula II. Fig. 62E is a kinetic dynamic vapor sorption curve of the compound of formula II. Fig. 62F is a ¹ H NMR spectrum of the compound of formula II in d ₆ -DMSO.

Figs. 63A-63B are diagrams of polymorphic Form A of the compound of formula III. Fig. 63A is an X-ray powder diffraction pattern of polymorphic Form A of the compound of formula III. Fig. 63B is a differential scanning calorimetry curve of polymorphic Form A of the compound of formula III.

Figs. 64A-64B are diagrams of polymorphic Form A of the compound of formula IV. Fig. 64A is an X-ray powder diffraction pattern of polymorphic Form A of the compound of formula IV. Fig. 64B is a differential scanning calorimetry curve of polymorphic Form A of the compound of formula IV.

Figs. 65A-64B are diagrams of polymorphic Form B of the compound of formula IV. Fig. 65A is an X-ray powder diffraction pattern of polymorphic Form B of the compound of formula IV. Fig. 65B is a differential scanning calorimetry curve of polymorphic Form B of the compound of formula IV.

Fig. 66 is a superposition of the X-ray powder diffraction patterns of polymorphs A and B of the compound of formula IV.

Detailed description

1. Definitions.

The term polymorph in this context refers to crystals of a single compound that have different physical properties due to the arrangement of the molecules in the crystal lattice. Different polymorphs of a single compound differ from each other in one or more chemical, physical, mechanical, electrical, thermodynamic and/or biological properties. The differences in physical properties exhibited by polymorphs may affect pharmaceutical parameters such as storage stability, compressibility, density (an important parameter in formulation and manufacturing of the product), dissolution rate (an important factor in determining bioavailability), solubility, melting point, chemical stability, physical stability, powder flowability, water sorption, caking and particle morphology. Differences in stability may result from changes in chemical reactivity (e.g. differential oxidation such that the dosage form of one polymorph discolors faster than the dosage form of another polymorph) or mechanical changes (e.g. crystalline changes on storage as a kinetically favored polymorph converts to a thermodynamically more stable polymorph), or both (e.g. one polymorph is more hygroscopic than another). Because of solubility/dissolution differences, some changes affect potency and/or toxicity. In addition, the physical properties of the crystal may be important for processing; for example, one polymorph may be more prone to solvate formation or may be more difficult to filter and wash out of free impurities (i.e., the shape and particle size distribution for one polymorph may differ from that of another polymorph). Polymorph as used herein does not include amorphous forms of the compound. As used herein, amorphous refers to a non-crystalline form of the compound, which may be a solid form of the compound or a solubilized form of the compound. For example, amorphous refers to a compound (such as a solid form of a compound) without a regularly repeating order of molecules or outer faces.

The term anhydrous in this context refers to a crystalline form of a compound of formula I-IV that contains 1% or less water by weight. For example, 0.5% or less, 0.25% or less, or 0.1% or less water by weight.

The term solvate as used herein refers to a crystalline form of a compound of formula IIV, such as a polymorphic form of the compound, in which the crystal lattice contains one or more solvents of crystallization.

The term hydrate or hydrated polymorph refers to a crystalline form of a compound of formula I-IV, such as a polymorphic form of the compound in which the crystal lattice contains water. Unless otherwise specified, the term hydrate as used herein refers to a stoichiometric hydrate. A stoichiometric hydrate contains water molecules as an integral part of the crystal lattice, with the removal of water molecules resulting in instability of the crystal lattice. In comparison, a non-stoichiometric hydrate contains water, but a change in water content does not cause a significant change in the crystal structure. Drying non-stoichiometric hydrates can remove a substantial portion of the water without significantly disrupting the crystal structure, and the crystals can then be rehydrated to yield the original non-stoichiometric hydrated crystalline form. Unlike stoichiometric hydrates, dehydration and rehydration of non-stoichiometric hydrates are not accompanied by a phase transition, and therefore all hydration states of a non-stoichiometric hydrate represent the same crystalline form.

- 13 041220

Purity with respect to a composition containing a polymorph of a compound of formula I-IV refers to the percentage of one particular polymorphic form relative to another polymorphic form or an amorphous form of a compound of formula I-IV in said composition. For example, a composition containing polymorphic form 1 having a purity of 90% contains 90 parts by weight of form 1 and 10 parts by weight of other polymorphic and/or amorphous forms of a compound of formula I-IV.

As used herein, a compound or composition is substantially free of one or more other components if the compound or composition does not contain a substantial amount of such other components. For example, the composition may contain less than 5, 4, 3, 2, or 1% by weight of other components. Such components may include starting materials, residual solvents, or any other impurities that may arise during the preparation and/or isolation of the compounds and compositions provided herein. In some embodiments, a polymorphic form provided herein is substantially free of other polymorphic forms. In some embodiments, a particular polymorph of a compound of Formula I-IV is substantially free of other polymorphs if the particular polymorph comprises at least 95% by weight of the compound of Formula I-IV present. In some embodiments, a particular polymorph of a compound of Formula I-IV is substantially free of other polymorphs if the particular polymorph comprises at least about 97%, about 98%, about 99%, or about 99.5% by weight of the compound of Formula I-IV present. In some embodiments, a particular polymorph of a compound of Formula I-IV is substantially free of water if the amount of water is no more than about 2%, about 1%, or about 0.5% by weight of the polymorph.

In this context, substantially pure with respect to a polymorphic form of the compound of formula I-IV means a sample of the polymorphic form of the compound having a purity of more than 90%, including more than 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99%, and also including exactly or about 100% of the compound based on the weight of the compound. The remaining material contains other form(s) of the compound and/or reactive impurities and/or process impurities arising during its preparation. For example, a polymorphic form of the compound of formula I-IV can be considered substantially pure if it has a purity of more than 90% of the polymorphic form of the compound of formula I-IV, measured in a manner currently known and generally accepted in the art, and the remaining less than 10% of the material contains other form(s) of the compound of formula I-IV and/or reactive impurities and/or process impurities. The presence of reaction impurities and/or process impurities can be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry or infrared spectroscopy.

The term near before the DSC, TGA, TG or DTA value, which is written in degrees Celsius, means the permissible deviation is ±5°C.

For the sake of brevity, some quantitative expressions in this context are stated as a range from about amount X to about amount Y. It should be understood that when a range is stated, the range is not limited to the stated upper and lower limits, but includes the entire range from about amount X to about amount Y, or any range in between.

Room temperature or rt refers to the ambient temperature in a typical laboratory, which is usually around 25°C.

As used herein, the terms subject, individual, or patient, when used interchangeably, refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the patient is a human. In some embodiments, the subject experiences and/or exhibits at least one symptom of a disease or disorder to be treated and/or prevented. In some embodiments, the subject has been identified or diagnosed with a cancer in which the RET gene, RET protein, or expression or activity or level of any of them is deregulated (RET-associated cancer) (e.g., as determined using an assay or kit approved by a regulatory agency, e.g., approved by the FDA). In some embodiments, the subject has a tumor that is positive for deregulation of the RET gene, RET protein, or expression or activity or level of any of them (e.g., as determined using an assay or kit approved by a regulatory agency). The subject may be a subject with a tumor(s) that is positive for deregulation of the RET gene, RET protein, or expression or activity, or level of any of them (e.g., determined to be positive using an assay or kit approved by a regulatory agency, such as one approved by the FDA). The subject may be a subject whose tumors are characterized by deregulation of the RET gene, RET protein, or expression or activity, or level of any of them (e.g., if the tumor is determined to be so using a kit or assay approved by a regulatory agency, such as one approved by the FDA). In some embodiments, the subject is predisposed to RET-associated cancer. In some embodiments, the subject has a medical history indicating that the subject suffers from a tumor characterized by deregulation of the RET gene, RET protein, or expression or activity, or level thereof (and optionally a medical history indicating that the subject is to be treated with any compositions provided herein). In some embodiments, the patient is a pediatric patient.

The term pediatric patient, as used herein, refers to a patient who is less than 21 years of age at the time of diagnosis or treatment. The term pediatric can be further classified into various subgroups, including: neonates (birth to the first month of life); infants (1 month to 2 years); children (2 years to 12 years); and adolescents (12 years to 21 years (but not including the twenty-second birthday)). Berhman RE, Kliegman R, Arvin AM, Nelson WE, Textbook of Pediatrics, 15th ed., Philadelphia: W.B. Saunders Company, 1996; Rudolph AM, et al., Rudolph's Pediatrics, 21st ed., New York: McGraw-Hill, 2002; and Avery MD, First LR, Pediatric Medicine, 2nd ed., Baltimore: Williams &Wilkins; 1994. In some embodiments, the pediatric patient is from birth to the first 28 days of life, from 29 days to less than two years, from two years to less than 12 years, or from 12 years to 21 years (but not including the twenty-second birthday). In some embodiments, the pediatric patient is from birth to the first 28 days of life, from 29 days to less than 1 year, from one month to less than four months, from three months to less than seven months, from six months to less than 1 year, from 1 year to less than 2 years, from 2 years to less than 3 years, from 2 years to less than seven years, from 3 years to less than 5 years, from 5 years to less than 10 years, from 6 years to less than 13 years, from 10 years to less than 15 years, or from 15 years to less than 22 years.

In this context, the terms treat or cure refer to therapeutic or palliative measures. The beneficial or desired clinical outcomes include, but are not limited to, complete or partial relief of symptoms associated with the disease or disorder or condition, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease, delay or slowing of the progression of the disease, improvement or alleviation of the disease state (e.g., one or more symptoms of the disease), and remission (partial or complete), whether apparent or not. Treatment may also mean prolongation of life expectancy compared to life expectancy without treatment.

The term administer or administering refers to the method of administering a dose of a compound or pharmaceutical composition to a vertebrate or invertebrate animal, including mammals, birds, fish or amphibians. The preferred route of administration may vary depending on various factors, such as the components of the pharmaceutical composition, the site of the disease and the severity of the disease.

The term pharmaceutically acceptable carrier or pharmaceutically acceptable excipient includes any and all solvents, cosolvents, complexing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are not biologically or otherwise undesirable. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where the conventional medium or agent is incompatible with the active ingredient, their use in the therapeutic compositions of the present invention is contemplated. Additional active ingredients can also be incorporated into the compositions. In addition, various excipients commonly used in the art can be used. These and other similar compounds are described in literature, for example, in the Merck Index, Merck & Company, Rahway, NJ. Incorporation of various components into pharmaceutical compositions is discussed, for example, in Gilman et al. (eds.) (2010); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th ed., The McGraw-Hill Companies.

A therapeutically effective amount or a pharmaceutically effective amount of a compound as provided herein is an amount that is effective in achieving the desired effect, and may vary according to the nature and severity of the disease state and the potency of the compound. A therapeutic effect is an alleviation to some extent of one or more symptoms of a disease, and may include a cure for the disease. A cure means the elimination of symptoms of an active disease. However, some long-term or permanent effect of the disease may exist even after a cure has been achieved (e.g., in the case of extensive tissue damage).

The term RET-associated disease or disorder, as used herein, refers to diseases or disorders associated with or characterized by deregulation of the RET gene, RET kinase (also referred to herein as RET kinase protein or RET kinase), or the expression or activity or level of any of them (e.g., one or more) (e.g., any type of deregulation of the RET gene, RET kinase, RET kinase domain, or the expression or activity or level of any of them, as described herein). Non-limiting examples of a RET-associated disease or disorder include, for example, cancer and gastrointestinal disorders such as irritable bowel syndrome (IBS).

The term RET-associated cancer as used herein refers to cancers associated with or characterized by deregulation of the RET gene, RET kinase (also referred to herein as RET kinase protein or RET kinase), or the expression or activity or level of any of them. Non-limiting examples of RET-associated cancers are described herein.

The expression "deregulation of the RET gene, RET kinase, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a translocation of the RET gene that results in the expression of a fusion protein, a deletion in the RET gene that results in the expression of a RET protein that contains a deletion of at least one amino acid compared to the wild-type RET protein, or a mutation in the RET gene that results in the expression of a RET protein with one or more point mutations, or an alternative splice version of the RET mRNA forming the RET protein that results in a deletion of at least one amino acid in the RET protein compared to the wild-type RET protein) or to an amplification of the RET gene that results in overexpression of the RET protein or autocrine activity due to overexpression of the RET gene in a cell that results in a pathogenic increase in the activity of the kinase domain of the RET protein (e.g., a constitutively active kinase domain of the RET protein RET) in a cell. As another example, deregulation of a RET gene, a RET protein, or the expression or activity or level of any of them can be a mutation in a RET gene that encodes a RET protein that is constitutively active or has increased activity compared to a protein encoded by a RET gene that does not contain such a mutation. For example, deregulation of a RET gene, a RET protein, or the expression or activity or level of any of them can be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of RET that contains a functional kinase domain and a second portion of a partner protein (i.e., a non-RET). In some examples, deregulation of a RET gene, a RET protein, or the expression or activity can be the result of a gene translocation of one RET gene with another non-RET gene. Non-limiting examples of fusion proteins are described in Table 1. Non-limiting examples of point mutations/insertions/deletions of the RET kinase protein are described in Table 2. Additional examples of RET kinase protein mutations (e.g., point mutations) are RET inhibitor resistance mutations. Non-limiting examples of RET inhibitor resistance mutations are described in Tables 3 and 4.

The term "wild type" describes a nucleic acid (e.g., a RET gene or RET mRNA) or a protein (e.g., a RET protein) that occurs in a subject that does not suffer from a RET-associated disease, such as a RET-associated cancer (and optionally also does not have an increased risk of developing a RET-associated disease and/or is not predisposed to a RET-associated disease), or that occurs in a cell or tissue obtained from a subject that does not suffer from a RET-associated disease, such as a RET-associated cancer (and optionally also does not have an increased risk of developing a RET-associated disease and/or is not predisposed to a RET-associated disease).

The term regulatory agency refers to a government agency that authorizes the medical use of pharmaceutical agents in a given country. For example, a non-limiting example of a regulatory agency is the Food and Drug Administration (FDA).

2. Polymorphs and pharmaceutically acceptable salts.

The present invention relates to compounds of formula I-IV and pharmaceutically acceptable salts thereof which exhibit rearranged transfection kinase inhibition (RET). In particular, provided herein are novel crystalline forms of 4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5a]pyridine-3-carbonitrile (formula I); 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (formula II); 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (formula III); and 6-(2-hydroxy-2methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5a]pyridine-3-carbonitrile (formula IV) and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing said compounds, methods for preparing said compounds and the use of said compounds in therapy. More particularly, the present invention relates to novel crystalline forms of Formulas I-IV and pharmaceutically acceptable salts thereof, which are useful for the treatment and prevention of diseases that can be treated with a RET kinase inhibitor, including RET-associated diseases and disorders.

- 16 041220

Formula I.

This document proposes a compound of formula I:

I including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula I provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods such as those described in US Publication No. 2017/0096425 can be used, and the entirety of this application is incorporated herein by reference.

Provided herein are polymorphic forms of the compound of formula I. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of the compound of formula I, including, for example, polymorphic form A. In some embodiments, the polymorphic form of the compound of formula I is a pharmaceutically acceptable salt. In some embodiments, the compound of formula I is a chloride salt. In some embodiments, the compound of formula I is a bromide salt. In some embodiments, the compound of formula I is an L-malate salt. In some embodiments, the compound of formula I is a D-malate salt.

Form A.

One such polymorph is a polymorph known as Form A. Form A is a polymorphic form of the compound of Formula I. In some embodiments, Form A has an XRD pattern obtained with CuKai radiation that has at least peaks at °2θ values of 4.4±0.2, 14.6±0.2, and 18.3±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 4.4±0.2, 13.5±0.2, 14.6±0.2, 18.3±0.2, and 18.8±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 4.4±0.2, 13.5±0.2, 14.6±0.2, 17.4±0.2, 18.3±0.2, 18.8±0.2, 21.0±0.2, and 24.6±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 4.4±0.2, 13.5±0.2, 14.6±0.2, 17.4±0.2, 18.3±0.2, 18.8±0.2, 21.0±0.2, and 24.6±0.2, 22.5±0.2, 24.6±0.2, and 27.7±0.2.

In some embodiments, provided herein is a composition comprising polymorph Form A. In some embodiments, the composition may be substantially pure. For example, the composition may have a purity of at least about 90%. In some embodiments, the composition may have a purity of at least about 95%. In some embodiments, the composition may have a purity of at least about 98%. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8%, or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition can contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I. For example, the composition can contain less than about 15% of the amorphous form.

In some embodiments, the present document provides a polymorphic form A that exhibits an endotherm observed at about 185-195°C, such as about 192.8°C, as measured by DSC, associated with sorbed water. In some embodiments, the present document provides a polymorphic form A that exhibits an endotherm observed at about 220-230°C, such as about 226.7°C, as measured by DSC, associated with sorbed water.

In some embodiments, the present document provides polymorphic Form A that exhibits a mass loss of about 1.1% from the onset of heating to about 238°C, as measured by TGA.

Provided herein are methods for preparing polymorph Form A. In some embodiments, polymorph Form A of the compound of Formula I is prepared by contacting 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile tetrahydrochloride, sodium triacetoxyborohydride, and triethylamine in a polar aprotic solvent. In some embodiments, the polar aprotic solvent is DMSO. In some embodiments, the proposed method further comprises heating a suspension comprising 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3yl)pyrazolo[1,5-a]pyridine-3-carbonitrile tetrahydrochloride, sodium triacetoxyborohydride, trimethylamine, and DMSO to about 30°C. ...

- 17 041220 but includes heating the slurry for about 10 hours to about 15 hours, such as about 13 hours. In some embodiments, the proposed method further includes cooling the slurry to about 19°C after about 13 hours of heating to about 30°C. In some embodiments, the proposed method further includes adding water to the slurry. For example, the proposed method may further include adding 2 volumes of water to the slurry. In some embodiments, the proposed method includes maturing a composition comprising said slurry and water. In some embodiments, the proposed method includes maturing a composition comprising said slurry and water for about 1 to about 10 hours, such as about 3.5 hours. In some embodiments, the proposed method includes isolating a solid by filtration. In some embodiments, the solid is dried. In some embodiments, the solid is dried under vacuum. In some embodiments, the solid is dried at about 45°C.

Salts of formula I.

In some embodiments, the compound of Formula I is a pharmaceutically acceptable salt. For example, pharmaceutically acceptable salts of the compound of Formula I can include, but are not limited to, chloride, bromide, sulfate, citrate, L-tartrate, D-tartrate, acetate, L-malate, D-malate, benzoate, propionate, and maleate salts. In some embodiments, the compound of Formula I is a chloride salt. In some embodiments, the chloride salt is formed in a solvent mixture. In some embodiments, the solvent is a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 1:1 by volume. In some embodiments, the chloride salt is formed in dimethylacetamide. In some embodiments, the compound of Formula I is a bromide salt. In some embodiments, the bromide salt is formed in a solvent mixture. In some embodiments, the solvent is a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the bromide salt is formed in dimethylacetamide. In some embodiments, the compound of Formula I is a sulfate salt. In some embodiments, the sulfate salt is formed in a mixture of solvents. In some embodiments, the sulfate salt is formed in a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 4:1 by volume. In some embodiments, the compound of Formula I is a citrate salt. In some embodiments, the citrate salt is formed in dichloromethane. In some embodiments, the compound of Formula I is an L-tartrate salt. In some embodiments, the L-tartrate salt is formed in dichloromethane. In some embodiments, the compound of Formula I is a D-tartrate salt. In some embodiments, the D-tartrate salt is formed in dichloromethane. In some embodiments, the compound of Formula I is an acetate salt. In some embodiments, the acetate salt is prepared in dichloromethane. In some embodiments, the compound of Formula I is an L-malate salt. In some embodiments, the L-malate salt is prepared in dichloromethane. In some embodiments, the L-malate salt is prepared in a mixture of solvents. In some embodiments, the L-malate salt is prepared in a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 3.3:1 by volume. In some embodiments, the compound of Formula I is a D-malate salt. In some embodiments, the D-malate salt is prepared in dichloromethane. In some embodiments, the D-malate salt is prepared in a mixture of solvents. In some embodiments, the D-malate salt is formed in a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the compound of formula I is a benzoate salt. In some embodiments, the benzoate salt is formed in dichloromethane. In some embodiments, the compound of formula I is a propionate salt. In some embodiments, the propionate salt is formed in dichloromethane. In some embodiments, the compound of formula I is a maleate salt. In some embodiments, the maleate salt is formed in dichloromethane.

Provided herein is a chloride salt of a compound of formula I. In some embodiments, the chloride salt has an O:free base ratio of about 1.1:1.

In some embodiments, provided herein is a composition comprising a chloride salt of a compound of Formula I. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some

- In some embodiments, the composition can have a purity of at least about 98%. For example, the composition can have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of Formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of Formula I. For example, the composition can contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of Formula I.

In some embodiments, provided herein is a chloride salt of a compound of formula I that exhibits an endotherm observed at about 230-245°C, such as about 241°C or 234°C, as measured by DSC, associated with sorbed water. In some embodiments, provided herein is a chloride salt of a compound of formula I that exhibits a melting point of about 241°C, as measured by DSC.

In some embodiments, the chloride salt of the compound of Formula I undergoes a mass loss of about 7.4% from the start of heating to about 255°C, as measured by TGA.

Provided herein are methods for producing a chloride salt of a compound of formula I. In some embodiments, the provided method comprises suspending a composition comprising a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of hydrochloric acid to the resulting mixture to obtain the chloride salt as a residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 1:1 by volume. In some embodiments, the provided method comprises suspending a composition comprising a compound of formula I in dimethylacetamide and adding a solution of hydrochloric acid to the resulting mixture to obtain the chloride salt as a residual solid. In some embodiments, the hydrochloric acid is added in an aqueous solution. In some embodiments, the solvent is used in an amount of about 46 volumes. In some embodiments, the solvent is used in an amount of about 50 volumes. In some embodiments, the solvent is used in an amount of about 75 volumes. In some embodiments, the temperature of the slurry is periodically changed from about 0 °C to about room temperature. In some embodiments, the temperature of the slurry is periodically changed from about 0 °C to about 30 °C. In some embodiments, the temperature of the slurry is periodically changed from about 40 °C to about room temperature. In some embodiments, the proposed method includes adding MTBE to the slurry. In some embodiments, the proposed method includes adding about 125 volumes of MTBE. In some embodiments, the proposed method includes maturing the slurry. In some embodiments, the proposed method includes maturing the slurry for about 3 hours. In some embodiments, the proposed method includes maturing the slurry for about 13 hours. In some embodiments, the proposed method includes maturing the slurry for about 20 to about 40 hours, such as about 30 hours. In some embodiments, the proposed method includes stirring the slurry. In some embodiments, the proposed method includes separating the solid by filtration.

Provided herein is a bromide salt of a compound of formula I. In some embodiments, the bromide salt has a Br:free base ratio of about 1.1:1.

In some embodiments, provided herein is a composition comprising a bromide salt of a compound of Formula I. In some embodiments, the composition can be substantially pure. For example, the composition can have a purity of at least about 90%. In some embodiments, the composition can have a purity of at least about 95%. In some embodiments, the composition can have a purity of at least about 98%. For example, the composition can have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition can contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I.

In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits an endotherm observed at about 235-250°C, such as about 238°C, as measured by DSC, associated with sorbed water. In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits an endotherm observed at about 220-235°C, such as about 225°C, as measured by DSC, associated with sorbed water. In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits a melting point of about 225°C, as measured by DSC. In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits a melting point of about 238°C, as measured by DSC.

In some embodiments, the bromide salt of the compound of Formula I undergoes a mass loss of about 10.3% from the start of heating to about 255°C, as measured by TGA.

Provided herein are methods for producing a bromide salt of a compound of formula I. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of hydrobromic acid to the resulting mixture to obtain the bromide salt as a residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of formula I in dimethylacetamide and adding a solution of hydrobromic acid to the resulting mixture to obtain the bromide salt as a residual solid. In some embodiments, the hydrobromic acid is added in an aqueous solution. In some embodiments, the solvent is used in an amount of 46 volumes. In some embodiments, the solvent is used in an amount of about 50 volumes. In some embodiments, the temperature of the suspension is periodically varied from about 0 °C to about room temperature. In some embodiments, the temperature of the slurry is periodically changed from about 30°C to about room temperature. In some embodiments, the temperature of the slurry is periodically changed from about 40°C to about room temperature. In some embodiments, the proposed method further comprises adding MTBE to the slurry. In some embodiments, the proposed method comprises adding about 150 volumes of MTBE. In some embodiments, the proposed method comprises maturing the slurry. In some embodiments, the proposed method comprises maturing the slurry for about 1 hour. In some embodiments, the proposed method comprises maturing the slurry for about 13 hours. In some embodiments, the proposed method comprises maturing the slurry for about 10 hours to about 30 hours, such as about 20 hours. In some embodiments, the proposed method comprises isolating the solid by filtration.

Provided herein is an L-malate salt of the compound of formula I. In some embodiments, the L-malate salt has a malate:free base ratio of about 0.97:1.

In some embodiments, provided herein is a composition comprising an L malate salt of a compound of Formula I. In some embodiments, the composition can be substantially pure. For example, the composition can have a purity of at least about 90%. In some embodiments, the composition can have a purity of at least about 95%. In some embodiments, the composition can have a purity of at least about 98%. For example, the composition can have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8%, or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition can contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I.

In some embodiments, provided herein is an L-malate salt of a compound of formula I that exhibits an endotherm observed at about 205-220°C, such as about 208°C, as measured by DSC, associated with sorbed water. In some embodiments, provided herein is an L-malate salt of a compound of formula I that exhibits a melting point of about 208°C, as measured by DSC.

In some embodiments, the L-malate salt of the compound of Formula I undergoes a mass loss of about 17.7% from the start of heating to about 253°C, as measured by TGA.

Provided herein are methods for producing an L-malate salt of a compound of formula I. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of L-malic acid to the resulting mixture to obtain the L-malate salt as a residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 3.3:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 6:1 by volume. In some embodiments, the L-malic acid is added in an ethanol solution. In some embodiments, the solvent is used in an amount of about 46 volumes. In some embodiments, the solvent is used in an amount of about 26 volumes. In some embodiments, the temperature of the suspension is periodically varied from about 0 °C to about room temperature. In some embodiments, the disclosed method includes maturing the suspension. In some embodiments, the proposed method includes maturing the suspension for about 5 hours to about 24 hours, such as about 13 hours. In some embodiments, the proposed method includes maturing the suspension for about 10 hours to about 30 hours, such as about 20 hours. In some embodiments, the proposed method includes stirring the suspension. In some embodiments, the proposed method includes isolating the solid by filtration.

Provided herein is a D-malate salt of the compound of formula I. In some embodiments, the D-malate salt has a malate:free base ratio of about 0.97:1.

In some embodiments, provided herein is a composition comprising a D-malate salt of a compound of Formula I. In some embodiments, the composition can be substantially pure. For example, the composition can have a purity of at least about 90%. In some embodiments, the composition can have a purity of at least about 95%. In some embodiments, the composition can have a purity of at least about 98%. For example, the composition can have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition can contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I.

In some embodiments, provided herein is a D-malate salt of a compound of formula I that exhibits an endotherm observed at about 205-215°C, such as about 208°C, as measured by DSC, associated with sorbed water. In some embodiments, provided herein is a D-malate salt of a compound of formula I that exhibits a melting point of about 209°C, as measured by DSC.

In some embodiments, the D-malate salt of the compound of Formula I undergoes a mass loss of about 18.4% from the start of heating to about 250°C, as measured by TGA.

Provided herein are methods for producing a D-malate salt of a compound of formula I. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of D-malic acid to the resulting mixture to obtain the D-malate salt as a residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is 3.3:1 by volume. In some embodiments, the D-malic acid is added in an ethanol solution. In some embodiments, the solvent is used in an amount of about 46 volumes. In some embodiments, the temperature of the suspension is periodically varied from about 0 °C to about room temperature. In some embodiments, the disclosed method includes maturing the suspension. In some embodiments, the proposed method includes maturing the suspension for about 13 hours. In some embodiments, the proposed method includes maturing the suspension for about 20 hours. In some embodiments, the proposed method includes isolating the solid by filtration.

Formula II.

The present invention provides a compound of formula II

including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula II provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods such as those described in U.S. Provisional Patent Application Serial Numbers 62/406,252; 62/447,850; 62/491,164; 62/554,817; or 62/566,093 can be used, and these applications are incorporated herein by reference in their entireties.

Provided herein are polymorphic forms of the compound of formula II. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of the compound of formula II, including, for example, polymorphic forms 1, 2, 7, and 8. In some embodiments, the polymorphic form of the compound of formula II is a pharmaceutically acceptable salt. In some embodiments, the compound of formula II is a phosphate salt.

Form 1.

One such polymorph is a polymorph known as Form 1. Form 1 is an anhydrous polymorph of the compound of Formula II. In some embodiments, Form 1 has an X-ray powder diffraction (XRPD) pattern obtained with CuKα1 radiation with at least peaks at °2θ values of 16.5±0.2, 18.9±0.2, and 26.0±0.2. In some embodiments, Form 1 has an XRPD pattern with at least peaks at °2θ values of 16.5±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2, and 26.0±0.2. In some embodiments, Form 1 has an XRD pattern with at least peaks at °2θ values of 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2, and 28.3±0.2. In some embodiments, Form 1 has an XRD pattern with at least peaks at °2θ values of 9.8±0.2, 16.5±0.2, 17.8±0.2, 18.9+0.2, 23.8±0.2, 25.0±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2, and 28.3±0.2.

In some embodiments, provided herein is a composition comprising Polymorph Form 1. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula II. For example, in some embodiments, the composition is substantially free of other anhydrous forms of the compound of Formula II. In some embodiments, the composition comprises less than about 15% by weight of other forms of the compound of Formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% of form 2, form 7, form 8, or a combination of two or more of them.

In some embodiments, the present document provides a polymorphic Form 1 that exhibits an endotherm observed at about 185-200°C, such as about 195°C, measured by differential scanning calorimetry (DSC) associated with sorbed water. In some embodiments, polymorphic Form 1 exhibits an endothermic event observed at about 200-210°C, such as about 207°C. In some embodiments, these endotherms are observed using a scan rate of 10°C per minute.

In some embodiments, provided herein is a polymorphic Form 1 that exhibits an endothermic event observed with an onset at about 190°C, as measured by thermogravimetric/differential thermal analysis (TG/DTA). In some embodiments, polymorphic Form 1 undergoes a mass loss of about 0.4% up to about 200°C, such as from about 190 to about 200°C. In some embodiments, polymorphic Form 1 exhibits an endothermic event with an onset at about 204°C. In some embodiments, said endothermic event is accompanied by a corresponding mass loss of about 0.2%.

The present invention provides methods for preparing polymorph Form 1. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of formula II in a solvent selected from the group consisting of 1,4-dioxane, 1-butanol, 1-propanol, acetone, anisole, chloroform, cyclohexane, cyclohexanone, dichloromethane, DMSO, ethanol, ethyl acetate, isopropyl alcohol, methyl ethyl ketone, methyl acetate, 2-ethoxyethanol, 2-methyl THF, methyl isobutyl ketone (MIBK), nitromethane, and THF to obtain polymorph Form 1 as a residual solid. In some embodiments, the solvent is ethyl acetate. In some embodiments, the solvent is present in a mixture with water, for example, the solvent can be a mixture of water and acetone or water and acetonitrile. In some embodiments, water is present in an amount of about 20% by weight. In some embodiments, water is present in an amount of about 50% by weight. In some embodiments, the temperature of the slurry is periodically changed from about 40°C to about room temperature. In some embodiments, the periodic temperature change is performed for about 60 hours to about 84 hours, such as about 72 hours. In some embodiments, the proposed method further includes collecting the residual solid. In some embodiments, the residual solid is collected by filtration. In some embodiments, the proposed method further includes drying the residual solid, such as under vacuum. In some embodiments, drying is performed at a temperature of about 30 to about 50°C, such as about 40°C.

In some embodiments, a method for producing polymorph Form 1 is provided. The method includes providing a composition comprising a compound of formula II in a solvent. In some embodiments, polymorph Form 1 can be obtained by evaporating the solvent from the composition comprising the compound of formula II to obtain polymorph Form 1 as a residual solid, wherein the solvent is selected from the group consisting of dichloromethane, DMSO, methyl acetate, 2-ethoxyethanol, nitromethane, and a mixture of acetonitrile and water (20%). In some embodiments, the method includes evaporating the solvent from the composition comprising the compound of formula II to obtain a mixture of polymorph Form 1 and another polymorph as a residual solid, wherein the solvent is selected from the group consisting of acetone, chloroform, and THF. In some embodiments, the residual solid is a mixture of Form 1 and Form 8.

In some embodiments, polymorph Form 1 can be prepared by cooling a solution containing a compound of Formula II in acetone to a temperature of about 5°C to precipitate polymorph Form 1 as a residual solid. In some embodiments, the residual solid is a mixture of Form 1 and Form 8.

In some embodiments, polymorphic Form 1 can be obtained by recrystallizing a composition comprising a compound of formula II to obtain polymorphic Form 1, wherein the recrystallization solvent is selected from the group consisting of a mixture of DMSO and water and a mixture of dichloromethane and heptane.

Form 2.

The present invention also provides a polymorph known as Form 2. Form 2 is a hydrated polymorphic form of the compound of Formula II. In some embodiments, Form 2 has an XRD pattern obtained with CuKα1 radiation with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, and 24.2±0.2. In some embodiments, Form 2 has an XRD pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2, and 24.2±0.2. In some embodiments, Form 2 has an XRD pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, and 24.6±0.2. In some embodiments, Form 2 has an XRD pattern with at least peaks at °2θ values of 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2, and 31.2±0.2.

In some embodiments, provided herein is a composition comprising Polymorph Form 2. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8%, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula II. In some embodiments, the composition comprises less than about 15% by weight of other forms of the compound of Formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% of form 1, form 7, form 8, or a combination of two or more of them.

In some embodiments, the present document provides a polymorphic form 2 that exhibits an endotherm observed at about 190-200°C, such as about 197.5°C, as measured by DSC, associated with sorbed water. In some embodiments, polymorphic form 2 exhibits an endothermic event observed at about 200-210°C, such as about 207.5°C. In some embodiments, these endotherms are observed using a scan rate of 10°C per minute.

In some embodiments, provided herein is a polymorphic Form 2 that exhibits a mass loss of about 0.7% from the onset of heating to about 165°C, as measured by TG/DTA. In some embodiments, polymorphic Form 2 exhibits an endothermic event observed with its onset at about 194°C. In some embodiments, polymorphic Form 2 undergoes a mass loss of about 0.2% up to about 200°C, such as from about 194°C to about 200°C. In some embodiments, polymorphic Form 2 exhibits an endothermic event with its onset at about 205°C.

Provided herein are methods for producing polymorphic Form 2. In some embodiments, the provided method comprises suspending a composition comprising a compound of formula II in a mixture of ethanol and water to obtain polymorphic Form 2 as a residual solid. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the temperature of the suspension is periodically changed from about 40°C to about room temperature. In some embodiments, the periodic temperature change is performed for from about 60 hours to about 84 hours, such as about 72 hours. In some embodiments, the provided method further comprises collecting the residual solid. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried on a filter bed.

Form 7.

The present invention provides a polymorph known as Form 7. Form 7 is a hydrated polymorphic form of the compound of formula II. In some embodiments, Form 7 has an X-ray diffraction pattern obtained with CuKα1 radiation with at least peaks at

- 23 041220 °2θ values of 16.6±0.2, 18.0±0.2, and 19.9±0.2. In some embodiments, form 7 has an XRD pattern with at least peaks at °2θ values of 16.6±0.2, 18.0±0.2, 19.3±0.2, 19.9±0.2, and 23.3±0.2. In some embodiments, form 7 has an XRD pattern with at least peaks at °2θ values of 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.9±0.2, 23.3±0.2, and 25.1±0.2. For example, in some embodiments, Form 7 has an XRD pattern with at least peaks at °2θ values of 15.8±0.2, 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.91±0.2, 21.4±0.2, 23.3±0.2, and 25.1±0.2.

In some embodiments, provided herein is a composition comprising polymorph Form 7. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula II. In some embodiments, the composition comprises less than about 15% by weight of other forms of the compound of Formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% of Form 1, Form 2, Form 8, or a combination of two or more of them.

In some embodiments, provided herein is a polymorphic form 7 that exhibits an endotherm observed at about 145-155°C, such as about 150°C, as measured by DSC, associated with sorbed water. In some embodiments, polymorphic form 7 exhibits an endotherm observed at about 190-205°C, such as about 201°C. In some embodiments, polymorphic form 7 exhibits an endothermic event observed at about 205-210°C, such as about 207°C. In some embodiments, these endotherms are observed using a scan rate of 10°C per minute.

In some embodiments, provided herein is a polymorphic form 7 that exhibits an endothermic event observed with its onset at about 147°C, as measured by TG/DTA. In some embodiments, polymorphic form 7 undergoes a mass loss of about 7% up to about 150°C, such as from about 145°C to about 155°C. In some embodiments, the mass loss is a loss of solvent. In some embodiments, the mass loss is about two equivalents of solvent compared to the amount of compound present in the sample. In some embodiments, the solvent is water. In some embodiments, polymorphic form 7 exhibits an endothermic event observed with its onset at about 196°C. In some embodiments, polymorphic Form 7 dehydrates upon heating to form polymorphic Form 1. In some embodiments, said endothermic event is associated with a transition observed in Form 1. In some embodiments, said transition is associated with an endothermic event of Form 1 observed from its onset at about 206°C.

Provided herein are methods for producing polymorph Form 7. In some embodiments, the provided method comprises suspending a composition comprising a compound of formula II in a mixture of 1,4-dioxane and water to obtain polymorph Form 7 as a residual solid. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the temperature of the suspension is periodically changed from about 40°C to about room temperature. In some embodiments, the periodic temperature change is performed for about 60 to about 84 hours, such as about 72 hours. In some embodiments, the provided method further comprises collecting the residual solid. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried on a filter bed.

Form 8.

The present invention provides a polymorph known as Form 8. Form 8 is a solvated polymorphic form of the compound of Formula II. Polymorphic Form 8 is an isopropyl alcohol solvate polymorphic form of the compound of Formula II. In some embodiments, Form 8 has an XRD pattern obtained with CuKα1 radiation with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, and 24.2±0.2. In some embodiments, Form 8 has an XRD pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2, and 24.2±0.2. In some embodiments, shape 8 has an XRD pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, and 24.6±0.2. In some embodiments, shape 8 has an XRD pattern with,

- 24 041220 at least peaks at °2θ values of 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2,

23.4±0.2, 24.2±0.2, 24.6±0.2 and 31.2±0.2.

In some embodiments, provided herein is a composition comprising polymorph Form 8. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula II. In some embodiments, the composition comprises less than about 15% by weight of other forms of the compound of Formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% of Form 1, Form 2, Form 7, or a combination of two or more of them.

In some embodiments, provided herein is a polymorphic form 8 that exhibits an endotherm observed at about 165-175°C, such as about 172°C, as measured by DSC, associated with sorbed water. In some embodiments, polymorphic form 8 exhibits an endotherm observed at about 185-200°C, such as about 196°C. In some embodiments, polymorphic form 8 exhibits an endothermic event observed at about 200-210°C, such as about 206°C. In some embodiments, these endotherms are observed using a scan rate of 10°C per minute.

In some embodiments, provided herein is a polymorphic Form 8 that exhibits an endothermic event observed at about 165°C as measured by TG/DTA. In some embodiments, polymorphic Form 8 undergoes a mass loss of about 4% up to about 165°C. In some embodiments, the mass loss is a loss of solvent. In some embodiments, the mass loss is about 0.5 equivalents of solvent. In some embodiments, the solvent is IPA. In some embodiments, polymorphic Form 8 exhibits an endothermic event observed with its onset at about 191°C. In some embodiments, the endothermic event is associated with a transition observed in Form 1. In some embodiments, the transition is associated with an endothermic event of Form 1 observed with its onset at about 205°C.

Provided herein are methods for producing polymorph Form 8. In some embodiments, the provided method comprises suspending a composition comprising a compound of Formula II in a solvent selected from the group consisting of IPA and 1-propanol to obtain polymorph Form 8 as a residual solid. In some embodiments, the temperature of the suspension is cycled from about 40°C to about room temperature. In some embodiments, the temperature is cycled for about 60 hours to about 84 hours, such as about 72 hours. In some embodiments, the provided method further comprises collecting the residual solid. In some embodiments, the residual solid is collected by filtration. In some embodiments, the provided method further comprises drying the residual solid, such as under vacuum. In some embodiments, the drying is performed at a temperature of about 30°C to about 50°C, such as about 40°C.

In some embodiments, a method for producing polymorph Form 8 is provided. The provided method includes providing a composition comprising a compound of formula II in a solvent. In some embodiments, the provided method includes evaporating the solvent from the composition comprising the compound of formula II, including its amorphous and polymorphic forms, to obtain a mixture of polymorph Form 8 and another polymorphic form as a residual solid. In some embodiments, the residual solid is a mixture of polymorph Form 8 and polymorph Form 1. In some embodiments, the solvent is acetone. In some embodiments, the solvent is chloroform. In some embodiments, the solvent is THF.

Salts of formula II.

In some embodiments, the compound of Formula II is a pharmaceutically acceptable salt. For example, pharmaceutically acceptable salts of the compound of Formula II can include, but are not limited to, sulfate, tosylate, naphthalene-2-sulfonate, oxalate, phosphate, tartrate, and fumarate salts. In some embodiments, the compound of Formula II is a sulfate salt. In some embodiments, the sulfate salt is formed in a solvent mixture. In some embodiments, the solvent is a mixture of IPA and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of Formula II is a tosylate salt. In some

- 25 041220 embodiments, the tosylate salt is formed in a mixture of solvents. In some embodiments, the solvent is a mixture of acetone and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of formula II is a naphthalene-2-sulfonate salt. In some embodiments, the naphthalene-2-sulfonate salt is formed in a mixture of solvents. In some embodiments, the solvent is a mixture of THF and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of formula II is an oxalate salt. In some embodiments, the oxalate salt is formed in a mixture of solvents. In some embodiments, the solvent is a mixture of 1,4-dioxane and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the oxalate salt is formed by evaporation from the mixture of solvents. In some embodiments, the solvent is a mixture of THF and water. In some embodiments, the compound of formula II is a tartrate salt. In some embodiments, the tartrate salt is formed in a solvent mixture. In some embodiments, the solvent is a mixture of IPA and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of formula II is a fumarate salt. In some embodiments, the fumarate salt is formed in a solvent mixture. In some embodiments, the solvent is a mixture of THF and water. In some embodiments, the compound of formula II is a phosphate salt. In some embodiments, the phosphate salt is formed in a solvent mixture. In some embodiments, the solvent is a mixture of acetone and water. In some embodiments, the solvent is a mixture of IPA and water. In some embodiments, water is present in an amount of about 10% by weight.

Provided herein is a phosphate salt of a compound of formula II. In some embodiments, the phosphate salt has a _PO4 :free base ratio of about 1.4:1. In some embodiments, the phosphate salt has an XRD pattern obtained with CuKα1 radiation with at least peaks at °2θ values of 3.6±0.2, 16.7±0.2, and 18.2±0.2. In some embodiments, the phosphate salt has an XRD pattern with at least peaks at °2θ values of 3.6±0.2, 15.9±0.2, 16.7±0.2, 17.8±0.2, and 18.2±0.2. In some embodiments, the phosphate salt has an XRD pattern with at least peaks at °2θ values of 3.6±0.2, 6.2±0.2, 15.9±0.2, 16.7±0.2, 17.8±0.2, 18.2±0.2, 20.3±0.2, and 25.5±0.2. For example, in some embodiments, the phosphate salt has an XRD pattern with at least peaks at °2θ values of 3.6±0.2, 6.2±0.2, 15.9±0.2, 16.7±0.2, 17.8±0.2, 18.2±0.2, 19.1±0.2, 20.3±0.2, 20.9±0.2, and 25.5±0.2.

In some embodiments, the invention provides a composition comprising a phosphate salt of a compound of Formula II. In some embodiments, the composition can be substantially pure. For example, the composition can be at least about 90% pure. In some embodiments, the composition can be at least about 95% pure. In some embodiments, the composition can be at least about 98% pure. For example, the composition can be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula II. In some embodiments, the composition includes less than about 15% by weight of other forms of the compound of Formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II.

In some embodiments, provided herein is a phosphate salt of a compound of formula II that exhibits an endotherm observed at about 165-175°C, such as about 170°C, as measured by DSC, associated with sorbed water. In some embodiments, said endotherm is observed using a scan rate of 10°C per minute.

In some embodiments, provided herein is a phosphate salt of a compound of formula II that exhibits a melting point of about 167°C as measured by TG/DTA. In some embodiments, the phosphate salt of a compound of formula II undergoes a mass loss of about 1.3% from the onset of heating to about 150°C. In some embodiments, the phosphate salt of a compound of formula II exhibits a second mass loss of about 1.2% with an onset at about 167°C.

Provided herein are methods for producing a phosphate salt of a compound of formula II. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of formula II in a mixture of water and IPA and adding a phosphoric acid solution to the resulting mixture to form the phosphate salt as a residual solid. In some embodiments, the water is present in an amount of about 10% by weight. In some embodiments, the acid is a 1 M phosphoric acid solution. In some embodiments, the temperature of the slurry is periodically varied from about 40°C to about room temperature. In some

- In embodiments, the periodic temperature change is carried out for about 12 to about 48 hours, such as about 24 hours. In some embodiments, the proposed method further comprises centrifuging the composition and collecting the residual solid. In some embodiments, the residual solid is washed with a solvent. In some embodiments, the solvent is IPA. In some embodiments, the proposed method further comprises drying the residual solid. In some embodiments, the residual solid is dried under vacuum. In some embodiments, drying is carried out at a temperature of about 30°C to about 50°C, such as about 40°C.

Formula III.

The present invention provides a compound of formula III:

O

III including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula III provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods such as those described in U.S. Provisional Patent Application Serial Numbers 62/406,252; 62/447,850; 62/491,164; 62/554,817; or 62/566,093 can be used, and these applications are incorporated herein by reference in their entirety.

Provided herein are polymorphic forms of the compound of formula III. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of the compound of formula III, including, for example, polymorphic form A. In some embodiments, the polymorphic form of the compound of formula III is a pharmaceutically acceptable salt.

Form A.

One such polymorph is a polymorph known as Form A. Form A is a polymorphic form of the compound of Formula III. In some embodiments, Form A has an XRD pattern obtained with CuKα1 radiation with at least peaks at °2θ values of 17.3±0.2, 19.2±0.2, and 23.9±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 4.7±0.2, 17.3±0.2, 18.8±0.2, 19.2±0.2, and 23.9±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 4.7±0.2, 6.8±0.2, 15.2±0.2, 17.3±0.2, 18.8±0.2, 19.2±0.2, 20.2±0.2, and 23.9±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 4.7±0.2, 6.8±0.2, 13.4±0.2, 15.2±0.2, 15.9±0.2, 17.3±0.2, 18.8±0.2, 19.2±0.2, 20.2±0.2, and 23.9±0.2.

In some embodiments, provided herein is a composition comprising Polymorph Form A. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula III. In some embodiments, the composition comprises less than about 15% by weight of other forms of the compound of Formula III. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula III. For example, the composition may contain less than about 15% of the amorphous form.

In some embodiments, the present document provides polymorphic form A that exhibits an endotherm observed at about 135-150°C, such as about 140.5°C or 146.5°C, as measured by DSC, associated with sorbed water.

The present invention provides methods for producing polymorph Form A. In some embodiments, the disclosed method comprises dissolving a compound of formula III in acetonitrile and adding water to obtain polymorph Form A as a solid. In some embodiments, the disclosed method comprises heating a composition comprising a compound of formula III in acetonitrile to reflux. In some embodiments, the ratio of acetonitrile to water is about 2:3 by volume. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried under

- 27 041220 high vacuum. In some embodiments, the residual solid is dried at about

40-45°C. In some embodiments, the residual solid is dried overnight.

Formula IV.

This document proposes a compound of formula IV

He

IV including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula IV provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods such as those described in U.S. Provisional Patent Application Serial Numbers 62/406,275; 62/447,849; 62/491,180; 62/531,690 or 62/566,030 can be used, and these applications are incorporated herein by reference in their entirety.

Provided herein are polymorphic forms of the compound of formula IV. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of the compound of formula IV, including, for example, polymorphic forms A and B. In some embodiments, the polymorphic form of the compound of formula IV is a pharmaceutically acceptable salt.

Form A.

One such polymorph is a polymorph known as Form A. Form A is a polymorphic form of the compound of Formula IV. In some embodiments, Form A has an XRD pattern obtained with CuKα1 radiation with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, and 21.9±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, 16.6±0.2, 19.4±0.2, and 21.9±0.2. In some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, 16.6±0.2, 19.4±0.2, 20.0±0.2, 20.5±0.2, 21.6±0.2, and 21.9±0.2. For example, in some embodiments, Form A has an XRD pattern with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, 16.6±0.2, 18.1±0.2, 18.8±0.2, 19.4±0.2, 20.0±0.2, 20.5±0.2, 21.6±0.2, and 21.9±0.2.

In some embodiments, provided herein is a composition comprising Polymorph Form A. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula IV. In some embodiments, the composition comprises less than about 15% by weight of other forms of the compound of Formula IV. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula IV. For example, the composition may contain less than about 15% of form B, the amorphous form, or a combination thereof.

In some embodiments, the present document provides polymorphic form A that exhibits an endotherm observed at about 145-155°C, such as about 149.9°C, as measured by DSC, associated with sorbed water.

The present invention provides methods for producing Polymorph Form A. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of formula IV in a solvent selected from the group consisting of acetone, acetonitrile, 2-butanol, chloroform, ethanol, ethyl acetate, heptane, hexane, isopropanol, MTBE, DMSO, THF, water, and combinations thereof to obtain Polymorph Form A as a residual solid. In some embodiments, the solvent is acetone, 2-butanol, or acetonitrile. In some embodiments, the solvent is present in a mixture with water, for example, the solvent can be a mixture of water and acetone, water and ethanol, or water and DMSO. In some embodiments, water is present in an amount of about 50% by weight. In some embodiments, water is present in an amount of about 40% by weight. In some embodiments, the solvent is present in a mixture with heptane, for example, the solvent can be a mixture of chloroform and heptane or heptane and acetone. In some embodiments, heptane is present in an amount of about 50% by weight. In some embodiments, heptane is present in an amount of about

- 28 041220

70% by weight. In some embodiments, Form A is prepared by adding an antisolvent to a solution of a compound of Formula IV in a solvent. In some embodiments, the antisolvent is heptane or water. In some embodiments, the solvent is DMSO and the antisolvent is water. In some embodiments, vapors of the antisolvent diffuse into the solution of the compound of Formula IV. In some embodiments, the proposed method further includes collecting the residual solid. In some embodiments, the residual solid is collected by filtration. In some embodiments, the proposed method includes washing the solid. In some embodiments, the proposed method includes washing the solid with water, MTBE, or a combination thereof. In some embodiments, the proposed method further includes drying the residual solid, such as under vacuum.

Form B.

One such polymorph is a polymorph known as Form B. Form B is a polymorphic form of the compound of Formula IV. In some embodiments, Form B has an XRD pattern obtained with CuKα1 radiation with at least peaks at °2θ values of 7.5±0.2, 13.7±0.2, and 16.9±0.2. In some embodiments, Form B has an XRD pattern with at least peaks at °2θ values of 7.5±0.2, 9.7±0.2, 13.7±0.2, 16.9±0.2, and 19.9±0.2. In some embodiments, Form B has an XRD pattern with at least peaks at °2θ values of 7.5±0.2, 9.7±0.2, 13.7±0.2, 14.5±0.2, 16.9±0.2, 19.4±0.2, 19.9±0.2, and 21.3±0.2. In some embodiments, Form B has an XRD pattern with at least peaks at °2θ values of 7.5±0.2, 9.7±0.2, 9.9±0.2, 13.7+0.2, 14.5+0.2, 16.9+0.2, 19.4+0.2, 19.9+0.2, 21.3±0.2, and 27.4±0.2.

In some embodiments, provided herein is a composition comprising Polymorph Form B. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may be at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% pure. In some embodiments, the composition is substantially free of other forms of the compound of Formula IV. In some embodiments, the composition comprises less than about 15% by weight of other forms of the compound of Formula IV. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula IV. For example, the composition may contain less than about 15% of Form A, the amorphous form, or a combination thereof.

In some embodiments, the present document provides polymorphic form B that exhibits an endotherm observed at about 160-170°C, such as about 164.6°C, as measured by DSC, associated with sorbed water.

Disclosed herein are methods for producing Polymorph Form B. In some embodiments, the disclosed method comprises suspending a composition comprising a compound of Formula IV in a mixture of ethanol and water to obtain Polymorph Form B as a residual solid. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the suspension is allowed to mature for about 24 to about 72 hours, such as about 36 hours. In some embodiments, the disclosed method further comprises collecting the residual solid. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried in a vacuum oven. In some embodiments, the residual solid is dried with a stream of nitrogen. In some embodiments, the residual solid is dried at room temperature. In some embodiments, the residual solid is dried for about 10 to about 20 hours, such as about 18 hours.

It should be understood that the 2-theta values in the XRPD diffraction patterns for the crystalline forms of the compounds of formula I-IV, for example, Form A of formula I, Forms 1, 2, 7 and 8 of formula I, Form A of formula III or Forms A and B of formula IV, as well as pharmaceutically acceptable salts thereof, for example, the chloride salt, the bromide salt, the malate salt and the phosphate salt, may vary somewhat depending on the instrument, as well as on variations in the preparation of the sample and variations from batch to batch, and, therefore, the stated values should not be understood as absolute. It should be understood that the peak positions in the XRPD diffraction pattern are indicated in terms of angular position (two theta) with an acceptable variability of ±0.2° 2θ. The variability of ±0.2° 2θ is used when comparing two powder XRPD diffraction patterns. In practice, if a diffraction peak in one diffraction pattern is assigned a range of angular positions (two theta) that represents the measured peak position of ±0.2°, and if these ranges of peak positions overlap, then the two peaks are considered to have the same angular position. For example, if a peak in one diffraction pattern is found to have a position of 11.0° 2θ, then for comparison purposes, the acceptable variability allows the peak to be assigned a position in the range 10.8°- 29 041220

11.2° 2θ. It should also be understood that the relative intensity of the peaks may vary depending on orientation effects, so that the intensity shown in the XRD patterns included herein is illustrative and should not be used for absolute comparison. Furthermore, it should be understood that for comparative purposes, some variability in the intensities of the peaks relative to their intensities shown in the XRD patterns is acceptable. Accordingly, it should be understood that the expression "essentially the same XRD pattern as shown in Fig. 1" means that for comparative purposes, at least 90% of the peaks shown in Fig. 1 are present.

The compounds provided herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms forming the compounds. That is, an atom, particularly when referred to in relation to a compound of formula I-IV, contains all isotopes and isotopic mixtures of that atom, such as naturally occurring isotopes in their natural abundance. For example, when referring to hydrogen, referring to 1H, ^2H , ^3H , or mixtures thereof is meant; when referring to carbon, referring to ^12C , ^13C , ^14C , or mixtures thereof is meant; when referring to nitrogen, referring to ^14N , ^15N , or mixtures thereof is meant; and when referring to oxygen, referring to ^16O , ^17O , ^18O , or mixtures thereof is meant. All isotopic variations of the compounds provided herein are considered to be within the scope of the invention.

For illustrative purposes, Schemes 1-6 provide general methods for preparing the compounds disclosed herein, as well as key intermediates. More detailed descriptions of individual reaction steps are provided, for example, in U.S. Provisional Patent Application Serial Numbers 62/329,895, 62/406,252, 62/447,850, 62/566,093, and 62/566,030, the entire contents of which are incorporated herein by reference. Those skilled in the art will recognize that other synthetic routes may be used to synthesize the compounds disclosed herein. Although specific starting materials and reagents are shown and described in the Schemes below, other starting materials and reagents may readily be substituted to prepare numerous derivatives and/or reaction conditions.

Scheme 1

Scheme 1 shows the general scheme for the synthesis of the compound of formula I (represented as compound

- 30 041220 and 13a for formula I in Scheme 1), wherein B is 1-methyl-1H-pyrazol-4-yl; X ¹ is N; X ² , X ³ and X ⁴ are CH; and D and E are represented by the structure where the wavy line denotes the point of attachment to the ring containing X ¹ , X ² , X ³ and X ⁴ .

Compound 2 is prepared by treating MSH reagent with 3-bromo-5-methoxypyridine, which is commercially available. The aminating reagent O-mesitylsulfonylhydroxylamine (MSH) can be prepared as described in Mendiola, J., et al., Org. Process Res. Dev. 2009, 13(2), 263-267. Compound 2 can be reacted with ethyl propiolate to give pyrazolo[1,5-a]pyridine, a mixture of compounds 3A and 3B, which are typically prepared in a ratio of about 2:1 to 9:1. A mixture of compounds 3A and 3B can be treated with 48% HBr at elevated temperatures, followed by recrystallization or chromatographic purification to isolate compound 4A as a minor isomer and compound 4B as a major isomer.

Isolated compound 4B can be functionalized with a formyl group using _POCl3 followed by purification to provide compound 5. The formyl group of compound 5 can be converted to an oxime group using NH2OH to provide compound 6. The oxime group of compound 6 can be converted to a nitrile group using acetic anhydride to provide compound 7. The B group can be installed by treating compound 7 with an appropriate boronic ester having the formula hetAr1-B( ^ORa )( ^ORb ), where ^hetAr1 is 1-methyl-1Hpyrazol-4-yl as described in formula I, using appropriate palladium catalyzed cross-coupling reaction conditions, such as Suzuki coupling conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, such as _Pcd2 (dba) ₃ , X-Phos and _Na2CO3 in dioxane at elevated temperatures) _. temperatures) to give compound 8, wherein B is 1-methyl-1H-pyrazol-4-yl as described in formula I. The methoxy group of compound 8 can be converted to a hydroxy group by treating compound 8 with aluminum trichloride to give compound 9. The free hydroxy group of compound 9 can be converted to a triflate group by treating compound 9 with a triflating reagent, for example, 1,1,1-trifluoro-Nphenyl-X-((trifluoromethyl)sulfonyl)methanesulfonamide, to give compound 10. Compound 12 can be prepared by condensation of compound 10 with the appropriate boronic ester compound 11, wherein ring D is where the wavy line represents the point of attachment of ring D to the ring containing X ¹ , X ² , X ³ and X ⁴ , and the asterisk represents the point of attachment to P ¹ ; X ¹ , X ² , X ³ and X ⁴ are as described above; P ¹ is an amino protecting group; Z is -B(OR ^x )(OR ^y ), Z is -B(OR ^a )(OR ^b ), and ^Ra and R ^b are H or C1-C6 alkyl, or ^Ra and R ^b together with the atoms to which they are bonded form a 5-6 membered ring optionally substituted with one to four C1-C ₃ alkyl groups, using appropriate palladium catalyzed cross-coupling reaction conditions, for example Suzuki coupling conditions (e.g. a palladium catalyst and optionally a ligand in the presence of an inorganic base, for example Pd ₂ (dba) ₃ , X-Phos and Na ₂ CO ₃ in dioxane at elevated temperatures). The protecting group on the D ring of compound 12 can be removed under standard conditions (e.g., the Boc protecting group can be removed by treating compound 12 under acidic conditions, such as with HCl). The deprotected D ring can be functionalized (i.e., reacted or treated with an appropriate reagent) to introduce an E group under standard conditions, such as those described above, to provide compound 13, where E is aju

Alternatively, compound 10 can be condensed with compound 14 using appropriate palladium-catalyzed cross-coupling conditions, such as Suzuki coupling conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, such as Pd( _PPh3 ) _{4 and Na2CO3} ), to provide compound 15. Compound 15 can be reacted with compound 16 under appropriate SNAr conditions (e.g., optionally in the presence of a base, such as _K2CO3 , and at elevated temperature) to provide compound 12a, wherein ring D of compound 16 is ^N1 , _the wavy line representing the point of attachment of ring D to the ring containing ^X1 , ^X2 , ^X3 , and ^X4 , and the asterisk representing the point of attachment to ^P1 ; ^X1 , ^X2 , ^X3 , and ^X4 are as described above;

- 31 041220

P ¹ is an amino protecting group; Z is -B(OR ^x )(OR ^y ), the second nitrogen atom being protected with a suitable amino protecting group before condensation. The protecting group on the D ring of 12a, if present, can be removed under standard conditions (e.g., the Boc group can be removed by treating 12a under acidic conditions, e.g., HCl). The deprotected D ring can be functionalized (i.e., reacted or treated with an appropriate reagent) to introduce an E group under standard conditions, such as those described above, to give 13a, where E is

Scheme 2

Scheme 2 shows an alternative method for synthesizing compound 13, wherein B, ^X1 , ^X2 , ^X3 , ^X4 , D, and E are as described in Scheme 1. Compound 4A (prepared as in Scheme 1) can be functionalized with a formyl group using _POCl3 to give compound 17. The formyl group can be converted to an oxime group using NH2OH to give compound 18. The oxime group can be converted to a nitrile group using acetic anhydride to give compound 19. The methoxy group of compound 19 can be converted to a hydroxy group by treating compound 19 with aluminum trichloride to give compound 20. Compound 21 can be prepared by condensation of compound 20 with the appropriate boronic ester compound 11, wherein ring D is

wherein the wavy line represents the point of attachment of ring D to the ring containing X ¹ , X ² , X ³ and X ⁴ , and the asterisk represents the point of attachment to P ¹ ; X ¹ , X ² , X ³ and X ⁴ are as described above; P ¹ is an amino protecting group; Z is -B(OR ^a )(OR ^b ), and ^Ra and R ^b are H or C1-C6 alkyl, or ^Ra and R ^b together with the atoms to which they are bonded form a 5-6 membered ring optionally substituted with one to four C1-C3 alkyl groups, using appropriate palladium catalyzed cross-coupling reactions, for example Suzuki coupling conditions (e.g. palladium catalyst and optionally ligand in the presence of an inorganic base, for example Pd(PPh ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperatures). The unsubstituted nitrogen atom in ring D is protected before condensation with a suitable amino protecting group. The free hydroxyl group of compound 21 can be converted into a triflate group by treating compound 21 with a triflating reagent, such as 1,1,1-trifluoro-N-phenyl-N((trifluoromethyl)sulfonyl)methanesulfonamide, to provide compound 22. The B group can be introduced by treating compound 22 with an appropriate boronic ester having the formula hetAr ¹ -B(OR ^a )(OR ^b ), wherein hetAr ¹ is 1-methyl-1H-pyrazol-4-yl as described in formula I, and R ^a and R ^b are H or C1-C6 alkyl, or R ^a and R ^b, together with the atoms to which they are bonded, form a 5-6 membered ring optionally substituted with one to four C1-C3 alkyl groups, using appropriate palladium catalyzed cross-coupling reaction conditions, such as Suzuki coupling conditions (e.g. a palladium catalyst and optionally a ligand in the presence of an inorganic base, e.g. Pd ₂ (dba) ₃ , X-Phos and Na ₂ CO ₃ in dioxane at elevated temperature) to provide compound 12, wherein B is 1-methyl-1H-32 041220 pyrazol-4-yl, as described in formula I. The protecting group on ring D of compound 12, if present, can be deprotected under standard conditions (e.g. the Boc group can be deprotected by treating compound 12 under acidic conditions, e.g. HCl in propan-2-ol). The deprotected ring D can be functionalized (e.g. reacted or treated with an appropriate reagent) to introduce a group E under standard conditions, such as those described above, to provide compound 13, wherein E is

Scheme 3

Scheme 3 shows a general scheme for the synthesis of a compound of formula II or formula III (represented as compound 12 for formula II or III in Scheme 3), where B is -CH ₂ C(CH ₃ ) ₂ OH; X ¹ is N; X ² , X ^{3 ,} and X ⁴ are CH; and D and E are represented by the structure

or respectively, where the wavy line denotes the point of attachment to the ring containing X ¹ , X ² , X ³ and X ⁴ .

Compound 2 is prepared by treating commercially available 3-bromo-5-methoxypyridine (compound 1) with O-(mesitylsulfonyl)hydroxylamine. O-mesitylsulfonylhydroxylamine can be prepared as described in Mendiola et al., Org. Process Res. Dev. (2009) 13(2):263–267. Compound 2 can be reacted with ethyl propiolate to give a mixture of compounds 3A and 3B, which are typically prepared in a ratio of about 2:1 to 9:1, respectively. The mixture of compounds 3A and 3B can be treated with 48% HBr at elevated temperature, followed by recrystallization or chromatographic purification to isolate compound 4A as a minor isomer and compound 4B as a major isomer. After isolation, compound 4A can be treated with POCl ₃ to give compound 5. The formyl group can be converted to an oxime group with NH ₂ OH to give compound 6. The oxime group can be converted to a nitrile group with acetic anhydride to give compound 7. The methoxy group of compound 7 can be converted to a hydroxy group by treating compound 7 with aluminum trichloride to give compound 8.

To prepare compound 9, compound 8 can be reacted with a reagent such as ^H0 X?x where X is a leaving atom or group (such as a halide or triflate), in the presence of a suitable base (e.g., an alkali metal carbonate such as potassium carbonate). Compound 11 can then be prepared by condensing compound 9 with the appropriate boronic ester compound 10 (where ring D is

, wherein the wavy line denotes the point of attachment of the D ring to the ring containing X ¹ , X ² , X ³ and X ⁴ , and the asterisk denotes the point of attachment to P ¹ ; X ¹ , X ² , X ³ and X ⁴ are as described above; P ¹ is an amino protecting group; Z is -B(OR ^x )(OR ^y ), and R ^z and R ^y are H or (16C)alkyl, or R ^x and R ^y together with the atoms to which they are bonded form a 5-6 membered ring optionally substituted with 1-4 substituents selected from (C ₁ -C ₃ -alkyl)), using appropriate palladium catalyzed cross-coupling reaction conditions, e.g. Suzuki coupling conditions (e.g. a palladium catalyst and optionally a ligand in the presence of an inorganic base, e.g. Pd(PPh ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperature). Compound 12 can then be prepared from compound 11 by removing the protecting group P ¹ under standard conditions (e.g. the Boc group can be removed by treating compound 11 under acidic conditions, e.g. HCl), followed by functionalization (i.e. reacting or treating compound 11 with an appropriate reagent) to introduce the E group

(for formula II) or (for formula III) under standard conditions.

Alternatively, compound 8 can be condensed with an appropriate boronic ester compound 10 to provide compound 11a using appropriate palladium-catalyzed cross-coupling reaction conditions, such as Suzuki coupling conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, such as Pd( _PPh3 ) _{4 and Na2CO3} in dioxane at elevated temperature). Compound 11a can then be reacted with a reagent such as ^HO X'X where X is a leaving atom or group (such as a halide or triflate) under Mitsunobu reaction conditions (e.g., PPh3 and diisopropyl azodicarboxylate) to provide compound 11. Compound 12 can then be prepared from compound 11 as described above.

Compound 9 (prepared, for example, as described in Scheme 3), in which B is such as

- 34 041220 described above can be condensed with the corresponding boronic ester 13 (where X ¹ , X ² , X ³ and X ⁴ are as described above; L ² is a leaving group such as a triflate or a halide); Z is -B(OR ^x )(OR ^y ), and R ^z and R ^y are H or (16C)alkyl, or R ^x and R ^y together with the atoms to which they are bonded form a 5-6 membered ring optionally substituted with 1-4 substituents selected from (C1-C3 alkyl)), using appropriate palladium catalyzed cross coupling reaction conditions, for example Suzuki coupling conditions (e.g. a palladium catalyst and optionally a ligand in the presence of an inorganic base, for example Pd(PPh3)4 and Na2CO3 in dioxane at elevated temperature), to give compound 14. Compound 16 can be prepared by condensing compound 14 with compound 15, wherein ring D is as defined above and P ¹ is an amino protecting group, under appropriate SNAr conditions (e.g. optionally in the presence of a base such as K2CO ₃ and at elevated temperature).

The protecting group P ¹ on the D ring of compound 16 can be removed under standard conditions (e.g., the Boc group can be removed by treating compound 16 under acidic conditions, such as HCl) to provide compound 12, where E is H (i.e., the D ring is deprotected). The deprotected D ring can then be functionalized (i.e., reacted or treated with an appropriate reagent) to introduce the E group under standard conditions, such as those described above, to provide compound 12, where E is as described above for Scheme 3.

Scheme 5

² FOR ZV

4A 4B 5 6

(R ^b )m

Scheme 5 shows the general scheme for the synthesis of a compound of formula IV (represented as compound 12 for formula IV in Scheme 5), where B is -CH ₂ C(CH ₃ ) ₂ OH; X ¹ is N; X ² , X ³ , and X ⁴ are CH; and D, E, (R ^a ) _n , and (R ^b ) m are represented by the structure

where the wavy line denotes the attachment point to the ring containing X ¹ , X ² , X ³ and X ⁴ .

Compound 2 is prepared by treating commercially available 3-bromo-5-methoxypyridine (compound

- 35 041220

1) O-(mesitylsulfonyl)hydroxylamine. O-mesitylsulfonylhydroxylamine can be prepared as described in Mendiola, J., et al., Org. Process Res. Dev. 2009, 13(2), 263-267. Compound 2 can be reacted with ethyl propiolate to give a mixture of compounds 3A and 3B, which are typically prepared in a ratio of about 2:1 to 9:1, respectively. The mixture of compounds 3A and 3B can be treated with 48% HBr at elevated temperature, followed by recrystallization or chromatographic purification to isolate compound 4A as a minor isomer and compound 4B as a major isomer. After isolation, compound 4A can be treated with POCl ₃ to give compound 5. The formyl group can be converted to an oxime group with NH ₂ OH to give compound 6. The oxime group can be converted to a nitrile group with acetic anhydride to give compound 7. The methoxy group of compound 7 can be converted to a hydroxy group by treating compound 7 with aluminum trichloride to give compound 8.

Compound 11a can then be reacted with a reagent such as ^H ^ ^χ where X is a leaving atom or group (such as a halide or triflate) under Mitsunobu reaction conditions (PPh ₃ and diisopropyl azodicarboxylate) to give compound 11. Compound 12 can then be prepared from compound 11 as described above.

Alternatively, compound 9 can be prepared by reacting compound 8 with a reagent such as ^Ηθ χ- _X where X is a leaving atom or group (such as a halide or triflate), in the presence of a base (e.g., an alkali metal carbonate such as potassium carbonate). Compound 11 can then be prepared by condensing compound 9 with the appropriate boronic ester compound 10 using appropriate palladium-catalyzed cross-coupling reaction conditions, such as Suzuki coupling conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, such as Pd( _PPh3 ) _{4 and Na2CO3} in dioxane at elevated temperature).

Scheme 6

Scheme 6 shows another general scheme for the synthesis of compound 12, where B, X ¹ , X ² , X ³ , X ⁴ , ring D and E are as described above for Scheme 5.

Compound 9 (prepared, for example, as described in Scheme 5), in which B is as described for Scheme 5, can be condensed with compound 13 (where X ¹ , X ² , X ³ and X ⁴ are as described for Scheme 5; L ² is a leaving group such as a triflate or a halide); Z is -B(OR ^x )(OR ^y ), and R ^z and R ^y are H or (1-6C)alkyl, or R ^x and R ^y together with the atoms to which they are bonded form a 5-6 membered ring optionally substituted with 1-4 substituents selected from (C1-C ₃ alkyl)), using appropriate palladium catalyzed cross-coupling reaction conditions, e.g. Suzuki coupling conditions (e.g. a palladium catalyst and optionally a ligand in the presence of an inorganic base, e.g. Pd(PPh ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperature), to provide compound 14. Compound 12 can be prepared by condensing compound 14 with compound 15 under appropriate SNAr conditions (e.g. optionally in the presence of a base, such as K ₂ CO ₃ , and at elevated temperature), wherein compound 15 is defined as

- 36 041220

or a salt thereof as described for formula IV.

An amino protecting group, as used herein, refers to a derivative of groups commonly used to block or protect an amino group while reactions are carried out on other functional groups on a compound. Examples of suitable protecting groups for use in any of the methods described herein include carbamates, amides, alkyl and aryl groups, imines, as well as many N-heteroatom derivatives that can be removed to restore the desired amino group. Non-limiting examples of amino protecting groups are acetyl, trifluoroacetyl, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (CBz), and 9-fluorenylmethyleneoxycarbonyl (Fmoc).

Additional examples of these groups and other protecting groups are presented in T. W. Greene et al., Greene's Protective Groups in Organic Synthesis (New York: Wiley Interscience, 2006).

Hydroxy groups can be protected with any convenient hydroxy protecting group, such as described in T. W. Greene et al., Greene's Protective Groups in Organic Synthesis. New York: Wiley Interscience, 2006. Examples include benzyl, trityl, silyl ethers, etc.

The nitrogen atoms in the compounds described in any of the above methods may be protected with any convenient nitrogen protecting group, such as those described in Greene & Wuts, eds., Protecting Groups in Organic Synthesis, 2nd ed. New York; John Wiley & Sons, Inc., 1991. Examples of nitrogen protecting groups include acyl and alkoxycarbonyl groups such as tert-butoxycarbonyl (BOC), phenoxycarbonyl, and [2-(trimethylsilyl)ethoxy]methyl (SEM).

3. Treatment methods.

The ability of a compound of formula I-IV, including its polymorphic forms and pharmaceutically acceptable salts, to act as a RET inhibitor can be demonstrated using the assays described in Examples 8 and 9.

In some embodiments, the compounds provided herein exhibit potent and selective inhibition of RET. For example, the compounds provided herein exhibit nanomolar potency against wild-type RET and RET kinase encoded by a RET gene containing an activating mutation or a RET kinase inhibitor resistance mutation, including, for example, the KIF5B-RET fusion, the ATP cleft front mutations G810R and G810S, the activating mutation M918T, and the gatekeeper mutations V804M, V804L, and V804E, with minimal activity against related kinases.

In some embodiments, the compounds provided herein exhibit nanomolar potency against an altered RET fusion protein encoded by a RET gene that encodes a RET fusion protein (e.g., any of the RET fusion proteins described herein, including, but not limited to, CCDC6-RET or KIF5B-RET), and said RET gene comprises a RET kinase inhibitor resistance mutation (e.g., any RET mutation described herein, including, but not limited to, V804M, V804L, or V804E), such that the altered RET protein is a RET fusion protein that exhibits RET kinase resistance due to the presence of an amino acid substitution or deletion that is responsible for resistance to a RET kinase inhibitor. Non-limiting examples include CCDC6-RET-V804M and KIF5B-RET-V804M. In some embodiments, the compounds provided herein exhibit nanomolar potency against an altered RET protein encoded by a RET gene that comprises a RET mutation (e.g., any RET mutation described herein, including, but not limited to, C634W or M918T) and that comprises a RET kinase inhibitor resistance mutation (e.g., any RET kinase inhibitor resistance mutation described herein, including, but not limited to, V804M, V804L, or V804E), such that the altered RET protein exhibits RET kinase resistance due to the presence of an amino acid substitution or deletion that is responsible for resistance to a RET kinase inhibitor.

In some embodiments, the compounds of formula I-IV or pharmaceutically acceptable salts, amorphous or polymorphic forms thereof are selectively directed to RET kinase. For example, a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof may selectively and specifically target RET kinase over other kinases or non-kinase targets.

In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, exhibits at least 30-fold selectivity for RET kinase over another kinase. For example, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, exhibits at least 40-fold selectivity; at least 50-fold selectivity; at least 60-fold selectivity; at least 70-fold selectivity; at least 80-fold selectivity; at least 90-fold selectivity; at least 100-fold selectivity; at

- 37 041220 at least 200-fold selectivity; at least 300-fold selectivity; at least 400-fold selectivity; at least 500-fold selectivity; at least 600-fold selectivity; at least 700-fold selectivity; at least 800-fold selectivity; at least 900-fold selectivity; or at least 1000-fold selectivity for RET kinase over another kinase. In some embodiments, the selectivity for RET kinase over another kinase is measured in a cellular assay (e.g., a cellular assay provided herein).

In some embodiments, the compounds provided herein may exhibit selectivity for RET kinase over KDR kinase (e.g., VEGFR2). In some embodiments, selectivity for RET kinase over KDR kinase is observed without loss of potency for RET kinase encoded by a RET gene that contains an activating mutation or a RET kinase inhibitor resistance mutation (e.g., a gatekeeper mutation). In some embodiments, the selectivity over KDR kinase is at least 10-fold (e.g., at least 40-fold selectivity; at least 50-fold selectivity; at least 60-fold selectivity; at least 70-fold selectivity; at least 80-fold selectivity; at least 90-fold selectivity; at least 100-fold selectivity; at least 150-fold selectivity; at least 200-fold selectivity; at least 250-fold selectivity; at least 300-fold selectivity; at least 350-fold selectivity; or at least 400-fold selectivity) compared to inhibition of KIF5B-RET (e.g., the disclosed compounds are more potent against KIF5B-RET than against KDR). In some embodiments, the selectivity for RET kinase over KDR kinase is about 30-fold. In some embodiments, the selectivity for RET kinase over KDR kinase is at least 100-fold. In some embodiments, the selectivity for RET kinase over KDR kinase is at least 150-fold. In some embodiments, the selectivity for RET kinase over KDR kinase is at least 400-fold. Without being bound by theory, effective inhibition of KDR kinase is believed to be a common feature of multikinase inhibitors (MKIs) that target RET and may account for the dose-limiting toxicity observed with such compounds.

In some embodiments, the inhibition of V804M is similar to that observed for wild-type RET. For example, the inhibition of V804M is within about 2-fold (e.g., about 5-fold, about 7-fold, about 10-fold) of the inhibition of wild-type RET (e.g., the provided compounds are equally effective against wild-type RET and V804M). In some embodiments, the selectivity for wild-type RET or V804M kinase over another kinase is measured in an enzyme assay (e.g., an enzyme assay provided herein). In some embodiments, the compounds provided herein exhibit selective cytotoxicity against RET mutant cells.

In some embodiments, the inhibition of G810S and/or G810R is similar to that observed for wild-type RET. For example, the inhibition of G810S and/or G810R is within about 2-fold (e.g., about 5-fold, about 7-fold, about 10-fold) of the inhibition of wild-type RET (e.g., compounds provided herein are equally effective against wild-type RET and G810S and/or G810R). In some embodiments, the selectivity for a wild-type RET or G810S and/or G810R kinase over another kinase is measured in an enzyme assay (e.g., an enzyme assay provided herein). In some embodiments, compounds provided herein exhibit selective cytotoxicity against RET mutant cells.

In some embodiments, the compounds provided herein exhibit penetration into the brain and/or the central nervous system (CNS). Such compounds can cross the blood-brain barrier and inhibit RET kinase in the brain and/or other CNS structures. In some embodiments, the compounds provided herein can cross the blood-brain barrier in a therapeutically effective amount. For example, treating a patient for cancer (e.g., a RET-associated cancer, such as a RET-associated brain cancer or a CNS cancer) can include administering to the patient (e.g., orally administering) a compound provided herein. In some such embodiments, the compounds provided herein are useful for treating a primary brain tumor or a metastatic brain tumor. For example, a RET-associated primary brain cancer or a metastatic brain cancer.

In some embodiments, the compounds of formula I-IV or pharmaceutically acceptable salts, amorphous or polymorphic forms thereof exhibit one or more of high GI absorption, low clearance, and low potential for drug-drug interactions.

Compounds of formula I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms are useful for the treatment of diseases and disorders that can be treated with a kinase inhibitor

- 38 041220

RET, such as RET-associated diseases and disorders, e.g., proliferative disorders such as cancers, including hematological cancers and solid tumors (e.g., advanced solid tumors and/or RET-positive solid tumors) and gastrointestinal disorders such as IBS.

In some embodiments, the compounds of formula I-IV or pharmaceutically acceptable salts, amorphous or polymorphic forms thereof are useful for preventing the diseases and disorders described herein (e.g., autoimmune diseases, inflammatory diseases and cancer). The term prevention in this context means completely or partially preventing the onset, recurrence or spread of a disease or pathological condition described herein, or a symptom thereof.

The term RET-associated disease or disorder, as used herein, refers to diseases or disorders associated with or characterized by deregulation of the RET gene, RET kinase (also referred to herein as RET kinase protein), or the expression or activity or level of any of them (e.g., one or more) (e.g., any type of deregulation of the RET gene, RET kinase, RET kinase domain, or the expression or activity or level of any of them, as described herein). Non-limiting examples of a RET-associated disease or disorder include, for example, cancer and gastrointestinal disorders such as irritable bowel syndrome (IBS).

The term RET-associated cancer as used herein refers to cancers associated with or characterized by deregulation of the RET gene, RET kinase (also referred to herein as RET kinase protein), or the expression or activity or level of any of them. Non-limiting examples of RET-associated cancers are described herein.

The term "deregulation of the RET gene, RET kinase, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising the RET kinase domain and a fusion partner, a mutation in the RET gene that results in the expression of a RET protein that contains a deletion of at least one amino acid compared to the wild-type RET protein, a mutation in the RET gene that results in the expression of a RET protein with one or more point mutations compared to the wild-type RET protein, a mutation in the RET gene that results in the expression of a RET protein with at least one inserted amino acid compared to the wild-type RET protein, a gene duplication that results in an increased level of RET protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of RET protein in a cell), an alternative splice version of mRNA RET that results in the formation of a RET protein having a deletion of at least one amino acid in the RET protein compared to a wild-type RET protein), or increased expression (e.g., increased level) of a wild-type RET kinase in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, deregulation of a RET gene, RET protein, or the expression or activity or level of any of them can be a mutation in a RET gene that encodes a RET protein that is constitutively active or has increased activity compared to a protein encoded by a RET gene that does not contain such a mutation. For example, deregulation of the RET gene, RET protein, or expression or activity or level of any of them can be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of RET that contains a functional kinase domain and a second portion of a partner protein (i.e., a non-RET). In some examples, deregulation of the RET gene, RET protein, or expression or activity or level of any of them can be the result of a gene translocation of one RET gene with another non-RET gene. Non-limiting examples of fusion proteins are provided in Table 1. Non-limiting examples of point mutations/insertions/deletions of the RET kinase protein are provided in Tables 2 and 2a. Additional examples of RET kinase protein mutations (e.g., point mutations) are RET inhibitor resistance mutations. Non-limiting examples of RET inhibitor resistance mutations are described in Tables 3 and 4.

In some embodiments, deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them may be caused by an activating mutation in a RET gene (see, e.g., chromosomal translocations that result in the expression of any of the fusion proteins listed in Table 1). In some embodiments, deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them may be caused by a genetic mutation that results in the expression of a RET kinase that has increased resistance to inhibition by a RET kinase inhibitor and/or a multikinase inhibitor (MKI), e.g., compared to a wild-type RET kinase (see, e.g., amino acid substitutions in Tables 3 and 4). In some embodiments, deregulation of the RET gene, RET kinase, or expression or activity or level of any of them may be caused by a mutation in a nucleic acid encoding an altered RET protein (e.g., a RET fusion protein or a RET protein having a mutation (e.g., a primary mutation)), resulting in

- 39 041220 expression of an altered RET protein that has increased resistance to inhibition by a RET kinase inhibitor and/or a multi-kinase inhibitor (MKI), for example, compared to wild-type RET kinase (see, for example, amino acid substitutions in Tables 3 and 4). The exemplary point mutations, insertions, and deletions of RET kinase provided in Tables 2 and 2a may be caused by an activating mutation and/or may result in the expression of RET kinase that has increased resistance to inhibition by a RET kinase inhibitor and/or a multi-kinase inhibitor (MKI).

The term "activating mutation" describes a mutation in a RET kinase gene that results in the expression of a RET kinase that has increased kinase activity, for example, compared to a wild-type RET kinase, for example, when assayed under the same conditions. For example, an activating mutation may result in the expression of a fusion protein that comprises a RET kinase domain and a fusion partner. In another example, an activating mutation may be a mutation in a RET kinase gene that results in the expression of a RET kinase that has one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions (e.g., any combination of any of the amino acid substitutions described herein) that has increased kinase activity, for example, compared to a wild-type RET kinase, for example, when assayed under the same conditions. In another example, an activating mutation may be a mutation in the RET kinase gene that results in the expression of a RET kinase having one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid deletions, e.g., compared to a wild-type RET kinase, e.g., when assayed under the same conditions. In another example, an activating mutation can be a mutation in a RET kinase gene that results in the expression of a RET kinase having at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20) inserted amino acids, compared to a wild-type RET kinase, e.g., an illustrative wild-type RET kinase described herein, e.g., when assayed under the same conditions. Additional examples of activating mutations are known in the art.

The term "wild type" describes a nucleic acid (e.g., a RET gene or RET mRNA) or a protein (e.g., a RET protein) that occurs in a subject that does not suffer from a RET-associated disease, such as a RET-associated cancer (and optionally also does not have an increased risk of developing a RET-associated disease and/or is not predisposed to a RET-associated disease), or that occurs in a cell or tissue obtained from a subject that does not suffer from a RET-associated disease, such as a RET-associated cancer (and optionally also does not have an increased risk of developing a RET-associated disease and/or is not predisposed to a RET-associated disease).

The term regulatory agency refers to a government agency that authorizes the medical use of pharmaceutical agents in a given country. For example, a non-limiting example of a regulatory agency is the Food and Drug Administration (FDA).

Provided herein is a method of treating cancer (e.g., a RET-associated cancer) in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. For example, provided herein are methods of treating RET-associated cancer in a patient in need of such treatment, comprising a) detecting deregulation of a RET gene, RET kinase, or the expression or activity or level of any of them in a sample obtained from the patient; and b) administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the deregulation of a RET gene, RET kinase, or the expression or activity or level of any of them comprises one or more fusion proteins. Non-limiting examples of RET gene fusion proteins are described in Table 1. In some embodiments, the fusion protein is KIF5B-RET. In some embodiments, the deregulation of the RET gene, RET kinase, or the expression or activity or level of any of them comprises one or more point mutations/insertions in the RET kinase protein. Non-limiting examples of point mutations/insertions/deletions in the RET kinase protein are described in Tables 2 and 2a. In some embodiments, the point mutations/insertions/deletions in the RET kinase protein are selected from the group consisting of M918T, M918V, C634W, V804L, V804M, G810S, and G810R. In some embodiments, the point mutations/insertions/deletions in the RET kinase protein are in a RET fusion protein (e.g., any of the RET gene fusion proteins described in Table 1). In some embodiments, the compound of Formula IIV is a polymorphic form. In some embodiments, the compound is polymorphic Form A of the compound of Formula I. In some embodiments, the compound is polymorphic Form 1 of the compound of Formula II. In some embodiments, the compound is polymorphic Form 2 of the compound of Formula II. In some variations

- 40 041220 In some embodiments, the compound is polymorphic form 7 of the compound of formula II. In some embodiments, the compound is polymorphic form 8 of the compound of formula II. In some embodiments, the compound is polymorphic form A of the compound of formula III. In some embodiments, the compound is polymorphic form A of the compound of formula IV. In some embodiments, the compound is polymorphic form B of the compound of formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is a chloride salt of the compound of Formula I. In some embodiments, the compound is a bromide salt of the compound of Formula I. In some embodiments, the compound is an L-malate salt of the compound of Formula I. In some embodiments, the compound is a D-malate salt of the compound of Formula I. In some embodiments, the compound is a phosphate salt of the compound of Formula II. In some embodiments, the phosphate salt is a sesquiphosphate salt (e.g., _PO4 :free base 1.4:1).

In some embodiments of any method or use described herein, the cancer (e.g., RET-associated cancer) is a hematological cancer. In some embodiments of any method or use described herein, the cancer (e.g., RET-associated cancer) is a solid tumor (e.g., an advanced solid tumor and/or a RET fusion-positive solid tumor). In some embodiments of any method or use described herein, the cancer (e.g., a RET-associated cancer) is lung cancer (e.g., small cell lung carcinoma or non-small cell lung carcinoma), thyroid cancer (e.g., papillary thyroid cancer, medullary thyroid cancer (e.g., sporadic medullary thyroid cancer or hereditary medullary thyroid cancer), differentiated thyroid cancer, recurrent thyroid cancer, or refractory differentiated thyroid cancer), thyroid adenoma, endocrine gland neoplasms, lung adenocarcinoma, pulmonary bronchiolar cell carcinoma, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, breast cancer, carcinoma breast, breast neoplasm, colorectal cancer (eg, metastatic colorectal cancer), papillary renal cell carcinoma, ganglioneuromatosis of the gastrointestinal mucosa, inflammatory myofibroblastic tumor, or cervical cancer. In some embodiments of any method or use described herein, the cancer (e.g., RET-associated cancer) is selected from the group: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, unknown primary carcinoma, cardiac tumors, cervical cancer, childhood cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, focal neoplasm, neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, cutaneous angiosarcoma, bile duct cancer, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumor, gestational trophoblastic disease, glioma, hairy cell tumor, hairy cell leukemia, head and neck cancer, thoracic neoplasms, head and neck neoplasms, CNS tumor, primary CNS tumor, cardiac cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, Kaposi's sarcoma, renal cell cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, oral and lip cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone, osteocarcinoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck, midline carcinoma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasms, focal neoplasms, neoplasms, myelogenous leukemia, myeloid leukemia, multiple myeloma, mycloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, cancer of the nasopharynx, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, lung neoplasm, respiratory tract cancer, lung neoplasm, respiratory tract neoplasm, bronchogenic carcinoma, bronchial neoplasms, oral cancer, lip cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pa

- 41 041220 pillomatosis, paraganglioma, cancer of the paranasal sinuses and nasal cavity, parathyroid gland cancer, penile cancer, laryngeal cancer, pheochromositoma, pituitary cancer, plasma cell neoplasm, pleuropulmonary blastoma, breast cancer in pregnancy, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, colon cancer, colon neoplasms, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, Spitz tumors, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, mimoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, unknown primary carcinoma, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer and Wilms' tumor.

In some embodiments, the hematological cancer (e.g., hematological cancers that are RET-associated cancers) is selected from the group consisting of leukemias, lymphomas (non-Hodgkin's lymphoma), Hodgkin's disease (also referred to as Hodgkin's lymphoma), and myeloma, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia (JMML), adult T-cell ALL, AML with trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), and multiple myeloma (MM). Additional examples of hematologic cancers include myeloproliferative disorders (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic primary myelofibrosis (IMF/IPF/PMF). In one embodiment, the hematologic cancer (e.g., a hematologic cancer that is a RET-associated cancer) is AML or CMML.

In some embodiments, the cancer (e.g., a RET-associated cancer) is a solid tumor. Examples of solid tumors (e.g., solid tumors that are RET-associated cancers) include, for example, thyroid cancer (e.g., papillary thyroid carcinoma, medullary thyroid carcinoma), lung cancer (e.g., lung adenocarcinoma, small cell lung carcinoma), pancreatic cancer, pancreatic ductal carcinoma, breast cancer, colon cancer, colorectal cancer, prostate cancer, renal cell carcinoma, head and neck tumors, neuroblastoma, and melanoma. See, for example, Nature Reviews Cancer, 2014, 14, 173-186.

In some embodiments, the cancer is selected from the group consisting of lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer, papillary renal cell carcinoma, ganglioneuromatosis of the gastrointestinal mucosa, and cervical cancer.

In some embodiments, the patient is a human.

Compounds of formula I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms are also useful for the treatment of RET-associated cancer.

Accordingly, provided herein is also a method of treating a patient diagnosed with or identified as having a RET-associated cancer, such as any of the illustrative RET-associated cancers described herein, comprising administering to said patient a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof, as described herein.

Deregulation of RET kinase, a RET gene, or expression or activity, or level of any (e.g., one or more) thereof, can promote tumorigenesis. For example, deregulation of RET kinase, a RET gene, or expression or activity, or level of any of them can be a translocation, overexpression, activation, amplification, or mutation of RET kinase, a RET gene, or a RET kinase domain. Translocation can include translocation of a gene resulting in expression of a fusion protein that comprises a RET kinase domain and a fusion partner. For example, the fusion protein can have increased kinase activity compared to a wild-type RET protein. In some embodiments, a mutation in a RET gene can include mutations in the RET ligand binding site, extracellular domains, kinase domain, and regions involved in protein:protein interactions and downstream signaling. In some embodiments, a mutation (e.g., an activating mutation) in the RET gene may result in the expression of a RET kinase having one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions (e.g., one

- 42,041,220 or more amino acid substitutions in the kinase domain (e.g., amino acid positions 7,231,012 in the wild-type RET protein), the gatekeeper amino acid (e.g., amino acid position 804 in the wild-type RET protein), the P-loop (e.g., amino acid positions 730-737 in the wild-type RET protein), the DFG motif (e.g., amino acid positions 892-894 in the wild-type RET protein), the amino acids of the solvent front of the ATP cleft (e.g., amino acid positions 758, 811, and 892 in the wild-type RET protein), the activation loop (e.g., amino acid positions 891-916 in the wild-type RET protein), the C-helix in the loop preceding the C-helix (e.g., amino acid positions 768-788 in the wild-type RET protein) and/or in an ATP-binding site (e.g., at amino acid positions 730-733, 738, 756, 758, 804, 805, 807, 811, 881, and 892 in the wild-type RET protein). In some embodiments, the mutation may be a gene amplification of the RET gene. In some embodiments, a mutation (e.g., an activating mutation) in the RET gene may result in the expression of a RET kinase or RET receptor that is missing at least one amino acid (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids) compared to a wild-type RET protein. In some embodiments, the deregulation of RET kinase may be increased expression (e.g., increased levels) of wild-type RET kinase in a mammalian cell due to aberrant cellular signaling and/or deregulated autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). In some embodiments, a mutation (e.g., an activating mutation) in the RET gene can result in the expression of a RET kinase or RET receptor that has at least one inserted amino acid (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids) compared to a wild-type RET protein. In some embodiments, the deregulation of RET kinase may be increased expression (e.g., increased levels) of wild-type RET kinase in a mammalian cell (e.g., compared to a control non-cancerous cell), such as due to aberrant cellular signaling and/or deregulated autocrine/paracrine signaling. Other deregulations may include splice variants of RET mRNA. In some embodiments, the wild-type RET protein is an exemplary wild-type RET protein described herein.

In some embodiments, deregulation of a RET gene, RET kinase, or expression or activity, or level of any of them comprises overexpression of a wild-type RET kinase (e.g., resulting in autocrine activation). In some embodiments, deregulation of a RET gene, RET kinase, or expression or activity, or level of any of them comprises overexpression, activation, amplification, or mutation in a chromosomal segment containing a RET gene or a portion thereof, including, for example, a portion of a kinase domain or a portion capable of exhibiting kinase activity.

In some embodiments, deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them comprises one or more chromosomal translocations or inversions resulting in a fusion of the RET gene. In some embodiments, deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them is the result of genetic translocations in which the expressed protein is a fusion protein comprising residues from a non-RET partner protein and comprises a minimum of a functional RET kinase domain.

Non-limiting examples of RET fusion proteins are presented in Table 1.

- 43 041220

Table 1

Illustrative RET Merger Partners and Cancers

Merger Partner Non-limiting examples of RET-associated cancer(s) BCR Chronic myelomonocytic leukemia (CM ML) CLIP1 Adenocarcinoma KIF5B NSCLC, ovarian cancer, spitzoid neoplasms; lung adenocarcinoma ^{3 4 14 28} ; adenosquamous carcinomas ¹⁵ CCDC6 (also called PTC1, D10S170 or H4) NSCLC, colon cancer, papillary thyroid cancer; adenocarcinomas; lung adenocarcinoma; metastatic colorectal cancer ⁵ ; adenosquamous carcinomas ¹⁵ , breast cancer ³⁰ RTC1ex9 (new CCDC6 permutation) Metastatic papillary thyroid cancer ² NCOA4 (also called RTSZ, ELE1 and RFG) Papillary thyroid cancer ²¹ , NSCLC, colon cancer, salivary gland cancer, metastatic colorectal cancer ⁵ ; lung adenocarcinoma ¹⁵ ; adenosquamous carcinomas ¹⁵ ; diffuse sclerosing variant of papillary thyroid cancer ¹⁰ , breast cancer ³⁰ , acinar- cell carcinoma ³² , mammary analogue of secretory carcinoma ³³ TRIM33 (also called RTC7, RFG7HTIF1G) NSCLC, papillary thyroid cancer, lung adenocarcinoma ⁴⁰ , miscellaneous ²² ERC1 (also called ELKS and RAB61P2) Papillary thyroid cancer, breast cancer FGFR1OP CMML, primary myelofibrosis with secondary acute myeloid leukemia MBD1 (also called PCM1) Papillary thyroid cancer PRKAR1A (also called PTC2) Papillary thyroid cancer TRIM24 (also called RTC6) Papillary thyroid cancer KTN1 (also called RTC8) Papillary thyroid cancer GOLGA5 (also called RTC5) Papillary thyroid cancer, spitzoid neoplasms NOOKZ Papillary thyroid cancer KIAA1468 (also called RTC9 and RFG9) Papillary thyroid cancer, lung adenocarcinoma ^{8 12} TRIM27 (also called RFP) Papillary thyroid cancer ACAR13 Papillary thyroid cancer FKBP15 Papillary thyroid cancer, acute myeloid leukemia ⁴⁰ SPECC1L Papillary thyroid cancer; thyroid carcinoma TBL1XR1 Papillary thyroid cancer; thyroid carcinoma

- 44 041220

CEP55 Diffuse gastric cancer ⁷ CUX1 Lung adenocarcinoma ACBD5 Papillary thyroid carcinoma MYH13 Medullary thyroid carcinoma ¹ Not described Inflammatory myofibroblastic tumor® PIBF1 Lung cell carcinoma of bronchioles ⁹ KIAA1217 (also called SKT) Papillary thyroid cancer ^{10 13} Lung adenocarcinoma ¹⁴ NSCLC ¹⁴ MPRIP NSCLC ¹¹ HRH4-RET Thyroid cancer and/or papillary thyroid carcinoma ¹⁷ Ria-RET Thyroid cancer and/or papillary thyroid carcinoma ¹⁷ RFG8 Papillary thyroid carcinoma ¹⁸ FOXP4 Lung adenocarcinoma ¹⁹ MYH10 Childhood myofibromatosis ²⁰ HTIF1 Various ²² H4L Various ²² RTS4 (new NCO4/ELE1 permutation) Papillary thyroid cancer ²³ FRMD4A NSCLC ²⁴ SQSTM1 Papillary thyroid carcinoma ²⁵ AFAP1L2 Papillary thyroid carcinoma ²⁵ AFAP1 NSCLC ³¹ PPFIBP2 Papillary thyroid carcinoma ²⁵ EML4 NSCLC PARD3 NSCLC ²⁷ RASGEF1A Breast cancer ³⁰ TEL (also called ETV6) In vitro ³⁴ , secretory carcinoma ⁵¹ RUFY1 Colon and Rectal Cancer ³⁵ OLFM4 Small intestine cancer ³⁸ UEVLD Papillary thyroid carcinoma ²⁹ DLG5 Non-anaplastic thyroid cancer (NAT) ³⁷ RRBP1 Colon cancer ³⁸ AN KZ Papillary thyroid carcinoma ³⁹ PICALM NSCLC ⁴⁰ MYO5C NSCLC ⁴¹ ERNA5 NSCLC ⁴⁰ RUFY2 Lung cancer ⁴² KIF13A Lung adenocarcinoma ⁴³ , NSCLC ⁴⁵ TNIP1 Colon and Rectal Cancer ⁴⁴ SNRNP70 Colon and Rectal Cancer ⁴⁴

- 45 041220

MRLN Thyroid carcinoma ⁴⁸ LMNA Spitzoid melanoma ⁴⁷ RUFY3 Papillary thyroid carcinoma TFG MYO5A Pigmented spindle cell nevus (PSCN) Reed ⁴⁸ ADD3 Lung adenocarcinoma ⁴⁹ JMJD1C NSCLC ⁵⁰ RBPMS DOCK1 TAF3 NCOA1 NSCLC ⁵²

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⁴¹ Lee et al. Annals of Oncology, 28(2), 292-297. doi: 10.1093/annonc/mdw559, 2016.

⁴² Zheng et al. Nature Medicine, 20(12), 1479-1484. doi: 10.1038/nm.3729, 2014.

⁴³ Zhang et al. Lung Cancer, 118, 27-29. doi: 10.1016/j.lungcan.2017.08.019, 2018.

⁴⁴ Morano et al. Molecular Cancer Therapeutics, (January 2018) Vol. 17, No. 1, suppl. Supplement. Abstract number: B049. Event information: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics 2017.

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⁴⁷ U.S. Patent Application Publication No. 2016/0010068.

⁴⁸ VandenBoom, et al. Am. J. Surg. Pathol. 42(8): 1042-1051, 2018. doi:

10.1097/PAS.00000000000001074 ⁴⁹ Cao, et al. Onco. Targets. Ther. 2018(11):2637–2646, 2018. doi: 10.2147/OTT.S155995 ⁵⁰ Luo, et al. Int. J. Cancer, 2018. Epubl. before print, doi: 10.1002/ijc.31542 ⁵¹ Guilmette, et al. Hum Pathol. pii: S0046-8177(18)30316-2, 2018. doi:

10.1016/j.humpath.2018.08.011 ⁵² Zhao, et al. Journal of Clinical Oncology Vol. 36, No. 15, suppl. [S], MA e21139.

In some embodiments, deregulation of a RET gene, RET kinase, or expression or activity, or level of any of them comprises one or more deletions (e.g., deletion of the amino acid at position 4), insertions, or point mutation(s) in RET kinase. In some embodiments, deregulation of a RET gene, RET kinase, or expression or activity, or level of any of them comprises deletion of one or more residues in RET kinase, resulting in constitutive activity of the RET kinase domain.

In some embodiments, deregulation of a RET gene, RET kinase, or expression or activity or level of any of them comprises at least one point mutation in the RET gene that results in the production of a RET kinase having one or more amino acid substitutions, insertions, or deletions compared to a wild-type RET kinase (see, for example, the point mutations listed in Table 2).

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Table 2

Amino acid substitutions/insertions/deletions in the RETA kinase protein

Amino acid position 2

Amino acid position 3

Amino acid position 4

Amino acid position 5

Amino acid position 6

Amino acid position 7

Amino acid position 8

Amino acid position 11

Amino acid position 12

Amino acid position 13

Amino acid position 20

Amino acid position 32 (eg S32L)

Amino acid position 34 (eg D34S)

Amino acid position 40 (eg L40P)

Amino acid position 45 (eg A45A) ³⁹

Amino acid position 56 (eg L56M) ³⁰

Amino acid position 64 (eg P64L)

Amino acid position 67 (eg R67H)

Amino acid position 77 (e.g. R77C)® ⁵

Amino acid position 114 (eg R114H)

Amino acid position 136 (eg, glutamic acid for stop codon)

Amino acid position 145 (eg V145G)

Amino acid position 177 (e.g. R177L)® ⁷

Amino acid position 180 (eg, arginine for stop codon)

Amino acid position 200

Amino acid position 270 (eg P270L)® ⁵

Amino acid position 278 (eg T278N) ⁵⁷

Amino acid position 292 (eg,

V292M)

Amino acid position 294

Amino acid position 321 (eg,

G321R)

Amino acid position 330 (eg R330Q)

Amino acid position 338 (eg T338I)

Amino acid position 360 (eg

R360W)

Amino acid position 373 (eg alanine for frameshifting)

Δ Amino acid positions 378 - 385 with insertion of one amino acid (eg, D378 G385>E)

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Amino acid position 393 (eg F393L)

Amino acid position 423 (eg G423R) ²⁷

Amino acid position 428 (eg E428K) ⁵⁷

Amino acid position 432 (eg A432A ³⁹ )

Amino acid position 446 (eg G446R) ²⁸

Δ Amino acid positions 505-506 (6 bp in-frame deletion of germline exon 7) ³

Amino acid position 510 (eg A510V)

Amino acid position 511 (eg E511K)

Amino acid position 513 (eg G513D) ⁷ *

Amino acid position 515 (eg C515S, C515W ⁴ )

Amino acid position 525 (eg R525W) ⁷ *

Amino acid position 531 (eg, C531R or 9 bp duplication ² )

Amino acid position 532 (eg, duplication) ²

Amino acid position 533 (eg G533C, G533S)

Amino acid position 534 (e.g. L534L)®

Amino acid position 550 (eg G550E)

Amino acid position 591 (eg V591I)

Amino acid position 593 (eg G593E)

Amino acid position 595 (e.g. E595D and E595A) ¹⁸

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Amino acid position 600 (eg,

R600Q)

Amino acid position 602 (eg I602V) ⁶

Amino acid position 603 (e.g. K603Q, K603E ² )

Amino acid position 606 (eg Y606C)

Amino acid position 609 (e.g. C609Y, C609S, C609G, C609R, C609F, C609W, C609C ³² )

Amino acid position 611 (e.g. C611R, C611S, C611G, C611Y, C611F, C611W)

Amino acid position 616 (eg E616Q) ²³

Δ Amino acid position 616 ^s4

Amino acid position 618 (C618S, C618Y, C618R, C618G, C618F, C618W, stop ⁵⁶ )

Amino acid position 619 (eg F619F)

Amino acid position 620 (e.g. C620S, C620W, C620R, C620G, C620L, C620Y, C620F, C620A ⁴⁷ )

Δ Amino acid positions 612-620 ⁷⁴

Amino acid position 622 (eg P622L) ⁶⁸

Amino acid position 623 (eg E623K)

Amino acid position 624 (eg D624N)

Amino acid position 628 (eg P628N) ⁷³

Amino acid positions 629-631 (e.g. L629-D631delinsH) ⁸⁰

Amino acid position 630 (e.g. C630A, C630R, C630S, C630Y, C630F, C630W)

Δ Amino acid position 630 ⁵⁶

Amino acid position 631 (e.g. D631N,

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D631Y, D631A, D631G, D631V, D631E)

Δ Amino acid position 631 ^s9

Amino acid positions 631-633>V (i.e.

residues 631-633 are replaced by one valine residue)

Amino acid positions 631-633>A (i.e.

residues 631-633 are replaced by one alanine residue)

Amino acid positions 631-633>E (i.e.

residues 631-633 are replaced by one glutamic acid residue)

Δ Amino acid positions 631-633 (eg D631 - L633)

Δ Amino acid positions 631-634 (eg D631-C634)

Amino acid position 632 (e.g. E632K, E632G ^{5 11} , E632V® ² , 632 for frameshift ⁴⁷ )

Amino acid positions 632-633>V (i.e.

residues 632 and 633 are replaced by one valine residue) ⁷⁴

Δ Amino acid positions 632-633 (e.g. E632 - L633 in somatic cells or a 6 bp in-frame germline deletion in exon 11 ⁹ )

Amino acid positions 632-639>HR (i.e. residues 632-639 are replaced by two residues, histidine and arginine)

Amino acid position 633 (e.g., L633R® ² , 9 bp duplication ² , L633delinsLCR ⁷¹ )

Amino acid position 634 (e.g., C634W, C634Y, C634S, C634R, C634F, C634G, C634L, C634A, or C634T, 9 bp deletion® ² , 9 bp duplication ⁵ ® , or 12 bp duplication ² ) (e.g., causing MTC)

Δ Amino acid position 634 ⁵ ® Amino acid position 632/633/634

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(E632V/L633R/634, 9 bp deletion) ⁶² Amino acid position 635 (e.g. R635G or ELCR insertion ² ) Amino acid position 636 T636P ² , T636M ⁴ ) (For example, Amino acid positions 636-637 T636-V637insCRT) ⁸⁰ (For example, Amino acid position 638 isoleucine for frameshift ⁴⁷ ) (For example, Amino acid position 640 (eg A640G) Amino acid position 634/640 (O634R/A640G) ⁵⁶ (For example, Amino acid position 641 (e.g. A641S, A641T ⁸ ) Amino acid position 634/641 (O634S/A641S) ⁵⁶ (For example, Amino acid position 639/641 (A639G/A641R) ⁵⁶ (For example, Amino acid position 644 (T644M) ⁵⁹ (For example, Amino acid position 648 (e.g. V648I) Amino acid positions 634/648 (O634R/V648I) ⁷⁷ (For example, Amino acid position 649 (S649L) ²⁸ (For example, Amino acid position 661 H661H) ⁶ (For example, Amino acid position 664 (e.g. A664D) Amino acid position 665 H665Q) (For example, Amino acid position 666 (e.g. K666E, K666M, K666N, K666R) Amino acid position 675 (T675T, silent

- 53 041220

nucleotide substitution) ¹⁸ Amino acid P679P) ⁶ position 679 (For example, Amino acid position 680 (eg A680T, frameshift alanine) ⁶ Amino acid position 686 (e.g. S686N) Amino acid S689T) ¹⁸ position 689 (For example, Amino acid position 691 (eg G691S) Amino acid R694Q) position 694 (For example, Amino acid position 700 (eg M700L) Amino acid position 706 (e.g. V706M, V706A) Amino acid position 713, splice variant (eg E713K (eg splice variant)) ⁶ Amino acid D714Y) ⁵⁷ position 714 (For example, Amino acid G727E) ⁶ position 727 (For example, Amino acid E732K) ²⁰ position 732 (For example, Amino acid E734K) ⁴⁸ position 734 (For example, Amino acid G736R) ⁶ position 736 (For example, Amino acid V738V) ⁶ position 738 (For example, Amino acid T742M) ⁵¹ position 742 (For example, Amino acid G748C) position 748 (For example, Amino acid position 749 (For example,

- 54 041220

R749T ³⁶ )

Amino acid position 750 (e.g. A750P, A750G®)

Amino acid position 752 (eg,

Y752Y)®

Amino acid position 751 (e.g. G751G)®

Amino acid position 762 (eg,

E762Q ³ ®)

Amino acid position 765 (eg S765P, S765F)

Amino acid position 766 (e.g. P766S, P766M®)

Amino acid position 768 (e.g. E768Q, E768D, E768N ⁴⁶ , E768G ⁷² )

Amino acid position 769 (eg, L769L®)

Amino acid position 770 (eg,

R770Q)

Amino acid position 771 (eg D771N)

Amino acid position 777 (eg N777S)

Amino acid position 778 (eg V778I)

Amino acid position 781 (eg,

Q781R)

Amino acid position 788 (e.g. I788I ³² , I788N ⁷⁸ )

Amino acid position 790 (eg L790F)

Amino acid position 768/790 (eg E768D/L790T) ⁴⁰

Amino acid position 791 (e.g. Y791F, Y791N ²⁴ )

Amino acid position 634/791 (eg C634Y/Y791F) ⁵⁵

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Amino acid position 790/791 (eg L790F/Y791F) ⁵⁵

Amino acid position 802

Amino acid position 804 (e.g. V804L ^{15 16} , V804M ^{15 16} , V804E ¹² ) (e.g., MTC-causing)

Amino acid position 778/804 ⁵⁰ (eg V778I/V804M ⁵⁴ )

Amino acid position 781/804 (eg, Q781R/V804M) ⁴¹

Amino acid position 805 (eg E805K)

Amino acid position 804/805 (eg V804M/E805K) ¹⁷

Amino acid position 806 (e.g., Y806F, Y806S ¹² , Y806G, Y806C ^{2 12 14} , Y806E ¹⁴ , Y806H ¹² , Y806N ¹² -Y806Y ³² )

Amino acid position 804/806 (eg V804M/Y806C) ³⁸

Amino acid position 810 (e.g. G810R ¹² , G810S ¹² , G810A ¹³ , G810C, G810V, and G810D)

Amino acid position 818 (eg E818K)

Amino acid position 819 (eg S819I)

Amino acid position 820 (eg R820L) ⁵⁷

Amino acid position 823 (eg G823E)

Amino acid position 826 (e.g. Y826M, Y826S) ¹⁰

Amino acid position 828 (eg G828R) ⁵⁷

Amino acid position 833 (eg R833C)

Amino acid position 836 (eg S836S) ¹⁹

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Amino acid position 841 (eg P841L, P841P)

Amino acid position 843 (eg E843D)

Amino acid position 844 (e.g. R844W, R844Q, R844L)

Amino acid position 804/844 (eg V804M/R844L) ⁷⁶

Amino acid position 845 (eg,

A845A)® ³

Amino acid position 848 (eg M848T)

Amino acid position 852 (eg I852M)

Amino acid position 853 (eg S853T) ⁵⁷

Amino acid position 865 (eg L865V) ¹²

Amino acid position 866 (eg A866W) ³³

Amino acid position 867 (eg E867K) ³⁷

Amino acid position 870 (eg L870F) ¹²

Amino acid position 873 (eg R873W, R873Q ⁴² )

Amino acid position 876 (eg A876V)

Amino acid position 881 (eg L881V)

Amino acid position 882

Amino acid position (e.g. A883F, A883S, A883T, A883Y ⁵³ , A883V)

Amino acid position 884 (e.g. E884K, E884V ³⁵ )

Amino acid position 886 (eg,

R886W)

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Amino acid position 891 (e.g. S891A, S891S ³² , S891L ³⁵ )

Amino acid position 893 (eg F893L) ⁴²

Amino acid position 894 (eg G894S) ⁴³

Amino acid position 897 (eg R897Q, R897P)

Amino acid position 898 (eg D898V, D898Y ⁶⁶ )

Δ Amino acid position 898

Δ Amino acid positions 898-902 ⁵⁸

Δ Amino acid positions 899-902 ⁴⁷

Δ Amino acid positions 898-901 ⁴⁷

Δ Amino acid positions 632-633/Δ

Amino acid positions 898-901 ⁴⁷

Amino acid position 900 (eg Y900F) ²²

Amino acid position 901 (eg E901K)

Amino acid position 904 (eg S904F, S904S, S904C ² , S904T ⁵⁷ )

Amino acid position 691/904 (eg G691S/S904S) ⁴⁹

Amino acid position 804/904 (eg V804M/S904C) ³⁸

Amino acid position 905 (eg Y905F) ²²

Amino acid position 907 (e.g. K907E, K907M)

Amino acid position 908 (eg R908K)

Amino acid position 911 (eg G911D, G911G (eg splice variant) ⁸ )

- 58 041220

Amino acid position 912 (eg R912P, R912Q)

Amino acid position 918 (e.g. M918T ² , M918V, M918L ⁶ ) (e.g., MTC-inducing)

Amino acid position 591/918 (eg V5911/M918T) ⁶¹

Amino acid position 620/918 (eg C620F/M918T) ⁴⁷

Amino acid position 891/918 (eg S891A/M918T) ⁴⁷

Δ Amino acid positions 898-901/M918T ⁴⁷

Amino acid position 919 (eg A919V, A919P ⁵² )

Amino acid position 768/919 ⁵⁴

Amino acid position 921 (e.g. E921K, E921D)

Amino acid position 911/918/921 (eg G911E/M918T/E921K) ⁶¹

Amino acid position 922 (eg S922P, S922Y)

Amino acid position 924 (e.g. F924S)®

Amino acid position 930 (eg T930M)

Amino acid position 961 (eg F961L)

Amino acid position 972 (eg R972G)

Amino acid position 973 (eg P973T) ⁵⁷

Amino acid position 977 (eg S977R) ³⁷

Amino acid position 981 (eg Y981F) ²²

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Amino acid R982C) ⁷⁰ position 982 (For example, Amino acid position (eg C634R/G691S/R982C) ⁴⁵ 634/691/982 Amino acid position 292/67/982 (e.g. V292M/ R67H/R982O) ⁷⁵ Amino acid position 634/292/67/982 (e.g. C634R/ V292M/ R67H/R982C) ⁷⁵ Amino acid M1009V) position 1009 (For example, Amino acid Y1015F) ²² position 1015 (For example, Amino acid D1017N) position 1017 (For example, Amino acid S1024F) ⁷⁹ position 1024 (For example, Amino acid V1041G) position 1041 (For example, Amino acid P1047S) ⁶⁵ position 1047 (For example, Amino acid A1051T) ⁵⁷ position 1051 (For example, L-amino acid position 1059 ⁵⁷ Amino acid M1064T) position 1064 (For example, Amino acid Y1096F) ²¹ position 1096 (For example, Amino acid A1105V) ⁵⁷ position 1105 (For example, Amino acid M1109T) ³⁴ position 1109 (For example, RET+3 ¹ (In-frame deletion in exons 6 and 11) ²⁵ (Deletion of 3 bp in reading frame in exon 15) ²⁶ Nucleotide ι 2136+2T>G) ²⁹ position 2136+2 (For example, (del632-636 ins6) ³¹ Amino acid Y791F + I852M) ³¹ Provisions 791 and 852 (for example, Amino acid positions 634 C634R + I852M) ³¹ I and 852 (for example, c.1893_1895del ⁴⁴

^A The presented RET kinase mutations may be activating mutations and/or may confer increased resistance of RET kinase to a RET kinase inhibitor and/or a multikinase inhibitor (MKI), for example, compared to wild-type RET kinase.

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⁶³ Elisei, et al, Journal of Genetic Syndromes & Gene Therapy, 5(1), 1, 2014. doi: 10.4172/21577412.1000214 ⁶⁴ Ahmed et al, The Journal of Molecular Diagnostics, 7(2), 283-288, 2005. doi: 10.1016/S15251578(10)60556-9 ⁶⁵ Oliveira, et al. J. Exp. Clin. Cancer Res. 37(84), 2018. doi: 10.1186/s13046-018-0746-y ⁶⁶ Yi, et al. Case Rep. Endocrinol. 2018:8657314, 2018. doi: 10.1155/2018/8657914 ⁶⁷ Huang, et al. Cell. 173(2): 355-370, 2018. doi: 10.1016/j.cell.2018.03.039 ⁶⁸ Bosic, et al. Pathology. 50(3):327-332, 2018. doi: 10.1016/j.pathol.2017.10.011 ⁶⁹ Yao, et al. Zhonghua YiXue Za Zhi. 87(28):1962-1965, 2007. PMID: 17923033 ⁷⁰ Quintela-Fandino, et al. Mol. Oncol. 8(8):1719-1728, 2014. doi: 10.1016/j.molonc.2014.07.005 ⁷¹ Urbini, et al. Int J Genomics 2018: 6582014. doi: 10.1155/2018/6582014 ⁷² Yu, et al. Clin Lung Cancer, pii: S1525-7304(18)30204-3, 2018. doi:

10.1016/j.cllc.2O18.08.010 ⁷³ Soca-Chafre, et al. Oncotarget 9(55):30499-30512, 2018. doi: 10.18632/oncotarget.25369 ⁷⁴ Kim, et al. BMC Urol 18(1):68, 2018. doi: 10.1186/s 12894-018-0380-1 ⁷⁵ Qi, et al. PLoS One 6(5):e20353, 2011. doi: 10.1371/journal.pone.0020353

- 62 041220 ⁷⁶ Bartsch, et al Exp Clin Endocrinol Diabetes 108(2):128-132, 2000. doi: 10.1055/S-2000-5806 ⁷⁷ Nunes, et al. J Clin Endocrinol Metab. 87(12):5658-5661, 2002. doi: 10.1210/jc.2002-020345 ⁷⁸ Plenkeret al., Sci. Transl. Med., 9(394), doi: 10.1126/scitranslmed.aah6144, 2017 ⁷⁹ Romei, et al., European Thyroid Journal, vol. 7, suppl. 1, cc. 63. Abstract Number: P1-07-69. Event Information: 41st Annual Meeting of the European Thyroid Association, ETA 2018. 15 Sep 2018-18 Sep 2018. doi: 10.1159/000491542 ⁸⁰ Ciampi, et al., European Thyroid Journal, vol 7, suppl 1, pp 63. Abstract number: OP-09-66. Event information: 41st Annual Meeting of the European Thyroid Association, ETA 2018. 15 Sep 2018-18 Sep 2018. doi : 10.1159/000491542

In some embodiments, deregulation of a RET gene, RET kinase, or expression or activity or level of any of them comprises at least one point mutation in the RET gene that results in the production of a RET kinase having one or more amino acid substitutions, insertions, or deletions compared to a wild-type RET kinase (see, for example, the point mutations listed in Table 2a).

Table 2a

Amino acid substitutions/insertions/deletions in the RET ^A kinase protein

Amino acid position 20

Amino acid position 32 (eg S32L)

Amino acid position 34 (eg D34S)

Amino acid position 40 (eg L40P)

Amino acid position 64 (eg P64L)

Amino acid position 67 (eg R67H)

Amino acid position 114 (eg R114H)

Amino acid position 145 (eg V145G)

Amino acid position 200

Amino acid position 292 (eg,

V292M)

Amino acid position 294

Amino acid position 321 (eg,

G321R)

Amino acid position 330 (eg,

R330Q)

Amino acid position 338 (eg T338I)

Amino acid position 360 (eg

- 63 041220

R360W)

Amino acid position 393 (eg F393L)

Amino acid position 432

Δ Amino acid residues 505-506 (6 bp in-frame deletion of germline exon 7)

Amino acid position 510 (eg A510V)

Amino acid position 511 (eg E511K)

Amino acid position 513 (eg G513D)

Amino acid position 515 (eg C515S, C515W ⁴ )

Amino acid position 525 (eg R525W)

Amino acid position 531 (eg, C531R or 9 bp duplication)

Amino acid position 532 (eg, duplication)

Amino acid position 533 (eg G533C, G533S)

Amino acid position 550 (eg G550E)

Amino acid position 591 (eg V591I)

Amino acid position 593 (eg G593E)

Amino acid position 595 (eg E595D

- 64 041220 and E595A)

Amino acid position 600 (eg R600Q)

Amino acid position 602 (eg I602V)

Amino acid position 603 (e.g. K603Q, K603E)

Amino acid position 606 (eg Y606C)

Amino acid position 609 (e.g. C609Y, C609S, C609G, C609R, C609F, C609W)

Amino acid position 611 (e.g. C611R, C611S, C611G, C611Y, C611F, C611W)

Amino acid position 616 (eg E616Q)

Amino acid position 618 (C618S, C618Y, C618R, C618G, C618F, C618W)

Amino acid position 620 (e.g. C620S, C620W, C620R, C620G, C620L, C620Y, C620F)

Amino acid position 623 (eg E623K)

Amino acid position 624 (eg D624N)

Amino acid position 630 (e.g. C630A, C630R, C630S, C630Y, C630F, C630W)

Amino acid position 631 (e.g. D631N, D631Y, D631A, D631G, D631V, D631E)

Amino acid position 632 (e.g. E632K,

E632G)

- 65 041220

Δ Amino acid residues 632-633 (6 bp in-frame deletion of germline exon 11)

Amino acid position 633 (eg, 9 base pair duplication)

Amino acid position 634 (e.g. C634W, C634Y, C634S, C634R, C634F, C634G, C634L, C634A, or C634T, or an ELCR insertion, or a 12-base pair duplication) (e.g., causing MTC)

Amino acid position 635 (eg R635G)

Amino acid position 636 (e.g. T636P, T636M)

Amino acid position 640 (eg A640G)

Amino acid position 641 (e.g. A641S, A641T)

Amino acid position 648 (eg V648I)

Amino acid position 649 (eg S649L)

Amino acid position 664 (eg A664D)

Amino acid position 665 (eg H665Q)

Amino acid position 666 (e.g. K666E, K666M, K666N, K666R)

Amino acid position 686 (eg S686N)

- 66 041220

Amino acid position 689 (eg S689T)

Amino acid position 691 (eg G691S)

Amino acid position 694 (eg R694Q)

Amino acid position 700 (eg M700L)

Amino acid position 706 (e.g. V706M,

V706A)

Amino acid position 713, splice variant (e.g. E713K)

Amino acid position 732 (eg E732K)

Amino acid position 736 (eg,

G736R)

Amino acid position 748 (eg,

G748C)

Amino acid position 750 (eg A750P)

Amino acid position 765 (eg S765P)

Amino acid position 766 (eg P766M)

Amino acid position 768 (eg E768Q, E768D)

Amino acid position 769 (eg L769L)

Amino acid position 770 (eg R770Q)

- 67 041220

Amino acid position 771 (eg D771N)

Amino acid position 777 (eg N777S)

Amino acid position 778 (eg V778I)

Amino acid position 781 (eg Q781R)

Amino acid position 790 (eg L790F)

Amino acid position 791 (e.g. Y791F, Y791N)

Amino acid position 802

Amino acid position 804 (e.g. V804L,

V804M, V804E) (for example, MTS callers)

Amino acid position 805 (eg E805K)

Amino acid position 804/805 (eg V804M/E805K)

Amino acid position 806 (e.g. Y806F, Y806S, Y806G, Y806C, Y806E, Y806H, Y806N)

Amino acid position 810 (e.g. G810R, G810S, G810A, G810C, G810V, and G810D)

Amino acid position 818 (eg E818K)

Amino acid position 819 (eg S819I)

Amino acid position 823 (eg G823E)

Amino acid position 826 (eg Y826M, Y826S)

- 68 041220

Amino acid position 833 (eg R833C)

Amino acid position 836 (eg S836S)

Amino acid position 841 (eg P841L, P841P)

Amino acid position 843 (eg E843D)

Amino acid position 844 (e.g. R844W, R844Q, R844L)

Amino acid position 848 (eg M848T)

Amino acid position 852 (eg I852M)

Amino acid position 865 (eg L865V)

Amino acid position 870 (eg L870F)

Amino acid position 873 (eg R873W)

Amino acid position 876 (eg A876V)

Amino acid position 881 (eg L881V)

Amino acid position 882

Amino acid position (eg A883F, A883S, A883T)

Amino acid position 884 (eg E884K)

Amino acid position 886 (eg R886W)

Amino acid position 891 (eg S891A)

- 69 041220

Amino acid position 897 (eg R897Q)

Amino acid position 898 (eg D898V)

Amino acid position 900 (eg Y900F)

Amino acid position 901 (eg E901K)

Amino acid position 904 (eg S904F, S904S, S904C)

Amino acid position 907 (e.g. K907E, K907M)

Amino acid position 908 (eg R908K)

Amino acid position 911 (eg,

G911D)

Amino acid position 912 (eg R912P, R912Q)

Amino acid position 918 (e.g. M918T, M918V, M918L) (e.g. MTC-causing)

Amino acid position 919 (eg A919V)

Amino acid position 921 (eg E921K)

Amino acid position 922 (eg S922P, S922Y)

Amino acid position 930 (eg T930M)

Amino acid position 961 (eg F961L)

Amino acid position 972 (eg,

- 70 041220

R972G)

Amino acid position 982 (eg R982C)

Amino acid position 1009 (eg,

M1009V)

Amino acid position 1015 (eg,

Y1015F)

Amino acid position 1017 (eg,

D1017N)

Amino acid position 1041 (eg,

V1041G)

Amino acid position 1064 (eg,

M1064T)

Amino acid position 1096 (eg,

Y1096F)

RET+3 (In-frame deletion in exons 6 and 11) (In-frame 3 bp deletion in exon 15) ^A The above RET kinase mutations may be activating mutations and/or may confer increased resistance of RET kinase to a RET inhibitor and/or a multikinase inhibitor (MKI), e.g., compared to wild-type RET kinase.

In some embodiments, deregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises a splice change in RET mRNA that results in expression of a protein that is an alternative splice variant of RET having at least one deleted residue (compared to wild-type RET kinase), resulting in constitutive activity of the RET kinase domain.

The RET kinase inhibitor described above includes any compound that exhibits RET inhibitory activity. In some embodiments, the RET kinase inhibitor is selective for RET kinase. Exemplary RET kinase inhibitors can exhibit an inhibitory activity (IC ₅₀ ) against RET kinase of less than about 1000 nM, less than about 500 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM, as measured in the assay described herein. In some embodiments, a RET kinase inhibitor may exhibit an inhibitory activity (IC50) against RET kinase of less than about 25 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM, as measured in the assay provided herein.

In this context, the first RET kinase inhibitor or the first RET inhibitor is a RET kinase inhibitor described herein, but which does not comprise a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof described herein. In this context, the second RET kinase inhibitor or the second RET inhibitor is a RET kinase inhibitor described herein, but which does not comprise a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof described herein. If the method provided herein includes both a first and a second RET inhibitor, the first and second RET kinase inhibitors are different.

In some embodiments, the deregulation of a RET gene, RET kinase, or the expression or activity or level of any of them comprises at least one point mutation in a RET gene that results in the production of a RET kinase having one or more amino acid substitutions or insertions or deletions in the RET gene that results in the production of a RET kinase having one or more amino acids inserted or deleted, compared to a wild-type RET kinase. In some cases, the resulting RET kinase is more resistant to inhibition of its phosphotransferase activity by one or more first RET inhibitor(s), compared to a wild-type RET kinase or to a RET kinase lacking such a mutation. Optionally, such mutations do not reduce the sensitivity of a cancer cell or tumor comprising a RET kinase to treatment with a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof (e.g., compared to a cancer cell

- 71 041220 a tumor that does not contain a particular RET inhibitor resistance mutation). In such embodiments, the RET inhibitor resistance mutation may result in a RET kinase that has one or more of an increased Vmax, a decreased Km for ATP, and an increased KD for a first RET kinase inhibitor in the presence of a first RET kinase inhibitor, compared to a wild-type RET kinase or a RET kinase that does not contain such a mutation in the presence of the same first RET kinase inhibitor.

In other embodiments, the deregulation of the RET gene, RET kinase, or the expression or activity or level of any of them comprises at least one point mutation in the RET gene that results in the production of a RET kinase that has one or more amino acid substitutions compared to a wild-type RET kinase and that has increased resistance to a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof compared to a wild-type RET kinase or a RET kinase that does not contain such a mutation. In such embodiments, the RET inhibitor resistance mutation may result in the formation of a RET kinase that has one or more of an increased Vmax, a decreased Km, and a decreased KD in the presence of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, compared to a wild-type RET kinase or a RET kinase that does not contain such a mutation in the presence of the same compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof.

Examples of RET inhibitor resistance mutations may, for example, include point mutations, insertions, or deletions within and near the ATP-binding site in the tertiary structure of RET kinase (e.g., amino acid positions 730-733, 738, 756, 758, 804, 805, 807, 810, 811, 881, and 892 of wild-type RET kinase, such as the exemplary wild-type RET kinase described herein) including, but not limited to, a gatekeeper residue (e.g., amino acid position 804 in wild-type RET kinase), P-loop residues (e.g., amino acid positions 730-737 in wild-type RET kinase), residues within or near the DFG motif (e.g., amino acid positions 888-898 in wild-type RET kinase), and amino acid residues of the solvent front of the ATP cleft (e.g., amino acid positions 758, 811, and 892 in wild-type RET kinase). Further examples of these types of mutations include changes in residues that can affect enzyme activity and/or drug binding, including, but not limited to, residues in the activating loop (e.g., amino acid positions 891-916 of wild-type RET kinase), residues near or interacting with the activating loop, residues involved in active or inactive conformations of the enzyme, changes including mutations, deletions, and insertions in the loop preceding the C-helix and within the C-helix (e.g., amino acid positions 768-788 in the wild-type RET protein). In some embodiments, the wild-type RET protein is an exemplary wild-type RET kinase described herein. Particular residues or regions of residues that may be altered (and constitute RET inhibitor resistance mutations) include, but are not limited to, those listed in Table 3, where the numbering is based on the sequence of the wild-type human RET protein (e.g., SEQ ID NO: 1). As will be appreciated by those skilled in the art, the amino acid position in the reference protein sequence that corresponds to a particular amino acid position in SEQ ID NO: 1 can be determined by aligning the reference protein sequence with SEQ ID NO: 1 (e.g., using software such as ClustalW2). Additional examples of RET inhibitor resistance mutation positions are provided in Table 4. Alterations to these residues may include single or multiple amino acid substitutions, insertions within or at the ends of sequences, and deletions within or at the ends of sequences. See also J. Kooistra, G. K. Kanev, O. P. J. Van Linden, R. Leurs, I. J. P. De Esch, and C. De Graaf, KLIFS: A structural kinase-ligand interaction database, Nucleic Acids Res., vol. 44, no. D1, pp. D365–D371, 2016), the entire contents of which are incorporated herein by reference.

Exemplary sequence of the mature human RET protein (SEQ ID NO: 1) MAKATSGAAG LRLLLLLLLP LLGKVALGLY FSRDAYWEKL YVDQAAGTPL LYVHALRDAP EEVPSFRLGQ HLYGTYRTRL HENNWICIQE DTGLLYLNRS LDHSSWEKLS VRNRGFPLLT VYLKVFLSPT SLREGECQWP GCARVYFSFF NTSFPACSSL KPRELCFPET RPSFRIRENR PPGTFHQFRL LPVQFLCPNI SVAYRLLEGE GLPFRCAPDS LEVSTRWALD REQREKYELV

AVCTVHAGAR EEVVMVPFPV TVYDEDDSAP TFPAGVDTAS AVVEFKRKED TWATLRVFD ADWPASGEL VRRYTSTLLP GDTWAQQTFR VEHWPNETSV QANGSFVRAT VHDYRLVLNR NLSISENRTM QLAVLVNDSD FQGPGAGVLL LHFNVSVLPV SLHLPSTYSL SVSRRARRFA QIGKVCVENC QAFSGINVQY KLHSSGANCS TLGWTSAED TSGILFVNDT KALRRPKCAE LHYMVVATDQ QTSRQAQAQL LVTVEGSYVA EEAGCPLSCA VSKRRLECEE CGGLGSPTGR CEWRQGDGKG ITRNFSTCSP STKTCPDGHC DWETQDINI CPQDCLRGSI VGGHEPGEPR GIKAGYGTCN CFPEEEKCFC EPEDIQDPLC DELCRTVIAA AVLFSFIVSV LLSAFCIHCY HKFAHKPPIS SAEMTFRRPA QAFPVSYSSS GARRPSLDSM ENQVSVDAFK ILEDPKWEFP RKNLVLGKTL GEGEFGKWK ATAFHLKGRA GYTTVAVKML KENASPSELR DLLSEFNVLK QVNHPHVIKL YGACSQDGPL LLIVEYAKYG SLRGFLRESR KVGPGYLGSG GSRNSSSLDH PDERALTMGD LISFAWQISQ GMQYLAEMKL VHRDLAARNI LVAEGRKMKI SDFGLSRDVY EEDSYVKRSQ GRIPVKWMAI ESLFDHIYTT QSDVWSFGVL LWEIVTLGGN PYPGIPPERL FNLLKTGHRM ERPDNCSEEM YRLMLQCWKQ EPDKRPVFAD ISKDLEKMMV KRRDYLDLAA STPSDSLIYD DGLSEEETPL VDCNNAPLPR ALPSTWIENK LYGMSDPNWP GESPVPLTRA DGTNTGFPRY PNDSVYANWM LSPSAAKLMD TFDS

In some embodiments, the RET inhibitor resistance mutation may include

- 72 041220 dysregulation of the MET gene, MET kinase, or expression, or activity, or level of any of them.

The expression "deregulation of the MET gene, MET kinase, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a translocation of the MET gene that results in the expression of a fusion protein, a deletion in the MET gene that results in the expression of a MET protein that contains a deletion of at least one amino acid compared to the wild-type MET protein, or a mutation in the MET gene that results in the expression of a MET protein with one or more point mutations, or an alternative splice version of the MET mRNA forming the MET protein that results in a deletion of at least one amino acid in the MET protein compared to the wild-type MET protein) or to an amplification of the MET gene that results in overexpression of the MET protein or autocrine activity due to overexpression of the MET gene in a cell that results in a pathogenic increase in the activity of the kinase domain of the MET protein (e.g., a constitutively active kinase domain of the MET protein MET) in a cell. As another example, deregulation of a MET gene, MET protein, or expression or activity or level of any of them can be a mutation in a MET gene that encodes a MET protein that is constitutively active or has increased activity compared to a protein encoded by a MET gene that does not contain such a mutation. For example, deregulation of a MET gene, MET protein, or expression or activity or level of any of them can be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of MET that contains a functional kinase domain and a second portion of a partner protein (i.e., non-MET). In some examples, deregulation of a MET gene, MET protein, or expression or activity can be the result of a gene translocation of one MET gene with another non-MET gene.

The term wild-type MET describes a nucleic acid (e.g., a MET gene or MET mRNA) or protein (e.g., a MET protein) that occurs in a subject that does not have a MET-associated cancer (and optionally also does not have an increased risk of developing a MET-associated cancer and/or is not predisposed to a MET-associated cancer), or that occurs in a cell or tissue obtained from a subject that does not have a MET-associated cancer (and optionally also does not have an increased risk of developing a MET-associated cancer and/or is not predisposed to a MET-associated cancer). The term MET-associated cancer, as used herein, refers to cancers associated with or characterized by deregulation of the MET gene, MET kinase, or the expression or activity or level of any of them.

In some embodiments, the compounds of Formula I-IV, or pharmaceutically acceptable salts, amorphous or polymorphic forms thereof, are useful for treating patients who develop cancers with RET inhibitor resistance mutations (e.g., that result in increased resistance to a first RET inhibitor, e.g., a substitution at amino acid position 804, e.g., V804M, V804L, or V804E, a substitution at amino acid position 810, e.g., G810S, G810R, G810C, G810A, G810V, and G810D, and/or with one or more RET inhibitor resistance mutations listed in Tables 3 and 4) by administering combination or sequential or additional (e.g., follow-on) therapy to existing drug therapy (e.g., other RET kinase inhibitors; e.g., the first and/or second RET kinase inhibitor). Illustrative first and second RET kinase inhibitors are described herein. In some embodiments, the first or second RET kinase inhibitor may be selected from the group consisting of cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667, and BLU6864.

In some embodiments, the compounds of Formula I-IV, or pharmaceutically acceptable salts, amorphous, or polymorphic forms thereof, are useful for treating a cancer that has been identified as having one or more RET inhibitor resistance mutations (which result in increased resistance to a first or second RET inhibitor, such as a substitution at amino acid position 804, such as V804M, V804L, or V804E, or, such as a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D). In some embodiments, the one or more RET inhibitor resistance mutations are in a nucleic acid sequence encoding a RET fusion protein (e.g., any of the RET gene fusion proteins described in Table 1), to form a RET fusion protein that exhibits resistance to a RET kinase inhibitor. In some embodiments, one or more RET inhibitor resistance mutations are in a nucleic acid sequence encoding a mutant RET protein (e.g., a mutant RET protein having one of the mutations described in Table 2), to form a mutant RET protein that exhibits RET kinase resistance. Non-limiting examples of RET inhibitor resistance mutations are listed in Tables 3 and 4.

- 73 041220

Table 3

RET inhibitor resistance mutations

Illustrative RET resistance mutations

Amino acid position 634 (eg C634W) ¹⁰

Amino acid position 732 (eg E732K) ⁷

Amino acid position 788 (eg I788N) ⁸

Amino acid position 790 (eg L790F) ⁹

Amino acid position 804 (e.g. V804M ¹² , V804L ¹ ' ² , V804E ⁶ )

Amino acid position 778/804 ¹³

Amino acid position 804/805 (eg V804M/E805K) ³

Amino acid position 806 (e.g. Y806C ⁴ ' ⁶ , Y806E ⁴ , Y806S ⁶ , Y806H ⁶ , Y806N ⁶ )

Amino acid position 804/806 (eg V804M/Y806C) ¹¹

Amino acid position 810 (e.g. G810A ⁵ , G810R ⁶ , G810S ⁶ , G810C, G810V, and G810D)

Amino acid position 865 (eg L865V ^S )

Amino acid position 870 (eg L870F ⁶ )

Amino acid position 891 (eg S891A) ¹⁰

Amino acid position 904 (eg S904F) ¹²

Amino acid position 804/904 (eg V804M/S904C) ¹¹

Amino acid position 918 (e.g., M918T) ^{10 1} Yoon et al., J. Med. Chem. 59(1):358–73, 2016.

² US Patent No. 8629135.

³ Cranston, et al., Cancer Res. 66(20):10179-87, 2006.

⁴ Carlomagno, et al., Endocr. Rei. Cancer 16(1):233-41,2009.

⁵ Huang et al, Mol. Cancer Then, 2016 Aug 5. pii: molcanther.0258.2016. [Email: publ. before publication].

⁶ Publication of PCT patent application No. WO 2016/127074.

⁷ Mamedova et al., Summer Undergraduate Research Programs (SURP) Student Abstracts, University of Oklahoma Health Sciences Center, 2016.

⁸ Plenker et al., Sci. Transl. Med., 9(394), doi: 10.1126/scitranslmed.aah6144, 2017.

⁹ Kraft et al, Cancer Research, 2017, Vol. 77, No. 13, suppl. Supplement 1. Abstract Number: 4882; American Association for Cancer Research Annual Meeting 2017. Washington, DC, USA. 01 Apr 2017–05 Apr 2017.

¹⁰ U.S. Patent Application Publication No. 2018/0022732.

¹¹ Roskoski and Sadeghi-Nejad, Pharmacol. Res., 128, 1-17. doi: 10.1016/j.phrs.2017.12.021, 2018.

¹² Nakaoku, et al. NatCommun, 9(1), 625. doi: 10.1038/S41467-018-02994-7, 2018.

¹³ Roy et al. Oncologist, 18(10): 1093-1100. doi: 10.1634/theoncologist.2013-0053, 2013.

- 74 041220

Table 4 Additional illustrative amino acid positions of RET inhibitor resistance mutations

RET amino acid and position Illustrative mutation Mechanistic rationale for resistance L730 R Steric hindrance and/or active conformational effect G731 V Steric hindrance and/or active conformational effect E732 TO Steric hindrance and/or active conformational effect G733 V Steric hindrance and/or active conformational effect E734 To Steric hindrance and/or active conformational effect L760 m Active conformational effect K761 E Active conformational effect E762 TO Active conformational effect N763 D Active conformational effect A764 V Active conformational effect S765 N Active conformational effect P766 A Active conformational effect S767 WITH Active conformational effect E768 TO Active conformational effect L779 M Steric hindrance and/or active conformational effect I788 M Steric hindrance and/or active conformational effect M868 R Steric hindrance and/or active conformational effect K869 E Steric hindrance and/or active conformational effect L870 Q Steric hindrance and/or active conformational effect V871 M Steric hindrance and/or active conformational effect N872 R Steric hindrance and/or active conformational effect R873 R Steric hindrance and/or active conformational effect D874 Y Steric hindrance and/or active conformational effect L881 R Steric hindrance and/or active conformational effect L895 M Active conformational effect S896 N Active conformational effect

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R897 C Active conformational effect D898 Y Active conformational effect V899 G Active conformational effect Y900 D Active conformational effect E901 TO Active conformational effect E902 TO Active conformational effect D903 Y Active conformational effect S904 WITH Active conformational effect Y905 D Active conformational effect V906 M Active conformational effect K907 E Active conformational effect R908 R Active conformational effect S909 Ο Active conformational effect Q910 R Active conformational effect G911 Ο Active conformational effect R912 R Active conformational effect

The oncogenic role of RET was first described for papillary thyroid carcinoma (PTC) (Grieco et al., Cell, 1990, 60, 557–63), which arises from thyroid follicular cells and is the most common thyroid malignancy. About 20–30% of PTCs have somatic chromosomal rearrangements (translocations or inversions) linking the promoter and 5' fragments of constitutively expressed, unrelated genes to the RET tyrosine kinase domain (Greco et al., Q. J. Nucl. Med. Mol. Imaging, 2009, 53, 440–54), driving its ectopic expression in thyroid cells. Fusion proteins resulting from such rearrangements are called RET/PTC proteins. For example, RET/PTC 1 is a fusion between CCDD6 and RET that is commonly found in papillary thyroid carcinomas. Similarly, RET/PTC3 and RET/PTC4 are fusions of ELE1 and RET that are highly prevalent in papillary thyroid carcinomas, although the fusion events that produce RET/PTC3 and RET/PTC4 result in different proteins with different molecular weights (see, e.g., Fugazzola et al., Oncogene, 13(5):1093-7, 1996). Some RET fusions associated with PTCs are not referred to as RET/PTC but are instead referred to as the fusion protein itself. For example, a fusion between RET and both ELKS and PCM1 occurs in PTC, but the fusion proteins are referred to as ELKS-RET and PCM1-RET (see, e.g., Romei and Elisei, Front. Endocrinol. (Lausanne), 3:54, doi: 10.3389/fendo.2012.00054, 2012). A role for RET-PTC rearrangements in PTC pathogenesis has been confirmed in transgenic mice (Santoro et al., Oncogene, 1996, 12, 1821-6). Various fusion partners have been identified to date, from PTC and other cancer types, all of which provide a protein/protein interaction domain that mediates ligand-independent RET dimerization and constitutive kinase activity (see, e.g., Table 1). Recently, a 10.6 Mb pericentric inversion on chromosome 10, where the RET gene maps, has been identified in about 2% of patients with lung adenocarcinoma, creating different variants of the KIF5B-RET chimeric gene (Ju et al., Genome Res., 2012, 22, 436–45; Kohno et al., 2012, Nature Med., 18, 375–7; Takeuchi et al., Nature Med., 2012, 18, 378–81; Lipson et al., 2012, Nature Med., 18, 382–4). The fusion transcripts are highly expressed, and all the resulting chimeric proteins contain the N-terminal portion of both the coiled-coil region of KIF5B that mediates homodimerization and the complete RET kinase domain. None of the RET-positive patients had other known oncogenic alterations (such as EGFR or K-Ras mutation, ALK translocation), supporting the possibility that the KIF5B-RET fusion may be a driving mutation in lung adenocarcinoma. The oncogenic potential of KIF5B-RET was confirmed by transfection of the fusion gene into cultured cell lines: similar to what was observed with RET-PTC fusion proteins, KIF5B-RET is constitutively phosphorylated and induces transformation of NIH-3T3 and IL-3-independent growth of BA-F3 cells. However, other RET fusion proteins have been identified in patients with lung adenocarcinoma, such as the CCDC6RET fusion protein, which was found to play a key role in the proliferation of the human lung adenocarcinoma cell line LC-2/ad (Journal of Thoracic Oncology, 2012, 7(12): 1872–1876). RET inhibitors have been shown to be useful in the treatment of lung cancers involving RET rearrangements (Drilon, A.E. et al. J Clin Oncol 33, 2015 (suppl; ref. 8007)). RET fusion proteins have also been identified in patients with colorectal cancer (Song Eun-Kee, et al. International Journal of Cancer, 2015, 136:

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(1967-1975).

In addition to RET sequence rearrangements, gain-of-function point mutations of the RET proto-oncogene are also leading oncogenic events, as demonstrated in medullary thyroid carcinoma (MTC), which arises from calcitonin-producing parafollicular cells (de Groot, et al., Endocrine Rev., 2006, 27, 535–60; Wells and Santoro, Clin. Cancer Res., 2009, 15, 7119–7122). About 25% of MTC cases are associated with multiple endocrine neoplasia type 2 (MEN2), a group of inherited cancer syndromes affecting neuroendocrine organs that are caused by activating germline point mutations in RET. In the MEN2 subtypes (MEN2A, MEN2B, and familial MTC/FMTC), RET gene mutations have a strong phenotype-genotype correlation, determining different MTC aggressiveness and clinical manifestations of the disease. Mutations in MEN2A syndrome involve one of six cysteine residues (mainly C634) located in the cysteine-rich extracellular region, which leads to ligand-independent homodimerization and constitutive activation of RET. Patients develop MTC at an early age (onset at 5-25 years) and may also develop pheochromocytoma (50%) and hyperparathyroidism. MEN2B is caused primarily by the M918T mutation located in the kinase domain. This mutation constitutively activates RET in its monomeric state and alters substrate recognition by the kinase. MEN2B syndrome is characterized by an early onset (<1 year) and is a highly aggressive form of MTC, pheochromocytoma (50% of patients) and ganglioneuroma. In FMTC, the only manifestation of the disease is MTC, usually occurring in adulthood. Many different mutations have been found, covering the entire RET gene. The remaining 75% of MTC cases are sporadic, and about 50% of these carry somatic RET mutations: the most common mutation is M918T, which, as in MEN2B, is associated with the most aggressive phenotype. Somatic point mutations of RET have also been described in other tumors, such as colorectal cancer (Wood et al., Science, 2007, 318, 1108-13) and small cell lung carcinoma (Jpn. J. Cancer Res., 1995, 86, 1127-30). In some embodiments, the MTC is a RET fusion-positive MTC.

RET signaling components have been shown to be expressed in primary breast tumors and to functionally interact with the estrogen receptor-cc cascade in breast tumor cell lines (Boulay et al., Cancer Res. 2008, 68, 3743-51; Plaza-Menacho et al., Oncogene, 2010, 29, 4648-57), and RET expression and activation by GDNF family ligands may play an important role in perineural invasion of various types of cancer cells (Ito et al., Surgery, 2005, 138, 78-894; Gil et al., J. Natl. Cancer Inst., 2010, 102, 107-18; Iwahashi et al., Cancer, 2002, 94, 167-74).

RET is also expressed in 30-70% of invasive breast cancers, with expression being relatively more common in estrogen receptor-positive tumors (Plaza-Menacho, I., et al., Oncogene, 2010, 29, 4648-4657; Esseghir, S., et al., Cancer Res., 2007, 67, 11732-11741; Morandi, A., et al., Cancer Res., 2013, 73, 3783-3795; Gattelli, A., EMBO Mol. Med., 2013, 5, 1335-1350).

Identification of RET rearrangements has been described in a subset of PDX (patient-derived xenografts) derived from colorectal cancer. Although the frequency of such events in colorectal cancer patients has not yet been established, these findings suggest a role for RET as a target in this indication (Gozgit et al., AACR Annual Meeting 2014). Various studies have shown that the RET promoter is frequently methylated in colorectal cancer, and heterozygous missense mutations that putatively reduce RET expression have been identified in 5-10% of cases, suggesting that RET may have some tumor suppressor properties in sporadic colorectal cancers (Luo, Y., et al., Oncogene, 2013, 32, 2037-2047; Sjoblom, T., et al., Science, 2006, 268-274; Cancer Genome Atlas Network, Nature, 2012, 487, 330-337).

An increasing number of tumor types have now been shown to express significant amounts of wild-type RET kinase, which may influence tumor development and dissemination. RET is expressed in 50-65% of pancreatic ductal carcinomas, and expression is more common in metastatic and higher-grade tumors (Ito, Y, et al., Surgery, 2005, 138, 788-794; Zeng, Q., et al., J. Int. Med. Res. 2008, 36, 656-664).

In neoplasms of hematopoietic lineages, RET is expressed in acute myeloid leukemia (AML) with monocytic differentiation, as well as in CMML (Gattei, V. et al., Blood, 1997, 89, 2925-2937; Gattei, V., et al., Ann. Hematol, 1998, 77, 207-210; Camos, M., Cancer Res. 2006, 66, 6947-6954). Recent studies have identified rare chromosomal rearrangements affecting RET in patients with chronic myelomonocytic leukemia (CMML). CMML is often associated with rearrangements of several tyrosine kinases that result in the expression of chimeric cytosolic oncoproteins that lead to the activation of RAS cascades (Kohlmann, A., et al., J. Clin. Oncol. 2010, 28, 2858-2865). In the case of RET, fusion proteins that link RET to BCR (BCR-RET) or to its oncogenic partner, fibroblast growth factor receptor 1 (FGFR1OP-RET), are transformed in early hematopoietic progenitor cells and can shift the maturation of these cells toward monocytic pathways, likely through the initiation of RET-mediated signaling

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RAS (Ballerini, P., et al., Leukemia, 2012, 26, 2384-2389).

RET expression has also been demonstrated in several other tumor types, including prostate cancer, small cell lung cancer, melanoma, renal cell carcinoma, and head and neck tumors (Narita, N., et al., Oncogene, 2009, 28, 3058-3068; Mulligan, L. M., et al., Genes Chromosomes Cancer, 1998, 21, 326-332; Flavin, R., et al., Urol. Oncol., 2012, 30, 900-905; Dawson, D. M., J Natl Cancer Inst, 1998, 90, 519-523).

In neuroblastoma, RET expression and its activation by GFL play a role in tumor cell differentiation, probably with the participation of other neurotrophic factor receptors, for the downregulation of N-Myc, the expression of which is a marker of poor prognosis (Hofstra, R. M., W., et al., Hum. Genet. 1996, 97, 362-364; Petersen, S. and Bogenmann, E., Oncogene, 2004, 23, 213-225; Brodeur, G. M., Nature Ref. Cancer, 2003, 3, 203-216).

Multitarget inhibitors that cross-react with RET are known (Borrello, M.G., et al., Expert Opin. Ther. Targets, 2013, 17(4), 403-419; International Patent Application Nos. WO 2014/141187, WO 2014/184069, and WO 2015/079251). Such multitarget inhibitors (or multikinase inhibitors, or MKIs) can also be associated with the development of RET inhibitor resistance mutations. See, e.g., Q. Huang et al., Preclinical Modeling of KIF5B-RET Fusion Lung Adenocarcinoma., Mol. Cancer Ther, 2016, 18, pp. 2521-2529; Yasuyuki Kaneta et al., Abstract B173: Preclinical characterization and antitumor efficacy of DS-5010, a highly potent and selective RET inhibitor, Mol Cancer Ther, January 1, 2018 (17) (1 suppl.) B173; DOE10.1158/1535-7163.TARG-17-B173, the entire contents of which are incorporated herein by reference.

Accordingly, provided herein are methods of treating a patient diagnosed with (or determined to have) a cancer, which comprise administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Also provided herein are methods of treating a patient identified with or diagnosed as having a RET-associated cancer, which comprise administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. In some embodiments, the patient is identified with or diagnosed as having a RET-associated cancer by using a regulatory agency-approved, e.g., FDA-approved, test or assay for identifying deregulation of the RET gene, RET kinase, or expression or activity or level of any of these in the patient or in a biopsy specimen obtained from the patient, or by performing any of the non-limiting examples of assays described herein. In some embodiments, the test or assay is provided in the form of a kit. In some embodiments, the cancer is a RET-associated cancer. For example, a RET-associated cancer can be a cancer that includes one or more RET inhibitor resistance mutations.

Also provided herein are methods of treating cancer in a patient in need thereof, comprising: (a) detecting a RET-associated cancer in the patient; and (b) administering to said patient a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. Some embodiments of the provided methods further comprise administering to the subject another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy). In some embodiments, the subject has previously been treated with a first RET inhibitor or has previously been treated with another anti-cancer agent, such as by at least partial tumor resection or radiation therapy. In some embodiments, a RET-associated cancer is identified in a patient by using a regulatory agency-approved, e.g., FDA-approved, test or assay to identify deregulation of a RET gene, RET kinase, or expression or activity or level of any of them in the patient or in a biopsy sample obtained from the patient, or by performing any of the non-limiting examples of assays described herein. In some embodiments, the test or assay is provided as a kit. In some embodiments, the cancer is a RET-associated cancer. For example, a RET-associated cancer can be a cancer that includes one or more RET inhibitor resistance mutations.

Also provided are methods of treating a patient that comprise performing an assay on a sample obtained from the patient to determine whether the patient has a deregulation of the RET gene, RET kinase, or expression or activity or level of any of them, and administering (e.g., specifically or selectively administering) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof to the patient who has been found to have a deregulation of the RET gene, RET kinase, or expression or activity or level of any of them. Some embodiments of the provided methods further comprise administering to the subject another anticancer agent (e.g., a second inhibitor

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RET, a second compound of formula I-IV, or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, or immunotherapy). In some embodiments of the methods provided, the subject has previously been treated with a first RET inhibitor or has previously been treated with another anti-cancer agent, such as by at least partial tumor resection or radiation therapy. In some embodiments, the patient is a patient suspected of having a RET-associated cancer, a patient exhibiting one or more symptoms of a RET-associated cancer, or a patient at increased risk of developing a RET-associated cancer. In some embodiments, the assay uses next-generation sequencing, pyrosequencing, immunohistochemistry, or a Break Apart FISH assay. In some embodiments, the assay is a regulatory agency-approved assay, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy. Additional, non-limiting assays that can be used in the methods are described herein. Additional assays are also known in the art. In some embodiments, the deregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations.

Also provided is a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof for use in the treatment of RET-associated cancer in a patient who has been found or diagnosed to have RET-associated cancer at the stage of performing an assay (e.g., an in vitro assay) on a sample obtained from the patient to determine whether the patient has a deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them, wherein the presence of a deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them determines the fact that the patient suffers from RET-associated cancer. Also proposed is the use of a compound of formula IIV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form for the manufacture of a medicament for the treatment of RET-associated cancer in a patient who has been diagnosed or has been diagnosed with RET-associated cancer at the stage of performing an analysis on a sample obtained from the patient to determine whether the patient has a deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them, wherein the presence of a deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them determines the fact that the patient suffers from RET-associated cancer. Some embodiments of any of the methods or uses described herein further comprise recording in the patient's medical record (e.g., on a machine-readable medium) that the patient has been found to have a deregulation of the RET gene, RET kinase, or expression or activity or level of any of them, by performing an assay, and is to be administered a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. In some embodiments, the assay uses next-generation sequencing, pyrosequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is an approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy. In some embodiments, the deregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations.

Also provided is a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof for use in treating cancer in a patient in need thereof or in a patient identified or diagnosed with a RET-associated cancer. Also provided herein is the use of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof for the manufacture of a medicament for treating cancer in a patient identified or diagnosed with a RET-associated cancer. In some embodiments, the cancer is a RET-associated cancer, such as a RET-associated cancer having one or more RET inhibitor resistance mutations. In some embodiments, the patient is identified or diagnosed with a RET-associated cancer by using a regulatory agency-approved, such as an FDA-approved, kit for identifying deregulation of a RET gene, RET kinase, or expression or activity, or level of any of these, in the patient or in a biopsy sample from the sample. As described herein, RET-associated cancer includes cancers described herein and known in the art.

In some embodiments of any of the methods or uses described herein, the patient is identified or diagnosed as having a cancer with deregulation of the RET gene, RET kinase, or the expression or activity or level of any of them. In some embodiments of any of the methods or uses described herein, the patient is identified as having a tumor that is positive for deregulation of the RET gene, RET kinase, or

- 79 041220 expression, or activity, or level of any of them. In some embodiments of any of the methods or uses, said patient may be a patient with a tumor(s) that is positive for deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them. In some embodiments of any of the methods or uses described herein, said patient may be a patient whose tumors have deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them. In some embodiments of any of the methods or uses described herein, the patient is predisposed to RET-associated cancer (e.g., a cancer that has one or more RET inhibitor resistance mutations). In some embodiments, provided herein are methods of treating a RET-associated cancer in a patient in need of such treatment, comprising a) detecting a deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the patient; and b) administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them comprises one or more fusion proteins. Non-limiting examples of RET gene fusion proteins are described in Table 1. In some embodiments, the fusion protein is KIF5B-RET. In some embodiments, the deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them comprises one or more point mutations/insertions/deletions in a RET kinase protein. Non-limiting examples of point mutations/insertions/deletions in the RET kinase protein are described in Tables 2 and 2a. In some embodiments, the point mutations/insertions/deletions in the RET kinase protein are selected from the group consisting of M918T, M918V, C634W, V804L, V804M, G810S, and G810R. In some embodiments, deregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations. Non-limiting examples of RET inhibitor resistance mutations are described in Tables 3 and 4. In some embodiments, the RET inhibitor resistance mutation is V804M. In some embodiments, the RET inhibitor resistance mutation is G810S. In some embodiments, the RET inhibitor resistance mutation is G810R. In some embodiments, a cancer with deregulation of the RET gene, RET protein, or expression or activity, or level of any of them is determined using an assay or kit approved by a regulatory agency, such as one approved by the FDA. In some embodiments, a tumor that is positive for deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them is a tumor positive for one or more RET inhibitor resistance mutations. In some embodiments, a tumor with deregulation of the RET gene, RET protein, or expression or activity, or level of any of them is determined using an assay or kit approved by a regulatory agency, such as one approved by the FDA.

In some embodiments of any of the methods or uses described herein, the patient has a medical history that indicates that the patient has a tumor that has a deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them (e.g., a tumor that has one or more RET inhibitor resistance mutations). In some embodiments, the medical history indicates that the patient is to be treated with one or more compounds of Formula I-IV, or pharmaceutically acceptable salts, amorphous or polymorphic forms, or compositions thereof, as provided herein. In some embodiments, a cancer with a deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them is a cancer with one or more RET inhibitor resistance mutations. In some embodiments, a cancer with a deregulation of the RET gene, RET protein, or expression or activity, or level of any of them is determined using an assay or kit approved by a regulatory agency, such as one approved by the FDA. In some embodiments, a tumor that is positive for deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, is a tumor that is positive for one or more RET inhibitor resistance mutations. In some embodiments, a tumor with deregulation of the RET gene, RET protein, or expression or activity, or level of any of them is determined using an assay or kit approved by a regulatory agency, such as one approved by the FDA.

Also provided are methods of treating a patient, which comprise administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof to a patient having a medical history indicating that the patient has a deregulation of the RET gene, RET kinase, or the expression or activity, or level of any of them. Also provided is the use of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof for the manufacture of a medicament for the treatment of RET-associated cancer in a patient having a medical history indicating that the patient has a deregulation of the RET gene, RET kinase, or the expression or activity, or level of any of them.

- 80 041220 of them. Some embodiments of the said methods and areas of application may further comprise: a step of performing an assay (e.g., an in vitro assay) on a sample obtained from a patient to determine whether the patient has a deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them, and recording the obtained information in the patient's clinical file (e.g., on a machine-readable medium) that the patient has been identified as having a deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them. In some embodiments, the assay is an in vitro assay. For example, an assay that uses next-generation sequencing, immunohistochemistry, or FISH assay with a Break Apart probe. In some embodiments, the assay is approved by a government regulatory agency, such as a kit approved by the FDA. In some embodiments, the assay is a liquid biopsy. In some embodiments, the deregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations.

A method of treating a subject is also provided herein. In some embodiments, the provided method comprises performing an assay on a sample obtained from the subject to determine whether the subject has a deregulation of the RET gene, the RET protein, or the expression or level of any of them. In some such embodiments, the provided method also comprises administering to the subject determined to have a deregulation of the RET gene, the RET protein, or the expression or activity or level of any of them, a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the provided method comprises determining whether the subject has a deregulation of the RET gene, the RET protein, or the expression or level of any of them by performing an assay on a sample obtained from the subject. In such embodiments, the provided method also comprises administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the deregulation of the RET gene, the RET kinase protein, or the expression or activity thereof is a gene or chromosomal translocation that results in the expression of a RET fusion protein (e.g., any of the RET fusion proteins described herein). In some embodiments, the RET fusion can be selected from the KIF5B-RET fusion and the CCDC6-RET fusion. In some embodiments, the deregulation of the RET gene, the RET kinase protein, or the expression or activity, or the level of any of them, is one or more point mutations in the RET gene (e.g., any one or more RET point mutations described herein). One or more point mutations in the RET gene can result, for example, in the translation of a RET protein having one or more of the following amino acid substitutions: M918T, M918V, C634W, V804L, V804M, G810S, and G810R. In some embodiments, the deregulation in the RET gene, RET kinase protein, or expression or activity or level of any of these is one or more RET inhibitor resistance mutations (e.g., any combination of one or more RET inhibitor resistance mutations described herein). Some embodiments of the provided methods further comprise administering to the subject another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy).

In some embodiments, the compounds provided herein exhibit penetration into the brain and/or the central nervous system (CNS). Such compounds can cross the blood-brain barrier and inhibit RET kinase in the brain and/or other CNS structures. In some embodiments, the compounds provided herein can cross the blood-brain barrier in a therapeutically effective amount. For example, treating a patient for cancer (e.g., a RET-associated cancer, such as a RET-associated brain cancer or a CNS cancer) can include administering to the patient (e.g., orally administering) a compound provided herein. In some such embodiments, the compounds provided herein are useful for treating a primary brain tumor or a metastatic brain tumor. For example, the disclosed compounds can be used in the treatment of one or more gliomas, such as glioblastoma (also known as glioblastoma multiforme), astrocytomas, oligodendrogliomas, ependymomas and mixed gliomas, meningiomas, medulloblastomas, gangliogliomas, schwannomas (neurilemmas), and craniopharyngiomas (see, e.g., the tumors listed in Louis, D.N. et al. Acta Neuropathol 131(6), 803-820 (June 2016)). In some embodiments, the brain tumor is a primary brain tumor. In some embodiments, the patient has previously been treated with another anti-cancer agent, such as another RET inhibitor (e.g., a compound that is not a compound of general formula I) or a multikinase inhibitor. In some embodiments, the brain tumor is a metastatic brain tumor. In some embodiments, the patient has previously been treated with another anti-cancer agent, such as another RET inhibitor (e.g., a compound that is not a compound of general formula I) or a multikinase inhibitor.

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Also provided are methods (e.g., in vitro methods) for selecting a treatment for a patient that has been determined or diagnosed to have a RET-associated cancer. Some embodiments may further comprise administering the selected treatment to the patient that has been determined or diagnosed to have a RET-associated cancer. For example, the selected treatment may comprise administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Some embodiments may further comprise the step of performing an assay on a sample obtained from the patient to determine whether the patient has a deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, and identifying and diagnosing, in a patient that has been determined to have a deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, the presence of a RET-associated cancer. In some embodiments, the cancer is a RET-associated cancer that has one or more RET inhibitor resistance mutations. In some embodiments, a patient is identified or diagnosed with a RET-associated cancer by using a regulatory agency approved, such as an FDA approved kit, for identifying deregulation of a RET gene, RET kinase, or expression or activity or level of any of these in the patient or in a biopsy sample from the patient. In some embodiments, the RET-associated cancer is a cancer described herein or known in the art. In some embodiments, the assay is an in vitro assay. For example, an assay that uses next generation sequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is regulatory agency approved, such as an FDA approved kit. In some embodiments, the assay is a liquid biopsy.

Also provided herein are methods for selecting a treatment for a patient, said methods comprising the step of performing an assay on a sample obtained from the patient to determine whether the patient has a deregulation of a RET gene, a RET kinase, or the expression or activity, or the level of any of them (e.g., one or more RET inhibitor resistance mutations), and identifying or diagnosing, in a patient determined to have a deregulation of a RET gene, a RET kinase, or the expression or activity, or the level of any of them, the presence of a RET-associated cancer. Some embodiments further comprise administering the selected treatment to a patient determined or diagnosed to have a RET-associated cancer. For example, the selected treatment may comprise administering a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, to a patient identified or diagnosed to have a RET-associated cancer. In some embodiments, said assay is an in vitro assay. For example, an assay that uses next-generation sequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy.

Methods of selecting a patient for treatment are also provided, wherein said methods comprise selecting, identifying or diagnosing a patient suffering from a RET-associated cancer, and selecting the patient for treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, identifying or diagnosing a patient as suffering from a RET-associated cancer may comprise the step of performing an analysis on a sample obtained from the patient to determine whether the patient has a deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, and identifying and diagnosing in a patient determined to have a deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, the presence of a RET-associated cancer. In some embodiments, the method of selecting a patient for treatment can be used as part of a clinical trial that includes administering various treatments for RET-associated cancer. In some embodiments, the RET-associated cancer is a cancer that has one or more RET inhibitor resistance mutations. In some embodiments, the assay is an in vitro assay. For example, an assay that uses next-generation sequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is approved by a government regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy. In some embodiments, deregulation of the RET gene, RET kinase, or expression or activity, or level of any of these includes one or more RET inhibitor resistance mutations.

In some embodiments of any of the methods or uses described herein, an assay used to determine whether a patient has a deregulation of the RET gene or RET kinase, or the expression or activity or level of any of them, using a sample obtained from the patient may include, for example, next generation sequencing,

- 82 041220 immunohistochemistry, fluorescence microscopy, FISH analysis with a Break Apart probe, Southern blotting, Western blotting, FACS analysis, Northern blotting and PCR-based amplification (e.g., RT-PCR and quantitative real-time RT-PCR). As is known in the art, these assays are typically performed, for example, using at least one labeled nucleic acid probe or at least one labeled antibody or antigen-binding fragment thereof. The assays may use other detection methods known in the art for detecting deregulation of the RET gene, RET kinase, or expression or activity, or the level of any of them (see, for example, the references cited herein). In some embodiments, the deregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations. In some embodiments, the sample is a biological sample or a biopsy sample (e.g., a paraffin-embedded biopsy sample) obtained from a patient. In some embodiments, the patient is a patient suspected of having a RET-associated cancer, a patient having one or more symptoms of a RET-associated cancer, and/or a patient at increased risk of developing a RET-associated cancer.

In some embodiments, deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them can be identified using a liquid biopsy (otherwise referred to as a liquid biopsy or liquid phase biopsy). See, e.g., Karachialiou et al., Realtime liquid biopsies become a reality in cancer treatment, Ann. Transl. Med, 3(3):36, 2016. Liquid biopsy methods can be used to detect overall tumor burden and/or deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them. Liquid biopsy can be performed on biological samples that are relatively easily obtained from a subject (e.g., through routine blood sampling), and is generally less invasive than traditional methods used to detect tumor burden and/or deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them. In some embodiments, a liquid biopsy can be used to detect the presence of deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them at an earlier stage than in traditional methods. In some embodiments, a biological sample to be used for a liquid biopsy can include, for example, blood, plasma, urine, cerebrospinal fluid, saliva, sputum, bronchoalveolar lavage, bile, lymphatic fluid, cystic fluid, stool, peritoneal effusion and combinations thereof. In some embodiments, a liquid biopsy can be used to detect circulating tumor cells (CTCs) in the blood. In some embodiments, a liquid biopsy can be used to detect cell-free DNA. In some embodiments, the cell-free DNA detected by a liquid biopsy is circulating tumor DNA (ctDNA) in the blood, formed from tumor cells. Analysis of ctDNA (e.g., using sensitive detection technologies such as, but not limited to, next-generation sequencing (NGS), conventional PCR, digital PCR, or microarray analysis) can be used to identify deregulation of the RET gene, RET kinase, or expression or activity or level of any of them.

In some embodiments, the liquid biopsy can detect ctDNA formed from a single gene. In some embodiments, the liquid biopsy can detect ctDNA formed from multiple genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more, or any number of genes in between). In some embodiments, ctDNA formed from multiple genes can be detected using any of a variety of commercially available test panels (e.g., commercially available test panels configured to detect deregulation of a RET gene, RET kinase, or expression or activity or level of any of them). Liquid biopsies can be used to detect deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, including, but not limited to, point mutations or single nucleotide variants (SNVs), copy number variants (CNVs), genetic fusions (e.g., translocations or rearrangements), insertions, deletions, or any combination thereof. In some embodiments, a liquid biopsy can be used to detect a gamete mutation. In some embodiments, a liquid biopsy can be used to detect a somatic mutation. In some embodiments, a liquid biopsy can be used to detect a primary genetic mutation (e.g., a primary mutation or primary fusion associated with the initial development of a disease, such as cancer). In some embodiments, a liquid biopsy can be used to detect a genetic mutation that develops after the development of the primary genetic mutation (e.g., a resistance mutation that occurs in response to a drug that is administered to a subject). In some embodiments, the deregulation of the RET gene, RET kinase, or expression or activity or level of any of them that is determined by the liquid biopsy is also present in a cancer cell that is in the subject's body (e.g., a tumor). In some embodiments, any type of gene deregulation can be detected by the liquid biopsy.

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RET, RET kinase, or expression or activity or level of any of them, as described herein. In some embodiments, a genetic mutation identified by a liquid biopsy can be used to identify a subject as potentially suitable for a particular treatment. For example, detection of deregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a subject can indicate that the subject will be responsive to a treatment that involves administering a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

A liquid biopsy may be performed at different time points during the course of diagnosis, the course of observation, and/or the course of treatment to determine one or more clinically significant parameters, including, but not limited to, disease progression, treatment efficacy, or the development of resistance mutations following administration of a drug to a subject. For example, a first liquid biopsy may be performed at a first time point, and a second liquid biopsy may be performed at a second time point during the course of diagnosis, the course of observation, and/or the course of treatment. In some embodiments, the first time point may be a time point before the subject is diagnosed with a disease (e.g., when the subject is healthy), and the second time point may be a time point after the subject has developed a disease (e.g., the second time point may be used to diagnose the subject with a disease). In some embodiments, the first time point may be a time point before the subject is diagnosed with a disease (e.g., when the subject is healthy), after which the subject is observed, and the second time point may be a time point after the subject is observed. In some embodiments, the first time point may be a time point after the subject is diagnosed with the disease, after which the subject is administered the treatment, and the second time point may be a time point after the treatment has been administered; in such cases, the second time point may be used to assess the effectiveness of the treatment (e.g., if the genetic mutation(s) detected at the first time point have decreased in abundance or are undetectable) or to determine the presence of a resistance mutation resulting from the treatment. In some embodiments, the treatment to be administered to the subject may comprise a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof.

In some embodiments, the efficacy of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, can be determined by analyzing the allele frequency of deregulated RET gene in cDNA obtained from a patient at different time points, such as cDNA obtained from a patient at a first time point and cDNA obtained from a patient at a second time point, wherein at least one dose of a compound of formula I-IV is administered to the patient between the first and second time points. Some embodiments of such methods can further comprise administering to the patient at least one dose of a compound of formula IIV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, between the first and second time points. For example, a reduction (e.g., a reduction from 1% to about 99%, a reduction from 1% to about 95% reduction, a reduction from 1% to about 90%, a reduction from 1% to about 85%, a reduction from 1% to about 80%, a reduction from 1% to about 75%, a reduction from 1% to about 70%, a reduction from 1% to about 65%, a reduction from 1% to about 60%, a reduction from 1% to about 55%, a reduction from 1% to about 50%, a reduction from 1% to about 45%, a reduction from 1% to about 40%, a reduction from 1% to about 35%, a reduction from 1% to about 30%, a reduction from 1% to about 25%, a reduction from 1% to about 20%, a reduction from 1% to about 15%, a reduction from 1% to about 10%, a reduction from 1% to about 5%, from about 5% to about 99%, from about 10% to about 99%, from about 15% to about 99%, from about 20% to about 99%, from about 25% to about 99%, from about 30% to about 99%, from about 35% to about 99%, from about 40% to about 99%, from about 45% to about 99%, from about 50% to about 99%, from about 55% to about 99%, from about 60% to about 99%, from about 65% to about 99%, from about 70% to about 99%, from about 75% to about 95%, from about 80% to about 99%, from about 90% to about 99%, from about 95% to about 99%, from about 5% to about 10%, from about 5% to about 25%, from about 10% to about 30%, from about 20% to about 40%, from about 25% to about 50%, from about 35% to about 55%, from about 40% to about 60%, from about 50% to about 75%, from about 60% to about 80%, or a decrease of from about 65% to about 85%) of the allele frequency (AF) of deregulated RET gene in scDNA obtained from the patient at a second time point, compared to the allele frequency (AF) of deregulated RET gene in scDNA obtained from the patient at a first time point, indicates that the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is effective in the subject. In some embodiments, the AF is reduced such that the level is below the detection limit of the instrument. Alternatively, an increase in the allele frequency (AF) of deregulated RET gene in the patient-derived scDNA at the second time point, compared to the allele frequency (AF) of deregulated RET gene in the patient-derived scDNA at the first time point, indicates that the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof is ineffective in the subject (e.g., the subject has developed a resistance mutation to

- 84 041220 compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof). Some embodiments of these methods may further comprise administering additional doses of the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof to a patient who has been shown to be effective with the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Some embodiments of these methods may further comprise administering another treatment (e.g., a treatment that does not include administering the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy) to a patient who has been shown to be ineffective with the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

In some examples of such methods, the time difference between the first and second points in time may be from about 1 day to about 1 year, from about 1 day to about 11 months, from about 1 day to about 10 months, from about 1 day to about 9 months, from about 1 day to about 8 months, from about 1 day to about 7 months, from about 1 day to about 6 months, from about 1 day to about 5 months, from about 1 day to about 4 months, from about 1 day to about 3 months, from about 1 day to about 10 weeks, from about 1 day to about 2 months, from about 1 day to about 6 weeks, from about 1 day to about 1 month, from about 1 day to about 25 days, from about 1 day to about 20 days, from about 1 day to about 15 days, from about 1 day to about 10 days, from about 1 day to about 5 days, from about 2 days to about 1 year, about 5 days to about 1 year, about 25 days to about 1 year, about 1 month to about 1 year, about 6 weeks to about 1 year, about 2 months to about 1 year, about 3 months to about 1 year, about 4 months to about 1 year, about 5 months to about 1 year, about 3 months to about 1 year, about 4 months to about 1 year, about 5 months to about 1 year, about 6 months to about 1 year, about 7 months to about 1 year, about 8 months to about 1 year, about 9 months to about 1 year, about 10 months to about 1 year, about 11 months to about 1 year, about 1 day to about 7 days, about 1 day to about 14 days, about 5 days to about 10 days, about 5 days to about 20 days, about 10 days to about 20 days, about 15 days to about 1 month, about 15 days to about 2 months, about 1 week to about 1 month, about 2 weeks to about 1 month, about 1 month to about 3 months, about 3 months to about 6 months, about 4 months to about 6 months, about 5 months to about 8 months, or about 7 months to about 9 months. In some embodiments of such methods, the patient may have previously been identified as having a cancer that has a deregulated RET gene (e.g., any of the examples of deregulated RET genes described herein). In some embodiments of such methods, the patient may have previously been diagnosed as having any of the types of cancers described herein. In some embodiments of such methods, the patient may have one or more metastases (e.g., one or more brain metastases).

In some of the above embodiments, the scDNA comprises a ctDNA, such as a RET-associated ctDNA. For example, the scDNA is a ctDNA, such as a RET-associated ctDNA. In some embodiments, at least a portion of the scDNA is determined to be a RET-associated ctDNA, such as a sequenced and/or quantified amount of total scDNA is determined to have a RET fusion mutation and/or a RET resistance mutation.

In the field of medical oncology, it is standard practice to use a combination of different forms of treatment to treat each patient suffering from cancer. In the field of medical oncology, other component(s) of such a combined treatment or therapy, in addition to the compositions provided herein, may be, for example, surgery, radiation therapy and chemotherapeutic agents such as other kinase inhibitors, signal transduction inhibitors and/or monoclonal antibodies. For example, the surgery may be an open surgery or a minimally invasive surgery. Thus, the compounds of formula I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms may also be useful as adjuvants for the treatment of cancer, i.e., they can be used in combination with one or more additional therapeutic agents or therapeutic agents, for example, a chemotherapeutic agent that acts by the same or a different mechanism of action. In some embodiments, a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof may be used prior to the administration of an additional therapeutic agent or additional therapy. For example, a patient in need thereof may be administered one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, over a period of time, and then subjected to at least partial tumor resection. In some embodiments, treatment with one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, may provide a reduction in tumor size (e.g., tumor burden) before at least partial tumor resection. In some embodiments, a patient in need thereof may be administered one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, over a period of time, and then subjected to one or more courses of radiation therapy. In some embodiments, treatment with one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof may provide a reduction in tumor size (e.g., tumor burden) prior to one or more courses of radiation therapy.

In some embodiments, the patient has a cancer (e.g., a locally advanced or metastatic tumor) that is resistant or intolerant to standard therapy (e.g., administration of a chemotherapeutic agent such as a first RET inhibitor or a multikinase inhibitor, immunotherapy, or radiation (e.g., radioactive iodine)). In some embodiments, the patient has a cancer (e.g., a locally advanced or metastatic tumor) that is resistant or intolerant to prior therapy (e.g., administration of a chemotherapeutic agent such as a first RET inhibitor or a multikinase inhibitor, immunotherapy, or radiation (e.g., radioactive iodine)). In some embodiments, the patient has a cancer (e.g., a locally advanced or metastatic tumor) for which there is no standard therapy. In some embodiments, the patient has not previously been treated with RET kinase inhibitors. For example, the patient has not previously been treated with a selective RET kinase inhibitor. In some embodiments, the patient has previously been treated with RET kinase inhibitors.

In some embodiments, the patient has been previously treated. In some embodiments, the patient suffering from NSCLC (e.g., RET fusion-positive NSCLC) has been previously treated with platinum-based chemotherapy, PD-1/PDL1 immunotherapy, or both prior to initiating treatment with a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form. In some embodiments, the patient suffering from thyroid cancer (e.g., RET fusion-positive thyroid cancer) has been previously treated with one or more of sorafenib, lenvatinib, and radioactive iodine prior to initiating treatment with a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form. In some embodiments, a patient suffering from colorectal cancer (e.g., RET fusion-positive colorectal cancer) has previously been treated with fluoropyrimidine-based chemotherapy, with or without targeted anti-VEGF therapy or targeted anti-EGFR therapy, prior to initiating treatment with a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a patient suffering from pancreatic cancer (e.g., RET fusion-positive pancreatic cancer) has previously been treated with one or more of fluoropyrimidine-based chemotherapy, gemcitabine-based chemotherapy, and S-1 chemotherapy prior to initiating treatment with a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a patient suffering from breast cancer (e.g., RET fusion-positive breast cancer) has previously been treated with one or more of an anthracycline, a taxane, a HER2-targeted therapy, and a hormonal therapy prior to initiating treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, a patient suffering from MTC (e.g., RET fusion-positive MTC cancer) has previously been treated with one or more of caboxantinib and vandetanib prior to initiating treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof.

In some embodiments of any of the methods described herein, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is administered in combination with a therapeutically effective amount of at least one additional therapeutic agent selected from one or more additional therapeutic agents or therapeutic (e.g., chemotherapeutic) agents.

Non-limiting examples of additional therapeutic agents include: other therapeutic agents directed at RET (i.e., a first or second RET kinase inhibitor), other kinase inhibitors (e.g., therapeutic agents directed at receptor tyrosine kinase (i.e., Trk inhibitors or EGFR inhibitors)), signal transduction pathway inhibitors, checkpoint inhibitors, apoptotic pathway modulators (e.g., obataclax); cytotoxic chemotherapeutic agents, therapeutic agents directed at angiogenesis, agents directed at the immune system, including immunotherapy, and radiation therapy.

In some embodiments, the other RET-targeted therapeutic agent is a multi-kinase inhibitor that exhibits RET inhibition activity. In some embodiments, the other RET-targeted therapeutic inhibitor is selective for RET kinase. Exemplary RET kinase inhibitors may exhibit an inhibitory activity (IC ₅₀ ) against RET kinase of less than about 1000 nM, less than about 500 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM, as measured in an assay described herein. In some embodiments, RET kinase inhibitors may exhibit an inhibitory activity

- 86 041220 activity (IC ₅₀ ) against RET kinase of less than about 25 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM, as measured in the assay provided herein.

Non-limiting examples of therapeutic agents that target RET (e.g., a first RET inhibitor or a second RET inhibitor) include alectinib (9-ethyl-6,6-dimethyl-8-[4-(morpholin4-yl)piperidin-1-yl]-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile); amuvatinib (MP470, HPK56) (N-(1,3-benzodioxol-5-ylmethyl)-4-([1]benzofuro[3,2-d]pyrimidin-4-yl)piperazine-1-carbothioamide); apatinib (YN968D1) (N-[4-(1-cyanocyclopentyl)phenyl-2-(4-picolyl)amino-3-nicotinamide methanesulfonate); cabozantinib (Cometriq XL-184) (N-(4-((6,7-dimethoxyquinolin-4yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); dovitinib (TKI258; GFKI-258; CHIR-258) ((3Z)-4-amino-5-fluoro-3-[5-(4-methylpiperazin-1-yl)-1,3-dihydrobenzimidazol-2-ylidene]quinolin-2-one); famitinib (5-[2-(diethylamino)ethyl]-2-[(Z)-(5-fluoro-2-oxo-1H-indol-3ylidene)methyl]-3-methyl-6,7-dihydro-1H-pyrrolo[3,2-c]pyridin-4-one); fedratinib (SAR302503, TG101348) (N-(2-methyl-2-propanyl)-3-{[5-methyl-2-({4-[2-(1-pyrrolidinyl)ethoxy]phenyl}amino)-4-pyrimidinyl]amino}benzenesulfonamide); foretinib (XL880, EXEL-2880, GSK1363089, GSK089) (N'[3-fluoro-4-[[6-methoxy-7-(3-morpholinopropoxy)-4-quinolyl]oxy]phenyl]-N1-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); fostamantinib (R788) (2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one, 6-[[5-fluoro2-[(3,4,5-trimethoxyphenyl)amino]-4-pyrimidinyl]amino]-2,2-dimethyl-4-[(phosphonooxy)methyl]-, sodium salt (1:2)); ilorasertiib (ABT-348) (1-(4-(4-amino-7-(1-(2-hydroxyethyl)-1H-pyrazol-4yl)thieno[3,2-c]pyridin-3-yl)phenyl)-3-(3-fluorophenyl)urea); lenvatinib (E7080, Lenvima) (4-[3chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide); motesanib (AMG 706) (N-(3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-[(pyridin-4-ylmethyl)amino]pyridin-3carboxamide); nintedanib (3-Z-[1-(4-(N-((4-methylpiperazin-1-yl)methylcarbonyl)-N-methylamino)anilino)-1-phenylmethylene]-6-methyloxycarbonyl-2-indolinone); ponatinib (AP24534) (3-(2-imidazo[1,2b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]benzamide); PP242 (torkinib) (2-[4-amino-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]-1Nindol-5-ol); quizartinib (1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-(7-(2-morpholinoethoxy)benzo[d]imidazo[2,1-b]thiazol-2-yl)phenyl)urea; regorfenib (BAY 73-4506, Stivarga) (4-[4-({[4-chloro-3(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide hydrate); RXDX-105 (CEP-32496, agerafenib) (1-(3-((6,7-dimethoxyquinazolin-4-yl)oxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea); semaxanib (SU5416) ((3Z)-3-[(3,5-dimethyl-1Hpyrrol-2-yl)methylidene]-1,3-dihydro-2H-indol-2-one); sitravatinib (MGCD516, MG516) (N-(3-fluoro-4{[2-(5-{[(2-methoxyethyl)amino]methyl}-2-pyridinyl)thieno[3,2-b]pyridin-7-yl]oxy}phenyl)-N-(4-fluorophenyl)-1,1-cyclopropanedicarboxamide); sorafenib (BAY 43-9006) (4-[4-[[[[4-chloro-3(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-2-pyridinecarboxamide); vandetanib (N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine); vatalanib (PTK787, PTK/ZK, ZK222584) (N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine); AD-57 (N-[4[4-amino-1-(1-methylethyl)-1 H-pyrazolo[3,4-d]pyrimidin-3-yl]phenyl]-N-[3-(trifluoromethyl)phenyl]urea); AD-80 (1-[4-(4-amino-1-propan-2-ylpyrazolo[3,4-d]pyrimidin-3-yl)phenyl]-3-[2-fluoro-5(trifluoromethyl)phenyl]urea); AD-81 (1-(4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3yl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea); ALW-II-41-27 (N-(5-((4-((4-ethylpiperazin-1yl)methyl)-3-(trifluoromethyl)phenyl)carbamoyl)-2-methylphenyl)-5-(thiophen-2-yl)nicotinamide); BPR1K871 (1-(3-chlorophenyl)-3-(5-(2-((7-(3 -(dimethylamino)propoxy)quinazolin-4-yl)amino)ethyl)thiazol-2yl)urea); CLM3 (1-phenethyl-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); EBI-907 (N-(2-chloro-3-(1-cyclopropoyl-8-methoxy-3H-pyrazolo[3,4-c]isoquinolin-7-yl)-4-fluorophenyl)-3-fluoropropane-1sulfonamide); NVP-AST-487 (N-[4-[(4-ethyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]-N'-[4-[[6(methylamino)-4-pyrimidinyl]oxy]phenyl]urea); NVP-BBT594 (BBT594) (5-((6-acetamidopyrimidin-4-yl)oxy)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)indolin-1-carboxamide); PD173955 (6-(2,6-dichlorophenyl)-8-methyl-2-(3-methylsulfanylanilino)pyrido[2,3-d]pyrimidin-7-one); PP2 (4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine); PZ1 (N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1yl)phenyl)acetamide); RPI-1 (1,3-dihydro-5,6-dimethoxy-3-[(4-hydroxyphenyl)methylene]-H-indol-2-one; (3E)-3-[(4-hydroxyphenyl)methylidene]-5,6-dimethoxy-1H-indol-2-one); SGI-7079 (3-[2-[[3-fluoro-4-(4-methyl-1-piperazinyl)phenyl]amino]-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]benzeneacetonitrile); SPP86 (1-isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); SU4984 (4-[4-[(E)-(2-oxo-1H-indol3-ylidene)methyl]phenyl]piperazine-1-carbaldehyde); sunitinib (SU11248) (N-(2-diethylaminoethyl)-5-[(Z)(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide); TG101209 (N-tert-butyl-3-(5-methyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide); withaferin A ((4β,5β,6β,22R)-4,27-dihydroxy-5,6:22,26-diepoxyergosta-2,24-dien-1,26-dione); XL-999 ((Z)-5-((1-ethylpiperidin-4-yl)amino)-3-((3-fluorophenyl)(5-methyl-1H-imidazol-2-yl)methylene)indolin-2one); BPR1J373 (5-phenylthiazol-2-ylamine-pyrimidine derivative); CG-806 (CG'806); DCC-2157; GTX-186; HG-6-63-01 ((E)-3-(2-(4-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)vinyl)-N-(4-((4-ethylpiperazin1-yl)methyl)-3-(trifluoromethyl)phenyl)-4-methylbenzamide); SW-01 (cyclobenzaprine hydrochloride);

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XMD15-44 (N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-4-methyl-3-(pyridin-3-ylethynyl)benzamide (derived from the structure)); Y078-DM1 (antibody drug conjugate consisting of RET antibody (Y078) linked to a derivative of the cytotoxic agent maytansine); Y078-DM4 (antibody drug conjugate consisting of RET antibody (Y078) linked to a derivative of the cytotoxic agent maytansine); ITRI-305 (D0N5TB, DIB003599); BLU-667 (((1 S,4R)-N-((S)-1 -(6-(4-fluoro-1 H-pyrazol-1 -yl)pyridin-3-yl)ethyl)-1 -methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide); BLU6864; DS-5010; GSK3179106; GSK3352589; NMS-E668 and TAS0286/HM05.

Additional examples of therapeutic agents targeting RET (e.g., a first RET kinase inhibitor or a second RET kinase inhibitor) include 5-amino-3-(5-cyclopropylisoxazol-3-yl)-1-isopropyl-1H-pyrazole-4-carboxamide; 3-(5-cyclopropylisoxazol-3-yl)1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine; 3-((6,7-dimethoxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol; N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(imidazo[1,2-a]pyridin-6-yl)phenyl)acetamide; N-(5-(tert-butyl)isoxazol-3-yl)-2-(3-(imidazo[1,2-b]pyridazin-6-yloxy)phenyl)acetamide; N-(2-Φτορ-5-trifluoromethylphenyl)-N'-{4'-[(2-benzamido)pyridin-4-ylamino]phenyl}urea; 2-amino-6-{[2-(4-chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-thienyl)pyridine-3,5-dicarbonitrile and 3-arylureidobenzylidenindolin-2-ones.

Additional examples of other RET kinase inhibitors include those described in U.S. Patent Nos. 9,150,517 and 9,149,464, and International Publication No. WO2014075035, which are incorporated herein by reference. For example, in some embodiments, the other RET inhibitor is a compound of Formula I

CI

J Ыъ

CG X—k ' Λ-ORt o ¹ where R1 is C ₆ -C ₂₄ alkyl or polyethylene glycol; or a pharmaceutically acceptable salt thereof. In some embodiments, the other RET inhibitor is 4-{5-[bis-(chloroethyl)amino]-1-methyl-1H-benzimidazol-2-yl}butyric acid dodecyl ester.

Further examples of other RET kinase inhibitors include those described in International Publication No. WO2016127074, the contents of which are incorporated herein by reference. For example, in some embodiments, the other RET inhibitor is a compound of formula (I), or a pharmaceutically acceptable salt thereof:

(O where rings A and B are each independently selected from aryl, heteroaryl, cycloalkyl and heterocyclyl; each L ¹ and L ² is independently selected from a bond, -(C1-C6-alkylene)-, -(C2-C6-alkenylene)-, -(C2-C6-alkynylene)-, -(C1-C6-haloalkylene)-, -(C1-C6-heteroalkylene)-, -C(O)-, -O-, -S-, -S(O), -S(O)2-, -N(R ¹ )-, -O-(C1-C6-alkylene)-, -(CrC ₆ -alkylene)-O-, -N(R ¹ )-C(O)-, -C(O)N(R1)-, -(C1-C6-alkylene)-N ¹ )-, -N(R1)-(CrC6alkylene)-, -N( ^R1 )-C(O)-(C1-C6alkylene)-, -(C1-C6alkylene)-N( ^R1 )-C(O)-, -C(O)-N( ^R1 )-(C1-C6alkylene)-, -(C1-C6alkylene)-C(O)-N( ^R1 )-, -N(R1)-S(O)2-, -S(O)2-N(R1)-, -N( ^R1 )-S(O)2-(C1-C6alkylene)- and -S(O)2-N(R1)(C1- _C6- alkylene)-; wherein each alkylene, alkenylene, alkynylene, haloalkylene and heteroalkylene is independently substituted with 0-5 R'groups; each RA and RB is independently selected from C1- _C6 alkyl, C1- _C6 alkoxy, halogen, C1- _C6 haloalkyl, C1- _C6 hydroxyalkyl, C1- _C6 heteroalkyl and -N(R1)(R1); wherein each alkyl, alkoxy, haloalkyl, hydroxyalkyl and hydroxyalkyl is independently substituted with 0-5 R ^a groups; each R ^C and RD are independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl, heterocyclylalkyl, nitro, cyano, -C(O)R1, -OC(O)R1, -C(O)OR1, -(C1-C6-alkylene)-C(O)R ¹ , -SR ¹ -S(O)2R ¹ , -S(O)2-N(R ¹ )(R ¹ ), -(C1-C6-αalkylene)-S(O)2R ¹ , -(CrCgalkylene)-S(O)2-N(R ¹ )(R ¹ ), -N(R1)(R1)-C(O)-N(R ¹ )(R ¹ )-N(R ¹ )-C(O)R ¹ , -N(R1)-C(O)OR1, -(C1-C6-αalkylene)N(R1)-C(O)R1, -N(R1)S(O)2R1 and -P(O)(R1)(R1); wherein each alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, haloalkyl, haloalkoxy, hydroxyalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl and heterocyclylalkyl is independently substituted with 0-5 R ^a groups; or 2 R ^C or 2 R ^D together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 R ^a groups; each R ¹ is independently selected from hydrogen, hydroxyl, halogen, thiol, C1-C ₆ alkyl, C1-C ₆ thioalkyl, C1-C ₆ alkoxy, C1-C _{6 haloalkyl, C1-C 6} hydroxyalkyl, C1-C 6 heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl

- 88 041220 and heterocyclylalkyl, wherein each alkyl, thioalkyl, alkoxy, haloalkyl, hydroxyalkyl, heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl and heterocyclylalkyl is independently substituted with 0-5 R ^b groups, or 2 R ¹ , together with the atom(s) to which they are attached, form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 R ^b groups; each ^Ra and R ^b is independently C1-C6 alkyl, halogen, hydroxyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 hydroxyalkyl, C1-C6 alkoxy, cycloalkyl, heterocyclyl or cyano, wherein each alkyl, haloalkyl, heteroalkyl, hydroxyalkyl, alkoxy, cycloalkyl and heterocyclyl is independently substituted with 0-5 R'groups; each R' is _CrC6 alkyl, _CrC6 heteroalkyl, halogen, hydroxyl, _CrC6 haloalkyl, _C1 -C6 hydroxyalkyl, cycloalkyl or cyano; or 2 R' together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring; m is 0, 1, 2 or 3; n is 0, 1 or 2; and p and q are each independently 0, 1, 2, 3 or 4.

For example, the RET inhibitor may be selected from the group consisting of:

- 89 041220

- 90 041220

- 91 041220

- 92 041220

or their pharmaceutically acceptable salts.

Further examples of other RET kinase inhibitors include those described in International Publication No. WO2016075224, the contents of which are incorporated herein by reference. For example, in some embodiments, the other RET inhibitor is a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein

R1 and R2 independently represent hydrogen or an optionally substituted group selected from straight or branched ( _C1 - _C6 )alkyl, ( _C3 - _C6 )cycloalkyl and COR', where R' is an optionally substituted group selected from straight or branched ( _C1C6 )alkyl and ( _C3 - _C6 )cycloalkyl;

R3 is hydrogen or an optionally substituted group selected from straight or branched ( _C1 - _C6 )alkyl, ( _C2 - _C6 )alkenyl, ( _C2 - _C6 )alkynyl, ( _C3 - _C6 )cycloalkyl, aryl, heteroaryl and a 3-7-membered heterocyclyl ring;

R4 is hydrogen or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, aryl, heteroaryl or heterocyclyl;

A represents a 5- or 6-membered heteroaryl ring or a phenyl ring;

B is a 5- or 6-membered ring selected from heteroaryl, (C ₅ -C ₆ )cycloalkyl and a heterocyclyl ring, or a phenyl ring; wherein ring A and ring B are fused together to form a bicyclic system containing a 6-membered aromatic or 5-6-membered heteroaromatic ring fused to a 6-membered aromatic or 5-6-membered heteroaromatic, (C ₅ -C ₆ )cycloalkyl or heterocyclyl ring;

Y represents carbon or nitrogen;

X is hydrogen, halogen, hydroxyl, cyano or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl and (C ₁ -C ₆ )alkoxyl; and R5 and R6 independently are hydrogen or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl, (C ₃ -C ₆ )cycloalkyl, heterocyclyl, aryl and heteroaryl.

Additional examples of other RET kinase inhibitors include those described in

- 93 041220 of International Publication No. WO 2015079251, the contents of which are incorporated herein by reference. For example, in some embodiments, the other RET inhibitor is a compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof, wherein it

X is NH, NRx, O or S, where Rx is (1-3C)alkyl;

R ₁ is selected from halogen (e.g. fluorine, chlorine or bromine), trifluoromethyl, (1-4C)alkyl (e.g. methyl), (1-4C)alkoxy or (3-6C)cycloalkyl, wherein the alkyl, alkoxy or cycloalkyl group is optionally substituted with one or more fluorine atoms;

R ₂ is selected from hydrogen, halogen (e.g. fluorine, chlorine or bromine), hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C)alkyl (e.g. methyl), (3-8C)cycloalkyl or (1-4C)alkoxy (e.g. OMe), wherein the alkyl, cycloalkyl or alkoxy group is optionally substituted with one or more fluorine atoms;

R ₃ is selected from hydrogen, halogen (e.g. fluorine, chlorine or bromine), hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C)alkyl (e.g. methyl), (3-8C)cycloalkyl or (1-4C)alkoxy (e.g. OMe), wherein the alkyl, cycloalkyl or alkoxy group is optionally substituted with one or more fluorine atoms;

R ₄ is selected from hydrogen, halogen (e.g. fluorine, chlorine or bromine), hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C)alkyl (e.g. methyl), (3-8C)cycloalkyl or (1-4C)alkoxy (e.g. OMe), wherein the alkyl, cycloalkyl or alkoxy group is optionally substituted with one or more fluorine atoms;

R5 is selected from hydrogen or a group defined by the formula

- O-L5-X5-Q5, where L5 is absent or represents a linear or branched (1-4C)alkylene;

X ₅ is absent or represents -C(O)O-, -O-, -C(O)-, -OC(O)-, -CH(QR _5L )-, -N(Rj)-, -N( R _5L )C(O)-, -N(R _5L )-C(O)O-, -C(O)-N(R _5L )-, -S-, -SO-, -SO2-, -S(O) ₂ N(R _5L )- or -N(R _5L )SO ₂ -, where R _5L is selected from hydrogen or methyl; and Q5 is (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, (38C)cycloalkyl, (3-8C)cycloalkyl-(1-4C)alkyl, aryl, aryl-(1 -4C)alkyl, heteroaryl, heteroaryl-(14C)alkyl, heterocyclyl or heterocyclyl-(1-4C)alkyl;

R ₆ is selected from hydrogen or a group defined by the formula

- O-L6-X6-Q6, where L ₆ is absent or represents a linear or branched (1-4C)alkylene;

X6 is absent or is selected from -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR6L)-, -N(R6L), -N(R6L)-C(O)-, -N(R6L)-C(O)O-, -C(O)-N(R6L)-, -S-, -SO-, -SO2-, -S(O)2N(R6L)- or -N(R6L)SO2-, where R6L is selected from hydrogen or (1-3C)alkyl;

Q ₆ is hydrogen, (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, (3-8C)cycloalkyl, (38C)cycloalkyl-(1-6C)alkyl, aryl, aryl-(1-6C)alkyl, heteroaryl, heteroaryl-(1-6C)alkyl, heterocyclyl, heterocyclyl-(1-6C)alkyl, or Q ₆ and R _L6 are linked so that together with the nitrogen atom to which they are attached they form a heterocyclic ring;

wherein R ₆ is optionally substituted (e.g. substituted on L ₆ and/or Q ₆ ) with one or more (1-6C)alkyl, (1-6C)alkanoyl, OR _6X , SR _6X , S(O)R6x, S(O) ₂ R _6X , C(O)OR _6X or C(O)NR _6X R' _6X , wherein R _6X and R' _6X are independently hydrogen, (1-8C)alkyl, or R _6X and R' _6X are linked such that together with the nitrogen atom to which they are attached they form a heterocyclic ring; and

R ₇ is selected from hydrogen, (1-6C)alkoxy or a group defined by the formula

- O-L7-X7-Q7-, where L7 is absent or represents a linear or branched (1-4C)alkylene;

X7 is absent or selected from -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR6L)-, -N(R7L)-, -N(R7L)-C(O)-, -N(R _7L )-C(O)O-, -C(O)-N(R _7L )-, -S-, -SO-, -SO2-, -S(O) ₂ N(R _7L )- or -N(R _7L )SO ₂ -, where R _7L is selected from hydrogen or (1-3C)alkyl;

Q7 is hydrogen, (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, (3-8C)cycloalkyl, (38C)cycloalkyl-(1-6C)alkyl, aryl, aryl-(1-6C)alkyl, heteroaryl, heteroaryl-(1-6C)alkyl, heterocyclyl, heterocyclyl-(1-6C)alkyl, or Q7 and R7L are linked so that together with the nitrogen atom to which they are attached they form a heterocyclic ring;

wherein R ₇ is optionally substituted (e.g. substituted on L ₇ and/or Q ₇ ) with one or more halogens, hydroxyls, nitro, cyano, (1-8C)alkyls, (1-8C)alkanoyls, OR _7X , SR _7X , S(O)R _7X , S(O) ₂ R _7X , C(O)OR _7X or C(O)NR _7X R' _7X , wherein R _7X and R' _7X independently represent hydrogen, (1-8C)alkyl, or R _7X and R' _7X

- 94 041220 are so linked that together with the nitrogen atom to which they are attached they form a heterocyclic ring; or

R7 is optionally substituted with one or more groups selected from oxo, (1-4C)haloalkyl, (1-4C)hydroxyalkyl, C(O)R7y or NR7 _y R'7 _y , where R7 _y and R'7 _y independently represent hydrogen or (1-8C)alkyl.

Further examples of other RET kinase inhibitors include those described in International Publication No. WO2017178845, the contents of which are incorporated herein by reference. For example, in some embodiments, the other RET inhibitor is a compound of formula (IV), or a pharmaceutically acceptable salt thereof, wherein

R1 is selected from hydrogen, (1-4C)haloalkyl, (1-4C)haloalkoxy or a group of the formula

-L-Y-Q, where L is absent or represents (1-5C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

Y is absent, O, S, SO, _SO2 , N( _Ra ), C(O), C(O)O, OC(O), C(O)N( _Ra ), N(Ra)C(O), N(Ra)C(O)N(Rb), N(Ra)C(O)O, OC(O)N(Ra), S(O^N(Ra) or N(Ra)SO2, where Ra and R1 are each independently selected from hydrogen or (1-4C)alkyl; and Q is hydrogen, (1-6C)alkyl, (26C)alkenyl, (2-6C)alkynyl, aryl, (3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl or heterocyclyl; wherein Q is optionally further substituted with one or more substituent groups independently selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino, (14C)aminoalkyl, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, NRcRd, ORc, C(O)Rc, C(O)ORc, OC(O)Rc, C(O)N(Rd)Rc, N(Rd)C(O)Rc, S(O)pRc (where p is 0, 1, or 2), SO2N(Rd)Rc, N(Rd)SO ₂ Rc, Si(Re)(R _d )R _c, or (CH ₂ )qNR _c R _d (where q is 1, 2, or 3); wherein Rc, Rd, and Re are each independently selected from hydrogen, (1-6C)alkyl, or (3-6C)cycloalkyl; or Rc and Rd are bonded such that, together with the atom nitrogen to which they are attached, they form a 4-7 membered heterocyclic ring which is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (14C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano or hydroxy; or Q is optionally substituted with a group of the formula

- Li-Lqi-Wi, where L1 is absent or represents (1-3C)alkylene, optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

- 95 041220

LQ is absent or selected from O, S, SO, SO2, N(R _f ), C(O), C(O)O, OC(O), C(O)N(R _f ), N(R _f )C(O),

N(R _f )C(O)N(R _g ), N(R _f )C(O)O, OC(O)N(R _f ), S(O) ₂ N(R _f ) or N(R _f )SO ₂ , where R _f and Rg are each independently selected from hydrogen or (1-2C)alkyl; and

W1 is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl, (3-8C)cycloalkyl, (38C)cycloalkenyl, heteroaryl or heterocyclyl; wherein W1 is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (14C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, aryl, heteroaryl, heterocyclyl, (3-6C)cycloalkyl, NRhRi, ORh, C(O)Rh, C(O)ORh, OC(O)Rh, C(O)N(Ri)Rh, N(Ri)C(O)Rh, S(O) _r Rh (where r is 0, 1 or 2), SO2N(Ri)Rh, N(Ri)SO2Rh or (CH2) _s NRiRh (where s is 1, 2 or 3); where Rh and Rj are each independently selected from hydrogen, (1-4C)alkyl or (36C)cycloalkyl;

each R _1a and R _1b is selected from H, (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl or mercapto;

W is selected from O, S or NR _W1 , where R _W1 is selected from H or (1-2C)alkyl; X1, _X2 , _X3 and _X4 are independently selected from CH, _CR2 or N;

R ₂ is selected from hydrogen, halogen, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)haloalkyl, (14C)haloalkoxy, amino, cyano, nitro, aryl, heteroaryl, heterocyclyl, cycloalkyl, (24C )alkynyl, NRjRk, ORj, C(O)Rj, C(O)OR _j , OC(O)Rj, C(O)N(R _k )Rj, N(R _k )C(O)R _j , N(R _k )C(O)N(Rj), S(O) _r1 R _k (where r1 is 0, 1 or 2), SO ₂ N(Rj)R _k , N(Rj)SO ₂ R _k or (CH ₂ ) _v NRjRk (where v is 1, 2 or 3); where Rj and R _k are each independently selected from hydrogen or (1-4C)alkyl; and wherein said (1-4C)alkyl, aryl, heteroaryl, heterocyclyl or cycloalkyl is optionally substituted with one or more substituents selected from halogen, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)haloalkyl, ( 1-4C)haloalkoxy, amino, cyano, nitro, phenyl, (2-4C)alkynyl, NR _j1 R _k1 , ORj1, C(O)R _jb C(O)OR _j1 , OC(O)R _j1 , C( O)N(R _k1 )R _j1 , N(R _k1 )C(O)R _j1 , S(O) _r2 Rh (where r ₂ is 0, 1 or 2), SO ₂ N(R _j1 )R _k1 , N(R _j1 )SO ₂ R _k1 or (CH ₂ ) _v1 NR _j1 R _k1 (where v1 is 1, 2 or 3); and where Rj1 and R _k1 are each independently selected from hydrogen or (1-4C)alkyl; and

R3 is selected from halogen, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino, cyano, nitro, (2-4C)alkynyl, NRlR _m , OR, C(O)Rl, C(O)ORl, OC(O)Rl, C(O)N(Rm)RN(Rm)C(O)R or (CH2)yNRlR _m (where y is 1, 2 or 3); wherein said (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy or halogen; and wherein Rl and R _m are each independently selected from hydrogen or (1-4C)alkyl.

Further examples of other RET kinase inhibitors include those described in International Publication No. WO2017178844, the contents of which are incorporated herein by reference. For example, in some embodiments, the other RET inhibitor is a compound of formula (V), or a pharmaceutically acceptable salt thereof, wherein

NO selected from the following:

- 96 041220

where ' denotes the attachment point;

R1 is selected from hydrogen, (1-4C)haloalkyl, (1-4C)haloalkoxy or a group of the formula

-L-Y-Q, where L is absent or represents (1-5C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

Y is absent or is O, S, SO, _SO2 , N(Ra), C(O), C(O)O, OC(O), C(O)N( _Ra ), N(Ra)C(O), N(Ra)C(O)N(Rb), N(Ra)C(O)O, OC(O)N(Ra), S(O)2N(Ra) or N(Ra)SO2, where Ra and Rb are each independently selected from hydrogen or (1-4C)alkyl; and Q is hydrogen, (1-6C)alkyl, (26C)alkenyl, (2-6C)alkynyl, aryl, (3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl or heterocyclyl; wherein Q is optionally further substituted with one or more substituent groups independently selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino, (14C)aminoalkyl, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, NRcRd, ORc, C(O)Rc, C(O)ORc, OC(O) _Rc , C(O)N(Rd)Rc, N(Rd)C(O)Rc, S(O)yR^ (where y is 0, 1, or 2), SO2N( _Rd ) _Rc , N( _{Rd ) SO2Rc} , Si(Rd)( _Rc )Re, or ( _CH2 ) _zNRcRd (where z is 1, 2, or 3); wherein Rc, Rd and Re are each independently selected from hydrogen, (1-6C)alkyl or (3-6C)cycloalkyl; or Rc and Rd may be linked so that together with the nitrogen atom to which they are attached they form a 4-7 membered heterocyclic ring which is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano or hydroxyl; or Q is optionally substituted with a group of the formula ^-L 1 ^-L Q1 ^-Z b wherein L ₁ is absent or is (1-3C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

L _Q1 is absent or selected from O, S, SO, _SO2 , N( _Rf ), C(O), C(O)O, OC(O), C(O)N( _Rf ), N( _Rf )C(O), N(Rg)C(O)N(Rf), N( _Rf )C(O)O, OC(O)N( _Rf ), S(O) _2N ( _Rf ) or N( _Rf ) _SO2 , where Rf and Rg are each independently selected from hydrogen or (1-2C)alkyl; and Z1 is hydrogen, (1-6C)alkyl, aryl, aryl(12C)alkyl, (3-8C)cycloalkyl, (3-8C)cycloalkenyl, heteroaryl or heterocyclyl; wherein Z1 is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, aryl, heteroaryl, heterocyclyl, (3-6C)cycloalkyl, NRhRi, ORh, C(O)Rh, C(O)ORh, OC(O)Rh, C(O)N(Ri)Rh, N(Ri)C(O)Rh, S(O)yaRh (where y ^a is 0, 1 or 2), SO2N(Ri)Rh, N(Ri)SO2Rh or (CH2)zaNRiRh (where z ^a is 1, 2 or 3); wherein Rh and Ri are each independently selected from hydrogen, (1-4C)alkyl or (3-6C)cycloalkyl;

each R _1a and R _1b is selected from hydrogen, (1-4C)alkyl, halogen, (1-4C)haloalkyl, (14C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl or mercapto;

W is selected from O, S or NRj, where Rj is selected from H or (1-2C)alkyl;

X1 and _X2 are each independently selected from N or CRk;

where R _k is selected from hydrogen, halogen, (1-4C)alkyl, (1-4C)alkoxy, amino, (1-4C)alkylamino, (14C)dialkylamino, cyano, (2C)alkynyl, C(O)R _k1 , C(O)OR _k1 , OC(O)R _k1 , C(O)N(R _k2 )R _k1 , N(R _k2 )C(O)R _k1 , S(O) _yb R _k1 (where y ^b is 0, 1, or 2), SO ₂ N(R _k2 )R _k1 , N(R _k2 )SO ₂ R _k1 or (CH ₂ ) _zb NRkiRk ₂ (where z ^b is 1, 2, or

- 97 041220

3); wherein said (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy or halogen; and

R _k1 and R _k2 are each independently selected from hydrogen or (1-4C)alkyl; X ₃ is selected from N or CRm; where

R _m is selected from hydrogen, halogen, (1-4C)alkyl, (1-4C)alkoxy, amino, (1-4C)alkylamino, (14C)dialkylamino, cyano, (2C)alkynyl, C(O)R _m1 , C(O)OR _m1 , OC(O)R _m1 , C(O)N(R _m2 )R _m1 , N(R _m2 )C(O)R _m1 , S(O)ycRm1 (where y ^c is 0, 1 or 2), SO2N(Rm2)Rm1, N(Rm2)SO2Rm1 or (CH2) _zc NRm1 Rm2 (where zc equal to 1, 2 or 3); wherein said (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy or halogen; and

Rm1 and Rm2 are each independently selected from hydrogen or (1-4C)alkyl;

Ro is selected from halogen, (1-4C)alkyl, (1-4C)alkoxy, amino, (1-4C)alkylamino, (14C)dialkylamino, cyano, (2C)alkynyl, C(O)Ro1, C(O)ORo1, OC(O)Ro1, C(O)N(Ro2)Ro1, N(Ro2)C(O)Ro1, S(O) _{ydR o1} (where ^yd is 0, 1 or 2), _SO2N (R _o2 )R _o1 , N(R _o2 ) _{SO2R o1} or ( _CH2 ) _{zdNR o1R o2} (where ^zd is 1, 2 or 3); wherein said (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy or halogen; And

R _o1 and R _o2 are each independently selected from hydrogen or (1-4C)alkyl; R ₂ is selected from hydrogen, (14C)alkyl or a group of the formula ^-L 2 ^-Y 2 ^-Q ₂ , wherein L ₂ is absent or is (1-3C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

Y ₂ is absent or represents C(O), C(O)O, C(O)N(Rp), where Rp is selected from hydrogen or (14C)alkyl; and

Q2 is hydrogen, (1-6C)alkyl, aryl, (3-8C)cycloalkyl, (3-8C)cycloalkenyl, heteroaryl or heterocyclyl; wherein Q2 is optionally further substituted with one or more substituent groups independently selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, NRqRr, ORq, where _Rq and _Rr are each independently selected from hydrogen, (1-4C)alkyl or (3-6C)cycloalkyl;

R ₃ is selected from the formula group

-Y3-Q3, where Y ₃ is C(O), C(O)N(R _y ), C(O)N(R _y )O, N(R _y )(O)C, C(O)O, OC(O), N(R _y )C(O)N(R _y1 ), SO ₂ N(R _y ), N(R _y )SO ₂ , oxazolyl, triazolyl, oxadiazolyl, thiazolyl, imidazolyl, thiadiazolyl, pyridinyl, pyrazolyl, pyrrolyl or tetrazolyl, where R _y and R _y1 are independently selected from hydrogen or (1-2C)alkyl; and

Q3 is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl, (3-8C)cycloalkyl, (38C)cycloalkenyl, heteroaryl or heterocyclyl; wherein Q3 is optionally further substituted with one or more substituent groups independently selected from (1-4C)alkyl, halogen, (14C)haloalkyl, (1-4C)haloalkoxy, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, NR _z R _aa , OR _z , where R _z and R _aa , each independently, are selected from hydrogen, (1-4C)alkyl or (36C)cycloalkyl; or Q ₃ is optionally substituted with a group of formula ^-L 4 ^-IQ 4 ^-Z 4 ^, where L ₄ is absent or is (1-3C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

Lq ₄ is absent or selected from O, S, SO, SO ₂ , N(R _ab ), C(O), C(O)O, OC(O), C(O)N(R _ab ), N(R _ab )C(O), N(Rac)C(O)N(Rab), N(Rab)C(O)O, OC(O)N(Rab), S(O)2N(Rab) or N(Rab)SO2, where Rab and Rac are each independently selected from hydrogen or (1-2C)alkyl; and

Z ₄ represents hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl, (3-8C)cycloalkyl, (38C)cycloalkenyl, heteroaryl or heterocyclyl; wherein Z4 is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (14C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, aryl, heteroaryl, heterocyclyl, (3-6C)cycloalkyl, NR _ad R _ae , OR _ad , C(O)R _ad , C(O)OR _ad , OC(O)Rad, C(O)N(Rae)Rad, N(Rae)C(O)Rad, S(O)yeRad (where y e ^is 0, 1 or 2), SO2N(Rae)Rad, N(Rae)SO2Rad or (CH2)zeNRadRae (where z ^e is 1, 2 or 3); where Rad and R _ae are each independently selected from hydrogen, (14C)alkyl or (3-6C)cycloalkyl; or

Q ₃ and R _y are linked so that together with the nitrogen atom to which they are attached they form a 4-7 membered heterocyclic ring which is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (14C)alkylamino, amino, cyano or hydroxyl;

provided that only one or two X ₂ or X ₃ can represent N.

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO2017145050, the contents of which are incorporated herein by reference. For example, in some embodiments, the other RET has Formula (VI) or is a pharmaceutically acceptable salt thereof.

- 98 041220

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO2016038552, the contents of which are incorporated herein by reference. For example, in some embodiments, the other RET has Formula (VII), or Formula (VIII), or is a pharmaceutically acceptable salt thereof.

(VIII)

Other therapeutic agents include RET inhibitors, such as those described, for example, in U.S. Patents 10,030,0005; 9,738,660; 9,801,880; 9,682,083; 9,789,100; 9,550,772; 9,493,455; 9,758,508; 9,604,980; 9,321,772; 9,522,910; 9,669,028; 9,186,318; 8,933,230; 9,505,784; 8,754,209; 8,895,744; 8,629,135; 8,815,906; 8,354,526; 8,741,849; 8,461,161; 8,524,709; 8129374; 8686005; 9006256; 8399442; 7795273; 7863288; 7465726; 8552002; 8067434; 8198298; 8106069; 6861509; 8299057; 9150517; 9149464; 8299057; and 7863288; in U.S. Pub. Nos. 2018/0009818; 2018/0009817; 2017/0283404; 2017/0267661; 2017/0298074; 2017/0114032; 2016/0009709; 2015/0272958; 2015/0238477; 2015/0099721; 2014/0371219; 2014/0137274; 2013/0079343;

2012/0283261; 2012/0225057; 2012/0065233; 2013/0053370; 2012/0302567; 2011/0189167; 2016/0046636;

2013/0012703; 2011/0281841; 2011/0269739; 2012/0271048; 2012/0277424; 2011/0053934; 2011/0046370;

2010/0280012; 2012/0070410; 2010/0081675; 2010/0075916; 2011/0212053; 2009/0227556; 2009/0209496;

2009/0099167; 2010/0209488; 2009/0012045; 2013/0303518; 2008/0234267; 2008/0199426; 2010/0069395;

2009/0312321; 2010/0173954; 2011/0195072; 2010/0004239; 2007/0149523; 2017/0281632; 2017/0226100;

2017/0121312; 2017/0096425; 2017/0044106; 2015/0065468; 2009/0069360; 2008/0275054; 2007/0117800;

2008/0234284; 2008/0234276; 2009/0048249; 2010/0048540; 2008/0319005; 2009/0215761; 2008/0287427;

2006/0183900; 2005/0222171; 2005/0209195; 2008/0262021; 2008/0312192; 2009/0143399; 2009/0130229;

2007/0265274; 2004/0185547; and 2016/0176865; and in International Publication Nos. WO 2018/149382; WO 2018/136796; WO 2017/079140; WO 2017/145050; WO 2017/097697; WO 2017/049462; WO 2017/043550; WO 2017/027883; WO 2017/013160; WO 2017/009644; WO 2016/168992; WO 2016/137060; WO 2016/127074; WO 2016/075224; WO 2016/038552; WO 2015/079251; WO 2014/086284; WO 2013/042137; WO 2013/036232; WO 2013/016720; WO 2012/053606; WO 2012/047017; WO 2007/109045; WO 2009/042646; WO 2009/023978; WO 2009/017838; WO 2017/178845; WO 2017/178844; WO 2017/146116; WO 2017/026718; WO 2016/096709; WO 2007/057397; WO 2007/057399; WO 2007/054357; WO 2006/130613; WO 2006/089298; WO 2005/070431; WO 2003/020698; WO 2001/062273; WO 2001/016169; WO 1997/044356; WO 2007/087245; WO 2005/044835; WO 2014/075035; and WO 2016/038519; and J. Med. Chem. 2012, 55(10), 4872-4876, the entire contents of which are incorporated herein by reference.

In some embodiments, the RET inhibitor (eg, a first RET inhibitor or a second RET inhibitor) is a compound of formula II:

- 99 041220 or a pharmaceutically acceptable salt or solvate thereof, wherein X ¹ is CH, CCH ₃ , CF, CCl or N; X ² is CH, CF or N; X ³ is CH, CF or N; X ⁴ is CH, CF or N;

where zero, one, or two X ¹ , X ² , X ³ , and X ⁴ represent N;

A is H, Cl, CN, Br, _CH3 , _CH2CH3 or cyclopropyl _;

B represents hetAr ¹ ;

hetAr ¹ is a 5-membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, S and O, said heteroaryl ring being optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-C6 alkyl, hydroxy-C1-C6 alkyl, fluoro-C1-C6 alkyl, difluoro-C1-C6 alkyl, trifluoro-C1-C6 alkyl, cyanoC1-C6 alkyl, (C1-C6 alkoxy)C1-C6 alkyl, (C1-C4 alkoxy)CH2C(=O)-, (C1-C4 alkoxy)C(=O)C1-C3 alkyl, C3-C6 cycloalkyl, (R ^a R ^b N)C1-C ₆ alkyl, (R ^a RbN)C(=O)C1-C ₆ -alkyl, (C1-C ₆ -alkyl 8O ₂ )C1-C ₆ alkyl, hetCyca and 4-methoxybenzyl;

R ^a and R ^b independently represent H or C1-C ₆ -alkyl;

hetCyca is a 4-6 membered heterocyclic ring containing a ring heteroatom selected from N and O, wherein said heterocyclic ring is optionally substituted with halogen, C1- _C6 alkyl, fluoro-C1- _C6 alkyl, difluoro-C1- _C6 alkyl, trifluoro-C1- _C6 alkyl, (C1- _C6 alkoxy)C1- _C6 alkyl, di( _C1 - _C3 alkyl) _NCH2C (=O), (C1- _C6 alkoxy)C(=O) or (C1-C6 alkoxy) _CH2C (=O);

D is hetCyc ¹ , hetCyc ² , hetCyc ^{3 ,} or hetCyc ⁹ ;

hetCyc ¹ is a 4-6 membered heterocyclic ring containing 1-2 ring atoms selected from N and O, wherein said heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of C1-C3 alkyl, fluoro-C1-C3 alkyl, difluoro-C1- _C3 alkyl, trifluoro-C1- _C3 alkyl and OH, or said heterocyclic ring is substituted with a C3-C6 cycloalkylidene ring, or said heterocyclic ring is substituted with an oxo group;

hetCyc ² is a 7-8 membered heterocyclic ring containing 1-3 ring heteroatoms independently selected from N and O, wherein said heterocyclic ring is optionally substituted with C1- _C3 alkyl;

hetCyc ³ is a 7-11 membered heterospirocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, said ring optionally substituted with C ₁ C3 alkyl;

hetCyc ⁹ is a fused 9-10 membered heterocyclic ring containing 1-3 ring nitrogen atoms and optionally substituted with an oxo group;

E represents:

(a) hydrogen, (b) OH, (c) R ^a R ^b N-, where R ^a is H or C1-C6 alkyl, and R ^b is H, C1-C6 alkyl or phenyl;

(d) C ₁ -C ₆ -alkyl optionally substituted by one to three fluorine atoms, (e) hydroxy-C ₁ -C ₆ -alkyl-, optionally substituted by one to three fluorine atoms, (f) C ₁ -C ₆ -alkoxy, optionally substituted by one to three fluorine atoms, (g) hydroxy(C 1-C ₆ -alkoxy)optionally substituted by one to three fluorine atoms, (h) (C ₁ -C ₆ -alkoxy)hydroxy C1-C ₆ -alkyl-, optionally substituted by one to three fluorine atoms, (i) (C ₁ -C ₆ -alkyl)C(=O)-, optionally substituted by one to three fluorine atoms, (j) (hydroxy C1-C ₆ -alkyl)C(=O)-, optionally substituted by one to three fluorine atoms, (k) (C1-C6-alkoxy)C(=O)-, (l) ( _C1 - _C6 -alkoxy)( _C1 - _C6 -alkyl)C(=O)-, (m) HC(=O)-, ⁽ⁿ⁾ Cyc ¹ , (o) Cyc ¹ C(=O)-, (p) Cyc ¹ (C1-C6-alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with one or more groups independently selected from the group consisting of OH, fluoro, C1-C3-alkoxy and R ^c R ^d N-, wherein R ^c and Rd are independently H or C1-C6-alkyl, (q) hetCyc ⁴ , (r) hetCyc ⁴ C(=O)-, (s) hetCyc ⁴ (C1-C3-alkyl)C(=O)-, (t) (hetCyc ⁴ )C(=O)C1-C2-alkyl-, (u) hetCyc ⁴ C(=O)NH-, (v) Ar ² , (w) Ar ² C(=O)-, (x) Ar ² C ₁ -C ₆ -alkyl-,

- 100 041220 (y) (Ar ² )hydroxy C ₂ -C 6 -alkyl-, (z) Ar ² (C 1 -C 3 -alkyl) C (= O) -, wherein said alkyl part is optionally substituted with one or two groups independently selected from the group consisting of OH, C 1 -C 6 -alkyl (optionally substituted with 1 to 3 fluorine atoms), hydroxy-C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy and R ^e R f N -, wherein R ^e and R f independently represent H or C 1 -C 6 -alkyl, or R ^e and R f together with the nitrogen atom to which they are attached form a 5- to 6-membered azacyclic ring optionally containing an additional ring heteroatom selected from N and O, (aa) hetAr ² C (= O) -, (bb) (hetAr ² )hydroxy-C ₂ -C ₆ -alkyl-, (cc) hetAr ² (C1-C3 alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with one or two groups independently selected from the group consisting of OH, C1-C6 alkyl, hydroxy-C1-C6 alkyl, C1-C6 alkoxy and R ^e RfN-, wherein R ^e and Rf are independently H or C1-C6 alkyl, or R ^e and R ^f together with the nitrogen atom to which they are attached form a 5- to 6-membered azacyclic ring optionally containing an additional ring heteroatom selected from N and O, (dd) R1R ² NC(=O)-, (ee) R ¹ R ² N(C ₁ -C ₃ alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with phenyl, (ff) R ¹ R ² NC(=O)C1-C2-alkyl-, (gg) R1R ² NC(=O)NH-, (hh) CH3SO2(C1-C6-alkyl)C(=O)-, (ii) (C1-C6-alkyl)SO2-, (jj) (C ₃ -C ₆ -cycloalkyl)CH ₂ SO ₂ -, (kk) hetCyc ⁵ -SO2-, (ll) R4R5NSO2-, (mm) R ⁶ C(=O)NH-, (nn) hetCyc ⁶ , (oo) hetAr ² C ₁ -C ₆ -alkyl-, (pp) (hetCyc ⁴ )C ₁ -C ₆ -alkyl-, (qq) (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl-, optionally substituted with 1 to 3 fluorine atoms, (rr) (C ₃ -C ₆ -cycloalkoxy)C ₁ -C ₆ -alkyl-, (ss) (C ₃ -C ₆ -cycloalkyl)C ₁ -C ₆ -alkyl-, wherein said cycloalkyl is optionally substituted with 1 to 2 fluorine atoms, (tt) (R ⁹ RhN)C1-C6 -alkyl-, wherein R ⁹ and R ^h independently represent H or C1-C6 -alkyl, (uu) Ar ² -O-, (vv) (C ₁ -C ₆ -alkyl-SO ₂ )C ₁ -C ₆ -alkyl-, (ww) (C ₁ -C ₆ -alkoxy)C(=O)NHC ₁ -C ₆ -alkyl-, (xx) (C ₃ -C ₆ -cycloalkoxy)C(=O)-, (yy) (C ₃ -C ₆ -cycloalkyl)SO ₂ -, wherein said cycloalkyl is optionally substituted with C1-C ₆ -alkyl, (zz) Ar ⁴ CH2OC(=O)-, (aaa) (N-(C ₁ -C ₃ -alkyl)pyridinonyl)C ₁ -C ₃ -alkyl-, and (bbb) (Ar ⁴ SO2)C1-C6-alkyl-;

Cys ¹ is _C3 - _C6 -cycloalkyl, wherein (a) said cycloalkyl is optionally substituted with one or more substituents independently selected from the group consisting of OH, halogen, _C1 - _C6- alkoxy, CN, hydroxy-C1- _C6 -alkyl, (C1- _C6 -alkoxy)C1- _C6 -alkyl, and C1- _C6- alkyl optionally substituted with 1-3 fluorine atoms, or (b) said cycloalkyl is substituted with phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1- _C3- alkyl, C1- _C3- alkoxy, and CF, or (c) said cycloalkyl is substituted with a 5-6-membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from both N and O, wherein said heteroaryl ring is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1- _C3- alkyl, C1- _C3 -alkoxy and _CF3 ;

Ar ² is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C6 alkyl, difluoro-C1-C6 alkyl, trifluoro-C1-C6 alkyl, CN, a 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, and RiR ^j N-, where R ¹ and R ^j are independently H or C1-C6 alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, and optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-C ₆ alkyl, C1-C ₆ alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C ₆ alkyl, difluoro-C1-C ₆ alkyl, trifluoro-C1-C ₆ alkyl, hydroxy-C1-C ₆ alkyl, (C ₃ -C ₆ ) cycloalkyl, (C1-C ₆ alkoxy)C1-C ₆ alkyl, CN, OH and R'RN-, where R' and R are independently H or C1-C ₃ alkyl;

hetCyc ⁴ is (a) a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N, O and S, wherein said S atom is optionally oxidized to

- 101 041220

SO2, (b) a 7-8-membered bridged heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, (c) a 6-12-membered fused bicyclic heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally independently substituted with 1-2 C1- _C6 alkyl substituents, or (d) a 7-10-membered spirocyclic heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, each of said heterocyclic rings optionally substituted with one or more substituents independently selected from the group consisting of halogen, OH, CN, _C1 - _C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1- _C6 alkoxy, ( _C1 - _C6 alkoxy)C ₁ - _C6 -alkyl, ( _C3 - _C6 )cycloalkyl, (C1-C6alkyl)C(=O)-, a 5-6-membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, and phenyl, wherein said phenyl is optionally substituted with one or more substituents selected from halogen, C1- _C6 -alkyl and C1- _C6 -alkoxy;

hetCyc ⁵ is a 5-6 membered heterocyclic ring containing a ring heteroatom selected from O and N;

hetCyc ⁶ is a 5-membered heterocyclic ring containing one or two ring heteroatoms independently selected from N and O, wherein said ring is substituted with an oxo group, and wherein said ring is further optionally substituted with one or more substituents independently selected from the group consisting of OH and C1-C ₆ alkyl;

R ¹ is H, C1-C ₆ -alkyl or (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl;

R ² is H, C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), (C1-C6alkoxy)C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), Cyc ³ , hydroxy-C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), C1-C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C _6- alkoxy)C(=O), hetCyc ⁷ , Ar ³ , Ar ³ C ₁ -C ₃ -alkyl-, hydroxy-C1-C ₆ -alkoxy or (3-6C cycloalkyl)CH2O-;

Sus ³ is a 3-6 membered carbocyclic ring optionally substituted with 1-2 groups independently selected from the group consisting of C1-C ₆ alkoxy, OH and halogen;

hetCyc ⁷ is a 5-6 membered heterocyclic ring containing a ring heteroatom selected from O and N, said ring optionally substituted with C1-C ₆ alkyl;

Ar ³ is phenyl optionally substituted with one or more substituents independently selected from halogen, C1-C ₃ -alkyl, C1-C ₃ -alkoxy, fluoro-C1-C ₃ -alkyl, difluoro-C1-C ₃ -alkyl and trifluoro-C 1-C ₃ -alkyl;

R ⁴ and R ⁵ independently represent H or C1-C ₆ -alkyl;

R ⁶ is C1-C ₆ -alkyl, hydroxy-C1-C ₆ -alkyl, C1-C ₆ -alkoxy, (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ alkyl, phenyl or hetCyc ⁸ ;

hetCyc ⁸ is a 5-6 membered heterocyclic ring containing a ring heteroatom selected from O and N, said heterocyclic ring being optionally substituted with C1-C6 alkyl; and

Ar ⁴ is phenyl optionally substituted by one or more halogen atoms.

In some embodiments, the RET inhibitor (eg, a first RET inhibitor or a second RET inhibitor) is a compound of formula III:

or a pharmaceutically acceptable salt or solvate thereof, wherein X ¹ is CH or N; X ² is CH or N; X ³ is CH or N; X ⁴ is CH or N; wherein one or two of X ¹ , X ² , X ³ and X ⁴ are N; A is CN; B is hetAr ¹ ;

hetAr ¹ is a 5-membered heteroaryl ring containing 1-3 ring nitrogen atoms, said heteroaryl ring being optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-C6 alkyl, hydroxy-C1-C6 alkyl, fluoro-C1-C6 alkyl, difluoro-C1-C6 alkyl, trifluoro-C1-C6 alkyl, cyano-C1-C6 alkyl, (C1-C6 alkoxy)C1-C6 alkyl, (C1-C4 alkoxy)CH2C(=O)-, (C1-C4 alkoxy)C(=O)C1-C3 alkyl, C3-C6 cycloalkyl, (R ^a R ^b N)C1-C6 alkyl, (R ^a R ^b N)C(=O)C _r C ₆ -alkyl, (C ₁ -C ₆ -alkyl-SO ₂ )C ₁ -C ₆ -alkyl and 4-methoxybenzyl;

R ^a and R ^b are independently H or C1-C ₆ -alkyl; D is hetCyc ¹ ;

hetCyc ¹ is a 4-6 membered heterocyclic ring containing 1-2 ring nitrogen atoms, wherein said heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of C1-C ₃ -alkyl, fluoro-C1-C ₃ -alkyl, difluoro-C1-C ₃ -alkyl, trifluoro-C1-C ₃ -alkyl and OH, or said heterocyclic ring is substituted with C ₃ - 102 041220

C6-cycloalkylidene ring, or said heterocyclic ring is substituted with an oxo group;

E represents:

(w) Ar ² C(=O)-, (x) Ar ² C ₁ -C ₆ -alkyl-, (z) Ar ² (C1-C3-alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with one or two groups independently selected from the group consisting of OH, C1-C6-alkyl (optionally substituted with 1 to 3 fluorine atoms), hydroxy-C1-C6-alkyl, C1-C6-alkoxy and R ^e R ^f N-, wherein R ^e and R ^f independently represent H or C1-C6-alkyl, or R ^e and R ^f together with the nitrogen atom to which they are attached form a 5- to 6-membered azacyclic ring optionally containing an additional ring heteroatom selected from N and O, (cc) hetAr ² (C1-C3-alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with one or two groups independently selected from the group consisting of OH, C1-C6 alkyl, hydroxy-C1-C6 alkyl, C1-C6 alkoxy and R ^e R ^f N-, wherein R ^e and R ^f independently represent H or C1-C6 alkyl, or R ^e and R ^f together with the nitrogen atom to which they are attached form a 5-6-membered azacyclic ring optionally containing an additional ring heteroatom selected from N and O, (dd) R1R ² NC(=O)-, (oo) hetAr ² C ₁ -C ₆ alkyl-,

Ar ² is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C6 alkyl, difluoro-C1-C6 alkyl, trifluoro-C1-C6 alkyl, CN, a 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, and RiRjN-, where R ¹ and Rj are independently H or C1-C6 alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-C ₆ alkyl, C1-C ₆ alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C ₆ alkyl, difluoro-C1-C ₆ alkyl, trifluoro-C1-C ₆ alkyl, hydroxy-C1-C ₆ alkyl, (C ₃ -C ₆ ) cycloalkyl, (C ₁ - _{C 6} alkoxy)C ₁ -C ₆ alkyl, CN, OH and R'RN-, where R' and R are independently H or C1-C ₃ alkyl;

R ¹ is H, C1-C ₆ -alkyl or (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl; and

R ² is H, C1-C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), (C1-C 6 -alkoxy)C1-C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1-C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), C1-C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C ₆ -alkoxy)C(=O), hydroxy-C1-C ₆ -alkoxy or (3-6C cycloalkyl)CH ₂ O.

In some embodiments, the RET inhibitor (e.g., a first RET inhibitor or a second RET inhibitor) is selected from the group consisting of: (S)-4-(6-(4-(2-hydroxy-3-phenylpropanoyl)piperazin1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(4-(2-(pyridin-2-yl)acetyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5a]pyridine-3-carbonitrile; 4-(6-(4-(2,6-difluorobenzoyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1Hpyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-carbonitrile 2,2,2-trifluoroacetate; 4-(5-(3-cyano-6-(1-methyl1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-N,N-diethylpiperαzine-1-carboxamide; 1(5-(3-cyαno-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-N-(2-methoxy-3methylbutyl)piperidine-4-carboxamide; 4-(6-(4-(2-(5-fluoropyridin-2-yl)acetyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile bis(2,2,2-trifluoroacetate); 4-(6-(4-(2,6-difluorobenzyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 4-(6-(4-(2-methoxybenzyl)piperazin-1-yl)pyridin-3-yl)-6(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(4(pyridin-2-ylmethyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 4-(6-(4-((6methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5a]pyridine-3-carbonitrile; or pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the RET inhibitor (eg, a first RET inhibitor or a second RET inhibitor) is a compound of formula IV:

IV or a pharmaceutically acceptable salt or solvate thereof, wherein

X ¹ , X ² , X ³ and X ⁴ independently represent CH, CF, CCH ₃ or N, where zero, one or two of X ¹ , X ² , X ³ and X ⁴ represent N;

- 103 041220

A is H, CN, Cl, _CH3- , CH3CH2-, cyclopropyl, -CH2CN or -CH(CN) _CH3 ;

B represents:

(a) hydrogen, (b) _C1 - _C6- alkyl optionally substituted with 1-3 fluorine atoms, (c) hydroxy- _C2 - _C6 -alkyl-, wherein the alkyl part is optionally substituted with 1-3 fluorine atoms or a _{C3- C6-} cycloalkylidene ring, (d) dihydroxy- _C3 - _C6 -alkyl-, wherein the alkyl part is optionally substituted with a _C3 - _C6 -cycloalkylidene ring, (e) ( _C1 - _C6 -alkoxy) _C1 - _C6 -alkyl-, optionally substituted with 1-3 fluorine atoms, (f) ( ^R1R2N ) _C1 - _C6 -alkyl-, wherein said alkyl part is optionally substituted with an OH group, ^and wherein ^R1 and ^R2 independently represent H or C1-C6-alkyl (optionally substituted 1-3 fluorine atoms);

(g) hetAr ¹ C1-C3-alkyl-, wherein hetAr ¹ is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, and optionally substituted with one or more independently selected C1-C6-alkyl substituents;

(h) ( _C3 - _C6- cycloalkyl) _C1 - _C3 -alkyl-, wherein said cycloalkyl is optionally substituted with an OH group, (i) (hetCyc ^a ) _C1 - _C3 -alkyl-, (j) hetCyca-, (k) _C3 - _C6 -cycloalkyl-, wherein said cycloalkyl is optionally substituted with an OH group, (l) ( _C1 - _C4 -alkyl)C(=O)O- _C1 - _C6 -alkyl-, wherein each C1- _C4 -alkyl and C1- _C6 -alkyl portion is optionally and independently substituted with 1 to 3 fluorine atoms, or (m) ( ^R1R2N )C(=O)C1-C6-alkyl-, ^{wherein R1} and ^R2 are independently H or C1-C6-alkyl (optionally substituted with 1 to 3 fluorine atoms);

hetCyca- is a 4-6-membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from OH, C1- _C6 -alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1- _C6 -alkyl-, C1- _C6- alkoxy, (C1- _C6- alkyl)C(=O)-, (C1- _C6 -alkoxy)C1- _C6 -alkyl- and fluorine, or wherein hetCyca is substituted with an oxo group;

ring D is (i) a saturated 4- to 7-membered heterocyclic ring containing two ring nitrogen atoms, (ii) a saturated 7- to 8-membered bridged heterocyclic ring containing two ring nitrogen atoms and optionally containing a third ring heteroatom which is oxygen, (iii) a saturated 7- to 11-membered heterospirocyclic ring containing two ring nitrogen atoms, or (iv) a saturated 9- to 10-membered bicyclic fused heterocyclic ring containing two ring nitrogen atoms, each of said rings being optionally substituted with (a) one to four groups independently selected from halogen, OH, C1- _C3 alkyl which is optionally substituted with 1-3 fluorine atoms, or C1- _C3 alkoxy which is optionally substituted with 1-3 fluorine atoms, (b) _C3 - _C6 - a cycloalkylidene ring, or (c) an oxo group;

E represents:

(a) hydrogen, (b) _C1 - _C6- alkyl optionally substituted with 1-3 fluorine atoms, (c) ( _C1 - _C6 -alkoxy) _C1 - _C6 -alkyl-, optionally substituted with 1-3 fluorine atoms, (d) ( _C1 - _C6 -alkyl)C(=O)-, wherein said alkyl part is optionally substituted with 1-3 fluorine atoms or a substituent R ^g R ^h N-, wherein R ^g and R ^h independently represent H or C1- _C6- alkyl, (e) (hydroxy-C2-C6-alkyl)C(=O)-, optionally substituted with 1-3 fluorine atoms, (f) (C1-C6-alkoxy)C(=O)-, (g) ( _C3 - _C6 -cycloalkyl)C(=O)-, wherein said cycloalkyl is optionally substituted with one or more substituents independently selected from C1-C ₆ -alkyl, C1-C ₆ -alkoxy, OH and (C1-C ₆ -alkoxy)C1-C ₆ alkyl-, or said cycloalkyl is substituted with a 5-6-membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N and O, (h) Ar ¹ C ₁ -C ₆ -alkyl-, (i) Ar ¹ (C1-C6 -alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with OH, hydroxy-C1-C6 -alkyl-, C1-C6 -alkoxy, R ^m R ⁿ N- or R ^m R ⁿ N-CH2-, wherein each R ^m and R ⁿ is independently H or C1-C ₆ -alkyl, (j) hetAr ² C ₁ -C ₆ -alkyl-, wherein said alkyl moiety is optionally substituted with 1-3 fluorine atoms, (k) hetAr ² (C ₁ -C ₆ -alkyl)C(=O)-, wherein said alkyl part is optionally substituted with an OH group, hydroxy-C1-C ₆ -alkyl- or C1-C ₆ -alkoxy, (l) hetAr ² C(=O)-, (m) hetCyc ¹ C(=O)-, (n) hetCyc ¹ C ₁ -C ₆ -alkyl-, (o) R ³ R ⁴ NC(=O)-, (p) Ar1N(R ³ )C(=O)-, (q) hetAr ² N(R ³ )C(=O)-,

- 104 041220 (r) (C ₁ -C ₆ -alkyl)SO ₂ -, wherein said alkyl part is optionally substituted by 1-3 fluorine atoms, (s) Ar ¹ SO2-, (t) hetAr ² SO2-, (u) N-(C ₁ -C ₆ -alkyl)pyridinonyl, (v)Ar ¹ C(=O)-;

(w) Ar'OC(=O)-, (x) ( _C3 - _C6- cycloalkyl)( _C1 - _C6 -alkyl)C(=O)-, (y) ( _C3 - _C6- cycloalkyl)( _C1 - _C6 -alkyl) _SO2- , wherein said alkyl part is optionally substituted with 13 fluorine atoms, (z) ^Ar1 (C1-C6-alkyl)SO2-, (aa) hetCyc'-OC(=O)-, (bb) hetCyc'CH2C(=O)-, (cc) ^hetAr2 or (dd) _C3 - _C6 -cycloalkyl;

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), R ^e R ^f N-, where R ^e and R ^f are independently H, C1-C6 alkyl, (R ^p R ^q N) C1-C6 alkoxy-, where R ^p and R ^q are independently H or C1-C6 alkyl, and (hetAr ^a ) C ₁ -C ₆ alkyl-, where hetAra is a 5-6 membered heteroaryl ring containing 1-2 ring nitrogen atoms, or Ar ¹ is a phenyl ring fused to a 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms, independently selected from N and O;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl ring containing 1-3 ring nitrogen atoms, wherein hetAr ² is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6 alkoxy)C1-C6 alkyl- (optionally substituted with 1-3 fluorine atoms), R ^e R ^f N-, wherein R ^e and R ^f are independently H or C1-C ₆ alkyl, OH, (C ₁ -C ₆ alkoxy)C ₁ -C ₆ -alkoxy- and C ₃ -C ₆ -cycloalkyl;

hetCyc ¹ is a 4-6 membered saturated heterocyclic ring containing 1-2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclic ring is optionally substituted with one or more substituents independently selected from C ₁ -C ₆ -alkoxy and halogen;

R ³ is H or C1-C6 alkyl; and R ⁴ is C1-C ₆ alkyl.

In some embodiments, the RET inhibitor (eg, a first RET inhibitor or a second RET inhibitor) is a compound of formula V:

or a pharmaceutically acceptable salt and solvate thereof, where

X ¹ , X ² , X ³ and X ⁴ independently represent CH or N, where zero, one or two of X ¹ , X ² , X ³ and X ⁴ represent N;

A represents CN; B represents:

(b) _C1 - _C6- alkyl optionally substituted with 1-3 fluorine atoms, (c) hydroxy- _C2 - _C6- alkyl-, wherein the alkyl part is optionally substituted with 1-3 fluorine atoms or a _{C3C6 -} cycloalkylidene ring, (e) ( _C1 - _C6 -alkoxy) _C1 - _C6 -alkyl-, optionally substituted with 1-3 fluorine atoms, (f) ( ^R1R2N )C1-C6-alkyl-, wherein said alkyl part is optionally substituted with an OH group, ^and wherein ^R1 and ^R2 independently represent H or C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms);

(g) hetAr ¹ C1-C3-alkyl-, where hetAr ¹ is a 5-6-membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, and optionally substituted with one or more independently selected C1- _C6 -alkyl substituents; or (i) (hetCyc ^a )C ₁ -C ₃ -alkyl-, hetCyca- is a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, and optionally substituted with one or more substituents independently selected from OH, C1-C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1-C ₆ -alkyl-, C1-C6-alkoxy, (C1-C6-alkyl)C(=O)-, (C ₁ - _{C 6} -alkoxy)C ₁ -C6-alkyl- and fluorine, or wherein hetCyc ^a is substituted with an oxo group;

ring D is (i) a saturated 4-7-membered heterocyclic ring containing two ring nitrogen atoms, or (ii) a saturated 7-9-membered bridged heterocyclic ring containing two ring nitrogen atoms and optionally containing a third ring heteroatom which is oxygen, each of said rings being optionally substituted with (a) one to four groups independently selected from halogen, OH, C1- _C3- alkyl which is optionally substituted with 1 to 3 fluorine atoms, or C1- _C3 -alkoxy which is optionally substituted with 1 to 3 fluorine atoms, (b) a _C3 - _C6 -cycloalkylidene ring, or (c) an oxo group;

E represents:

(h) Ar ¹ C ₁ -C ₆ -alkyl-, (j) hetAr ² C1-C6-alkyl-, wherein the alkyl part is optionally substituted with 1-3 fluorine atoms, or (l) hetAr ² C(=O)-,

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), ^Re R ^f N-, where Re and R ^f are independently H or C1-C6 alkyl, (R ^p R ^q N)C1-C6 alkoxy-, where R ^p and R ^q are independently H or C1-C6 alkyl, and (hetAr ^a )C ₁ -C ₆ alkyl-, where hetAra is a 5-6 membered heteroaryl ring containing 1-2 ring nitrogen atoms, or Ar ¹ is a phenyl ring fused to a 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O; and hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl ring containing 1-3 ring nitrogen atoms, wherein hetAr ² is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6 alkoxy)C1-C6 alkyl- (optionally substituted with 1-3 fluorine atoms), R ^e R ^f N-, wherein R ^e and R ^f are independently H or C1-C6 alkyl, OH, (C ₁ -C ₆ alkoxy)C ₁ -C ₆ -alkoxy- and C ₃ -C ₆ -cycloalkyl.

In some embodiments, the RET inhibitor (e.g., a first RET inhibitor or a second RET inhibitor) is selected from the group consisting of: 4-(6-(4-benzylpiperazin-1-yl)pyridin-3-yl)-6-(2-morpholinoethoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-hydroxyethoxy)-4-(6-(6-((6methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; (R)-6-(2-hydroxypropoxy)-4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1 yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-methoxyethoxy)-4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-(dimethylamino)ethoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-morpholinoethoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile; 4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-((1-methyl-1H-imidazol-4-yl)methoxy)pyrazolo[1,5a]pyridine-3-carbonitrile; and 6-ethoxy-4-(5-(6-((5-fluoro-6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyrazin-2-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; or pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the RET inhibitor (eg, a first RET inhibitor or a second RET inhibitor) is a compound of formula VI:

or a pharmaceutically acceptable salt or solvate thereof, wherein

X ¹ , X ² , X ³ and X ⁴ independently represent CH, CCH ₃ , CF or N, where zero, one or two of X ¹ , X ² , X ³ and X ⁴ represent N;

A represents H, CN, Cl, methyl, ethyl or cyclopropyl;

B represents:

(a) hydrogen, (b) C ₁ -C ₆ -alkyl, optionally substituted with 1-3 fluorine atoms,

- 106 041220 (c) hydroxy- _C2 -C6-alkyl-, wherein the alkyl part is optionally substituted with a _C3 - _C6- cycloalkylidene ring, (d) dihydroxy- _C3 - _C6 -alkyl-, wherein the alkyl part is optionally substituted with a _C3 - _C6 -cycloalkylidene ring ^, (e) ( _C1 - _C6 -alkoxy) _C1 - _C6 -alkyl-, optionally substituted with 1-3 fluorine atoms, (f) ( ^R1R2N )C1-C6-alkyl-, wherein ^R1 and ^R2 are independently selected from H, C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), ( _C1 - _C6 -alkoxy) _C1 - _C6 -alkyl-, (C1- _C6 -alkyl)C(=O)- and (C1-C ₆ -alkoxy)C(=O)-;

(g) hetAr ¹ C1-C3-alkyl-, wherein hetAr ¹ is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, and optionally substituted with one or more independently selected C1-C6-alkyl substituents;

(h) ( _C3 - _C6 -cycloalkyl) _C1 - _C3 -alkyl-, wherein said cycloalkyl is optionally substituted with an OH group, (i) (hetCyc ^a ) _C1 - _C3 -alkyl-, (j) hetCyca, (k) ^{(R1R2N )} C(=O)C1-C6-alkyl-, wherein ^R1 and ^R2 are independently selected from H and C1-C6-alkyl;

(l) (R1R2N)C(=O)-, where ^R1 and ^R2 are independently selected from H and C1- _C6- alkyl, or (m) hetCyc ^a C(=O) _C1 - _C6 -alkyl-;

hetCyca- is a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from OH, C1- _C6 alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1- _C6 alkyl-, halogen, (C1- _C6 alkyl)C(=O)-, C1- _C6 alkoxy, oxo and (C1- _C6 alkoxy)C(=O)-;

Ring D represents:

(i) a saturated monocyclic 4- to 7-membered heterocyclic ring containing one ring heteroatom that is nitrogen, (ii) a saturated 7- to 8-membered bridged heterocyclic ring containing one ring heteroatom that is nitrogen, or (iii) a saturated 7- to 11-membered heterospirocyclic ring system containing one ring heteroatom that is nitrogen;

each R ^a is independently C1- _C6 -alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1- _C6- alkyl, or (C1- _C6 -alkoxy)C1- _C6 -alkyl-;

R ^b represents:

(a) hydroxy, (b) cyclopropyl, (c) hetCyc ^b CH2-, (d) R'R'NC(=O)CH ₂ OCH ₂ - where Ri and R ^j are independently H or C1-C ₆ -alkyl, (e) RcRdN-, (f) R ^c RdNCH2-, (g) C ₁ -C ₆ -alkoxy-, (h) (C ₁ -C4-alkyl)-C(=O)NH-, wherein said alkyl moiety is optionally substituted with hetCyc ^b , hetAra, C ₁ -C ₆ -alkoxy- or R'RN-, or said alkyl moiety is optionally substituted with two substituents independently selected from R'RN- and OH, wherein each R' and R is independently hydrogen or C1-C ₆ -alkyl, (i) (R'RN)C ₁ -C ₆ -alkoxy(CH ₂ ) _n -, where n is 0 or 1, and R' and R are independently hydrogen or C1-C ₆ -alkyl, (j) hetCyc ^b (C ₁ -C ₃ -alkyl)OCH ₂ -, (k) hetCyc ^b C(=O)NH- or (l) hetAr ^a C(=O)NH-;

hetCycb is a 4-6 membered heterocyclic ring, a 7-8 membered bridged heterocyclic ring or a 7-10 membered heterospirocyclic ring, each ring containing 1-2 ring heteroatoms independently selected from N and O, wherein hetCyc ^b is optionally substituted with one or more substituents independently selected from OH, fluoro, C1- _C6 alkyl (optionally substituted with 1-3 fluoro atoms), hydroxy-C1- _C6 alkyl- (optionally substituted with 1-3 fluoro atoms), (C1- _C6 alkoxy)C1- _C6 alkyl-, (C1- _C6 alkoxy)C(=O)-, C1- _C6 alkoxy and R'RN-, where R' and R are independently hydrogen or C1- _C6 alkyl;

hetAr ^a is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, wherein hetAr ^a is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C ₁ -C ₆ alkyl (optionally substituted with 1-3 fluorine atoms) and C 1 -C ₆ alkoxy (optionally substituted with 1-3 fluorine atoms),

R ^c is hydrogen or C1-C ₆ -alkyl;

R ^d is hydrogen, C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), (C1C6-alkoxy)C(=O)-, hydroxy-C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), (hydroxy-C1C6-alkyl)C(=O)-, (C1-C6-alkyl)C(=O)-, (R ^k R ^l N)C1-C ₆ -alkyl-, where R ^k and R ¹ independently represent H or C1-C6-alkyl, R ^m R ⁿ NC(=O)C1-C ₆ -alkyl-, where R ^m and R ⁿ independently represent H or C1-C ₆

- 107 041220 alkyl, PhCH ₂ -, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C ₁ -C ₆ alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ alkoxy (optionally substituted with 1-3 fluorine atoms), (C 1 -C ₆ alkoxy) C 1 -C ₆ alkyl- (optionally substituted with 1-3 fluorine atoms), C ₃ -C ₆ cycloalkyl, hydroxy-C 1 -C ₆ alkyl, (C ₁ -C ₆ alkyl) SO ₂ -, R ^e R f N- and (R ^e R ^f N) C 1 -C 6 alkyl-, wherein each R ^e and R f independently represents H or C 1 -C 6 alkyl, (C ₁ -C ₆ alkoxy) C ₁ -C ₆ -alkyl- or hetCycc, where hetCycc is a 4-6-membered heterocyclic ring containing a ring heteroatom selected from N and O and optionally substituted with C1-C ₆ -alkyl;

n is 0, 1,2, 3, 4, 5 or 6;

m is 0 or 1;

E represents:

(a) hydrogen, (b) hydroxy, (c) C1-C6 _- alkyl optionally substituted with 1-3 fluorine atoms, (d) ^Ar1C1 - _{C6 -} alkyl-, wherein said alkyl part is optionally substituted with 1-3 fluorine atoms, ( ^e ) hetAr2C1- _{C6 -} alkyl-, (f) ( _C1 - _C6 -alkoxy) _C1 - _C6 -alkoxy-, (g) Ar1O-, (h) hetAr2 ^- O-, (i) Ar1NR ^g -, where R ^g is H or C1-C6-alkyl, (j) ^{hetAr2NR g} -, where R ^g is H or C1-C6-alkyl, (k) ^R3C (=O)NR ^g -, where R ^g is H or C1-C6-alkyl; (l) Ar1C(=O)NR ^g -, where R ^g is H or C1-C6 alkyl, (m) hetAr ² C(=O)NR ^g (CH2)p-, where p is 0 or 1, and R ^g is H or C1-C6 alkyl, (n) R ⁴ R ⁵ NC(=O)-, (o) Ar1NR ^g C(=O)-, where R ^g is H or C1-C ₆ alkyl, (p) hetAr ² NR ^g C(=O)-, where R ^g is H or C1-C ₆ alkyl, (q) Ar ¹ (C ₁ -C ₆ alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with OH, hydroxy(C1-C ₆ alkyl), C1-C ₆ alkoxy or NH2, (r) hetCyc ⁵ C(=O)-, (s) R ⁴ R ⁵ NC(=O)NR ^g -, where R ^g is H or C1-C ₆ -alkyl, or (t) (C ₁ -C ₆ alkyl)sO ₂ -;

(u) Ar ¹ (C1-C6 alkyl)C(=O)NR ^g -, where R ^g is H or C1-C6 alkyl, (v) hetAr ⁴ C(=O)NR ^g -, where R ^g is H or C1-C ₆ alkyl, (w) hetAr ² -S(=O)-, (x) (C ₃ -C ₆ cycloalkyl)CH ₂ SO ₂ -, (y) Ar ¹ (C ₁ -C ₆ alkyl)sO ₂ -, (z) hetAr ² SO2-, (aa)Ar ¹ , (bb) hetAr ² , (cc) hetCyc ⁵ , (dd) C ₁ -C ₆ alkoxy, (ee) Ar ¹ (C ₁ -C ₆ alkyl)-O-, (ff) hetAr ² (C ₁ -C ₆ alkyl)-O-, (gg) hetAr ² -OC ₁ -C ₆ -alkyl-, (hh) Ar ¹ (C1-C6 alkyl)NR ^g -, where R ^g is H or C1-C6 alkyl, (ii ) hetAr ² -S-, (jj) Ar ² SO2NR ^g (CH2)p-, where p is 0 or 1, and R ^g is H or C1-C ₆ -alkyl, (kk) (C1-C6 alkoxy)C(=O)-, (ll) (C ₁ -C ₆ alkyl)NR ^g C(=O)O-, where R ^g is H or C1-C ₆ -alkyl, (mm) (C ₁ -C ₆ alkyl)NR ^g SO2-, where R ^g is H or C1-C6 alkyl, (nn) hetCyc ⁵ C(=O)NR ^g -, where R ^g is H or C1-C ₆ -alkyl, (oo) Q-NR ^h (C1-C3-alkyl)C(=O)NR ^g -, where R ^g and R ^h independently represent H or C1-C6-alkyl, Q represents H, C1- _C6 -alkyl or (C1- _C6 -alkyl)OC(=O)(pp)

where R ^g and R ^h are independently H or C1-C ₆ -alkyl, Q is H, C1-C 6 -alkyl or (C1-C ₆ -alkyl)OC(=O)-, and r is 1, 2, 3 or 4, (qq)

- 108 041220

R ^h O where R ^g and R ^h independently represent H or C1-C ₆ -alkyl, and Q represents H, C1-C 6 -alkyl or (C1-C ₆ -alkyl)OC(=O)-, (rr)

where R ^g is H or C1-C ₆ alkyl, and Q is H, C1-C ₆ alkyl, or (C1-C 6 alkyl)OC(=O)-, or (ss) R ^g R ^h N-, where R ^g and R ^h are independently H or C1-C ₆ alkyl, (tt) (C ₃ -C ₆ cycloalkyl)C(=O)NR ^s -, where the cycloalkyl is optionally and independently substituted with one or more halogen atoms, (uu) (C ₁ -C ₆ alkyl)C(=O)NR ^s CH2-, where R ^g is H or C1-C 6 alkyl, or (vv) (C ₁ -C ₆ alkyl)SO ₂ NR ^s -, where R ^g is H or C1-C ₆ alkyl;

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6 alkoxy)C1-C6 alkyl- (optionally substituted with 1-3 fluorine atoms), C3-C6 cycloalkyl, hydroxy-C1-C6 alkyl, (C1-C6 alkyl)SO2-, R ^e RfN- and (R ^e Rf ^N )C1- _C6 alkyl-, wherein each R ^e and Rf independently represents H or C1-C6 alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl containing 1-2 ring nitrogen atoms, wherein hetAr ² is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C ₁ -C ₆ alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ alkoxy (optionally substituted with 1-3 fluorine atoms), (C 1 -C ₆ alkoxy)C 1 -C ₆ alkyl- (optionally substituted with 1-3 fluorine atoms) and hydroxy-C ₁ -C ₆ alkoxy-;

hetCyc ⁵ is a 4-6 membered saturated heterocyclic ring containing 1-2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclic ring is optionally substituted with one or more substituents independently selected from C1-C6 alkoxy and oxo;

R ³ is C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1C6 alkyl-, C1-C6 alkoxy, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)CH2-, (C3-C6 cycloalkyl)O-, (C3-C6 cycloalkyl)CH2O-, hetCyc ⁷ O-, Ph-O- or (C1-C6 alkoxy)C1-C ₆ alkyl-; wherein each of said C ₃ -C ₆ cycloalkyl moieties is optionally substituted with C ₁ -C ₆ alkyl (optionally substituted with 1-3 fluorine atoms), C1-C ₆ alkoxy, OH or R'RN-, wherein R' and R independently represent hydrogen or C1-C ₆ alkyl;

R ⁴ is H or C1-C ₆ -alkyl;

R ⁵ is Ar ² , hetAr ³ , Ar ² CH2-, hetCyc ⁶ -CH2-, hydroxy-C1-C ₆ -alkyl-, (C ₃ -C ₆ cycloalkyl)CH ₂ - or C1-C ₆ -alkyl optionally substituted with 1-3 fluorine atoms;

Ar ² is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6 alkoxy)C1-C6 alkyl- (optionally substituted with 1-3 fluorine atoms), C3-C6 cycloalkyl and R ^g R ^h N-, where R ^g and R ^h are independently H or C1-C ₆ alkyl, or Ar ² is phenyl fused to a 6-membered heterocyclic ring containing a ring nitrogen atom and optionally substituted with C ₁ -C ₆ alkyl;

hetAr ³ is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C ₁ -C ₆ alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ alkoxy (optionally substituted with 1-3 fluorine atoms) and (C 1 -C ₆ alkoxy)C 1 -C ₆ alkyl- (optionally substituted with 1-3 fluorine atoms);

hetAr ⁴ is pyridin-4(1H)-onyl or pyridin-2(1H)-onyl optionally substituted with one or more substituents independently selected from C1-C6 alkyl and halogen;

hetCyc ⁶ is a 5-7 membered heterocyclic ring containing 1-3 ring heteroatoms independently selected from N, O and S; and hetCyc ⁷ is a 5-7 membered heterocyclic ring containing 1-3 ring heteroatoms independently selected from N, O and S.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or a second

- 109 041220 RET inhibitor) is a compound of formula VII:

VII or a pharmaceutically acceptable salt or solvate thereof, wherein

X ¹ , X ² , X ³ and X ⁴ independently represent CH or N, where zero, one or two of X ¹ , X ² , X ³ and X ⁴ represent N;

A represents CN; B represents:

(b) C ₁ -C ₆ -alkyl optionally substituted with 1-3 fluorine atoms, (c) hydroxy-C ₂ -C ₆ -alkyl-, wherein the alkyl part is optionally substituted with a C ₃ -C ₆ -cycloalkylidene ring, or (i) (hetCyc ^a )C ₁ -C ₃ -alkyl-;

hetCyca- is a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from OH, C1- _C6 alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy- _C1 - _C6 alkyl-, halogen, (C1- _C6 alkyl)C(=O)-, C1- _C6 alkoxy, oxo and ( _C1 - _C6 alkoxy)C(=O)-;

Ring D is a saturated monocyclic 4-7 membered heterocyclic ring containing one ring heteroatom, which is nitrogen;

each R ^a is independently C1- _C6 -alkyl (optionally substituted with 1-3 fluorine atoms);

R ^b represents (a) hydroxy;

n is 0 or 1;

m is 0 or 1;

E represents:

(e) ^hetAr2C1 - _{C6 -} alkyl-, (h) hetAr2 ^- O-, (k) ^R3C (=O) ^NRg- , where ^Rg is H or C1- _C6- alkyl, (l) Ar1C(=O) ^NRg- , where ^Rg is H or C1- _C6- alkyl, or (m) ^hetAr2C (=O) ^NRg (CH2)P-, where p is 0 or 1 and ^Rg is H or C1-C6-alkyl;

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6 alkoxy)C1-C6 alkyl- (optionally substituted with 1-3 fluorine atoms), C3-C6 cycloalkyl, hydroxy-C1-C6 alkyl, (C1-C6 alkyl)SO2-, R ^e R ^f N- and (R ^e R ^f N)C1-C ₆ alkyl-, where each R ^e and R ^f independently represents H or C ₁ -C ₆ alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl containing 1-2 ring nitrogen atoms, wherein hetAr ² is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1- _C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1- _C6 alkoxy (optionally substituted with 1-3 fluorine atoms), ( _C1 - _C6 alkoxy) _C1 - _C6 alkyl- (optionally substituted with 1-3 fluorine atoms) and hydroxy- _C1 - _C6 alkoxy-; and

R ³ is C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1C6-alkyl-, C1-C6-alkoxy, C3-C6-cycloalkyl, (C3-C6-cycloalkyl)CH2-, (C3-C6-cycloalkyl)O-, (C3-C6cycloalkyl)CH2O-, hetCyc ⁷ O-, Ph-O- or (C1-C ₆ -alkoxy)C ₁ -C ₆ -alkyl-; wherein each of said C ₃ -C ₆ cycloalkyl moieties is optionally substituted with C1-C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), C1-C ₆ -alkoxy, OH or R'RN-, wherein R' and R independently represent hydrogen or C1-C ₆ -alkyl.

In some embodiments, the RET inhibitor (e.g., a first RET inhibitor or a second RET inhibitor) is selected from the group consisting of N-(1-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-4-methylpiperidin-4-yl)benzamide; 6-ethoxy-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(3-(pyridin-2-yloxy)azetidin-1-yl)pyridin-3yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-((6-methoxypyridazin-3-yl)oxy)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; (S)-6-(2hydroxy-2-methylpropoxy)-4-(6-(3-(pyridin-2-yloxy)pyrrolidin-1-yl)pyridin-3-yl)pyrazolo[1,5a]pyridine-3-carbonitrile; N-(1-(5-(3-cyano-6-((3-fluoro-1-methylazetidin-3-yl)methoxy)pyrazolo[1,5a]pyridin-4-yl)pyridin-2-yl)-4-methylpiperidin-4-yl)-5-fluoro-2-methylbenzamide; 3-chloro-N-(1-(5-(3- 110 041220 cyano-6-((3-fluoro-1-methylazetidin-3-yl)methoxy)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-4-methylpiperidin-4-yl)picolinamide; N-((38,48)-1-(5-(3-cyano-6-ethoxypyrazolo[1,5-a]pyridin-4yl)pyridin-2-yl)-3-hydroxypiperidin-4-yl)-3-methylbutanamide; 6-(2-hydroxy-2-methylpropoxy)-4(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; and 3-chloro-N-((38,48)-1-(5-(3-cyano-6-ethoxypyrazolo[1,5-a]pyridin-4-yl)pyrazin-2-yl)-3hydroxypiperidin-4-yl)picolinamide; or pharmaceutically acceptable salts or solvates thereof.

Non-limiting examples of therapeutic agents that target receptor tyrosine kinases (e.g., Trk) include afatinib, cabozantinib, cetuximab, crizotinib, dabrafenib, entrectinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, pazopanib, panitumumab, pertuzumab, sunitinib, trastuzumab, I-((38,4R)-4-(3-fluorophenyl)-I-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methylpyrimidin-5-yl)-1-phenyl-1H-pyrazol-5yl)urea, AG 879, AR-772, AR-786, AR-256, AR-618, AZ-23, AZ623, D8-6051, G6 6976, GNF-5837, GTx-186, GW 441756, LOXO-101, MGCD516, PLX7486, RXDX101, VM-902A, TPX-0005, and T8R-011. Additional Trk targeting therapeutic agents include those described in U.S. Patent Nos. 8,450,322; 8,513,263; 8,933,084; 8,791,123; 8,946,226; 8,450,322; 8,299,057; and 8,912,194; and U.S. Publication Nos. 2016/0137654; 2015/0166564; 2015/0051222; 2015/0283132; and 2015/0306086; in International Publication Nos. WO 2010/033941; WO 2010/048314; WO 2016/077841; WO 2011/146336; WO 2011/006074; WO 2010/033941; WO 2012/158413; WO 2014078454; WO 2014078417; WO 2014078408; WO 2014078378; WO 2014078372; WO 2014078331; WO 2014078328; WO 2014078325; WO 2014078323; WO 2014078322; WO 2015175788; WO 2009/013126; WO 2013/174876; WO 2015/124697; WO 2010/058006; WO 2015/017533; WO 2015/112806; WO 2013/183578; and WO 2013/074518, the entire contents of which are incorporated herein by reference.

Additional examples of Trk inhibitors are provided in U.S. Patent No. 8,637,516, International Publication No. WO 2012/034091, U.S. Patent No. 9,102,671, International Publication No. WO 2012/116217, U.S. Publication No. 2010/0297115, International Publication WO 2009/053442, U.S. Patent No. 8,642,035, International Publication WO 2009092049, U.S. Patent No. 8,691,221, International Publication WO2006131952, the entire contents of which are incorporated herein by reference. Exemplary Trk inhibitors include GNF-4256, described in Cancer Chemother. Pharmacol. 75(1): 131–141, 2015; and GNF-5837 (N-[3-[[2,3-dihydro-2-oxo-3-(1H-pyrrol2-ylmethylene)-1H-indol-6-yl]amino]-4-methylphenyl]-N'-[2-fluoro-5-(trifluoromethyl)phenyl]urea), described in AC8 Med. Chem. Lett. 3(2): 140–145, 2012, the entire contents of which are incorporated herein by reference.

Additional examples of Trk inhibitors include those described in U.S. Publication No. 2010/0152219, U.S. Patent No. 8,114,989, and International Publication No. WO 2006/123113, the entire contents of which are incorporated herein by reference. Illustrative Trk inhibitors include AZ623, described in Cancer 117(6):1321-1391, 2011; AZD6918, described in Cancer Biol. Ther. 16(3):477-483, 2015; AZ64, described in Cancer Chemother. Pharmacol. 70:477-486, 2012; AZ-23 ((8)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1Hpyrazol-3-yl)pyrimidine-2,4-diamine), described in Mol. Cancer Ther. 8:1818-1827, 2009; and AZD7451; the entire contents of which are incorporated herein by reference.

The Trk inhibitor may include the agents described in U.S. Patents 7,615,383; 7,384,632; 6,153,189; 6,027,927; 6,025,166; 5,910,574; 5,877,016; and 5,844,092, the entire contents of which are incorporated herein by reference.

Additional examples of Trk inhibitors include CEP-751, described in Int. J. Cancer 72:672–679, 1997; CT327, described in Acta Derm. Venereol. 95:542–548, 2015; the compounds described in International Publication No. WO 2012/034095; the compounds described in U.S. Patent No. 8,673,347 and International Publication No. WO 2007/022999; the compounds described in U.S. Patent No. 8,338,417; the compounds described in International Publication No. WO 2016/027754; the compounds described in U.S. Patent No. 9,242,977; the compounds described in U.S. Publication No. 2016/0000783; sunitinib (N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide), described in PLo8 One 9:e95628, 2014; compounds described in International Publication No. WO 2011/133637; compounds described in U.S. Pat. No. 8,637,256; compounds described in Expert. Opin. Ther. Pat. 24(7):731-744, 2014; compounds described in Expert Opin. Ther. Pat. 19(3):305-319, 2009; (R)-2-phenylpyrrolidine-substituted imidazopyridazines, such as GNF-8625, (R)-1-(6-(6-(2-(3-fluorophenyl)pyrrolidin-1yl)imidazo[1,2-b]pyridazin-3-yl)-[2,4'-bipyridin]-2'-yl)piperidin-4-ol, described in AC8 Med. Chem. Lett. 6(5):562–567, 2015; GTx-186 and others described in PLo8 One 8(12):e83380, 2013; K252a ((98-(9α, 10β, 12a))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one), described in Mol. Cell Biochem. 339(1-2):201–213, 2010; 4-aminopyrazolylpyrimidines, for example AZ-23 (((8)5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidin-2,4-diamine)), described in J. Med. Chem. 51(15):4672-4684, 2008; PHA-739358 (danusertib), described in

- 111 041220 of Mol. Cancer Ther. 6:3158, 2007; Go 6976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12-nindolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile), described in J. Neurochem. 72:919924, 1999; GW441756 ((3Z)-3-[(1-methylindol-3-yl)methylidene]-1H-pyrrolo[3,2-b]pyridin-2-one), described in IJAE 115:117, 2010; milciclib (PHA-848125AC), described in J.

Carcinog. 12:22, 2013; AG-879 ((2E)-3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-2-cyano-2propanethioamide); altiratinib (N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-2,5difluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); cabozantinib (N-(4-((6,7dimethoxyquinolin-4-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); lestaurtinib ((5S,6S,8R)-6-hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5, 8methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-ac-indacene-13(6H)-one); dovatinib (4-amino-5-fluoro-3-[6(4-methylpiperazin-1-yl) -1H-benzimidazol-2-yl]quinolin-2(1H)-one mono-2-hydroxypropanoate hydrate); sitravatinib (N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7yl)oxy)phenyl )-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); ONO-5390556; regorafenib (4-[4({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy ]-N-methylpyridine-2-carboxamide hydrate; and VSR-902A; all of the above references are incorporated herein by reference in their entirety.

The ability of a Trk inhibitor to act as an inhibitor of TrkA, TrkB and/or Trk C can be tested using the assays described in Examples A and B of U.S. Patent No. 8,513,263, the contents of which are incorporated herein by reference.

In some embodiments, the receptor tyrosine kinase inhibitor is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. For example, EGFR inhibitors can include osimertinib (merelectinib, Tagrisso), erlotinib (Tarceva), gefitinib (Iressa), cetuximab (Erbitux), necitumumab (Portrazza), neratinib (Nerlynx), lapatinib (Tykerb), panitumumab (Vectibix), and vandetanib (Caprelsa). In some embodiments, the EGFR inhibitor is osimertinib.

In some embodiments, signal transduction pathway inhibitors include Ras-Raf-MEK-ERK pathway inhibitors (e.g., binimetinib, selumetinib, encorafinib, sorafenib, trametinib, and vemurafenib), PI3K-Akt-mTOR-S6K pathway inhibitors (e.g., everolimus, rapamycin, perifosine, temsirolimus), and other kinase inhibitors such as baricitinib, brigatinib, capmatinib, danusertib, ibrutinib, milciclib, quercetin, regorafenib, ruxolitinib, semaxanib, AP32788, BLU285, BLU554, INCB39110, INCB40093, INCB50465, INCB52793, INCB54828, MGCD265, NMS-088, NMS-1286937, PF 477736 ((R)-amino-N-[5,6-dihydro-2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-1Hpyrrolo[4,3,2-ef][2,3]benzodiazepin-8-yl]cyclohexaneacetamide), PLX3397, PLX7486, PLX8394,

PLX9486, PRN1008, PRN1371, RXDX103, RXDX106, RXDX108 and TG101209 (N-tert-butyl 3-(5-methyl-2(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide).

Non-limiting examples of checkpoint inhibitors include ipilimumab, tremelimumab, nivolumab, pidilizumab, MPDL3208A, MEDI4736, MSB0010718C, BMS-936559, BMS-956559, BMS-935559 (MDX-1105), AMP-224, and pembrolizumab.

In some embodiments, the cytotoxic chemotherapeutic agents are selected from arsenic trioxide, bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan, lomustine, methotrexate, mitomycin C, oxaliplatin, paclitaxel, pemetrexed, temozolomide, and vincristine.

Non-limiting examples of therapeutic agents that target angiogenesis include aflibercept and bevacizumab.

The term immunotherapy refers to an agent that modulates the immune system. In some embodiments, immunotherapy can enhance the expression and/or activity of an immune system regulator. In some embodiments, immunotherapy can reduce the expression and/or activity of an immune system regulator. In some embodiments, immunotherapy can engage and/or enhance the activity of an immune cell.

In some embodiments, the immunotherapy is cellular immunotherapy (e.g., adoptive T cell therapy, dendritic cell therapy, natural killer cell therapy). In some embodiments, the cellular immunotherapy is sipuleucel-T (APC8015; Provenge™; Plosker (2011) Drugs 71(1): 101-108). In some embodiments, the cellular immunotherapy includes cells that express a chimeric antigen receptor (CAR). In some embodiments, the cellular immunotherapy is CAR-T cell therapy. In some embodiments, the CAR-T cell therapy is tisagenlecleucel (Kymriah™).

In some embodiments, the immunotherapy is an antibody therapy (e.g., a monoclonal antibody, a conjugated antibody). In some embodiments, the antibody therapy is bevacizumab (Mvasti™, Avastin®), trastuzumab (Herceptin®),

- 112 041220 avelumab (Bavencio®), rituximab (MabThera™, Rituxan®), edrecolomab (Panorex), daratumumab (Darzalex®), olaratumab (Lartruvo™), ofatumumab (Arzerra®), alemtuzumab (Campath®), cetuximab (Erbitux®), oregovomab, pembrolizumab (Keytruda®), dinutiximab (Unituxin®), obinutuzumab (Gazyva®), tremelimumab (CP-675.206), ramucirumab (Cyramza®), ublituximab (TG-1101), panitumumab (Vectibix®), elotuzumab (Empliciti™), avelumab (Bavencio®), necitumumab (Portrazza™), cirmtuzumab (UC961), ibritumomab (Zevalin®), isatuximab (SAR650984), nimotuzumab, frezolimumab (GC1008), lirilumab (INN), mogamulizumab (Poteligeo®), ficlatuzumab (AV-299), denosumab (Xgeva®), ganitumab, urelumab, pidilizumab, or amatuximab.

In some embodiments, the immunotherapy is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate is gemtuzumab ozogamicin (Mylotarg™), inotuzumab ozogamicin (Besponsa®), brentuximab vedotin (Adcetris®), ado-trastuzumab emtansine (TDM-1; Kadcyla®), mirvetuximab soravtansine (IMGN853), or anetumab ravtansine.

In some embodiments, the immunotherapy includes blinatumomab (AMG103; Blincyto®) or midostaurin (Rydapt).

In some embodiments, the immunotherapy comprises a toxin. In some embodiments, the immunotherapy is denileukin diftitox (Ontak®).

In some embodiments, the immunotherapy is a cytokine therapy. In some embodiments, the cytokine therapy is interleukin 2 (IL-2) therapy, interferon alpha (IFNa) therapy, granulocyte colony stimulating factor (G-CSF) therapy, interleukin 12 (IL-12) therapy, interleukin 15 (IL-15) therapy, interleukin 7 (IL-7) therapy, or erythropoietin alpha (EPO) therapy. In some embodiments, the IL-2 therapy is aldesleukin (Proleukin®). In some embodiments, the IFNa therapy is IntronA® (Roferon-A®). In some embodiments, the GCSF therapy is filgrastim (Neupogen®).

In some embodiments, the immunotherapy is an immune checkpoint inhibitor. In some embodiments, the immunotherapy comprises one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab (Yervoy®) or tremelimumab (CP-675206). In some embodiments, the PD-1 inhibitor is pembrolizumab (Keytruda®) or nivolumab (Opdivo®). In some embodiments, the PD-L1 inhibitor is atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi™).

In some embodiments, the immunotherapy is an mRNA-based immunotherapy. In some embodiments, the mRNA-based immunotherapy is CV9104 (see, e.g., Rausch et al. (2014) Human Vaccin Immunother 10(11): 3146-52; and Kuble et al. (2015) J. Immunother Cancer 3:26).

In some embodiments, the immunotherapy is bacillus Calmette-Guerin (BCG) therapy.

In some embodiments, the immunotherapy is an oncolytic viral therapy. In some embodiments, the oncolytic viral therapy is talimogene laherparepvec (T-VEC; Imlygic®).

In some embodiments, the immunotherapy is a cancer vaccine. In some embodiments, the cancer vaccine is a human papillomavirus (HPV) vaccine. In some embodiments, the HPV vaccine is Gardasil®, Gardasil9®, or Cervarix®. In some embodiments, the cancer vaccine is a hepatitis B virus (HBV) vaccine. In some embodiments, the HBV vaccine is Engerix-B®, Recombivax HB®, or GI-13020 (Tarmogen®). In some embodiments, the cancer vaccine is Twinrix® or Pediarix®. In some embodiments, the cancer vaccine is BiovaxlD®, Oncophage®, GVAX, ADXS11-001, ALVAC-CEA, PROSTVAC®, Rindopepimut®, CimaVax-EGF, lapuleucel-T (APC8024; Neuvenge™), GRNVAC1, GRNVAC2, GRN-1201, hepcortespenlizimut-L (Hepko-V5), DCVAX®, SCIB1, BMT CTN 1401, PrCa VBIR, PANVAC, ProstAtak®, DPX-Survivac, or viagenpumatucel-L (HS-110).

In some embodiments, the immunotherapy is a peptide vaccine. In some embodiments, the peptide vaccine is nelipepimut-S (E75) (NeuVax™), IMA901, or SurVaxM (SVN53-67). In some embodiments, the cancer vaccine is an immunogenic individual neoantigen vaccine (see, e.g., Ott et al. (2017) Nature 547: 217-221; Sahin et al. (2017) Nature 547: 222-226). In some embodiments, the cancer vaccine is RGSH4K or NEO-PV-01. In some embodiments, the cancer vaccine is a DNA-based vaccine. In some embodiments

- 113 041220 DNA-based vaccine is a mammaglobulin A DNA-based vaccine (see, for example,

Kim et al. (2016) Oncolmmunology 5(2): e1069940).

In some embodiments, the immune system targeting agents are selected from aldesleukin, interferon alpha-2b, ipilimumab, lambrolizumab, nivolumab, prednisone, and sipuleucel-T.

Non-limiting examples of radiation therapy include radioiodide therapy, external beam radiation therapy, and radium-223 therapy.

Additional kinase inhibitors include those described, for example, in U.S. Patent Nos. 7,514,446; 7,863,289; 8,026,247; 8,501,756; 8,552,002; 8,815,901; 8,912,204; 9,260,437; 9,273,051; U.S. Publication No. 2015/0018336; International Publication Nos. WO 2007/002325; WO 2007/002433; WO 2008/080001; WO 2008/079906; WO 2008/079903; WO 2008/079909; WO 2008/080015; WO 2009/007748; WO 2009/012283; WO 2009/143018; WO 2009/143024; WO WO 2009/014637; 2009/152083; WO 2010/111527; WO 2012/109075; WO 2014/194127; WO 2015/112806; WO 2007/110344; WO 2009/071480; WO 2009/118411; WO 2010/031816; WO 2010/145998; WO 2011/092120; WO 2012/101032; WO 2012/139930; WO 2012/143248; WO 2012/152763; WO 2013/014039; WO 2013/102059; WO 2013/050448; WO 2013/050446; WO 2014/019908; WO 2014/072220; WO 2014/184069; and WO 2016/075224, the entire contents of which are incorporated herein by reference.

Further examples of kinase inhibitors include those described in, for example, WO 2016/081450; WO 2016/022569; WO 2016/011141; WO 2016/011144; WO 2016/011147; WO 2015/191667; WO 2012/101029; WO 2012/113774; WO 2015/191666; WO 2015/161277; WO 2015/161274; WO 2015/108992; WO 2015/061572; WO 2015/058129; WO 2015/057873; WO 2015/017528; WO/2015/017533; WO 2014/160521; and WO 2014/011900, the entire contents of which are incorporated herein by reference.

Additional examples of kinase inhibitors include luminespib (AUY-922, NVP-AUY922) (5(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide) and doramapimod (BIRB-796) (1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4ylethoxy)naphthalen-1-yl]urea).

Accordingly, the present document also provides a method of treating cancer, comprising administering to a patient in need thereof a pharmaceutical combination for treating cancer, which comprises (a) a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, (b) an additional therapeutic agent, and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate or sequential use for treating cancer, wherein the amounts of the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent together are effective for treating cancer.

In some embodiments, the additional therapeutic agent(s) comprises any of the above therapeutic agents or therapeutic agents that are standard treatments for cancer, wherein the cancer is characterized by deregulation of the RET gene, RET protein, or expression or activity, or level of any of them.

Said additional therapeutic agents can be administered with one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof, in the same or in different dosage forms, by the same or different routes of administration and/or by the same or different administration schedules in accordance with standard pharmaceutical practice known to those skilled in the art.

The present document also provides (i) a pharmaceutical combination for treating cancer in a patient in need thereof, which comprises (a) a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, (b) at least one additional therapeutic agent (e.g., any of the illustrative additional therapeutic agents described herein or known in the art), and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate, or sequential use in treating cancer, wherein the amounts of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and the additional therapeutic agent together are effective for treating cancer; (ii) a pharmaceutical composition comprising such a combination; (iii) the use of such a combination for the preparation of a medicament for treating cancer; and (iv) a commercial package or article comprising such a combination as a combination preparation for simultaneous, separate, or sequential use; as well as a method of treating cancer in a patient in need thereof. In one embodiment, the patient is a human. In some embodiments, the cancer is a RET-associated cancer. For example, a RET-associated cancer that has one or more RET inhibitor resistance mutations.

The term pharmaceutical combination in this context refers to a pharmaceutical therapy obtained by mixing or combining more than one active ingredient, and

- 114 041220 includes both fixed and non-fixed combinations of active ingredients. The term fixed combination means that a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and at least one additional therapeutic agent (e.g. a chemotherapeutic agent) are administered to a patient simultaneously, in the form of a single composition or dosage form. The term non-fixed combination means that a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and at least one additional therapeutic agent (e.g. a chemotherapeutic agent) are formulated as separate compositions or dosage forms so that they can be administered to a patient in need thereof simultaneously, in parallel or sequentially at different intermediate time intervals, such administration providing an effective maintenance of two or more compounds in the patient's body. This also applies to cocktail therapies, for example the administration of three or more active ingredients.

Accordingly, the present document also provides a method of treating cancer comprising administering to a patient in need thereof a pharmaceutical combination for treating cancer comprising (a) a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, (b) an additional therapeutic agent, and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate or sequential use for treating cancer, wherein the amounts of the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent together are effective for treating cancer. In one embodiment, the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent are administered simultaneously as separate dosage forms. In one embodiment, the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent are administered as separate dosage forms sequentially, in any order, in a total therapeutically effective amount, such as in daily or intermediate doses. In one embodiment, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and an additional therapeutic agent are administered simultaneously in a combined dosage form. In some embodiments, the cancer is a RET-associated cancer. For example, a RET-associated cancer that has one or more RET inhibitor resistance mutations. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, the patient is administered one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, prior to administration of said pharmaceutical composition. In some embodiments, the cancer is lung cancer (e.g., RET-associated lung cancer).

Also provided herein is a method of treating a disease or disorder mediated by RET in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form, or pharmaceutical composition thereof. In some embodiments, the disease or disorder mediated by RET is a deregulation of a RET gene, a RET kinase, or the expression or activity, or the level of any of them. For example, a deregulation of a RET gene, a RET kinase, or the expression or activity, or the level of any of them comprises one or more RET inhibitor resistance mutations. A disease or disorder mediated by RET can include any disease, disorder, or pathological condition directly or indirectly associated with RET expression or activity, including overexpression and/or abnormal levels of activity. In another embodiment, the disease is cancer (e.g., RET-associated cancer). In one embodiment, the cancer is any cancer or RET-associated cancer described herein. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, the patient is administered one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof prior to administration of the pharmaceutical composition. In some embodiments, the cancer is lung cancer (e.g., RET-associated lung cancer).

Although the genetic basis of tumorigenesis may vary among different types of cancer, the cellular and molecular mechanisms required for metastasis are the same for all types of solid tumors. During the metastatic cascade, cancer cells lose growth inhibitory responses, undergo changes in adhesiveness, and produce enzymes that can degrade extracellular matrix components. This leads to tumor cell detachment from the original tumor, infiltration into the bloodstream through newly formed vasculature, migration, and extravasation of tumor cells to favorable distant sites,

- 115 041220 where they can form colonies. Numerous genes have been identified as promoters or suppressors of metastasis. For example, overexpression of glial cell-derived neurotrophic factor (GDNF) and its receptor tyrosine kinase RET correlates with cancer proliferation and metastasis. See, for example, Zeng, Q. et al. J. Int. Med. Res. (2008) 36(4): 656–64.

Accordingly, provided herein are also methods of suppressing, preventing, facilitating the prevention of, or reducing the symptoms of cancer metastasis in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. Such methods can be used in the treatment of one or more cancers described herein. See, e.g., U.S. Publication No. 2013/0029925; International Publication No. WO 2014/083567; and U.S. Patent No. 8,568,998. See also, e.g., Hezam K et al., Rev Neurosci 2018 Jan 26;29:93-98; Gao L, et al., Pancreas 2015 Jan;44:134-143; Ding K et al., J Biol Chem 2014 Jun 6; 289:16057-71; and Amit M et al., Oncogene 2017 Jun 8; 36:3232-3239. In some embodiments, the cancer is RET-associated cancer. In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is used in combination with an additional therapy or another therapeutic agent, including a chemotherapeutic agent, such as a kinase inhibitor. For example, a first or second RET kinase inhibitor. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, the patient is administered one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, prior to administration of the pharmaceutical composition. In some embodiments, the cancer is lung cancer (eg, RET-associated lung cancer).

The term metastasis is a well-known term and means the formation of an additional tumor (e.g., a solid tumor) at a site distant from the primary tumor in the body of a subject or patient, wherein the additional tumor contains the same or similar cancer cells as the primary tumor.

Also provided are methods for reducing the risk of developing metastasis or additional metastasis in a patient suffering from RET-associated cancer, which include: selecting, identifying or diagnosing a patient as having a RET-associated cancer, and administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof to a patient who has been selected, identified or diagnosed as having a RET-associated cancer. Also provided are methods for reducing the risk of developing metastasis or additional metastasis in a patient suffering from a RET-associated cancer, which include administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof to a patient suffering from a RET-associated cancer. The reduction in the risk of developing metastasis or additional metastasis in a patient suffering from a RET-associated cancer can be compared to the risk of developing metastasis or additional metastasis in the patient before treatment, or compared to a patient or group of patients having a similar or the same RET-associated cancer who are not receiving treatment or are receiving a different treatment. In some embodiments, the RET-associated cancer is a RET-associated cancer that has one or more RET inhibitor resistance mutations. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, the patient is administered one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, prior to administration of said pharmaceutical composition. In some embodiments, the cancer is lung cancer (e.g., RET-associated lung cancer).

The expression risk of developing metastasis means the risk that a subject or patient with a primary tumor will develop an additional tumor (e.g., a solid tumor) at a site distant from the primary tumor in the subject's or patient's body over a specified period of time, wherein the additional tumor contains the same or similar cancer cells as the primary tumor. Methods for reducing the risk of developing metastasis in a subject or patient suffering from cancer are described herein.

The expression risk of developing additional metastasis means the risk that a subject or patient having a primary tumor and one or more additional tumors at sites distant from the primary tumor (wherein one or more additional tumors contain the same or similar cancer cells as the primary tumor) will develop one or more additional tumors distant from the primary tumor, wherein the additional tumors contain the same or similar cancer cells as the primary tumor. Methods for reducing the risk of developing additional metastasis are described herein.

In some embodiments, the presence of one or more inhibitor resistance mutations

- 116 041220

RET in a tumor causes the tumor to be resistant to treatment with a first RET inhibitor. The following describes methods useful when a RET inhibitor resistance mutation causes the tumor to be more resistant to treatment with a first RET inhibitor. For example, provided herein are methods of treating a subject suffering from cancer, which comprise: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and administering to the identified subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is administered in combination with a first RET inhibitor. Also provided are methods of treating a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations, which comprise administering to the subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is administered in combination with a first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more RET inhibitor resistance mutations listed in Tables 3 and 4. For example, the one or more RET inhibitor resistance mutations can include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

For example, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting deregulation of a RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, wherein the first RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668. In some embodiments, the methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that has at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the first RET inhibitor of step (b) if said subject has a cancer cell that does not have a RET inhibitor resistance mutation.

In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting deregulation of a RET gene, RET kinase, or expression or activity, or level of any of them in a sample obtained from the subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, wherein the first RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668. In some embodiments, the methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that has at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the first RET inhibitor of step (b) if said subject has a cancer cell that does not have a RET inhibitor resistance mutation.

In some embodiments, the compound of Formula I-IV is a polymorphic form. In some embodiments, the compound is Polymorphic Form A of the compound of Formula I. In some embodiments, the compound is Polymorphic Form 1 of the compound of Formula II. In some embodiments, the compound is Polymorphic Form 2 of the compound of Formula II. In some embodiments, the compound is Polymorphic Form 7 of the compound of Formula II. In some embodiments, the compound is Polymorphic Form 8 of the compound of Formula II. In some embodiments, the compound is Polymorphic Form A of the compound of Formula III. In some embodiments, the compound is Polymorphic Form A of the compound of Formula IV. In some embodiments, the compound is Polymorphic Form B of the compound of Formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is a chloride salt of the compound of Formula I. In some embodiments, the compound is a bromide salt of the compound of Formula I. In some embodiments, the compound is an L-malate salt of the compound of Formula I. In some embodiments, the compound is a D-malate salt of the compound of Formula I. In some embodiments, the compound is a phosphate salt of the compound of Formula II. In some embodiments, the phosphate salt is a sesquiphosphate salt (e.g., _PO4 :free base 1.4:1).

In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1 and/or one or more point mutations/insertions/deletions in a RET kinase protein from Tables 2 and 2a in a sample obtained from the subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, wherein the first RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179W6, GSK3352589 and NMS-E668. In some embodiments, the methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation from Table 3 or 4; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that has at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the first RET inhibitor of step (b) if said subject has a cancer cell that does not have a RET inhibitor resistance mutation.

In some embodiments, the compound of formula I-IV is a polymorph. In some embodiments, the compound is polymorph Form A of the compound of formula I. In some embodiments, the compound is polymorph Form 1 of the compound of formula II. In some embodiments, the compound is polymorph Form 2 of the compound of formula II. In some embodiments, the compound is polymorph Form 7 of the compound of formula II. In some embodiments, the compound is polymorph Form 8 of the compound of formula II. In some embodiments, the compound is polymorph Form A of the compound of formula III. In some embodiments, the compound is polymorph Form A of the compound of formula IV. In some embodiments, the compound is polymorph Form B of the compound of formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is a chloride salt of the compound of Formula I. In some embodiments, the compound is a bromide salt of the compound of Formula I. In some embodiments, the compound is an L-malate salt of the compound of Formula I. In some embodiments, the compound is a D-malate salt of the compound of Formula I. In some embodiments, the compound is a phosphate salt of the compound of Formula II. In some embodiments, the phosphate salt is a sesquiphosphate salt (e.g., _PO4 :free base 1.4:1).

In some embodiments, provided herein are methods for treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, wherein the first RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((3)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668. In some embodiments, the methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has a V804M, G810S, or G810R RET inhibitor resistance mutation; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof selected from the group consisting of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that has at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the first RET inhibitor of step (b) if said subject has a cancer cell that does not have a RET inhibitor resistance mutation.

In some embodiments, the compound of formula I-IV is a polymorph. In some embodiments, the compound is polymorph Form A of the compound of formula I. In some embodiments, the compound is polymorph Form 1 of the compound of formula II. In some embodiments, the compound is polymorph Form 2 of the compound of formula II. In some embodiments, the compound is polymorph Form 7 of the compound of formula II. In some embodiments, the compound is polymorph Form 8 of the compound of formula II. In some embodiments, the compound is polymorph Form A of the compound of formula III. In some embodiments, the compound is polymorph Form A of the compound of formula IV. In some embodiments, the compound is polymorph Form B of the compound of formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is a chloride salt of the compound of Formula I. In some embodiments, the compound is a bromide salt of the compound of Formula I. In some embodiments, the compound is an L-malate salt of the compound of Formula I. In some embodiments, the compound is a D-malate salt of the compound of Formula I. In some embodiments, the compound is a phosphate salt of the compound of Formula II. In some embodiments, the phosphate salt is a sesquiphosphate salt (e.g., _PO4 :free base 1.4:1).

As another example, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject a second RET inhibitor, wherein the second RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU667 ((1S,4R)-N-((3)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010,

GSK3179106, GSK3352589, and NMS-E668, as a monotherapy or in combination with another anti-cancer agent, wherein said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (b), wherein said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting deregulation of a RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the disclosed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject a second RET inhibitor, wherein the second RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010,

GSK3179106, GSK3352589, and NMS-E668, as monotherapy or in combination with another anti-cancer agent, wherein said subject has a cancer cell that comprises at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, of step (b), wherein said subject has a cancer cell that does not comprise a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins of Table 1 and/or one or more point mutations/insertions/deletions in a RET kinase protein from Tables 2 and 2a in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the disclosed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation from Table 3 or 4; and (d) administering to the subject a second RET inhibitor, wherein the second RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1Hpyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668, as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the disclosed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has a RET inhibitor resistance mutation V804M, G810S, or G810R; and (d) administering to the subject a second RET inhibitor, wherein the second RET inhibitor is selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668, as a monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

As another example, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of crizotinib and osimertinib, as monotherapy or in combination with a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, if said subject has a cancer cell that comprises at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, of step (b), if said subject has a cancer cell that does not comprise a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins of Table 1 and/or one or more point mutations/insertions in a RET kinase protein of Table 1. 2 in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the methods provided further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation from Table 3 or 4; and (d) administering to said subject a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of crizotinib and osimertinib, as monotherapy or in combination with a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments listed above, the RET-associated cancer is lung cancer.

In some embodiments, the presence of one or more inhibitor resistance mutations

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RET in a tumor causes the tumor to be resistant to treatment with a first RET inhibitor. The following describes methods useful when a RET inhibitor resistance mutation causes the tumor to be more resistant to treatment with a first RET inhibitor. For example, provided herein are methods of treating a subject suffering from cancer, which comprise: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and administering to the identified subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is administered in combination with a first RET inhibitor. Also provided are methods of treating a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations, which comprise administering to the subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is administered in combination with a first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more RET inhibitor resistance mutations listed in Tables 3 and 4. For example, the one or more RET inhibitor resistance mutations can include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

In some embodiments provided herein, circulating tumor DNA can be used to monitor the responsiveness of a patient to a particular therapy (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof). For example, prior to initiating treatment with a therapy described herein (e.g., using a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof), a biological sample can be obtained from the subject and the level of circulating tumor DNA in the biological sample can be determined. Such a sample is considered a baseline sample. The subject may then be administered one or more doses of the therapy described herein (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof), and circulating tumor DNA levels may be monitored (e.g., after the first dose, second dose, third dose, etc., or after one week, two weeks, three weeks, four weeks, etc.). If the level of circulating tumor DNA is lower than that of the baseline sample (e.g., a decrease of 1% to about 99%, a decrease of 1% to about 95%, a decrease of 1% to about 90%, a decrease of 1% to about 85%, a decrease of 1% to about 80%, a decrease of 1% to about 75%, a decrease of 1% to about 70%, a decrease of 1% to about 65%, a decrease of 1% to about 60%, a decrease of 1% to about 55%, a decrease of 1% to about 50%, a decrease of 1% to about 45%, a decrease of 1% to about 40%, a decrease of 1% to about 35%, a decrease of 1% to about 30%, a decrease of 1% to about 25%, a decrease of 1% to about 20%, a decrease of 1% to a decrease of about 15%, a decrease of 1% to a decrease of about 10%, from 1% to about 5%, a decrease of about 5% to about 99%, a decrease of about 10% to about 99%, a decrease of about 15% to about 99%, a decrease of about 20% to about 99%, a decrease of about 25% to about 99%, a decrease of about 30% to about 99%, a decrease of about 35% to about 99%, a decrease of about 40% to about 99%, a decrease of about 45% to about 99%, a decrease of about 50% to about 99%, a decrease of about 55% to about 99%, a decrease of about 60% to about 99%, a decrease of about 65% to about 99%, a decrease of about 70% to about 99%, a decrease of about 75% to about 95%, a decrease of about 80% to about 99%, a decrease from about 90% to about 99%, a decrease from about 95% to about 99%, a decrease from about 5% to about 10%, a decrease from about 5% to about 25%, a decrease from about 10% to about 30%, a decrease from about 20% to about 40%, a decrease from about 25% to about 50%, a decrease from about 35% to about 55%, a decrease from about 40% to about 60%, a decrease from about 50% to about 75%, a decrease from about 60% to about 80%, or a decrease from about 65% to about 85%, etc.), then this is indicative of susceptibility to therapy. In some embodiments, the level of circulating tumor DNA is reduced such that the level becomes below the detection limit of the device. In some embodiments, the level of circulating tumor DNA in a biological sample obtained from a patient (n) is compared to a sample taken immediately before (n-1). If the level of circulating tumor DNA in sample n is lower than in sample n-1 (e.g., a decrease of from 1% to about 99%, a decrease of from 1% to about 95%, a decrease of from 1% to about 90%, a decrease of from 1% to about 85%, a decrease of from 1% to about 80%, a decrease of from 1% to about 75%, a decrease of from 1% to about 70%, a decrease of from 1% to about 65%, a decrease of from 1% to about 60%, a decrease of from 1% to about 55%, a decrease of from 1% to about 50%, a decrease of from 1% to about 45%, a decrease of from 1%

- 121 041220 up to a decrease of about 40%, a decrease of 1% to a decrease of about 35%, a decrease of 1% to a decrease of about 30%, a decrease of 1% to a decrease of about 25%, a decrease of 1% to a decrease of about 20%, a decrease of 1% to a decrease of about 15%, a decrease of 1% to a decrease of about 10%, a decrease of 1% to about 5%, a decrease of about 5% to about 99%, a decrease of about 10% to about 99%, a decrease of about 15% to about 99%, a decrease of about 20% to about 99%, a decrease of about 25% to about 99%, a decrease of about 30% to about 99%, a decrease of about 35% to about 99%, a decrease of about 40% to about 99%, a decrease of about 45% to about 99%, a decrease of about 50% to about 99%, a decrease from about 55% to about 99%, a decrease from about 60% to about 99%, a decrease from about 65% to about 99%, a decrease from about 70% to about 99%, a decrease from about 75% to about 95%, a decrease from about 80% to about 99%, a decrease from about 90% to about 99%, a decrease from about 95% to about 99%, a decrease from about 5% to about 10%, a decrease from about 5% to about 25%, a decrease from about 10% to about 30%, a decrease from about 20% to about 40%, a decrease from about 25% to about 50%, a decrease from about 35% to about 55%, a decrease from about 40% to about 60%, a decrease from about 50% to about 75%, a decrease from about 60% to about 80%, or a decrease from about 65% to a decrease of about 85%, etc.), then this is an indicator of responsiveness to therapy. In some embodiments, the level of circulating tumor DNA is reduced such that the level is below the detection limit of the device. If responsive to therapy, the subject can be administered one or more doses of therapy and continue to monitor circulating tumor DNA.

If the level of circulating tumor DNA in a sample is higher than at baseline (e.g., an increase of 1% to about 99%, an increase of 1% to about 95%, an increase of 1% to about 90%, an increase of 1% to about 85%, an increase of 1% to about 80%, an increase of 1% to about 75%, an increase of 1% to about 70%, an increase of 1% to about 65%, an increase of 1% to about 60%, an increase of 1% to about 55%, an increase of 1% to about 50%, an increase of 1% to about 45%, an increase of 1% to about 40%, an increase of 1% to about 35%, an increase of 1% to about 30%, an increase of 1% to about 25%, an increase of 1% to about 20%, an increase of 1% to about 15% increase, 1% increase to about 10% increase, 1% increase to about 5% increase, about 5% increase to about 99% increase, about 10% increase to about 99% increase, about 15% increase to about 99% increase, about 20% increase to about 99% increase, about 25% increase to about 99% increase, about 30% increase to about 99% increase from about 35% to about 99%, increase from about 40% to about 99%, increase from about 45% to about 99%, increase from about 50% to about 99%, increase from about 55% to about 99%, increase from about 60% to about 99%, increase from about 65% to about 99%, increase from about 70% to about 99%, increase from about 75% to about 95%, an increase from about 80% to about 99%, an increase from about 90% to about 99%, an increase from about 95% to about 99%, an increase from about 5% to about 10%, an increase from about 5% to about 25%, an increase from about 10% to about 30%, an increase from about 20% to about 40%, an increase from about 25% to about 50%, an increase from about 35% to about 55%, an increase from about 40% to about 60%, an increase from about 50% to about 75%, an increase from about 60% to about 80%, or an increase from about 65% to about 85%, etc.), then this may indicate resistance to therapy. If the level of circulating tumor DNA in sample n is higher than that in sample n-1 (e.g., an increase of 1% to about 99%, an increase of 1% to about 95%, an increase of 1% to about 90%, an increase of 1% to about 85%, an increase of 1% to about 80%, an increase of 1% to about 75%, an increase of 1% to about 70%, an increase of 1% to about 65%, an increase of 1% to about 60%, an increase of 1% to about 55%, an increase of 1% to about 50%, an increase of 1% to about 45%, an increase of 1% to about 40%, an increase of 1% to about 35%, an increase of 1% to about 30%, an increase of 1% to about 25%, an increase of 1% to about 20%, an increase of 1% to about 15%, an increase of 1% to about 10%, increase from 1% to about 5%, increase from about 5% to about 99%, increase from about 10% to about 99%, increase from about 15% to about 99%, increase from about 20% to about 99%, increase from about 25% to about 99%, increase from about 30% to about 99%, increase from about 35% to about 99%, increase from about 40% to about 99%, increase from about 45% to about 99%, increase from about 50% to about 99%, increase from about 55% to about 99%, increase from about 60% to about 99%, increase from about 65% to about 99%, increase from about 70% to about 99%, increase from about 75% to about 95%, increase from about 80% to about 99%, increasing from about 90% to about 99%, an increase from about 95% to about 99%, an increase from about 5% to about 10%, an increase from about 5% to about 25%, an increase from about 10% to about 30%, an increase from about 20% to about 40%, an increase from about 25% to about 50%, an increase from about 35% to about 55%, an increase from about 40% to about 60%, an increase from about 50% to about 75%, an increase from about 60% to about 80%, or an increase from about 65% to about 85%, etc.), then this may indicate resistance to therapy. If resistance to therapy is suspected, the subject may be referred for one or more of imaging tests, biopsy, surgery, or other diagnostic tests. In some embodiments, when resistance to therapy is suspected, a compound capable of treating resistance to a RET inhibitor (e.g., a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as provided herein) can be administered to the subject (as monotherapy or in combination with prior therapy). See, e.g., Cancer Discov; 7(12); 1368-70 (2017); and

Cancer Discov; 7(12); 1394-403 (2017).

In some embodiments provided herein, a protein biomarker can be used to monitor the responsiveness of a patient to a particular therapy (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof). For example, prior to initiating treatment with a therapy described herein (e.g., using a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof), a biological sample can be obtained from a subject and the level of a protein biomarker can be determined in the biological sample. Such a sample is considered a baseline sample. The subject may then be administered one or more doses of the therapy described herein (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof), and protein biomarker levels may be monitored (e.g., after the first dose, second dose, third dose, etc., or after one week, two weeks, three weeks, four weeks, etc.). If the level of a protein biomarker is lower than that of the baseline sample (e.g., a decrease of 1 to about 99%, a decrease of 1 to about 95%, a decrease of 1 to about 90%, a decrease of 1 to about 85%, a decrease of 1 to about 80%, a decrease of 1 to about 75%, a decrease of 1% to about 70%, a decrease of 1% to about 65%, a decrease of 1% to about 60%, a decrease of 1% to about 55%, a decrease of 1% to about 50%, a decrease of 1% to about 45%, a decrease of 1% to about 40%, a decrease of 1% to about 35%, a decrease of 1% to about 30%, a decrease of 1% to about 25%, a decrease of 1% to about 20%, a decrease of 1% to about 15%, from 1% to about 10% reduction, from 1 to about 5%, from about 5 to about 99% reduction, from about 10 to about 99% reduction, from about 15 to about 99% reduction, from about 20 to about 99% reduction, from about 25 to about 99% reduction, from about 30 to about 99% reduction, from about 35 to about 99% reduction, from about 40 to about 99% reduction, from about 45 to about 99% reduction, from about 50 to about 99% reduction, from about 55 to about 99% reduction, from about 60 to about 99% reduction, from about 65 to about 99% reduction, from about 70 to about 99% reduction, from about 75 to about 95% reduction, from about 80 to about 99% reduction, from about 90 to about 99% reduction, from about 95 to about 99%, a decrease of about 5 to about 10%, a decrease of about 5 to about 25%, a decrease of about 10 to about 30%, a decrease of about 20 to about 40%, a decrease of about 25 to about 50%, a decrease of about 35 to about 55%, a decrease of about 40 to about 60%, a decrease of about 50 to about 75%, a decrease of about 60 to about 80%, or a decrease of about 65 to about 85%, etc.), then this indicates responsiveness to the therapy. In some embodiments, the level of the protein biomarker decreases such that the level becomes below the detection limit of the device. In some embodiments, the level of the protein biomarker in a biological sample obtained from a patient (n) is compared with a sample taken immediately before it (n-1). If the level of a protein biomarker in sample n is lower than in sample n-1 (e.g., decrease from 1 to about 99%, decrease from 1 to about 95%, decrease from 1 to about 90%, decrease from 1 to about 85%, decrease from 1 to about 80%, decrease from 1 to about 75%, decrease from 1% to about 70% decrease, decrease from 1% to about 65% decrease, decrease from 1% to about 60% decrease, decrease from 1% to about 55% decrease, decrease from 1% to about 50% decrease, decrease from 1% to about 45% decrease, decrease from 1% to about 40% decrease, decrease from 1% to about 35% decrease, decrease from 1% to about 30% decrease, decrease from 1% to about 25% decrease, decrease from 1% to about 20% decrease, decrease from 1% to about 15%, a decrease from 1% to a decrease of about 10%, a decrease from 1 to about 5%, a decrease from about 5 to about 99%, a decrease from about 10 to about 99%, a decrease from about 15 to about 99%, a decrease from about 20 to about 99%, a decrease from about 25 to about 99%, a decrease from about 30 to about 99%, a decrease from about 35 to about 99%, a decrease from about 40 to about 99%, a decrease from about 45 to about 99%, a decrease from about 50 to about 99%, a decrease from about 55 to about 99%, a decrease from about 60 to about 99%, a decrease from about 65 to about 99%, a decrease from about 70 to about 99%, a decrease from about 75 to about 95%, a decrease from about 80 to about 99%, a decrease from about 90 to about 99%, a decrease from about 95 to about 99%, a decrease from about 5 to about 10%, a decrease from about 5 to about 25%, a decrease from about 10 to about 30%, a decrease from about 20 to about 40%, a decrease from about 25 to about 50%, a decrease from about 35 to about 55%, a decrease from about 40 to about 60%, a decrease from about 50% to a decrease of about 75%, a decrease from about 60% to a decrease of about 80%, or a decrease from about 65% to a decrease of about 85%, etc.), then this is an indicator of responsiveness to therapy. In some embodiments, the level of the protein biomarker decreases such that the level is below the detection limit of the device. In the case of responsiveness to therapy, the subject can be administered one or more doses of the therapy and continue to monitor the protein biomarker.

If the protein biomarker level in the nth sample is above the baseline (e.g., increase from 1 to about 99%, increase from 1 to about 95%, increase from 1 to about 90%, increase from 1 to about 85%, increase from 1 to about 80%, increase from 1 to about 75%, increase from 1 to about 70%,

- 123 041220 increase from 1 to about 65%, increase from 1 to about 60%, increase from 1 to about 55%, increase from 1 to about 50%, increase from 1 to about 45%, increase from 1 to about 40% , increase from 1 to about 35%, increase from 1 to about 30%, increase from 1 to about 25%, increase from 1 to about 20%, increase from 1 to about 15%, increase from 1 to about 10%, increase from 1 to about 5%, increase from about 5 to about 99%, increase from about 10 to about 99%, increase from about 15 to about 99%, an increase from about 20 to about 99%, an increase from about 25 to about 99%, an increase from about 30 to about 99%, an increase from about 35 to about 99%, increase from about 40 to about 99%, increase from about 45 to about 99%, increase from about 50 to about 99%, increase from about 55 to about 99%, increase from about 60 to about 99%, increase from about 65 to about 99%, increase from about 70 to about 99%, increase from about 75 to about 95%, increase from about 80 to about 99%, increase from about 90 to about 99%, increase from about 95 to about 99%, increase from about 5 to about 10%, increase from about 5 to about 25%, increase from about 10 to about 30%, increase from about 20 to about 40%, increase from about 25 to about 50%, increase from about 35 to about 55%, increase from about 40 to about 60%, increase from about 50 to about 75%, an increase from about 60 to about 80%, or an increase from about 65 to about 85%, etc.), then this may indicate resistance to therapy. If the level of a protein biomarker in sample n is higher than in sample n-1 (e.g., increase from 1 to about 99%, increase from 1 to about 95%, increase from 1 to about 90%, increase from 1 to about 85%, increase from 1 to about 80%, increase from 1 to about 75%, increase from 1 to about 70%, increase from 1 to about 65%, increase from 1 to about 60%, increase from 1 to about 55%, increase from 1 up to about 50%, increase from 1 to about 45%, increase from 1 to about 40%, increase from 1 to about 35%, increase from 1 to about 30%, increase from 1 to about 25%, increase from 1 to about 20%, increase from 1 to about 15%, increase from 1 to about 10%, increase from 1 to about 5%, increase from about 5 to about 99%, increase from about 10 to about 99%, increase from about 15 to about 99%, increase from about 20 to about 99%, increase from about 25 to about 99%, increase from about 30 to about 99 %, increase from about 35 to about 99%, increase from about 40 to about 99%, increase from about 45 to about 99%, increase from about 50 to about 99%, increase from about 55 to about 99%, increase from about 60 to about 99%, increase from about 65 to about 99%, increase from about 70 to about 99%, increase from about 75 to about 95% , increase from about 80 to about 99%, increase from about 90 to about 99%, increase from about 95 to about 99%, increase from about 5 to about 10%, increase from about 5 to about 25%, increase from about 10 to about 30%, increase from about 20 to about 40%, increase from about 25 to about 50%, increase from about 35 to about 55%, increase from about 40 to about 60% , an increase from about 50 to about 75%, an increase from about 60 to about 80%, or an increase from about 65 to about 85%, etc.), then this may indicate resistance to therapy. If resistance to therapy is suspected in a subject, it is possible refer for one or more imaging tests, biopsy, surgery, or other diagnostic tests. In some embodiments, when resistance to therapy is suspected, a subject may be administered (as monotherapy or in combination with prior therapy) a compound capable of treating resistance to a RET inhibitor (e.g., a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as proposed in this document).

In some embodiments, one or more protein biomarkers are monitored. The specific protein biomarkers to be monitored may depend on the type of cancer and can be readily determined by one of skill in the art. Non-limiting examples of protein biomarkers include: CA 125, carcinoembryonic antigen (CEA), calcitonin, adrenocorticotropic hormone (ACTH), cortisol, CA 19-9, prolactin, hepatocyte growth factor, osteopontin, myeloperoxidase, tissue inhibitor of metalloproteinase 1, angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase-3-like protein-1 (YKL-40), galectin-3 (GAL-3), CYFRA 21-1 (cytokeratins), EPCAM (epithelial cell adhesion molecule), ProGRP (progastrin-releasing peptide), and CEACAM (carcinoembryonic antigen). See, for example, Cohen JD, Li L, Wang Y, et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science; published online Jan 18, 2018. pii: eaar3247. DOI: 10.1126/science.aar3247; Fawaz M Makki et al. Serum biomarkers of papillary thyroid cancer. J Otolaryngol Head Neck Surg. 2013; 42(1): 16; and Tatiana N. Zamay et al. Current and Prospective Protein Biomarkers of Lung Cancer. Cancers (Basel). 2017 Nov; 9(11): 155. In some embodiments, the biomarkers include one or more of CEA, calcitonin, thyroglobulin, ACTH, and cortisol. In some embodiments, the cancer is medullary thyroid cancer and the protein biomarkers include CEA and calcitonin. In some embodiments, the cancer is non-medullary thyroid cancer and the protein biomarker includes thyroglobulin. In some embodiments, the biomarkers are ACTH and cortisol (e.g., if the patient suffers from Cushing's disease due to their cancer).

Also provided herein are methods for treating a RET-associated cancer in a subject, which comprise (a) administering one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a first RET kinase inhibitor to a subject who has

- 124 041220 identified or diagnosed with a RET-associated cancer (e.g., any type of RET-associated cancer described herein) (e.g., identified or diagnosed with a RET-associated cancer using any illustrative method described herein or known in the art); (b) after step (a), determining the level of circulating tumor DNA in a biological sample (e.g., in a biological sample containing blood, serum, or plasma) obtained from the subject; (c) administering a therapeutically effective amount of a second RET inhibitor or a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anti-cancer agent to a subject that has been identified as having about the same or an increased level of circulating tumor DNA compared to a reference level of circulating tumor DNA (e.g., any reference level of circulating tumor DNA described herein). In some examples of these methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample obtained from the subject prior to step (a). Some embodiments of the disclosed methods further comprise determining the level of circulating tumor DNA in a biological sample obtained from the subject prior to step (a). In some examples of the disclosed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects who have the same RET-associated but are receiving an ineffective treatment or placebo, or are not yet receiving a therapeutic treatment, or the level of circulating tumor DNA in the body of a subject who has the same RET-associated cancer but is receiving an ineffective treatment or placebo, or is not yet receiving a therapeutic treatment. In some examples of the disclosed methods, the first RET inhibitor is selected from the group: cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667 and BLU6864.

Also provided herein are methods for treating a RET-associated cancer in a subject that comprise administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, to a subject (i) that has been identified or diagnosed with a RET-associated cancer (e.g., any type of RET-associated cancer described herein) (e.g., that has been identified or diagnosed with a RET-associated cancer using any of the illustrative methods described herein or known in the art), (ii) that has previously been administered one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a second RET kinase inhibitor, and (ii) that, following administration of the one or more doses of the second RET kinase inhibitor, has been found to have about the same or an increased level of circulating tumor DNA, compared to a reference level of circulating tumor DNA (e.g., any reference level of circulating tumor DNA, tumor DNA described herein or known in the art). In some embodiments of the disclosed methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (e.g., a biological sample comprising blood, plasma, or serum) obtained from the subject prior to administration of the one or more doses of the second RET kinase inhibitor. Some embodiments of the disclosed methods further comprise determining the level of circulating tumor DNA in a biological sample obtained from the subject prior to administration of the one or more doses of the second RET kinase inhibitor. In some examples of the disclosed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects who have the same RET-associated but are receiving an ineffective treatment or placebo, or are not yet receiving a therapeutic treatment, or the level of circulating tumor DNA in the body of a subject who has the same RET-associated cancer but is receiving an ineffective treatment or placebo, or is not yet receiving a therapeutic treatment. In some embodiments of such methods, the second RET kinase inhibitor is selected from the group consisting of: cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667, and BLU6864.

Also provided herein are methods of treating a RET-associated cancer in a subject comprising: (a) administering one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy to a subject identified or diagnosed as having a RET-associated cancer (e.g., any type of RET-associated cancer described herein) (e.g., a subject identified or diagnosed as having a RET-associated cancer using any method described herein or known in the art); (b) after step (a), determining the level of circulating tumor DNA in a biological sample (e.g., a biological sample comprising blood, serum, or plasma) obtained from the subject; (c) administering a therapeutically eff

- 125 041220 an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and an additional therapeutic agent or treatment (e.g., any additional therapeutic agents or treatments for RET-associated cancer described herein or known in the art) to a subject who has been identified as having about the same or an increased level of circulating tumor DNA, compared to a reference level of circulating tumor DNA (e.g., any illustrative reference level of circulating tumor DNA described herein or known in the art). In some embodiments of the disclosed methods, the additional therapeutic agent is a second RET kinase inhibitor (e.g., a RET kinase inhibitor selected from the group: cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667, and BLU6864). In some examples of any of the provided methods, the additional therapeutic agent or treatment comprises one or more of: radiation therapy, a chemotherapeutic agent (e.g., any illustrative chemotherapeutic agent described herein or known in the art), a checkpoint inhibitor (e.g., any illustrative checkpoint inhibitor described herein or known in the art), surgery (e.g., at least partial resection of a tumor), and one or more other kinase inhibitors (e.g., any illustrative kinase inhibitor described herein or known in the art). In some examples of these methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (e.g., a biological sample comprising blood, serum, or plasma) obtained from the subject prior to step (a). In some examples of the disclosed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects who have the same RET-associated cancer but are receiving an ineffective treatment or placebo, or are not yet receiving therapeutic treatment, or the level of circulating tumor DNA in the body of a subject who has the same RET-associated cancer but is receiving an ineffective treatment or placebo, or is not yet receiving therapeutic treatment.

Also provided herein are methods of treating a RET-associated cancer in a subject comprising: administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and an additional therapeutic agent or treatment to a subject (i) who has been identified or diagnosed as having a RET-associated cancer (e.g., any type of RET-associated cancer described herein) (e.g., a subject who has been identified or diagnosed as having a RET-associated cancer using any of the methods described herein or known in the art), (ii) who has previously been administered one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy, and (ii) who, after administration of one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy, is identified as having the same or an elevated level of circulating tumor DNA, as compared to a reference level of circulating tumor DNA (e.g., any illustrative reference level of circulating tumor DNA described herein). In some embodiments of such methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample obtained from the subject prior to administration of one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy. Some embodiments of the disclosed methods further comprise determining the level of circulating tumor DNA in a biological sample obtained from the subject prior to administration of one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy. In some examples of the disclosed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects who have the same RET-associated cancer but are receiving an ineffective treatment or placebo, or are not yet receiving a therapeutic treatment, or the level of circulating tumor DNA in the body of a subject who has the same RET-associated cancer but is receiving an ineffective treatment or placebo, or is not yet receiving a therapeutic treatment. In some embodiments of the disclosed method, the additional therapeutic agent is a second RET kinase inhibitor (e.g., a second RET kinase inhibitor selected from the group consisting of cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667, and BLU6864). In some embodiments of such methods, the additional therapeutic agent

- 126 041220 or the treatment comprises one or more of radiation therapy, a chemotherapeutic agent (e.g., any illustrative chemotherapeutic agent described herein or known in the art), a checkpoint inhibitor (e.g., any illustrative checkpoint inhibitor described herein or known in the art), surgery (e.g., at least partial resection of a tumor), and one or more other kinase inhibitors (e.g., any kinase inhibitor described herein or known in the art).

Also provided herein are methods for selecting a treatment for a subject, comprising: selecting a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, for a subject (i) who has been identified or diagnosed with a RET-associated cancer (e.g., any RET-associated cancer described herein) (e.g., a subject who has been identified or diagnosed with a RET-associated cancer using any of the methods described herein or known in the art), (ii) who has previously been administered one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a second RET kinase inhibitor (e.g., any of the RET kinase inhibitors described herein or known in the art), and (ii) who, following administration of the one or more doses of the second RET kinase inhibitor, is identified with about the same or an increased level of circulating tumor DNA, compared to a reference level. circulating tumor DNA. In some embodiments of any of the disclosed methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (e.g., a biological sample containing blood, serum, or plasma) obtained from the subject prior to administration of the one or more doses of the second RET kinase inhibitor. Some embodiments of the disclosed methods further comprise determining the level of circulating tumor DNA in a biological sample obtained from the subject prior to administration of the one or more doses of the second RET kinase inhibitor. In some examples of the disclosed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects who have the same RET-associated cancer but are receiving an ineffective treatment or placebo, or are not yet receiving a therapeutic treatment, or the level of circulating tumor DNA in the body of a subject who has the same RET-associated cancer but is receiving an ineffective treatment or placebo, or is not yet receiving a therapeutic treatment. In some embodiments of such methods, the second RET kinase inhibitor is selected from the group of cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667 and BLU6864.

Also provided herein are methods of selecting a treatment for a subject, which comprise selecting a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and an additional therapeutic agent or treatment for a subject (i) who has been identified or diagnosed as having a RET-associated cancer (e.g., any RET-associated cancer described herein or known in the art) (e.g., a subject who has been diagnosed or identified as having a RET-associated cancer using any of the methods described herein or known in the art), (ii) who has previously been administered one or more doses (e.g., two or more, three or more, four or more, five or more, or ten or more) of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy, and (ii) who, following administration of one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is identified as having the same or an increased level of circulating tumor DNA, compared to a reference level of circulating tumor DNA. In some embodiments of such methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (e.g., a biological sample comprising blood, serum, or plasma) obtained from the subject prior to administration of one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy. Some embodiments further comprise determining the level of circulating tumor DNA in a biological sample obtained from the subject prior to administration of one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy. In some examples of the disclosed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects who have the same RET-associated cancer but are receiving an ineffective treatment or placebo, or who are not yet receiving therapeutic treatment, or the level of circulating tumor DNA in the body of a subject who has the same RET-associated cancer but is receiving an ineffective treatment or placebo,

- 127 041220 or not yet receiving therapeutic treatment. In some embodiments of any of the methods provided, the additional therapeutic agent is a second RET kinase inhibitor (e.g., a second RET kinase inhibitor selected from the group: cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667, and BLU6864). In some embodiments of any of the methods described herein, the additional therapeutic agent or treatment comprises one or more of radiation therapy, a chemotherapeutic agent (e.g., any of the examples of chemotherapeutic agents described herein or known in the art), a checkpoint inhibitor (e.g., any checkpoint inhibitor described herein or known in the art), surgery (e.g., at least partial resection of the tumor), and one or more other kinase inhibitors (e.g., any of the other kinase inhibitors described herein or known in the art).

Also provided herein are methods for determining the effectiveness of a treatment in a subject, comprising: (a) determining a first level of circulating tumor DNA in a biological sample (e.g., a biological sample comprising blood, serum, or plasma) obtained from a subject identified or diagnosed as having a RET-associated cancer at a first time point; (b) administering to the subject a treatment comprising one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, after the first time point and before a second time point; (c) determining a second level of circulating tumor DNA in a biological sample (e.g., a biological sample comprising blood, serum, or plasma) obtained from the subject at the second time point; and (d) determining that the treatment is effective in a subject determined to have a reduced second level of circulating tumor DNA, compared to the first level of circulating tumor DNA; or determining that the treatment is ineffective in a subject who is determined to have about the same or an increased second level of circulating tumor DNA, compared to the first level of circulating tumor DNA. In some embodiments of such methods, the first time point and the second time point are between about 1 week and about 1 year (e.g., between about 1 week and about 10 months, between about 1 week and about 8 months, between about 1 week and about 6 months, between about 1 week and about 4 months, between about 1 week and about 3 months, between about 1 week and about 2 months, between about 1 week and about 1 month, or between about 1 week and about 2 weeks).

Also provided herein are methods of determining whether a subject has developed resistance to a treatment, comprising: (a) determining a first level of circulating tumor DNA in a biological sample (e.g., a biological sample comprising blood, serum, or plasma) obtained from a subject identified or diagnosed as having a RET-associated cancer, at a first time point; (b) administering to the subject a treatment comprising one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, after the first time point and before a second time point; (c) determining a second level of circulating tumor DNA in a biological sample obtained from the subject at the second time point; and (d) determining that the subject has not developed resistance to the treatment if a second level of circulating tumor DNA is decreased compared to a first level of circulating tumor DNA; or determining that the subject has developed resistance to the treatment if the subject has about the same or an increased second level of circulating tumor DNA compared to the first level of circulating tumor DNA. In some embodiments of such methods, the first time point and the second time point are between about 1 week and about 1 year (e.g., between about 1 week and about 10 months, between about 1 week and about 8 months, between about 1 week and about 6 months, between about 1 week and about 4 months, between about 1 week and about 3 months, between about 1 week and about 2 months, between about 1 week and about 1 month, or between about 1 week and about 2 weeks).

Illustrative methods for detecting circulating tumor DNA are described in Moati et al., Clin. Res. Hepatol. Gastroenterol. April 4, 2018; Oussalah et al., EBioMedicine March 28, 2018; Moon et al., Adv. Drug Deliv. Rev. April 4, 2018; Solassaol et al., Clin. Chem. Lab. Med. April 7, 2018; Arriola et al., Clin. Transl. Oncol. April 5, 2018; Song et al., J. Circ. Biomark. March 25, 2018; Aslibekyan et al., JAMA Cardiol. April 4, 2018; Isbell et al., J. Thorac. Cardiovasc. Surg. March 13, 2018; Boeckx et al., Clin. Colorectal Cancer February 22, 2018; Anunobi et al., J. Surg. Res. March 28, 2018; Tan et al., Medicine 97(13):eO197, 2018; Reithdorf et al., Transl. Androl. Urol. 6(6):1090–1110, 2017; Volckmar et al., Genes Chromosomes Cancer 57(3): 123–139, 2018; and Lu et al., Chronic Dis. Transl. Med. 2(4):223–230, 2016. Additional methods for determining circulating tumor DNA levels are known in the art.

In some embodiments, provided herein are methods of treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a

- 128 041220 regulating the RET gene, RET kinase, or the expression or activity or level of any of them in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a multikinase inhibitor, wherein the multikinase inhibitor is selected from vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the multikinase inhibitor from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a first multikinase inhibitor, wherein said multikinase inhibitor is selected from the group consisting of: vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of formula IIV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the multikinase inhibitor of step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1 and/or one or more point mutations/insertions/deletions in a RET kinase protein from Tables 2 and 2a in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a multikinase inhibitor, wherein the multikinase inhibitor is selected from the group consisting of: vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation from Table 3 or 4; and (d) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the multikinase inhibitor of step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a multikinase inhibitor, wherein the multikinase inhibitor is selected from the group consisting of vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has a RET inhibitor resistance mutation V804M, G810S, or G810R; and (d) administering to said subject a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof selected from the group consisting of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the multikinase inhibitor of step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

As another example, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) establishing

- 129 041220 determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a multikinase inhibitor (e.g., vandetanib or cabozantinib) as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a multi-kinase inhibitor (e.g., vandetanib or cabozantinib) as monotherapy or in combination with another anti-cancer agent if said subject has a cancer cell that has at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, of step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment comprising (a) detecting one or more fusion proteins from Table 1 and/or one or more point mutations/insertions/deletions in a RET kinase protein from Tables 2 and 2a in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation from Table 3 or 4; and (d) administering to said subject a multikinase inhibitor (e.g., vandetanib or cabozantinib) as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has a RET inhibitor resistance mutation V804M, G810S, or G810R; and (d) administering to said subject a multikinase inhibitor (e.g., vandetanib or cabozantinib) as monotherapy or in combination with another anti-cancer agent if said subject has a cancer cell that has at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (b) if said subject has a cancer cell that does not have a RET inhibitor resistance mutation.

Also provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, of step (b), as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, or immunotherapy) or with an anti-cancer therapy (e.g., surgery or radiation) if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting

- 130 041220 deregulation of the RET gene, RET kinase, or the expression or activity or level of any of them in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the disclosed methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, of step (b), as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, or immunotherapy) or with an anti-cancer therapy (e.g., surgery or radiation) if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins of Table 1 and/or one or more point mutations/insertions/deletions in a RET kinase protein of Tables 2 and 2a in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, selected from the group consisting of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation from Table 3 or 4; and (d) administering to the subject additional doses of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy) or with an anti-cancer therapy (e.g., surgery or radiation), if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1 carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administered in step (d). In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has the RET inhibitor resistance mutation V804M, G810S, or G810R; and (d) administering to the subject additional doses of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy) or with an anti-cancer therapy (e.g., surgery or radiation), if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administered in step (d).

Also provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) detecting at least one RET inhibitor resistance mutation in a cancer cell in the sample obtained from the subject; and (d) administering to the subject additional doses of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, from step (b), as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy) or with an anti-cancer therapy (e.g., surgery or radiation). In some

- 131 041 220 embodiments provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment comprising (a) detecting deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) detecting at least one RET inhibitor resistance mutation in a cancer cell in the sample obtained from the subject; and (d) administering to the subject additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, of step (b), as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of Formula IIV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, or immunotherapy) or with an anti-cancer therapy (e.g., surgery or radiation). In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins of Table 1 and/or one or more point mutations/insertions/deletions in a RET kinase protein of Tables 2 and 2a in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, selected from the group consisting of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the methods provided further comprise (after (b)) (c) detecting at least one RET inhibitor resistance mutation from Table 3 or 4 in a cancer cell in a sample obtained from the subject; and (d) administering to the subject additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof from step (b), as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, or immunotherapy) or with an anti-cancer therapy (e.g., surgery or radiation). In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((3)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administered in step (d). In some embodiments, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) detecting a V804M, G810S, or G810R RET inhibitor resistance mutation in a cancer cell in the sample obtained from the subject; and (d) administering to the subject additional doses of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, from step (b) as monotherapy or in combination with another anticancer agent (e.g., a second RET inhibitor, a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy) or with anticancer therapy (e.g., surgery or radiation). In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administered in step (d).

This document further provides a method of treating lung cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, crizotinib, osimertinib, or any combination thereof.

In some embodiments, the lung cancer is a RET-associated cancer. For example, a method provided may comprise: (a) detecting deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from a subject; and (b) administering to said subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the methods provided further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation (e.g., deregulation of MET, such as amplification of the MET gene); and (d) administering to said subject a second therapeutic agent, wherein the second therapeutic agent is crizotinib, as monotherapy or in combination with a compound of formula I-IV or its

- 132 041220 a pharmaceutically acceptable salt, amorphous or polymorphic form, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some such embodiments, the proposed method comprises (a) detecting one or more fusion proteins from Table 1 and/or one or more point mutations/insertions in the RET kinase protein from Table 2 in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In additional embodiments, the methods provided further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation (e.g., MET deregulation, such as MET gene amplification); and (d) administering to said subject a second therapeutic agent, wherein the second therapeutic agent is crizotinib, as monotherapy or in combination with a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (b), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

In some embodiments, the lung cancer is an EGFR-associated cancer. For example, a method provided may comprise: (a) detecting a deregulation of an EGFR gene, an EGFR kinase, or the expression or activity or level of any of them in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of an EGFR inhibitor (e.g., osimertinib). In some embodiments, the methods provided further comprise (after (b)) (c) determining whether a cancer cell in the sample obtained from the subject has at least one deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them (e.g., a RET fusion protein); and (d) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with an EGFR inhibitor (e.g., osimertinib), if the subject has a cancer cell that has at least one deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them (e.g., a RET fusion protein); or (e) administering to said subject additional doses of the EGFR inhibitor (e.g., osimertinib) of step (b), if the subject has a cancer cell that does not have a deregulation of the RET gene, RET kinase, or expression or activity, or level of any of them (e.g., a RET fusion protein). In some such embodiments, the provided method comprises (a) detecting a deregulation of the EGFR gene, EGFR kinase, or expression or activity, or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of osimertinib. In additional embodiments, the disclosed method further comprises (after (b)) (c) determining whether a cancer cell in a sample obtained from the subject has one or more fusion proteins from Table 1 and/or one or more point mutations/insertions in the RET kinase protein from Table 2; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with osimertinib, if the subject has a cancer cell that contains one or more fusion proteins from Table 1 and/or one or more point mutations/insertions in the RET kinase protein from Table 2; or (e) administering to said subject additional doses of osimertinib from step (b) if the subject has a cancer cell that does not contain one or more fusion proteins from Table 1 and/or one or more point mutations/insertions in the RET kinase protein from Table 2.

The term EGFR-associated cancer as used herein refers to cancers associated with or characterized by deregulation of the EGFR gene, EGFR kinase, or the expression or activity or level of any of them.

The expression deregulation of the EGFR gene, EGFR kinase or expression or activity or level of any of them refers to a genetic mutation (e.g., a translocation of the EGFR gene that results in the expression of a fusion protein, a deletion in the EGFR gene that results in the expression of an EGFR protein that contains a deletion of at least one amino acid compared to the wild-type EGFR protein, or a mutation in the EGFR gene that results in the expression of an EGFR protein with one or more point mutations, or an alternative splice version of the EGFR mRNA forming the EGFR protein that results in a deletion of at least one amino acid in the EGFR protein compared to the wild-type EGFR protein) or to an amplification of the EGFR gene that results in overexpression of the EGFR protein or autocrine activity due to overexpression of the EGFR gene in a cell that results in a pathogenic increase in the activity of the kinase domain of the EGFR protein (e.g., a constitutively active kinase domain of the EGFR protein) in a cell. As another example, deregulation of the EGFR gene, EGFR protein, or the expression or activity or level of any of them can be a mutation in the EGFR gene that encodes an EGFR protein that is constitutively active or has increased activity

- 133 041220 deregulation compared to a protein encoded by an EGFR gene that does not contain such a mutation. For example, deregulation of the EGFR gene, EGFR protein, or expression or activity, or the level of any of them, can be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of EGFR that contains a functional kinase domain and a second portion of a partner protein (i.e., not EGFR). In some examples, deregulation of the EGFR gene, EGFR protein, or expression or activity can be the result of a gene translocation of one EGFR gene with another non-EGFR gene. In some embodiments, the EGFR mutation is a T790M mutation. In some embodiments, the EGFR mutation is a C797S mutation.

The term wild-type EGFR describes a nucleic acid (e.g., an EGFR gene or EGFR mRNA) or a protein (e.g., an EGFR protein) that occurs in a subject that does not have an EGFR-associated cancer (and optionally also does not have an increased risk of developing an EGFR-associated cancer and/or is not predisposed to an EGFR-associated cancer), or that occurs in a cell or tissue obtained from a subject that does not have an EGFR-associated cancer (and optionally also does not have an increased risk of developing an EGFR-associated cancer and/or is not predisposed to an EGFR-associated cancer).

Also provided herein are methods of selecting a treatment for a subject suffering from a cancer, comprising: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and selecting a treatment, which comprises administering a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with a first RET inhibitor. In some embodiments, the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is administered in combination with a first RET inhibitor. Also provided herein are methods of selecting a treatment for a subject suffering from a cancer, comprising: selecting a treatment, which comprises administering a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, to a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations. Also provided are methods of selecting a subject suffering from a cancer for a treatment that does not include a first RET inhibitor as a monotherapy, comprising: identifying a subject having a cancer cell that comprises one or more RET inhibitor resistance mutations; and selecting the identified subject for a treatment that includes a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. Also provided are methods of selecting a subject suffering from a cancer for a treatment that does not include a first RET inhibitor as a monotherapy, comprising: selecting a subject identified as having a cancer cell that comprises one or more RET inhibitor resistance mutations for a treatment that includes administering a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more RET inhibitor resistance mutations listed in Table 3 and 4. In some embodiments, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods for determining the likelihood that a subject suffering from a cancer (e.g., a RET-associated cancer) will exhibit a positive response to treatment with a first RET inhibitor as a monotherapy, comprising: determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and determining that a subject having a cancer cell that has one or more RET inhibitor resistance mutations has a reduced likelihood of a positive response (i.e., an increased likelihood of a negative response) to treatment with the first RET inhibitor as a monotherapy. Also provided are methods for determining the likelihood that a subject suffering from a cancer (e.g., a RET-associated cancer) will exhibit a positive response to treatment with a first RET inhibitor as a monotherapy, comprising: determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and determining that a subject that does not have a cancer cell that contains one or more RET inhibitor resistance mutations has an increased likelihood of responding favorably to treatment with the first RET inhibitor as a monotherapy, compared to a subject that has a cancer cell that has one or more RET inhibitor resistance mutations. Also provided are methods of predicting the effectiveness of treatment with a first RET inhibitor as a monotherapy in a subject suffering from cancer, the methods comprising: determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and determining that treatment with the first RET inhibitor as a monotherapy is less likely to be effective in a subject that has a cancer cell in a sample obtained from the subject that contains one or more RET inhibitor resistance mutations. Also provided are methods of predicting the effec

- 134 041220 effectiveness of treatment with a first RET inhibitor as a monotherapy in a subject suffering from cancer, which comprises: determining that treatment with the first RET inhibitor as a monotherapy is less likely to be effective in a subject having a cancer cell in a sample obtained from the subject that contains one or more RET inhibitor resistance mutations. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more RET inhibitor resistance mutations listed in Table 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods of treating a subject suffering from cancer, which comprise: (a) administering to the subject one or more doses of a first RET inhibitor over a certain period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) administering to the subject a compound of formula IIV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (d) administering to the subject additional doses of the first RET inhibitor from step (a), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of the first RET inhibitor of step (a), the subject may also be administered another anti-cancer agent (e.g., a second RET inhibitor or a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or an immunotherapy). In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (c), the other RET inhibitor may be the first RET inhibitor administered in step (a). In some embodiments, the one or more RET inhibitor resistance mutations confers increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods of treating a subject suffering from cancer, which comprise: (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) administering to the subject a second RET inhibitor as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (d) administering to the subject additional doses of the first RET inhibitor of step (a), if said subject has a cancer cell that does not have a RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of the first RET inhibitor of step (a), then the subject may also be administered another anti-cancer agent. In some embodiments, the one or more RET inhibitor resistance mutations confers increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more of the RET inhibitor resistance mutations listed in Tables 3 and 4. For example, the one or more RET inhibitor resistance mutations can comprise a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof). In some embodiments, the additional anti-cancer agent is an immunotherapy.

Also provided are methods for treating a subject suffering from a cancer (e.g., a RET-associated cancer), comprising: (a) determining whether a cancer cell in a sample obtained from a subject suffering from a cancer and previously receiving one or more doses of a first RET inhibitor has one or more RET inhibitor resistance mutations; and (b) administering to the subject a compound

- 135 041220 of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (c) administering to the subject additional doses of the previously administered first RET inhibitor, if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of the first RET inhibitor previously administered to the subject, the subject may also be administered another anti-cancer agent (e.g., a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy). In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more of the RET inhibitor resistance mutations listed in Tables 3 and 4. For example, the one or more RET inhibitor resistance mutations can comprise a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (b), the other anti-cancer agent can be the first RET inhibitor administered in step (a).

Also provided are methods of treating a subject suffering from a cancer, which comprise: (a) determining whether a cancer cell in a sample obtained from a subject suffering from a cancer and previously receiving one or more doses of a first RET inhibitor has one or more RET inhibitor resistance mutations; and (b) administering to the subject a second RET inhibitor as a monotherapy or in combination with another anti-cancer agent if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (c) administering to the subject additional doses of a first RET inhibitor previously administered to the subject if the subject has a cancer cell that does not have a RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of a first RET inhibitor previously administered to the subject, the subject may also be administered another anti-cancer agent. In some embodiments, the one or more RET inhibitor resistance mutations confers increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more of the RET inhibitor resistance mutations listed in Tables 3 and 4. For example, the one or more RET inhibitor resistance mutations can comprise a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (b), the other anti-cancer agent may be the first RET inhibitor administered in step (a).

In some embodiments, the RET-associated cancer described herein may occur in a subject in conjunction with deregulation of another gene, another protein, or expression or activity, or level of any of them.

For example, a RET-associated cancer that exhibits a RET fusion may occur in a subject in conjunction with one or more of: deregulation of the MET gene, MET protein, or expression or activity, or level of any of them; deregulation of the PIK3CA gene, PIK3CA protein, or expression or activity, or level of any of them; deregulation of the KRAS gene, KRAS protein, or expression or activity, or level of any of them; deregulation of the EGFR gene, EGFR protein, or expression or activity, or level of any of them (e.g., amplification of the EGFR gene); deregulation of the FGFR2 gene, FGFR2 protein, or expression or activity, or level of any of them (e.g., a fusion of the FGFR2 gene or FGFR2 protein); deregulation of the CDK4 gene, CDK4 protein, or expression or activity, or level of any of them (e.g., amplification of the CDK4 gene); deregulation of the mTOR gene, the mTOR protein, or the expression or activity, or the level of any of them; deregulation of the CDKN2A gene, the CDKN2A protein, or the expression or activity, or the level of any of them (e.g., deletions in the CDKN2A gene or in the CDKN2A protein); deregulation of the CDKN2B gene, the CDKN2B protein, or the expression or activity, or the level of any of them (e.g., deletions in the CDKN2B gene or in the CDKN2B protein); deregulation of the NF1 gene, the NF1 protein, or the expression or activity, or the level of any of them; deregulation of the MYC gene, the MYC protein, or the expression or activity, or the level of any of them (e.g., amplifications in the MYC gene); de- 136 041220 regulation of the MDM2 gene, MDM2 protein, or expression or activity, or level of any of them (e.g., amplification in the MDM2 gene); deregulation of the GNAS gene, GNAS protein, or expression or activity, or level of any of them; deregulation of the BRCA2 gene, BRCA2 protein, or expression or activity, or level of any of them.

In some embodiments, a RET-associated cancer that exhibits a mutation in the RET gene and/or RET protein may occur in a subject in conjunction with one or more of: deregulation of the PIK3CA gene, PIK3CA protein, or expression or activity, or level of any of them; deregulation of the KRAS gene, KRAS protein, or expression or activity, or level of any of them; deregulation of the EGFR gene, EGFR protein, or expression or activity, or level of any of them; deregulation of the FGFR1 gene, the FGFR1 protein, or the expression or activity, or level of any of them (e.g., amplification of the FGFR1 gene); deregulation of the FGFR2 gene, the FGFR2 protein, or the expression or activity, or level of any of them (e.g., amplification of the FGFR2 gene); deregulation of the FGFR3 gene, the FGFR3 protein, or the expression or activity, or level of any of them (e.g., a fusion of the FGFR3 gene or the FGFR3 protein); deregulation of the ERBB2 gene, the ERBB2 protein, or the expression or activity, or level of any of them (e.g., amplification of the ERBB2 gene); and deregulation of the KIT gene, the KIT protein, or the expression or activity, or level of any of them.

In some embodiments, a RET-associated cancer that exhibits RET gene amplification may occur in a patient along with one or more additional kinase amplifications. For example, with amplification in the FGFR1 gene; with amplification in the FGFR2 gene; with amplification in the FGFR3 gene; with amplification in the FGFR4 gene; with amplification in the CDK4 gene; and with amplification in the CDK6 gene.

In some embodiments, if a RET-associated cancer described herein may occur in a subject in conjunction with a deregulation of another kinase, the methods described herein may further comprise administering an additional therapeutic agent that targets and/or treats the deregulation of the other kinase. For example, provided herein are methods of treating a RET-associated cancer in a subject in need of such treatment comprising (a) detecting a deregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the provided method further comprises (c) detecting a deregulation of the other kinase in a sample obtained from the subject; and (d) administering to the subject a therapeutic agent that targets and/or treats a deregulation in another kinase. In some embodiments, the administration of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, is performed simultaneously, sequentially, or serially. In some embodiments, the detection steps (a) and (c) may be performed simultaneously or sequentially in any order.

Additional therapeutic agents that target and/or treat dysregulation of another kinase may include any known inhibitor of the other kinase. Examples of such agents are listed below:

Illustrative PARP inhibitors include: 3-aminobenzamide (INO-1001), 5-aminoisoquinoline, ABT472, ABT767, AG140361, AG14032, ANG2864, ANG3186, AZD2281, AZD2461, BGP-15, BSI101, BSI401, CEP6800, CEP8983, CK102, CEP9722 (prodrug of CEP8983), CPH101 with CPH102, DR2313, E7016 (GPI-21016), E7449, GP16150, IMP4297, IMP04149, INO1002, INO1003, JPI283, JPI289, KU0687, KU58948, niraparib (MK-4827), NT125, olaparib (AZD2281), ONO-1924H, ONO2231, pamiparib (BGB290), PJ-34, rucaparib (AG014699), SC10914, SOMCL9112, talazoparib (BMN-673) and veliparib (ABT888).

Illustrative CDK 4/6 inhibitors include: palbociclib (PD0332991), abemaciclib (LY2835219), ribociclib (LEE011), trilaciclib (G1T28), voruciclib, and G1T38.

Illustrative ERBB2 (HER2/neu) inhibitors include: afatinib, afatinib, dacomitinib (PF-00299804), DS8201-a, erlotinib, gefitinib, KU004, lapatinib, lapatinib ditosylate, MM-111, mubritinib (TAK-165), neratinib, pyrotinib (HTI-1001), tucatinib (ONT-380, ARRY-380), 7C3, cetuximab, HER2-BsAb, hersintuzumab, margetuximab, MI130004, NeuVax, paytumumab, pertuzumab, SYD985, trastuzumab, and trastuzumab emtansine.

Illustrative amplified ERBB2 (HER2/neu) inhibitors include dacomitinib (PF-00299804), lapatinib, neratinib, pertuzumab, trastuzumab, and trastuzumab emtansine.

Exemplary EGFR inhibitors include: AC0010, afatinib, AP26113, ASP8273, avatinib, avitinib, AZD3759, BMS-690514, brigatinib, canertinib, Cap-701, CHMFL-EGFR-202, CUDC-101, dacomitinib, EAI045, EGF816, erlotinib, erlotinib, gefitinib, GNS-1481, GNS-1486, G66976, HS10296, icotinib, KU004, lapatinib, nazartinib, neratinib, olmutinib (HM61713, BI 1482694), osimertinib, osimertinib (AZD9291), pelitinib, PF-06747775, PKC412, pyrotinib (HTI-1001), rocilentinib, vandetanib, varlitinib, XL647, 7C3, cetuximab, depatuxizumab mafodotin (ABT-414), matuzumab, nimotuzumab, panitumumab, and zalutumumab.

- 137 041220

Illustrative wild-type EGFR inhibitors include: afatinib, BMS-690514, canertinib,

CUDC-101, dacomitinib, erlotinib, gefitinib, lapatinib, neratinib, pelitinib, vandetanib, varlitinib, XL647, cetuximab, matuzumab, nimotuzumab, panitumumab and zalutumumab.

Illustrative mutated EGFR inhibitors include: AC0010, afatinib, AP26113, ASP8273, avatinib, avitinib, AZD3759, BMS-690514, brigatinib, canertinib, Cap-701, CHMFL-EGFR202, CUDC-101, dacomitinib, EAI045, EGF816, GNS-1481, GNS-1486, G66976, HS-10296, icotinib, nazartinib, neratinib, olmutinib (HM61713, BI 1482694), osimertinib (AZD9291), PF-06747775, PKC412, rocilentinib, vandetanib, varlitinib and cetuximab.

An illustrative inhibitor of amplified EGFR is depatuxizumab mafodotin (ABT-414).

Illustrative FGFR inhibitors include: ASP5878, AZD4547, BGJ398, BLU9931, brivatinib, cediranib, DEBIO 1347, derazantinib (ARQ-087), dovitinib (CHIR258), E7090, ENMD-2076, erdafitinib (JNJ-42756293), FGF 401, FIIN-1, FRIN-1, INCB054828, L16H50, lenvatinib, lucitanib, LY2874455, nintedanib, NP603, orantinib (SU6668), pazopanib, PBI05204, PD173074, ponatinib, PRN1371, regorafenib, rgaratinib (BAY-1163877), S49076, SOMCL-085, SU5402, sunitinib, TAS-120, FP-1039, GAL-F2, GAL-FR21, GAL-FR22, GAL-FR23, GP369, hLD1.vb, LD1, MFGR1877S, MM-161, PRO001 and R3Mab.

Illustrative FGFR fusion inhibitors include: BGJ398, DEBIO 1347, derazantinib (ARQ087), E7090, erdafitinib (JNJ-42756293), lucitanib, and TAS-120.

Illustrative FGFR1, FGFR2, and FGFR3 inhibitors include: AZD4547, BGJ398, DEBIO 1347, E7090, INCB054828, S49076, SOMCL-085, and TAS-120.

Illustrative FGF4 inhibitors include: BLU-554, BLU9931, NVP-FGF401, and hLD1.vb.

Illustrative inhibitors of amplified FGFR1 include: AZD4547, BGJ398, DEBIO 1347, derazantinib (ARQ-087), erdafitinib (JNJ-42756293), INCB054828, and lucitanib.

Illustrative amplified FGFR2 inhibitors include: AZD4547, DEBIO 1347, derazantinib (ARQ-087), lucitanib, regorafenib, and TAS-120.

An illustrative inhibitor of amplified FGFR3 is AZD4547.

Illustrative MEK inhibitors include: AZD8330 (ARRY-424704), AZD6244 (ARRY142866), BI-847325, binimetinib, BIX02188, BIX02189, CH4987655, CH5126766, CI-1040, cobemetinib (GDC-0973), EBI-1051, G-573, G8935, GDC-0623, myricetin, nobiletin, PD0325901, PD184161, PD318088, PD98059, PD334581, pimasertib (AS-703026), refametinib (RDEA119, BAY 869766), selumentinib (AZD6244), SL-327, TAK-733, trametinib and U0126.

Illustrative KRAS inhibitors include: 0375-0604, a covalent quinazoline switch pocket II (SUP) compound, ARS-1620, AZD4785, and LP1.

Illustrative PI3K inhibitors include: 3-methyladenine, A66, alpelisib (BYL719), AMG319, apitolisib (GDC-0980, RG7422), AS-252424, AS-604850, AS-605240, AZD6842, AZD8186, AZD8835, BGT226 (NVP-BGT226), buparlisib (BKM120), CAY10505, CH5132799, copanlisib (BAY 80-6946), CUDC-907, CZC24832, dactolisib (BEZ235, NVP-BEZ235), DS7423, duvelisib (IPI-145, INK1197), GDC-0032, GDC-0084, GDC-0326, gedatolisib (PF-05212384, PKI-5587), GNE-317, GS-9820, GSK1059615, GSK2292767, GSK2636771, HS-173, IC-87114, idelalisib (CAL-101, GS-1101), IPI-145, IPI-3063, IPI-549, LY294002, LY3023414, nemiralisib (GSK2269557), omipalisib (GSK2126458, GSK458), PF-04691502, PF-4989216, PI-103, PI-3065, pictilisib (GDC-0941), PIK-293, PIK-294, PIK-75, PIK-90, PIK-93, PIK-III, pilaralisib (XL147), PKI-587, PP-110, PQR309, PQR309, PW-12, PX-866, quercetin, S14161, SAR245409 (XL765), SAR260301, SAR405, serabelisib (INK-1117, MLN-1117, TAK1117), SF-1126, SF-2523, SN32976, taselisib (GDC-0032), TV101110, TG100-115, TG100-713, TGR-1202, TGX-221, umbralisib (TGR-1202), voxtalisib (XL765, SAR245409), VPS34-IN1, VS-5584 (SB2343), WJD008, wortmannin and ZSTK474.

Illustrative KIT inhibitors include: AMG 706, amuvatinib (MP-470), APcK110, axitinib (AG-013736), AZD2932, dasatinib (BMS-354825), dovitinib (TKI-258, CHIR-258), EXEL-0862, imatinib, KI-328, masitinib (AB1010), midostaurin, MLN518, motesanib, N3-(6-aminopyridin-3-yl)-N1-(2-cyclopentylethyl)-4-methylisophthalamide, nilotinib, OSI-930, pazopanib (GW786034), pexidartinib (PLX3397), PKC412, PLX647, PP1, quizartinib (AC220), regorafenib (BAY 73-4506), semaxinib (SU 5416), sitravatinib (MGCD516), sorafenib, STI571, SU11248, SU9529, sunitinib, telatinib, tivozanib (AV-951), tirphostin AG 1296, VX-322 and WBZ_4.

Illustrative MDM2 inhibitors include: (-)-parthenolide, ALRN6924, AM-8553, AMG232, CGM-097, DS-3032b, GEM240, HDM201, HLI98, idasanutlin (RG-7338), JapA, MI-219, MI-219, MI-319, MI-77301 (SAR405838), MK4828, MK-8242, MX69, NSC 207895 (XI-006), nutlin-3, nutlin-3a, nutlin3b, NVP-CFC218, NVP-CGM097, PXn727/822, RG7112, RO2468, RO5353, RO5503781, serdemethane (JNJ26854165), SP-141 and YH239-EE.

Illustrative inhibitors of amplified MDM2 include: AM-8553, AMG232, DS3032b, MI-77301 (SAR405838), NSC 207895 (XI-006), nutlin-3a, NVP-CFC218, NVP-CGM097, and RG7112.

Illustrative MET inhibitors include: (-)-oleocanthal, ABBV-399, AMG-208, AMG-337,

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AMG-458, BAY-853474, BMS-754807, BMS-777607, BMS-794833, cabozantinib (XL184, BMS-907351), capmatinib (INCB28060), crizotinib (PF-02341066), DE605, foretinib (GSK1363089, XL880), glesatinib (MGCD265), golvatinib (E7050), INCB028060, JNJ-38877605, KRC-408, merestinib (LY2801653), MK-2461, MK8033, NPS-1034, NVP-BVU972, PF-04217903, PHA-665752, S49076, savolitinib (AZD6094, HMPL-504), SGX-523, SU11274, TAS-115, tepotinib (EMD 1214063, MSC2156119J), volitinib, CE355621 and onartuzumab.

Illustrative mTOR inhibitors include: anthracimine, apitolisib (GDC-0980, RG7422), AZD-8055, BGT226 (NVP-BGT226), CC-223, CZ415, dactolisib (BEZ235, NVP-BEZ235), DS7423, everolimus (RAD001), GDC-0084, GDC-0349, gedatolisib (PF-05212384, PKI-5587), GSK1059615, INK128, KU-0063794, LY3023414, MLN0128, omipalisib (GSK2126458, GSK458), OSI-027, OSU-53, palomid 529 (P529), PF-04691502, PI-103, PKI-587, PP242, PQR309, ridafarolimus (AP-23573), sapanisertib (INK 128, MLN0128), SAR245409 (XL765), SF-1126, SF2523, sirolimus (rapamycin), SN32976, TAK228, temsirolimus (CCI-779, NSC 683864), torin 1, torin 2, torkinib (PP242), umirolimus, vistusertib (AZD2014), voxtalisib (XL765, SAR245409), VS-5584, VS-5584 (SB2343), WAY-600, WYE-125132 (WYE-132), WYE-354, WYE-687, XL388 and zotarolimus (ABT-578).

Illustrative MYC inhibitors include: 10058-F4, 10074-G5, and KSI-3716.

The term "deregulation of a gene, protein, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising a kinase domain and a fusion partner, a mutation in a gene that results in the expression of a protein that contains a deletion of at least one amino acid compared to the wild-type protein, a mutation in a gene that results in the expression of a protein with one or more point mutations compared to the wild-type protein, a mutation in a gene that results in the expression of a protein with at least one inserted amino acid compared to the wild-type protein, a gene duplication that results in an increased level of a protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of a protein in a cell), an alternative splice version of an mRNA that results in the formation of a protein having a deletion of at least one amino acid in the protein compared to the wild-type protein), or an increased expression (e.g., increased level) of a wild-type protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, deregulation of a gene, protein, or expression or activity or level of any of them can be a mutation in a gene that encodes a protein that is constitutively active or has increased activity compared to a protein encoded by a gene that does not contain such a mutation. For example, deregulation of a gene, protein, or expression or activity or level of any of them can be the result of a gene or chromosomal translocation that results in expression of a fusion protein comprising a first portion of the protein that contains a functional kinase domain and a second portion of a partner protein (i.e., which is not the primary protein). In some examples, deregulation of a gene, protein, or expression or activity or level of any of these may result from gene translocation of one gene to another gene.

Treatment of a patient with cancer with a multikinase inhibitor (MKI) or target-specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, or RAS inhibitor) may result in deregulation of the RET gene, RET kinase, or their expression or activity or level in the cancer and/or resistance to the RET inhibitor. See, e.g., Bhinge et al., Oncotarget 8:27155–27165, 2017; Chang et al., Yonsei Med. J. 58:9–18, 2017; and Lopez-Delisle et al., doi: 10.1038/s41388-017-0039-5, Oncogene 2018.

Treating a patient with cancer with a RET inhibitor in combination with a multi-kinase inhibitor or a target-specific kinase inhibitor (e.g., a BRAF inhibitor, an EGFR inhibitor, a MEK inhibitor, an ALK inhibitor, a ROS1 inhibitor, a MET inhibitor, an aromatase inhibitor, a RAF inhibitor, or a RAS inhibitor) may provide increased therapeutic efficacy compared to treating the same patient or the same patient with a RET inhibitor alone, or with a multi-kinase inhibitor or target-specific kinase inhibitor alone. See, e.g., Tang et al., doi: 10.1038/modpathol.2017.109, Mod. Pathol. 2017; Andreucci et al., Oncotarget 7:80543–80553, 2017; Nelson-Taylor et al., Mol. Cancer Ther. 16:1623-1633, 2017; and Kato et al., Clin. Cancer Res. 23:1988-1997, 2017.

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has previously been administered a multi-kinase inhibitor (MKI) or target-specific kinase inhibitor (e.g., a BRAF inhibitor, an EGFR inhibitor, a MEK inhibitor, an ALK inhibitor, a ROS1 inhibitor, a MET inhibitor, an aromatase inhibitor, a RAF inhibitor, or a RAS inhibitor) (e.g., as a monotherapy), which comprises: administering to the patient (i) a therapeutically effective dose of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy, or (ii) a therapeutically effective dose

- 139 041220 compounds of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and a therapeutically effective dose of a previously administered MKI or a previously administered target-specific kinase inhibitor.

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has been previously administered an MKI or a target-specific kinase inhibitor (e.g., a BRAF inhibitor, an EGFR inhibitor, a MEK inhibitor, an ALK inhibitor, a ROS1 inhibitor, a MET inhibitor, an aromatase inhibitor, a RAF inhibitor, or a RAS inhibitor) (e.g., as a monotherapy) that comprises: identifying a patient having a cancer cell that contains a deregulation of the RET gene, the RET kinase, or the expression or activity or level of any of them; and administering to the identified patient (i) a therapeutically effective dose of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy, or (ii) a therapeutically effective dose of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and a therapeutically effective dose of a previously administered MKI or a previously administered target-specific kinase inhibitor.

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) comprising: administering to the patient a therapeutically effective amount of an MKI or a target-specific kinase inhibitor (e.g., a BRAF inhibitor, an EGFR inhibitor, a MEK inhibitor, an ALK inhibitor, a ROS1 inhibitor, a MET inhibitor, an aromatase inhibitor, a RAF inhibitor, or a RAS inhibitor) (e.g., as a monotherapy) for a first period of time; after said period of time, identifying the patient having a cancer cell that has a deregulation of the RET gene, RET kinase, or their expression or activity or level; and administering to the identified patient (i) a therapeutically effective dose of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy or (ii) a therapeutically effective dose of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and a therapeutically effective dose of a previously administered MKI or a previously administered target-specific kinase inhibitor.

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of the BRAF gene, BRAF kinase, or their expression or activity or level, which comprise administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of a BRAF inhibitor (e.g., any BRAF inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of a BRAF gene, a BRAF kinase, or the expression or activity or level thereof; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a BRAF inhibitor (e.g., any BRAF inhibitor described herein or known in the art).

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of the EGFR gene, EGFR protein, or their expression or activity or level, which comprise administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of an EGFR inhibitor (e.g., any EGFR inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of the EGFR gene, EGFR protein, or expression or activity or level thereof; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of an EGFR inhibitor (e.g., any EGFR inhibitor described herein or known in the art).

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of a MEK gene, a MEK protein, or their expression or activity or level, which comprise administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of a MEK inhibitor (e.g., any MEK inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of a MEK gene, a MEK protein, or the expression or activity or level thereof; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and (ii) a therapeutically effective amount of a MEK inhibitor (e.g., any MEK inhibitor described herein or known in the art).

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of an ALK gene, an ALK protein, or their expression or activity or level, which comprise administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of an ALK inhibitor (e.g., any ALK inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of an ALK gene, an ALK protein, or their expression or activity or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and (ii) a therapeutically effective amount of an ALK inhibitor (e.g., any ALK inhibitor described herein or known in the art).

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of a ROS gene, ROS protein, or their expression or activity or level, which comprise administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of a ROS inhibitor (e.g., any ROS inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of a ROS gene, a ROS protein, or their expression or activity or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of a ROS inhibitor (e.g., any ROS inhibitor described herein or known in the art).

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of a MET gene, a MET protein, or their expression or activity or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of a MET inhibitor (e.g., any MET inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of a MET gene, a MET protein, or their expression or activity or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a MET inhibitor (e.g., any MET inhibitor described herein or known in the art).

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of an aromatase gene, an aromatase protein, or their expression or activity or level, which comprise administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of an aromatase inhibitor (e.g., any aromatase inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of an aromatase gene, an aromatase protein, or their expression or activity or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, and (ii) a therapeutically effective amount of an aromatase inhibitor (e.g., any aromatase inhibitor described herein or known in the art).

- 141 041220

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of a RAF gene, a RAF protein, or their expression or activity or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of a RAF inhibitor (e.g., any RAF inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of a RAF gene, a RAF protein, or the expression or activity or level thereof; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a RAF inhibitor (e.g., any RAF inhibitor described herein or known in the art).

The present invention provides methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that has a deregulation of a RAS gene, a RAS protein, or their expression or activity or level, which comprise administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and (ii) a therapeutically effective amount of a RAS inhibitor (e.g., any RAS inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) that comprise: identifying a patient having a cancer cell that has a deregulation of a RAS gene, a RAS protein, or their expression or activity or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a RAS inhibitor (e.g., any RAS inhibitor described herein or known in the art).

The term "deregulation of the BRAF gene, BRAF protein, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising a BRAF kinase domain and a fusion partner, a mutation in the BRAF gene that results in the expression of a BRAF protein that contains a deletion of at least one amino acid compared to the wild-type BRAF protein, a mutation in the BRAF gene that results in the expression of a BRAF protein with one or more point mutations compared to the wild-type BRAF protein, a mutation in the BRAF gene that results in the expression of a BRAF protein with at least one inserted amino acid compared to the wild-type BRAF protein, a gene duplication that results in an increased level of BRAF protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of BRAF protein in a cell), an alternatively spliced version of BRAF mRNA that results in a BRAF protein having a deletion of at least one amino acid in the BRAF protein compared to the wild-type BRAF protein), or increased expression (e.g., increased level) of the wild-type BRAF protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, the deregulation of the BRAF gene, BRAF protein, or the expression or activity or level of either can be a mutation in a BRAF gene that encodes a BRAF protein that is constitutively active or has increased activity compared to a protein encoded by a BRAF gene that does not contain such a mutation. For example, deregulation of the BRAF gene, BRAF protein, or expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of the BRAF protein that contains a functional kinase domain and a second portion of a partner protein (i.e., which is not BRAF). In some examples, deregulation of the BRAF gene, BRAF protein, or expression or activity or level of any of them may be the result of a gene translocation of one BRAF gene with another non-BRAF gene.

Non-limiting examples of a BRAF inhibitor include dabrafenib, vemurafenib (also called RG7204 or PLX4032), sorafenib tosylate, PLX-4720, GDC-0879, BMS-908662 (Bristol-Meyers Squibb), LGX818 (Novartis), PLX3603 (Hofmann-LaRoche), RAF265 (Novartis), RO5185426 (HofmannLaRoche), and GSK2118436 (GlaxoSmithKline). Additional examples of a BRAF inhibitor are known in the art.

The term deregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a hybrid protein containing the EGFR kinase domain and a fusion partner, a mutation in the EGFR gene that results in the expression of an EGFR protein that contains a deletion of at least

- 142 041 220 one amino acid compared to the wild-type EGFR protein, a mutation in the EGFR gene that results in the expression of an EGFR protein with one or more point mutations compared to the wild-type EGFR protein, a mutation in the EGFR gene that results in the expression of an EGFR protein with at least one inserted amino acid compared to the wild-type EGFR protein, a gene duplication that results in an increased level of EGFR protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of EGFR protein in a cell), an alternative splice version of EGFR mRNA that results in the formation of an EGFR protein having a deletion of at least one amino acid in the EGFR protein compared to the wild-type EGFR protein), or increased expression (e.g., increased level) of the wild-type EGFR protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, deregulation of the EGFR gene, EGFR protein, or expression or activity or level of either can be a mutation in the EGFR gene that encodes an EGFR protein that is constitutively active or has increased activity compared to the protein encoded by an EGFR gene that does not contain such a mutation. For example, deregulation of the EGFR gene, EGFR protein, or expression or activity or level of either can be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of the EGFR protein that contains a functional kinase domain and a second portion of a partner protein (i.e., not EGFR). In some examples, deregulation of the EGFR gene, EGFR protein, or expression or activity, or level of any of them may result from gene translocation of one EGFR gene with another non-EGFR gene.

Non-limiting examples of EGFR inhibitor include gefitinib, erlotinib, brigatinib, lapatinib, neratinib, icotinib, afatinib, dacomitinib, poziotinib, vandetanib, afatinib, AZD9291, CO-1686, HM61713, AP26113, CI-1033, PKI-166, GW-2016, EKB-569, PDI-168393, AG-1478, CGP-59326A. Additional examples of EGFR inhibitor are known in the art.

The term "deregulation of a MEK gene, a MEK protein, or the expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising a MEK kinase domain and a fusion partner, a mutation in a MEK gene that results in the expression of a MEK protein that contains a deletion of at least one amino acid compared to the wild-type MEK protein, a mutation in a MEK gene that results in the expression of a MEK protein with one or more point mutations compared to the wild-type MEK protein, a mutation in a MEK gene that results in the expression of a MEK protein with at least one inserted amino acid compared to the wild-type MEK protein, a gene duplication that results in an increased level of MEK protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of MEK protein in a cell), an alternative splice version of MEK mRNA, which results in the formation of a MEK protein having a deletion of at least one amino acid in the MEK protein compared to a wild-type MEK protein), or increased expression (e.g., increased level) of the wild-type MEK protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, the deregulation of a MEK gene, a MEK protein, or the expression or activity or level of any of them can be a mutation in a MEK gene that encodes a MEK protein that is constitutively active or has increased activity compared to a protein encoded by a MEK gene that does not contain such a mutation. For example, deregulation of a MEK gene, a MEK protein, or the expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of a MEK protein that contains a functional kinase domain and a second portion of a partner protein (i.e., not MEK). In some examples, deregulation of a MEK gene, a MEK protein, or the expression or activity or level of any of them may be the result of a gene translocation of one MEK gene with another non-MEK gene.

Non-limiting examples of a MEK inhibitor include mekinist, trametinib (GSK1120212), cobimetinib (XL518), binimetinib (MEK162), selumetinib, PD-325901, CI-1040, PD035901, TAK-733, PD098059, U0126, AS703026/MSC1935369, XL-518/GDC-0973, BAY869766/RDEA119, and GSK1120212. Additional examples of a MEK inhibitor are known in the art.

The term "deregulation of the ALK gene, ALK protein, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a hybrid protein comprising the ALK kinase domain and a fusion partner, a mutation in the ALK gene that results in the expression of an ALK protein that contains a deletion of at least one amino acid compared to the wild-type ALK protein, a mutation in the ALK gene that results in the expression of an ALK protein with one or more point mutations compared to the wild-type ALK protein, a mutation in the ALK gene that results in the expression of an ALK protein with at least one inserted amino acid compared to the wild-type ALK protein, a gene duplication that results in an increased level of ALK protein in a cell, or a mutation in a regulatory sequence (e.g.,

- 143 041220 promoter and/or enhancer) that results in an increased level of ALK protein in a cell), an alternative splice version of ALK mRNA that results in the formation of an ALK protein having a deletion of at least one amino acid in the ALK protein compared to the wild-type ALK protein), or increased expression (e.g., increased level) of the wild-type ALK protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, deregulation of the ALK gene, ALK protein, or the expression or activity or level of any of them can be a mutation in the ALK gene that encodes an ALK protein that is constitutively active or has increased activity compared to the protein encoded by an ALK gene that does not contain such a mutation. For example, deregulation of an ALK gene, an ALK protein, or the expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of an ALK protein that contains a functional kinase domain and a second portion of a partner protein (i.e., not ALK). In some examples, deregulation of an ALK gene, an ALK protein, or the expression or activity or level of any of them may be the result of a gene translocation of one ALK gene with another non-ALK gene.

Non-limiting examples of an ALK inhibitor include crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa), dalantercept, ACE-041 (brigatinib) (AP26113), entrectinib (NMSE628), PF-06463922 (Pfizer), TSR-011 (Tesaro), CEP-37440 (Teva), CEP-37440 (Teva), X-396 (Xcovery), and ASP-3026 (Astellas). Additional examples of an ALK inhibitor are known in the art.

The expression "deregulation of the ROS1 gene, ROS1 protein, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising the ROS1 kinase domain and a fusion partner, a mutation in the ROS1 gene that results in the expression of a ROS1 protein that contains a deletion of at least one amino acid compared to the wild-type ROS1 protein, a mutation in the ROS1 gene that results in the expression of a ROS1 protein with one or more point mutations compared to the wild-type ROS1 protein, a mutation in the ROS1 gene that results in the expression of a ROS1 protein with at least one inserted amino acid compared to the wild-type ROS1 protein, a gene duplication that results in an increased level of ROS1 protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of ROS1 protein in a cell), an alternative splice version of ROS1 mRNA that results in the formation of a ROS1 protein having a deletion of at least one amino acid in the ROS1 protein compared to the wild-type ROS1 protein), or increased expression (e.g., increased level) of the wild-type ROS1 protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, the deregulation of a ROS1 gene, ROS1 protein, or the expression or activity or level of any of them can be a mutation in a ROS1 gene that encodes a ROS1 protein that is constitutively active or has increased activity compared to a protein encoded by a ROS1 gene that does not contain such a mutation. For example, deregulation of a ROS1 gene, ROS1 protein, or expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of a ROS1 protein that contains a functional kinase domain and a second portion of a partner protein (i.e., not ROS1). In some examples, deregulation of a ROS1 gene, ROS1 protein, or expression or activity or level of any of them may be the result of a gene translocation of one ROS1 gene with another non-ROS1 gene.

Non-limiting examples of a ROS1 inhibitor include crizotinib, ectrectinib (RXDX-101), lorlatinib (PF-06463922), certinib, TPX-0005, DS-605, and cabozantinib. Additional examples of a ROS1 inhibitor are known in the art.

The term "deregulation of the MET gene, MET protein, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising a MET kinase domain and a fusion partner, a mutation in the MET gene that results in the expression of a MET protein that contains a deletion of at least one amino acid compared to the wild-type MET protein, a mutation in the MET gene that results in the expression of a MET protein with one or more point mutations compared to the wild-type MET protein, a mutation in the MET gene that results in the expression of a MET protein with at least one inserted amino acid compared to the wild-type MET protein, a gene duplication that results in an increased level of MET protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of MET protein in a cell), an alternative splice version of MET mRNA, which results in the formation of a MET protein having a deletion of at least one amino acid in the MET protein, as compared to a wild-type MET protein), or increased expression (e.g., increased level) of the wild-type MET protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., as compared to a control non-cancerous cell). As another example, deregulation of the MET gene, MET protein, or the expression or activity or level of any of

- 144 041220 may be a mutation in a MET gene that encodes a MET protein that is constitutively active or has increased activity compared to a protein encoded by a MET gene that does not contain such a mutation. For example, deregulation of a MET gene, MET protein, or the expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of a MET protein that contains a functional kinase domain and a second portion of a partner protein (i.e., non-MET). In some examples, deregulation of a MET gene, MET protein, or the expression or activity or level of any of them may be the result of a gene translocation of one MET gene with another non-MET gene.

Non-limiting examples of a MET inhibitor include crizotinib, cabozantinib, JNJ-38877605, PF-04217903 (Pfizer), MK-2461, GSK 1363089, AMG 458 (Amgen), tivantinib, INCB28060 (Incyte), PF02341066 (Pfizer), E7050 (Eisai), BMS-777607 (Bristol-Meyers Squibb), JNJ-38877605 (Johnson & Johnson), ARQ197 (ArQule), GSK/1363089/XL880 (GSK/Exelixis), and XL174 (BMS/Exelixis). Additional examples of a MET inhibitor are known in the art.

The term "deregulation of an aromatase gene, an aromatase protein, or the expression or activity or level of any of them" refers to a genetic mutation (e.g., a mutation in an aromatase gene that results in the expression of an aromatase protein that contains a deletion of at least one amino acid compared to a wild-type aromatase protein, a mutation in an aromatase gene that results in the expression of an aromatase protein with one or more point mutations compared to a wild-type aromatase protein, a mutation in an aromatase gene that results in the expression of an aromatase protein with at least one inserted amino acid compared to a wild-type aromatase protein, a gene duplication that results in an increased level of aromatase protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of aromatase protein in a cell), an alternative splice version of aromatase mRNA that results in the formation of an aromatase protein having a deletion of at least one amino acid in the aromatase protein compared to wild-type aromatase protein), or increased expression (e.g., increased level) of wild-type aromatase protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, deregulation of an aromatase gene, aromatase protein, or the expression or activity or level of any of them can be a mutation in an aromatase gene that encodes an aromatase protein that is constitutively active or has increased activity compared to the protein encoded by an aromatase gene that does not contain such a mutation.

Non-limiting examples of an aromatase inhibitor include arimidex (anastrozole), aromasin (exemestane), femara (letrozole), teslac (testolactone), and formestane. Additional examples of an aromatase inhibitor are known in the art.

The expression "deregulation of the RAF gene, the RAF protein, or the expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising a RAF kinase domain and a fusion partner, a mutation in the RAF gene that results in the expression of a RAF protein that contains a deletion of at least one amino acid compared to the wild-type RAF protein, a mutation in the RAF gene that results in the expression of a RAF protein with one or more point mutations compared to the wild-type RAF protein, a mutation in the RAF gene that results in the expression of a RAF protein with at least one inserted amino acid compared to the wild-type RAF protein, a gene duplication that results in an increased level of RAF protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of RAF protein in a cell), an alternative splice version of RAF mRNA, which results in the formation of a RAF protein having a deletion of at least one amino acid in the RAF protein compared to a wild-type RAF protein), or increased expression (e.g., increased level) of the wild-type RAF protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, the deregulation of a RAF gene, a RAF protein, or the expression or activity or level of any of them can be a mutation in a RAF gene that encodes a RAF protein that is constitutively active or has increased activity compared to a protein encoded by a RAF gene that does not contain such a mutation. For example, deregulation of a RAF gene, a RAF protein, or the expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of a RAF protein that contains a functional kinase domain and a second portion of a partner protein (i.e., which is not RAF). In some examples, deregulation of a RAF gene, a RAF protein, or the expression or activity or level of any of them may be the result of a gene translocation of one RAF gene with another non-RAF gene.

Non-limiting examples of RAF inhibitors include sorafenib, vemurafenib, dabrafenib, BMS-908662/XL281, GSK2118436, RAF265, RO5126766, and RO4987655. Additional examples of inhi

- 145 041220 RAF inhibitors are known in the art.

The term "dysregulation of the RAS gene, RAS protein, or expression or activity or level of any of them" refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein comprising the RAS kinase domain and a fusion partner, a mutation in the RAS gene that results in the expression of a RAS protein that contains a deletion of at least one amino acid compared to the wild-type RAS protein, a mutation in the RAS gene that results in the expression of a RAS protein with one or more point mutations compared to the wild-type RAS protein, a mutation in the RAS gene that results in the expression of a RAS protein with at least one inserted amino acid compared to the wild-type RAS protein, a gene duplication that results in an increased level of RAS protein in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of RAS protein in a cell), an alternative splice version of RAS mRNA, which results in the formation of a RAS protein having a deletion of at least one amino acid in the RAS protein compared to a wild-type RAS protein), or increased expression (e.g., increased level) of the wild-type RAS protein in a mammalian cell due to aberrant cellular signaling and/or deregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, the deregulation of a RAS gene, RAS protein, or the expression or activity or level of any of them can be a mutation in a RAS gene that encodes a RAS protein that is constitutively active or has increased activity compared to a protein encoded by a RAS gene that does not contain such a mutation. For example, deregulation of a RAS gene, a RAS protein, or the expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein comprising a first portion of a RAS protein that contains a functional kinase domain and a second portion of a partner protein (i.e., not RAS). In some examples, deregulation of a RAS gene, a RAS protein, or the expression or activity or level of any of them may be the result of a gene translocation of one RAS gene with another non-RAS gene.

Non-limiting examples of a RAS inhibitor include Kobe0065 and Kobe2602. Additional examples of a RAS inhibitor are known in the art.

Non-limiting examples of multikinase inhibitors (MKIs) include dasatinib and sunitinib.

In some embodiments, provided herein are methods of treating a subject suffering from a cancer, comprising: (a) administering to the subject one or more doses of a first RET inhibitor or a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, over a period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has at least one deregulation of a gene, protein, or expression or activity or level of any of the above, wherein said gene or protein is selected from the group consisting of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2; and (c) administering 1) a second RET inhibitor as monotherapy or in combination with another anticancer agent, 2) administering additional doses of the first RET inhibitor or a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof in combination with an inhibitor that targets a gene or protein (e.g., an inhibitor of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA and HER2), or 3) discontinuing administration of the RET inhibitor from step a) and administering to the subject an inhibitor that targets a gene or protein (e.g., an inhibitor of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA and HER2), if said subject has a cancer cell that has at least one deregulation of a gene, protein, or its expression or activity or level, wherein said gene or protein is selected from the group consisting of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA and HER2; or (d) administering to the subject additional doses of the first RET inhibitor of step (a) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, one or more deregulations of a gene, protein, or expression or activity or level of any of them, wherein said gene or protein is selected from the group consisting of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA and HER2, provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor or a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the tumor is a NSCLC tumor and the one or more deregulated genes, proteins, or expression or activity or level of any of these are selected from targetable mutations in EGFR or MET, targetable rearrangements involving ALK or ROS1, or activating mutations in KRAS. In some embodiments, the tumor is a thyroid (non-MTC) tumor and the one or more deregulated genes, proteins, or expression or activity or level of any of these are selected from targetable mutations in BRAF or activating mutations in RAS. In some embodiments, the tumor is an MTC tumor and the one or more deregulated genes, proteins, or expression or activity or level of any of these are selected from targetable mutations in ALK or activating mutations in RAS. In some embodiments, the tumor is a pancreatic tumor

- 146 041220 gland, and one or more deregulations of a gene, protein, or expression, or activity, or level of any of them are activating mutations in KRAS. In some embodiments, the tumor is a colorectal tumor, and the one or more deregulations of a gene, protein, or expression, or activity, or level of any of them are selected from targetable mutations in BRAF or PIK3CA or an activating mutation in RAS. In some embodiments, the tumor is a breast tumor, and the one or more deregulations of a gene, protein, or expression, or activity, or level of any of them are selected from targetable mutations in PIK3CA or alterations in HER2.

Also provided are methods for selecting a treatment for a subject suffering from cancer, which comprise (a) administering to the subject one or more doses of a first RET inhibitor over a certain period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) selecting for the subject a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy or in combination with another anti-cancer agent, if said subject has a cancer cell that contains one or more RET inhibitor resistance mutations; or (d) selecting for the subject additional doses of the first RET inhibitor of step (a), if the subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, when selecting additional doses of the first RET inhibitor of step (a), the provided method may further comprise selecting doses of another anti-cancer agent for the subject. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Tables 3 and 4. For example, the one or more RET inhibitor resistance mutations can include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (c), the other RET inhibitor may be the first RET inhibitor introduced in step (a).

Also provided are methods for selecting a treatment for a subject suffering from cancer, which comprise (a) administering to the subject one or more doses of a first RET inhibitor over a certain period of time; (b) after (a), determining whether a cancer cell in a sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) selecting for the subject a second RET inhibitor as monotherapy or in combination with another anti-cancer agent if the subject has a cancer cell that contains one or more RET inhibitor resistance mutations; or (d) selecting for the subject additional doses of the first RET inhibitor from step (a) if the subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, when selecting additional doses of the first RET inhibitor from step (a), the provided method may further comprise selecting doses of another anti-cancer agent for the subject. In some embodiments, the one or more RET inhibitor resistance mutations confers increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more of the RET inhibitor resistance mutations listed in Tables 3 and 4. For example, the one or more RET inhibitor resistance mutations can comprise a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof). In some embodiments, the additional anticancer agent is an immunotherapy. In some embodiments, the other RET may be the first RET inhibitor administered in step (a).

Also provided are methods for selecting a treatment for a subject suffering from cancer, which comprise (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously receiving one or more doses of a first RET inhibitor has one or more RET inhibitor resistance mutations; (b) selecting for the subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (c) selecting for the subject additional doses of a first RET inhibitor previously administered to the subject, if the subject has a cancer cell that does not contain

- 147 041220 a RET inhibitor resistance mutation. In some embodiments, if additional doses of the first RET inhibitor previously administered to the subject are selected for the subject, the proposed method may further comprise selecting doses of another anticancer agent (e.g., a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof) for the subject. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more RET inhibitor resistance mutations listed in Table 3 and 4. For example, the one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (c), the other RET inhibitor may be the first RET inhibitor administered in step (a).

Also provided are methods for selecting a treatment for a subject suffering from cancer, which comprise (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously receiving one or more doses of a first RET inhibitor has one or more RET inhibitor resistance mutations; (b) selecting for the subject a second RET inhibitor as monotherapy or in combination with another anticancer agent if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (c) selecting for the subject additional doses of a first RET inhibitor previously administered to the subject if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, if additional doses of a first RET inhibitor previously administered to the subject are selected for the subject, the method may further comprise selecting doses of another anti-cancer agent (e.g., a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof) for the subject. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more of the RET inhibitor resistance mutations listed in Table 3 and 4. For example, the one or more RET inhibitor resistance mutations can include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET can be the first RET inhibitor administered in step (a).

Also provided are methods for determining the risk of a subject developing a cancer that has a certain resistance to a first RET inhibitor, which include: determining whether a cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and identifying a subject having a cell that has one or more RET inhibitor resistance mutations as a subject with an increased likelihood of developing a cancer that has a certain resistance to the first RET inhibitor. Also provided are methods for determining the risk of a subject developing a cancer that has a certain resistance to a first RET inhibitor, which include: identifying a subject having a cell that contains one or more RET inhibitor resistance mutations as a subject with an increased likelihood of developing a cancer that has a certain resistance to the first RET inhibitor. Also provided are methods for determining the presence of a cancer that has a certain resistance to a first RET inhibitor, which include: determining whether a cell in a sample obtained from a subject has one or more RET inhibitor resistance mutations; and determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations suffers from a cancer that has a certain resistance to the first RET inhibitor. Also provided are methods for determining the presence of a cancer that has a certain resistance to a first RET inhibitor in a subject, which include: determining that a subject having a cancer cell that has one or more RET inhibitor resistance mutations suffers from a cancer that has a certain resistance to the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations comprise one or more RET inhibitor resistance mutations listed in Table 3 and

4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

In some embodiments of any method described herein, the RET inhibitor resistance mutation that renders the cancer cell or tumor increasingly resistant to treatment with the first RET inhibitor may be any of the RET inhibitor resistance mutations listed in Table 3 or 4 (e.g., a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D).

In some embodiments, the presence of one or more RET inhibitor resistance mutations in a tumor causes the tumor to be more resistant to treatment with a compound of Formula IIV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. Methods useful when the RET inhibitor resistance mutation causes the tumor to be more resistant to treatment with a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, are described below. For example, provided herein are methods of treating a subject suffering from cancer, which comprise: identifying a subject having a cancer cell that comprises one or more RET inhibitor resistance mutations; and administering to the identified subject a treatment that does not include a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy (e.g., a second RET kinase inhibitor). Also provided are methods for treating a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations, which comprise administering to said subject a treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy (e.g., a second RET kinase inhibitor). In some embodiments, the one or more RET inhibitor resistance mutations render the cancer cell or tumor increased resistant to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

The present document also provides methods for selecting a treatment for a subject suffering from a cancer, which comprise: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and selecting a treatment that does not include a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy for the identified subject (e.g., a second RET kinase inhibitor). The present document also provides methods for selecting a treatment for a subject suffering from a cancer, which comprise: selecting a treatment that does not include a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy (e.g., a second RET kinase inhibitor) for a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations. Also provided are methods for selecting a subject suffering from cancer for treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy (e.g., a second RET kinase inhibitor), which comprise: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and selecting the identified subject for treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy (e.g., a second RET kinase inhibitor). Also provided are methods for selecting a subject suffering from cancer for a treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof as a monotherapy (e.g., a second RET kinase inhibitor), which comprise: selecting a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations for a treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof as a monotherapy. In some embodiments, the one or more RET inhibitor resistance mutations render the cancer cell or tumor increased resistant to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof.

Also provided are methods for determining the likelihood that a subject suffering from cancer will exhibit a positive response to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy, which include: determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations has a reduced likelihood of a positive response to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy. Also provided are methods for determining the likelihood that a subject suffering from cancer will exhibit a positive response to treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy, comprising: determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations has a reduced likelihood of a positive response to treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy. Also provided are methods for predicting the effectiveness of treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as a monotherapy, in a subject suffering from cancer, comprising: determining that a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and determining that treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy is less likely to be effective in a subject having a cancer cell in a sample obtained from the subject that contains one or more RET inhibitor resistance mutations. Also provided are methods for predicting the effectiveness of treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy in a subject suffering from cancer, which comprise: determining that treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as a monotherapy is less likely to be effective in a subject having a cancer cell in a sample obtained from the subject that contains one or more RET inhibitor resistance mutations. In some embodiments, one or more RET inhibitor resistance mutations confer increased resistance to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

Also provided are methods of treating a subject suffering from cancer, which comprise: (a) administering one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof over a certain period of time; (b) after (a) determining that a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and (c) administering a second RET inhibitor or a second compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy or in combination with another anticancer agent to a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; or (d) administering additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (a) to a subject having a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (a), the subject can also be administered another anti-cancer agent or a second compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor increasingly resistant to treatment with the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET can be the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, administered in step (a).

Also provided are methods of treating a subject suffering from cancer, which comprise: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously receiving one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, has one or more RET inhibitor resistance mutations; (b) administering a second RET inhibitor or a second compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy or in combination with another anticancer agent to a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; or (c) administering additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, previously administered to a subject having a cancer cell that does not contain a RET resistance mutation. In some embodiments, if the subject is administered additional doses of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (a), then the subject may also be administered another anti-cancer agent. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the additional anti-cancer agent is

- 150 041220 is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, administered in step (a).

Also provided herein are methods for selecting a treatment for a subject suffering from a cancer, which comprise: (a) administering to the subject one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, over a period of time; (b) after (a), determining that a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and (c) selecting a second RET inhibitor or a second compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anticancer agent for the subject if said subject has a cancer cell that contains a RET inhibitor resistance mutation; or (d) selecting additional doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof from step (a) for the subject if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, if additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (a) are selected for the subject, the method may further comprise further selecting another anti-cancer agent. In some embodiments, the one or more RET inhibitor resistance mutations render the cancer cell or tumor increasingly resistant to treatment with the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, administered in step (a).

Also provided herein are methods for selecting a treatment for a subject suffering from a cancer, which comprise: (a) determining whether a cancer cell in a sample obtained from a subject suffering from a cancer and previously receiving one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, has one or more RET inhibitor resistance mutations; (b) selecting a second RET inhibitor or a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, as monotherapy or in combination with another anticancer agent for the subject if said subject has a cancer cell that has a RET inhibitor resistance mutation; or (c) selecting additional doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, previously administered to the subject, if said subject has a cancer cell that does not have a RET resistance mutation. In some embodiments, if additional doses of the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, from step (a) are selected for the subject, the method may further comprise further selecting another anti-cancer agent. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor increased resistant to treatment with the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof. In some embodiments, the additional anti-cancer agent is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (e.g., a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be the compound of Formula IIV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, administered in step (a).

Also provided are methods for determining the risk of a subject developing a cancer that has a particular resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, comprising: determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and identifying the subject, if the subject has a cancer cell that contains one or more RET inhibitor resistance mutations, as having an increased likelihood of developing a cancer that has a particular resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form. Also provided are methods for determining the risk of a subject developing a cancer that has a particular resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, comprising: identifying the subject having

- 151 041220 having a cell that contains one or more RET inhibitor resistance mutations as having an increased likelihood of developing a cancer that has a certain resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form. Also provided are methods for determining the presence of a cancer that has a certain resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, which include: determining whether a cancer cell in a sample obtained from a subject has one or more RET inhibitor resistance mutations; and determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations suffers from a cancer that has a certain resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form. Also provided are methods for determining whether a subject has a cancer that has a certain resistance to a compound of formula IIV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, comprising: determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations suffers from a cancer that has a certain resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form. In some embodiments, the one or more RET inhibitor resistance mutations render the cancer cell or tumor increased resistant to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

In some embodiments of any of the methods described herein, the RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistant to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof may be any of the RET inhibitor resistance mutations listed in Table 3 or 4.

Methods for determining the degree of resistance of a cancer cell or tumor to a RET inhibitor (e.g., any RET inhibitor described herein or known in the art) can be determined using methods known in the art. For example, the degree of resistance of a cancer cell to a RET inhibitor can be assessed by determining the IC ₅₀ of a RET inhibitor (e.g., any RET inhibitor described herein or known in the art) on the viability of the cancer cell. In other examples, the degree of resistance of a cancer cell to a RET inhibitor can be assessed by determining the growth rate of the cancer cell in the presence of a RET inhibitor (e.g., any RET inhibitor) described herein. In other examples, the degree of resistance of a tumor to a RET inhibitor can be assessed by determining the mass or size of one or more tumors in a subject's body during treatment with a RET inhibitor (e.g., any RET inhibitor described herein). In other examples, the degree of resistance of a cancer cell or tumor to a RET inhibitor can be indirectly assessed by determining the activity of a RET kinase containing one or more RET inhibitor resistance mutations (i.e., the same RET kinase expressed in a cancer cell or tumor in a subject). The degree of resistance of a cancer cell or tumor containing one or more RET inhibitor resistance mutations to a RET inhibitor is expressed relative to the degree of resistance of a cancer cell or tumor that does not contain a RET inhibitor resistance mutation (e.g., a cancer cell or tumor that does not contain the same RET inhibitor resistance mutations, a cancer cell or tumor that does not contain any RET inhibitor resistance mutations, or a cancer cell or tumor that expresses a wild-type RET protein). For example, the determined resistance rate of a cancer cell or tumor containing one or more RET inhibitor resistance mutations can be greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, greater than about 8%, greater than about 9%, greater than about 10%, greater than about 11%, greater than about 12%, greater than about 13%, greater than about 14%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater than greater than about 110%, greater than about 120%, greater than about 130%, greater than about 140%, greater than about 150%, greater than about 160%, greater than about 170%, greater than about 180%, greater than about 190%, greater than about 200%, greater than about 210%, greater than about 220%, greater than about 230%, greater than about 240%, greater than about 250%, greater than about 260%, greater than about 270%, greater than about 280%, greater than about 290%, or greater than about 300% of the degree of resistance of a cancer cell or tumor that does not contain a RET inhibitor resistance mutation (e.g., a cancer cell or tumor that does not contain any RET inhibitor resistance mutations, a cancer cell or tumor that does not contain any RET inhibitor resistance mutations, or resistance to a RET inhibitor, or a cancer cell or tumor that expresses wild-type RET protein).

RET is thought to play an important role in the development and survival of afferent nociceptors in the skin and gut. RET kinase knockout mice lack enteric neurons.

- 152 041220 and other abnormalities of the nervous system, suggesting a requirement for a functional RET kinase protein product during development (Taraviras, S. et al., Development, 1999, 126:27852797). Additionally, population-based studies of patients with Hirschsprung disease, characterized by colonic obstruction due to lack of normal colonic innervation, have shown a higher proportion of both inherited and sporadic loss-of-function RET mutations (Butler Tjaden N., et al., Transl. Res., 2013, 162: 1-15). Irritable bowel syndrome (IBS) is a common disorder affecting 10-20% of people in developed countries and is characterized by abnormal bowel habits, bloating, and visceral hypersensitivity (Camilleri, M., N. Engl. J. Med., 2012, 367: 1626-1635). Although the etiology of IBS is unknown, it is thought to result from a disorder between the brain and the gastrointestinal tract, a disruption in the gut microbiome, or increased inflammation. The resulting gastrointestinal changes interfere with normal intestinal transit, leading to diarrhea or constipation. Additionally, in many patients with IBS, sensitization of the peripheral nervous system leads to visceral hypersensitivity or allodynia (Keszthelyi, D., Eur. J. Pain, 2012, 16: 1444-1454). See, for example, US Publication No. 2015/0099762.

Accordingly, the present invention provides methods for treating a patient diagnosed with (or identified to have) irritable bowel syndrome (IBS), including diarrhea-predominant, constipation-predominant or alternating bowel pattern disease, functional bloating, functional constipation, functional diarrhea, non-specific functional bowel disorder, functional abdominal pain syndrome, chronic idiopathic constipation, functional esophageal disorders, functional gastroduodenal disorders, functional anorectal pain and inflammatory bowel disease, which comprise administering to the patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

Also provided are methods for treating a patient identified or diagnosed with RET-associated irritable bowel syndrome (IBS) (e.g., a patient identified or diagnosed with RET-associated irritable bowel syndrome (IBS) using a regulatory agency-approved, e.g., FDA-approved, kit for identifying deregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a patient, or in a biopsy specimen obtained from a patient), which comprise administering to said patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

Also provided herein are methods for treating pain associated with IBS, which comprise administering to a patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof is administered in combination with a first other therapeutic agent useful for treating one or more symptoms of IBS.

Also provided are methods for treating irritable bowel syndrome (IBS) in a patient in need thereof, comprising: (a) determining whether the irritable bowel syndrome (IBS) in the patient is RET-associated IBS (e.g., using a regulatory agency-approved, e.g., FDA-approved, kit for identifying deregulation of the RET gene, RET kinase, or expression or activity or level of any of them in the patient or in a biopsy specimen obtained from the patient, or by performing any of the non-limiting examples of assays described herein); and (b) if the IBS is determined to be RET-associated IBS, administering to said patient a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

In some embodiments, the compounds of this invention are useful for treating irritable bowel syndrome (IBS) in combination with one or more additional therapeutic agents or therapies effective for treating irritable bowel syndrome that act by the same or a different mechanism of action. At least one additional therapeutic agent can be administered with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, in the same or different dosage forms, by the same or different routes of administration, and by the same or different administration schedules in accordance with standard pharmaceutical practice known to those skilled in the art.

Non-limiting examples of adjunctive therapies for the treatment of irritable bowel syndrome (IBS) include probiotics, dietary fiber supplements (e.g., psyllium, methylcellulose), antidiarrheals (e.g., lopermide), bile acid binders (e.g., cholestyramine, colestipol, colesevelam), anticholinergics and antispasmodics (e.g., hyoscyamine, dicyclomine), antidepressants (e.g., tricyclic antidepressants such as imipramine or notriptyline, or selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine or paroxetine), antibodies (e.g., rifaximin), alosetron, and lubiprostone.

Accordingly, also provided herein are methods for treating irritable bowel syndrome (IBS), comprising administering to a patient in need thereof a pharmaceutical combination for treating IBS, which comprises (a) a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, (b) an additional therapeutic agent, and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate or sequential use for treating IBS, wherein the amounts of the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent together are effective for treating IBS. In one embodiment, the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent are administered simultaneously as separate dosage forms. In one embodiment, the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent are administered as separate dosage forms sequentially, in any order, in a total therapeutically effective amount, such as in daily or intermediate doses. In one embodiment, the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent are administered simultaneously as a combined dosage form.

The present document also provides (i) a pharmaceutical combination for the treatment of irritable bowel syndrome in a patient in need thereof, comprising (a) a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, (b) at least one additional therapeutic agent (e.g., any of the illustrative additional therapeutic agents described herein for the treatment of irritable bowel syndrome, or known in the art), and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate or sequential use for the treatment of irritable bowel syndrome, wherein the amounts of the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and the additional therapeutic agent together are effective for the treatment of irritable bowel syndrome; (ii) a pharmaceutical composition comprising such a combination; (iii) the use of such a combination for the preparation of a medicament for the treatment of irritable bowel syndrome; and (iv) a commercial package or product containing such a combination as a combination preparation for simultaneous, separate or sequential use; as well as a method for treating irritable bowel syndrome in a patient in need thereof. In one embodiment, the patient is a human.

The term pharmaceutical combination as used herein refers to a pharmaceutical therapy obtained by mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term fixed combination means that a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and at least one additional therapeutic agent (e.g. an agent effective for treating irritable bowel syndrome) are administered to a patient simultaneously, as a single composition or dosage form. The term non-fixed combination means that a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and at least one additional therapeutic agent (e.g. an agent effective for treating irritable bowel syndrome) are formulated as separate compositions or dosage forms such that they can be administered to a patient in need thereof simultaneously, in parallel or sequentially at different intermediate time intervals, wherein such administration provides an effective maintenance of two or more compounds in the patient's body. In one embodiment, the compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and the additional therapeutic agent are formulated as separate unit dosage forms, wherein the separate dosage forms are suitable for sequential or simultaneous administration. This also applies to cocktail therapies, for example, the administration of three or more active ingredients.

In some embodiments, a compound provided herein can be used as a maintenance therapy agent for a patient undergoing anti-cancer treatment. For example, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, can be useful in reducing one or more symptoms associated with treatment with one or more anti-cancer therapies, such as complications of diarrhea or constipation and/or abdominal pain. See, for example, U.S. Publication No. 2015/0099762 and Hoffman, J. M. et al. Gastroenterology (2012) 142:844-854. Accordingly, the compound or pharmaceutically acceptable salt thereof, or composition provided herein can be administered to a patient to control one or more complications associated with cancer treatment (e.g., gastrointestinal complications such as diarrhea, constipation, or abdominal pain).

In some embodiments, a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, can be administered to a patient undergoing anti-cancer treatment (e.g., a patient experiencing an adverse event associated with cancer treatment, such as an immune adverse event or a gastrointestinal complication, including diarrhea, constipation, and abdominal pain). For example, a compound provided herein or a pharmaceutically acceptable salt thereof can be used to treat colitis or IBS associated with administration of a checkpoint inhibitor; see, e.g., Postow, M.A. et al. Journal of Clinical Oncology (2015) 33: 1974-1982. In some such embodiments, a compound provided herein or a pharmaceutically acceptable salt thereof can be formulated to provide low bioavailability and/or targeted delivery to the gastrointestinal tract. See, for example, U.S. Patent No. 6,531,152.

Also provided is a method of inhibiting RET kinase activity in a cell, comprising contacting said cell with a compound of formula I. In one embodiment, the contacting is performed in vitro. In one embodiment, the contacting is performed in vivo. In one embodiment, the contacting is performed in vivo, wherein said method comprises administering an effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, to a subject having a cell that has RET kinase activity. In some embodiments, the cell is a cancer cell. In one embodiment, the cancer cell is any cancer described herein. In some embodiments, the cancer cell is a RET-associated cancer cell. In some embodiments, the cell is a gastrointestinal tract cell.

Also provided is a method of inhibiting RET kinase activity in a mammalian cell, comprising contacting the cell with a compound of formula I. In one embodiment, the contacting is performed in vitro. In one embodiment, the contacting is performed in vivo. In one embodiment, the contacting is performed in vivo, wherein said method comprises administering an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof to a mammal having a cell that has RET kinase activity. In some embodiments, the mammalian cell is a mammalian cancer cell. In one embodiment, the mammalian cancer cell is any cancer described herein. In some embodiments, the mammalian cancer cell is a RET-associated cancer cell. In some embodiments, the mammalian cell is a gastrointestinal cell.

In this context, the term "contacting" refers to combining the moieties in an in vitro system or in an in vivo system. For example, contacting RET kinase with a compound provided herein includes administering the compound provided herein to an individual or patient, such as a human, that has RET kinase, as well as, for example, administering the compound provided herein to a sample containing a cellular or purified preparation containing RET kinase.

This document also provides a method for inhibiting cell proliferation in vitro or in vivo, comprising contacting a cell with an effective amount of a compound of formula IIV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition described herein.

The expression "effective amount" means that amount of the compound which, when administered to a patient in need of such treatment, is sufficient to (i) treat a disease or disorder associated with RET kinase, (ii) alleviate, improve or eliminate one or more symptoms of a particular disease, pathological condition or disorder, or (iii) delay the onset of one or more symptoms of a particular disease, pathological condition or disorder described herein. The amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof which corresponds to such an amount varies depending on such factors as the particular compound, the disease state and its severity, the properties (e.g., weight) of the patient in need of treatment, but can nevertheless be determined in a routine manner by one skilled in the art.

4. Pharmaceutical compositions and administration.

When used as a pharmaceutical, the compound of formula I-IV, including polymorphic forms and pharmaceutically acceptable salts thereof, can be administered in the form of pharmaceutical compositions. Such compositions can be prepared by methods well known in the pharmaceutical arts, and can be administered in a variety of ways, depending on whether local or systemic treatment is required, as well as the area to be treated. Administration can be local (including transdermal, epidermal, ophthalmic and mucosal, including intranasal, vaginal,

- 155 041220 oral and rectal delivery), pulmonary (e.g. by inhalation or insufflation of powders or aerosols, including administration via a nebulizer; intratracheal or intranasal), oral or parenteral. Oral administration may include a dosage form formulated for once-daily or twice-daily (BID) administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injections or infusions; or intracranial, e.g. intrathecal or intraventricular administration. Parenteral administration may be in the form of a single bolus dose or, for example, via a continuous perfusion dispenser. Pharmaceutical compositions and dosage forms for local administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Standard pharmaceutical carriers, aqueous, powder or oily bases, thickening agents and the like may be necessary or desirable.

The present document also provides pharmaceutical compositions that contain as an active ingredient a compound of formula I-IV or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable carriers (excipients). For example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form. In some embodiments, the composition is suitable for topical administration. In preparing the compositions provided in this invention, the active ingredient is typically mixed with an excipient, diluted with an excipient, or introduced into such a carrier in the form of, for example, a capsule, sachet, paper or other packaging. If the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a filler, carrier, or medium for the active ingredient. Thus, the compositions of the present invention can be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions or sterile packaged powders. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is a solid oral dosage form. In some embodiments, the composition is formulated in the form of a tablet or capsule.

The document further provides pharmaceutical compositions comprising a compound of formula I-IV or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising a compound of formula I-IV or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient can be prepared by intimately mixing a compound of formula I-IV or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof, with a pharmaceutical carrier, in accordance with conventional pharmaceutical formulation techniques. The carrier can take various forms depending on the desired route of administration (e.g., oral, parenteral). In some embodiments, the composition is a solid oral composition.

Suitable pharmaceutically acceptable carriers are well known in the art. A description of such pharmaceutically acceptable carriers is provided in The Handbook of Pharmaceutical Excipients, published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain.

Methods for formulating pharmaceutical compositions are described in many publications such as Pharmaceutical Dosage Forms: Tablets, Second Edition, Revised and Supplemented, Volumes 1-3, edited by Lieberman et al; Pharmaceutical Dosage Forms: Parenteral Medications, Volumes 1-2, edited by Avis et al; and Pharmaceutical Dosage Forms: Disperse Systems, Volumes 1-2, edited by Lieberman et al; published by Marcel Dekker, Inc.

In preparing compositions in oral dosage forms, any conventional pharmaceutical medium may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents, etc.; for solid oral preparations such as powders, capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrating agents include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Solid oral preparations may also be coated with substances such as sugars, or may be enteric coated to alter the primary site of their absorption. For parenteral administration, the carrier usually consists of sterile water, and other ingredients may be added to aid solubility or to aid preservation. Suspensions or solutions for

- 156 041220 injections can also be prepared using aqueous vehicles together with appropriate additives. The pharmaceutical compositions of the present invention contain, per unit form, for example, a tablet, capsule, powder, injection, teaspoonful, etc., such an amount of the active ingredient as is necessary to deliver the effective dose described herein.

Compositions containing a compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof may be formulated as unit dosage forms, each dose containing from about 5 to about 1000 mg (1 g), more usually from about 100 to about 500 mg of the active ingredient. The term unit dosage form refers to physically discrete units suitable as single doses for humans or other patients, each unit containing a predetermined quantity of the active material (i.e., a compound of formula I-IV or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof) calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

In some embodiments, the compositions provided herein comprise from about 5 mg to about 50 mg of the active ingredient. Those skilled in the art will appreciate that the compounds or compositions of these embodiments comprise from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, or from about 45 mg to about 50 mg of the active ingredient.

In some embodiments, the compositions provided herein comprise from about 50 mg to about 500 mg of the active ingredient. Those skilled in the art will appreciate that the compounds or compositions of these embodiments comprise from about 50 mg to about 100 mg, from about 100 mg to about 150 mg, from about 150 mg to about 200 mg, from about 200 mg to about 250 mg, from about 250 mg to about 300 mg, from about 350 mg to about 400 mg, or from about 450 mg to about 500 mg of the active ingredient. In some embodiments, the compositions provided herein comprise about 10 mg, about 20 mg, about 80 mg, or about 160 mg of the active ingredient.

In some embodiments, the compositions provided herein comprise from about 500 mg to about 1000 mg of the active ingredient. Those skilled in the art will appreciate that the compounds or compositions of these embodiments comprise from about 500 mg to about 550 mg, from about 550 mg to about 600 mg, from about 600 mg to about 650 mg, from about 650 mg to about 700 mg, from about 700 mg to about 750 mg, from about 750 mg to about 800 mg, from about 800 mg to about 850 mg, from about 850 mg to about 900 mg, or from about 900 mg to about 950 mg, or from about 950 mg to about 1000 mg of the active ingredient.

The daily dose of a compound of formula I-IV or a polymorphic form or pharmaceutically acceptable salt thereof may vary widely from 1.0 to 10,000 mg per adult per day or more, or any range therebetween.

For oral administration, the proposed compositions are preferably presented in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 10.0, 15.0, 25.0, 50.0, 100, 150, 160, 200, 250 and 500 mg of the active ingredient for symptomatic titration of the dose for a particular patient to be treated. An effective amount of the drug is typically administered at a dose of about 0.1 mg/kg to about 1000 mg/kg of body weight per day, or any range therebetween. Preferably, the range is from about 0.5 to about 500 mg/kg of body weight per day, or any range therebetween. More preferably, from about 1.0 to about 250 mg/kg of body weight per day, or any range therebetween. More preferably, from about 0.1 to about 100 mg/kg body weight per day, or any range therebetween. In one example, the range may be from about 0.1 to about 50.0 mg/kg body weight per day, or any amount or range therebetween. In another example, the range may be from about 0.1 to about 15.0 mg/kg body weight per day, or any range therebetween. In another example, the range may be from about 0.5 to about 7.5 mg/kg body weight per day, or any amount or range therebetween. Pharmaceutical compositions containing a compound of formula I-IV or a polymorphic form thereof, or a pharmaceutically acceptable salt, may be administered in a regimen of 1 to 4 times per day or as a single daily dose.

The active compound can be effective over a wide range of doses, and is typically administered in a pharmaceutically effective amount. Optimum doses for administration can be readily determined by one skilled in the art. It should therefore be understood that the actual amount of compound administered is typically determined by a physician and varies with relevant circumstances, including the route of administration, the actual compound administered, the potency of the drug, the condition being treated, and the severity of the disease state. In addition, factors associated with the particular patient being treated, including the patient's response, age, weight, diet, time of administration, and severity of the patient's symptoms, may also necessitate dosage adjustments.

In some embodiments, the compounds provided herein can be administered in an amount of about 1 mg/kg to about 100 mg/kg. In some embodiments, the compound provided herein can be administered in an amount of about 1 mg/kg to about 20 mg/kg, about

- 157 041220 mg/kg to about 50 mg/kg, from about 10 mg/kg to about 40 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 20 mg/kg to about 60 mg/kg kg or from about 40 mg/kg to about 70 mg/kg. For example, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg. In some embodiments, such administration may be once daily or twice daily (BID) administration.

In some embodiments, the compounds provided herein can be administered in an amount of about 10 mg twice daily (BID), 20 mg BID, about 40 mg BID, about 60 mg BID, about 80 mg BID, about 120 mg BID, about 160 mg BID, and about 240 mg BID. In some embodiments, each dose is administered at least 6 hours after the previous dose. In some embodiments, each dose is administered at least 12 hours after the previous dose.

In some embodiments, the compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof exhibits pH-dependent solubility at lower pH values. Accordingly, patients are also administered proton pump inhibitors (PPIs) and/or may require antacids to adjust the dose of the compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof (e.g., increasing the dose of the compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof). In some embodiments, the cytochrome P450 (CUP) isoform that metabolizes the compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof is CYP3A4. Accordingly, patients who are also taking agents that inhibit or induce CYP3A4 may require a dose adjustment of the compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof (e.g., an increase in the dose of the compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof in the case of a CYP3A4 inducer, or a decrease in the dose of the compound of formula I-IV or a polymorphic form or a pharmaceutically acceptable salt thereof in the case of a CYP3A4 inhibitor).

Those skilled in the art will appreciate that in vivo and in vitro testing using suitable, known and accepted cellular and/or animal models are predictive of the ability of an experimental compound to treat or prevent a given disorder.

Those skilled in the art will also appreciate that clinical trials in humans, including first-in-human trials, dose-finding trials, and efficacy trials, in healthy patients and/or in patients suffering from a given disorder, can be conducted according to methods well known in the clinical and medical arts.

5. Sets.

The present invention provides pharmaceutical kits suitable, for example, for the treatment of RET-associated diseases or disorders, such as cancer or irritable bowel syndrome (IBS), which comprise one or more containers containing a pharmaceutical composition that contains a therapeutically effective amount of a compound as provided herein. Such kits may further comprise, if desired, one or more different components of conventional pharmaceutical kits, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those skilled in the art. The kit may also include instructions, either in the form of inserts or labels, indicating the amount of components to be administered, instructions for administration, and/or instructions for mixing the components.

Examples

The following examples illustrate the present invention.

Example 1. Synthesis of a compound of formula I.

Intermediate connection A1 and A2.

6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and 4-bromo-6-methoxypyrazolo[1,5-a]pyridine (A2) N=\ N=\ a a

Part A. Preparation of O-(mesitylsulfonyl)hydroxylamine (intermediate R1).

Step 1. Preparation of tert-butyl (mesitylsulfonyl)oxycarbamate. TEA (14.46 mL, 48.01 mmol) was added dropwise with stirring to a solution of 2,4,6-trimethylbenzene-1-sulfonyl chloride (10.0 g, 45.72 mmol) and tert-butyl hydroxycarbamate (6.088 g, 45.72 mmol) in MTBE (100 mL) cooled to 0 °C. The resulting suspension was stirred at 0 °C for an additional 30 min and then warmed to room temperature. The reaction mixture was then diluted with water (100 mL) and adjusted to pH 4 with 1 N HCl( _aq ). The organic layer was dried ( _Na2SO4 ), filtered and concentrated to give the title compound initially as a yellowish oil which, when dried overnight under high vacuum, became _a white solid (12.89 g, 89% yield).

1H NMR ( _CDCl3 ): δ 7.66 (br s, 1H), 6.98 (s, 2H), 2.67 (s, 6H), 2.32 (s, 3H), 1.31 (s, 9H).

Step 2. Preparation of O-(mesitylsulfonyl)hydroxylamine (intermediate R1). To TFA (117 mL, 1521 mmol) at 0 °C was slowly added tert-butyl (mesitylsulfonyl)oxycarbamate (39.0 g, 124 mmol) over 25 min. The reaction mixture was stirred at 0 °C for 1.5 h and then quenched by successive addition of crushed ice and water. The resulting thick suspension was stirred vigorously at room temperature for 5 min. Avoiding drying of the filter cake, the solid was collected by careful filtration under vacuum, then washed with water (4 L) until the filtrate reached a pH of 6 (caution: risk of explosion of the dry compound at room temperature). The wet cake was then dissolved in dichloromethane (150 ml) and the resulting two-phase solution was separated. The dichloromethane layer was dried over _MgSO4 for 30 min and then filtered and washed with dichloromethane (420 ml) to give the title compound as a 0.22 M solution in dichloromethane.

Part B. Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and 4-bromo-6methoxypyrazolo[1,5-a]pyridine (A2).

Step 1. Preparation of 1-amino-3-bromo-5-methoxypyridine-1-ium-2,4,6-trimethylbenzenesulfonate. To a solution of O-(mesitylsulfonyl)hydroxylamine (intermediate R1) (26.6 g, 117 mmol) in DCM (570 mL) cooled to 0 °C was added 3-bromo-5-methoxypyridine (22.1 g, 117 mmol) portionwise. The reaction mixture was stirred for 1 h at 0 °C, then treated with additional 3-bromo-5-methoxypyridine (250 mg, 1.39 mmol) and stirred for an additional 2 h at 0 °C. The reaction mixture was diluted with _Et2O (600 mL), stirred at 0°C for 10 min, and then filtered under vacuum, washing with _Et2O (3x250 mL). After the volume had been reduced by about 1/3, an additional precipitate was obtained from the filtrate, which was collected by filtration. Both filter cakes were dried in vacuo to give the title compound (39.3 g, 83% yield).

1H NMR (CDCl ₃ ) δ 9.25 (brs, 1H), 8.99 (m, 1H), 8.74 (m, 1H), 7.46 (m, 1H), 6.83 (s, 2H ), 3.92 (s, 3H), 2.65 (s, 6H), 2.22 (s, 3H).

Step 2. Preparation of ethyl 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate and ethyl 4-bromo6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate. To a magnetically stirred white suspension of 1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate (33.24 g, 82.42 mmol) in DMF (82 mL) at room temperature was added TEA (22.98 mL, 164.8 mmol), followed by dropwise addition of ethyl propiolate (16.71 mL, 164.8 mmol). After vigorous stirring for 2 days, the reaction mixture was slowly quenched by addition in portions to rapidly stirred ice water (820 mL). The mixture was stirred at room temperature for 10 min and then filtered under vacuum. The collected solid was washed with water and air dried to give the title compounds as an orange solid with an isomeric ratio of about 4:1 (by 1H NMR) with the 6-Br isomer as the major isomer (21 g). The wet solid isomeric mixture (about 75% w/w) was used directly in step 3 without further purification. MS (atm. pressure chemical ionization, apci) m/z = 298.9, 300.9 (M+H). The regioisomeric ratio was determined by the chemical shift of MeO on 1H NMR ( _CDCl3 ) δ 3.98 (6-Br isomer) versus 3.83 (4-Br isomer).

Step 3: Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and 4-bromo-6-methoxypyrazolo[1,5-a]pyridine (A2): An isomeric mixture of ethyl 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate and ethyl 4-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate from step 2 (15 g, 50.1 mmol) was added to 48% HBr (114 mL) with stirring, then heated at 80 °C for 90 min, then stirred at room temperature overnight. The resulting suspension was vacuum filtered and washed with water. The aqueous filtrate and filter cake were processed independently. The filter cake was dissolved in MTBE and filtered under vacuum to remove insoluble impurities. _The MTBE filtrate was dried over anhydrous _Na2SO4 , filtered and concentrated in vacuo to give 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (intermediate A1) as a beige solid (ca. 98:2 6-/4- Br; 5.08 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1H NMR ( _CDCl3 ) δ 8.26 (m, 1H), 7.82 (d, 1H), 6.61 (m, 1H), 6.43 (m, 1H), 3.94 (s, 3H).

The initial aqueous filtrate of the reaction mixture was independently extracted with EtOAc (2x500 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. The crude residue was dissolved in DCM (50 mL) and then filtered to remove insoluble solids. Concentration of the DCM filtrate in vacuo followed by chromatography on silica (0 to 50% EtOAc/hexanes) provided a second crop of 6-bromo-4-methoxypyrazolo[1,5a]pyridine (Intermediate A1) as a white solid (upper spot R _f , 2.06 g) and the minor isomer of the title compound 4-bromo-6-methoxypyrazolo[1,5a]pyridine (Intermediate A2), also as a white solid (lower spot R f , 1.32 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1H NMR (CDCl3) δ 8.02 (m, 1H), 7.85 (d, 1H), 7.17 (d, 1H), 6.55 (m, 1H), 3.80 (s, 3H).

Intermediate connection A3

- 159 041220

6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde.

To a cooled to 0 °C solution of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (intermediate A1, 0.75 g, 3.303 mmol) in DMF (33 mL) was slowly added _POCl3 (0.92 mL, 9.909 mmol). The reaction mixture was warmed to room temperature and stirred for 4 h and then diluted with H2O (30 mL). The resulting suspension was basified to pH 9-10 with 1 M NaOH( _aq ), then stirred for 1 h and vacuum filtered, then washed successively with H2O (25 mL) and MTBE (50 mL) to give the title compound (0.76 g, 90% yield). MS (apci) m/z = 256.9 (M+H).

Intermediate connection A4 No.\ jCT

Step 1. Preparation of (E)-6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime. To a suspension of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (intermediate A3, 0.76 g, 3.0 mmol) and hydroxylamine hydrochloride (0.31 g, 4.5 mmol) in EtOH (40 mL) was added water (20 mL) and the reaction mixture was stirred at 50 °C for 4 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. The residue was suspended in water, then treated with saturated NaHCO ₃ ( _aq ) and filtered under vacuum. The solid was washed successively with _H2O (25 mL) and MTBE (50 mL) to give the title compound (0.68 g, 84% yield). MS (apci) m/z = 271.9 (M+H).

Step 2. Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile. A solution of (E)-6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (17.15 g, 63.50 mmol) in acetic anhydride (707 mL, 7.49 mol) was heated at 120 °C overnight. After subsequent distillation to remove acetic anhydride, the resulting residue was dried in vacuo to give the title compound (15.92 g, 99.4% yield).

1H NMR (CDCI3) δ 8.32 (m, 1H), 8.12 (s, 1H), 6.74 (m, 1H), 4.03 (s, 3H).

Intermediate connection A5

3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate.

Step 1. Preparation of 4-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile. To a solution of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile (intermediate A4, 50 g, 198.4 mmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (49.53 g, 238.0 mmol) in dioxane (660 mL) was added ₂ M _Na2CO3 ( _aq ) (297.5 mL, 595.1 mmol). The reaction mixture was purged with nitrogen for 20 min, then Pd( _PPh3 ) ₄ (4.584 g, 3.967 mmol) was added, followed by purging with nitrogen for an additional 5 min. The reaction mixture was heated at 80 °C for 18 h, then cooled to room temperature and stirred vigorously for 2 h. The suspension was vacuum filtered, washed successively with _H2O (2 x 300 mL) and MTBE (3 x 300 mL), then dried in vacuo overnight to give the title compound, which was used in the next step without further purification (52.62 g). MS (apci) m/z = 254.1 (M+H).

Step 2. Preparation of 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile. To a suspension of 4-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (52.62 g, 207.8 mmol) in DCE (2 L) was added _AlCl3 (92.86 g, 696.42 mmol) and the reaction mixture was stirred at 80 °C for 3 h. Additional AlCl3 (2.5 g, 18.75 mmol) was added and the reaction mixture was refluxed overnight. After cooling to room temperature, the reaction mixture was diluted with DCE (1 L) and then quenched with _H2O (5 x 500 mL). The mixture was stirred at room temperature for 3 h, then the resulting suspension was filtered under vacuum and the filter cake was dried in a vacuum oven (40 °C) to give the title compound, which was used in the next step without further purification (43.69 g). MS (apci) m/z = 239.9 (M+H).

1H NMR (d ⁶ -DMSO) δ 11.38 (s, 1H), 8.74 (d, 1H), 8.50 (s, 1H), 8.21 (s, 1H), 7.94 (s, 1H), 6.96 (d, 1H), 3.88 (s, 3H).

Step 3. Preparation of 3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate. To a suspension of 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5a]pyridine-3-carbonitrile (43.69 g, 182.6 mmol) in DMA (365 mL) was added DIEA (63.6 mL, 365.3 mmol), followed by 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (71.77 g, 200.9 mmol). The resulting solution was stirred at room temperature for 2 h and then slowly poured into H ₂ O (4 L). The resulting suspension was stirred for 2 h, then filtered under vacuum. The filter cake was washed with _H2O (3 x 500 mL) and air dried overnight. The filter cake was then dissolved in DCM (1.6 L) and the phases of the resulting biphasic mixture were separated. The organic layer was dried over anhydrous _MgSO4 , filtered through Celite® and washed with DCM. The combined organic layers were concentrated to afford the title compound as 90% pure light brown solid (64.3 g, 95% yield). The purity of the title compound can be further improved to >95% by silica chromatography (0-90% acetone/hexanes). ¹⁹ F NMR ( _CDCl3 ) δ -72.0.

1H NMR (CDCl ₃ ) δ 8.66 (d, 1H), 8.29 (s, 1H), 7.77 (d, 1H), 7.70 (s, 1H), 7.55 (d, 1H), 4.01 (s, 3H). Intermediate A6

tert-butyl-4-(5-(3-cyano-6-(1-methyl-1H-πyrazol-4-yl)πyrazolo[1,5-a]πpyridin-4-yl)πpyridin-2yl)piperazin-1 -carboxylate.

To a mixture of 3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate (intermediate A5; 10.0 g, 26.9 mmol) and tert-butyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperazine-1-carboxylate (12.6 g, 32.3 mmol) in dioxane (250 mL) was added 2 M _Na2CO3 (in _Odn ) (14.3 g, 135 mmol) and the reaction mixture was purged with nitrogen for 15 min, then _Pd2 ( _dba ) ₃ (1.23 g, 1.35 mmol) was added and X-Phos (2.57 g, 5.39 mmol). The mixture was purged with nitrogen for an additional 5 min and then heated at 80 °C overnight. After cooling to room temperature, the reaction mixture was poured into H ₂ O (1.5 L) and stirred for 2 h. The resulting suspension was filtered and washed successively with H ₂ O (3 x 200 mL), MTBE (4 x 100 mL), and hexanes (4 x 100 mL) to give the title compound as a solid after drying in vacuo overnight (12 g, 92% yield). MS (apci) m/z = 485.2 (M+H).

Intermediate connection A7

6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile dihydrochloride.

To a solution of tert-butyl 4-(5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4yl)pyridin-2-yl)piperazine-1-carboxylate (A6, 12.0 g, 24.77 mmol) in MeOH (12 mL) and DCM (50 mL) was added HCl (5-6 M in iPrOH, 49.53 mL, 247.7 mmol). After stirring at room temperature for 21 h, the reaction mixture was diluted with MeOH (50 mL) and DCM (50 mL). The suspension was stirred at room temperature until LCMS indicated the reaction was complete. The reaction mixture was filtered, washed with _Et2O (5x50 mL) and then dried for 19 h in a vacuum oven at 45°C to give the title compound (9.97 g, 88% yield). MS (apci) m/z = 385.1 (M+H).

Compound of formula I

4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4yl)pyrazolo[1,5-a]pyridine-3-carbonitrile.

Method 1. A room temperature solution of 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile dihydrochloride (A7; 0.125 g, 0.273 mmol) in dry DMA (2.5 mL) was treated with TEA (114 μL, 0.820 mmol), Me ₄ N (AcO) ₃ BH (144 mg, 0.547 mmol), and 6-methoxynicotinaldehyde (0.0750 g, 0.547 mmol). The resulting mixture was stirred overnight.

- 161 041220 at room temperature, then quenched with water and CHCl ₃ and left stirring for 30 min. The resulting two-phase mixture was filtered through a sintered PS filter and the aqueous layer was washed

CHCl ₃ . The organic filtrate was concentrated in vacuo and the C18 residue was purified by reverse phase chromatography (5-90% ACN/water as gradient eluent) to give the title compound (56 mg, 41% yield). MS (apci) m/z = 506.0 (M+H).

Method 2. A reaction vessel was charged with 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile tetrahydrochloride (A7; 19.0 g) and 6-methoxynicotinaldehyde (7.37 g). DMSO (247 mL) was added followed by triethylamine (15 mL). The yellow suspension was stirred at room temperature for ~2.5 h and then sodium triacetoxyborohydride (15.2 g) was added in one portion and the reaction mixture was warmed to 30 °C and stirred overnight. The reaction was deemed complete by HPLC and the mixture was cooled in an ice/water bath to 19 °C. Water (550 mL) was added slowly, maintaining the temperature below 30 °C. The suspension was stirred for 3.5 h and then filtered and the filter cake was washed with water (2 x 285 mL). The solid was dried in a vacuum oven at 45 °C to give the title compound (18.0 g, 99.4%). The crystalline solid was analyzed by X-ray diffraction to give a diffraction pattern consistent with Form A of formula I.

Example 2. Synthesis of compounds of formula II-IV.

Intermediate connection B1

N=\ ί T

4-bromo-6-hydroxypyrazolo[1,5-α]pyridine-3-carbonitrile.

Step 1. Preparation of 1-amino-3-bromo-5-methoxypyridine-1-ium-2,4,6-trimethylbenzenesulfonate. To a solution of O-(mesitylsulfonyl)hydroxylamine (intermediate R1, 26.6 g, 117 mmol) in dichloromethane (570 mL) cooled to 0 °C was added 3-bromo-5-methoxypyridine (22.1 g, 117 mmol) portionwise. The reaction mixture was stirred for 1 h at 0 °C, then treated with additional 3-bromo-5-methoxypyridine (250 mg, 1.39 mmol) and stirred for an additional 2 h at 0 °C. The reaction mixture was diluted with _Et2O (600 mL), stirred at 0°C for 10 min, and then filtered under vacuum and washed with _Et2O (3x250 mL). After the volume had been reduced by about 1/3, an additional amount of precipitate was obtained from the filtrate, which was collected by filtration. Both filter cakes were dried in vacuo to give the title compound (39.3 g, 83% yield).

^1H NMR ( _CDCl3 ): δ 9.25 (brs, 1H), 8.99 (m, 1H), 8.74 (m, 1H), 7.46 (m, 1H), 6.83 (s , 2H), 3.92 (s, 3H), 2.65 (s, 6H), 2.22 (s, 3H).

Step 2. Preparation of ethyl 6-bromo-4-methoxypyrazolo[1,5-α]pyridine-3-carboxylate and ethyl 4-bromo6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate. To a magnetically stirred white suspension of 1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate (33.24 g, 82.42 mmol) in DMF (82 mL) at room temperature was added TEA (22.98 mL, 164.8 mmol), followed by dropwise addition of ethyl propiolate (16.71 mL, 164.8 mmol). After vigorous stirring for 2 days, the reaction mixture was slowly quenched by addition in portions to rapidly stirred ice water (820 mL). The mixture was stirred at room temperature for 10 min and then filtered under vacuum. The collected solid was washed with water and air dried to give the title compounds as an orange solid with an isomeric ratio of about 4:1 (by 1H NMR) with the 6-Br isomer as the major isomer (21 g). The wet solid isomeric mixture (about 75% w/w) was used directly in step 3 without further purification. MS (apci) m/z = 298.9, 300.9 (M+H). The regioisomeric ratio was determined by the chemical shift of MeO on 1H NMR ( _CDCl3 ) δ 3.98 (6-Br isomer) versus 3.83 (4-Br isomer).

Step 3. Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (Intermediate B1) and 4-bromo-6-methoxypyrazolo[1,5-a]pyridine. An isomeric mixture of ethyl 6-bromo-4-methoxypyrazolo[1,5a]pyridine-3-carboxylate and ethyl 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate from step 2 (15 g, 50.1 mmol) was added to 48% HBr (114 mL) with stirring, then heated at 80 °C for 90 min, then stirred at room temperature overnight. The resulting suspension was vacuum filtered and washed with water. The aqueous filtrate and filter cake were worked up independently. The filter cake was dissolved in MTBE and filtered under vacuum to remove insoluble impurities. _The MTBE filtrate was dried over anhydrous _Na2SO4 , filtered and concentrated in vacuo to give 6-bromo-4-methoxypyrazolo[1,5-a]pyridine as a beige solid (ca. 98:2 6-/4- Br; 5.08 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1H NMR ( _CDCl3 ): δ 8.26 (m, 1H), 7.82 (d, 1H), 6.61 (m, 1H), 6.43 (m, 1H), 3.94 (s, 3H).

The initial aqueous filtrate of the reaction mixture was independently extracted with EtOAc ( _2x500 mL). The combined organic layers were dried ( _Na2SO4 ), filtered and concentrated in vacuo. The crude residue was dissolved in DCM (50 mL) and then filtered to remove insoluble solids.

- 162 041220 substances. Concentration of the DCM filtrate in vacuo followed by chromatography on silica (0 to 50% EtOAc/hexanes) gave a second crop of 6-bromo-4-methoxypyrazolo[1,5a]pyridine (intermediate 1) as a white solid (upper spot Rf, 2.06 g) and a minor isomer of the title compound 4-bromo-6-methoxypyrazolo[1,5-a]pyridine, also as a white solid (lower spot Rf, 1.32 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1H NMR ( _CDCl3 ): δ 8.02 (m, 1H), 7.85 (d, 1H), 7.17 (d, 1H), 6.55 (m, 1H), 3.80 (s, 3H).

Step 4. Preparation of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde. A solution of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine (5.0 g, 22 mmol) in DMF (220 mL) was cooled to 0 °C and then slowly treated with POCl ₃ (6.2 mL, 66 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture was cooled to 0 °C, quenched with water (220 mL), and basified with 6 M NaOH ( _BODH ) to pH 9-10. The reaction mixture was stirred for 1 h and then vacuum filtered. The solid was washed successively with water (3 x 50 mL) and MTBE (3 x 50 mL). The collected solid was suspended in DCM (500 mL) and washed in a sonication bath for 30 min, then filtered under vacuum. The filtrate was saved, and the filter cake was dissolved in water (300 mL) and extracted with _DCM . The organic extracts as well as the saved DCM filtrate were combined and dried over anhydrous _Na2SO4 , then filtered and concentrated in vacuo to give the title compound (4.84 g, 86% yield). MS (apci) m/z = 256.9 (M+H).

Step 5. Preparation of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime. To a suspension of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (4.84 g, 19.0 mmol) in EtOH (253 mL) at room temperature were added water (127 mL) and hydroxylamine hydrochloride (1.98 g, 28.5 mmol). After stirring at 50 °C overnight, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was suspended in water (150 mL) and then slowly quenched with saturated NaHCO ₃ ( _BODH ) solution (30 mL). After stirring for 1 h at room temperature, the suspension was filtered under vacuum and the filter cake was washed successively with _H2O (500 mL) and MTBE (100 mL) to give the title compound as a 2:1 E/Z mixture (5.13 g, quantitative yield), which was used in the next step without further purification. MS (apci) m/z = 271.9 (M+H).

Step 6. Preparation of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile. An E/Z mixture of 4-bromo6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (4.95 g, 18.33 mmol) in acetic anhydride (172.9 mL, 1833 mmol) was stirred at 140 °C for 25 h and then cooled to room temperature. The resulting suspension was further cooled in an ice bath for 15 min and then vacuum filtered and washed successively with water (200 mL) and MTBE (300 mL) to give the title compound (3.74 g, 81% yield).

1H NMR (d ⁶ -DMSO): δ 8.70 (s, 1H), 8.60 (s, 1H), 7.78 (s, 1H), 3.83 (s, 3H).

Step 7. Preparation of 4-bromo-6-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile: A suspension of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile (50.0 g, 198.4 mmol) in DCE (500 mL) was treated with AlCl ₃ (79.34 g, 595.1 mmol). Under N ₂ ( _Γ ) atmosphere, the resulting mixture was stirred for 19 h at 76 °C, then cooled to room temperature. Using THF (1750 mL) as washing solvent, the reaction mixture was poured into a mechanically stirred suspension of sodium sulfate decahydrate (10 equiv, 639 g) in THF (1000 mL). After stirring overnight at room temperature, the resulting suspension was filtered and the solid was washed with additional THF (2x250 mL). The filtrate was concentrated in vacuo and the resulting solid was dried under high vacuum for 3 days to give the title compound (46.18 g, 98% yield) in sufficient purity for further use.

¹ H NMR (d ⁶ -DMSO): δ 10.48 (s, 1H), 8.58 (s, 1H), 8.38 (d, 1H), 7.64 (3, 1H).

Intermediate connection B2

4-bromo-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile.

In a pressure flask, a mixture of 4-bromo-6-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile (intermediate B1; 10.0 g, 42.0 mmol) and K ₂ CO ₃ ( _mв ) (17.4 g, 126 mmol) in DMF (50 mL) was treated with 2,2-dimethyloxirane (36.9 mL, 420 mmol). After sealing the flask, the reaction mixture was stirred for 12 h at 60 °C, then for 12 h at 85 °C. The mixture was allowed to cool to room temperature. The room temperature mixture was poured into water (400 mL), then stirred for 1 h at room temperature. The resulting suspension was filtered under vacuum and the filter cake was washed with water. The solid was collected and dried in vacuo to afford the pure title compound (11 g, 84% yield).

- 163 041220

Intermediate connection B3

4-(6-fluoropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-3-carbonitrile.

A mixture of 4-bromo-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile (intermediate B2; 10.0 g, 32.2 mmol), 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)pyridine (10.8 g, 48.4 mmol) and Pd(PPh ₃ ) ₄ (1.12 g, 0.967 mmol) in dioxane (200 mL) was treated with 2 M Na ₂ CO ₃ ( _Bo 2 _H ) (64.5 mL, 129 mmol). The resulting mixture was bubbled with Ar _(g) and then stirred for 12 h at 85 °C under N ₂ ( _G ) atmosphere. After cooling to room temperature, the resulting mixture was poured into cold water (1.5 L). The pH of the mixture was adjusted to about pH 6 by adding 10% citric acid. After stirring for 1 h at room temperature, the resulting suspension was filtered under vacuum. The solid was collected and dried in vacuo to neatly afford the title compound (10 g, 95% yield).

Intermediate connection B4

4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile dihydrochloride.

Step 1. Preparation of tert-butyl 3-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5a]pyridin-4-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate. A mixture of 4-(6-fluoropyridin3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile (Intermediate B3; 1.70 g, 8.55 mmol), 3,6-diazabicyclo[3.1.1]heptane-6-carboxylic acid tert-butyl ester (1.70 g, 8.55 mmol) and _K2CO3 ( _{s )} (7.88 g, 57.0 mmol) in DMSO (7 mL) was stirred for 12 h at 90 °C. The resulting thick suspension was diluted with additional DMSO (2 mL) and stirred for 12 h at 90 °C. The mixture was cooled to room temperature and diluted with water (100 mL). The aqueous mixture was washed with DCM. The combined organic extracts were dried over anhydrous _MgSO4 ( _s ), filtered and concentrated in vacuo. The crude residue was purified by silica chromatography (30-80% EtOAc/hexanes as gradient eluent system) to afford the pure title compound (2.87 g, 100% yield). MS (apci) m/z = 505.2 (M+H).

Step 2. Preparation of 4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile dihydrochloride.

A solution of tert-butyl 3-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-4yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (see step 1; 3.05 g, 6.04 mmol) in DCM (20 mL) was treated with 4 N HCl in dioxanes (15.1 mL, 60.4 mmol). The resulting mixture was stirred for 12 h at room temperature and then concentrated in vacuo. The crude residue was diluted with DCM and toluene, sonicated, then concentrated in vacuo to give the title compound as the dihydrochloride salt (2.44 g, quantitative yield). MS (apci) m/z = 405.2 (M+H).

Compound of formula II

6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6diazabicyclo[3.1.1]heptan-3-yl)pyridin-3 -yl)pyrazolo[1,5-a]pyridin-3-carbonitrile.

To a mixture of 4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile dihydrochloride (Intermediate B4, 6.0 g, 12.6 mmol) in DCM (30 mL) was added triethylamine (5.3 mL, 37.7 mmol), followed by 6-methoxynicotinaldehyde (2.59 g, 18.9 mmol) and then sodium triacetoxyborohydride (5.33 g, 25.1 mmol) in one portion with stirring. Additional DCM (30 mL) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into water (200 mL) and extracted with DCM (200 mL). After phase separation, the organic layer was washed with water (2x200 mL). The combined aqueous washings were again extracted with DCM (200 mL). The volume

- 164 041220 The combined organic extracts were washed with brine (250 mL), passed through a fritted phase separator, and treated with activated charcoal (Darco G-60, 6 g). After stirring at room temperature for 2 h, the mixture was vacuum filtered, washing with DCM (3 x 10 mL). The filtrate was treated with 3-(trimethoxysilyl)propane-1-thiol (11 g, 7.4 mmol) and stirred on a magnetic stirrer overnight. The mixture was vacuum filtered and concentrated on a rotary evaporator to about 200 mL. To the DCM solution obtained above, heptane (150 mL) was added portionwise with stirring until a cloudy solution was obtained. After 90 min of stirring, the white suspension was filtered under vacuum, washed with heptane (200 mL) to give the title product as a fine crystalline white powder (3.0 g, 59%).

Recrystallization of compound of formula II in DMSO/water mixture was carried out as follows. Compound of formula II (10.1 g) and DMSO (110 mL) were charged into a reaction flask. The mixture was heated to 50 °C until the solid was completely dissolved. The mixture was cooled to 25 °C and filtered through a fine filter. DMSO (10 mL) was charged through the filter as a washing solution. The resulting solution was heated to 45 °C and water (5 mL) was slowly added. The mixture was stirred for 30 min to obtain a seed layer. Water (25 mL) was added over 1 h and the suspension was left to mature at 45 °C for another h. Then the suspension was allowed to cool to 25 °C and stirred for 2 h. The suspension was filtered and the filter cake was washed with water (20 mL x 3), MeOH (20 mL x 2), and MTBE (20 mL x 2). The filter cake was dried at room temperature in a vacuum oven to yield 9.35 g (74%) of the title compound. MS (apci) m/z = 526.2 (M+H).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 8.64 (d, 1H, J=2.3 Hz), 8.55 (s, 1H), 8.38 (d, 1H, J=2.3 Hz), 8.04 (d, 1H, J=2.3 Hz), 7.80 (dd, 1H, J=8.6, 2.3 Hz), 7.64 (dd, 1H, J=8.6, 2.3 Hz), 7.27 (d, 1H, J=2.0 Hz), 6.76 (d, 1H, J=8.6 Hz), 6.73 (d, 1H, J=8.2 Hz), 4.67 (s, 1H), 3.85 (s, 2H), 3.79 (s, 3H), 3.72 (d, 2H, J=12.5 Hz), 3.64 (d, 2H, J=5.9 Hz), 3.51 (shd, 2H), 3.47 (s, 2H), 2.47 (m, 1H), 1.55 (d, 1H), 1.20 (s, 6H).

Compound of formula III

O

6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3 yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile.

To a mixture of 4-(6-(3,6-diazabicyclo[3.1.1 ]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile dihydrochloride (intermediate B4, 4.50 g, 9.43 mmol) in DMSO (63 mL) was added 6-methoxynicotinic acid (1.73 g, 11.3 mmol), followed by Hunig's base (5.25 mL, 30.2 mmol) and HATU (4.30 g, 11.3 mmol). The mixture was stirred at room temperature for 1 h, then poured into a mixture of water (500 mL) and saturated _NaHCO3 (50 mL), then stirred for an additional 4 h. The suspension was vacuum filtered and the collected solid was first triturated with MTBE (100 mL) then purified by flash chromatography on silica (1-15% MeOH in DCM with 1% NH4OH) to give the title product. The resulting product was recrystallized by dissolving in a minimum amount of DCM then slowly adding MTBE to form a suspension. Filtration followed by drying under high vacuum at 40-45 °C for 2 days gave the title product as a crystalline white powder (4.0 g, 78.6%).

Further recrystallization of the title product in _CH3CN /water was carried out as follows. A mixture of the title product (37 g) in CH3CN (222 mL) was heated to reflux to give a clear solution. Water (333 mL) was then added slowly at the same temperature with stirring. After the addition, heating was stopped and the mixture was allowed to cool to room temperature and stirred overnight. The solid was collected by vacuum filtration and dried under high vacuum at 40-45 °C overnight to give the title product as a crystalline white powder (24 g, 65%).

1H NMR (DMSO-d ⁶ ) δ 8.60-8.65 (d, 1H), 8.53 (s, 1H), 8.49-8.51 (m, 1H), 8.28-8.31 (d, 1H), 7.91-7.95 (m, 1H), 7.73-7.78 (m, 1H), 7.23-7.25 (m, 1H), 6.81-6.85 (m, 1H), 6.65-6.69 (d, 1H), 4.84-4.94 (bm, 1H), 4.66 (s, 1H), 4.51-4.63 (bm, 1H), 4.04-4.20 (bm, 1H), 3.88 (s, 3H), 3.83 (s, 2H), 3.60-3.63 (m, 2H), 3.42-3.53 (sm, 1H), 2.75-2.85 (m, 1H), 1.63-1.69 (m, 1H), 1.18 (s, 6H). MS (apci) m/z = 540.2 (M+H).

- 165 041220

Compound of formula IV

6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile.

To a suspension of 4-(6-fluoropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile (intermediate B3, 90 mg, 0.276 mmol) in DMSO (2 mL) was added DIEA (193 μL, 1.10 mmol), followed by the addition of 4-(pyridin-2-ylmethyl)piperidin-4-ol hydrochloride (69 mg, 0.303 mmol). The reaction mixture was stirred at 90 °C for 60 h then directly purified by C18 reversed-phase chromatography (using 5-95% acetonitrile in water with 0.1% TFA as a gradient eluent). Fractions containing the desired product were combined, partially concentrated in vacuo to remove ACN, then partitioned between saturated _NaHCO3 ( _bog ) and DCM. The biphasic mixture was extracted with additional DCM (2x). The combined organic extracts were dried over anhydrous _MgSO4 ( _s ), filtered, and concentrated in vacuo. The residue was sonicated in _Et2O (2 mL) and then concentrated in vacuo to afford the title compound (48 mg, 35% yield). MS (apci) m/z = 499.2 (M+H).

Further recrystallization and isolation of Form A: The compound of formula IV (10 g) was added to the reaction vessel, DMSO (50 mL) was added and the mixture was stirred. After 10 min, the compound of formula IV was completely dissolved and then water (4 x 5 mL, total 20 mL) was added dropwise. After stirring for 10 min, a fine suspension was formed. Additional water (3 x 5 mL, total 15 mL) was added and after stirring for 30-60 min, the suspension was filtered. The filter cake was washed with water (10 mL), MTBE (50 mL) and then the solid was dried at room temperature under vacuum to yield 9.2 g of the compound of formula IV. The crystalline solid was analyzed by DSC and XRD to give the corresponding endotherm and the diffraction pattern of Form A for the compound of formula IV.

Example 3. Selection of polymorphic salts for a compound of formula I.

A. Solubility tests of the free base of the compound of formula I.

The solubility of the free base of the compound of formula I was tested in 13 solvents at 25 and 60°C. The solubility in mixed solvent systems such as DCM/EtOH, DMSO/EtOH, DMSO/water, THF/EtOH and THF/water was also determined.

Solubility tests were performed as follows. 10-20 mg of the free base were stirred in 1 mL of solvent overnight at room temperature; the solubility at 25°C was recorded. The samples were then heated to 60°C. If the solid dissolved upon heating, additional free base was added in 3-5 mg increments. The solubility test results are presented in Tables 5-10 and Figs. 1A-1E.

Table 5

The total solubility of the free base of the compound of formula I is

Solvent Solubility at 25 °C (mg/ml) Does the solid dissolve at 60°C? EtOAc 0.36 No iPAc 0.25 No MeOH 0,07 No EtOH 0,04 No toluene 0.35 No acetone 0.44 No IPA 0 No IEC 0.69 No THF 2.59 Yes (10.2-13.0 mg/ml) ACN 0.33 No DMA 12.9 >40 mg/ml dhe 4.6 Not studied DXM 15,5 N/A water 0,07 N/A 0.1 MNS1 1,5 Not studied

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The free base exhibited solubility of 2.59 mg/mL, 12.9 mg/mL and 15.5 mg/mL in THF,

DMA and DCM, respectively. For most of the solvent systems studied, the free base did not dissolve after heating to 60°C. 10-13 mg of the free base dissolved in THF at 60°C, although the solution became milky at 13 mg. The solubility study in DMA at 60°C was stopped after a total of 40 mg of free base had been added.

Table 6

Solubility of the free base of compound of formula I in DCM/EtOH at room temperature

% DXM in EtOH Solubility (mg/ml) 0 0,05 10 0,06 20 0.26 30 0.87 40 2.53 50 7.42 60 16.6 70 22.7 80 29,0 90 34.2 100 17.3

The solubility of the free base in DCM/EtOH mixtures is shown in Fig. 1A and Table 6, above.

Table 7

Solubility of the free base of compound of formula I in DMSO/EtOH at room temperature

% DMSO in EtOH Solubility (mg/ml) 0 0,05 10 0.09 20 0.18 30 0.39 40 0.61 50 1D4 60 1.80 70 2.57 80 4.28 90 4.44 100 6.96

The solubility of the free base in DMSO/EtOH mixtures is shown in Fig. 1B and Table 7, above.

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Table 8

Solubility of the free base of the compound of formula I in DMSO/H2O at room temperature

% DMSO in _H2O Solubility (mg/ml) 0 0 10 0.09 20 0,08 30 0.09 40 0,10 50 0.09 60 0,10 70 0,15 80 0.35 90 1.52 100 6.96

The solubility of the free base in DMSO/H2O mixtures is shown in Fig. 1C and Table 8 above.

Table 9

Solubility of the free base of compound of formula I in THF/EtOH at room temperature

% THF in EtOH Solubility (mg/ml) 10 0.29 20 0.34 30 0.55 40 0.82 50 1.38 60 1.97 70 2.51 80 3.09 90 3.18 100 2.59

The solubility of the free base in THF/EtOH mixtures is shown in Fig. 1D and Table 9, above.

Table 10

Solubility of the free base of the compound of formula I in THF/H2O at room temperature

% THF in EtOH Solubility (mg/ml) 10 0,15 20 0.13 30 0.35 40 1.36 50 2.50 60 3.76 70 6.17 80 6.65 90 6.56 100 2.59

The solubility of the free base in THF/ _H2O mixtures is shown in Fig. 1E and Table 10, above.

Research was conducted to determine the conditions for possible recrystallization of free os

- 168 041220 novelties of the compound of formula I, as shown in Table 11.

Table 11

Attempts to recrystallize the free base of the compound of formula I

Scale (g) Solvent (vol.)// Antisolvent (vol.) Temperature (°C) Time (hrs.) LTF (mg/ml) Notes 0,05 DMSO (18)// EtOH 200 proof (27) from 70 °C to room temperature 16 1,20 The free base was dissolved in DMSO at 70 °C. EtOH was added slowly. A solid formed slowly after 1 h of cooling. Cooled overnight. Filtered. Isolated 78.6%. 0,10 DMSO (18)// EtOH 200 proof (25) from 70 °C to room temperature 16 1,20 The free base was dissolved in DMSO at 70 °C. EtOH was added slowly until turbid. A solid formed rapidly at 70 °C. Cooled overnight. Filtered. Isolated 83.6%. 0,10 DMSO (18)// EtOH 200 proof (18) from 70 °C to room temperature 16 2.26 The free base was dissolved in DMSO at 70 °C. EtOH was added slowly until turbid. Cooled overnight. Filtered. Isolated 80.5%.

5.0 DMSO (20)// EtOH 200 proof (20) from 70 °C to room temperature 16 1,6 The free base was dissolved in DMSO at 70 °C. EtOH was added slowly until turbid. Cooled overnight. Filtered. 88% was recovered. 0,12 DMSO (18)//EUN 200 proof (33) from 70 °C to room temperature 16 1,00 The free base was dissolved in DMSO at 70 °C. EtOH was added slowly until turbid. Cooled overnight. Filtered. 85% was recovered. 0,10 DMSO (18)//EUN 200 proof (28) from 70 °C to room temperature 16 1.03 The free base was dissolved in DMSO at 70 °C. Cooled to 40 °C, the solution became cloudy, EtOH was added slowly. A solid formed immediately. 78.2% was isolated. 0,10 DMSO (18)//EUN 190 proof (28) from 70 °C to room temperature 72 0.70 The free base was dissolved in DMSO at 70 °C. EtOH was added slowly until turbid. Cooled overnight. Do not filter.

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0,10 90% DCM/EtOH (25)// EtOH 200 proof (19) from 40 °C to room temperature 16 2.38 The free base was dissolved in 90% DCM in EtOH at 40 °C. EtOH was added slowly until turbid. The solid slowly disintegrated at 40 °C. Cooled overnight. 0,10 66% DCM/EtOH (30)// EtOH 200 proof (20) from 40 °C to room temperature 16 3.70 The free base was dissolved in 66% DCM in EtOH at 40 °C. EtOH was added slowly, the solution remained a solution. Cooled overnight. A solid was observed in the morning. 1,0 DMSO (6)//H2O (24) from 100 °C to room temperature 24 1,1 The free base was dissolved in DMSO at 100 °C. H ₂ O was added slowly to give a suspension. Heat for 10 h at 100 °C, then cooled overnight to room temp. Wash with 20 volumes of H ₂ O. Oven dried. Yield - TBD. 1,0 DMSO (10)//EtOH 200 proof (20) from 100 °C to room temperature 24 0.9 The free base was dissolved in DMSO at 100 °C. Cooled to 75 °C. EtOH was added slowly to obtain a suspension. Stirred in for 10 hours at 75 °C, then cooled overnight to room temp. Filtered. Washed with 20 volumes of H ₂ O. Oven dried. Yield - to be determined. 1,0 EtOH 200 proof (20) from 75 °C to room temperature 24 0,6 The free base was suspended in EtOH at 75 °C. Heat for 24 h at 75 °C. Cooled to room temperature and filtered. Washed with 20 volumes of _H2O . Oven dried. Yield - TBD.

The free base of the compound of formula I was found to be soluble in 18 volumes of DMSO at 70°C and in 6 volumes of DMSO at 100°C. EtOH was added to the DMSO solution at 70°C; the solution became cloudy after ~18 volumes of EtOH (1:1 DMSO/EtOH). Additional EtOH resulted in the solid being destroyed at 70°C.

B. Characterization of the free base of the compound of formula I.

The compound of formula I, prepared as described above in Example 1, was analyzed by polarized light microscopy (PLM), XRPD, DSC, TGA, DSP, FTIR, and 1H NMR and identified as Form A (Figs. 2A-2G). The X-ray powder diffraction pattern is shown in Fig. 2A. The peaks in the X-ray powder diffraction pattern are shown below in

- 170 041220 Table 12. An overlay of the X-ray powder diffraction patterns from different lots of the free base of the compound of formula I is shown in Fig. 2B. A small endothermic event was observed on the DSC with an onset at about 187 °C, followed by an endothermic event with an onset at about 224 °C (Fig. 2C). The isothermal (25 °C) DSP curve is shown in Fig. 2D. TGA analysis showed a mass loss of 1.1% from the onset of heating to about 238 °C (Fig. 2E). The FTIR spectrum is shown in Fig. 2F. The NMR spectrum (in DMSO-d ⁶ ) is shown in Fig. 2G.

Table 12

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24.12 3.69 5.20 5.60 24.57 3.62 30,30 33,00 24.94 3.57 1.80 2,00 25,32 3.51 5,00 5.40 25,60 3.48 2.50 2.70 26.45 3.37 8,00 8.70 26.65 3.34 8.90 9.60 27,17 3.28 2.50 2.70 27.72 3.22 15.70 17,10 28,25 3.16 1.30 1.40 28,56 3.12 3.80 4.10 29,31 3.04 1.60 1.70 29.64 3.01 3.40 3.70 30.16 2.96 0.80 0.80 30.75 2.91 3.50 3.80 31.24 2.86 2,00 2.20 31,72 2.82 1,00 1,10 32,19 2.78 0,50 0,60 33,16 2.70 1.30 1.40 34.05 2.63 2.30 2.50 34,40 2.60 1,10 1,20 35,02 2.56 0.80 0.90 35,44 2.53 1.40 1.50

C. Initial selection of salts.

An initial screening of salts was performed for the compound of formula I using inorganic and organic acid counterions and DCM or DMA as the solvent (solubility of the free base of the compound of formula I in DCM = 15.5 mg/mL; solubility of the free base of the compound of formula I in DMA = 12.9 mg/mL). The acid and solvent combinations are presented below in Table 13.

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Table 13

Results of the primary selection of salts

Acid Solvent Appearance Original ³ In 13 hours. After heating to 40 °C for 4.5 hours. After heating to 40°C and cooling overnight After evaporation After dilution with MTBE Maleic acid ¹ DXM Hard with a pink tint Solid with a slight pink tint N/A N/A N/A N/A L-Lactic acid DXM Transparent, no solid substance Transparent, without hard wax, with a pink tint N/A N/A A brown, sticky, opaque substance that becomes sticky when dry. N/A Acetic acid DXM Transparent, no solid substance Transparent, without hard wax, with a yellow tint N/A N/A Beige solid when dry II N/A Adipic acid DXM A little cloudy, some "solid matter" on the walls of the flask A little cloudy, hard to tell if there is any solid matter A little cloudy, hard to tell if there is any solid matter Slightly cloudy, small particles floating in the solution N/A N/A Fumaric acid DXM Muddy Cloudy, small particles of solid matter Cloudy, small particles of solid matter Cloudy, small particles of solid matter N/A N/A D- Glutamic acid DXM Muddy A little cloudy, hard to tell if there is any solid matter Cloudy, milky Cloudy, milky N/A N/A D-Malic acid ¹ DXM Solid particles slightly pink in color Solid particles slightly pink in color N/A N/A N/A N/A

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L-Malic acid ¹ DXM White solid particles Solid particles, white (with a pink tint) N/A N/A N/A N/A MSOH DXM White solid particles Transparent, brown solid on the walls Transparent, milky particles on the walls (solvent loss due to a loose lid) Transparent milky solid on the walls N/A N/A _pTsOH.H2O DXM Cloudy, milky Cloudy, milky Cloudy, milky Cloudy, milky N/A N/A Benzenesulfonic acid DXM Transparent, with a light yellow tint Transparent, light pink, some solid matter A little solid on the walls, milky solution A little solid on the walls, milky solution N/A N/A Benzoic acid DXM Transparent, with a light yellow tint Transparent, light yellow N/A N/A Beige solid when dry II N/A Malic acid DXM Transparent, with a light yellow tint White, cloudy, some solid material on the walls of the flask A little solid matter on the walls, small particles A little solid matter on the walls, small particles N/A N/A HBr/ _H2O DXM Cloudy, pink "sticky" substance on the walls of the flask Cloudy, milky, pink "sticky" substance on the walls of the flask Cloudy, milky, pink solid on the walls of the flask Cloudy, milky, pink solid on the walls of the flask N/A N/A

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Succinic acid DXM Transparent, with a light yellow tint Very fine particles Small particles, some solid matter on the walls Very fine particles N/A n/a L-Tartaric acid ² DXM Cloudy, small particles Cloudy, can't tell if solids are being filtered Pink-tinted solid particles Pink-tinted solid particles N/A n/a НС1/ _Н2О DXM Muddy Cloudy, milky Brown "sticky" solid on the walls, milky Brown "sticky" solid on the walls, milky n/a n/a

Sulfuric acid DXM Transparent, some solid substance stuck to the walls of the flask Transparent, some solid substance stuck to the walls of the flask Solid substance on the walls of the flask, some solid substance floating Clear solution/white "solid" on the walls of the flask n/a N/A Phosphoric acid DXM Transparent, with a yellow tint Transparent, with a yellow tint Clear yellow solution, brown solid stuck to flask Clear yellow solution, brown solid stuck to flask n/a n/a Citric acid ² DXM Cloudy, small particles Solid particles of white color N/A N/A n/a n/a Propionic acid DXM Transparent Transparent, with a yellow tint N/A N/A Beige solid when dry II n/a D-Tartaric acid ² DXM Cloudy, some solid matter Solid particles of white color n/a n/a N/A n/a

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L- Glutamic acid DXM Solid particles float in solution A little cloudy, hard to tell if there is any solid matter A little cloudy, hard to tell if there is any solid matter Cloudy, milky N/A n/a Maleic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I L-Lactic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I Acetic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I Adipic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I Fumaric acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I D- Glutamic acid DMA Muddy Cloudy emulsion Cloudy emulsion Cloudy emulsion n/a Cloudy emulsion I D-Malic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I L-Malic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I Methanesulfonic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I _pTsOH.H2O DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I Benzenesulfonic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I Benzoic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion I

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Malic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I NVg/NgO DMA Solid particles of dirty white color filtered white solid slow filtration N/A N/A N/A N/A Succinic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I L-Tartaric acid3 DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Transparent solution Propionic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I Sulfuric acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I

Phosphoric acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I Citric acid DMA Transparent solution Transparent solution Transparent solution Transparent solution N/A Cloudy emulsion I НС1/ _Н2О DMA White solid particles A white solid was filtered off. N/A N/A N/A N/A Propionic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I D-Tartaric acid ² DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion I L- Glutamic acid DMA Cloudy emulsion Cloudy emulsion Cloudy emulsion Cloudy emulsion N/A Cloudy emulsion I НС1/dioxane DMA Emulsion I Emulsion Emulsion I Emulsion N/A Emulsion I

¹ Acids that yielded solids after 13 h that could be filtered.

² After heating and cooling, the solid was filtered off.

³ Observations for experiments in DCM were recorded after setting up all experiments (~1 h after the first flask).

Samples that yielded a solid after 13 h at room temperature were filtered and analyzed. Samples that did not yield a solid after stirring overnight and were clear solutions were evaporated to dryness (DCM reactions) or had MTBE added (DMA reactions). The resulting solids were isolated and analyzed. Samples that yielded unfilterable solids were subjected to heat cycling; the solid (if any) was isolated by filtration after heating and cooling. The appearance of the solids is listed above in Table 13.

The solids formed from the following acids were filtered and analyzed: maleic acid, D-malic acid, L-malic acid, citric acid, L-tartaric acid, D-tartaric acid, HBr, and HCl. The solids obtained after evaporation of DCM were also analyzed for the following acids: acetic acid, benzoic acid, and propionic acid.

In the initial selection, eleven salts were obtained for further analysis (MPS, DSC, etc.). Salts were obtained from the following acids: maleic acid, acetic acid, D-malic acid, L-malic acid, citric acid, L-tartaric acid, D-tartaric acid, benzoic acid, propionic acid, HCl and HBr.

Additional screening of sulfuric and phosphoric acid salts was also performed, as shown below in Table 14.

Table 14

Screening conditions for sulfuric and phosphoric acid salts

Scale Solvent (vol.) Acid (eq.) Temperature Time (hrs.) Notes 20 mg THF (100) _H2SO4 / _H2O ( _1.1 ) 25 °C 24 Did not become a solution. Milky solution overnight, solid substance stuck to the walls of the flask. 20 mg EtOH (100) _H2SO4 / _H2O ( _1.1 ) 25 °C 24 When acid was added, a thick substance formed, turned into a solid gel. Milky solution overnight. 20 mg 1:1 DHM/EtOH (100) _H2SO4 / _H2O ( _1.1 ) 25 °C 24 When acid was added, the solid mostly dissolved. Sticky substance on the walls. Milky solution overnight.

20 mg 4:1 DHM/EtOH (SO) _H2SO4 / _H2O ( _1.1 ) 25 °C 24 The free base was completely dissolved before the acid was added. The acid formed almost immediately. Filtered solid overnight. 20 mg THF (100) H ₃ PO ₄ /H ₂ O (1.1) 25 °C 24 Didn't become a solution, first a thick substance formed. Milky solution overnight. 20 mg EtOH (100) H ₃ PO ₄ /H ₂ O (1.1) 25 °C 24 Did not become a solution, a thick substance formed. Milky solution overnight. 20 mg 1:1 DHM/EtOH (100) H ₃ PO ₄ /H ₂ O (1.1) 25 °C 24 Sticky substance on the walls of the flask, became thicker over time. Milky solution overnight. 20 mg 4:1 DHM/EtOH (SO) H ₃ PO ₄ /H ₂ O (1.1) 25 °C 24 Free base was completely dissolved before acid was added. Acid formed almost immediately. Gel overnight.

The free base of the compound of formula I was found to be soluble in 4:1 DCM/EtOH, showing limited solubility in THF, EtOH, and 1:1 DCM/EtOH. The conditions listed above in Table 14 did not yield a filterable phosphoric acid salt. Use of sulfuric acid in 4:1 DCM/EtOH yielded a filterable solid after stirring overnight.

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The solids obtained from the acids listed in Table 15 were analyzed by MPS and

DSC; the appearance and melting point of the indicated substances, determined by DSC, are presented in Table 15. The DSC curves are shown in Figs. 3-14.

Table 15

Appearance and melting point of the isolated salts

Acid Appearance of a solid after filtration or evaporation of the solvent Melting point (DSC) N/A Free base 229.23 °C Maleic acid Grey/light brown solid 226.84 °C D-Malic acid Grey/light brown solid 212.69 °C L-Malic acid Grey/light brown solid 213.52 °C Citric acid Grey/light brown solid 197.53 °C L-Tartaric acid Grey/light brown solid 222.66 °C D-Tartaric acid Grey/light brown solid 219.24 °C Acetic acid Beige solid 229.06 °C Benzoic acid Beige solid 229.16 °C Propionic acid Beige solid 228.77 °C NVg Beige solid 225.25 °C NS1 Dirty white solid 241.25 °C _H2SO4 Dirty white solid 285.95 °C

The DSC thermogram for the maleate salt identified during the initial salt screening is shown in Fig. 3. An endothermic event was observed in the DSC with an onset at about 224°C. A small endothermic event was observed in the DSC with an onset at about 208°C.

The DSC thermogram for the acetic acid salt identified during the initial salt screening is shown in Fig. 4. An endothermic event was observed in the DSC with an onset at about 227°C.

The DSC thermogram for the D-malic acid salt identified during the initial salt screening is shown in Fig. 5. An endothermic event was observed in the DSC with an onset at about 211°C.

The DSC thermogram for the benzoic acid salt identified during the initial salt screening is shown in Fig. 6. An endothermic event was observed in the DSC with an onset at about 228°C.

The DSC thermogram for the L-tartaric acid salt identified during the initial salt screening is shown in Fig. 7. An endothermic event was observed in the DSC with an onset at about 216°C.

The DSC thermogram for the citric acid salt identified during the initial salt screening is shown in Fig. 8. An endothermic event was observed in the DSC with an onset at about 190°C.

The DSC thermogram for the propionic acid salt identified during the initial salt screening is shown in Fig. 9. An endothermic event was observed in the DSC with an onset at about 226°C. An endothermic event was observed in the DSC with an onset at about 148°C.

The DSC thermogram for the D-tartaric acid salt identified during the initial salt screening is shown in Fig. 10. An endothermic event was observed in the DSC with an onset at about 210°C. A small endothermic event was observed in the DSC with an onset at about 159°C.

The DSC thermogram for the L-malic acid salt identified during the initial salt screening is shown in Fig. 11. An endothermic event was observed in the DSC with an onset at about 211°C.

The DSC thermogram for the sulfuric acid salt identified during the initial salt screening is shown in Fig. 12. An endothermic event was observed in the DSC with an onset at about 277°C. A small endothermic event was observed in the DSC with an onset at about 35°C.

C. Scaling up and optimizing selected salts.

The yield of hydrochloric acid (HCl) and hydrobromic acid (HBr) salts obtained during the initial salt collection was scaled as shown in Table 16 for further analysis.

Table 16

Increasing the scale of production of HCl and HBr salts

Scale b (g) Acids (eq.) Volume m DMA Max, temperature (°C) Anti-solvent volume (MTBE) Ripening time The isolated solid substance 0.2 g 36% НС1 (1.1) 75 28 °C 125 During the night, while stirring, 152 mg, yield 71%. Found 6.2% C1 (expected value 6.5%) 0.2 g 48% HBg (1.1) 50 40 °C 150 Overnight without stirring 215 mg, yield 86%. Found 12% Bg (expected value 13.5%)

The DSC thermogram for the hydrochloric acid salt prepared at the ~200 mg scale is shown in Fig. 13. An endothermic event with an onset at about 233°C was observed in the DSC.

The DSC thermogram for the hydrobromic acid salt prepared at the ~200 mg scale is shown in Fig. 14. An endothermic event was observed in the DSC with an onset at about 195°C.

Alternative conditions for the formation of the HCl salt were studied. For example, the volume of DMA was reduced to 50 volumes and the reaction mixture was heated to 40°C, as shown in Table 17 (row 1). At this temperature, HCl was added; the resulting mixture was then cooled to room temperature. The DSC thermogram of the hydrochloric acid salt prepared in this manner is shown in Fig. 15A. An endothermic event was observed in the DSC with an onset at about 230°C.

The HCl salt was also prepared using a mixed solvent system of 1:1 DCM/EtOH in 50 volumes at 30°C. The DSC thermogram of the hydrochloric acid salt prepared in this manner is shown in Fig. 15B. An endothermic event was observed on DSC with an onset at about 224°C.

Table 17

Alternative conditions for obtaining HCl salt

Scale (g) Acid (eq.) DMA volume Max. temperature (°C) Volume of antisolvent (MTBE/vol.) Ripening time Isolated solid 0.2 g 36% НС1 (1.1) 75 28 °C 125 Overnight with stirring 152 mg, yield 71%. Found 6.2% C1 (expected value 6.5%) 0.2 g 48% HBg (1.1) 50 40 °C 150 Overnight without stirring 215 mg, yield 86%. Found 12% Br(expected value 13.5%)

Four salts (HCl, HBr, L- and D-malic acid salts) and the free base of the compound of formula I were prepared in 2 g scale (Table 18). The four salts were analyzed and characterized by MPS, DSC, TGA, DSP, X-ray diffraction, FT-IR and 'H NMR (Figs. 16A-16F, 17A-17D and 18A-18H).

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Table 18

Scale-up of production of HCl, HBr, L-malic and D-malic acid salts

Scale b (g) Solvent (vol.) Acid (eq.) Time (hours) Tempo Melting point (DSC) Actual yield (%) Notes 2.0 DMA (50) НС1 (1,1) Zch. from 40 °C to room temperature 240.61 °C 1.88 g (87.8%) HCl was added at 40 °C. Filtered and. White solid.

2.0 3.6:1 DXM/EUH (46) НС1 (1,1) 20 h. 25 °C 237.99 °C 1.93 g (90.1%) Chlorine analysis showed 1 equiv. HCl 2.0 3.6:1 DXM/EUH (46) NVg (1,1) 20 h. 25 °C 237.91 °C 2.26 g (97.4%) Bromine analysis showed 1 equiv. HBr 2.0 3.6:1 DXM/EUH (46) LMalic acid (0.97) 20 h. 25 °C 208.31 °C 1.64 g 66.8% 2.0 3.6:1 DXM/EUH (46) DMalic acid (0.97) 20 h. 25 °C 208.25 °C 1.60 g 65.2% 2,2 3.6:1 DXM/EUH (46) LMalic acid (0.97) 20 h. 25 °C N/A 2.11 g 75.9% 2,5 3.6:1 DXM/EUH (46) DMalic acid (0.97) 20 h. 25 °C N/A 2.41 g 76.2%

Analysis of chlorine and bromine confirmed the formation of the corresponding mono-HX salts. Salts of L- and D-malic acid were obtained with a yield of 75-78%; the HCl salt was obtained with a yield of 90%.

For the HCl salt (DMA), an endothermic event with an onset at about 233 °C was observed on DSC (Fig. 16A); a small exothermic event with an onset at about 253 °C was observed on DSC. For the HCl salt (EtOH/DCM), an endothermic event with an onset at about 223 °C was observed on DSC; a small endothermic event with an onset at about 71 °C was observed on DSC (Fig. 16D). Isothermal (25 °C) DSC analysis showed that the HCl salt (DMA) is hygroscopic (Fig. 16B). At 80% relative humidity, an increase in mass of about 5% was observed. Some hysteresis was observed. When removed from the chamber, the HCl salt sample remained a free-flowing powder; no spreading due to absorption of moisture from the air was observed. TGA analysis of the HCl salt (DMA) showed a mass loss of 7.4% from the start of heating to about 255 °C (Fig. 16C). TGA analysis of the HCl salt (EtOH/DCM, mixed solvent system) showed a mass loss of 8.2% from the start of heating to about 255 °C (Fig. 16D). An overlay of the X-ray powder diffraction patterns of the HCl salt (prepared in DMA, from different batches, before/after DSP) and the HCl salt (EtOH/DCM) is shown in Fig. 16E. The ¹ H NMR spectrum of the HCl salt (prepared in EtOH/DCM) in DMSO-66 is shown in Fig. 16F.

For the HBr salt, an endothermic event was observed on DSC with an onset at about 215°C; on DSC, a small endothermic event was observed with an onset at about 34°C (Fig. 17A). TGA analysis of the salt

- 181 041220

HBr showed a mass loss of about 10.3% from the start of heating to about 255°C (Fig. 17A). The X-ray powder diffraction pattern of the HBr salt is shown in Fig. 17B. The FTIR spectrum

HBr salt is shown in Fig. 17C. 1H NMR spectrum of HBr salt in DMSO-d6 is shown in Fig. 17D.

For L-malate salt, an endothermic event was observed on DSC with an onset at about 205°C (Fig. 18A). For D-malate salt, an endothermic event was observed on DSC with an onset at about 206°C (Fig. 18B). TGA analysis of L-malate salt showed a mass loss of about 17.7% from the start of heating to about 253°C (Fig. 18A). TGA analysis of D-malate salt showed a mass loss of about 18.4% from the start of heating to about 250°C (Fig. 18B). Isothermal (25°C) DSP analysis showed that both L-malate and D-malate salts are hygroscopic (Figs. 18C and 18D). At 80% RH, a mass increase of about 2.8% was observed. Some hysteresis was observed. When removed from the chamber, the samples of salts with L-malic and D-malic acids remained as a free-flowing powder; no spreading was observed due to absorption of moisture from the air. The superposition of the X-ray powder diffraction patterns of the salts of L- and D-malic acids is shown in Fig. 18E. The superposition of the FT-IR spectra of the salts of L- and D-malic acids is shown in Fig. 18F. The 1H NMR spectra of the salts of L- and D-malic acids in DMSO-d ⁶ are shown in Figs. 18G-18H, respectively.

Two lots of L-malic acid salt were tested by DSC, TGA, and XRPD analysis. For L-malic acid salt (Lot A), an endothermic event was observed by DSC with an onset at about 210 °C (Figure 18I). TGA analysis of L-malic acid salt (Lot A) showed a mass loss of about 18.0% from the onset of heating to about 250 °C (Figure 18J). For L-malic acid salt (Lot B), an endothermic event was observed by DSC with an onset at about 217 °C (Figure 18K). TGA analysis of L-malic acid salt (Lot B) showed a mass loss of about 17.7% from the onset of heating to about 250 °C (Figure 18L). An overlay of the XRPD patterns of L-malic acid salts (Lots A and B) and the free base of the compound of formula I is shown in Figure 1. 18M.

The solubility of HCl, HBr, L-malic acid and D-malic acid salts of the compound of formula I in various solvent systems was studied.

The solubility test of the HCl salt was carried out as follows. About 10-20 mg of the HCl salt was stirred in 1 ml of the solvent. In each solvent system, some solid was observed after 2 h. The samples were then heated to 65 °C for 3 h, after which the appearance was recorded. The resulting mixture was then allowed to cool to room temperature overnight. The results of the solubility test are presented in Table 19.

Table 19 Solubility of HCl salt

Solvent Appearance at 25 °C after 2 hours Does the solid dissolve at 65°C? Solubility at 25 °C (mg/ml) Water Solid particles No 0.24 MeOH Solid particles No 0.58 EtOH Solid particles No 0,12 IPA Solid particles No 0 EtOAc Solid particles No 0 iPAc Solid particles No 0 THF Solid particles No 0 ACN Solid particles No 0 Toluene Solid particles No 0 DXM Solid particles n/a 0,10 Acetone Solid particles N/A 0 IEC Solid particles No 0 MTBE Solid particles No 0 2-methyl-THF Solid particles No 0 DCE Solid particles No 0 dmso Solid particles Close to 18.7 mg/ml 5.94 0DMNS1 Solid particles Not studied 2,18

The HCl salt was found to be insoluble in IPA, EtOAc, iPAc, THF, ACN, toluene, acetone, MEK, MTBE, 2-methyl-THF, and DCE. Solubility of <1 mg/mL was observed in water, MeOH, EtOH, and DCM. Solubility of about 6 mg/mL was observed in DMSO.

The solubility test of the HBr salt was carried out as follows. About 10-20 mg of the HBr salt was stirred in 1 ml of the solvent. In each solvent system, some solid was observed after 2 h. The samples were then heated to 65°C for 3 h, after which the appearance was recorded. The resulting mixture was then allowed to cool to room temperature overnight. The results of the solubility test are presented in Table 20.

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Table 20 Solubility of HBr salt

Solvent Appearance at 25 °C after 2 hours Does the solid dissolve at 65°C? Solubility at 25 °C (mg/ml) water Solid particles No 0 MeOH Solid particles No 0 EtOH Solid particles No 0 IPA Solid particles No 0 EtOAc Solid particles No 0 iPAc Solid particles No 0 THF Solid particles No 0 ACN Solid particles No 0 DXM Solid particles No 0 Acetone Solid particles No 0 IEC Solid particles No 0 MTBE Solid particles No 0 2-methyl-THF Solid particles No 0 dhe Solid particles No 0 DMSO Solid particles No 1.95

The HBr salt exhibited low solubility in the solvent systems studied; the solid did not dissolve when heated to 65°C. A solubility of 1.95 mg/mL in DMSO was observed.

The solubility test of L-malic acid salt was carried out as follows. About 10-20 mg of L-malic acid salt was stirred in 1 ml of solvent at room temperature overnight. The results of the solubility test are presented in Table 21.

Table 21

Solubility of L-malic acid salt

Solvent Batch 1: Solubility at 25 °C (mg/mL) Batch 2: Solubility at 25 °C (mg/mL) water 0.26 0.23 MeOH 0.52 0.58 EtOH 0.16 0,15 IPA 0 0 EtOAc 0,20 1.58 iPAc 0 0.33 THF 2.53 5.42 ACN 0.28 0.85 Toluene 0.29 0.62 DXM 0.24 5.50 Acetone 0.61 1.57 IEC 0.51 2.31 MTBE 0 0 2-methyl-THF 0.61 1,21 Dhe 0.13 4.97 DMSO 14.2 15.4 0.1 MNS1 2.30 Not studied

The solubility test was performed on two lots of L-malic acid salt. Some variability in solubility between the lots was observed. A solubility of 14-15 mg/mL in DMSO was observed.

A second solubility test to determine the solubility of the L-malate salt at elevated temperatures was conducted as follows. About 10-20 mg of the L-malate salt was stirred in 1 mL of solvent at room temperature for 30 min. The samples were heated to 60°C for 3.0 h; the solubility was recorded. The samples were then heated to 70°C for about 3.0 h; the solubility was recorded. The results of the second solubility test are presented in Table 22.

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Table 22

Solubility of L-malic acid salt at elevated temperatures Solvent Does the solid dissolve Does the solid dissolve

substance at 60 °C? substance at 70 °C? water No No MeOH No N/A EtOH No No IPA No No EtOAc No No iPAc No No THF at 65 °C almost 12.3 mg/ml N/A ACN No No Toluene No No DXM N/A N/A Acetone No N/A IEC No No MTBE No No 2-methyl-THF No No dhe No No DMSO Yes (>36 mg/ml) Yes (>56 mg/ml) 0.1 MNS1 Yes (>13.4 mg/ml) N/A

The solubility test of D-malic acid salt was carried out as follows. About 10-20 mg of D-malic acid salt was stirred in 1 ml of solvent overnight. For the solubility test at elevated temperatures, about 10-20 mg of D-malic acid salt was stirred in 1 ml of solvent at room temperature for 30 minutes. The samples were heated to 60°C for 3.0 h; the solubility was recorded. The samples were then heated to 70°C for about 3.0 h; the solubility was recorded. The results of the solubility test are presented in Table 23.

Table 23

Solubility of D-malate salt

Solvent Solubility at 25 °C (mg/ml) Does the solid dissolve at 60°C? Does the solid dissolve at 70°C? water 0.35 No No MeOH 0.49 No N/A EtOH 0.14 No No IPA 0 No No EtOAc 0.21 No No iPAc 0,12 No No THF 2.65 At 65 °C ~10 mg/ml N/A ACN 0.23 No No Toluene 0 No No DXM 0.17 N/A N/A Acetone 0.65 No n/a IEC 0.61 No No MTBE 0 No No 2-methyl-THF 0.62 No No Dhe 0.14 No No DMSO 16.7 Yes (>40 mg/ml) Yes (>56 mg/ml) 0.1 MNS1 2.90 Yes (>15 mg/ml) N/A

The scale of production of L-malic acid salt and HCl salt was further increased. L-malic acid salt was obtained with a yield of 77.7%, and HCl salt was obtained with a yield of 91%. Then, the conditions for producing L-malic acid salt were optimized to increase the yield, as shown in Table 24.

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Table 24

Scale-up of production of HCl salt and L-malic acid salt

Scale (g) Solvent (vol.) Acid (eq.) Time (hrs.) Temp. Actual yield (%) Notes 10 3.6:1 DXM/EtOH (46) LMalic acid (0.97) 20 h. 25 °C 9.83 g (77.7%) 10 3.6:1 DXM/EtOH (46) НС1 (1,1) 20 h. 25 °C 10.05 g (91.4%) 2.0 -3.3:1 DCM/EtOH (31) LMalic acid (0.97) 20 h. 25 °C 2.01 g (79.4%) The free base was dissolved at room temperature in 30 vol. 4:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol.). 1,5 -6:1 DCM/EtOH (26 vol.) L- Malic acid (1.05) 20 h. 25 °C 1.64 g (86.2%) The free base was dissolved at room temperature in 30 vol. 4:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol.). 8.0 -6:1 DCM/EtOH (26 vol.) L- Malic acid (1.05) 20 h. 25 °C 8.70 g (86.0%) The free base was dissolved at room temperature in 25 vol. 9:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol.).

8.0 -6:1 DCM/EtOH (26 vol.) L- Malic acid (1.05) 20 h. 25 °C 8.86 g (87.5%) The free base was dissolved at rt in 25 vol of 9:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol). The suspension was then cooled to 0 °C for 1.5 h and then filtered.

The solvent volume was reduced to 30 volumes using a 4:1 DCM/EtOH ratio. The addition of L-malic acid (0.97 equiv) in 1 volume of EtOH increased the total solvent volume to 31 volumes, and the solvent ratio was about 3.3:1 DCM/EtOH. These conditions increased the yield of the L-malate salt to 79.4%.

In a 9:1 DCM/EtOH solvent system, the volume was reduced to 25 volumes. L-malic acid (1.05 equiv.) was dissolved in 1 volume of EtOH, resulting in a total solvent of 26 volumes and a final solvent ratio of about 6:1 DCM/EtOH. These conditions increased the yield of the L-malate salt to 86.2%. The malate salt was obtained in 86% yield when these conditions were scaled up to 8 grams. A second reaction on an 8 gram scale was performed under the same conditions, but the suspension was cooled to 0 °C for 1.5 h before filtration. This reaction gave a yield of 87.5%.

D. Stability screening of the free base and selected salts.

The stability of the free base of formula I and the HCL salt, L-malic acid salt and D-malic acid salt was tested by weekly HPLC analysis for 4 weeks. The samples were stored at 40°C and 75% RH. The stability test results are presented in Table 25.

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Example 4. Selection of polymorphic salts for the compound of formula II A. Instruments and methods for conducting analysis.

The instruments and analytical methods used to screen the polymorphic forms described below in Examples 2 and 3 were as follows.

X-ray powder diffraction (XPD).

XRD analysis was performed on a Panalytical X'pert Pro instrument scanning samples from 3 to 35° 2θ. The material was carefully ground and loaded into a multiwell plate with a Kapton or Mylar polymer film to hold the sample. The multiwell plate was then inserted into a Panalytical diffractometer operating in transmission mode and analyzed using the following experimental conditions.

Origin of source data: XRDML dimension.

Scanning axis: gonio.

Initial position [°2θ]: 3.0066.

Final position [°2θ]: 34.9866.

Step size [°2θ]: 0.0130.

Scan step time [s]: 18.8700.

Scan type: continuous.

PSD Mode: Scan PSD Length [°2θ]: 3.35.

Divergence [°2θ]: 0.0000.

Type of divergence gap: fixed.

Slit size [°]: 1.0000.

Measurement temperature [°C]: 25.00.

Anode material: Cu K-alpha1 [A]: 1.54060.

K-alpha2 [A]: 1.54443 K-beta [A]: 1.39225.

Ratio K-A2/K-A1: 0.50000.

Generator settings: 40 mA, 40 kV.

Goniometer radius [mm]: 240.00.

Diet, focus-divergence gap [mm]: 91.00.

Incident Beam Monochromator: No.

Rotation: No.

Polarized light microscopy (PLM).

The presence of crystallinity (birefringence) was determined using an Olympus BX50 polarizing microscope equipped with a Motic camera and video image capture software (Motic Images Plus 2.0). All images were recorded using a 20x objective unless otherwise stated.

Thermogravimetric analysis (TGA).

Approximately 5 mg of material was weighed into an open aluminum crucible and loaded into a combined thermogravimetric/differential thermal analyzer (TG/DTA) and maintained at room temperature. The sample was then heated at a rate of 10°C/min from 20°C to 300°C, during which time the change in sample mass was recorded, as well as any differential thermal events (DTA). Nitrogen was used as a purge gas at a flow rate of 300 ^cm3 /min.

Differential scanning calorimetry (DSC).

About 5 mg of the sample was weighed into an aluminum DSC crucible and sealed loosely with an aluminum lid with a pierced hole. The crucible with the sample was then placed in a Seiko DSC6200 instrument (equipped with a cooling device), cooled, and maintained at 20 °C. After a stable heat flux response was achieved, the sample and the control sample were heated at a scanning speed of 186 041220

10°C/min and the response heat flux was recorded.

Nuclear magnetic resonance (NMR).

NMR experiments were performed on a Bruker AVIIIHD spectrometer equipped with a DCH cryoprobe operating at 500.12 MHz for protons. Experiments were performed in deuterated solvents and each sample was obtained at a concentration of about 10 mM.

Dynamic vapor sorption (DVA).

Approximately 10 mg of sample was placed on a mesh pan of a vapor sorption balance and mounted on a Surface Measurement Systems DVS-1 Dynamic Vapor Sorption Balance. The sample was subjected to a step-by-step cycling of conditions from 40-90% relative humidity (RH) in 10% increments, holding the sample at each step until a stable mass was reached (99.5% completion). After completion of the sorption cycle, the sample was dried using the same procedure to 0% RH, followed by a second sorption cycle to 40% RH. The change in mass during the sorption/desorption cycles was plotted, indicating the hygroscopic nature of the sample. XRD analysis was then performed on the remaining solid.

Gravimetric vapor sorption (GVS).

Approximately 10-20 mg of sample was placed on a mesh pan of a vapor sorption balance and mounted on the balance of a Hiden Analytical IGASorp moisture sorption analyzer. The sample was subjected to a linear profile change from 40-90% RH in 10% increments, holding the sample at each stage until a stable mass was reached (stage completed at 98%). After completion of the sorption cycle, the sample was dried using the same procedure to 0% RH, and finally returned to the original state of 40% RH. The change in mass during the sorption/desorption cycles was plotted, determining the hygroscopic nature of the sample.

High performance liquid chromatography with ultraviolet detection (HPLC-UV)

Device. HPLC with UV detector.

Column: ACE Excel 3 Super C18, column 75 mm x 4.6 mm.

Column temperature: 40°C.

Automatic sampler temperature: 25°C.

UV wavelength: 235 nm.

Injection volume: 5.0 QL.

Flow rate: 1.0 ml/min.

Mobile phase A: 0.1% TFA in water.

Mobile phase B: 0.1% TFA in acetonitrile.

Gradient program:

Time (minutes) Solvent B (%) 0,00 10 15,00 90 15,10 10 20,00 10

Charged aerosol (CAP) detection.

Device. HPLC with charged aerosol detection.

Column: Thermo Acclaim P2 50x2.1 mm, 3.0 QL.

Temperature: 30°C.

Automatic sampler temperature: room temperature.

Sample injection volume: 10 QL.

Flow rate: 0.5 ml/min.

Mobile phase A: deionized water.

Mobile phase B: 10 mM ammonium formate buffer, pH 3.65.

Gradient program:

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Time (minutes) Solvent B (%) 0,00 35 2 35 12 65 15.3 65 18.6 40 19.3 35 23 35

B. Initial characteristics.

The compound of formula II, prepared as described above in Example 2, was analyzed by XRPD, TG/DTA, DSC, DSP, MPS, ^1H NMR, and HPLC and identified as Form 1. The material was highly crystalline as determined by XRPD (Fig. 62A). MPS analysis showed agglomerates of small needle-like crystals. The TG/DTA curve (Fig. 62C) showed a small endotherm with an onset near 189°C, then a sharp endotherm with an onset near 199°C, associated with a melting transition. This event was followed by thermal decomposition. The initial endotherm was probably due to a transition between solid phases, followed by melting of the more stable solid form. DSC showed a slight endotherm with an onset at about 184°C followed by a sharp endotherm with an onset at about 199°C (Fig. 62B). The material was moderately hygroscopic as determined by DSP (Figs. 62D and 62E) with a mass gain of about 3.6% at 90% RH. No change in this form was observed after DSP. The purity of the material was 99.2% as determined by HPLC. ¹ H NMR in 66-DMSO showed trace amounts of DCM (0.04 equiv) and MeOH (0.06 equiv) in the material (Fig. 62F).

C. Primary selection of salts.

A primary salt screen was performed for the compound of formula II using the acidic counterions and solvent systems presented below in Tables 26 and 27.

Table 26

Proven Acids

Hydrochloric acid Sulfuric acid 1,2- Etand isul phokis ota Methanesulfonic acid Naphthalene-2-sulfonic acid Benzenesulfonic acid 2- Hydroxyethanesulfonic acid L-Aspartic acid Maleic acid Ethanesulfonic acid L-Glutamic acid L-Tartaric acid Citric acid D-Glucuronic acid L-Malic acid D-Gluconic acid L-Lactic acid L-Ascorbic acid Succinic acid Oxalic acid Phosphoric acid l-T oluenesulfonic acid Fumaric acid Hippuric acid Benzoic acid

Table 27 Solvent systems Solvent system

THF/water (1%)

1,4-Dioxane/water (10%)

MeCN/water (20%)

Acetone/water (10%)

IPA/water (10%)

EUN/water (10%)

The salt screening was carried out as follows. A basic aqueous solution of the acid was added to

- 188 041220 approximately 30 mg of compound of formula II in each of the solvent systems indicated above in Table 27.

If stock solutions could not be obtained, the acid was added undiluted to a solution of the compound of formula II. The masses and volumes of acids used to obtain stock solutions are presented below in Table 28.

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Table 28

Initial solutions of counterions for primary screening of salts

Acid Basic acid solution Known suitable solvent Amount of added acid per tube/well Other Amount of acid Volume of solvent (ml) Hydrochloric acid 83.5 µl 1 Water 57.1 µl Sulfuric acid 56.1 µl 1 Water 57.1 µl 1.2 Ethanesulfonic acids 234.2 mg 1 EtOH 57.1 µl added 1 equiv. HCI p- Toluene sulfonic acid 190.2 mg 1 Water 57.1 µl Methanesulfonic acid 64.9 µl 1 Water 57.1 µl

Naphthalene-2-sulfonic acid 255.8 mg 1 EtOH 57.1 µl added 1 equiv. HCI Benzenesulfonic acid 168.3 mg 1 Water 57.1 µl Oxalic acid 90.0 mg 1 Water 57.1 µl 2- Hydroxyethanesulfonic acid 148.1 mg 1 Water 57.1 µl added 1 equiv. HCI L-Aspartic acid N/A N/A N/A 7.7 mg Added undiluted Maleic acid 116.1 mg 1 Water 57.1 µl Phosphoric acid 98.0 mg 1 Water 57.1 µl Ethanesulfonic acid 81.6 1 Water 57.1 µl L-Glutamic acid N/A N/A N/A 8.5 mg Added undiluted L-Tartaric acid 150.1 mg 1 Water 57.1 µl Fumaric acid N/A n/a N/A 6.5 mg Added undiluted Citric acid 192.1 mg 1 Water 57.1 µl D-Glucuronic acid 194.1 mg 1 Water 57.1 µl L-Malic acid 134.1 mg 1 Water 57.1 µl Hippuric acid N/A n/a N/A 10.3 mg Added undiluted D-Gluconic acid 319.0 µl 1 Water 57.1 µl

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L-Lactic acid 76.0 µl 1 Water 57.1 µl L-Ascorbic acid 176.1 mg 1 Water 57.1 µl Benzoic acid 122.1 mg 1 IPA 57.1 µl Succinic acid 118.1 mg 1 MeOH 57.1 µl

The samples were then cycled between room temperature (rt) and 40°C in 4-hour cycles for 24 hours. Each mixture was filtered and the isolated solids (wet) were analyzed by X-ray diffraction to determine crystallinity and shape. Samples in which no solid was observed were stored uncovered to allow the solvent to evaporate. The remaining samples were placed in an oven at 40°C and 75% RH overnight. The samples were then reanalyzed by X-ray diffraction to determine changes in shape or crystallinity. The observations and results, including the various shapes of the diffraction patterns observed during the screening, are presented below in Tables 29–53 and Figs. 24A–24B through 47A–47B.

Table 29

Observations for hydrochloric acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension Suspension I Suspension I Suspension I Suspension I After temperature cycling White solid Pale orange solid White solid White solid White solid Pale yellow solid RPD after temperature cycling Form 2 Form 2 Form 2 Form 2 Form 2 Form 2 RPD after stability testing Form 2 Form 2 Form 2 Form 2 Form 2 Form 2

The diffraction patterns after temperature cycling and after stability testing for hydrochloric acid are shown in Fig. 24A and 24B, respectively.

Table 30

Observations for sulfuric acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension Suspension I Suspension I Suspension I After temperature cycling Yellow resinous substance Pale yellow solution Colorless solution Pale yellow solution White solid in yellow solution Pale yellow solution RPD after temperature cycling No solid matter No solid matter No solid matter No solid matter Form 3 No solid matter RPD after stability testing No solid matter No solid matter No solid matter No solid matter Form 5 No solid matter

The diffraction patterns after temperature cycling and after stability testing for sulfuric acid are shown in Figs. 25A and 25B, respectively.

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Table 31

Observations for 1,2-ethanedisulfonic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before the cyclic change of temperature Suspension I Suspension I Suspension Suspension I Suspension Suspension I After cyclic temperature change Pale yellow solution Pale yellow solution Colorless solution Pale yellow solution Colorless solution Pale yellow solution RPD after cyclic temperature change No solid matter No solid matter No solid matter No solid matter No solid matter No solid matter RPD after stability testing No solid matter No solid matter No solid matter No solid matter No solid matter No solid matter

Table 32

Observations for p-toluenesulfonic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension Suspension Suspension Suspension Suspension Suspension After temperature cycling Yellow solution Yellow solution White solid White solid in yellow solution White solid in yellow solution White solid in yellow solution RPD after temperature cycling Weak diffraction No solid matter No solid matter Form 4 Amorphous Amorphous RPD after stability testing Form 4 No solid matter No solid matter Form 4 Form 4 Amorphous

The diffraction patterns after temperature cycling and after stability testing for p-toluenesulfonic acid are shown in Figs. 26A and 26B, respectively.

Table 33

Observations for methanesulfonic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension Suspension I Suspension After temperature cycling Yellow solution Yellow solution Yellow solution White solid White solid White solid RPD after temperature cycling No solid matter No solid matter No solid matter Amorphous Form 2 Amorphous RPD after stability testing No solid matter No solid matter No solid matter Amorphous Form 2 Amorphous

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The diffraction patterns after temperature cycling and after stability testing for methanesulfonic acid are shown in Figs. 27A and 27B, respectively.

Table 34

Observations for naphthalene-2-sulfonic acid and XRD results

Solvent THF/water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EUN/ water (10%) Before temperature cycling Suspension Suspension Suspension Suspension Suspension Suspension After temperature cycling Yellow solution Yellow solution Pale yellow solution White solid White solid in yellow solution White solid in yellow solution RPD after temperature cycling Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/Form 5 Form 2 RPD after stability testing Form 2/Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/Form 5 Form 2

The diffraction patterns after temperature cycling and after stability testing for naphthalene-2-sulfonic acid are shown in Fig. 28A and 28B, respectively.

Table 35

Observations for benzenesulfonic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension Suspension I Suspension I After temperature cycling Pale yellow solution White solid Pale yellow solution White solid White solid White solid RPD after temperature cycling No solid matter No solid matter No solid matter Weak diffraction Form 2 Form 2 RPD after stability testing No solid matter No solid matter No solid matter Weak diffraction Form 2 Form 2

The diffraction patterns after temperature cycling and after stability testing for benzenesulfonic acid are shown in Figs. 29A and 29B, respectively.

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Table 36

Observations for oxalic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EUN/ water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension Suspension Suspension After temperature cycling Pale yellow solid White solid in yellow solution White solid White solid White solid White solid RPD after temperature cycling No solid matter Form 6 No solid matter Amorphous Amorphous Amorphous RPD after stability testing No solid matter Form 6 No solid matter Form 2/Form 6 Form 6 Form 6

The diffraction patterns after temperature cycling and after stability testing for oxalic acid are shown in Figs. 30A and 30B, respectively.

Table 37

Observations for 2-hydroxyethanesulfonic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPAZ water (10%) EUN/ water (10%) Before the cyclic change of temperature Suspension Suspension I Suspension Suspension Suspension I Suspension I After cyclic temperature change White solid White solid White solid White solid White solid White solid RPD after cyclic temperature change Form 1 (free base) / Form 2 Form 7 Form 1 (free base) Form 1 (free base) Form 2/ Form 7 Form 7 RPD after stability testing Form 1 (free base) / Form 2 Form 7 Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 2/ Form 7 Form 7

The diffraction patterns after temperature cycling and after stability testing for 2-hydroxyethanesulfonic acid are shown in Figs. 31A and 31B, respectively.

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Table 38

Observations for L-aspartic acid and RPD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before cyclical temperature changes Suspension I Suspension Suspension Suspension Suspension I Suspension After cyclic temperature changes Yellow solid Pale yellow solution Colorless solution White solid White solid White solid RPD after cyclic temperature changes Form 8 Form 1 (free base)/ Form 8 Form 1 (free base) / Form 8 Form 1 (free base)/ Form 8 Form 8 Form 1 (free base)/ Form 8 RPD after stability testing Form 8 Form 1 (free base)/ Form 8 Form 8 Form 14 Form 8 Form 1 (free base)/ Form 8

The diffraction patterns after temperature cycling and after stability testing for L-aspartic acid are shown in Fig. 32A and 32B, respectively.

Table 39

Observations for maleic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before the cyclic change of temperatures Suspension I Suspension Suspension Suspension I Suspension I Suspension I After cyclic temperature changes Yellow solid Colorless solution Colorless solution White solid White solid White solid RPD after cyclic temperature change Weak diffraction No solid matter No solid matter Amorphous Amorphous Amorphous RPD after stability testing Form 15 No solid matter No solid matter Amorphous Amorphous Amorphous

The diffraction patterns after temperature cycling and after stability testing for maleic acid are shown in Fig. 33A and 33B, respectively.

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Table 40

Observations for phosphoric acid and RPD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EUN/ water (10%) Before temperature cycling Suspension Suspension I Suspension I Suspension I Suspension I Suspension After temperature cycling Yellow resinous substance Yellow solution Pale yellow resinous substance Pale yellow resinous substance White solid Yellow resinous substance RPD after temperature cycling Amorphous No solid matter No solid matter Form 9 Form 10 Weak diffraction RPD after stability testing Form 10 No solid matter No solid matter Form 9/ Form 10 Form 10 Form 10

The diffraction patterns after temperature cycling and after stability testing for phosphoric acid are shown in Figs. 34A and 34B, respectively.

Table 41

Observations for ethanesulfonic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EUN/ water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension I Suspension Suspension I After temperature cycling Yellow solution Yellow resinous substance Pale yellow resinous substance Pale yellow resinous substance White solid Yellow solution RPD after temperature cycling No solid matter No solid matter No solid matter No solid matter Amorphous No solid matter RPD after stability testing No solid matter No solid matter No solid matter No solid matter Amorphous No solid matter

The diffraction patterns after temperature cycling and after stability testing for ethanesulfonic acid are shown in Figs. 35A and 35B, respectively.

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Table 42

Observations for L-glutamic acid and RPD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before the cyclic change of temperature Suspension I Suspension I Suspension Suspension Suspension Suspension After cyclic temperature change White solid in yellow solution White solid White solid White solid White solid White solid RPD after cyclic temperature change Form 8 Form 7 Form 1 (free base) Form 1 (free base) Form 1 (free base) / Form 2 Form 1 (free base) / Form 2 RPD after stability testing Form 8 Form 7 Form 1 (free base) Form 1 (free base) Form 1 (free base) / Form 2 Form 1 (free base)

The diffraction patterns after temperature cycling and after stability testing for L-glutamic acid are shown in Fig. 36A and 36B, respectively.

Table 43

Observations for L-tartaric acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) YUN/ water (10%) Before temperature cycling Suspension Suspension Suspension I Suspension Suspension I Suspension After temperature cycling White solid in yellow solution White solid White solid White solid White solid White solid RPD after temperature cycling Weak diffraction Weak diffraction No solid matter Weak diffraction Form 11 Weak diffraction RPD after stability testing Weak diffraction Weak diffraction No solid matter Weak diffraction Form 11 Weak diffraction

The diffraction patterns after temperature cycling and after stability testing for L-tartaric acid are shown in Fig. 37A and 37B, respectively.

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Table 44

Observations for fumaric acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension Suspension I Suspension I Suspension I After temperature cycling Pale yellow solid White solid Colorless solution White solid White solid White solid RPD after temperature cycling Form 12 No solid matter No solid matter No solid matter Form 8 Form 8 RPD after stability testing Form 12 No solid matter No solid matter No solid matter Form 8 Form 8

The diffraction patterns after temperature cycling and after stability testing for fumaric acid are shown in Fig. 38A and 38B, respectively.

Table 45

Observations for citric acid and RPD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EUN/ water (10%) Before the cyclic change of temperature Suspension Suspension I Suspension Suspension Suspension Suspension I After cyclic temperature change Yellow solid Pale yellow solution Colorless solution Pale yellow resinous substance White solid White solid RPD after cyclic temperature change Weak diffraction No solid matter No solid matter Weak diffraction Form 1 (free base) No solid matter RPD after stability testing Weak diffraction No solid matter No solid matter Weak diffraction Form 1 (free base) No solid matter

The diffraction patterns after temperature cycling and after stability testing for citric acid are shown in Fig. 39A and 39B, respectively.

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Table 46

Observations for D-glucuronic acid and RPD results

Solvent THF/water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension Suspension I Suspension I Suspension After temperature cycling Pale yellow solution Pale yellow solution Pale yellow solution White solid White solid Colorless solution RPD after temperature cycling No solid matter No solid matter Form 1 (free base) No solid matter Form 8 Form 8 RPD after stability testing No solid matter No solid matter Form 1 (free base) No solid matter Form 8 Form 1 (free base)

The diffraction patterns after temperature cycling and after stability testing for D-glucuronic acid are shown in Fig. 40A and 40B, respectively.

Table 47

Observations for L-malic acid and XRD results

Solution THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension Suspension I Suspension I Suspension Suspension I Suspension After temperature cycling Yellow solid Pale yellow solution White solid White solid White solid White solid RPD after temperature cycling Amorphous No solid matter No solid matter Form 1 (free base) Form 8 Form 8 RPD after stability testing Amorphous No solid matter No solid matter Form 1 (free base) Form 8 Form 1 (free base)

The diffraction patterns after temperature cycling and after stability testing for L-malic acid are shown in Fig. 41A and 41B, respectively.

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Table 48

Observations for hippuric acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before cyclical temperature changes Suspension I Suspension Suspension Suspension Suspension Suspension After cyclic temperature changes White solid in yellow solution White solid White solid White solid White solid White solid RPD after cyclic temperature changes Form 8 Form 1 (free base)/ Form 8 Form 1 (free base) Form 1 (free base) Form 8 Form 8 RPD after stability testing Form 8 Form 1 (free base)/ Form 8 Form 1 (free base) Form 1 (free base) Form 1 (free base)/ Form 8 Form 1 (free base)/ Form 8

The diffraction patterns after temperature cycling and after stability testing for hippuric acid are shown in Fig. 42A and 42B, respectively.

Table 49

Observations for D-gluconic acid and RPD results

Solution THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before the cyclic change of temperature Suspension I Suspension I Suspension Suspension Suspension Suspension After cyclic temperature change Yellow solid White solid White solid White solid White solid White solid RPD after cyclic temperature change Form 13 Form 7 Form 1 (free base) Form 1 (free base) Form 8 Form 1 (free base) / Form 8 RPD after stability testing Form 13 Form 7 Form 1 (free base) Form 1 (free base) Form 1 (free base) / Form 8 Form 1 (free base) / Form 8

The diffraction patterns after temperature cycling and after stability testing for D-gluconic acid are shown in Fig. 43A and 43B, respectively.

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Table 50

Observations for L-lactic acid and RPD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) YUN/ water (10%) Before the cyclic change of temperature Suspension I Suspension Suspension Suspension Suspension I Suspension After cyclic temperature change Pale yellow solid White solid White solid White solid White solid White solid RPD after cyclic temperature change Form 13 Form 1 (free base) Form 1 (free base) Form 1 (free base) Form 8 Form 8 RPD after stability testing Form 13 Form 1 (free base) Form 1 (free base) Form 1 (free base) Form 8 Form 1 (free base) / Form 8

The diffraction patterns after temperature cycling and after stability testing for L-lactic acid are shown in Fig. 44A and 44B, respectively.

Table 51

Observations for L-ascorbic acid and RPD results

Solution THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) YUN/ water (10%) Before the cyclic change of temperatures Suspension I Suspension Suspension Suspension Suspension Suspension After cyclic temperature changes Yellow solution White solid in yellow solution Orange solid Orange solid Yellow solid Yellow solid RPD after cyclic temperature change No solid matter Form 1 (free base) Form 1 (free base) Form 1 (free base) Form 8 Form 1 (free base)/ Form 8 RPD after stability testing No solid matter Form 1 (free base)/ Form 2 Form 1 (free base) Form 1 (free base)/ Form 2 Form 1 (free base)/ Form 8 Form 1 (free base)/ Form 8

The diffraction patterns after temperature cycling and after stability testing for L-ascorbic acid are shown in Fig. 45A and 45B, respectively.

Table 52

Observations for benzoic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before the cyclic change of temperatures Suspension Suspension Suspension Suspension I Suspension Suspension

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After cyclic temperature changes White solid White solid White solid White solid White solid White solid RPD after cyclic temperature change Form 8 Form 1 (free base)/ Form 8 Form 1 (free base)/ Form 8 Form 1 (free base) Form 8 Form 8 RPD after stability testing Form 1 (free base)/ Form 8 Form 1 (free base)/ Form 8 Form 1 (free base)/ Form 8 Form 1 (free base) Form 1 (free base)/ Form 8 Form 1 (free base)/ Form 8

The diffraction patterns after temperature cycling and after stability testing for benzoic acid are shown in Figs. 46A and 46B, respectively.

Table 53

Observations for succinic acid and XRD results

Solvent THF/ water (1%) Dioxane/water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before the cyclic change of temperatures Suspension Suspension Suspension I Suspension Suspension Suspension After cyclic temperature changes White solid in yellow solution White solid in yellow solution White solid White solid White solid White solid RPD after cyclic temperature change Form 1 (free base)/ Form 8 Form 1 (free base)/ Form 8 Form 8 Form 1 (free base) Form 8 Weak diffraction RPD after stability testing Form 1 (free base)/ Form 8 Form 1 (free base) Form 8 Form 1 (free base) Form 1 (free base) Form 1 (free base)

The diffraction patterns after temperature cycling and after stability testing for succinic acid are shown in Figs. 47A and 47B, respectively.

Based on the results of the primary salt screening, the primary suitable salts were identified and are presented below in Table 54.

Table 54

Primary suitable salts, according to RPD data

1 Sulfate obtained in IPA/water mixture (10%) 2 Tosylate obtained in a mixture of acetone/water (10%) 3 Naphthalene-2-sulfonate obtained in a mixture of THF/water (10%) 4 Oxalate obtained in a mixture of 1,4-dioxane/water (10%) 5 Oxalate obtained by evaporation from a THF/water mixture 6 Phosphate obtained in a mixture of acetone/water (10%) 7 Phosphate obtained in a mixture of IPA/water (10%) 8 Tartrate obtained in a mixture of IPA/water (10%) 9 Fumarate obtained in a THF/water mixture

Each salt was thermally analyzed by TG/DTA to identify possible salt forms. Eight of the nine matched salts were found to be solvates or hydrates.

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The following results were obtained.

Sulphate (IPA/water (10%)): weight loss was observed from the start of heating. No obvious thermal transitions were observed. Thermal decomposition was observed at about 257°C. The TG/DTA curve of the sulphate salt is shown in Fig. 48.

Tosylate (acetone/water (10%)): A mass loss of about 6% was observed from the onset of heating, followed by a second mass loss of 3% due to loss of solvent. A broad endotherm was observed with an onset at about 28°C, followed by a broad exotherm with an onset at about 158°C. Thermal decomposition was observed at about 192°C. The TG/DTA curve of the tosylate salt is shown in Fig. 49.

Naphthalene-2-sulfonate (THF/water (10%)): mass loss was observed from the onset of heating. No obvious thermal transitions were observed. The TG/DTA curve of naphthalene-2-sulfonate salt is shown in Fig. 50.

Oxalate (1,4-dioxane/water (10%)): A mass loss of about 0.6% was observed from the start of heating. A broad endotherm was observed with an onset at 194°C, followed by thermal decomposition. The TG/DTA curve of the oxalate salt is shown in Fig. 51.

Oxalate (evaporation from THF/water): A broad endotherm was observed with an onset of about 31°C, with a corresponding mass loss of about 6.2%. A broad endotherm was observed with an onset of about 100°C, with a corresponding mass loss of about 1.6%. Thermal decomposition was observed at about 150°C. DSC analysis showed a broad endotherm with an onset of about 27°C. The TG/DTA curve of the oxalate salt is shown in Fig. 52.

Phosphate (acetone/water (10%)): a mass loss of about 3% was observed from the start of heating. The TG/DTA curve of the phosphate salt is shown in Fig. 53.

Phosphate (IPA/water (10%)): A mass loss of about 2.5% was observed from the start of heating. A slight endotherm was observed with an onset of about 162°C. Thermal decomposition was observed after 200°C. The TG/DTA curve of the phosphate salt is shown in Fig. 54.

Tartrate (IPA/water (10%)): A mass loss of about 3.7% was observed from the start of heating. A slight endotherm was observed with an onset of about 173°C, followed by thermal decomposition. The TG/DTA curve of the tartrate salt is shown in Fig. 55.

Fumarate (THF/water): A mass loss of 5.2% was observed with an onset at 128°C, corresponding to a small endotherm at the same temperature. A second small endotherm was observed with an onset near 190°C, with a corresponding mass loss of 2.2%. A sharp melting endotherm was observed with an onset at 202°C, associated with melting of Form 2 (the free base). The TG/DTA curve of the fumarate salt is shown in Fig. 56.

D. Secondary selection of salts.

The phosphate salt obtained in IPA/water (10%) in the initial salt screening was prepared on a larger scale and further characterized. The scaled-up method is described below. 300 mg of the compound of formula II were suspended in 3 mL of IPA/water (10%) (10 mL/g), then 1.0 equiv of 1 M phosphoric acid stock solution was added. The mixture was shaken and temperature cycled in 4-hour increments at 40 °C overnight. The resulting solid was filtered and washed with IPA.

The phosphate salt prepared on a larger scale was crystalline as determined by XRPD and MSP. The XRPD peaks of the phosphate salt are shown below in Table 5 and in Fig. 23A. The diffraction pattern was consistent with that obtained during the primary run. Thermal analysis indicated a melting point of about 167°C (Fig. 23C). A mass loss of about 1.3% was observed from the onset of heating. A second mass loss of about 1.2% was observed with an onset of about 167°C. The material was very hygroscopic as determined by GSP (Figs. 23D and 23E), with a mass gain of about 14% but no change in shape or crystallinity as determined by post-GSP analysis. A broad water peak was observed in the H NMR spectrum, indicating formation of a salt (Fig. 23F). The purity of the salt was 97.7% as determined by HPLC. Collision-activated dissociation (CAD) analysis showed that the salt has a 1.4:1 _PO4 :free base ratio.

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Table 55

XRD peaks of phosphate salt

Position [°2-theta] ПШПВ left [°2Θ] Area [imp*°20] distance d [A] Height [imp.] Rel. INT. [%] 3.61 0,10 151.75 24.51 1503.25 100,00 6.20 0,15 87.25 14.26 576.19 38,33 7.19 0,15 57.90 12.30 382.36 25,44 8.91 0,15 35.23 9.93 232.63 15.48 9.54 0,10 40.79 9.27 404.11 26.88 10.31 0,15 69.31 8.58 457,72 30,45 12.50 0,10 41.74 7.08 413,47 27.51 13,00 0,15 39.81 6.81 262.90 17.49 14.61 0,10 44.88 6.06 444.57 29.57 15,12 0,10 35.26 5.86 349.27 23,23 15.89 0,15 133,60 5.58 882.27 58,69 16.72 0,10 101.45 5.30 1004.92 66.85 17.84 0,10 92.25 4.97 913,84 60.79 18,22 0,10 108.09 4.87 1070,70 71.23 19,13 0,10 48,49 4.64 480.33 31.95 20,28 0.13 108.57 4.38 860.39 57.24 20.92 0,10 56.42 4.25 558,88 37,18 22,15 0.18 82,62 4.01 467,65 31,11 23.05 0,10 39,45 3.86 390,79 26,00 23.88 0,10 41.78 3.73 413.88 27.53 24,21 0.13 39.71 3.68 314.69 20.93 24.61 0,15 30.06 3.62 198.52 13.21 25,10 0.13 37.82 3.55 299.73 19.94 25,52 0.13 75,71 3.49 599.97 39.91 27.04 0,10 26.96 3.30 267,03 17.76 27.30 0.26 78,12 3.27 309,55 20.59 28,52 0.18 61,69 3.13 349.18 23,23 29,21 0,15 16.56 3.06 109.36 7.28 30.07 0,20 21,19 2.97 104.97 6.98 31,22 0,15 18,15 2.87 119.87 7.97 34.03 0,20 8.71 2.63 43,12 2.87

Portions of the phosphate salt form were weighed into 3 glass vials to study the stability of the salt. Each vial was stored for 7 days under the following conditions: 1) room temperature and light; 40°C and 75% RH; or 3) 80°C. After 7 days, each sample was analyzed by XRPD to determine crystallinity and form. The material was stable under stress conditions with no change in form or crystallinity observed after the 7-day stability test under any of the conditions tested. The material stored at room temperature was 98.03% pure. The material stored at 40°C/75% RH was 97.44% pure. The material stored at 80°C was 64.96% pure. One major impurity, most likely a degradation product, was identified.

The thermodynamic solubility of the phosphate salt was studied at pH 1, pH 4.5 and pH 6.8. The description below

- 204 041220 san method for studying thermodynamic solubility. About 10 mg of the phosphate salt was weighed into a 2 mL glass test tube. 2 mL of the required buffer solution was added to the tube and the resulting slurry was stirred at room temperature overnight. Each solution was centrifuged and the resulting solid collected. There was insufficient solid collected to analyze the phosphate salts from the pH 1 and pH 4.5 buffers. The phosphate salt from the pH 6.8 buffer was analyzed by XRPD and shown to be Form 1. The mother liquor from each experiment was analyzed by HPLC to determine the solubility of each salt at each pH. The results are presented in Table 56 below.

Table 56 Thermodynamic solubility

Salt pH buffer Concentration (mg/ml) Results of RPD Phosphate 1 4.1 Not enough solids 4,5 0.97 Not enough solids 6.8 <0.02 Form 1

Example 5. Screening of polymorphs of the compound of formula II A. Instruments and methods for conducting the analysis.

The tools and methods of analysis used for the screening of polymorphic forms described in Example 5 are described above in Example 4.

B. Screening of solubility in solvents.

Solvent solubility screening was performed in 32 solvent systems as follows. About 10 mg of the compound of formula II was placed in each of 32 test tubes and 5 volumetric aliquots of the appropriate solvent system were added to the corresponding tubes. Dissolution of the mixture was checked before each addition. If no visible dissolution was observed, the mixture was heated to about 40°C and checked again. This procedure was repeated until visible dissolution or until 100 volumes of solvent had been added. Undissolved material from the solubility test was isolated and analyzed by XRPD. The solvent systems and the approximate solubility of the compound of formula II in each solvent system tested are presented below in Table 57, where (P) is the partial solubility at 100 volumes.

Table 57

List of solvents for solubility screening

Solvent Approximate solubility (mg/ml) 1 1,4-Dioxane <5 (R) 2 1-Butanol <5 3 1-Propanol <5 4 Acetone <5 5 Acetone:water (50%) <5 6 Anisole <5 (R) 7 CHCh <5 (R) 8 Cyclohexane <5

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9 Cyclohexanone <5 (R) 10 DXM <5 (R) 11 tert-Butyl methyl ether N/A 12 DMSO 41 13 Ethanol <5 14 EtOAc <5 15 Heptane N/A 16 IPA <5 17 Isopropyl acetate N/A 18 Isopropyl ether N/A 19 MeCN N/A 20 MeCN: water (20%) 5 21 IEC <5 22 MeOAs <5 23 MeOH N/A 24 2-Ethoxyethanol <5 (R) 25 2-Methyl-THF <5 (R) 26 MIBK <5 27 Nitromethane <5 (R) 28 NMP 17 29 THF <5 (R) 30 THF:water (1%) 48 31 Toluene N/A 32 Water <5

This substance was found to have only low solubility in most of the solvent systems studied, with moderate solubility observed in only 3 solvent systems (DMSO, NMP, and THF/water (1%)).

The resulting solids were filtered and analyzed by X-ray powder diffraction to determine shape and crystallinity. The results are presented in Table 58 below. Figure 57 shows the X-ray powder diffraction patterns of the resulting solids.

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Table 58

Results of RPD of the obtained solids

Solvent Form 1 1,4-Dioxane Not enough solids 2 1-Butanol Form 7 3 1-Propanol Form 8 4 Acetone Amorphous 5 Acetone:water (50%) Amorphous 6 Anisole Not enough solids 7 SNS1z Form 1/Form 13 8 Cyclohexane Form 1 9 Cyclohexanone Not enough solids 10 DXM Not enough solids 11 tert-Butyl methyl ether Form 1 12 DMSO Amorphous 13 Ethanol Form 8 14 EtOAc Weak diffraction 15 Heptane Form 1 16 IPA Form 2 17 Isopropyl acetate Form 1 18 Isopropyl ether Form 1 19 MeCN Form 1 20 MeSM:water (20%) Form 2/Form 13 21 IEC Not enough solids 22 MeOAs Form 7 23 MeOH Form 1 24 2-Ethoxyethanol Not enough solids 25 2-Methyl-THF Form 7 26 MIBK Form 1 27 Nitromethane Form 1 28 NMP Not enough solids 29 THF Form 13 30 THF:water (1%) Form 8 31 Toluene Form 1 32 Water Form 2/Form 13

C. Primary polymorphic screening.

Primary polymorphic screening was performed using the solvent systems presented in Table 59 below, which were selected based on the solvent solubility screening data described above.

Table 59

List of solvents for primary polymorphic screening

Solvent 1 Acetone 2 Acetone/water (50%) 3 Anisole 4 1-Butanol 5 2-Butanone 6 Chloroform 7 Cyclohexane 8 Cyclohexanone 9 Dichloromethane 10 1,4-Dioxane 11 Ethanol 12 Ethyl acetate 13 Dimethyl sulfoxide 14 MeCN/water (20%) 15 Methyl acetate 16 2-Ethoxyethanol 17 Nitromethane 18 Methyl isobutyl ketone 19 2-Methyl-THF 20 2-Propanol 21 1-Propanol 22 T etragide rofuran 23 L/-Methylpyrrolidone 24 THF/water (1%) 25 Water

1. Experiments with cyclic temperature changes (ripening).

Suspensions for temperature cycling (maturation) experiments were prepared using the solvent systems from Table 59 above as follows. About 40 mg of the crystalline compound of formula II was suspended in the appropriate solvent system. If complete dissolution occurred, additional material was added until a suspension was obtained. The resulting suspensions were subjected to successive 4-hour heating-cooling cycles from 40°C to room temperature over a period of 72 hours.

After temperature cycling, each mixture was filtered and the recovered solids (wet) were analyzed by X-ray diffraction to determine crystallinity and shape. Samples were then stored at 40°C and 75% RH overnight and reanalyzed by X-ray diffraction to determine possible changes in shape or crystallinity. The results are presented below in Table 60 and Figs. 58A-58D.

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Table 60

Experiments with temperature cycling (ripening)

Solvent Observations After temperature cycling After stability tests 1,4-Dioxane Solid matter Weak diffraction Form 1 1-Butanol Solid matter Weak diffraction Form 1 1-Propanol Solid matter Form 8 Form 1 Acetone Solid matter Weak diffraction Form 1 Acetone:water (50%) Solid matter Form 1 Form 1 Anisole Solid matter Form 1 Form 1 SNS1 ₃ Solid matter Amorphous Form 1 Cyclohexane Solid matter Form 1 Form 1 Cyclohexanone Solid matter Weak diffraction Form 1 DXM Solid matter Amorphous Form 1 DMSO Solid matter Form 1 Form 1 Ethanol Solid matter Weak diffraction Form 1 EtOAc Solid matter Form 1 Form 1 IPA Solid matter Form 8 Form 1 MeSM:water (20%) Solid matter Weak diffraction Form 1 IEC Solid matter Weak diffraction Form 1 MeOAs Solid matter Weak Form 1 diffraction 2-Ethoxyethanol Solid matter Weak diffraction Form 1 2-Methyl-THF Solid matter Weak diffraction Form 1 MIBK Solid matter Form 1 Form 1 Nitromethane Solid matter Weak diffraction Form 1 NMP Not enough solids Not enough solids Not enough solids THF Solid matter Weak diffraction Form 1 THF:water(1%) Solid matter Weak diffraction Form 1/Form 8 Water Solid matter Form 1 Form 1

The supernatant from each experiment was divided equally into 3 tubes for evaporation, quenching, and antisolvent addition experiments (described below).

2. Evaporation experiments.

For the evaporation experiments, a portion of the supernatant samples from the suspensions containing the compound of formula II were transferred to test tubes. The tubes were then opened and allowed to evaporate at room temperature. The recovered material was analyzed by X-ray diffraction without prior drying. The results are presented below in Table 61 and Fig. 59.

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Table 61

Evaporation Experiments

Solvent Solid matter Observations Form 1,4-Dioxane Yes Pale yellow solid Form 17 1-Butanol No N/A N/A 1-Propanol No N/A N/A Acetone Yes White solid Form 1/Form 8 Acetone:water (50%) Yes White needle-shaped crystals N/A Anisole Yes White solid N/A SNS1 ₃ Yes Yellow solid Form 1/Form 8 Cyclohexane No N/A N/A Cyclohexanone No N/A N/A DXM Yes White solid Form 1 DMSO Yes White solid Form 1 Ethanol Yes White solid N/A EtOAc Yes White solid Amorphous IPA No N/A N/A MES:water (20%) Yes White needle-shaped crystals Form 1 IEC Yes White solid Amorphous MeOAs Yes White solid Form 1 2-Ethoxyethanol Yes White solid Form 1 2-Methyl-THF Yes White solid N/A MIBK No N/A N/A Nitromethane Yes Pale yellow solid Form 1 NMP No N/A N/A THF Yes Yellow solid Form 1/Form 8 THF:water(1%) No N/A N/A Water No N/A N/A

3. Rapid cooling experiments.

The quenching experiments were carried out at 5°C and -20°C. A portion of the supernatant samples from the suspensions containing the compound of formula II were transferred to test tubes and allowed to cool at 5°C in a refrigerator. After sufficient material had been obtained (by precipitation), the substance was analyzed without prior drying, using X-ray diffraction. The samples in which no sediment had formed were transferred to a freezer and allowed to cool at -20°C. After sufficient material had been obtained, the substance was analyzed without prior drying, using X-ray diffraction. The results of the quenching experiments carried out at 5°C are presented below in Table 62 and Fig. 60. No solid substance was observed in the samples stored at -20°C after 10 days.

Table 62

Rapid cooling experiments (5°C)

Solvent Solid matter Temperature Observations Form 1,4-Dioxane Yes 2 °C White solid Amorphous 1-Butanol No N/A N/A N/A 1-Propanol No N/A N/A N/A Acetone Yes 2 °C White solid Form 1/ Form 8 Acetone:water (50%) No N/A Anisole Yes 2 °C White solid Form 18 SNS1 ₃ No N/A N/A N/A Cyclohexane No N/A N/A N/A Cyclohexanone Yes 2 °C White solid Amorphous DXM Yes 2 °C White solid N/A DMSO N/A N/A N/A N/A Ethanol No N/A N/A N/A EtOAc Yes 2 °C White solid Amorphous IPA No N/A N/A N/A MeSM:water (20%) Yes 2 °C White needle-shaped crystals Form 1 IEC Yes 2 °C White solid Amorphous MeOAs Yes 2 °C White solid Amorphous 2-Ethoxyethanol Yes 2 °C White solid Amorphous 2-Methyl-THF No N/A N/A N/A MIBK No N/A N/A N/A Nitromethane Yes 2 °C White needle-shaped crystals Amorphous NMP No N/A N/A N/A THF No N/A N/A N/A THF:water(1%) No N/A N/A N/A Water No N/A N/A N/A

4. Experiments with the addition of antisolvent.

The antisolvent addition experiments were performed by transferring a portion of the supernatant samples from the suspensions containing the compound of formula II into test tubes and adding the antisolvent stepwise to the saturated solutions. Once sufficient material was obtained, the substance was analyzed without prior drying using XRPD. The solvents used in the antisolvent addition experiments are presented below in Table 63. The results of the antisolvent addition experiments are presented below in Table 64 and in Fig. 61.

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Table 63

Solvents and antisolvents for experiments with the addition of an antisolvent

Sample Solvent Anti-solvent 1 Acetone MTBE 2 Acetone/water (50%) MTBE 3 Anisole MTBE 4 1-Butanol MTBE 5 2-Butanone MTBE 6 Chloroform MTBE 7 Cyclohexane MTBE 8 Cyclohexanone Heptane 9 Dichloromethane MTBE 10 1,4-Dioxane DIPE 11 Ethanol DIPE 12 Ethyl acetate MTBE 13 Dimethyl sulfoxide N/A 14 MeCN/water (20%) MTBE 15 Methyl acetate MTBE 16 2-Ethoxyethanol Water 17 Nitromethane MTBE 18 Methyl isobutyl ketone DIPE 19 2-Methyl-THF Water 20 2-Propanol MTBE 21 1-Propanol MTBE 22 Tetrahydrofuran MTBE

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Table 64

Experiments with the addition of antisolvent

Solvent Antisolvent Volume of antisolvent Solid substance o Observed by IYA Form 1,4-Dioxane DIPE 0,5 Yes White solid Form 1 1-Butanol MTBE 1 Yes White solid n/a 1-Propanol MTBE 1,5 No n/a N/A Acetone MTBE 1 Yes White solid Form 1/ Form 8 Acetone: water (50%) MTBE 1 Yes White needle-shaped crystals n/a Anisole Water 1 Yes White solid n/a SNS1 ₃ MTBE 1,5 Yes White solid Form 1/ Form 8 Cyclohexane MTBE 1 No n/a n/a Cyclohexane it Heptane 1 Yes White solid Amorphous DXM MTBE 1 Yes White solid n/a DMSO n/a n/a No n/a n/a Ethanol DIPE 1 No n/a n/a EtOAc MTBE 1 No n/a N/A IPA MTBE 1 No n/a N/A MES:water (20%) MTBE 1 No n/a N/A IEC MTBE 1 Yes White solid N/A MeOAs MTBE 1 Yes White solid Amorphous 2Ethoxyethane l Water 1 Yes White solid Amorphous 2-Methyl-THF Water 1 No n/a n/a MIBK DIPE 1 No n/a n/a Nitromethane MTBE 1 No n/a n/a NMP Water 1 Yes Yellow needle-shaped crystals Form 1 THF MTBE 1 Yes White solid Amorphous THF:water (1%) MTBE 1 No n/a n/a Water MTBE 1 No n/a n/a

D. Secondary polymorphic screening.

During the initial screening of polymorphs in solvents, four forms were identified: Form 1, Form 2, Form 7, and Form 8. Each form was prepared at 300 mg scale and fully characterized by XRD, TG/DTA, DSC, GSP with XRD after GSP, 1H NMR spectroscopy, HPLC-UV, and MPS.

Seven-day stability testing was also conducted as follows. A portion of each polymorphic form was weighed into 3 glass vials. Each vial was stored for 7 days under the following conditions: 1) room temperature and light; 40°C and 75% RH; or 3) 80°C. After 7 days, each sample was analyzed by XRPD to determine crystallinity and form. Further testing showed that Form 1 was the most stable form of the compound of formula II identified during the polymorphic screening. Forms 2 and 7 were found to be hydrated forms of the compound of formula II which dehydrated to Form 1 upon heating. Form 8 was found to be a solvate of IPA which desolvated to Form 1 upon heating. No change in appearance was observed after 7 days for any of the samples, and no change in shape, as determined by XRD, was observed after 7 days for any of the samples, although the Form 8 sample stored at 40°C/75% RH appeared to have a higher amorphous content.

1. Form 1.

Form 1 is the most stable form of the compound of formula II identified during polymorphic screening. Form 1 was prepared on a 300 mg scale as follows. About 500 mg of the free base of compound of formula II was weighed into a 20 mL scintillation vial. Ethyl acetate (4 mL) was added to the vial and the resulting suspension was subjected to temperature cycling from 40°C to room temperature for 72 h. The sample was filtered and the collected material was dried overnight under vacuum at 40°C.

XRD pattern of the material after drying (40°C under vacuum) showed Form 1. The XRD peaks of Form 1 are shown below in Table 65 and in Fig. 19A. The material was crystalline. The MPS data showed agglomerates of particles with strong birefringence. A small endothermic event was observed, as determined by DSC (Fig. 19B), with an onset near 190°C. A melting transition was observed with an onset near 203°C. A small endothermic event was observed, as determined by TG/DTA (Fig. 19C), with an onset near 190°C, with a corresponding mass loss of 0.4%. A melting transition was observed with an onset near 204°C, with a corresponding mass loss of 0.2%. Decomposition was observed after the melting transition. The material was moderately hygroscopic by GSP (Figs. 19D and 19E) with a mass gain of 2.6% at 90% RH. No shape change was observed by XRP after GSP analysis. Trace amounts of solvent were observed by ¹ H NMR (Fig. 19F). The spectra were consistent with those of the material obtained (compound of formula II).

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Table 65

RPD peaks of form 1

Position [°2-theta] PSHPV left [°2θ] Area [imp*°20] distance d [A] Height [imp.] Rel. INT. [%] 4.95 0.19 24.33 17.84 97.48 6.05 8.23 0.19 61.56 10.74 246.62 15.32 9.75 0.19 82.45 9.07 330,34 20,52 12.77 0.19 41.33 6.93 165.59 10.28 13.80 0.19 35.96 6.41 144.06 8.95 14.77 0.19 46.81 5.99 187.52 11.65 15.51 0.19 79,80 5.71 319,71 19.86 16.53 0,12 267.93 5.36 1610,16 100,00 17.11 0.19 44.95 5.18 180.08 11,18 17.82 0,12 93.39 4.97 561.24 34.86 18.86 0.16 220.48 4.70 1060,02 65.83 22,34 0.19 29.93 3.98 119.89 7.45 22.81 0.19 29.57 3.89 118.47 7.36 23.75 0.19 145.46 3.74 582.78 36.19 24.99 0,12 52.20 3.56 313,71 19.48 25,32 0,12 99.61 3.52 598,63 37,18 25.59 0.19 132.87 3.48 532.35 33,06 25.96 0.19 158.21 3.43 633,84 39,36 26.56 0,12 46,49 3.35 279.36 17.35 27.07 0.37 96.42 3.29 193.14 12,00 28,28 0,12 55,67 3.15 334.57 20.78 28.86 0.19 64.48 3.09 258.35 16.04

2. Form 2.

Form 2 was isolated from Form 7 and was considered to be a hydrated form of the compound of formula II, which dehydrated to Form 1 upon heating. Form 2 was prepared on a 300 mg scale as follows. About 500 mg of the free base of the compound of formula II was weighed into a 20 mL scintillation vial. Ethanol/water (4 mL, 10%) was added to the vial and the resulting suspension was subjected to temperature cycling from 40°C to room temperature over 72 h. The sample was filtered and the collected material was dried overnight on the filter.

The XRD pattern of the suspension material (obtained at a larger scale) was consistent with the small scale data observed in the initial salt screening, with a pattern consistent with Form 7. XRD pattern analysis performed after drying (on a filter bed) the material indicated Form 1. The XRD peaks of Form 2 are shown below in Table 66 and in Fig. 20A. The material was crystalline. The MSP data showed agglomerates of particles with strong birefringence. A small endothermic event was observed, with an onset of about 192°C by DSC (Fig. 20B). A melting transition of Form 1 was observed with an onset of about 204°C. The TG/DTA data (Fig. 20C) showed a mass loss of 0.7% from the start of heating. A small endothermic event was observed with an onset at about 194°C, with a corresponding mass loss of 0.2%. A solid-state transition and a melting transition of Form 1 were observed with an onset at about 205°C. Decomposition was observed after the melting transition. The material was moderately hygroscopic as determined by GSP (Figs. 20D and 20E) with a mass gain of 2% at 90% RH. No shape change was observed after GSP analysis as determined by XRPD. Trace amounts of solvent were observed by ¹ H NMR (not shown). The spectra were consistent with those of the material obtained (compound of formula II).

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Table 66

RPD peaks of form 2

Position [°2-theta] PSHPV left [°20] Area [imp*°20] distance d[A] Height [name.] Rel. INT. [%] 6.19 0,15 92.45 14.28 610,55 9.20 8.91 0,10 34.21 9.93 338,85 5.11 10.29 0,15 65,01 8.60 429.31 6.47 13.43 0.41 18.88 6.59 46.76 0,70 13.87 0,15 11.28 6.38 74.48 1,12 14.68 0,15 76.40 6.03 504.57 7.60 15,12 0,10 194.68 5.86 1928,53 29.06 15.87 0,15 16,00 5.58 105.68 1.59 16.50 0,15 76.75 5.37 506,86 7.64 16.79 0,10 39.53 5.28 391,55 5.90 17,14 0,10 28,80 5.17 285.27 4.30 17.42 0.13 43.66 5.09 346,03 5.21 17.82 0,10 669,86 4.98 6635,61 100,00 18,14 0,10 64.77 4.89 641,58 9.67 18.72 0,15 11.62 4.74 76,73 1,16 19.09 0,15 11,11 4.65 73.40 1,11 20.38 0,10 80.18 4.36 794.26 11.97 21,08 0,15 100.83 4.21 665,90 10.04 22,10 0,15 85,85 4.02 566.98 8.54 22.81 0,10 44.29 3.90 438,70 6.61 23.37 0,10 62.75 3.81 621,59 9.37 24.20 0,10 182.81 3.68 1810,90 27,29 24.61 0,10 61.83 3.62 612,45 9.23 25,00 0.13 45,50 3.56 360,61 5.43 25.48 0,10 27.06 3.50 268,10 4.04 26.14 0,10 10.42 3.41 103.22 1.56 27,21 0,10 49.21 3.28 487,46 7.35 27.40 0,10 29.76 3.26 294.85 4.44 27.97 0.13 22.74 3.19 180,20 2.72 29.03 0,15 32,60 3.08 215,30 3.24 29,36 0.13 49.11 3.04 389.19 5.87 29.63 0.13 56.71 3.01 449.38 6.77 29.98 0.13 44.37 2.98 351,62 5.30 30,50 0,10 13.45 2.93 133.23 2.01 31,20 0,10 58.94 2.87 583.89 8.80 31,66 0,10 19.31 2.83 191.32 2.88 32,22 0.13 43.31 2.78 343,20 5.17 32,61 0,20 9.60 2.75 47,53 0.72 34.09 0.13 20,42 2.63 161.81 2.44 34,46 0,10 25.96 2.60 257.14 3.88

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3. Form 7.

Form 7 was considered to be a hydrated form of the compound of formula II, which dehydrated to Form 1 upon heating. Form 7 was prepared on a 300 mg scale as follows. About 500 mg of the free base of the compound of formula II was weighed into a 20 mL scintillation vial. 4 mL of 1,4-dioxane/water (10%) was added to the vial and the resulting suspension was subjected to temperature cycling from 40°C to room temperature for 72 h. The sample was filtered and the collected material was dried overnight on the filter.

X-ray diffraction analysis of the suspension material was consistent with the small scale material identified in the initial salt screening. X-ray diffraction after drying (on a filter bed) of the material showed Form 1.

RPD peaks.

Form 7 is shown below in Table 67 and in Fig. 21A. The material was crystalline. The MSP data showed agglomerates of particles with strong birefringence. An endotherm was observed by DSC (Fig. 21B) with an onset near 147°C. A small endotherm was observed with an onset near 196°C, associated with an observed transition to Form 1. A melting transition of Form 1 was observed with an onset near 205°C. The TG/DTA data (Fig. 21C) showed an endotherm with an onset near 147°C with a corresponding mass loss of about 7%, associated with loss of solvent. The mass loss was equivalent to about 2 equivalents of water present. A small endotherm was observed with an onset near 196°C, associated with an observed transition to Form 1. A melting transition of Form 1 was observed with an onset near 206°C. The material was slightly hygroscopic by GSP (Figs. 21D and 21E), with a mass increase of 0.8% at 90% RH, and no shape change was observed by XRP analysis after GSP. Trace amounts of solvent were observed by 1H NMR (Fig. 21F).

Table 67

RPD peaks of form 7

Position [°2-theta] PSHPV left [°20] Area [imp*°29] distance d[A] Height [imp.] Rel. INT. [%] 4.88 0,15 124.64 18,10 823,10 14.40 6.19 0,15 19.85 14.28 131,10 2.29 8.59 0,15 142.39 10.30 940,35 16.45 9.87 0,15 176.65 8.96 1166,58 20,41 10.31 0,15 28,54 8.58 188,50 3.30 11.62 0,10 34.57 7.62 342,41 5.99 12.58 0,10 36,56 7.03 362.18 6.34 14,14 0,15 140.39 6.27 927,13 16.22 14.84 0,15 143.38 5.97 946,87 16.57 15.77 0.26 452,10 5.62 1791,38 31,34 16.58 0,15 865,48 5.35 5715,58 100,00 17.26 0,10 199.94 5.14 1980,57 34.65 18.04 0,10 290,40 4.92 2876,67 50.33 18.97 0,10 237,60 4.68 2353,62 41,18 19.34 0,15 399,14 4.59 2635,93 46.12 19.91 0,10 334.71 4.46 3315,58 58,01 21.35 0,10 180.69 4.16 1789,93 31,32 21.84 0,10 138.81 4.07 1375,00 24.06 23,34 0,10 258.86 3.81 2564,23 44.86 24,28 0.13 108.82 3.67 862.39 15.09 25.12 0,10 217,06 3.55 2150,16 37,62 25.73 0,10 158.86 3.46 1573,65 27.53

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4. Form 8.

Form 8 was identified as the isopropyl alcohol (IPA) solvate, which desolvated to Form 1 upon heating. Form 8 was prepared on a 300 mg scale as follows. About 500 mg of the free base of compound of formula II was weighed into a 20 mL scintillation vial. 4 mL of IPA was added to the vial and the resulting suspension was subjected to temperature cycling from 40°C to room temperature for 72 h. The sample was filtered and the collected material was dried overnight under vacuum at 40°C.

XRPD analysis of the suspension material was consistent with the small scale data observed in the initial polymorph and salt screening. XRPD analysis after drying (40°C under vacuum) of the material yielded Form 8. The XRPD peaks of Form 8 are shown below in Table 68 and in Fig. 22A. The material was crystalline. The MSP data showed agglomerates of particles with strong birefringence. An endotherm was observed by DSC (Fig. 22B) with an onset of about 168°C. A small endotherm was observed with an onset of about 190°C, associated with the apparent transition to Form 1. The melting transition of Form 1 was observed with an onset of about 203°C. The TG/DTA data (Fig. 22C) showed an endotherm with an onset of about 165°C with a corresponding mass loss of about 4%, associated with solvent loss. The mass loss corresponded to approximately 0.5 equivalents of IPA present in the sample. A small endotherm was observed with an onset at approximately 191 °C, associated with the observed transition to Form 1. A melting transition of Form 1 was observed with an onset at approximately 205 °C. The material was moderately hygroscopic as determined by GSP (Figs. 22D and 22E) with a mass gain of 2.6% at 90% RH. Increased amorphous content was observed by XRP data after GSP analysis. 0.5 equivalents of IPA were observed by 1H NMR (Fig. 22F).

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Table 68

RPD peaks of form 8

Position [°2-theta] PSHPV left [°2θ] Area [imp.*°20] distance d[A] Height [imp.] Rel. INT. [%] 6.19 0,15 92.45 14.28 610,55 9.20 8.91 0,10 34.21 9.93 338,85 5.11 10.29 0,15 65,01 8.60 429.31 6.47 13.43 0.41 18.88 6.59 46.76 0,70 13.87 0,15 11.28 6.38 74.48 1,12 14.68 0,15 76.40 6.03 504.57 7.60 15,12 0,10 194.68 5.86 1928,53 29.06 15.87 0,15 16,00 5.58 105.68 1.59 16.50 0,15 76.75 5.37 506,86 7.64 16.79 0,10 39.53 5.28 391,55 5.90 17,14 0,10 28,80 5.17 285.27 4.30 17.42 0.13 43,66 5.09 346,03 5.21 17.82 0,10 669,86 4.98 6635,61 100,00 18,14 0,10 64.77 4.89 641,58 9.67 18.72 0,15 11.62 4.74 76,73 1,16 19.09 0,15 11,11 4.65 73.40 1,11 20.38 0,10 80.18 4.36 794.26 11.97 21,08 0,15 100.83 4.21 665,90 10.04 22,10 0,15 85,85 4.02 566.98 8.54 22.81 0,10 44.29 3.90 438,70 6.61 23.37 0,10 62.75 3.81 621,59 9.37 24.20 0,10 182.81 3.68 1810,90 27,29 24.61 0,10 61.83 3.62 612,45 9.23 25,00 0.13 45,50 3.56 360,61 5.43

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25.48 0,10 27.06 3.50 268,10 4.04 26.14 0,10 10.42 3.41 103.22 1.56 27,21 0,10 49.21 3.28 487,46 7.35 27.40 0,10 29.76 3.26 294.85 4.44 27.97 0.13 22.74 3.19 180,20 2.72 29.03 0,15 32,60 3.08 215,30 3.24 29,36 0.13 49.11 3.04 389.19 5.87 29.63 0.13 56.71 3.01 449.38 6.77 29.98 0.13 44.37 2.98 351,62 5.30 30,50 0,10 13.45 2.93 133.23 2.01 31,20 0,10 58.94 2.87 583.89 8.80 31,66 0,10 19.31 2.83 191.32 2.88 32,22 0.13 43.31 2.78 343,20 5.17 32,61 0,20 9.60 2.75 47,53 0.72 34.09 0.13 20,42 2.63 161.81 2.44 34,46 0,10 25.96 2.60 257.14 3.88

Example 6. Polymorph of the compound of formula III.

The compound of formula III, prepared as described in Example 2, was analyzed by XRPD, DSC, and 1H NMR and found to be Form A. The XRPD pattern of Form A is shown in Fig. 63A. The XRPD peaks of Form A are shown in Table 69. An endothermic event with an onset at about 83°C was observed by DSC (Fig. 63B).

Table 69

X-ray peaks of Form A of compound of formula III

2-Theta Intensity 2-Theta Intensity 2.02 4930 13.42 3244 2.74 2840 14 1121 3.12 2219 14.5 1495 4.24 2468 15,24 3953 4.66 5782 15.86 2721 5.1 1247 16.68 1776 6.24 1061 17.3 9498 6.76 3997 17.72 1523 7.16 905 18.76 5838 7.74 827 19.2 9152 8.7 811 20,24 3605 9.22 1144 20.96 1217 9.34 1419 21.92 2319 9.72 815 22.1 1894 10.2 776 23.2 1306 10.56 755 23.86 5921 11,5 740 24.1 2517 11.86 715 25,24 1657 12.66 1246 25.48 2717 13.24 1713 25.7 1941

Example 7. Polymorph of the compound of formula IV.

A. Initial characteristics.

The compound of formula IV, prepared as described in Example 2, was analyzed using

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XRD, DSC and 1H NMR were performed and it was found to be Form A. The XRD pattern of Form A is shown in Fig. 64A. The XRD peaks of Form A are shown in Table 70. An endothermic event with an onset at about 149°C was observed by DSC (Fig. 64B).

Table 70

X-ray peaks of Form A of compound of formula IV

2-Theta d(A) Height N% 8.32 10.62 373,20 100,00 9.05 9.77 8.90 2.40 10,11 8.74 0.90 0,20 10.65 8.30 1,20 0,30 11.49 7.70 30,90 8.30 12.25 7.22 27,50 7.40 13.03 6.79 24.40 6.50 13.51 6.55 3.30 0.90 14.08 6.29 0,70 0,20 14.38 6.16 1.40 0,40 14.68 6.03 4.30 1,20 15,60 5.68 3.00 0.80 16.27 5.44 265,60 71.20 16.64 5.32 94.40 25.30 16.89 5.25 5,00 1.40 17.57 5.04 24.40 6.50 18.08 4.90 42,00 11.30 18.61 4.76 39,00 10.50 18.85 4.70 41,00 11,00 19,15 4.63 23.80 6.40

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19.35 4.58 92,10 24.70 19.80 4.48 3.60 1,00 20,04 4.43 49.20 13.20 20,28 4.37 30,50 8.20 20.45 4.34 59,60 16,00 20,90 4.25 0,30 0,10 21.59 4.11 68.30 18.30 21.92 4.05 120,80 32,40 22.69 3.92 13.90 3.70 23,01 3.86 4.10 1,10 23,34 3.81 6.80 1.80 23.81 3.73 11.40 3.10 24.41 3.64 26,50 7,10 25,19 3.53 11.20 3.00 25.71 3.46 7.30 2,00 26.06 3.42 17.40 4.70 27.04 3.29 12.40 3.30 28,21 3.16 3.40 0.90 28,78 3.10 1,10 0,30 29.56 3.02 1,20 0,30 30,41 2.94 17.90 4.80 31,04 2.88 3.00 0.80 31.87 2.81 8.40 2.20 32,10 2.79 21,40 5.70 33,12 2.70 1.30 0,40

33.61 2.66 3.60 1,00 34.16 2.62 5.80 1.50 34.81 2.58 4.10 1,10 35.11 2.55 6.20 1.60 36.76 2.44 2.40 0,60 37,00 2.43 2.40 0,60 37,42 2.40 1.80 0,50 37.84 2.38 2,10 0,60 39,36 2.29 2,00 0,50

B. Solubility screening.

The solubility of the compound of formula IV (free base) was tested in various solvent mixtures as shown in Table 71.

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Table 71

Solubility of compound of formula IV (free base) at about 25°C

Solvent mg/ml Acetone 42.9 Acetone: water (1:1) 45.8 Acetone: heptane (1:1) 6.1 Butanol 16.6 MTBE 5.5 Ethanol 5.2 EUN:water(1:1) 51 EUN:water (90:10) 55 Ethyl acetate 21,0 Heptane 0,0 IEC 30.7 IPA 7.0 ACN 21.2 Chloroform >59 Chloroform: heptane (1:1) P,7 DXM >70 p-dioxane >62 Hexane 0,0 Methanol >74 THF >37 THF:heptane (30:70) 0,6 Water 0,007

C. Polymorphic screening.

Additional polymorphic screening was performed using crystallization techniques such as cooling and solvent/antisolvent precipitation. The results are presented in Table 72.

Table 72

Polymorphic screening of compound of formula IV

Solvent Crystallization technology Results of RPD Acetone Suspended at ~25°C for ~3 days Form A Acetone/water Diffusion of vapors at room temperature Form A Acetone:water (1:1) Suspended at ~25°C for ~3 days Form A Acetone/heptane Diffusion of vapors at room temperature Form A Acetone:heptane (2:1) The API was dissolved at ~55 °C, slowly cooled to room temperature without stirring. Form A Acetone: heptane (1:1) Suspended at ~25°C for ~3 days Form A CAN Suspended at ~25°C for ~3 days Form A 2-Butanol Suspended at ~25°C for ~3 days Form A 2-Butanol Suspended at ~5°C for ~3 days Form A

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Chloroform Slow evaporation at room temperature -- Chloroform/heptane Diffusion of vapors at room temperature Form A Chloroform:heptane (1:1) Suspended at ~25 °C for -3 days Form A DXM Slow evaporation at room temperature — p-dioxane Rapid evaporation at room t- Re EtOH Suspended at ~25 °C for -3 days Form A EUN:water (1:1) Suspended at ~25 °C for -3 days Form A EUN:water (90:10) Suspended at ~25 °C for -3 days Form B EtOAc Suspended at ~25 °C for -3 days Form A EtOAc Suspended at -5 °C for -3 days Form A EtOAc The API was dissolved at ~70 °C, slowly cooled to room temperature. Form A Heptane Suspended at ~25 °C for -3 days Form A Hexane Suspended at ~25 °C for -3 days Form A IPA Suspended at ~25 °C for -3 days Form A IPA The API was dissolved at ~70°C, slowly cooled to room temperature without stirring. Form A MeOH Slow evaporation at room temperature —

MeOH/water Diffusion of vapors at room temperature, oily substance -- IEC Suspended at ~25°C for -3 days, insufficient solids MTBE Suspended at -25 °C for -3 days Form A THF Slow evaporation at room temperature -- THF: heptane (30:70) Suspended at -25 °C for -3 days Form A THFteptane (1:4) Precipitation using solvent/antisolvent, observed formation of precipitate and oily substance, left stirring at room temperature for -1 day Form A TNB:water (1:4) Precipitation using solvent/antisolvent, formation of precipitate and small amount of oily substance observed, stirred at -5 °C, oily substance persisted THF/heptane Diffusion of vapors at room temperature Form A Water Suspended at -25 °C for -3 days Form A

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Form B. A new XRD pattern was observed for the substance from a suspension in ethanol/water (90:10) as shown above in Table 72. The substance with the new XRD pattern was designated Form B.

Form B was prepared at ~65 mg scale by suspending a composition containing Form A and 1 mL of 9:1 EtOH: _H2O at 25°C for ~36 h. The solid was isolated by centrifugal filtration (nylon filter, benchtop minicentrifuge, 8000 rpm, five minutes) and then dried in a vacuum oven under _N2 flow at room temperature overnight (~18 h), followed by XRPD and DSC analysis.

Form B was further analyzed by XRPD and DSC. The XRPD data are shown in Fig. 65A and Table 73. The DSC thermogram of Form B is shown in Fig. 65B. An endothermic event was observed, as determined by DSC, with an onset at about 162°C, which was about 12°C higher than that of Form A. An overlay of the XRPD patterns of polymorphs A and B of the compound of formula IV is shown in Fig. 66.

Table 73

X-ray peaks of form B of compound of formula IV

2-Theta d(A) Height N% 4.85 18,19 20,60 5.30 7.45 11.86 390,20 100,00 7.69 11.48 24.90 6.40 9.70 9,11 127.30 32,60 9.94 8.89 88,80 22.80 13.74 6.44 142.80 36,60 14.54 6.09 94,00 24.10 14.89 5.94 1.90 0,50 15.43 5.74 19.40 5,00 15.76 5.62 3.30 0.90 16.31 5.43 20,70 5.30 16.89 5.24 149,00 38.20 17,15 5.17 14.30 3.70 17.39 5.09 10.60 2.70 17.79 4.98 10.80 2.80 18.08 4.90 25,60 6.60 18.48 4.80 20,60 5.30 19.44 4.56 126,00 32.30 19.90 4.46 127,60 32.70 20,80 4.27 2.50 0,60 21,31 4.17 100,00 25,60 21,31 4.17 100,00 25,60 21,44 4.14 62,10 15.90 22,21 4.00 2.60 0,70

- 225 041220

22,41 3.96 15.80 4.00 22.80 3.90 27.70 7,10 23.05 3.86 9.80 2.50 23.53 3.78 9.50 2.40 24,25 3.67 23.30 6.00 24.50 3.63 27,00 6.90 24.91 3.57 10,00 2.60 25,35 3.51 3.80 1,00 25.71 3.46 0.90 0,20 26.31 3.39 15.70 4.00 26.74 3.33 2.60 0,70 26.94 3.31 4.20 1,10 27,39 3.25 31,00 7.90 28,24 3.16 13,10 3.40 28.92 3.08 9.20 2.40 29.56 3.02 0,70 0,20 30.04 2.97 3.70 0.90 31,12 2.87 2,10 0,50 31.76 2.82 5.70 1.50 31.99 2.80 2,10 0,50 32.27 2.77 1,00 0,30 32.98 2.71 2.80 0,70 33.61 2.66 10,20 2.60 34.19 2.62 2,00 0,50 34.68 2.58 1,20 0,30

35.11 2.55 1.70 0,40 35,66 2.52 1.70 0,40 35.84 2.50 2.80 0,70 36,32 2.47 1.30 0,30 38.05 2.36 2.90 0,70 39,00 2.31 1.80 0,50 39.21 2.30 3.00 0.80

Example 8: RET enzyme assay.

Compounds of formula I-IV were tested for their ability to inhibit wild-type RET kinase and the V804M mutant RET kinase using CisBio's HTRF® KinEASE™-TK assay technology. Briefly, the N-terminal cytoplasmic domain of the GST-tagged human recombinant RET (aa 658-end) from Eurofins (0.25 nM RET; catalog #14-570M) or the N-terminal cytoplasmic domain of the V804M mutant human recombinant RET with a GST-tagged (aa 658-end) from Millipore (0.25 nM enzyme; catalog #14760) were incubated with 250 nM biotin TK substrate (CisBio, catalog #62TKOPEC) and 1 mM ATP together with the experimental compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM _MgCl2 , 0.01% Triton X-100, and 2% DMSO in a volume of 8 μl. Compound was typically prepared in three-fold serial dilutions in DMSO and added to the assay to give the appropriate final concentration. After 30 min incubation at 22°C, the reaction was quenched by adding 8 μl of quench solution containing 31.25 nM Sa-XL665 and 1X TK-ab-cryptate in HTRF detection buffer (all from CisBio, cat. #62TKOPEC). After 1 h of incubation at 22°C, reaction progress was determined using a PerkinElmer EnVision multi-mode plate reader via dual wavelength HTRF detection, and percent control (PCO) was calculated using the log emissivity. 100 PCO was determined without the use of experimental compound, and 0 PCO was determined using pre-quenched control reactions. The ROS values were fitted to a 4-parameter logistic curve and the IC ₅₀ was determined as the inhibitor concentration at which the ROS equaled 50 for the fitted curve. It was found that the compounds

- 226 041220 The compound of formula I has IC ₅₀ values of 4.5 nM and 18.9 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively. The compound of formula II has been found to have IC ₅₀ values of 14.0 nM and 24.1 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively. The compound of formula III has been found to have IC ₅₀ values of 29.9 nM and 59.0 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively. The compound of formula IV has been found to have IC ₅₀ values of 17.0 nM and 121.7 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively.

Example 9. Cellular analysis of RET.

The cellular potency of a compound inhibiting RET kinase was determined in HEK293 cells expressing the Kif5b-RET fusion protein. Briefly, HEK-293 cells expressing the Kif5b-RET fusion protein were plated at 50,000 cells/well in poly-D-lysine-coated 96-well plates the day before the assay. Cells were incubated for 1 h with the experimental compound in DMEM at a final DMSO concentration of 0.5%. Compound of formula I-IV was typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After 1 h, the medium was removed, cells were fixed with 3.8% formaldehyde for 20 min, washed with PBS and permeabilized for 10 min with 100% methanol. The plates were then washed with PBS-0.05% Tween20 and blocked with LI-COR blocking solution (LI-COR, cat. #927-40000) for 1 h. The plates were washed with PBS-0.05% Tween20, then incubated with anti-phospho-RET (Tyr1062) antibody (Santa Cruz, cat. #sc-20252-R) and anti-GAPDH antibody (Millipore, cat. #MAB374) for 2 h. The plates were washed with PBS-0.05% Tween20 and incubated with anti-rabbit 680 (Molecular Probes, cat. #A21109) and anti-mouse 800 (LI-COR, cat. # 926-32210) for 1 h. All antibodies were diluted in LI-COR blocking solution containing 0.05% Tween. Plates were washed with PBS-0.05% Tween20, 100 µl PBS was added to each well and the plates were read on a LI-COR Aerius fluorescence plate reader. The phospho-RET signal was normalized to the GAPDH signal. 100 ROS (percentage of control) was determined without experimental compounds, and 0 ROS was determined using 1 µM control inhibitor. ROS values were fitted to a 4-parameter logistic curve. The IC ₅₀ value is the point where the curve intersects 50 ROS. The compound of formula I was found to have an _IC50 value of 4.0 nM for inhibiting RET kinase in HEK-293 cells expressing the Kif5b-RET fusion protein. The compound of formula II was found to have an _IC50 value of 4.2 nM for inhibiting RET kinase in HEK-293 cells expressing the Kif5b-RET fusion protein. The compound of formula III was found to have an _IC50 value of 10.9 nM for inhibiting RET kinase in HEK-293 cells expressing the Kif5b-RET fusion protein. The compound of formula IV was found to have an _IC50 value of 16.5 nM for inhibiting RET kinase in HEK-293 cells expressing the Kif5b-RET fusion protein.

Claims (9)

CLAIM 1. A crystalline form of a compound of formula II having the formula wherein said crystalline form is form 1 characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at °2θ values: a) 16.5±0.2, 18.9±0.2 and 26.0±0.2; b) 16.5±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2 and 26.0±0.2; c) 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2 and 28.3±0.2; or d) 9.8±0.2, 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0.2, 25.0±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2 and 28.3±0.2. 2. The crystalline form of a compound of formula II having the formula wherein said crystalline form is form 2 characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at °2θ values: a) 15.1±0.2, 17.8±0 .2 and 24.2±0.2; b) 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2 and 24.2±0.2; - 227 041220 c) 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2 and 24.6±0.2; or d) 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2 and 31.2±0.2. 3. The crystalline form of the compound of formula II having the formula where the specified crystalline form is form 7, characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at values of °2θ: a) 16.6±0.2, 18.0±0.2 and 19.9±0.2; b) 16.6±0.2, 18.0±0.2, 19.3±0.2, 19.9±0.2 and 23.3±0.2; c) 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.9±0.2, 23.3±0.2 and 25.1±0.2; or d) 15.8±0.2, 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.91±0.2, 21.4±0.2, 23.3±0.2 and 25.1±0.2. 4. The crystalline form of the compound of formula II having the formula N=\ N^zAcN And νΑ>| where the specified crystalline form is form 8 solvate with isopropyl alcohol, characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at values of °2θ: a) 15.1±0.2, 17.8±0.2 and 24.2±0.2; b) 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2 and 24.2±0.2; c) 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2 and 24.6±0.2; or d) 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2 and 31.2±0.2. 5. Solid oral pharmaceutical composition containing a pharmaceutically acceptable carrier and a crystalline form according to any one of claims 1-4. 6. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 1 and said composition contains less than about 15% by weight of other forms of the compound. 7. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 2 and said composition contains less than about 15% by weight of other forms of the compound. 8. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 7 and said composition contains less than about 15% by weight of other forms of the compound. 9. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 8 and said composition contains less than about 15% by weight of other forms of the compound.