WO2024222894A1

WO2024222894A1 - Benzofuran compounds and their use as pi3k alpha inhibitors

Info

Publication number: WO2024222894A1
Application number: PCT/CN2024/090120
Authority: WO
Inventors: Ming Li; Ruipeng ZHANG; Chun-Yen Chen; Xiang-Ju Justin Gu
Original assignee: Laekna Pharmaceutical Ningbo Co., Ltd.
Priority date: 2023-04-27
Filing date: 2024-04-26
Publication date: 2024-10-31

Abstract

Provided herein are certain multicyclic compounds, such as a compound of Formula (I), as PI3Kα inhibitors, pharmaceutical compositions comprising the compounds, and method of use of the compounds or pharmaceutical compositions in the treatment of diseases or disorders.

Description

BENZOFURAN COMPOUNDS AND THEIR USE AS PI3K ALPHA INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to International Patent Application No. PCT/CN2023/091375 filed on April 27, 2023 and International Patent Application No. PCT/CN2023/115780 filed on August 30, 2023, the entirety of each of which is incorporated herein by reference.

SEQUENCE LISTING

This application is submitted concurrently with a computer readable Sequence Listing in XML file format, the content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted is entitled “14668-028-228_SEQLISTING. xml” , was created on April 16, 2024, and is 3, 295 bytes in size.

FIELD

Provided herein are certain multicyclic compounds as PI3K alpha (PI3Kα) inhibitors, pharmaceutical compositions comprising the compounds, and method of use of the compounds or pharmaceutical compositions in the treatment of diseases or disorders.

BACKGROUND

Lipid phosphoinositides in cell membranes are master regulators of membrane signaling events for membrane trafficking, metabolism, growth, signaling and autophagy, with alterations in phosphoinositide metabolism being causative for many human diseases (Dickson, E.J., Hille, B., Biochem. J. 2019, 476 (1) , 1–23) . Phosphoinositides are generated from phosphatidylinositol (PI) by the action of lipid phosphoinositide kinases (19 unique genes in mammals) and are degraded by the action of phosphoinositide phosphatases (Schink, K.O. et al., Annu. Rev. Cell Dev. Biol. 2016, 32, 143–171) .

Class I phosphoinositide 3-kinases (PI3Ks) are involved in signaling pathways downstream of tyrosine kinases (RTKs) , G protein-coupled receptors (GPCRs) , and GTPases such as RAS, RAC, and CDC42, regulating a range of cellular activities, including metabolism, proliferation, and migration (Vanhaesebroeck, B. et al., Nat Rev Drug Discov 2021, 20, 741–769) . Class I PI3Ks comprise heterodimers formed by p110 catalytic subunits (α, β, γ or δ) and p85 regulatory subunits, among which PI3Kα and PI3Kβ have a wide tissue distribution, while PI3Kγ and PI3Kδ are more abundant in leukocytes. At the cellular level, a key function of PI3Kα is to convert growth factor stimulation into activation of anabolic processes and concomitant inhibition of catabolic processes (Hammond, G. R. V. &Burke, J.E., Curr. Opin. Cell Biol. 2020, 63, 57–67) . Two key effectors of PI3Kαinvolved in this response are AKT and mTOR, these are serine/threonine kinases with a myriad of substrates and pleiotropic functions. Combined with AKT/mTOR, PI3Kα pathway endows the widespread transcriptional changes for energy generation and biosynthetic activity, key requisites for cell proliferation and survival (Lee, J.V. et al., Cell Metab. 2014, 20, 306–319) .

The human p110α protein is encoded by the PIK3CA gene. PIK3CA is a 34 kb gene located on chromosome 3q26.3 that consists of 20 exons coding for 1068 amino acids yielding a 124 kDa size protein. PIK3CA is one of the most frequently mutated kinase gene in solid tumors. Oncogenic mutations are present across PIK3CA, apart from the RAS-binding domain, also highly enriched for hotspot mutations in the helical (E542K, E545K) and kinase (H1047R) domains, having the strongest biological impact in experimental cell model systems compared with other PIK3CA mutations (Zhang, Y. et al., Cancer Cell 2017, 31, 820–832 e823) . Mutations in PIK3CA mimic and enhance dynamic events in the natural activation process of the auto-inhibited p85–p110 heterodimer (Burke, J. E. et al., Oncotarget 2013, 4, 180–181) . PIK3CA mutation has multiple impacts, including the reduction of growth factor dependence, emergence of stem cell-like properties, the toleration of chromosomal instability, potentially driving tumor evolution (Vanhaesebroeck, B. et al., Biomolecules 9, 331) . Transcriptional profiling of a PIK3CA-mutated derivative of the MCF10A breast cell line indicated the expression of PI3K-driven, nuclear factor-κB (NF-κB) -dependent target genes enriched in cytokines, chemokines or secreted proteins (Hutti, J. E. et al., Cancer Res. 2012, 72, 3260–3269) . PIK3CA mutation in cancer cells also create an immunosuppressive stromal environment by induction of high glycolysis in cancer cells, leading to a high demand for glucose and subsequent depletion of metabolic fuels in the stroma, thus contributing to immune suppression (Hao, Y. et al., Nat. Commun. 2016, 7, 11971; Biswas, S. K., Immunity 2015, 43, 435–449) .

Because of the PI3Kα pathway’s role in oncogenesis, a variety of PI3Kαinhibitors have been developed to attempt to improve cancer control. Isoform-selective PI3Kα inhibitors have a good potency for the ATP pocket and became a main rationale for the use in oncology to target cancer cell-intrinsic PI3K pathway (Tarantelli, C. et al., Clin. Cancer Res. 2018, 24, 120–129) . Main challenge in the therapeutic exploitation of PI3Kαinhibitors is toxicity and pathway reactivation. Feedback can counteract PI3Kα inhibition by both cell-intrinsic and systematic mechanisms (Burke, J.E. et al. Nat Rev Drug Discov 2022, DOI: 10.1038/s41573-022-00582-5) . Inhibition of PI3K leads to a decreased activation of AKT, relieving suppression of receptor tyrosine kinase (RTK) expression and reactivating the PI3K pathway (Chakrabarty, A. et al., Proc. Natl Acad. Sci. USA 2012, 109, 2718–2723) . In clinical, PI3Kα inhibition may lead to hyperglycemia, causing the reactivation of the pathway, which hampers the clinical dosing and therapeutic window. These events suggest an opportunity to combine PI3K inhibition with dietary or pharmacological interventions to lower blood glucose levels (Hopkins, B. D. et al., Nature 2018, 560, 499–503) . Therefore, a major focus in PI3Kα drug development is the mutant-selective inhibitors. Targeting PI3Kαdominant mutations (E542K, E545K, H1047R) may improve the therapeutic window, enabling sufficient target inhibition in tumor as well as avoiding the dose-limiting toxicity.

SUMMARY

In one embodiment, provided herein are multicyclic compounds as PI3Kαinhibitors.

In one embodiment, provided herein is a compound of Formula (I) :

or a stereoisomer, or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein X¹, X², R^a3, R^a4, R^a5, R^a6, Ring A, Ring B, L, R and R¹ are as defined herein or elsewhere.

Also provided herein are pharmaceutical compositions comprising a compound provided herein and a pharmaceutically acceptable excipient.

Also provided herein are methods of inhibiting a PI3Kα protein, comprising contacting the PI3Kα protein with a compound provided herein or a pharmaceutical composition provided herein.

Also provided herein are methods of treating PI3Kα associated diseases or cancer, comprising administering to a subject having the disease or cancer a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein.

DETAILED DESCRIPTION

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

As used herein, and in the specification and the accompanying claims, the indefinite articles “a” and “an” and the definite article “the” include plural as well as single referents, unless the context clearly indicates otherwise.

As used herein, the terms “comprising” and “including” can be used interchangeably. The terms “comprising” and “including” are to be interpreted as specifying the presence of the stated features or components as referred to, but does not preclude the presence or addition of one or more features, or components, or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples encompassed by the term “consisting of” . Consequently, the term “consisting of” can be used in place of the terms “comprising” and “including” to provide for more specific embodiments.

As used herein, the term “or” is to be interpreted as an inclusive “or” meaning any one or any combination. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C” . An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

As used herein, the phrase “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone) ; and B (alone) . Likewise, the phrase “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .

It should be noted that if there is a discrepancy between a depicted structure and a name for that structure, the depicted structure is to be accorded more weight.

It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

As used herein, and unless otherwise specified, the term “alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated. In one embodiment, the alkyl group has, for example, from one to twenty-four carbon atoms (C₁-C₂₄ alkyl) , four to twenty carbon atoms (C₄-C₂₀ alkyl) , six to sixteen carbon atoms (C₆-C₁₆ alkyl) , six to nine carbon atoms (C₆-C₉ alkyl) , one to fifteen carbon atoms (C₁-C₁₅ alkyl) , one to twelve carbon atoms (C₁-C₁₂ alkyl) , one to eight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl) , n-butyl, n-pentyl, 1, 1-dimethylethyl (t-butyl) , 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, an alkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkenyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds. The term “alkenyl” also embraces radicals having “cis” and “trans” configurations, or alternatively, “E” and “Z” configurations, as appreciated by those of ordinary skill in the art. In one embodiment, the alkenyl group has, for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkenyl) , four to twenty carbon atoms (C₄-C₂₀ alkenyl) , six to sixteen carbon atoms (C₆-C₁₆ alkenyl) , six to nine carbon atoms (C₆-C₉ alkenyl) , two to fifteen carbon atoms (C₂-C₁₅ alkenyl) , two to twelve carbon atoms (C₂-C₁₂ alkenyl) , two to eight carbon atoms (C₂-C₈ alkenyl) or two to six carbon atoms (C₂-C₆ alkenyl) and which is attached to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1, 4-dienyl, and the like. Unless otherwise specified, an alkenyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkynyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon triple bonds. In one embodiment, the alkynyl group has, for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkynyl) , four to twenty carbon atoms (C₄-C₂₀ alkynyl) , six to sixteen carbon atoms (C₆-C₁₆ alkynyl) , six to nine carbon atoms (C₆-C₉ alkynyl) , two to fifteen carbon atoms (C₂-C₁₅ alkynyl) , two to twelve carbon atoms (C₂-C₁₂ alkynyl) , two to eight carbon atoms (C₂-C₈ alkynyl) or two to six carbon atoms (C₂-C₆ alkynyl) and which is attached to the rest of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specified, an alkynyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which is saturated. Cycloalkyl group may include fused, bridged, or spiro ring systems. In one embodiment, the cycloalkyl has, for example, from 3 to 15 ring carbon atoms (C₃-C₁₅ cycloalkyl) , from 3 to 10 ring carbon atoms (C₃-C₁₀ cycloalkyl) , or from 3 to 8 ring carbon atoms (C₃-C₈ cycloalkyl) . The cycloalkyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include, but are not limited to, adamantyl, norbornyl, decalinyl, 7, 7-dimethyl-bicyclo [2.2.1] heptanyl, and the like. Unless otherwise specified, a cycloalkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkenyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which includes one or more carbon-carbon double bonds. Cycloalkenyl may include fused, bridged, or spiro ring systems. In one embodiment, the cycloalkenyl has, for example, from 3 to 15 ring carbon atoms (C₃-C₁₅ cycloalkenyl) , from 3 to 10 ring carbon atoms (C₃-C₁₀ cycloalkenyl) , or from 3 to 8 ring carbon atoms (C₃-C₈ cycloalkenyl) . The cycloalkenyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyl radicals include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise specified, a cycloalkenyl group is optionally substituted. Similarly, as used herein, and unless otherwise specified, the term “cycloalkynyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which includes one or more carbon-carbon triple bonds.

As used herein, and unless otherwise specified, the term “heteroalkyl” refers to an alkyl radical that has one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, and phosphorus, or combinations thereof. A numerical range can be given to refer to the chain length in total. For example, a -CH₂OCH₂CH₃ radical is referred to as a “C₄” heteroalkyl. Connection to the parent molecular structure can be through either a heteroatom or a carbon in the heteroalkyl chain. One or more heteroatom (s) in the heteroalkyl radical can be optionally oxidized. One or more nitrogen atoms, if present, can also be optionally quaternized. Unless otherwise specified, a heteroalkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “aryl” refers to a monocyclic aromatic group and/or multicyclic aromatic group that contain at least one aromatic hydrocarbon ring. In certain embodiments, the aryl has from 6 to 18 ring carbon atoms (C₆-C₁₈ aryl) , from 6 to 14 ring carbon atoms (C₆-C₁₄ aryl) , or from 6 to 10 ring carbon atoms (C₆-C₁₀ aryl) . Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. The term “aryl” also refers to bicyclic, tricyclic, or other multicyclic hydrocarbon rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl) . Unless otherwise specified, an aryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “heteroaryl” refers to a monocyclic aromatic group and/or multicyclic aromatic group that contains at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from O, S, and N. The heteroaryl may be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. The term “heteroaryl” also refers to bicyclic, tricyclic, or other multicyclic rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrollinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise specified, a heteroaryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “heterocyclyl” refers to a monocyclic and/or multicyclic non-aromatic group that contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorous, and sulfur. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom. A heterocyclyl group can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other multicyclic ring system, wherein the multicyclic ring systems can be a fused, bridged or spiro ring system. Heterocyclyl multicyclic ring systems can include one or more heteroatoms in one or more rings. A heterocyclyl group can be saturated or partially unsaturated. Saturated heterocyclyl groups can be termed “heterocycloalkyl” . Partially unsaturated heterocyclyl groups can be termed “heterocycloalkenyl” if the heterocyclyl contains at least one double bond, or “heterocycloalkynyl” if the heterocyclyl contains at least one triple bond. In one embodiment, the heterocyclyl has, for example, 3 to 18 ring atoms (3-to 18-membered heterocyclyl) , 4 to 18 ring atoms (4-to 18-membered heterocyclyl) , 3 to 12 ring atoms (3-to 12-membered heterocyclyl) , 5 to 18 ring atoms (5-to 18-membered heterocyclyl) , 4 to 8 ring atoms (4-to 8-membered heterocyclyl) , or 5 to 8 ring atoms (5-to 8-membered heterocyclyl) . Examples of heterocyclyl groups include, but are not limited to, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuryl, and piperidinyl. Unless otherwise specified, a heterocyclyl group is optionally substituted.

Whenever it appears herein, a numerical range such as “3 to 18” refers to each integer in the given range; e.g., a heterocyclyl with “3 to 18 ring atoms” means that the heterocyclyl group can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc., up to and including 18 ring atoms. Similarly, a C₁-C₆ alkyl means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms.

As used herein and unless otherwise specified, a “cycloalkylalkyl” group is a radical of the formula: -alkyl-cycloalkyl, wherein alkyl and cycloalkyl are defined above. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl, or both the alkyl and the cycloalkyl portions of the group. Representative cycloalkylalkyl groups include but are not limited to cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl, cyclohexylethyl, cyclopentylpropyl, cyclohexylpropyl and the like.

As used herein and unless otherwise specified, an “aralkyl” group is a radical of the formula: -alkyl-aryl, wherein alkyl and aryl are defined above. Substituted aralkyl groups may be substituted at the alkyl, the aryl, or both the alkyl and the aryl portions of the group. Representative aralkyl groups include but are not limited to benzyl and phenethyl groups and aralkyl groups wherein the aryl group is fused to a cycloalkyl group such as indan-4-yl ethyl.

As used herein and unless otherwise specified, other similar composite terms mirror the above description for “cycloalkylalkyl” and “aralkyl” . For example, a “heterocyclylalkyl” group is a radical of the formula: -alkyl-heterocyclyl, wherein alkyl and heterocyclyl are defined above. A “heteroarylalkyl” group is a radical of the formula: -alkyl-heteroaryl, wherein alkyl and heteroaryl are defined above. A “heterocycloalkylalkyl” group is a radical of the formula: -alkyl-heterocycloalkyl, wherein alkyl and heterocycloalkyl are defined above.

As used herein, and unless otherwise specified, the term “halogen” , “halide” or “halo” refers to fluorine, chlorine, bromine, and/or iodine. As used herein, and unless otherwise specified, the terms “haloalkyl, ” “haloalkenyl, ” “haloalkynyl, ” and “haloalkoxy” refer to alkyl, alkenyl, alkynyl, and alkoxy structures that are substituted with one or more halo groups or with combinations thereof.

As used herein, and unless otherwise specified, the term “alkoxy” refers to -O- (alkyl) , wherein alkyl is defined above. As used herein, and unless otherwise specified, the term “aryloxy” refers to -O- (aryl) , wherein aryl is defined above.

As used herein, and unless otherwise specified, the term “alkyl sulfonyl” refers to –SO₂-alkyl, wherein alkyl is defined above.

As used herein, and unless otherwise specified, the term “carboxyl” and “carboxy” refers to -COOH.

As used herein, and unless otherwise specified, the term “alkoxycarbonyl” refers to -C (=O) O- (alkyl) , wherein alkyl is defined above. As used herein, and unless otherwise specified, the term “arylalkyloxy” refers to -O- (alkyl) - (aryl) , wherein alkyl and aryl are defined above. As used herein, and unless otherwise specified, the term “cycloalkyloxy” refers to -O- (cycloalkyl) , wherein cycloalkyl is defined above. As used herein, and unless otherwise specified, the term “cycloalkylalkyloxy” refers to -O- (alkyl) - (cycloalkyl) , wherein cycloalkyl and alkyl are defined above.

As used herein, and unless otherwise specified, the term “acyl” refers to –C (O) -R^a, wherein R^a can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, R^a may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term “acyloxy” refers to –O-C (O) -R^a, wherein R^a can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, R^a may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term “amino” refers to –N (R^#) (R^#) , wherein each R^#independently can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. When a -N (R^#) (R^#) group has two R^#other than hydrogen, they can be combined with the nitrogen atom to form a ring. In one embodiment, the ring is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring. In one embodiment, one or more ring atoms are heteroatoms independently selected from O, S, or N. The term “amino” also includes N-oxide (–N⁺ (R^#) (R^#) O^-) . In certain embodiments, each R^#or the ring formed by -N (R^#) (R^#) independently may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term “amide” or “amido” refers to –C (O) N (R^#) ₂ or –NR^#C (O) R^#, wherein each R^#independently can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. When a –C (O) N (R^#) ₂ group has two R^#other than hydrogen, they can be combined with the nitrogen atom to form a ring. In one embodiment, the ring is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring. In one embodiment, one or more ring atoms are heteroatoms independently selected from O, S, or N. In certain embodiments, each R^#or the ring formed by -N (R^#) (R^#) independently may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term “aminoalkyl” refers to - (alkyl) - (amino) , wherein alkyl and amino are defined above. As used herein, and unless otherwise specified, the term “aminoalkoxy” refers to -O- (alkyl) - (amino) , wherein alkyl and amino are defined above.

As used herein, and unless otherwise specified, the term “alkylamino” refers to -NH (alkyl) or -N (alkyl) (alkyl) , wherein alkyl is defined above. Examples of such alkylamino groups include, but are not limited to, -NHCH₃, -NHCH₂CH₃, -NH (CH₂) ₂CH₃, -NH (CH₂) ₃CH₃, -NH (CH₂) ₄CH₃, -NH (CH₂) ₅CH₃, -N (CH₃) ₂, -N (CH₂CH₃) ₂, -N ( (CH₂) ₂CH₃) ₂, -N (CH₃) (CH₂CH₃) , and the like.

As used herein, and unless otherwise specified, the term “arylamino” refers to -NH (aryl) or -N (aryl) (aryl) , wherein aryl is defined above. As used herein, and unless otherwise specified, similar composite terms such as “arylalkylamino” and “cycloalkylamino” mirrors the descriptions above for “alkylamino” and “arylamino” .

As used herein, and unless otherwise specified, the term “sulfanyl” , “sulfide” , or “thio” refers to -S-R^a, wherein R^a can be, but is not limited to, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, R^a may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term “sulfoxide” refers to –S (O) -R^a, wherein R^a can be, but is not limited to, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, R^a may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term “sulfonyl” or “sulfone” refers to –S (O) ₂-R^a, wherein R^a can be, but is not limited to, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, R^a may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term “sulfonamido” or “sulfonamide” refers to –S (=O) ₂–N (R^#) ₂ or –N (R^#) –S (=O) ₂–R^#, wherein each R^#independently can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. When a –S (=O) ₂– N (R^#) ₂ group has two R^#other than hydrogen, they can be combined with the nitrogen atom to form a ring. In one embodiment, the ring is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring. In one embodiment, one or more ring atoms are heteroatoms independently selected from O, S, or N. In certain embodiments, each R^#or the ring formed by -N (R^#) (R^#) independently may be unsubstituted or substituted with one or more substituents.

“Azide” refers to a –N₃ radical. “Cyano” refers to a –CN radical. “Nitro” refers to the –NO₂ radical. “Oxa” refers to the –O–radical. “Oxo” refers to the =O radical.

As used herein, and unless otherwise specified, the term “optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

When the groups described herein are said to be “substituted, ” they may be substituted with any appropriate substituent or substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as halogen (chloro, iodo, bromo, or fluoro) ; alkyl; alkenyl; alkynyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxyl amine; alkoxyamine; aryloxyamine, aralkoxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxo (═O) ; B (OH) ₂, O (alkyl) aminocarbonyl; cycloalkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) , or a heterocyclyl, which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiazinyl) ; monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl) aryloxy; aralkyloxy; heterocyclyloxy; and heterocyclyl alkoxy.

As used herein, and unless otherwise specified, the term “isomer” refers to different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Atropisomers” are stereoisomers from hindered rotation about single bonds. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any proportion can be known as a “racemic” mixture. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry can be specified according to the Cahn-Ingold-Prelog R-Ssystem. When a compound is an enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro-or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. However, the sign of optical rotation, (+) and (-) , is not related to the absolute configuration of the molecule, R and S. Certain compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry at each asymmetric atom, as (R) -or (S) -. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically substantially pure forms and intermediate mixtures. Optically active (R) -and (S) -isomers can be prepared, for example, using chiral synthons or chiral reagents, or resolved using conventional techniques.

As used herein, and unless otherwise specified, the term “enantiomeric purity” or “enantiomer purity” refers to a qualitative or quantitative measure of a purified enantiomer. The enantiomeric purity of compounds described herein may be described in terms of enantiomeric excess (ee) , which indicates the degree to which a sample contains one enantiomer in greater amounts than the other. A racemic mixture has an ee of 0%, while a single completely pure enantiomer has an ee of 100%. Examples of the enantiomeric purity include an ee of at least about 10%, at least about 12%, at least about 14%, at least about 16%, at least about 18%, at least about 20%, at least about 22%, at least about 24%, at least about 26%, at least about 28%, at least about 30%, at least about 32%, at least about 34%, at least about 36%, at least about 38%, at least about 40%, at least about 42%, at least about 44%, at least about 46%, at least about 48%, at least about 50%, at least about 52%, at least about 54%, at least about 56%, at least about 58%, at least about 60%, at least about 62%, at least about 64%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about or at least about 99%. Similarly, “diastereomeric purity” may be described in terms of diasteriomeric excess (de) , which indicates the degree to which a sample contains one diastereoisomers in greater amounts than the other (s) .

As used herein, and unless otherwise specified, the term “substantially purified enantiomer” refers to a compound wherein one enantiomer has been enriched over the other. In one embodiment, the other enantiomer represents less than about 20%, less than about 10%, less than about 5%, or less than about 2%of the enantiomer. In one embodiment, a substantially purified enantiomer has an enantiomeric excess of S enantiomer of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%or at least about 99.9%. In one embodiment, a substantially purified enantiomer has an enantiomeric excess of R enantiomer of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%or at least about 99.9%.

As used herein, and unless otherwise specified, “Stereoisomers” can also include E and Z isomers, or a mixture thereof, and cis and trans isomers or a mixture thereof. In certain embodiments, a compound described herein is isolated as either the E or Z isomer. In other embodiments, a compound described herein is a mixture of the E and Z isomers.

As used herein, and unless otherwise specified, the term “tautomer” or “tautomeric form” refers to isomeric forms of a compound that are in equilibrium with each other. In one embodiment, a tautomer is formed by the migration of a proton from one atom of a molecule to another atom of the same molecule (known as proton tautomers, such as keto-enol tautomerization or imine-enamine tautomerization) . The concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, pyrazoles may exhibit the following isomeric forms, which are referred to as tautomers of each other:

All tautomers of the compounds described herein are within the scope of the present application.

As used herein, and unless otherwise specified, the term “pharmaceutically acceptable salt” includes both acid and base addition salts.

Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

Examples of pharmaceutically acceptable base addition salt include, but are not limited to, salts prepared from addition of an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. In one embodiment, the inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. In one embodiment, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

As used herein, and unless otherwise specified, the term “subject” refers to an animal, including, but not limited to, a primate (e.g., human) , cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

As used herein, and unless otherwise specified, the terms “treat, ” “treating, ” and “treatment” refer to the eradication or amelioration of a disease or disorder, or of one or more symptoms associated with the disease or disorder. In general, treatment occurs after the onset of the disease or disorder. In certain embodiments, the terms refer to minimizing the spread or worsening of the disease or disorder resulting from the administration of one or more prophylactic or therapeutic agents to a subject with such a disease or disorder.

As used herein, and unless otherwise specified, the terms “prevent, ” “preventing, ” and “prevention” refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In general, prevention occurs prior to the onset of the disease or disorder.

As used herein, and unless otherwise specified, the terms “manage, ” “managing, ” and “management” refer to preventing or slowing the progression, spread or worsening of a disease or disorder, or of one or more symptoms thereof. Sometimes, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or disorder.

As used herein, and unless otherwise specified, the term “therapeutically effective amount” are meant to include the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. The term “therapeutically effective amount” also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.

As used herein, and unless otherwise specified, the term “IC₅₀” refers an amount, concentration, or dosage of a compound that is required for 50%inhibition of a maximal response in an assay that measures such response.

As used herein, and unless otherwise specified, the term “PI3Kα-associated disease or disorder” refers to diseases or disorders associated with or having a dysregulation of a PIK3CA gene, a PI3Kα protein, or the expression or activity or level of any (e.g., one or more) of the same (e.g., any of the types of dysregulation of a PIK3CA gene, or a PI3Kαprotein, or the expression or activity or level of any of the same described herein) . Non-limiting examples of a PI3Kα-associated disease or disorder include, for example, PIK3CA-related overgrowth syndromes (PROS) , brain disorders (e.g., as macrocephaly-capillary malformation (MCAP) and hemimegalencephaly) , congenital lipomatous (e.g., overgrowth of vascular malformations) , epidermal nevi and skeletal/spinal anomalies (e.g., CLOVES syndrome) and fibroadipose hyperplasia (FH) , or cancer (e.g., PI3Kα-associated cancer) .

As used herein, and unless otherwise specified, the term “PI3Kα-associated cancer” refers to cancers associated with or having a dysregulation of a PIK3CA gene, a PI3Kα protein, or expression or activity, or level of any of the same.

As used herein, and unless otherwise specified, the term “dysregulation of a PIK3CA gene, a PI3Kα protein, or the expression or activity or level of any of the same” refers to a genetic mutation (e.g., a mutation in a PIK3CA gene that results in the expression of a PI3Kα that includes a deletion of at least one amino acid as compared to a wild type PI3Kα, a mutation in a PIK3CA gene that results in the expression of PI3Kα with one or more point mutations as compared to a wild type PI3Kα, a mutation in a PIK3CA gene that results in the expression of PI3Kα with at least one inserted amino acid as compared to a wild type PI3Kα, a gene duplication that results in an increased level of PI3Kα in a cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of PI3Kα in a cell) , an alternative spliced version of PI3Kα mRNA that results in PI3Kαhaving a deletion of at least one amino acid in the PI3Kα as compared to the wild type PI3Kα) , or increased expression (e.g., increased levels) of a wild type PI3Kα in a mammalian cell due to aberrant cell signaling and/or dysregulated autocrine/paracrine signaling (e.g., as compared to a control non-cancerous cell) . In one embodiment, a dysregulation of a PIK3CA gene, a PI3Kα protein, or expression or activity, or level of any of the same, is a mutation in PIK3CA gene that encodes a PI3Kα that is constitutively active or has increased activity as compared to a protein encoded by a PIK3CA gene that does not include the mutation. Non-limiting examples of PI3Kα point mutations/substitutions/insertions/deletions are described in Table 2.

As used herein, and unless otherwise specified, the term “activating mutation” in reference to PI3Kα describes a mutation in a PIK3CA gene that results in the expression of PI3Kα that has an increased kinase activity, e.g., as compared to a wild type PI3Kα, e.g., when assayed under identical conditions. In one embodiment, an activating mutation is a mutation in a PIK3CA gene that results in the expression of a PI3Kα 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, e.g., as compared to a wild type a PI3Kα, e.g., when assayed under identical conditions. In one embodiment, an activating mutation is a mutation in a PIK3CA that results in the expression of a PI3Kα that has one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acids deleted, e.g., as compared to a wild type PI3Kα, e.g., when assayed under identical conditions. In one embodiment, an activating mutation is a mutation in a PIK3CA gene that results in the expression of a PI3Kα that has 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) amino acid inserted as compared to a wild type PI3Kα, e.g., the exemplary wild type PI3Kα described herein, e.g., when assayed under identical conditions.

As used herein, and unless otherwise specified, the term “wild type” or “wild-type” refers to a nucleic acid (e.g., a PIK3CA gene or a PI3Kα mRNA) or protein (e.g., a PI3Kα) sequence that is typically found in a subject that does not have a disease or disorder related to the reference nucleic acid or protein.

As used herein, and unless otherwise specified, the term “wild type PI3Kα” or “wild-type PI3Kα” describes a normal PI3Kα nucleic acid (e.g., PIK3CA or PI3Kα mRNA) or protein that is found in a subject that does not have a PI3Kα-associated disease, e.g., a PI3Kα-associated cancer (and optionally also does not have an increased risk of developing a PI3Kα-associated disease and/or is not suspected of having a PI3Kα-associated disease) , or is found in a cell or tissue from a subject that does not have a PI3Kα-associated disease, e.g., a PI3Kα -associated cancer (and optionally also does not have an increased risk of developing a PI3Kα -associated disease and/or is not suspected of having a PI3Kα-associated disease) .

As used herein, and unless otherwise specified, the term “pharmaceutically acceptable carrier, ” “pharmaceutically acceptable excipient, ” “physiologically acceptable carrier, ” or “physiologically acceptable excipient” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams &Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition, Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition, Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004.

Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. Examples of isotopes that can be incorporated into compounds provided herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, e.g., ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. For example, provided herein are compounds having the present structures except for the replacement or enrichment of a hydrogen by deuterium or tritium at one or more atoms in the molecule, or the replacement or enrichment of a carbon by ¹³C or ¹⁴C at one or more atoms in the molecule. In one embodiment, provided herein are isotopically labeled compounds having one or more hydrogen atoms replaced by or enriched by deuterium. In one embodiment, provided herein are isotopically labeled compounds having one or more hydrogen atoms replaced by or enriched by tritium. In one embodiment, provided herein are isotopically labeled compounds having one or more carbon atoms replaced or enriched by ¹³C. In one embodiment, provided herein are isotopically labeled compounds having one or more carbon atoms replaced or enriched by ¹⁴C.

As used herein, and unless otherwise specified, the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05%of a given value or range.

COMPOUNDS

In one embodiment, provided herein are multicyclic compounds as PI3Kαinhibitors. In one embodiment, provided herein are multicyclic compounds comprising a benzofuran core and a urea moiety as PI3Kα inhibitors.

In one embodiment, provided herein is a compound of formula (I) :

or a stereoisomer, or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein:

X¹ is CR^a1 or N;

X² is NR^a2, O, or S;

R^a1 is hydrogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl and alkoxy are optionally substituted;

R^a2 is hydrogen or C₁-C₆ alkyl, and wherein the alkyl is optionally substituted;

R^a3, R^a4, R^a5, and R^a6 are each independently hydrogen, halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 10-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted;

R is C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkoxy, cycloalkyl, and heterocyclyl are optionally substituted;

Ring A is C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 10-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted;

Ring B is C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 12-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted;

L is C₁-C₆ alkylene or C₃-C₈ cycloalkylene, and wherein the alkylene, and cycloalkylene are optionally substituted;

R¹ is optionally substituted 3 to 12-membered heterocyclyl, -SO₂R^c, -S (=O) ₂NR^bR^c, -SO₂NH₂, -S (=O) (=NR^b) R^c, -C (=O) NR^bR^c, -C (=O) NH₂, -NR^b (C=O) R^c, OR^c, or -NR^bR^c;

R^b is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 12-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; and

R^c is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 12-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted.

In one embodiment, R¹ is optionally substituted 3 to 12-membered heterocyclyl.

In one embodiment, R¹ is -SO₂R^c, -S (=O) ₂NR^bR^c, -SO₂NH₂, -S (=O) (=NR^b) R^c, -C (=O) NR^bR^c, -C (=O) NH₂, -NR^b (C=O) R^c, or OR^c.

In one embodiment, X¹ is N.

In one embodiment, X¹ is CR^a1. In one embodiment, X¹ is CH. In one embodiment, X¹ is C- (C₁-C₆ alkyl) . In one embodiment, X¹ is C-CH₃. In one embodiment, X¹ is C-C₂H₅. In one embodiment, X¹ is C- (C₃ alkyl) . In one embodiment, X¹ is C- (C₄ alkyl) . In one embodiment, X¹ is C- (C₅ alkyl) . In one embodiment, X¹ is C- (C₆ alkyl) .

In one embodiment, X¹ is C- (C₁-C₆ alkoxy) . In one embodiment, X¹ is C-methoxy. In one embodiment, X¹ is C-ethoxy. In one embodiment, X¹ is C- (C₃ alkoxy) . In one embodiment, X¹ is C- (C₄ alkoxy) . In one embodiment, X¹ is C- (C₅ alkoxy) . In one embodiment, X¹ is C- (C₆ alkoxy) .

In one embodiment, R^a1 is H. In one embodiment, R^a1 is C₁-C₆ alkyl. In one embodiment, R^a1 is C₁-C₃ alkyl. In one embodiment, R^a1 is methyl. In one embodiment, R^a1 is ethyl. In one embodiment, R^a1 is C₃ alkyl. In one embodiment, R^a1 is C₄ alkyl. In one embodiment, R^a1 is C₅ alkyl. In one embodiment, R^a1 is C₆ alkyl. In one embodiment, the alkyl is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkoxy.

In one embodiment, R^a1 is C₁-C₆ alkoxy. In one embodiment, R^a1 is C₁-C₃ alkoxy. In one embodiment, R^a1 is methoxy. In one embodiment, R^a1 is ethoxy. In one embodiment, R^a1 is C₃ alkoxy. In one embodiment, R^a1 is C₄ alkoxy. In one embodiment, R^a1 is C₅ alkoxy. In one embodiment, R^a1 is C₆ alkoxy. In one embodiment, the alkoxy is unsubstituted. In one embodiment, the alkoxy is substituted. In one embodiment, the alkoxy is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, X² is O. In one embodiment, X² is S.

In one embodiment, X² is NR^a2. In one embodiment, X² is N- (C₁-C₆ alkyl) . In one embodiment, X² is NH. In one embodiment, X² is N-CH₃. In one embodiment, X² is N-C₂H₅. In one embodiment, X² is N- (n-propyl) or N- (iso-propyl) . In one embodiment, X² is N- (n-butyl) , N- (iso-butyl) , or N- (tert-butyl) . In one embodiment, X² is N- (C₅ alkyl) . In one embodiment, X¹ is N- (C₆ alkyl) .

In one embodiment, R^a2 is H. In one embodiment, R^a2 is C₁-C₆ alkyl. In one embodiment, R^a2 is C₁-C₃ alkyl. In one embodiment, R^a2 is methyl. In one embodiment, R^a2 is ethyl. In one embodiment, R^a2 is C₃ alkyl. In one embodiment, R^a2 is C₄ alkyl. In one embodiment, R^a2 is C₅ alkyl. In one embodiment, R^a2 is C₆ alkyl. In one embodiment, the alkyl is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkoxy.

In one embodiment, R^a3 is hydrogen. In one embodiment, R^a3 is halogen (F, Cl, Br, or I) . In one embodiment, R^a3 is fluorine. In one embodiment, R^a3 is nitro. In one embodiment, R^a3 is cyano. In one embodiment, R^a3 is C₁-C₆ alkyl. In one embodiment, R^a3 is C₁-C₃ alkyl. In one embodiment, R^a3 is methyl. In one embodiment, R^a3 is ethyl. In one embodiment, R^a3 is C₃ alkyl. In one embodiment, R^a3 is C₄ alkyl. In one embodiment, R^a3 is C₅ alkyl. In one embodiment, R^a3 is C₆ alkyl.

In one embodiment, R^a3 is C₁-C₆ alkoxy. In one embodiment, R^a3 is C₁-C₃ alkoxy. In one embodiment, R^a3 is methoxy. In one embodiment, R^a3 is ethoxy. In one embodiment, R^a3 is C₃ alkoxy. In one embodiment, R^a3 is C₄ alkoxy. In one embodiment, R^a3 is C₅ alkoxy. In one embodiment, R^a3 is C₆ alkoxy.

In one embodiment, R^a3 is C₃-C₈ cycloalkyl. In one embodiment, R^a3 is C₃-C₆ cycloalkyl. In one embodiment, R^a3 is cyclopropyl. In one embodiment, R^a3 is cyclobutyl. In one embodiment, R^a3 is cyclopentyl. In one embodiment, R^a3 is cyclohexyl.

In one embodiment, R^a3 is C₆-C₁₀ aryl. In one embodiment, R^a3 is C₆-C₈ aryl. In one embodiment, R^a3 is phenyl. In one embodiment, R^a3 is naphthyl.

In one embodiment, R^a3 is 5 to 10-membered heteroaryl. In one embodiment, R^a3 is 5 to 8-membered heteroaryl. In one embodiment, R^a3 is 5-membered heteroaryl. In one embodiment, R^a3 is 6-membered heteroaryl. In one embodiment, R^a3 is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a3 is 3 to 8-membered heterocyclyl. In one embodiment, R^a3 is 3 to 6-membered heterocyclyl. In one embodiment, R^a3 is a 3-membered heterocyclyl. In one embodiment, R^a3 is a 4-membered heterocyclyl. In one embodiment, R^a3 is a 5-membered heterocyclyl. In one embodiment, R^a3 is a 6-membered heterocyclyl. In one embodiment, R^a3 is a 3-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a3 is unsubstituted. In one embodiment, R^a3 is substituted. In one embodiment, R^a3 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, R^a4 is hydrogen. In one embodiment, R^a4 is halogen (F, Cl, Br, or I) . In one embodiment, R^a4 is nitro. In one embodiment, R^a4 is cyano. In one embodiment, R^a4 is C₁-C₆ alkyl. In one embodiment, R^a4 is C₁-C₃ alkyl. In one embodiment, R^a4 is methyl. In one embodiment, R^a4 is ethyl. In one embodiment, R^a4 is C₃ alkyl. In one embodiment, R^a4 is C₄ alkyl. In one embodiment, R^a4 is C₅ alkyl. In one embodiment, R^a4 is C₆ alkyl.

In one embodiment, R^a4 is C₁-C₆ alkoxy. In one embodiment, R^a4 is C₁-C₃ alkoxy. In one embodiment, R^a4 is methoxy. In one embodiment, R^a4 is ethoxy. In one embodiment, R^a4 is C₃ alkoxy. In one embodiment, R^a4 is C₄ alkoxy. In one embodiment, R^a4 is C₅ alkoxy. In one embodiment, R^a4 is C₆ alkoxy.

In one embodiment, R^a4 is C₃-C₈ cycloalkyl. In one embodiment, R^a4 is C₃-C₆ cycloalkyl. In one embodiment, R^a4 is cyclopropyl. In one embodiment, R^a4 is cyclobutyl. In one embodiment, R^a4 is cyclopentyl. In one embodiment, R^a4 is cyclohexyl.

In one embodiment, R^a4 is C₆-C₁₀ aryl. In one embodiment, R^a4 is C₆-C₈ aryl. In one embodiment, R^a4 is phenyl. In one embodiment, R^a4 is naphthyl.

In one embodiment, R^a4 is 5 to 10-membered heteroaryl. In one embodiment, R^a4 is 5 to 8-membered heteroaryl. In one embodiment, R^a4 is 5-membered heteroaryl. In one embodiment, R^a4 is 6-membered heteroaryl. In one embodiment, R^a4 is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a4 is 3 to 8-membered heterocyclyl. In one embodiment, R^a4 is 3 to 6-membered heterocyclyl. In one embodiment, R^a4 is a 3-membered heterocyclyl. In one embodiment, R^a4 is a 4-membered heterocyclyl. In one embodiment, R^a4 is a 5-membered heterocyclyl. In one embodiment, R^a4 is a 6-membered heterocyclyl. In one embodiment, R^a4 is a 3-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a4 is unsubstituted. In one embodiment, R^a4 is substituted. In one embodiment, R^a4 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, R^a5 is hydrogen. In one embodiment, R^a5 is halogen (F, Cl, Br, or I) . In one embodiment, R^a5 is fluorine. In one embodiment, R^a5 is nitro. In one embodiment, R^a5 is cyano. In one embodiment, R^a5 is C₁-C₆ alkyl. In one embodiment, R^a5 is C₁-C₃ alkyl. In one embodiment, R^a5 is methyl. In one embodiment, R^a5 is ethyl. In one embodiment, R^a5 is C₃ alkyl. In one embodiment, R^a5 is C₄ alkyl. In one embodiment, R^a5 is C₅ alkyl. In one embodiment, R^a5 is C₆ alkyl.

In one embodiment, R^a5 is C₁-C₆ alkoxy. In one embodiment, R^a5 is C₁-C₃ alkoxy. In one embodiment, R^a5 is methoxy. In one embodiment, R^a5 is ethoxy. In one embodiment, R^a5 is C₃ alkoxy. In one embodiment, R^a5 is C₄ alkoxy. In one embodiment, R^a5 is C₅ alkoxy. In one embodiment, R^a5 is C₆ alkoxy.

In one embodiment, R^a5 is C₃-C₈ cycloalkyl. In one embodiment, R^a5 is C₃-C₆ cycloalkyl. In one embodiment, R^a5 is cyclopropyl. In one embodiment, R^a5 is cyclobutyl. In one embodiment, R^a5 is cyclopentyl. In one embodiment, R^a5 is cyclohexyl.

In one embodiment, R^a5 is C₆-C₁₀ aryl. In one embodiment, R^a5 is C₆-C₈ aryl. In one embodiment, R^a5 is phenyl. In one embodiment, R^a5 is naphthyl.

In one embodiment, R^a5 is 5 to 10-membered heteroaryl. In one embodiment, R^a5 is 5 to 8-membered heteroaryl. In one embodiment, R^a5 is 5-membered heteroaryl. In one embodiment, R^a5 is 6-membered heteroaryl. In one embodiment, R^a5 is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a5 is 3 to 8-membered heterocyclyl. In one embodiment, R^a5 is 3 to 6-membered heterocyclyl. In one embodiment, R^a5 is a 3-membered heterocyclyl. In one embodiment, R^a5 is a 4-membered heterocyclyl. In one embodiment, R^a5 is a 5-membered heterocyclyl. In one embodiment, R^a5 is a 6-membered heterocyclyl. In one embodiment, R^a5 is a 3-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a5 is unsubstituted. In one embodiment, R^a5 is substituted. In one embodiment, R^a5 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, R^a6 is hydrogen. In one embodiment, R^a6 is halogen (F, Cl, Br, or I) . In one embodiment, R^a6 is nitro. In one embodiment, R^a6 is cyano. In one embodiment, R^a6 is C₁-C₆ alkyl. In one embodiment, R^a6 is C₁-C₃ alkyl. In one embodiment, R^a6 is methyl. In one embodiment, R^a6 is ethyl. In one embodiment, R^a6 is C₃ alkyl. In one embodiment, R^a6 is C₄ alkyl. In one embodiment, R^a6 is C₅ alkyl. In one embodiment, R^a6 is C₆ alkyl.

In one embodiment, R^a6 is C₁-C₆ alkoxy. In one embodiment, R^a6 is C₁-C₃ alkoxy. In one embodiment, R^a6 is methoxy. In one embodiment, R^a6 is ethoxy. In one embodiment, R^a6 is C₃ alkoxy. In one embodiment, R^a6 is C₄ alkoxy. In one embodiment, R^a6 is C₅ alkoxy. In one embodiment, R^a6 is C₆ alkoxy.

In one embodiment, R^a6 is C₃-C₈ cycloalkyl. In one embodiment, R^a6 is C₃-C₆ cycloalkyl. In one embodiment, R^a6 is cyclopropyl. In one embodiment, R^a6 is cyclobutyl. In one embodiment, R^a6 is cyclopentyl. In one embodiment, R^a6 is cyclohexyl.

In one embodiment, R^a6 is C₆-C₁₀ aryl. In one embodiment, R^a6 is C₆-C₈ aryl. In one embodiment, R^a6 is phenyl. In one embodiment, R^a6 is naphthyl.

In one embodiment, R^a6 is 5 to 10-membered heteroaryl. In one embodiment, R^a6 is 5 to 8-membered heteroaryl. In one embodiment, R^a6 is 5-membered heteroaryl. In one embodiment, R^a6 is 6-membered heteroaryl. In one embodiment, R^a6 is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a6 is 3 to 8-membered heterocyclyl. In one embodiment, R^a6 is 3 to 6-membered heterocyclyl. In one embodiment, R^a6 is a 3-membered heterocyclyl. In one embodiment, R^a6 is a 4-membered heterocyclyl. In one embodiment, R^a6 is a 5-membered heterocyclyl. In one embodiment, R^a6 is a 6-membered heterocyclyl. In one embodiment, R^a6 is a 3-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms.

In one embodiment, R^a6 is unsubstituted. In one embodiment, R^a6 is substituted. In one embodiment, R^a6 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, In one embodiment, the displayed R^a3, R^a4, R^a5, and R^a6 are not hydrogen.

In one embodiment, R^a4 and R^a6 are both hydrogen. In one embodiment, R^a3 and R^a5 are both halogen. In one embodiment, R^a3 and R^a5 are both fluorine. In one embodiment, R^a3 is hydrogen and R^a5 is fluorine. In one embodiment, R^a4 and R^a6 are both hydrogen, and R^a3 and R^a5 are both halogen. In one embodiment, R^a3, R^a4, and R^a6 are all hydrogen, and R^a5 is halogen. In one embodiment, R^a3, R^a4, and R^a6 are all hydrogen, and R^a5 is fluorine. In one embodiment, R^a3, R^a4, and R^a6 are all hydrogen, and R^a5 is C₁-C₆ alkoxy. In one embodiment, R^a3, R^a4, and R^a6 are all hydrogen, and R^a5 is methoxy.

In one embodiment, the compound is a compound of Formula (II) :

or a stereoisomer, or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. In one embodiment of Formula (II) (or a sub-formula thereof) , both R^a3 and R^a5 are not hydrogen. In one embodiment, R^a3 is hydrogen, and R^a5 is not hydrogen. In one embodiment, R^a3 is hydrogen, and R^a5 is halogen. In one embodiment, R^a3 is hydrogen, and R^a5 is C₁-C₆ alkoxy.

In one embodiment, Ring A is C₃-C₈ cycloalkyl. In one embodiment, Ring A is C₃-C₆ cycloalkyl. In one embodiment, Ring A is cyclopropyl. In one embodiment, Ring A is cyclobutyl. In one embodiment, Ring A is cyclopentyl. In one embodiment, Ring A is cyclohexyl. In one embodiment, the cycloalkyl is unsubstituted. In one embodiment, the cycloalkyl is substituted.

In one embodiment, Ring A is C₆-C₁₀ aryl. In one embodiment, Ring A is C₆-C₈ aryl. In one embodiment, Ring A is phenyl. In one embodiment, the phenyl is unsubstituted. In one embodiment, the phenyl is substituted.

In one embodiment, Ring A is 5 to 10-membered heteroaryl. In one embodiment, Ring A is 5 to 8-membered heteroaryl. In one embodiment, Ring A is 5-membered heteroaryl. In one embodiment, Ring A is 6-membered heteroaryl. In one embodiment, Ring A is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, Ring A is a 5-or 6-membered nitrogen-containing heteroaryl. In one embodiment, Ring A is 5 or 6-membered nitrogen-containing heteroaryl, and nitrogen is the only type of heteroatom contained in the heteroaryl. In one embodiment, the heteroaryl is unsubstituted. In one embodiment, the heteroaryl is substituted.

In one embodiment, Ring A is 3 to 8-membered heterocyclyl. In one embodiment, Ring A is 3 to 6-membered heterocyclyl. In one embodiment, Ring A is 3-membered heterocyclyl. In one embodiment, Ring A is 4-membered heterocyclyl. In one embodiment, Ring A is 5-membered heterocyclyl. In one embodiment, Ring A is 6-membered heterocyclyl. In one embodiment, Ring A is 4-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, Ring A is 4-to 6-membered nitrogen-containing heterocyclyl. In one embodiment, Ring A is 4-to 6-membered oxygen-containing heterocyclyl. In one embodiment, Ring A is 4-to 6-membered nitrogen-containing heterocyclyl, and nitrogen is the only type of heteroatom contained in the heteroaryl. In one embodiment, Ring A is 4-to 6-membered oxygen-containing heterocyclyl, and oxygen is the only type of heteroatom contained in the heteroaryl. In one embodiment, the heterocyclyl is unsubstituted. In one embodiment, the heterocyclyl is substituted.

In one embodiment, Ring A is imidazolyl. In one embodiment, Ring A is pyridyl. In one embodiment, Ring A is pyrazolyl. In one embodiment, Ring A is pyridazinyl. In one embodiment, Ring A is pyrimidinyl. In one embodiment, Ring A is triazinyl. In one embodiment, Ring A is pyrazinyl. In one embodiment, Ring A is triazolyl. In one embodiment, Ring A is oxazolyl. In one embodiment, Ring A is thiazolyl. In one embodiment, Ring A is pyrrolidinyl. In one embodiment, Ring A is piperidinyl. In one embodiment, Ring A is morpholinyl. In one embodiment, Ring A is oxazolyl.

In one embodiment, Ring A is wherein the attachment to the left is to the urea moiety, and the attachment to the right is to Ring B. In one embodiment, Ring A iswherein the attachment to the left is to the urea moiety, and the attachment to the right is to Ring B.

In one embodiment, Ring A is unsubstituted. In one embodiment, Ring A is substituted. In one embodiment, Ring A is substituted with one or more halogen, nitro, cyano, hydroxyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In one embodiment, Ring A is substituted with one or more halogen, hydroxyl, nitro, cyano, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, Ring B is C₃-C₈ cycloalkyl. In one embodiment, Ring B is C₃-C₆ cycloalkyl. In one embodiment, Ring B is cyclopropyl. In one embodiment, Ring B is cyclobutyl. In one embodiment, Ring B is cyclopentyl. In one embodiment, Ring B is cyclohexyl. In one embodiment, the cycloalkyl is unsubstituted. In one embodiment, the cycloalkyl is substituted.

In one embodiment, Ring B is C₆-C₁₀ aryl. In one embodiment, Ring B is C₆-C₈ aryl. In one embodiment, Ring B is phenyl. In one embodiment, the phenyl is unsubstituted. In one embodiment, the phenyl is substituted.

In one embodiment, Ring B is 5 to 10-membered heteroaryl. In one embodiment, Ring B is 5 to 8-membered heteroaryl. In one embodiment, Ring B is 5-membered heteroaryl. In one embodiment, Ring B is 6-membered heteroaryl. In one embodiment, Ring B is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, Ring B is a 5-or 6-membered nitrogen-containing heteroaryl. In one embodiment, Ring B is 5 or 6-membered nitrogen-containing heteroaryl, and nitrogen is the only type of heteroatom contained in the heteroaryl. In one embodiment, the heteroaryl is unsubstituted. In one embodiment, the heteroaryl is substituted.

In one embodiment, Ring B is 3 to 8-membered heterocyclyl. In one embodiment, Ring B is 4 to 6-membered heterocyclyl. In one embodiment, Ring B is 3-membered heterocyclyl. In one embodiment, Ring B is 4-membered heterocyclyl. In one embodiment, Ring B is 5-membered heterocyclyl. In one embodiment, Ring B is 6-membered heterocyclyl. In one embodiment, Ring B is 3-to 8-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, Ring B is a nitrogen-containing 3 to 8-membered heterocyclyl. In one embodiment, Ring B is 4 to 6-membered nitrogen-containing heterocyclyl. In one embodiment, Ring B is 4 to 6-membered oxygen-containing heterocyclyl. In one embodiment, Ring B is 4 to 6-membered nitrogen-containing heterocyclyl, and nitrogen is the only type of heteroatom contained in the heteroaryl. In one embodiment, Ring B is 4 to 6-membered oxygen-containing heterocyclyl, and oxygen is the only type of heteroatom contained in the heteroaryl. In one embodiment, the heterocyclyl is unsubstituted. In one embodiment, the heterocyclyl is substituted.

In one embodiment, Ring B is azetidinyl. In one embodiment, Ring B is pyrrolidinyl. In one embodiment, Ring B is piperidinyl. In one embodiment, Ring B is piperazinyl. In one embodiment, Ring B is morpholinyl. In one embodiment, Ring B is thiazolyl. In one embodiment, Ring B is oxazolyl. In one embodiment, Ring B is imidazolyl.

In one embodiment, Ring B is In one embodiment, Ring B isIn one embodiment, Ring B isIn these embodiments, the attachment to the left is to the Ring A, and the attachment to the right is to L.

In one embodiment, Ring B is unsubstituted. In one embodiment, Ring B is substituted. In one embodiment, Ring B is substituted with one or more halogen, nitro, cyano, hydroxyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In one embodiment, Ring B is substituted with one or more halogen, hydroxyl, nitro, cyano, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, Ring A is 5 to 6-membered heteroaryl, and Ring B is 4 to 6-membered heterocyclyl. In one embodiment, Ring A is 5 to 6-membered nitrogen-containing heteroaryl, and Ring B is 4 to 6-membered nitrogen-containing heterocyclyl. In one embodiment, Ring A is 6-membered nitrogen-containing heteroaryl, and Ring B is 4-membered nitrogen-containing heterocyclyl. In one embodiment, Ring A is phenyl, and Ring B is 4 to 6-membered heterocyclyl. In one embodiment, Ring A is phenyl, and Ring B is 4 to 6-membered nitrogen containing heterocyclyl. In one embodiment, Ring A is phenyl, and Ring B is azetidinyl. In one embodiment, Ring A is pyrimidinyl, and Ring B is azetidinyl.

In one embodiment, the compound is a compound of Formula (III-A) , or (III-B) :

or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt thereof, wherein:

Ring B is a nitrogen containing 3 to 8-membered heterocyclyl;

X³ is CR^a7 or N;

X⁴ is CR^a8 or N;

X⁵ is CR^a9 or N;

X⁶ is CR^a10 or N; and

R^a7, R^a8, R^a9, and R^a10 are each independently hydrogen, halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl and alkoxy are optionally substituted.

In one embodiment, X³ is CR^a7. In one embodiment, X³ is N.

In one embodiment, R^a7 is hydrogen. In one embodiment, R^a7 is halogen (F, Cl, Br, or I) . In one embodiment, R^a7 is nitro. In one embodiment, R^a7 is cyano. In one embodiment, R^a7 is C₁-C₆ alkyl. In one embodiment, R^a7 is C₁-C₃ alkyl. In one embodiment, R^a7 is methyl. In one embodiment, R^a7 is ethyl. In one embodiment, R^a7 is C₃ alkyl. In one embodiment, R^a7 is C₄ alkyl. In one embodiment, R^a7 is C₅ alkyl. In one embodiment, R^a7 is C₆ alkyl. In one embodiment, R^a7 is C₁-C₆ alkoxy. In one embodiment, R^a7 is C₁-C₃ alkoxy. In one embodiment, R^a7 is methoxy. In one embodiment, R^a7 is ethoxy. In one embodiment, R^a7 is C₃ alkoxy. In one embodiment, R^a7 is C₄ alkoxy. In one embodiment, R^a7 is C₅ alkoxy. In one embodiment, R^a7 is C₆ alkoxy. In one embodiment, R^a7 is unsubstituted. In one embodiment, R^a7 is substituted. In one embodiment, R^a7 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, X⁴ is CR^a8. In one embodiment, X³ is N.

In one embodiment, R^a8 is hydrogen. In one embodiment, R^a8 is halogen (F, Cl, Br, or I) . In one embodiment, R^a8 is nitro. In one embodiment, R^a8 is cyano. In one embodiment, R^a8 is C₁-C₆ alkyl. In one embodiment, R^a8 is C₁-C₃ alkyl. In one embodiment, R^a8 is methyl. In one embodiment, R^a8 is ethyl. In one embodiment, R^a8 is C₃ alkyl. In one embodiment, R^a8 is C₄ alkyl. In one embodiment, R^a8 is C₅ alkyl. In one embodiment, R^a8 is C₆ alkyl. In one embodiment, R^a8 is C₁-C₆ alkoxy. In one embodiment, R^a8 is C₁-C₃ alkoxy. In one embodiment, R^a8 is methoxy. In one embodiment, R^a8 is ethoxy. In one embodiment, R^a8 is C₃ alkoxy. In one embodiment, R^a8 is C₄ alkoxy. In one embodiment, R^a8 is C₅ alkoxy. In one embodiment, R^a8 is C₆ alkoxy. In one embodiment, R^a8 is unsubstituted. In one embodiment, R^a8 is substituted. In one embodiment, R^a8 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, X⁵ is CR^a9. In one embodiment, X³ is N.

In one embodiment, R^a9 is hydrogen. In one embodiment, R^a9 is halogen (F, Cl, Br, or I) . In one embodiment, R^a9 is nitro. In one embodiment, R^a9 is cyano. In one embodiment, R^a9 is C₁-C₆ alkyl. In one embodiment, R^a9 is C₁-C₃ alkyl. In one embodiment, R^a9 is methyl. In one embodiment, R^a9 is ethyl. In one embodiment, R^a9 is C₃ alkyl. In one embodiment, R^a9 is C₄ alkyl. In one embodiment, R^a9 is C₅ alkyl. In one embodiment, R^a9 is C₆ alkyl. In one embodiment, R^a9 is C₁-C₆ alkoxy. In one embodiment, R^a9 is C₁-C₃ alkoxy. In one embodiment, R^a9 is methoxy. In one embodiment, R^a9 is ethoxy. In one embodiment, R^a9 is C₃ alkoxy. In one embodiment, R^a9 is C₄ alkoxy. In one embodiment, R^a9 is C₅ alkoxy. In one embodiment, R^a9 is C₆ alkoxy. In one embodiment, R^a9 is unsubstituted. In one embodiment, R^a9 is substituted. In one embodiment, R^a9 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, X⁶ is CR^a10. In one embodiment, X³ is N.

In one embodiment, R^a10 is hydrogen. In one embodiment, R^a10 is halogen (F, Cl, Br, or I) . In one embodiment, R^a10 is nitro. In one embodiment, R^a10 is cyano. In one embodiment, R^a10 is C₁-C₆ alkyl. In one embodiment, R^a10 is C₁-C₃ alkyl. In one embodiment, R^a10 is methyl. In one embodiment, R^a10 is ethyl. In one embodiment, R^a10 is C₃ alkyl. In one embodiment, R^a10 is C₄ alkyl. In one embodiment, R^a10 is C₅ alkyl. In one embodiment, R^a10 is C₆ alkyl. In one embodiment, R^a10 is C₁-C₆ alkoxy. In one embodiment, R^a10 is C₁-C₃ alkoxy. In one embodiment, R^a10 is methoxy. In one embodiment, R^a10 is ethoxy. In one embodiment, R^a10 is C₃ alkoxy. In one embodiment, R^a10 is C₄ alkoxy. In one embodiment, R^a10 is C₅ alkoxy. In one embodiment, R^a10 is C₆ alkoxy. In one embodiment, R^a10 is unsubstituted. In one embodiment, R^a10 is substituted. In one embodiment, R^a10 is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, X³ is CR^a7, and X⁶ is CR^a10. In one embodiment, X³ and X⁶ are both CH. In one embodiment, X⁴ and X⁵ are both nitrogen. In one embodiment, X³ is CR^a7, X⁴ is nitrogen, and X⁶ is CR^a10. In one embodiment, X³ is CR^a7, X⁵ is nitrogen, and X⁶ is CR^a10.

In one embodiment, the compound is a compound of Formula (IV-A) , (IV-B) , (IV-C) , or (IV-D) :

R^a11 is hydrogen, hydroxyl, halogen, nitro, cyano, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl, and alkoxy are optionally substituted; and

each instance of R^a12 is independently hydrogen, halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl, and alkoxy are optionally substituted.

In one embodiment, each instance of R^a12 is independently hydrogen, or C₁-C₆ alkyl. In one embodiment, all of the R^a12 are hydrogen. In one embodiment, one of the R^a12 is C₁-C₆ alkyl, and the rest of R^a12 are hydrogen. In one embodiment, one of the R^a12 is methyl, and the rest of R^a12 are hydrogen. In one embodiment, two of the R^a12 are C₁-C₆ alkyl, and the rest of R^a12 are hydrogen. In one embodiment, when a carbon connected to a R^a12 is a chiral center, it has S-configuration. In one embodiment, when a carbon connected to a R^a12 is a chiral center, it has R-configuration.

In one embodiment, L is C₁-C₆ alkylene. In one embodiment, L is C₁-C₃ alkylene. In one embodiment, L is -CH₂-. In one embodiment, L is -CH₂CH₂-. In one embodiment, L is -CH (CH₃) -. In one embodiment, L is C₃ alkylene, such as -C (CH₃) ₂-, -CH (CH₃) CH₂-, or -CH₂CH₂CH₂-. In one embodiment, L is C₄ alkylene. In one embodiment, L is C₅ alkylene. In one embodiment, L is C₆ alkylene. In one embodiment, the alkylene is unsubstituted. In one embodiment, the alkylene is substituted. In one embodiment, the alkylene is substituted with one or more hydroxyl, halogen, or C₁-C₆ alkoxy.

In one embodiment, L is C₃-C₈ cycloalkylene. In one embodiment, L is C₃-C₆ cycloalkylene. In one embodiment, L is C₃ cycloalkylene. In one embodiment, L is C₄ cycloalkylene. In one embodiment, L is C₅ cycloalkylene. In one embodiment, L is C₆ cycloalkylene. In one embodiment, L is C₇ cycloalkylene. In one embodiment, L is C₈ cycloalkylene. In one embodiment, the cycloalkylene is substituted. In one embodiment, the cycloalkylene is substituted with one or more hydroxyl, halogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R¹ is -SO₂R^c. In one embodiment, R¹ is -SO₂CH₃.

In one embodiment, R¹ is -S (=O) ₂NR^bR^c. In one embodiment, R¹ is -S (=O) ₂NHR^c. In one embodiment, R¹ is -S (=O) ₂NHCH₃. In one embodiment, R¹ is -SO₂NH₂.

In one embodiment, R¹ is -S (=O) (=NR^b) R^c. In one embodiment, R¹ is -S (=O) (=NH) R^c. In one embodiment, R¹ is -S (=O) (=NH) CH₃.

In one embodiment, R¹ is -C (=O) NR^bR^c. In one embodiment, R¹ is -C (=O) NHR^c. In one embodiment, R¹ is -C (=O) NHCH₃. In one embodiment, R¹ is -C (=O) NH₂.

In one embodiment, R¹ is -NR^b (C=O) R^c. In one embodiment, R¹ is -NH (C=O) R^c. In one embodiment, R¹ is -NH (C=O) CH₃.

In one embodiment, R¹ is -OR^c. In one embodiment, R¹ is -OCH₃. In one embodiment, R¹ is -OC₂H₅.

In one embodiment, R¹ is -NR^bR^c. In one embodiment, R¹ is -NHR^c. In one embodiment, R¹ is -NH (C_1-6 alkyl) . In one embodiment, R¹ is -N (C_1-6 alkyl) ₂. In one embodiment, R¹ is -NH (CH₃) . In one embodiment, R¹ is -N (CH₃) ₂.

In one embodiment, R¹ is an optionally substituted 3 to 12-membered heterocyclyl. In one embodiment, R¹ is an optionally substituted 3 to 10-membered heterocyclyl. In one embodiment, R¹ is an optionally substituted 3 to 8-membered heterocyclyl. In one embodiment, R¹ is an optionally substituted 3 to 6-membered heterocyclyl. In one embodiment, R¹ is an optionally substituted 3 to 6-membered monocyclic heterocyclyl. In one embodiment, R¹ is an optionally substituted 3 to 6-membered nitrogen-containing monocyclic heterocyclyl. In one embodiment, R¹ is an optionally substituted 6 to 12-membered bicyclic heterocyclyl. In one embodiment, R¹ is an optionally substituted 6 to 12-membered nitrogen-containing bicyclic heterocyclyl. In one embodiment, R¹ is an optionally substituted 6 to 12-membered spiro heterocyclyl. In one embodiment, R¹ is an optionally substituted 6 to 12-membered fused heterocyclyl. In one embodiment, R¹ is an optionally substituted 6 to 12-membered bridged heterocyclyl. In one embodiment, R¹ is optionally substituted with one or more R^a13. In one embodiment, the heterocyclyl R¹ is Ring C as described herein and elsewhere.

In one embodiment, the compound is a compound of Formula (V-A) , (V-B) , (V-C) , or (V-D) , (V-E) , (V-F) , (V-G) , (V-H) , (V-I) , (V-J) , (V-K) , (V-L) , (V-M) , or (V-N) :

or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3, 4, 5, or 6;

Ring C is a 3 to 12-membered heterocyclyl optionally substituted with one or more R^a13; and

each instance of R^a13 is independently halogen, hydroxyl, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, -C (=O) (C₁-C₆ alkyl) , -C (=O) NH (C₁-C₆ alkyl) , -C (=O) NH₂, -NH (C=O) (C₁-C₆ alkyl) , -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂, and wherein the alkyl and alkoxy are optionally substituted with one or more halogen, oxo or hydroxyl.

In one embodiment, Ring C is a 3 to 6-membered monocyclic heterocyclyl. In one embodiment, Ring C is a 3 to 6-membered nitrogen-containing monocyclic heterocyclyl. In one embodiment, Ring C is a 5 or 6-membered monocyclic heterocyclyl. In one embodiment, Ring C is a 5 or 6-membered nitrogen-containing heterocyclyl.

In one embodiment, Ring C is a 6 to 12-membered spiro heterocyclyl. In one embodiment, Ring C is a 6 to 12-membered nitrogen-containing spiro heterocyclyl. In one embodiment, Ring C is a 6 to 12-membered fused heterocyclyl. In one embodiment, Ring C is a 6 to 12-membered nitrogen-containing fused heterocyclyl. In one embodiment, Ring C is a 6 to 12-membered bridged heterocyclyl. In one embodiment, Ring C is a 6 to 12-membered nitrogen-containing bridged heterocyclyl.

In one embodiment, Ring C is azetidinyl. In one embodiment, Ring C is pyrrolidinyl. In one embodiment, Ring C is piperidinyl. In one embodiment, Ring B is piperazinyl. In one embodiment, Ring C is morpholinyl. In one embodiment, Ring C is thiazolyl. In one embodiment, Ring C is oxazolyl. In one embodiment, Ring C is imidazolyl.

In one embodiment, Ring C is:
wherein the point of attachment is to L, and each of which is optionally substituted.

In one embodiment, Ring C is unsubstituted. In one embodiment, Ring C is substituted with one or more R^a13. In one embodiment, Ring C is substituted with one R^a13. In one embodiment, Ring C is substituted with two R^a13. In one embodiment, Ring C is substituted with three R^a13.

In one embodiment, R^a13 is halogen (i.e. F, Cl, Br, or I) . In one embodiment, R^a13 is fluorine. In one embodiment, R^a13 is oxo (i.e. =O) . In one embodiment, R^a13 is OH.

In one embodiment, R^a13 is -C (=O) (C₁-C₆ alkyl) . In one embodiment, R^a13 is -C (=O) (C₁-C₃ alkyl) . In one embodiment, R^a13 is -C (=O) (CH₃) . In one embodiment, R^a13 is -C (=O) (C₂H₅) . In one embodiment, the alkyl (in R^a13) is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or oxo.

In one embodiment, R^a13 is C₁-C₆ alkyl. In one embodiment, R^a13 is C₁-C₃ alkyl. In one embodiment, R^a13 is methyl. In one embodiment, R^a13 is ethyl. In one embodiment, R^a13 is C₃ alkyl. In one embodiment, R^a13 is C₄ alkyl. In one embodiment, R^a13 is C₅ alkyl. In one embodiment, R^a13 is C₆ alkyl. In one embodiment, the alkyl (in R^a13) is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or oxo.

In one embodiment, R^a13 is C₁-C₆ alkoxy. In one embodiment, R^a13 is C₁-C₃ alkoxy. In one embodiment, R^a13 is methoxy. In one embodiment, R^a13 is ethoxy. In one embodiment, R^a13 is C₃ alkoxy. In one embodiment, R^a13 is C₄ alkoxy. In one embodiment, R^a13 is C₅ alkoxy. In one embodiment, R^a13 is C₆ alkoxy. In one embodiment, the alkoxy is unsubstituted. In one embodiment, the alkoxy (in R^a13) is substituted. In one embodiment, the alkoxy is substituted with one or more halogen, hydroxyl, or oxo.

In one embodiment, R^a13 is -C (=O) NH (C₁-C₆ alkyl) . In one embodiment, R^a13 is -C (=O) NH (C₁-C₃ alkyl) . In one embodiment, R^a13 is -C (=O) NH (CH₃) . In one embodiment, R^a13 is -C (=O) NH (C₂H₅) . In one embodiment, R^a13 is -C (=O) NH₂. In one embodiment, R^a13 is -NH (C=O) (C₁-C₆ alkyl) . In one embodiment, R^a13 is -NH (C=O) (C₁-C₃ alkyl) . In one embodiment, R^a13 is -NH (C=O) (CH₃) . In one embodiment, R^a13 is -NH (C=O) (C₂H₅) . In one embodiment, R^a13 is -NH (C₁-C₆ alkyl) . In one embodiment, R^a13 is -N (C₁-C₆ alkyl) ₂. In one embodiment, R^a13 is -NH (C₁-C₃ alkyl) . In one embodiment, R^a13 is is -NH (CH₃) . In one embodiment, R^a13 is -NH (C₂H₅) . In one embodiment, R^a13 is -N (CH₃) ₂. In one embodiment, R^a13 is -N (CH₃) (C₂H₅) . In one embodiment, the alkyl (in R^a13) is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or oxo.

In one embodiment, Ring C is:

wherein the point of attachment is to L.

In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is 3. In one embodiment, n is 4. In one embodiment, n is 5. In one embodiment, n is 6.

In one embodiment, R^a11 is hydrogen. In one embodiment, R^a11 is hydroxyl. In one embodiment, R^a11 is halogen. In one embodiment, R^a11 is fluorine. In one embodiment, R^a11 is nitro. In one embodiment, R^a11 is cyano.

In one embodiment, R^a11 is C₁-C₆ alkyl. In one embodiment, R^a11 is C₁-C₃ alkyl. In one embodiment, R^a11 is methyl. In one embodiment, R^a11 is ethyl. In one embodiment, R^a11 is C₃ alkyl. In one embodiment, R^a11 is C₄ alkyl. In one embodiment, R^a11 is C₅ alkyl. In one embodiment, R^a11 is C₆ alkyl. In one embodiment, the alkyl (in R^a11) is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkoxy.

In one embodiment, R^a11 is C₁-C₆ alkoxy. In one embodiment, R^a11 is C₁-C₃ alkoxy. In one embodiment, R^a11 is methoxy. In one embodiment, R^a11 is ethoxy. In one embodiment, R^a11 is C₃ alkoxy. In one embodiment, R^a11 is C₄ alkoxy. In one embodiment, R^a11 is C₅ alkoxy. In one embodiment, R^a11 is C₆ alkoxy. In one embodiment, the alkoxy is unsubstituted. In one embodiment, the alkoxy (in R^a11) is substituted. In one embodiment, the alkoxy is substituted with one or more halogen, hydroxyl, C₁-C₆ alkoxy, or C₁-C₆ alkyl.

In one embodiment, when the carbon connected to R^a11 is a chiral center, it has R-configuration. In one embodiment, when the carbon connected to R^a11 is a chiral center, it has S-configuration.

In one embodiment, R^b is hydrogen. In one embodiment, R^b is C₁-C₆ alkyl. In one embodiment, R^b is C₁-C₃ alkyl. In one embodiment, R^b is methyl. In one embodiment, R^b is hydroxymethyl. In one embodiment, R^b is ethyl. In one embodiment, R^b is 2-hydroxyethyl. In one embodiment, R^b is 2-dimethylaminoethyl. In one embodiment, R^b is 2- methoxyethyl. In one embodiment, R^b is C₃ alkyl. In one embodiment, R^b is C₄ alkyl. In one embodiment, R^b is C₅ alkyl. In one embodiment, R^b is C₆ alkyl. In one embodiment, R^b is C₁-C₆ alkyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂. In one embodiment, R^b is C₂-C₆ alkyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂. In one embodiment, R^b is ethyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂. In one embodiment, R^b is - (C₁-C₆ alkylene) -OH. In one embodiment, R^b is - (C₁-C₆ alkylene) - (C₁-C₆ alkoxy) . In one embodiment, R^b is - (C₁-C₆ alkylene) -OCH₃. In one embodiment, R^b is - (C₁-C₆ alkylene) -NH₂. In one embodiment, R^b is - (C₁-C₆ alkylene) -NH(C₁-C₆ alkyl) . In one embodiment, R^b is - (C₁-C₆ alkylene) -NHCH₃. In one embodiment, R^b is - (C₁-C₆ alkylene) -N (C₁-C₆ alkyl) ₂. In one embodiment, R^b is - (C₁-C₆ alkylene) -N (CH₃) ₂. In one embodiment, R^b is - (C₂-C₆ alkylene) -OH. In one embodiment, R^b is - (C₂-C₆ alkylene) - (C₁-C₆ alkoxy) . In one embodiment, R^b is - (C₂-C₆ alkylene) -OCH₃. In one embodiment, R^b is - (C₂-C₆ alkylene) -NH₂. In one embodiment, R^b is - (C₂-C₆ alkylene) -NH (C₁-C₆ alkyl) . In one embodiment, R^b is - (C₂-C₆ alkylene) -NHCH₃. In one embodiment, R^b is - (C₂-C₆ alkylene) -N (C₁-C₆ alkyl) ₂. In one embodiment, R^b is - (C₂-C₆ alkylene) -N (CH₃) ₂. In one embodiment, the alkyl (in R^b) is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkoxy. In one embodiment, the alkylene (in R^b) is unsubstituted. In one embodiment, the alkylene is substituted with one or more halogen. In one embodiment, the alkylene is - (CH₂) _2-6-. In one embodiment, the alkylene is -CH₂CH₂-.

In one embodiment, R^b is C₂-C₆ alkenyl. In one embodiment, R^b is ethenyl (other name: vinyl) . In one embodiment, R^b is C₃ alkenyl. In one embodiment, R^b is prop-1-enyl. In one embodiment, R^b is allyl. In one embodiment, R^b is C₄ alkenyl. In one embodiment, R^b is but-1-enyl. In one embodiment, R^b is C₅ alkenyl. In one embodiment, R^b is pent-1-enyl. In one embodiment, R^b is penta-1, 4-dienyl. In one embodiment, R^b is C₆ alkenyl. In one embodiment, the alkenyl is unsubstituted. In one embodiment, the alkenyl is substituted. In one embodiment, the alkenyl is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkoxy.

In one embodiment, R^b is C₃-C₈ cycloalkyl. In one embodiment, R^b is C₃-C₆ cycloalkyl. In one embodiment, R^b is cyclopropyl. In one embodiment, R^b is cyclobutyl. In one embodiment, R^b is cyclopentyl. In one embodiment, R^b is cyclohexyl. In one embodiment, the cycloalkyl is unsubstituted. In one embodiment, the cycloalkyl is substituted. In one embodiment, the cycloalkyl is substituted with one or more halogen, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^b is C₆-C₁₀ aryl. In one embodiment, R^b is C₆-C₈ aryl. In one embodiment, R^b is phenyl. In one embodiment, the phenyl is unsubstituted. In one embodiment, the phenyl is substituted. In one embodiment, the phenyl is substituted with one or more halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^b is 5 to 10-membered heteroaryl. In one embodiment, R^b is 5 to 8-membered heteroaryl. In one embodiment, R^b is 5-membered heteroaryl. In one embodiment, R^b is 6-membered heteroaryl. In one embodiment, R^b is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, R^b is a 5 or 6-membered nitrogen-containing heteroaryl. In one embodiment, the heteroaryl is unsubstituted. In one embodiment, the heteroaryl is substituted. In one embodiment, the heteroaryl is substituted with one or more halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^b is 3 to 8-membered heterocyclyl. In one embodiment, R^b is 3 to 6-membered heterocyclyl. In one embodiment, R^b is a 3-membered heterocyclyl. In one embodiment, R^b is a 4-membered heterocyclyl. In one embodiment, R^b is a 5-membered heterocyclyl. In one embodiment, R^b is a 6-membered heterocyclyl. In one embodiment, R^b is a 3-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, R^b is a 3-to 6-membered oxygen-containing heterocyclyl. In one embodiment, R^b is a 3-to 6-membered nitrogen-containing heterocyclyl. In one embodiment, the heterocyclyl is unsubstituted. In one embodiment, the heterocyclyl is substituted. In one embodiment, the heterocyclyl is substituted with one or more halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^b is oxiran-2-yl. In one embodiment, R^b is oxetan-2-yl. In one embodiment, R^b is oxetan-3-yl. In one embodiment, R^b is tetrahydrofuran-2-yl. In one embodiment, R^b is tetrahydrofuran-3-yl. In one embodiment, R^b is tetrahydro-2H-pyran-4-yl. In one embodiment, R^b is tetrahydro-2H-pyran-3-yl. In one embodiment, R^b is tetrahydro-2H-pyran-2-yl. In one embodiment, R^b is azetidinyl (e.g., 1-azetidinyl, or 3-azetidinyl) . In one embodiment, R^b is pyrrolidinyl (e.g., pyrrolidin-1-yl) . In one embodiment, R^b is piperidinyl (e.g., piperidin-1-yl) . In one embodiment, R^b is piperazinyl (e.g., piperazin-1-yl, or 4- (C₁-C₆ alkyl) piperazin-1-yl) . In one embodiment, R^b is morpholinyl (e.g., 4-morpholinyl) . In one embodiment, R^b is thiazolyl (e.g., thiazol-2-yl) . In one embodiment, R^b is oxazolyl (e.g., oxazol-2-yl) . In one embodiment, R^b is imidazolyl (e.g., 1H-imidazol-2-yl) . In one embodiment, R^b is pyridyl (e.g., pyridine-2-yl, pyridine-3-yl, or pyridine-4-yl) .

In one embodiment, R^c is C₁-C₆ alkyl. In one embodiment, R^c is C₁-C₃ alkyl. In one embodiment, R^c is methyl. In one embodiment, R^c is hydroxymethyl. In one embodiment, R^c is ethyl. In one embodiment, R^c is 2-hydroxyethyl. In one embodiment, R^c is 2-dimethylaminoethyl. In one embodiment, R^c is 2-methoxyethyl. In one embodiment, R^c is C₃ alkyl. In one embodiment, R^c is C₄ alkyl. In one embodiment, R^c is C₅ alkyl. In one embodiment, R^c is C₆ alkyl. In one embodiment, R^c is C₁-C₆ alkyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂. In one embodiment, R^c is C₂-C₆ alkyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂. In one embodiment, R^c is ethyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂. In one embodiment, R^c is - (C₁-C₆ alkylene) -OH. In one embodiment, R^c is - (C₁-C₆ alkylene) - (C₁-C₆ alkoxy) . In one embodiment, R^c is - (C₁-C₆ alkylene) -OCH₃. In one embodiment, R^c is - (C₁-C₆ alkylene) -NH₂. In one embodiment, R^c is - (C₁-C₆ alkylene) -NH (C₁-C₆ alkyl) . In one embodiment, R^c is - (C₁-C₆ alkylene) -NHCH₃. In one embodiment, R^c is - (C₁-C₆ alkylene) -N (C₁-C₆ alkyl) ₂. In one embodiment, R^c is - (C₁-C₆ alkylene) -N (CH₃) ₂. In one embodiment, the alkyl (in R^c) is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkoxy. In one embodiment, the alkylene (in R^c) is unsubstituted. In one embodiment, the alkylene is substituted with one or more halogen. In one embodiment, the alkylene is - (CH₂) _2-6-. In one embodiment, the alkylene is -CH₂CH₂-.

In one embodiment, R^c is C₂-C₆ alkenyl. In one embodiment, R^c is ethenyl (other name: vinyl) . In one embodiment, R^c is C₃ alkenyl. In one embodiment, R^c is prop-1-enyl. In one embodiment, R^c is allyl. In one embodiment, R^c is C₄ alkenyl. In one embodiment, R^c is but-1-enyl. In one embodiment, R^c is C₅ alkenyl. In one embodiment, R^c is pent-1-enyl. In one embodiment, R^c is penta-1, 4-dienyl. In one embodiment, R^c is C₆ alkenyl. In one embodiment, the alkenyl is unsubstituted. In one embodiment, the alkenyl is substituted. In one embodiment, the alkenyl is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkoxy.

In one embodiment, R^c is C₃-C₈ cycloalkyl. In one embodiment, R^c is C₃-C₆ cycloalkyl. In one embodiment, R^c is cyclopropyl. In one embodiment, R^c is cyclobutyl. In one embodiment, R^c is cyclopentyl. In one embodiment, R^c is cyclohexyl. In one embodiment, the cycloalkyl is unsubstituted. In one embodiment, the cycloalkyl is substituted. In one embodiment, the cycloalkyl is substituted with one or more halogen, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^c is C₆-C₁₀ aryl. In one embodiment, R^c is C₆-C₈ aryl. In one embodiment, R^c is phenyl. In one embodiment, the phenyl is unsubstituted. In one embodiment, the phenyl is substituted. In one embodiment, the phenyl is substituted with one or more halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^c is 5 to 10-membered heteroaryl. In one embodiment, R^c is 5 to 8-membered heteroaryl. In one embodiment, R^c is 5-membered heteroaryl. In one embodiment, R^c is 6-membered heteroaryl. In one embodiment, R^c is a 5 or 6-membered heteroaryl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, R^c is a 5 or 6-membered nitrogen-containing heteroaryl. In one embodiment, the heteroaryl is unsubstituted. In one embodiment, the heteroaryl is substituted. In one embodiment, the heteroaryl is substituted with one or more halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^c is 3 to 8-membered heterocyclyl. In one embodiment, R^c is 3 to 6-membered heterocyclyl. In one embodiment, R^c is a 3-membered heterocyclyl. In one embodiment, R^c is a 4-membered heterocyclyl. In one embodiment, R^c is a 5-membered heterocyclyl. In one embodiment, R^c is a 6-membered heterocyclyl. In one embodiment, R^c is a 3-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, R^c is a 3-to 6-membered oxygen-containing heterocyclyl. In one embodiment, R^c is a 3-to 6-membered nitrogen-containing heterocyclyl. In one embodiment, the heterocyclyl is unsubstituted. In one embodiment, the heterocyclyl is substituted. In one embodiment, the heterocyclyl is substituted with one or more halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R^c is oxiran-2-yl. In one embodiment, R^c is oxetan-2-yl. In one embodiment, R^c is oxetan-3-yl. In one embodiment, R^c is tetrahydrofuran-2-yl. In one embodiment, R^c is tetrahydrofuran-3-yl. In one embodiment, R^c is tetrahydro-2H-pyran-4-yl. In one embodiment, R^c is tetrahydro-2H-pyran-3-yl. In one embodiment, R^c is tetrahydro-2H-pyran-2-yl. In one embodiment, R^c is azetidinyl (e.g., 1-azetidinyl, or 3-azetidinyl) . In one embodiment, R^c is pyrrolidinyl (e.g., pyrrolidin-1-yl) . In one embodiment, R^c is piperidinyl (e.g., piperidin-1-yl) . In one embodiment, R^c is piperazinyl (e.g., piperazin-1-yl, or 4- (C₁-C₆ alkyl) piperazin-1-yl) . In one embodiment, R^c is morpholinyl (e.g., 4-morpholinyl) . In one embodiment, R^c is thiazolyl (e.g., thiazol-2-yl) . In one embodiment, R^c is oxazolyl (e.g., oxazol-2-yl) . In one embodiment, R^c is is imidazolyl (e.g., 1H-imidazol-2-yl) . In one embodiment, R^c is pyridyl (e.g., pyridine-2-yl, pyridine-3-yl, or pyridine-4-yl) .

In one embodiment, R is C₁-C₆ alkyl. In one embodiment, R is C₁-C₆ alkyl substituted with one or more halogens. In one embodiment, R is C₁-C₃ alkyl. In one embodiment, R is methyl. In one embodiment, R is fluoromethyl. In one embodiment, R is difluoromethyl. In one embodiment, R is trifluoromethyl. In one embodiment, R is ethyl. In one embodiment, R is C₃ alkyl. In one embodiment, R is isopropyl. In one embodiment, R is C₄ alkyl. In one embodiment, R is C₅ alkyl. In one embodiment, R is C₆ alkyl. In one embodiment, the alkyl (in R) is unsubstituted. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is substituted with one or more hydroxyl. In one embodiment, the alkyl is substituted with one or more C₁-C₆ alkoxy.

In one embodiment, R is C₁-C₆ alkoxy. In one embodiment, R is C₁-C₃ alkoxy. In one embodiment, R is methoxy. In one embodiment, R is ethoxy. In one embodiment, R is C₃ alkoxy. In one embodiment, R is C₄ alkoxy. In one embodiment, R is C₅ alkoxy. In one embodiment, R is C₆ alkoxy. In one embodiment, the alkoxy (in R) is unsubstituted. In one embodiment, the alkoxy is substituted. In one embodiment, the alkoxy is substituted with one or more halogen, hydroxyl, or C₁-C₆ alkyl.

In one embodiment, R is C₃-C₈ cycloalkyl. In one embodiment, R is C₃-C₆ cycloalkyl. In one embodiment, R is cyclopropyl. In one embodiment, R is cyclobutyl. In one embodiment, R is cyclopentyl. In one embodiment, R is cyclohexyl. In one embodiment, the cycloalkyl is unsubstituted. In one embodiment, the cycloalkyl (in R) is substituted. In one embodiment, the cycloalkyl is substituted with one or more halogen, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R is 3 to 8-membered heterocyclyl. In one embodiment, R is 3 to 6-membered heterocyclyl. In one embodiment, R is a 3-membered heterocyclyl. In one embodiment, R is a 4-membered heterocyclyl. In one embodiment, R is a 5-membered heterocyclyl. In one embodiment, R is a 6-membered heterocyclyl. In one embodiment, R is a 3-to 6-membered heterocyclyl containing one or more nitrogen, oxygen, or sulfur ring atoms. In one embodiment, R is a 3-to 6-membered oxygen-containing heterocyclyl. In one embodiment, R is a 4-membered oxygen-containing heterocyclyl. In one embodiment, R is a 5-membered oxygen-containing heterocyclyl. In one embodiment, R is a 6-membered oxygen-containing heterocyclyl. In one embodiment, R is a 3-to 6-membered nitrogen-containing heterocyclyl. In one embodiment, the heterocyclyl (in R) is unsubstituted. In one embodiment, the heterocyclyl is substituted. In one embodiment, the heterocyclyl is substituted with one or more halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy.

In one embodiment, R is oxiran-2-yl. In one embodiment, R is oxetan-2-yl. In one embodiment, R is oxetan-3-yl. In one embodiment, R is tetrahydrofuran-2-yl. In one embodiment, R is tetrahydrofuran-3-yl. In one embodiment, R is tetrahydro-2H-pyran-4-yl. In one embodiment, R is tetrahydro-2H-pyran-3-yl. In one embodiment, R is tetrahydro-2H-pyran-2-yl. In one embodiment, R is azetidinyl (e.g., 1-azetidinyl, or 3-azetidinyl) . In one embodiment, R is pyrrolidinyl (e.g., pyrrolidin-1-yl) . In one embodiment, R is piperidinyl (e.g., piperidin-1-yl) . In one embodiment, R is piperazinyl (e.g., piperazin-1-yl, or 4- (C₁-C₆ alkyl) piperazin-1-yl) . In one embodiment, R is morpholinyl (e.g., 4-morpholinyl) .

In one embodiment, the carbon connected to R has S-configuration. In one embodiment, the carbon connected to R has R-configuration.

In one embodiment, the compounds provided herein are single enantiomers. In one embodiment, the compounds provided herein are single diastereoisomers. In one embodiment, the compounds provided herein are mixtures of enantiomers. In one embodiment, the compounds provided herein are mixtures of diastereoisomers. In one embodiment, the compounds provided herein are racemic compounds.

In one embodiment, the compounds provided herein have an enantiomeric excess (ee) of at least about 50%. In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 80%. In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 90%. In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 95%. In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 97%. In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 99%. In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 99.5%. In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 99.9%.

In one embodiment, the compounds provided herein have a diastereomeric excess (de) of at least about 50%. In one embodiment, the compounds provided herein have a diastereomeric excess of at least about 80%. In one embodiment, the compounds provided herein have a diastereomeric excess of at least about 90%. In one embodiment, the compounds provided herein have a diastereomeric excess of at least about 95%. In one embodiment, the compounds provided herein have a diastereomeric excess of at least about 97%. In one embodiment, the compounds provided herein have a diastereomeric excess of at least about 99%. In one embodiment, the compounds provided herein have a diastereomeric excess of at least about 99.5%. In one embodiment, the compounds provided herein have a diastereomeric excess of at least about 99.9%.

In one embodiment, the compound is a compound in Table 1, or a pharmaceutically acceptable salt thereof.

Table 1.

In one embodiment, the compounds provided herein are PI3Kα inhibitors that reduce the level of PI3Kα protein and/or inhibit or reduce at least one biological activity (e.g., enzymatic activity) of PI3Kα protein. In one embodiment, the expression level of the PI3Kαprotein is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or 99%. In one embodiment, the biological activity of the PI3Kα protein is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or 99%.

The ability of test compounds to act as inhibitors of PI3Kα may be demonstrated by assays known in the art. The activity of the compounds and compositions provided herein as PI3Kα inhibitors can be assayed in vitro, in vivo, or in a cell line. In vitro assays include assays that determine inhibition of the kinase. Alternate in vitro assays quantitate the ability of the inhibitor to bind to the protein kinase and can be measured either by radio labeling the compound prior to binding, isolating the compound/kinase complex and determining the amount of radio label bound, or by running a competition experiment where new compounds are incubated with the kinase bound to known radio ligands.

Potency of a PI3Kα inhibitor as provided herein can be determined by EC₅₀ value. A compound with a lower EC₅₀ value, as determined under substantially similar conditions, is a more potent inhibitor relative to a compound with a higher EC₅₀ value. In some embodiments, the substantially similar conditions comprise determining a PI3Kα-dependent phosphorylation level, in vitro or in vivo (e.g., in tumor cells, A594 cells, U2OS cells, A431 cells, Ba/F3 cells, or 3T3 cells expressing a wild type PI3Kα, a mutant PI3Kα, or a fragment of any thereof) .

Potency of a PI3Kα inhibitor as provided herein can also be determined by IC₅₀ value. A compound with a lower IC₅₀ value, as determined under substantially similar conditions, is a more potent inhibitor relative to a compound with a higher IC₅₀ value. In some embodiments, the substantially similar conditions comprise determining a PI3Kα-dependent phosphorylation level, in vitro or in vivo (e.g., in tumor cells, SKOV3, T47D, CAL33, BT20, HSC2, OAW42, NCI, HCC1954, NCIH1048, Detroit562, A594 cells, U2OS cells, A431 cells, A594 cells, U2OS cells, Ba/F3 cells, or 3T3 cells expressing a wild type PI3Kα, a mutant PI3Kα, or a fragment of any thereof) .

In one embodiment, the compounds provided herein bind to a PI3Kα protein with an affinity in the range of about 1 pM to about 100 μM, about 1 pM to about 1 μM, about 1 pM to about 500 nM, or about 1 pM to about 100 nM. In some embodiment, the compounds provided herein bind to a PI3Kα protein with an affinity of about 100 nM to about 1 μM, about 100 nM to about 900 nM, about 100 nM to about 800 nM, about 100 nM to about 700 nM, about 100 nM to about 600 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, about 100 nM to about 300 nM, about 100 nM to about 200 nM, about 200 nM to about 1 μM, about 300 nM to about 1 μM, about 400 nM to about 1 μM, about 500 nM to about 1 μM, about 600 nM to about 1 μM, about 700 nM to about 1 μM, about 800 nM to about 1 μM, about 900 nM to about 1 μM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, or about 900 nM. In some embodiment, the compounds provided herein bind to a PI3Kα protein with an affinity of about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 20 nM to about 100 nM, about 30 nM to about 100 nM, about 40 nM to about 100 nM, about 50 nM to about 100 nM, about 60 nM to about 100 nM, about 70 nM to about 100 nM, about 80 nM to about 100 nM, about 90 nM to about 100 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM. In some embodiment, the compounds provided herein bind to a PI3Kα protein with an affinity of less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. In one embodiment, the compounds provided herein bind to a PI3Kα protein with an affinity of less than 1 nM. In one embodiment, the affinity is characterized by an IC₅₀ value. In one embodiment, the affinity is characterized by an EC₅₀ value. In one embodiment, the PI3Kα protein is wild type PI3Kα. In one embodiment, the PI3Kα protein has one or more mutations, e.g., the mutations in Table 2.

In one embodiment, the compounds provided herein exhibit selective inhibition of PI3Kα. In one embodiment, the compounds provided herein selectively target PI3Kα over another isoform of PI3K (e.g., PI3Kβ, PI3Kδ, or PI3Kγ) . In one embodiment, the compounds provided herein is capable of binding to the helical or kinase domain of PI3Kα. The helical or kinase domain of PI3Kα is known in the art (e.g. Zhao et al., Proc Natl Acad Sci. 2008, 105: 2652–2657) . In one embodiment, the compounds provided herein bind to an allosteric site in the kinase domain. In one embodiment, the compounds provided herein exhibits picomolar, nanomolar, or micromolar potency against a PI3Kα kinase with one or more mutations, with minimal activity against related kinases (e.g., wild type PI3Kα) . Inhibition of wild type PI3Kα can cause undesirable side effects (e.g., hyperglycemia and skin rashes) that can impact quality of life and compliance, or lead to dose limiting toxicities. See, e.g., Hanker, et al., Cancer Disc. 2019, 9 (4) : 482-491. Mutant-selective inhibitors may reduce the risk of such dose limiting toxicities, including hyperglycemia, observed with inhibitors of wild type PI3Kα. In one embodiment, the compounds provided herein exhibits higher inhibition of a mutant PI3Kα than wild type PI3Kα. In one embodiment, the compounds provided herein exhibit at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher inhibition of a mutant PI3Kα than wild type PI3Kα. In one embodiment, a compound provided herein exhibits from about 2-fold to about 10-fold greater inhibition of PI3Kα containing one or more mutations as described herein relative to inhibition of wild type PI3Kα. In one embodiment, a compound provided herein exhibits from about 10-fold to about 100-fold greater inhibition of PI3Kα containing one or more mutations as described herein relative to inhibition of wild type PI3Kα. In one embodiment, a compound provided herein exhibits from about 100-fold to about 1000-fold greater inhibition of PI3Kα containing one or more mutations as described herein relative to inhibition of wild type PI3Kα. In one embodiment, the mutant PI3Kα has one or more mutations, e.g., the mutations in Table 2.

The selectivity between wild type PI3Kα and PI3Kα containing one or more mutations as described herein can also be measured using in vitro assays such as surface plasmon resonance and fluorence-based binding assays, and cellular assays such as the levels of pAKT, a biomarker of PI3Kα activity, or proliferation assays where cell proliferation is dependent on mutant PI3Kα kinase activity.

METHODS OF USE

In one embodiment, provided herein is a method of treating diseases or conditions by inhibiting a PI3Kα protein comprising administering to a subject in need thereof a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein.

In one embodiment, provided herein is a method of treating a PI3Kα-associated disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein. In one embodiment, the disease or disorder is PIK3CA-related overgrowth syndromes (PROS) . In one embodiment, the disease or disorder is a proliferative disease (e.g., cancer) .

In one embodiment, provided herein is a method of treating a cancer, comprising administering to a subject having the cancer a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein. In one embodiment, the cancer is a PI3Kα-associated cancer. In one embodiment, the PI3Kα-associated cancer has one or more mutations described in Table 2.

In one embodiment, the cancer is a hematological cancer. In one embodiment, the cancer is a solid tumor. In one embodiment the cancer is breast cancer (including both HER2+ and HER2-breast cancer, ER+ breast cancer, and triple negative breast cancer) , colon cancer, rectal cancer, colorectal cancer, ovarian cancer, lymphangioma, meningioma, head and neck squamous cell cancer (including oropharyngeal squamous cell carcinoma) , melanoma (including uveal melanoma) , kidney cancer, pancreatic neuroendocine neoplasms (pNETs) , stomach cancer, esophageal cancer, acute myeloid leukemia, relapsed and refractory multiple myeloma, pancreatic cancer, lung cancer (including adenocarcinoma lung cancer and squamous cell lung carcinoma) , glioma, esophageal squamous cell carcinoma, esophagastric adenocarcinoma, or endometrial cancer. In one embodiment, the cancer is head and neck cancer, brain cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, liver cancer, stomach cancer, hematological cancer, thyroid cancer, colon cancer, or gastric cancer

In one embodiment, the cancer is head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC) , or oropharyngeal squamous cell carcinoma) . In one embodiment, the cancer is brain cancer (e.g., glioblastoma) . In one embodiment, the cancer is breast cancer (e.g., triple negative breast cancer, ER-positive breast cancer, HER2-positive breast cancer, or HER2-negative breast cancer) . In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is cervical cancer. In one embodiment, the cancer is lung cancer (e.g., adenocarcinoma lung cancer, and squamous cell lung carcinoma) . In one embodiment, the cancer is kidney cancer. In one embodiment, the cancer is bladder cancer. In one embodiment, the cancer is liver cancer. In one embodiment, the cancer is sarcoma. In one embodiment, the cancer is a hematological cancer (e.g., leukemia, lymphoma, or myeloma) . In one embodiment, the cancer is thyroid cancer. In one embodiment, the cancer is colon cancer. In one embodiment, the cancer is gastric cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is endometrial cancer. In one embodiment, the cancer is an advanced or metastatic.

In one embodiment, the cancer is associated with or has a dysregulation of a PIK3CA gene, a PI3Kα protein, or expression or activity, or level of any of the same. In one embodiment, the cancer is associated with or has a dysregulation of a PIK3CA gene. In one embodiment, the cancer is associated with or has one or more mutations in the PIK3CA gene. In one embodiment, the cancer is associated with or has a dysregulation of a PI3Kα protein. In one embodiment, the cancer is associated with or has one or more mutations in a PI3Kα protein. In one embodiment, the mutation in a PI3Kα protein comprises one or more PI3Kα protein substitutions, point mutations, and insertions. Non-limiting examples of PI3Kα protein mutations (e.g. substitutions, insertions, or deletions) are described in Table 2.

In one embodiment, the dysregulation of a PIK3CA gene, a PI3Kα protein, or expression or activity or level of any of the same, includes a splice variation in a PI3KαmRNA which results in an expressed protein that is an alternatively spliced variant of PI3Kα having at least one residue deleted (as compared to the wild type PI3Kα protein) resulting in a constitutive activity of a PI3Kα protein domain.

In one embodiment, the dysregulation of a PIK3CA gene, a PI3Kα protein, or expression or activity or level of any of the same, includes at least one point mutation in a PIK3CA gene that results in the production of a PI3Kα protein that has one or more amino acid substitutions or insertions or deletions in a PIK3CA gene that results in the production of a PI3Kα protein that has one or more amino acids inserted or removed, as compared to the wild type PI3Kα protein. In one embodiment, the resulting mutant PI3Kα protein has increased activity, as compared to a wild type PI3Kα protein or a PI3Kα protein not including the same mutation. In one embodiment, the compounds described herein selectively inhibit the resulting mutant PI3Kα protein relative to a wild type PI3Kα protein or a PI3Kα protein not including the same mutation.

In one embodiment, the PI3Kα protein mutation is E542A, E542G, E542K, E542Q, E542V, E545A, E545D, E545G, E545K, E545Q, M1043I, M1043L, M1043T, M1043V, H1047L, H1047Q, H1047R, H1047Y, or G1049R, or a combinations thereof. In one embodiment, the PI3Kα protein mutation is H1047X, where X is any amino acid. In one embodiment, the PI3Kα protein mutation is H1047R.

In one embodiment, the PIK3CA mutation comprises a modification in a codon that encodes an amino acid substitution at a specific position selected from a group consisting of G118, C420, E542, E545, Q546, H1047, and any combination thereof, in the corresponding PI3Kα protein. In one embodiment, the PIK3CA mutation results in the translation of a PI3Kα protein having one or more mutations in the adaptor-binding domain (ABD) , C2 domain, helicase domain, or kinase domain. In one embodiment, the PI3Kα protein has a mutation in the helicase domain, for example, in Exon 7 (e.g., C420R) , or in Exon 9 (e.g., E542K, E545A, E545D, E545G, E545K, Q546E, or Q546R) . In one embodiment, the PI3Kα protein has a mutation in the kinase domain, for example, in Exon 20 (e.g., H1047L, H1047R, or H1047Y) . In one embodiment, the PI3Kα protein has mutations in both the helical domain and kinase domain. In one embodiment, the PIK3CA mutations and PI3Kα protein mutations are those described in Mangone et al., Clinics. 2012; 67 (11) : 1285-1290; Ligresti et al., Cell Cycle, 2009, 8 (9) : 1352–1358; Zhao et al., Proc Natl Acad Sci. 2008, 105: 2652–2657, the entirely of each of which is incorporated herein by reference.

In one embodiment, a compound provided herein is used to treat a cancer, wherein the cancer is a PIK3CA mutant cancer. In one embodiment, the cancer is colon cancer having one or more mutations selected from the group consisting of C311G, G317T, G323C, del332–334, G353A, G365A, C370A, T1035A, T1258C, G1357C, C1616G, A1625G, A1634G, G1635T, C1636A, A1637C, C1981A, G2702T, T2725C, T3022C, A3073G, C3074A, G3129T, C3139T, and A3140T in the coding exons of PIK3CA. In one embodiment, the PIK3CA mutant cancer is glioblastomas having one or more mutations selected from the group consisting of T1132C, G1048C, A2102C, and G3145A in the coding exons of PIK3CA. In one embodiment, the PIK3CA mutant cancer is gastric cancer having G2702T, or A3140G mutation in the coding exons of PIK3CA. In one embodiment, the PIK3CA mutant cancer is lung cancer having G1633A mutation in the coding exons of PIK3CA. In one embodiment, the PIK3CA mutant cancer is breast cancer having one or more mutations selected from the group consisting of C1241T, T1258C, del1352–1366, G1624A, G1633A, C1636G, A3140G, A3140T, G1624A, G1633A, A1634G, C3075T, A3140T, and A3140G in the coding exons of PIK3CA. In one embodiment, the PIK3CA mutant cancers are those described in Ligresti et al., Cell Cycle, 2009, 8 (9) : 1352–1358, the entirely of which is incorporated herein by reference.

In one embodiment, provided herein is a method of treating a PI3Kα-associated cancer in a subject comprising (a) diagnosing the cancer in the subject as a PI3Kα-associated cancer, and then (b) administering a therapeutically effective amount of a compound provided herein to the subject. In one embodiment, the diagnosing of PI3Kα-associated cancer involves liquid biopsy. In one embodiment, the diagnosing of PI3Kα-associated cancer involves tumor biopsy. In one embodiment, the diagnosing of PI3Kα-associated cancer involves genetic testing (e.g. DNA sequencing) .

In one embodiment, provided herein is a method of treating a subject having a dysregulation of a PIK3CA gene or PI3Kα protein by administering a compound provided herein to the subject. In one embodiment, the subject has been identified or diagnosed as having a cancer with a dysregulation of a PIK3CA gene or a PI3Kα protein. In one embodiment, the subject has a tumor that is positive for a dysregulation of a PIK3CA gene or a PI3Kα protein. In one embodiment, the subject has a tumor that is positive for a mutation in the coding exons of PIK3CA. In one embodiment, the subject has a tumor that is positive for a mutation in the amino acid sequence of PI3Kα. In one embodiment, the one or more mutations in a PIK3CA gene can result, e.g., in the translation of an PI3Kα protein having one or more of the following amino acids: 542, 545, 1043, and 1047 and 1049. In one embodiment, PI3Kα protein has one or more mutations selected from the groups consisting of E542A, E542G, E542K, E542Q, E542V, E545A, E545D, E545G, E545K, E545Q, M1043I, M1043L, M1043T, M1043V, H1047L, H1047Q, H1047R, H1047Y, and G1049R. In one embodiment, the cancer with a dysregulation of a PIK3CA gene, a PI3Kα protein, or expression or activity or level of any of the same is determined using a regulatory agency-approved, e.g., FDA-approved, assay or kit. In one embodiment, the assay utilizes next generation sequencing, pyrosequencing, immunohistochemistry, or break apart FISH analysis. In one embodiment, the assay is a regulatory agency-approved assay, e.g., FDA-approved kit. In one embodiment, the assay is a liquid biopsy. In one embodiment, the biological sample to be used in a liquid biopsy includes, e.g., blood, plasma, urine, cerebrospinal fluid, saliva, sputum, broncho-alveolar lavage, bile, lymphatic fluid, cyst fluid, stool, ascites, or a combination thereof. In one embodiment, a liquid biopsy is used to detect circulating tumor cells (CTCs) . In one embodiment, a liquid biopsy is used to detect cell-free DNA. In one embodiment, cell-free DNA detected using a liquid biopsy is circulating tumor DNA (ctDNA) that is derived from tumor cells. In one embodiment, analysis of ctDNA (e.g., using sensitive detection techniques such as next-generation sequencing (NGS) , traditional PCR, digital PCR, or microarray analysis) is used to identify dysregulation of a PIK3CA gene, a PI3Kα protein, or the expression or activity or level of any of the same. Additional assays are also known in the art.

In one embodiment, compounds provided herein are provided for use as a medicament or are provided for use in preparing a medicament, e.g., for the treatment of cancer. In some embodiment, compounds provided herein are provided for use in a method for the treatment of cancer.

In one embodiment, compounds provided herein are provided for use in a method for the treatment of diseases or conditions by inhibiting PI3Kα protein.

Exemplary sequence of human phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit isoform alpha (UniProtKB entry P42336) (SEQ ID NO: 1) is listed below.

Table 2. Exemplary mutations (substitutions/insertions/deletions) in the amino acid sequence of PI3Kα protein

PHARMACEUTICAL COMPOSITIONS

The pharmaceutical composition provided herein is administered by various routes to mammals, including rodents and humans. In one embodiment, the administration is intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, or intradermal administration. In one embodiment, the administration is intravenous administration. In one embodiment, the administration is intramuscular administration.

In one embodiment, the administration is oral administration. In one embodiment, a pharmaceutical composition provided herein is orally administered in an orally acceptable dosage form including capsules, tablets, aqueous suspensions or solutions.

In one embodiment, compounds provided herein are administered to a mammal in the form of a raw chemical without any other components present. In one embodiment, compounds provided herein are administered to a mammal as part of a pharmaceutical composition containing the compound combined with a suitable pharmaceutically acceptable carrier (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003) ; Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004) ; Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000) ) . Non-limiting examples of pharmaceutically suitable carriers include solids and/or liquids such as water, alcohol and glycerol. Pharmaceutically acceptable excipients and diluents include, but are not limited to buffers, preservatives, binders, fillers, disintegrants, lubricants, wetting agents, antioxidants, flavorings, thickeners, coloring agents, emulsifiers, suspending agents and the like. Non-limiting examples of excipients and diluents also include sucrose, lactose, dextrose, sorbitol, mannitol, erythritol, maltitol, starch, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.

In one embodiment, a pharmaceutical composition provided herein is prepared as liquid suspensions or solutions using a liquid, such as an oil, water, an alcohol, and combinations of these.

In one embodiment, a pharmaceutical composition provided herein is prepared as a sterile injectable, which may be aqueous or oleaginous suspensions. The suspension is formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Polysorbate) . In one embodiment, the sterile injectable formulation is a sterile injectable solution or suspension in a diluent or solvent. In one embodiment, , sterile fixed oils are employed as a solvent or suspending medium. Pharmaceutically acceptable natural oils or fatty acids may also be used in the preparation of injectable formulations.

In one embodiment, a pharmaceutical composition provided herein is administered in the form of suppositories for rectal administration.

In one embodiment, a pharmaceutical composition provided herein is administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Topical application for the lower intestinal tract is effected in a rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches may also be used. For topical applications, the pharmaceutical compositions is formulated in a suitable ointment, lotion, or cream containing the active component suspended or dissolved in one or more carriers.

In one embodiment, a pharmaceutical composition provided herein is administered ophthalmically and formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzyl alkonium chloride. In one embodiment, for ophthalmic uses, the pharmaceutical compositions is formulated in an ointment such as petrolatum.

In one embodiment, a pharmaceutical composition provided herein is administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

In one embodiment, the pharmaceutical compositions to be used for in vivo administration can be sterile. In one embodiment, this is accomplished by filtration through, e.g., sterile filtration membranes.

In one embodiment, a pharmaceutical composition provided herein is administered to a patient that may experience the beneficial effects of a compound provided herein. In one embodiment, the patients are mammals, e.g., humans and companion animals. In one embodiment, the patient is a human.

In one embodiment, also provided herein are kits which comprise a compound provided herein (or a composition comprising a compound provided herein) packaged in a manner that facilitates their use to practice methods provided herein. In one embodiment, the kit includes a compound provided herein (or a composition comprising a compound provided herein) packaged in a container, such as a sealed vial, with a label affixed to the container or included in the kit that describes use of the compound or composition to practice the method provided herein. In one embodiment, the compound or composition is packaged in a unit dosage form. In one embodiment, the kit further includes a device suitable for administering the compound or composition according to the intended route of administration. In one embodiment, the kit comprises a compound provided herein, and instructions for administering the compound to a patient having cancer.

EXAMPLES

Methods for preparing the compounds provided herein are illustrated in the following examples. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification, or alternatively can be synthesized by a skilled person by using well-known methods.

As used herein and unless otherwise specified, when the stereochemical configuration for a chiral center in a compound provided herein is drawn stereo specifically (e.g., with widget and/or dash bonds) , either without additional designation or being designated “R” (or “ (R) ” ) or “S’ (or “ (S) ” ) , it means the mixture (s) was separated and absolute stereochemistry was known, or only one enantiomer was obtained and absolute stereochemistry was known. For some compounds, the stereochemical configuration at indicated centers has been designated as “*R” (first eluted from the column in case the column conditions of the separation are described in the synthesis protocol and when only one stereocenter present or indicated) or “*S” (second eluted from the column in case the column conditions of the separation are described in the synthesis protocol and when only one stereocenter present or indicated) when the absolute stereochemistry is undetermined (even if the bonds are drawn stereo specifically) although the compound itself has been isolated as a single stereoisomer and is enantiomerically pure. In case a compound designated as “*R” is converted into another compound, the “*R” indication of the resulting compound is derived from its starting material.

Preparation of intermediates

For intermediates that were used in a next reaction step as a crude or as a partially purified intermediate, in some cases no molar amounts are mentioned for such intermediate in the next reaction step or alternatively estimated molar amounts or theoretical molar amounts for such intermediate in the next reaction step are indicated in the reaction protocols described below.

Preparation of intermediate 1

To a solution of 1- (3, 5-difluoro-2-hydroxy-phenyl) ethanone (22 g, 127.81 mmol) in DMF (200 mL) was added K₂CO₃ (35.33 g, 255.62 mmol) and 2- (trifluoromethyl) oxirane (15.75 g, 140.59 mmol) . The mixture was stirred at 25 ℃ for 16 hr. The reaction mixture was diluted with H₂O (500 mL) and extracted with EtOAc (500 mL x 4) . The combined organic layers were washed with aqueous LiCl solution (500 mL) and aqueous NaCl solution (1000 mL) , dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluents: A, petroleum ether; B, ethyl acetate, 0%B to 26%B in A) to afford intermediate 1 (17.33 g, 51.64 mmol, 40.40%yield) as a yellow liquid.

The following intermediates were synthesized by an analogous method as described above for intermediate 1.

Preparation of intermediate 3

To a stirred solution of intermediate 1 (17.33 g, 60.98 mmol) in DCM (200 mL) was added DMP (38.80 g, 91.47 mmol, 28.34 mL) in portions at 0 ℃ under nitrogen atmosphere. The resulting mixture was stirred at 30 ℃ for 5 h under nitrogen atmosphere. The mixture was washed with saturated Na₂S₂O₃ (200 mL x 2) and saturated NaHCO₃ (200 mL x 2) and extracted with DCM (400 mL x 2) . The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 26%B in A) to afford intermediate 3 (15.66 g, 42.02 mmol, 68.90%yield) as a yellow liquid.

The following intermediates were synthesized by an analogous method as described above for intermediate 3.

Preparation of intermediate 5

To a solution of intermediate 3 (15.66 g, 55.50 mmol) in Ac₂O (200 mL) was added NaOAc (6.83 g, 83.25 mmol) . The mixture was stirred at 50 ℃ for 3 hr. The reaction mixture was basified with saturated NaHCO₃ to pH = 7 and extracted with EtOAc (200 mL x 3) . The combined organic layers were washed with aqueous NaCl 1000 mL, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 34%B in A) to afford intermediate 5 (11.82 g, 44.74 mmol, 80.61%yield) as a white solid.

The following intermediates were synthesized by an analogous method as described above for intermediate 5.

Preparation of intermediate 7

To a solution of intermediate 5 (8.35 g, 31.62 mmol) in EtOH (120 mL) was added hydroxylamine hydrochloride (10.99 g, 158.12 mmol) and sodium acetate (12.97 g, 158.12 mmol) . The mixture was stirred at 100 ℃ for 6 hr. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to get a residue, which was diluted with H₂O 200 mL and extracted with EtOAc (200 mL x 3) . The combined organic layers were washed with aqueous NaCl (200 mL x 2) and saturated NaHCO₃ (200 mL x 2) , dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford intermediate 7 (9 g, crude) which was used into the next step without further purification.

The following intermediates were synthesized by an analogous method as described above for intermediate 7.

Preparation of intermediate 9

To a solution of intermediate 7 (9 g, 31.62 mmol, crude) in EtOH (120 mL) was added NH₄Cl (16.91 g, 316.20 mmol) and Zn (21.16 g, 323.60 mmol) . The mixture was stirred at 80 ℃ for 1 hr. The reaction mixture was cooled to room temperature, filtered and the filter cake was washed with EtOAc (500 mL) . Then, the filtrate was concentrated under reduced pressure to give a residue. The residue was diluted with H₂O 100 mL and extracted with EtOAc 200 mL (100 mL x 2) . The combined organic layers were washed with aqueous NaCl 100 mL, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 31%B in A) to afford intermediate 9 (4.73 g, 17.25 mmol, 54.55%yield) as a yellow oil.

The following intermediates were synthesized by an analogous method as described above for intermediate 9.

Preparation of intermediates 9a and 9b

The intermediate 9 (5 g, 18.85 mmol) was separated by chiral HPLC (separation condition: Column: IH-3.0 cm; Mobile Phase: Hex: EtOH = 75: 25, at 25 mL/min; Temp: 40 ℃; Wavelength: 254 nm) . The first fraction was collected as intermediate 9a (2.0 g, yield: 40%, 100 %ee at RT = 4.221 min) and the second fraction was collected as intermediate 9b (2.0 g, yield: 40%, 100 %ee at RT = 5.629 min) .

Preparation of intermediate 11

To a solution of 2-chloropyrimidin-5-amine (5 g, 38.60 mmol) in THF (25 mL) was added phenyl chloroformate (6.04 g, 38.60 mmol, 4.84 mL) . The mixture was stirred at 25 ℃ for 2 hr. The reaction mixture was concentrated under reduced pressure to give phenyl N- (2-chloropyrimidin-5-yl) carbamate, which was dissolved in pyridine (30 mL) and added intermediate 9 (2.7 g, 10.18 mmol) . The mixture was stirred at 80 ℃ for 1 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 30%B in A) to afford intermediate 11 (3.98 g, 8.70 mmol, 85.45%yield) as a yellow solid.

Preparation of intermediates 11a and 11b

Intermediate 11 (5 g, 13.31 mmol) was separated by supercritical fluid chromatography (column: DAICEL CHIRALPAK AD (250mm*50mm, 10 um) ; mobile phase: [CO₂-EtOH (0.1%NH₃H₂O) ] ; B%: 30%, isocratic elution mode) . The first fraction was collected as intermediate 11a (2.81 g, 5.43 mmol, 40.81%yield, 81.21%purity) as a yellow solid. The second fraction was collected as intermediate 11b (1.75 g, 4.07 mmol, 30.57%yield, 98.09%purity) as a yellow solid.

The following intermediates were synthesized by an analogous method as described above for intermediate 11.

Preparation of intermediate 13

To a solution of 1- (3, 5-difluoro-2-hydroxylphenyl) ethan-1-one (1.0 g, 5.8 mmol) , 1-bromo-3-methylbutan-2-one (1.0 g, 6.4 mmol) and K₂CO₃ (0.96 g, 7.0 mmol) in MeCN (20 mL) was heated at 80 ℃ for 16 hr. The resulting mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the crude product which was purified by silica gel column chromatography (eluent with PE/EtOAc from 10%to 50%) to afford intermediate 13 (650 mg, 47%yield) as a white solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 13.

Preparation of intermediate 15

To a solution of intermediate 13 (1.0 g, 4.54 mmol) in MeOH (20 mL) was added NH₄OAc (3.49 g, 45.4 mmol) and Na₂SO₄ (200 mg, 1.58 mmol) . After stirred at r. t. for 1hr, NaBH₃CN (285.3 mg, 4.54 mmol) was added, and the reaction mixture was heated up to 80 ℃ and stirred for 18 hours. After cooled down to r. t., the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3) . The combined organic layers were washed with brine, dried over anhydrous sodium sulphate, filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE = 0-50%) to afford intermediate 15 (700 mg, 3.16 mmol, 69.6%yield) as a yellow solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 15.

Preparation of intermediate 19

To a solution of 2, 4-dimethoxybenzylamine (10 g, 59.81 mmol, 8.98 mL) in DCM (100 mL) was added dropwise TEA (12.10 g, 119.61 mmol, 16.65 mL) and MsCl (8.37 g, 73.07 mmol, 5.66 mL) at 0 ℃. The resulting mixture was stirred at 25 ℃ for 16 hr. The reaction mixture was quenched by addition aqueous NaHCO₃ 100 mL at 0 ℃, and then diluted with H₂O 100 mL and extracted with DCM (100 mL x 2) . The combined organic layers were washed with aqueous NaCl 200 mL, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give intermediate 19 as a crude product, which was used into the next step without further purification.

Preparation of intermediate 20

To a solution of intermediate 19 (3 g, 12.23 mmol) in THF (30 mL) was added dropwise n-BuLi (2.5 M, 15.17 mL) at -70 ℃ under N₂ atmosphere. After addition, the mixture was stirred at -70 ℃ for 1 hr and then tert-butyl 3-oxoazetidine-1-carboxylate (4.19 g, 24.46 mmol) in THF (30 mL) was added dropwise at -70 ℃. After addition, the mixture was stirred at this temperature for 1 hr, and then the resulting mixture was stirred at 25 ℃ for 16 h under N₂ atmosphere. The reaction mixture was quenched by addition of MeOH 30 mL at 0 ℃ and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 48%B in A) to afford intermediate 20 (2.29 g, 4.72 mmol, 38.57%yield) as a yellow oil.

Preparation of intermediate 21

To a solution of intermediate 20 (700 mg, 1.68 mmol) in DCM (14 mL) was added TFA (1.07 g, 9.42 mmol, 0.7 mL) . The mixture was stirred at 25 ℃ for 16 hr. The reaction mixture was filtered, and the filter cake redissolved in methanol, filtered and the filtrate was concentrated under vacuum to give intermediate 21 as a crude product, which was used in the next step without further purification.

The following intermediates were synthesized by an analogous method as described above for intermediate 21.

Preparation of intermediate 22

To a solution of methylsulfonylmethane (1.72 g, 18.25 mmol, 1.48 mL) in THF (10 mL) was added n-BuLi (2.5 M, 7.30 mL) at -45 ℃ under N₂, and the mixture was stirred at -45℃ for 0.5 hr. Tert-butyl 3-oxoazetidine-1-carboxylate (500 mg, 2.92 mmol) was added and the mixture was stirred at 25℃ for 3.5 hr. The reaction mixture was quenched with saturated aqueous solution of NH₄Cl (10 mL) at 0 ℃, the mixture was poured into H₂O (30 mL) and extracted with ethyl acetate (20 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the crude product. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 1/5) to afford intermediate 22 (872 mg, 2.69 mmol, 92.27%yield) as a white solid.

Preparation of intermediate 24

To a solution of tert-butyl 3- (methylsulfonyloxymethyl) azetidine-1-carboxylate (1 g, 3.77 mmol) in DMF (10 mL) was added potassium ethanethioate (860.89 mg, 7.54 mmol) . The solution was degassed under vacuum, purged with N₂ atmosphere for three times and heated up to 80 ℃ and stirred at 80 ℃ for 8 hr. Water (30 mL) was added to the mixture dropwise and stirred at 25 ℃ for 5 min. The resulting mixture was extracted with ethyl acetate (20 mL x 3) . The combined organic layers were washed with saturated aqueous solution of LiCl (40 mL) , dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue, which was purified by silica gel column chromatography (Petroleum Ether/Ethyl Aetate=1/0 to 0/1) to afford intermediate 24 (843 mg, 3.43 mmol, 90.9%yield) as a yellow oil.

The following intermediate was synthesized by an analogous method as described above for intermediate 24.

Preparation of intermediate 25

At 0℃, to a solution of intermediate 24 (400 mg, 1.63 mmol) in MeCN (4 mL) and HCl (2 M, 1.22 mL) was added N-chlorosuccinimide (870.85 mg, 6.52 mmol) and the resulting mixture was stirred at 0 ℃ for 2 hr. Water (30 mL) was added to the mixture dropwise at 0 ℃. The mixture was extracted with ethyl acetate (20 mL x 3) . The combined organic layers were washed with saturated NaCl, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to get a crude product, which was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 0/1) to afford intermediate 25 (330 mg, 1.17 mmol, 71.63%yield) as a yellow solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 25.

Preparation of intermediate 26

Under an ice bath condition, to a solution of intermediate 25 (330 mg, 1.22 mmol) in DCM (5 mL) was added methylamine (2 M, 1.84 mL) dropwise. Then TEA (185.69 mg, 1.84 mmol, 255.42 uL) was added. The mixture was stirred at 25℃ for 2 hours. The reaction mixture was diluted with H₂O (30 mL) and extracted with EtOAc (20 mL x 3) . The combined organic layers were washed with saturated NaCl (30 mL) , dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 0/1) to afford intermediate 26 (311 mg, 1.11 mmol, 90.52%yield) as a yellow solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 26.

Preparation of intermediate 27

A mixture of intermediate 26 (150 mg, 567.45 μmol) was added HCl/dioxane (2 mL) . The mixture was stirred at 25℃ for 1hr. The reaction mixture was concentrated under reduced pressure to give intermediate 27 as a crude product, which was used for next step without further purification.

The following intermediates were synthesized by an analogous method as described above for intermediate 27.

Preparation of intermediate 28

To a solution of tert-butyl 3- (hydroxymethyl) -3-methyl-azetidine-1-carboxylate (1 g, 4.97 mmol) in DCM (10 mL) was added TEA (1.01 g, 9.94 mmol, 1.38 mL) , the mixture was cooled to 0℃, and then methanesulfonyl chloride (1.02 g, 8.90 mmol, 689.19 μL) was added dropwise. The mixture was stirred at 25 ℃ for 4 hr. The reaction mixture was quenched with the saturated solution of sodium bicarbonate (20 mL) , washed with H₂O (20 mL) , and extracted with dichloromethane (20 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 1/3) to afford intermediate 28 (1.4 g, 4.86 mmol, 97.84%yield) as a colorless oil.

The following intermediate was synthesized by an analogous method as described above for intermediate 28.

Preparation of intermediate 29

To a solution of intermediate 28 (1.2 g, 4.30 mmol) in MeCN (15 mL) was added sodium thiomethoxide (1.2 g, 17.12 mmol, 1.09 mL) . The reaction mixture was stirred at 85 ℃ for 6 hr. The reaction mixture was quenched with the saturated aqueous solution of NH₄Cl (20 mL) , the mixture was poured into H₂O (60 mL) and extracted with dichloromethane (40 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=0/1 to 1/1) to afford intermediate 29 (982 mg, 3.95 mmol, 91.89%yield) as a colorless oil.

The following intermediate was synthesized by an analogous method as described above for intermediate 29.

Preparation of intermediate 30

To a solution of intermediate 29 (682 mg, 2.95 mmol) in DCM (12 mL) was added m-CPBA (1.80 g, 8.84 mmol, 85%purity) at 0℃, and then the mixture was stirred at 25℃ for 4hr. The reaction mixture was quenched with saturated aqueous solution of Na₂SO₃ (15 mL) at 0 ℃, poured into H₂O (30 mL) and extracted with ethyl acetate (20 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 0/1) to afford intermediate 30 (792 mg, 3.00 mmol, 88.62%yield) as a colorless oil.

The following intermediate was synthesized by an analogous method as described above for intermediate 30.

Preparation of intermediate 32

To a solution of methylamine hydrochloride (43.80 mg, 648.66 μmol) in DMF (4 mL) was added DIEA (279.45 mg, 2.16 mmol, 376.62 μL) , 2- (1-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) acetic acid (100 mg, 432.44 μmol) and HATU (328.85 mg, 864.88 μmol) . The solution was stirred at 50 ℃ for 1 h. The reaction mixture was concentrated under reduced pressure to get a residue. The residue was purified by flash column chromatography on 4 g silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 75%B in A, 10 mL/min) to afford intermediate 32 (177 mg, crude) as a pink solid.

Preparation of intermediate 34

To a solution of tert-butyl 1-oxa-5-azaspiro [2.3] hexane-5-carboxylate (200 mg, 1.08 mmol) in MeOH (5 mL) was added NaOMe (243.06 mg, 1.35 mmol, 30%purity) . After stirred at 25 ℃ for 16 hr, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate from 1/0 to 1/1) to afford intermediate 34 (200 mg, 920.55 μmol, 76.73%yield) as yellow oil.

The following intermediates were synthesized by an analogous method as described above for intermediate 34.

Preparation of intermediate 36

To a solution of tert-butyl 3- (aminomethyl) -3-hydroxy-azetidine-1-carboxylate (1 g, 4.94 mmol) and TEA (1.00 g, 9.89 mmol, 1.38 mL) in DCM (10 mL) was added acetyl acetate (1.01 g, 9.89 mmol, 928.73 μL) dropwise at 0 ℃. Then the mixture was stirred at 20 ℃ for 12 h. The reaction mixture was concentrated under reduced pressure to get a residue, which was diluted with H₂O (50 mL) and extracted with EtOAc (30 mL x 3) . The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to get a residue. The residue was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 100%B in A, 20 mL/min) to afford intermediate 36 (597 mg, 2.44 mmol, 49.43%yield) as a light-yellow gum.

Preparation of intermediate 38

To a solution of tert-butyl 3- (aminomethyl) azetidine-1-carboxylate (1 g, 5.37 mmol) in DCM (10 mL) was added TEA (1.63 g, 16.11 mmol, 2.24 mL) and Ac₂O (548.12 mg, 5.37 mmol, 504.25 μL) . The mixture was stirred at 25 ℃ for 1 hr. The reaction mixture was quenched by addition of aqueous solution of NH₄Cl (10 mL) at 25 ℃, diluted with H₂O 30 mL and extracted with DCM (20 mL x 3) . The combined organic layers were washed with aqueous NaCl 30 mL, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give intermediate 38 as crude product, which was used in the next step without further purification.

Preparation of intermediate 40

To a solution of intermediate 20 (300 mg, 720.31 μmol) in DMF (6 mL) were added Cs₂CO₃ (704.07 mg, 2.16 mmol) and MeI (102.24 mg, 720.31 μmol, 44.84 μL) at 0℃. The reaction mixture was stirred at 0℃ for 0.5 hr. The reaction mixture was diluted with H₂O 30 mL and extracted with EtOAc 30 mL (10 mL x 3) . The combined organic layers were washed with aqueous solution of LiCl (10 mL) and aqueous solution of NaCl (10 mL) , dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give intermediate 40 (411.8 mg, 788.68 μmol, 82.12%yield) as crude product which was used in next step without further purification.

Preparation of intermediate 42

To a solution of methylsulfonylmethane (3.49 g, 37.12 mmol, 3.01 mL) in THF (20 mL) was added n-BuLi (2.5 M, 14.85 mL) at -45 ℃ under N₂. The mixture was stirred at -45℃ for 0.5 hr and then tert-butyl 3-oxopyrrolidine-1-carboxylate (1.1 g, 5.94 mmol) was added to the mixture, which was stirred at 25 ℃ for 3.5 hr. The reaction mixture was quenched with saturated aqueous solution of NH₄Cl (20 mL) at 0 ℃, poured into H₂O (50 mL) and extracted with ethyl acetate (50 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 1/5) to afford intermediate 42 (1.7 g, 5.78 mmol, 97.35%yield) as a white solid.

Preparation of intermediate 43

A mixture of intermediate 42 (1.7 g, 6.09 mmol) in HCl/EtOAc (10 mL) was stirred at 25 ℃ for 1hr. The reaction mixture was concentrated under reduced pressure to give intermediate 43 (1.5 g, HCl salt) as crude product, which used into next step without further purification.

Preparation of intermediate 44

Under an ice bath condition, to a solution of benzyl 4-(chlorosulfonylmethyl) piperidine-1-carboxylate (500 mg, 1.51 mmol) in DCM (5 mL) was then added methylamine (2 M in EtOH, 2.26 mL dropwise. Then TEA (228.72 mg, 2.26 mmol, 314.60 μL) was added. The reaction mixture was stirred at 25 ℃ for 0.5 h. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (15 ml x 4) . The combined organic layers were washed with the saturated brine (30 mL) , dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 0/1) to afford intermediate 44 (356 mg, 1.05 mmol, 69.93%yield) as a white solid.

Preparation of intermediate 45

A mixture of intermediate 44 (100 mg, 306.36 μmol) in conc. HCl (2 mL) was stirred at 25 ℃ for 0.5 h. The reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give intermediate 45 (55 mg, crude) , which was used in the next step without further purification.

Preparation of intermediate 46

To a solution of tert-butyl 3- (iodomethyl) azetidine-1-carboxylate (4.00 g, 13.46 mmol) in THF (14 mL) was added K₂CO₃ (5.58 g, 40.39 mmol) and 2-sulfanylethanol (1.36 g, 17.41 mmol, 1.22 mL) . The reaction mixture was stirred at 20 ℃ for 12 h. The reaction solution was quenched with H₂O (20 mL) and extracted with EtOAc (15 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford intermediate 46 (3.8 g, crude) , which was used in the next step without further purification.

Preparation of intermediate 47

To a solution of intermediate 46 (3.8 g, 15.36 mmol) in DCM (60 mL) was added m-CPBA (9.36 g, 46.09 mmol, 85%purity) at 0 ℃. The reaction mixture was stirred at 20 ℃ for 4 h. The reaction mixture was quenched with saturated Na₂SO₃ (30 mL) at 0 ℃, and then concentrated under reduced pressure to remove DCM. The mixture was extracted with EtOAc (20 mL x 3) . The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the crude product, which was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 100%B in A, 50 mL/min. ) to give intermediate 47 (2.13 g, 7.55 mmol, 49.14%yield) as a light-yellow oil.

The following intermediate was synthesized by an analogous method as described above for intermediate 47.

Preparation of intermediate 48

To a solution of intermediate 47 (300 mg, 1.07 mmol) in THF (10 mL) was added NaH (85.90 mg, 2.15 mmol, 60%purity) at 0 ℃ under N₂. The reaction mixture was stirred at 0 ℃ for 0.5 h. MeI (304.86 mg, 2.15 mmol, 133.71 μL) was added at 0 ℃ and the mixture was stirred at 25 ℃ for 3.5 h. The reaction mixture was quenched by addition saturated NH₄Cl (50 mL) , diluted with H₂O (50 mL) and extracted with EtOAc (80 mL x 2) . The combined organic layers were washed with brine (100 mL) , dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a crude product, which was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 76%B in A. ) to afford intermediate 48 (120 mg, 395.37 μmol, 36.82%yield) as a colorless oil.

Preparation of intermediate 50

To a solution of intermediate 20 (340 mg, 816.35 μmol) in DMF (2 mL) was added Cs₂CO₃ (797.95 mg, 2.45 mmol) and MeI (231.74 mg, 1.63 mmol, 101.64 μL) . The reaction mixture was stirred at 25 ℃ for 16 hr. The reaction mixture was diluted with H₂O 30 mL and extracted with EtOAc 40 mL (20 mL x 2) . The combined organic layers were washed with aqueous LiCl 30 mL and aqueous NaCl 30 mL, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A, petroleum ether; B, ethyl acetate, 0%B to 50%B in A) to give intermediate 50 (294 mg, 656.61 μmol, 80.43%yield) as colorless oil.

Preparation of intermediate 52

To a solution of tert-butyl 3- (methylsulfonyloxymethyl) azetidine-1-carboxylate (1 g, 3.77 mmol) in MeCN (10 mL) was added (methylsulfanyl) sodium (1.05 g, 14.98 mmol, 954.55 μL) . The reaction mixture was stirred at 85 ℃ for 6 hr. The reaction mixture was quenched with a saturated solution of NH₄Cl (20 mL) , poured into H₂O (50 mL) and extracted with dichloromethane (30 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 3/1) to afford intermediate 52 (800 mg, 3.68 mmol, 97.67%yield) as a white solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 52.

Preparation of intermediate 53

To a solution of intermediate 53 (400 mg, 1.84 mmol) in EtOH (4 mL) was added PhI (OAc) ₂ (1.78 g, 5.52 mmol) and NH₄OAc (567.49 mg, 7.36 mmol) . The reaction mixture was stirred at 25 ℃ for 2 hr. The reaction mixture was diluted with H₂O 50 mL and extracted with EtOAc (30 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give intermediate 53 (600 mg, crude) which was used in the next step without further purification.

Preparation of intermediate 56

To a solution of tert-butyl 3- (1-hydroxyethyl) azetidine-1-carboxylate (0.5 g, 2.48 mmol) in DCM (10 mL) was added TEA (301.66 mg, 2.98 mmol, 414.94 μL) . Then MsCl (600 mg, 5.24 mmol, 405.41 μL) was added to the reaction mixture slowly at 0 ℃ under N₂. The reaction mixture was warmed slowly to 25 ℃ and stirred at 25 ℃ for 2 h. The reaction mixture was quenched with the saturated solution of sodium bicarbonate (20 mL) at 0 ℃, then H₂O (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give intermediate 56 (700 mg, crude) , which was used in the next step without further purification.

Preparation of intermediate 60

To a suspension of activated zinc powder (9.24 g, 141.29 mmol) in THF (150 mL) were added TMSCl (767.50 mg, 7.06 mmol, 896.61 μL) and 1, 2-dibromoethane (1.33 g, 7.06 mmol, 532.99 μL) and the suspension was stirred at 60 ℃ for 15 min. A solution of tert-butyl 3-iodoazetidine-1-carboxylate (20 g, 70.64 mmol) in DMA (150 mL) was added and the reaction mixture, which was stirred at 60 ℃ for 15 min. The reaction mixture was cooled down to 25 ℃ and 5-bromo-2-iodo-pyrimidine (20.13 g, 70.64 mmol) , Pd (dppf) Cl₂·CH₂Cl₂ (5.77 g, 7.06 mmol) and CuI (1.35 g, 7.06 mmol) were added. The reaction mixture was stirred at 80 ℃ for 2 hr. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (200 mL) and water (500 mL) and then filtered. The filtrate was extracted with ethyl acetate (3 x 300 mL) . The combined organic layers were washed with aq. NaCl solution (500 mL) , dried over Na₂SO_4, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=10/1 to 2/1) to afford intermediate 60 (5.5 g, 17.51 mmol, 24.78%yield) as yellow solid.

Preparation of intermediate 61

To a solution of intermediate 60 (2.5 g, 7.96 mmol) and diphenylmethanimine (4.33 g, 23.87 mmol, 4.01 mL) in toluene (20 mL) were added BINAP (495.48 mg, 795.73 μmol) and t-BuONa (2.29 g, 23.87 mmol) . The suspension was degassed under vacuum and purged with N₂ atmosphere for three times, and then Pd₂ (dba) ₃ (728.66 mg, 795.73 μmol) was added. The mixture was degassed under vacuum and purged with N₂ atmosphere for three times and stirred at 110 ℃ for 16 h. The reaction mixture was cooled to room temperature. H₂O (50 mL) was added, the mixture was extracted with ethyl acetate (30 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=10/1 to 3/1) to afford intermediate 61 (3.1 g, 7.48 mmol, 93.99%yield) as brown oil.

Preparation of intermediate 62

To a solution of intermediate 61 (3.1 g, 7.48 mmol) in THF (50 mL) was added HCl (0.5 M, 44.87 mL) and stirred at 25 ℃ for 0.5 h. H₂O (50 mL) was added, the mixture was extracted with ethyl acetate (80 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/1 to 0/1) to afford intermediate 62 (210 mg, 839.01 μmol, 11.22%yield) as brown oil.

Preparation of intermediate 63

To a solution of intermediate 62 (210 mg, 839.01 μmol) in THF (10 mL) was added phenyl chloroformate (131.36 mg, 839.01 μmol, 105.26 μL) and stirred at 25 ℃ for 16 h. The reaction mixture concentrated under reduced pressure to give intermediate 63 (311 mg, crude) , which was used in the next step without further purification.

Preparation of intermediate 64

To a solution of intermediate 63 (311 mg, 839.63 μmol) in pyridine (10 mL) was added intermediate 10 (207.55 mg, 839.63 μmol) . The reaction mixture was stirred at 80 ℃ for 16 h. The reaction mixture was cooled to room temperature. H₂O (50 mL) was added, the mixture was extracted with ethyl acetate (30 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=5/1 to 1/1) to afford intermediate 64 (360 mg, 687.71 μmol, 73.72%yield) as white solid.

Preparation of intermediate 65

To a solution of intermediate 64 (130 mg, 248.34 μmol) in DCM (2 mL) was added TFA (2.00 g, 17.50 mmol, 1.30 mL) . The reaction mixture was stirred at 25 ℃ for 0.25 h. The reaction mixture was concentrated under reduced pressure to give a residue. aq. NaOH (10 %) was added to basified to pH = 14, the mixture was extracted with DCM (30 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give intermediate 65 (130 mg, crude) , which was used in the next step without further purification.

Preparation of intermediate 66

A mixture of methyl 2- (azetidin-3-yl) acetate (362.30 mg, TFA salt) in DMSO (2 mL) were added TEA (1.88 g, 18.62 mmol, 2.59 mL, 25 eq) and intermediate 12 (300 mg, 744.92 μmol) and then the mixture was stirred at 80 ℃ for 12 hr. The reaction mixture was cooled to room temperature. H₂O (30 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3) . The combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (4 gSilica Flash Column, Eluent of 0～48%Ethyl acetate/Petroleum ether gradient @30 mL/min) to afford intermediate 66 (280 mg, 565.17 μmol, 75.87%yield) as a yellow solid.

Preparation of intermediate 67

A mixture of intermediate 66 (280 mg, 565.17 μmol) in MeOH (0.5 mL) was added LiOH (2 M in H₂O, 3 mL) and then the mixture was stirred at 25 ℃ for 1hr. The reaction mixture was adjusted pH to around 5, and then extracted with EtOAc (20 mL x 3) . The combined organic layers were concentrated under reduced pressure to give intermediate 67 (260 mg, crude) , which was used in the next step without further purification.

Preparation of intermediate 76

To a solution of intermediate 47 (200 mg, 715.94 μmol) and TEA (217.34 mg, 2.15 mmol, 298.95 μL) in DCM (2 mL) was added MsCl (460 mg, 4.02 mmol, 310.81 μL) at 0 ℃ and the mixture was stirred at 25 ℃ for 2 hr. The reaction mixture was quenched with H₂O (30 mL) under 0 ℃ and extracted with EtOAc (20 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to get a residue, which was purified by flash column chromatography silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0%B to 60%B in A, 10 mL/min) to give intermediate 76 (176 mg, 660.46 μmol, 92.25%yield, 98.07%purity) as a colorless oil.

Preparation of intermediate 77

To a solution of intermediate 76 (175 mg, 669.63 μmol) in DCM (2 mL) were added N-methylmethanamine hydrochloride (109.21 mg, 1.34 mmol) and TEA (237.16 mg, 2.34 mmol, 326.21 μL) and the mixture was stirred at 25 ℃ for 3 hr. The reaction mixture was diluted with H₂O (20 mL) and extracted with DCM (15 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to get a residue, which was purified by flash column chromatography on silica gel (eluent: A: Dichloromethane, B: Methanol, 0%B to 9%B in A, 10 mL/min) to give intermediate 77 (184 mg, 599.70 μmol, 89.56%yield, 99.87%purity) as a colorless oil.

Preparation of intermediate 79

To a solution of intermediate 22 (500 mg, 1.88 mmol) in DMF (10 mL) were added Cs₂CO₃ (1.84 g, 5.65 mmol) and MeI (267.48 mg, 1.88 mmol, 117.32 μL) . The mixture was stirred at 0 ℃ for 1 hr. The reaction mixture was diluted with H₂O (50 mL) and extracted with EtOAc (30 mL *3) . The combined organic layers were washed with sat. LiCl (50 mL *3) , dried over Na₂SO₄, filtered, and concentrated under reduced pressure to get a residue, which was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0%B to 80%B in A, 20 mL/min. ) to give intermediate 79 (306 mg, 1.05 mmol, 55.85%yield, 96.09%purity) as a colorless oil.

Preparation of intermediate 81

To a solution of 4-bromo-2-fluorophenol (2.9 mL, 26.18 mmol) in DCM (50 mL) were added TEA (5.5 mL, 39.27 mmol) and acetyl chloride (2.3 mL, 31.41 mmol) at 0 ℃. The reaction mixture was stirred for 2 hr at room temperature. The reaction mixture was diluted with water (50 mL) and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (EA in PE = 0-30 %) to afford intermediate 81 (5.8 g, yield: 95.07 %) as a yellow liquid.

The following intermediate was synthesized by an analogous method as described above for intermediate 81.

Preparation of intermediate 82

A mixture of intermediate 81 (3.8 g, 16.31 mmol) and AlCl₃ (8.70 g, 65.23 mmol) was heated to 130 ℃. The reaction mixture was stirred for 3 hr at 130 ℃. The reaction mixture was cooled down to room temperature and diluted with DCM (20 mL) . The reaction mixture was poured into ice water (30 mL) and filtrated. The filtrate was extracted with DCM (20 mL x 3) and the combined organic layers were washed with brine, dried over anhydrous sodium sulphate. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by flash chromatography (EA in PE = 0-20 %) to give intermediate 82 (2.2 g, yield: 57.89 %) as a yellow solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 82.

Preparation of intermediate 88

To a solution of intermediate 87 (710 mg, 2.18 mmol) in DCM (20 mL) was added Boc₂O (1.398 mL, 6.53 mmol) , DMAP (53.24 mg, 0.44 mmol) and TEA (879.63 mg, 8.71 mmol) at room temperature. The reaction mixture was stirred for 1 hr. The reaction was diluted with water (20 mL) . The reaction mixture was extracted with DCM (20 mL x 3) and the combined organic layers were washed with brine, dried over anhydrous sodium sulphate. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by flash chromatography (EA in PE = 0-30 %) to give intermediate 88 (430 mg, yield: 37.52 %) as a yellow gum.

Preparation of intermediate 89

To a solution of intermediate 88 (430 mg, 0.82 mmol) in dioxane (20 mL) was added 4, 4, 5, 5-tetramethyl-2- (tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (249 mg, 0.98 mmol) , potassium acetate (160 mg, 1.63 mmol) and Pd (dppf) Cl₂ (60 mg, 0.082 mmol) . The reaction mixture was heated to 90 ℃ under nitrogen atmosphere and stirred for 16 hr. The reaction was diluted with water (20 mL) . The reaction mixture was extracted with EtOAc (20 mL x 3) and the combined organic layers were washed with brine, dried over anhydrous sodium sulphate. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by flash chromatography (EA in PE = 0-30 %) to give intermediate 89 (330 mg, yield: 70.44 %) as a yellow solid.

Preparation of intermediate 90

To a solution of intermediate 89 (330 mg, 0.58 mmol) in MeOH (20 mL) was added urea hydrogen peroxide (135 mg, 1.44 mmol) at room temperature. The reaction mixture was stirred for 4 hr. The reaction was diluted with water (20 mL) . The reaction mixture was extracted with EtOAc (20 mL x 3) and the combined organic layers were washed with brine, dried over anhydrous sodium sulphate. The mixture was filtered, and the filtrate was evaporated under reduced pressure to give intermediate 90 (230 mg, yield: 86.24 %) , which was used for next step and without further purification.

Preparation of intermediate 91

To a solution of intermediate 90 (230 mg, 0.50 mmol) in DMF (10 mL) was added iodomethane (211 mg, 1.49 mmol) and K₂CO₃ (206 mg, 1.49 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hr. The reaction was diluted with water (20 mL) . The reaction mixture was extracted with EtOAc (20 mL x 3) and the combined organic layers were washed with brine, dried over anhydrous sodium sulphate. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by flash chromatography (EA in PE = 0-30 %) to give intermediate 91 (120 mg, yield: 50.67 %) as a yellow solid.

Preparation of intermediate 92

A solution of intermediate 91 (120 mg, 0.25 mmol) in HCl/EtOAc (5 mL) was stirred for 2 hr at room temperature. The reaction mixture was concentrated in vacuum. The residue was dissolved in EtOAc and washed with Na₂CO₃ solution. The organic layer was concentrated in vacuum. The residue was purified by flash chromatography (EA in PE = 0-30 %) to afford intermediate 92 (50 mg, yield: 71.76 %) as a white solid.

Preparation of intermediate 99

To a stirred solution of intermediate 98 (800 mg, 2.47 mmol) in dioxane (20 mL) was added 4, 4, 5, 5-tetramethyl-2- (tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (937 mg, 3.69 mmol) , potassium acetate (126 mg, 1.29 mmol) and Pd (dppf) Cl₂ (59 mg, 0.082 mmol) . The reaction mixture was heated to 90 ℃ under nitrogen atmosphere and stirred overnight. The reaction was diluted with water (20 mL) . The reaction mixture was extracted with EtOAc (20 mL x 3) and the combined organic layers were washed with brine, dried over anhydrous sodium sulphate. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by flash chromatography (EA in PE = 0-30%) to afford intermediate 99 (560 mg, 61.18%) as a white solid.

Preparation of intermediate 100

To a solution of intermediate 99 (110 mg, 0.38 mmol) in EtOH (10 mL) was added urea hydrogen peroxide (104 mg, 0.76 mmol) at 0 ℃ and stirred for 2 hr. The reaction mixture was diluted with water and extracted with DCM twice. The combined organic layers were washed with brine, dried over anhydrous sodium sulphate, filtered, and concentrated to give a residue, which was purified by flash chromatography (EA in PE = 0-30%) to afford intermediate 100 (70 mg, yelid: 64.70%) as a yellow solid.

Preparation of intermediate 101

To a solution of intermediate 100 (50 mg, 0.191 mmol) in DMF (10 mL) was added iodomethane (41 mg, 0.29 mmol) and K₂CO₃ (53 mg, 0.38 mmol) . The reaction mixture was stirred at r. t. for 16 hr under a nitrogen atmosphere. The reaction mixture was diluted with water and extracted with DCM (10 ml x 3) , the combined organic layers were washed with brine, dried over anhydrous sodium sulphate, filtered, and concentrated to give a residue, which was purified by flash chromatography (EA in PE = 0-30%) to give intermediate 101 (40 mg, 75.93%) as a yellow solid.

Preparation of intermediate 105

To a solution of intermediate 24 (90 mg, 0.37 mmol) in DCM (1.5 mL) was added TFA (1.5 mL) . The reaction mixture was stirred at r. t. for 3 hr. The reaction mixture was concentrated under reduced pressure to afford intermediate 105 (50 mg, crude) , which was used directly for next step without further purification.

Preparation of intermediate 106

To a solution of intermediate 105 (50 mg, 0.34 mmol) and 2-chloro-5-nitropyrimidine (55 mg, 0.34 mmol) in EtOH (5 mL) was added DIEA (133 mg, 1.03 mmol) . The reaction mixture was stirred at 80 ℃ for 3 hr. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography on silica gel eluted with DCM/MeOH (25/1) to afford intermediate 106 (88 mg, yield: 95.27%) as a yellow solid.

Preparation of intermediate 107

To a solution of intermediate 106 (88 mg, 0.33 mmol) in CH₃CN (4 mL) was added HCl (1 mL, 1 N) and NCS (175 mg, 1.31 mmol) at 0 ℃ and stirred at r. t. for 1 hr. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (50 mL x 2) . The combined organic layers were washed with saturated NaHCO₃ and brine. The organic layer was dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure to afford intermediate 107 (90 mg, crude) as a light-yellow solid, which was used for the next step without further purification.

Preparation of intermediate 108

To a solution of 2-methoxyethan-1-amine (46 mg, 0.62 mmol) in DCM (4 mL) was added pyridine (49 mg, 0.62 mmol) and intermediate 107 (90 mg, 0.31 mmol) . The reaction mixture was stirred at r. t. for 2 hr. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography on silica gel eluted with DCM/MeOH (10/1) to afford intermediate 108 (60 mg, yield: 58.89%) as a white solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 108.

Preparation of intermediate 109

To a solution of intermediate 108 (60 mg, 0.18 mmol) in MeOH (5 mL) was added Pd/C (19 mg, 0.18 mmol) . The reaction mixture was stirred at r. t. for 3 hr under H₂ atmosphere. The reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give intermediate 109 (54 mg, crude) as a yellow solid, which was used directly for next step without further purification.

The following intermediate was synthesized by an analogous method as described above for intermediate 109.

Preparation of intermediate 110

To a solution of intermediate 9 (500 mg, 1.886 mmol) , DIEA (0.312 mL, 1.886 mmol) in THF (10 mL) was added phenyl chloroformate (0.237 mL, 1.886 mmol) at 0 ℃. The reaction mixture was stirred at r. t. for 30 min. The reaction mixture was concentrated to give intermediate 110 (crude) as white solid, which was used in the next step without further purification.

The following intermediate was synthesized by an analogous method as described above for intermediate 110.

Preparation of intermediate 113

To a solution of 5, 7-difluoro-3-methyl-1-benzofuran (200 mg, 1.19 mmol) in DCM (5 mL) were added cyclobutanecarbonyl chloride (169 mg, 1.43 mmol) and AlCl₃ (238 mg, 1.78 mmol) at 0 ℃. The reaction mixture was stirred at r. t. for 2 hr under nitrogen atmosphere. The reaction mixture was quenched with water. The reaction mixture was extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford a residue, which was purified by silica gel column chromatography (PE: EA = 20: 1) to afford intermediate 113 (271 mg, yield: 91.04%) as a white solid.

The following intermediate was synthesized by an analogous method as described above for intermediate 113.

Preparation of intermediate 121

To a solution of 1- (3, 5-difluoro-2-hydroxyphenyl) ethan-1-one (3 g, 17.43 mmol) in DCM (30 mL) was added TEA (2.9 mL, 20.91 mmol) and slowly added Tf₂O (2.9 mL, 17.43 mmol) at 0 ℃. The reaction mixture was stirred at r. t. for 3 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel eluted with PE: EA (10: 1) to afford intermediate 121 (4.3 g, yield: 81.11%) as a yellow oil.

Preparation of intermediate 122

To a solution of intermediate 121 (4.3 g, 14.14 mmol) and (4-methoxyphenyl) methanethiol (1.9 mL, 14.14 mmol) in dioxane (40 mL) was added Xant Phos (0.41 g, 0.71 mmol) , Pd₂ (dba) ₃ (0.65 g, 0.71 mmol) and DIEA (4.7 mL, 28.27 mmol) . The reaction mixture was stirred at 110℃ for 2 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel eluted with PE: EA (10: 1) to afford intermediate 122 (4 g, yield: 91.77%) as a yellow oil.

Preparation of intermediate 123

To a solution of intermediate 122 (4 g, 12.97 mmol) in toluene (10 mL) was added AlCl₃ (3.46 g, 25.96 mmol) at 0 ℃. The reaction mixture was stirred at 0℃ for 18 hr. The mixture was poured into ice water and extracted with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure to give a residue, which was purified by silica gel column chromatography eluted with PE/EA = 10: 1 to afford intermediate 123 (1.5 g, yield: 61.44%) as a yellow oil.

Preparation of intermediate 124

To a solution of intermediate 123 (1.5 g, 7.97 mmol) and 1-bromo-3-methylbutan-2-one (1.1 mL, 8.77 mmol) in MeCN (20 mL) was added K₂CO₃ (3.30 g, 23.91 mmol) . The reaction mixture was stirred at 80℃ for 18 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel eluted with PE: EA (20: 1) to afford intermediate 124 (1.5 g, yield: 74.01%) as a yellow solid.

Preparation of intermediate 128

To a solution of 2, 3, 5-trifluorobenzoic acid (5 g, 28.39 mmol) in DMSO (50 mL) was added NaOH (1.82 g, 45.43 mmol) . The mixture was stirred at 130 ℃ for 18 hr. The reaction mixture was poured into ice water (200 mL) , and adjusted to pH = 1 with 1 N HCl. The mixture was extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous sodium sulphate. The mixture was filtered and the filtrate was evaporated under reduced pressure to give intermediate 128 (4.8 g, yield: 97.10%) as a yellow solid, which was used for the next step without further purification.

Preparation of intermediate 129

To a solution of intermediate 128 (4.6 g, 26.42 mmol) in MeOH (60 mL) was added H₂SO₄ (10 mL, 28.15 mmol) . The mixture was stirred for 4 hr under reflux condition. The reaction mixture was poured into ice water (200 mL) and extracted with EtOAc (100 mL x 2) . The combined organic layers were dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure to give a residue, which was purified by column chromatography on silica gel eluted with PE: EA (5: 1) to afford intermediate 129 (3 g, yield: 60.35%) as a white solid.

Preparation of intermediate 130

To a solution of intermediate 129 (1.7 g, 9.03 mmol) in MeCN (25 mL) was added K₂CO₃ (2.50 g, 18.07 mmol) and 1-bromo-3-methylbutan-2-one (1.2 mL, 10.84 mmol) . The mixture was stirred at 80 ℃ for 3 hr. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to afford intermediate 130 (2.4 g, yield: 97%) as a yellow solid, which was used for the next step without further purification.

Preparation of intermediate 131

To a solution of intermediate 130 (2.4 g, 8.81 mmol) in THF (15 mL) was added t-BuOK (13.2 mL, 13.22 mmol, 1N in THF) under N₂ atmosphere. The reaction mixture was stirred at 0 ℃ for 2 hr. The reaction mixture was quenched with NH₄Cl aq. solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography PE: EA (5: 1) to afford intermediate 131 (1.8 g, yield: 85.00%) as a brown solid.

Preparation of intermediate 132

To a solution of intermediate 131 (500 mg, 2.08 mmol) in acetone (10 mL) was added K₂CO₃ (863 mg, 6.24 mmol) and dimethyl sulfate (0.3 mL, 2.70 mmol) . The mixture was stirred at 60 ℃ for 6 hr. The reaction mixture was filtered and concentrated in vacuo to give a residue, which was purified by column chromatography on silica gel eluted with PE/EA (5/1) to afford intermediate 132 (450 mg, yield: 85.07%) as a yellow solid.

Preparation of intermediate 140

To a solution of 2- (1-tert-butoxycarbonyl-3-methyl-azetidin-3-yl) acetic acid (150 mg, 654.24 μmol) in DMF (5 mL) was added DIEA (422.78 mg, 3.27 mmol, 569.79 μL) , methanamine hydrochloride (66.26 mg, 981.37 μmol) and HATU (497.53 mg, 1.31 mmol) . The solution was stirred at 50 ℃ for 1 hr. The reaction mixture was diluted with H₂O (30 mL) and extracted with EtOAc (20 mL x 3) . The combined organic layers were washed with sat. LiCl (50 mL x 2) , dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to get a residue, which was purified by flash column chromatography silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0%B to 70%B in A, 10 mL/min) to give intermediate 140 (240 mg, crude) as a brown oil.

Preparation of intermediate 142

To a solution of tert-butyl 3-formylazetidine-1-carboxylate (350 mg, 1.89 mmol) and 1-piperazin-1-ylethanone (242.20 mg, 1.89 mmol) in MeOH (4 mL) was added AcOH (226.95 mg, 3.78 mmol, 216.35 μL) . The mixture was stirred at 25 ℃ for 15 min before NaBH₃CN (237.49 mg, 3.78 mmol) was added. The reaction mixture was stirred at 40 ℃ for 1 hr. The reaction mixture was diluted with dichloromethane (30 mL) , basified to pH=8 with the saturated solution of sodium bicarbonate (30 mL) and then the mixture was diluted with H₂O (20 mL) and extracted with dichloromethane (30 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO₂, DCM/MeOH=1/0 to 10/1) to afford intermediate 142 (550 mg, 1.85 mmol, 97.87%yield, 100%purity) as a white solid.

The following intermediates were synthesized by an analogous method as described above for intermediate 142.

Preparation of intermediate 143

A solution of intermediate 142 (550 mg, 1.85 mmol) in HCl/dioxane (5 mL) was stirred at 25 ℃ for 1 hr. The reaction mixture was concentrated under reduced pressure to give intermediate 143 (550 mg, crude, HCl salt) , which was used for next step without further purification.

The following intermediates were synthesized by an analogous method as described above for intermediate 143.

Preparation of intermediate 146

To a solution of 2-chloro-5-nitro-pyrimidine (1 g, 6.27 mmol) and azetidin-3-ylmethanol hydrochloride (774.65 mg, 6.27 mmol) in DMF (10 mL) was added DIEA (4.05 g, 31.34 mmol, 5.46 mL) . The resulting mixture was stirred at 50 ℃ for 8 hr. Water (80 mL) was added and the mixture was extracted with ethyl acetate (60 mL x 3) . The combined organic layers were washed with the saturated solution of lithium chloride (100 mL x 3) . The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue, which was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 0/1) to afford intermediate 146 (960 mg, 4.52 mmol, 72.08%yield, 98.92%purity) as a yellow solid.

Preparation of intermediate 147

To a stirred solution of intermediate 146 (300 mg, 1.43 mmol) in anhydrous DCM (6 mL) at 0 ℃ was added DMP (908.06 mg, 2.14 mmol, 663.30 μL) . The reaction mixture was stirred at 25 ℃ for 8 h. The resulting mixture was diluted with DCM (50 mL) . The mixture was washed with sat. Na₂S₂O₃ (30 mL x 2) . The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 0/1) to afford intermediate 147 (200 mg, 864.66 μmol, 60.58%yield, 90%purity) as a yellow solid.

Preparation of intermediate 148

To a solution of intermediate 147 (150 mg, 720.55 μmol) in MeOH (2 mL) were added 2-oxa-6-azaspiro [3.3] heptane (85.71 mg, 864.66 μmol) and AcOH (86.54 mg, 1.44 mmol, 82.50 μL) . The mixture was stirred at 25 ℃ for 10 min before addition of NaBH₃CN (90.56 mg, 1.44 mmol) was. The reaction mixture was stirred at 25 ℃ for 40 min. The reaction mixture was diluted with dichloromethane (40 mL) , basified to pH=8 with the saturated solution of sodium bicarbonate (30 mL) and extracted with dichloromethane (20 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 10/1) to afford intermediate 148 (150 mg, 514.92 μmol, 48.36%yield, 90%purity) as a yellow solid.

Preparation of intermediate 149

To a solution of intermediate 148 (70 mg, 240.30 μmol) in EtOH (0.5 mL) , H₂O (0.2 mL) and THF (0.5 mL) were added Fe (67.10 mg, 1.20 mmol) and NH₄Cl (25.71 mg, 480.60 μmol) . The reaction mixture was stirred at 80 ℃ for 1 hr. The reaction was quenched with H₂O (30 mL) and extracted with EtOAc (20 mL x 3) . The combined organic layers were washed with saturated NaCl (50 mL) , dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a crude product, which was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) to afford intermediate 149 (40 mg, 130.11 μmol, 54.14%yield, 85%purity) as a yellow oil.

Preparation of intermediate 155

To a solution of tert-butyl 3-acetylazetidine-1-carboxylate (1 g, 5.02 mmol) in THF (10 mL) was added morpholine (218.62 mg, 2.51 mmol, 220.83 μL) and Ti (i-PrO) ₄ (4.28 g, 15.06 mmol, 4.44 mL) . The reaction mixture was stirred at 50 ℃ for 3 h. Then the reaction mixture was cooled down to 25 ℃ and NaBH (OAc) ₃ (3.19 g, 15.06 mmol, 3 eq) was added and stirred at 25 ℃ for 1 h. The reaction mixture was diluted with dichloromethane (80 mL) , basified to pH=8 with the saturated solution of sodium bicarbonate (100 mL) and then the mixture was extracted with dichloromethane (60 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO₂, DCM/MeOH=1/0 to 10/1) to afford intermediate 155 (1 g, crude) as a colorless oil.

Preparation of intermediate 177

To the mixture of intermediate 174 (100 mg, 0. 448 mmol) in EtOH (15 mL) was added 2-chloro-5-nitropyrimidine (78 mg, 0.493 mmol) and TEA (0.2 mL, 1.34 mmol) at 0℃. The reaction mixture was stirred at 80℃ overnight. The reaction mixture was diluted with water, and the resulting mixture was extracted with EA, washed with brine, dried over by Na₂SO₄, filtered, and concentrated in vacuum to give intermediate 177 (120 mg, yield: 77.36%) as a yellow solid.

The following intermediates were synthesized by an analogous method as described above for intermediate 177.

Preparation of intermediate 178

The mixture of intermediate 177 (100 mg, 0.289 mmol) and Pd/C 10% (10 mg) in MeOH (25 mL) was stirred at room temperature for 1 hour under H₂ atmosphere. The reaction mixture was filtered and concentrated in vacuum to give intermediate 178 (80 mg, yield: 87.58%) as yellow solid.

The following intermediates were synthesized by an analogous method as described above for intermediate 178.

Preparation of intermediate 189

To the mixture of tert-butyl 3-oxopiperazine-1-carboxylate (334 mg, 1.7mmol) in DMF (10 mL) was added NaH (80 mg, 2.0 mmol) at 0 ℃. The reaction mixture was stirred at this temperature for 1 hr before the addition of benzyl 3- ( ( (methylsulfonyl) oxy) methyl) azetidine-1-carboxylate (500 mg, 1.7 mmol) . The reaction mixture was stirred at room temperature overnight. The reaction was quenched with water, and the resulting mixture was extracted with EA, washed by brine, dried over by Na₂SO₄, filtered, and concentrated in vacuum to give a residue, which was purified by silica gel column chromatography (DCM/MeOH = 4/1) to give intermediate 189 (223 mg, yield: 33.1%) as colorless oil.

The following intermediate was synthesized by an analogous method as described above for intermediate 189.

Preparation of intermediate 190

A mixture of intermediate 189 (223 mg, 0.55 mmol) , Pd/C 10% (59 mg, 0.06 mmol) in MeOH (20 mL) was stirred at room temperature for 1 hour under H₂ atmosphere. The reaction mixture was filtered and concentrated in vacuum to give intermediate 190 (82 mg, yield: 55.0%) as colorless oil.

The following intermediate was synthesized by an analogous method as described above for intermediate 190.

Preparation of intermediate 193

To the mixture of intermediate 150 (50 mg, 0.13 mmol) in DMF (4 mL) was added DIEA (46 mg, 0.36 mmol) and intermediate 192 (44 mg, 0.12 mmol) . The reaction mixture was stirred at room temperature overnight and concentrated in vacuum to give a residue, which was purified by silica column chromatography (DCM/MeOH = 4/1) to give intermediate 193 (42 mg, yield: 49.5%) as a white solid.

Preparation of Compounds

Preparation of Compound 1

To a solution of intermediate 12 (300 mg, 744.92 μmol) in DMSO (5 mL) were added TEA (1.88 g, 18.62 mmol, 2.59 mL) and 3- (methylsulfonylmethyl) azetidine hydrochloride (207.46 mg, 1.12 mmol) . The mixture was stirred at 80 ℃ for 16 hr. The reaction mixture was cooled to room temperature. H₂O (30 mL) was added, and the mixture was extracted with EtOAc (20 mL x 3) . The combined organic layers were washed with H₂O (20 mL *3) , dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (Petroleum Ether/Ethyl Acetate=1/0 to 0/1) . The pure fractions were collected, and the solvent was evaporated under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL) . The mixture was lyophilized to dryness to give Compound 1 (130 mg, 239.96 μmol, 32.21%yield) as a yellow solid.

Preparation of Compound 2 &3

Compound 1 was separated by SFC (column: (s, s) WHELK-O1 (250mm*30mm, 5um) ; mobile phase: [CO₂-i-PrOH (0.1%NH₃H₂O) ] ; B%: 50%, isocratic elution mode) . The pure fractions were collected, and the solvent was evaporated under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL) . The mixture was lyophilized to dryness to give the desired compound. The first fraction was obtained as Compound 2 (43.31 mg, 84.02 μmol, 43.31%yield) , and the second fraction was obtained as Compound 3 (41.26 mg, 78.68 μmol, 40.56%yield) .

Preparation of Compound 4

To a solution of intermediate 11 (200 mg, 475.37 μmol) in DMSO (2 mL) was added TEA (144.31 mg, 1.43 mmol, 198.50 μL) and 3- (methylsulfonylmethyl) azetidine hydrochloride (176.53 mg, 950.75 μmol) . The mixture was stirred at 80 ℃ for 16 hr. The reaction mixture was diluted with H₂O 20 mL and extracted with EtOAc (10 mL x 3) . The combined organic layers were washed with aqueous NaCl 15 mL, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA) -ACN] ; gradient: 32%-62%B over 10 min) . The pure fractions were collected, and the solvent was evaporated under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL) . The mixture was lyophilized to dryness to give Compound 4 (80.8 mg, 151.46 μmol, 31.86%yield) as an off-white solid.

Preparation of Compound 5 &6

Compound 4 was separated by SFC, basic condition; column: (s, s) WHELK-O1 (250mm*30mm, 10um) ; mobile phase: [CO₂-EtOH (0.1%NH₃H₂O) ] ; B%: 35%, isocratic elution mode. The pure fractions were collected, and the solvent was evaporated under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL) . The mixture was lyophilized to dryness to give the desired compound. The first fraction was collected as Compound 5 (38.94 mg, 71.28 μmol, 25.35%yield) , and the second fraction was collected as Compound 6 (42.56 mg, 77.33 μmol, 27.50%yield) .

The following Compounds were synthesized by an analogous method as described above for Compound 4.

Preparation of Compound 18

To a solution of intermediate 57 (120 mg, 249.27 μmol) and methylamine (33.66 mg, 498.55 μmol, 2 eq, HCl) in DCM (3 mL) was added HATU (142.17 mg, 373.91 μmol) and DIEA (96.65 mg, 747.82 μmol, 130.25 μL) . The mixture was stirred at 0-25 ℃ for 2 hr. The mixture was concentrated to afford the crude product. The crude was purified by flash chromatography (4gSilica Flash Column, EtOAc of 100%, PE/EtOAc @40mL/min) to afford the product. The product was further purified by prep. HPLC (Column: Waters xbridge 150*25mm 10um, Mobile Phase A: [water (NH₄HCO₃) -ACN] , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 30%B to 60%) . The pure fractions were collected, and the volatiles were removed under vacuum. The residue was partitioned between ACN (40 mL) and water (20 mL) . The solution was lyophilized to afford Compound 18 (2.99 mg, 5.98 μmol, 2.40%yield) as a white solid.

The following Compound was synthesized by an analogous method as described above for Compound 18.

Preparation of Compound 26

To a solution of intermediate 65 (130 mg, 307.06 μmol) in DCM (5 mL) was added a solution of N, N-dimethylcarbamoyl chloride (29.72 mg, 276.36 μmol, 25.36 μL) in DCM (5 mL) dropwise. The reaction mixture was stirred at 25 ℃ for 1 h. H₂O (30 mL) was added, the mixture was extracted with ethyl acetate (30 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge C18 150*50mm*10um; mobile phase: [water (NH₃H₂O) -ACN] ; gradient: 32%-62%B over 11 min) to afford Compound 26 (9.58 mg, 18.29 μmol, 97.09%purity) as white solid.

Preparation of Compound 47

To a solution of Compound 39 (60 mg, 106.48 μmol) and TEA (32.32 mg, 319.43 μmol, 44.46 μL) in DCM (3 mL) was added MsCl (200 mg, 1.75 mmol, 135.14 μL) at 0 ℃ and the mixture was stirred at 25 ℃ for 2 hr. The reaction mixture was quenched by H₂O (20 mL) under 0 ℃, and extracted with DCM (15 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to get a residue, which was purified by preparative-HPLC (Column: Waters xbridge (150 *25 mm 5 um) , Mobile Phase A: water (NH₃·H₂O) , Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient condition from 34%B to 64%) . The pure fractions were collected, and the solvent was evaporated under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL) . The mixture was lyophilized to dryness to give Compound 47 (30 mg, 53.80 μmol, 50.53%yield, 97.83%purity) as a white solid.

Preparation of Compound 52

To a solution of phenyl intermediate 110 (70 mg, 0.18 mmol) in pyridine (5 mL) was added intermediate 109 (54 mg, 0.18 mmol) . The reaction mixture was stirred at 80℃ for 2 h. The reaction was concentrated under reduced pressure to give a residue, which was purified by column chromatography on silica gel eluted with DCM/MeOH (15/1) to afford a crude product. The crude product was further purified by prep-HPLC (GiLSON-Xbridge C18 (5 μm 19 *150 mm) , Mobile Phase A: Water (0.1%NH₄CO₃) , Mobile Phase B: acetonitrile, UV: 214 nm, Flowrate: 15 mL /min, Temperature: rt, Gradient: 25 -70% (%B) ) to afford Compound 52 (16.1 mg, yield: 14.79%) as a white solid.

The following Compound was synthesized by an analogous method as described above for Compound 52.

Preparation of Compound 60 and 61

To a solution of intermediate 137 (150 mg, 894.81 μmol) in MeOH (2 mL) was added TEA (727.00 mg, 7.18 mmol, 1 mL) and intermediate 11b (100 mg, 237.69 μmol) . The mixture was stirred at 40 ℃ for 16 hr. The reaction mixture was cooled down to room temperature and diluted with H₂O 40 mL and extracted with EtOAc (20 mL x 3) . The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 0/1) to afford the racemic product, which was further separated by SFC (column: DAICEL CHIRALPAK IK (250 mm*30 mm, 10 um) ; mobile phase: [CO₂-MeOH (0.1%NH₃H₂O) ] ; B%: 30%, isocratic elution mode) . The first fraction was collected as Compound 60 (52.23 mg, 100.92 μmol, 42%yield, 99.59%purity) as a white solid and the second fraction was collected as Compound 61 (50.28 mg, 97.30 μmol, 40%yield, 99.74%purity) as a white solid.

The following Compounds were synthesized by an analogous method as described above for Compound 60.

Preparation of Compound 68

To a solution of intermediate 143 (200 mg, 855.66 μmol, HCl salt) and intermediate 11b (100 mg, 237.69 μmol) in MeOH (1 mL) was added TEA (24.05 mg, 237.69 μmol, 33.08 μL) . The mixture was stirred at 40 ℃ for 12 hr. The reaction mixture was cooled down to room temperature and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, EtOAc: MeOH=1/0 to 10/1) to afford Compound 68 (40 mg, 67.64 μmol, 28.46%yield, 98.34%purity) as a white solid.

The following Compounds were synthesized by an analogous method as described above for Compound 68.

Preparation of Compound 69

To a solution of intermediate 149 (40 mg, 153.07 μmol) and intermediate 150 (58.97 mg, 153.07 μmol) in THF (3 mL) was added TEA (46.47 mg, 459.20 μmol, 63.92 μL) . The reaction mixture was stirred at 25 ℃ for 30 min. The reaction mixture was quenched with H₂O (30 mL) and extracted with EtOAc (20 mL x 3) . The combined organic layers were washed with saturated NaCl (50 mL) , dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a crude product, which was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) to give the pure fractions and the solvent was evaporated under vacuum. The residue was partitioned between CH₃CN (2 mL) and water (10 mL) . The mixture was lyophilized to dryness to give Compound 69 (5.04 mg, 8.74 μmol, 5.71%yield, 95.76%purity) as a white solid.

The following Compounds were synthesized by an analogous method as described above for Compound 69.

Preparation of Compound 84

To the mixture of intermediate 193 (42 mg, 0.06 mmol) in DCM (2 mL) was added TFA (2 mL) at 0 ℃. The reaction mixture was stirred at room temperature for 2 hours. And then it was evaporated in vacuum to give a residue. The residue was diluted with NaHCO₃ aqueous and extracted with DCM. The combined organic layers were washed with brine and evaporated to give a residue, which was purified by C18 column (Column: spherical C18, 20-35 μm, 25 g, Mobile Phase A: water, Mobile Phase B: acetonitrile, gradient condition from 20%B to 60%B) to give Compound 84 (5.8 mg, yield: 16.7%) as a white solid.

LCMS (Liquid chromatography/Mass spectrometry)

General procedure

The High-Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods. Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g., scanning range, dwell time…) in order to obtain ions to allow the identification of the compound’s nominal monoisotopic molecular weight (MW) . Data acquisition was performed with appropriate software.

Compounds are described by their experimental retention times (Rt) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M+H] ⁺ (protonated molecule) and/or [M-H] ^- (deprotonated molecule) . All results were obtained with experimental uncertainties that are commonly associated with the method used. Method 1

Mobile phase: Ramp from 30%ACN (0.018%TFA) in water (0.037%TFA) to 90%ACN in 2.00 min, Flow rate is set at 1.5 mL/min; then ramp from 90%ACN in water to 100%ACN in 1.70 min. Flow rate is set at 1.5 mL/min; return to 30%ACN in water and hold for 0.30 min. Flow rate is set at 2.0 mL/min. Column temperature at 50℃ and detector wavelength from 210 nm to 265 nm. The column is ofEVO C18 4.6 x 50 mm, 5 μm.

Method 2

Mobile phase: Ramp from 5%ACN (0.01875%TFA) in water (0.0375%TFA) to 95%ACN in 2.40 min, Flow rate is set at 2.0 mL/min; then hold at 95%ACN for 0.30 minutes. Flow rate is set at 2.0 mL/min; return back to 5%ACN in water and hold for 0.30 min. Flow rate is set at 2.0 mL/min. Column temperature at 50℃. The column is of EVO C18 4.6x50mm, 5 μm.

Method 3

Mobile phase: Ramp from 5%ACN (0.01875%TFA) in water (0.0375%TFA) to 95%ACN in 3.20 min, Flow rate is set at 1.5 mL/min; then hold at 95%ACN for 0.30 minutes. Flow rate is set at 1.5 mL/min; return to 5%ACN in water and hold for 0.30 min. Flow rate is set at 2.0 mL/min. Column temperature at 50℃. The column is of EVO C18 4.6 x 50 mm, 5 μm.

Method 4

Mobile phase: Ramp from 5%ACN in water (0.025%NH₃·H₂O) to 95%ACN in 3.00 min, Flow rate is set at 0.6 mL/min; then hold at 95%ACN for 0.70 minutes Flow rate is set at 0.6 mL/min; return to 5%ACN in water and hold for 0.30 min. Flow rate is set at 1.2 mL/min. Column temperature at 40℃ and detector wavelength from 210 nm to 265 nm. The column isXBridge C18 2.1 x 30 mm, 3.5 μm.

Method 5

Mobile phase: Ramp from 5%ACN (0.01875%TFA) in water (0.0375%TFA) to 95%ACN in 4.8 min, Flow rate is set at 0.6 mL/min; then hold at 95%ACN for 0.60 minutes. Flow rate is set at 1.0 mL/min; return to 5%ACN in water and hold for 0.60 min. Flow rate is set at 1.0 mL/min. Column temperature at 50 ℃. The column is Kinetex EVO C18 2.1*50mm, 1.7 μm.

Method 6

Mobile phase: Ramp from 5%ACN (0.01875%TFA) in water (0.0375%TFA) to 95%ACN in 3.20 min, Flow rate is set at 1.5 mL/min; then hold at 95%ACN for 0.30 minutes. Flow rate is set at 1.5 mL/min; return to 5%ACN in water and hold for 0.30 min. Flow rate is set at 2.0 mL/min. Column temperature at 50℃. The column is of EVO C18 4.6 x50 mm, 5 μm.

Method 7

Mobile phase: Ramp from 5%ACN (0.01875%TFA) in water (0.0375%TFA) to 95%ACN in 2.40 min, Flow rate is set at 2.0 mL/min; then hold at 95%ACN for 0.30 minutes Flow rate is set at 2.0 mL/min; return to 5%ACN in water and hold for 0.30 min. Flow rate is set at 2.0 mL/min. Column temperature at 50 ℃. The column is of EVO C18 4.6 x 50 mm, 5 μm.

Method 8

Mobile phase: Ramp from 5%ACN (0.01875%TFA) in water (0.0375%TFA) to 95%ACN in 3.20 min, Flow rate is set at 1.5 mL/min; then hold at 95%ACN for 0.30 minutes. Flow rate is set at 1.5 mL/min; return to 5%ACN in water and hold for 0.30 min. Flow rate is set at 2.0 mL/min. Column temperature at 50 ℃. The column is of EVO C18 4.6 x 50 mm, 5 μm.

Method 9

Mobile phase: Ramp from 5%ACN (0.018%TFA) in water (0.037%TFA) to 95%ACN in 3.0 min, Flow rate is set at 1.0 mL/min; then hold at 95%ACN for 0.60 minutes. Flow rate is set from 1.0 mL/min to 1.5 mL/min; return to 5%ACN in water and hold for 0.40 min. Flow rate is set at 1.5 mL/min. Column temperature at 50℃. The column is of Shim-pack Velox SP-C18 3.0 x 30 mm, 2.7 μm.

Method 10

Mobile phase: Ramp from 5%ACN in water (0.025%NH₃·H₂O) to 95%ACN in 2.60 min, Flow rate is set at 0.6 mL/min; then hold at 95%ACN for 0.25 minutes. Flow rate is set at 0.8 mL/min; return to 5%ACN in water and hold for 0.15 min. Flow rate is set at 1.2 mL/min. Column temperature at 40℃ and detector wavelength from 210 nm to 265 nm. The column is ofXBridge C18 2.1 x 30 mm, 3.5 μm.

Method 11

Mobile phase: Ramp from 5%ACN (0.01875%TFA) in water (0.0375%TFA) to 95%ACN in water in 0.60 min, Flow rate is set at 2.0 mL/min; then hold at 95%ACN for 0.18 minutes. Flow rate is set at 2.0 mL/min; return back to 5%ACN in water and hold for 0.02 min. Flow rate is set at 2.0 mL/min. Column temperature at 50℃. The column is ofEVO C18 2.1 x 30 mm, 5 μm.

Method 12

Mobile phase: Ramp from 5%ACN in water (0.025%NH₃·H₂O) to 95%ACN in 3.00 min, Flow rate is set at 0.9 mL/min; then hold at 95%ACN for 0.70 minutes. Flow rate is set at 0.9 mL/min; return to 5%ACN in water and hold for 0.30 min. Flow rate is set at 1.2 mL/min. Column temperature at 40℃ and detector wavelength from 210 nm to 265 nm. The column is ofXBridge C18 3.0 x 50 mm, 5 μm.

Analytical data

The LCMS analytical information listed in Table 3 below.

Table 3

NMR Methods

NMR experiments were carried out using a Bruker Advance III 400 spectrometer at ambient temperature (298.6 K) , using internal deuterium lock, and equipped with BBO 400 MHz S1 5 mm probe head with z gradients and operating at 400 MHz for the proton and 100 MHz for carbon. Chemical shifts (δ) are reported in parts per million (ppm) . J values are expressed in Hz.

The NMR analytical information in the Tables below.

Biochemical PI3Kα (PIK3CA/PIK3R1) kinase assay

PI3Kα kinase activity and the determination of inhibitors IC₅₀ was determined by ADP-Glo^TM Kinase Assay (V9102, Promega) . Recombinant, Full length human PI3Kα wild-type or H1047R mutant protein were purchased as 1: 1 complex of N-terminal 6x his-tagged PIK3CA (p110α, catalytic subunit) and untagged PIK3R1 (p85α, regulatory subunit) from Viva Biotech. L-α-phosphatidylinositol from Glycine max (Soy PI, Cat. L130328) was used for the lipid substrate by dissolving in the ddH₂O to a final concentration of 1 mM. 10 mM stock compounds in DMSO were serially diluted as 1: 4 ratio to generate a 12-point then dispensed into 384-well low volume plate (Cat. 784076, Greiner) using liquid handler system (mosquito LV, SPT Labtech) . The kinase buffer was prepared in 50 mM HEPES, 10 mM MgCl₂, 1 mM EGTA, 2 mM DTT, and 0.015%Brij-35.5 nM PI3Kα proteins plus 2 μM Soy PI were pre-incubated with compounds in plate at RT for 30 min. After the pre-incubation, the reaction was initiated by adding a final concentration of 100 μM ATP for 2 h. After that time, an equal volume of ADP-Glo reagent was added to stop the reaction and deplete the remaining ATP at RT for 1 h. Then, an equal volume of detection reagent was added to the mixture for 1 h, to achieve a simultaneous reaction of conversion of remaining ADP to ATP and consumption of newly synthesized ATP by the luciferase reaction. After the reaction, generated luminescence was measured by a microplate reader (VICTORPerkinElmer) with 500 ms integration time. All of measured IC₅₀ values were analyzed in GraphPad Prism 8.0.2 (La Jolla California USA, www. graphpad. com) using four parameters dose-response inhibition model.

PI3Kα (PIK3CA) activity in vitro cell based assay

The human breast cancer cells T-47D with PICKCA mutation H1047R were maintained in RPMI 1640 (Gibco, 11875093) supplemented with 10%Fetal Bovine Serum, heat inactivated (Invitrogen, 10091-148) . Cultures were maintained in a humidified incubator at 37℃ under 5%CO₂/95%air. For compound testing, T-47D cells were seeded at a density of 2x10E4 cells per well in 96-well plates in 100 μL of RPMI 1640 Media with 0.1%FBS, incubated overnight. Compounds dissolved in 10 mM stock solutions in DMSO were serially diluted 1: 5 in DMSO to generate a 10-point dilution series. To initiate compounds treatment, the supernatant of cells was aspirated from 96-well plate, and 100 μL dilution series of compounds in RPMI 1640 Media with 0.1%FBS were added to the cell plate to final concentrations ranging from 20 μM to 0.0000102 μM in 0.2%DMSO. 0.2%DMSO alone was used to establish the maximum (MAX) signal and Alpelisib was used as a reference compound. After 1 hour treatment, the medium was removed, and the cells lysed in 40 μL of freshly prepared 1 x Lysis Buffer with shaking (～350 rpm) for 20 minutes at room temperature. Then 10 μL of the lysate were transferred to a 384-well Optiplate^TM for AlphaLisa assay with phospho-AKT (1/2/3) AlphaLISA kit (PerkinElmer, ALSU-PAKT-B10K) . The Acceptor Mix (Reaction Buffer 1 + Reaction Buffer 2 + Activation Buffer +AlphaLISA CaptSure Acceptor Beads) was prepared by diluting Activation buffer 25-fold in combined Reaction Buffer 1 and Reaction Buffer 2. The Acceptor beads were diluted 50-fold in the combined Reaction Buffers. 2.5 μL of Acceptor Mix was added to each well, the plate was sealed and covered with foil and incubated for 1 hour at room temperature. The Donor Mix (Dilution Buffer + Alpha Streptavidin Donor Beads) was prepared by diluting Donor Beads 50-fold in dilution buffer. 2.5 μL of the Donor Mix was added to each well and the plate sealed and covered with foil and incubated for 1 hour at room temperature in the dark. The plates were read on a Spark multimode plate reader instrument from Tecan using standard AlphaLisa settings.

Cell proliferation assay

The human breast cancer cells with PI3KCA mutations, T-47D (PI3KCA H1047R/WT) , MDA-MB-453 (PI3KCA H1047R/WT) , were employed to test the activity of compounds on cell proliferation. The breast cancer cell line SK-BR-3, with wild-type PI3KCA, was used as control cell line. The T-47D, MDA-MB-453 and SK-BR3 cells were maintained in RPMI 1640 (Gibco, 11875093) , DMEM (Gibco, 11965092) or McCoy's 5A (Gibco, 16600082) medium respectively, supplemented with 10%Fetal Bovine Serum, heat inactivated (Invitrogen, 10091-148) . Cultures were maintained in a humidified incubator at 37℃ under 5%CO₂/95%air. To investigate the effect of various compounds on cell growth, T-47D, MDA-MB-453 or SK-BR-3 cells were seeded at a density of 500 cells per well in 384-well plates in 40 μL of growth medium. The plate was then incubated at 37℃ with 5%CO₂ for adhesion. Once the cells adhered to the plate, compounds at a 2X top concentration (20 μM) were prepared in growth medium and 40 μL of the compound solution was added to each well, then the plate was incubated at 37℃ for 5 days. On the fourth day, 1/10th volume of (10×) Alamar blue reagent (Thermo, A50100) was added directly to cells in culture medium, and the plate was incubated overnight at 37℃ with 5%CO₂. Fluorescence was measured by plate reader (Perkin Elmer Victor Nivo 5F) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

Biological Data

The biological activities of certain compounds using the assays described above are shown in Table 4. For PI3Kα ADP-Glo IC₅₀ (nM) : A denotes < 20 nM; B denotes 20 nM ≤ IC₅₀ < 50 nM; C denotes 50 nM ≤ IC₅₀ < 100 nM; D denotes IC₅₀ ≥ 100 nM. For T47D p-AKT IC₅₀ (nM) : A denotes < 20 nM; B denotes 20 nM ≤ IC₅₀ < 50 nM; C denotes 50 nM ≤ IC₅₀ < 100 nM; D denotes IC₅₀ ≥ 100 nM. For T47D anti-proliferation IC₅₀ (nM) and MB-453 anti-proliferation IC₅₀ (nM) : A denotes < 100 nM; B denotes 100 nM ≤ IC₅₀ < 500 nM; C denotes 500 nM ≤ IC₅₀ < 1000 nM; D denotes IC₅₀ ≥ 1000 nM.

Table 4

The embodiments described above are intended to be merely exemplary, and those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the invention and are encompassed by the appended claims.

Claims

A compound of Formula (I) :

or a stereoisomer, or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein:

X¹ is CR^a1 or N;

X² is NR^a2, O, or S;

R^a1 is hydrogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl and alkoxy are optionally substituted;

R^a2 is hydrogen or C₁-C₆ alkyl, and wherein the alkyl is optionally substituted;

R^a3, R^a4, R^a5, and R^a6 are each independently hydrogen, halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 10-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted;

R is C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₈ cycloalkyl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkoxy, cycloalkyl, and heterocyclyl are optionally substituted;

Ring A is C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 10-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted;

Ring B is C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 12-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted;

L is C₁-C₆ alkylene or C₃-C₈ cycloalkylene, and wherein the alkylene, and cycloalkylene are optionally substituted;

R¹ is optionally substituted 3 to 12-membered heterocyclyl, -SO₂R^c, -S (=O) ₂NR^bR^c, -SO₂NH₂, -S (=O) (=NR^b) R^c, -C (=O) NR^bR^c, -C (=O) NH₂, -NR^b (C=O) R^c, OR^c, or -NR^bR^c;

R^b is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 12-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; and

R^c is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5 to 12-membered heteroaryl, or 3 to 8-membered heterocyclyl, and wherein the alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted.
The compound of claim 1, wherein X¹ is CR^a1.
The compound of claim 1 or 2, wherein X² is O.
The compound of any one of claims 1 to 3, wherein R^a4 is hydrogen.
The compound of any one of claims 1 to 4, wherein R^a6 is hydrogen.
The compound of claim 1, which is a compound of Formula (II) :

or a stereoisomer, or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.
The compound of any one of claims 1 to 6, wherein Ring A is 5 to 10-membered heteroaryl.
The compound of claim 7, wherein Ring A is 5 or 6-membered heteroaryl.
The compound of claim 8, wherein Ring A is 5 or 6-membered nitrogen-containing heteroaryl, and nitrogen is the only type of heteroatom contained in the heteroaryl.
The compound of claim 8, wherein Ring A is imidazolyl, pyridyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, pyrazinyl, triazolyl, oxazolyl, or thiazolyl.
The compound of claim 10, wherein Ring A is pyrimidinyl.
The compound of any one of claims 1 to 6, wherein Ring A is C₆-C₁₀ aryl.
The compound of any one of claim 1 to 12, wherein Ring A is unsubstituted.
The compound of any one of claims 1 to 6, wherein Ring A is wherein the attachment to the left is to the urea moiety, and the attachment to the right is to Ring B.
The compound of any one of claims 1 to 14, wherein Ring B is 3 to 8-membered heterocyclyl.
The compound of claim 15, wherein Ring B is 4 to 6-membered heterocyclyl.
The compound of claim 16, wherein Ring B is 4 to 6-membered nitrogen-containing heterocyclyl, and nitrogen is the only type of heteroatom contained in the heterocyclyl.
The compound of claim 17, wherein Ring B is azetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl.
The compound of any one of claims 1 to 14, wherein Ring B is C₃-C₈ cycloalkyl.
The compound of any one of claims 1 to 19, wherein Ring B is substituted with one or more halogen, hydroxyl, nitro, cyano, C₁-C₆ alkyl, or C₁-C₆ alkoxy.
The compound of any one of claims 1 to 14, wherein Ring B is wherein the attachment to the left is to the Ring A, and the attachment to the right is to L.
The compound of claim 1, which is a compound of Formula (III-A) or (III-B) :

or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt thereof, wherein:

Ring B is a nitrogen containing 3 to 8-membered heterocyclyl;

X³ is CR^a7 or N;

X⁴ is CR^a8 or N;

X⁵ is CR^a9 or N;

X⁶ is CR^a10 or N; and

R^a7, R^a8, R^a9, and R^a10 are each independently hydrogen, halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl and alkoxy are optionally substituted.
The compound of claim 22, wherein X³ is CR^a7.
The compound of claim 23, wherein R^a7 is hydrogen.
The compound of any one of claims 22 to 24, wherein X⁴ is N.
The compound of any one of claims 22 to 25, wherein X⁵ is N.
The compound of any one of claims 22 to 26, wherein X⁶ is CR^a10.
The compound of claim 27, wherein R^a10 is hydrogen.
The compound of claim 1, which is a compound of Formula (IV-A) , (IV-B) , (IV-C) , or (IV-D) :

or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt thereof, wherein:

R^a11 is hydrogen, hydroxyl, halogen, nitro, cyano, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl, and alkoxy are optionally substituted; and

each instance of R^a12 is independently hydrogen, halogen, nitro, cyano, hydroxyl, C₁-C₆ alkyl, or C₁-C₆ alkoxy, and wherein the alkyl, and alkoxy are optionally substituted.
The compound of claim 29, wherein each instance of R^a12 is independently hydrogen or C₁-C₆ alkyl.
The compound of any one of claims 1 to 30, wherein R¹ is -SO₂R^c.
The compound of any one of claims 1 to 30, wherein R¹ is -S (=O) ₂NR^bR^c.
The compound of any one of claims 1 to 30, wherein R¹ is -C (=O) NR^bR^c.
The compound of any one of claims 1 to 30, wherein R¹ is -NR^b (C=O) R^c.
The compound of any one of claims 1 to 30, wherein R¹ is -NR^bR^c.
The compound of any one of claims 1 to 30, wherein R¹ is OR^c.
The compound of any one of claims 1 to 30, wherein R¹ is an optionally substituted 3 to 12-membered heterocyclyl.
The compound of any one of claims 1 to 37, wherein L is -CH₂-, -CH₂CH₂-, or -CH (CH₃) -.
The compound of claim 29, which is a compound of Formula (V-A) , (V-B) , (V-C) , or (V-D) , (V-E) , (V-F) , (V-G) , (V-H) , (V-I) , (V-J) , (V-K) , (V-L) , (V-M) , or (V-N) :

or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt thereof, wherein:

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

Ring C is a 3 to 12-membered heterocyclyl optionally substituted with one or more R^a13; and

each instance of R^a13 is independently halogen, hydroxyl, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, -C (=O) (C₁-C₆ alkyl) , -C (=O) NH (C₁-C₆ alkyl) , -C (=O) NH₂, -NH (C=O) (C₁-C₆ alkyl) , -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂, and wherein the alkyl and alkoxy are optionally substituted with one or more halogen, oxo, or hydroxyl.
The compound of claim 39, wherein Ring C is a 3 to 6-membered monocyclic heterocyclyl.
The compound of claim 39, wherein Ring C is a 6 to 12-membered spiro, bridged or fused heterocyclyl.
The compound of claim 39, wherein Ring C is:
wherein the point of attachment is to L, and wherein Ring C is optionally substituted with one or more R^a13.
The compound of any one of claims 39 to 42, wherein R^a13 is halogen, oxo, C₁-C₃ alkyl, C₁-C₃ alkoxy, or -C (=O) (C₁-C₃ alkyl) .
The compound of any one of claims 29 to 43, wherein R^a11 is hydrogen, hydroxyl, fluorine, methyl, or methoxy.
The compound of any one of claims 29 to 44, wherein when the carbon connected to R^a11 is a chiral center, it has R-configuration.
The compound of any one of claims 29 to 44, wherein when the carbon connected to R^a11 is a chiral center, it has S-configuration.
The compound of any one of claims 1 to 46, wherein R^b is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, or 3 to 6-membered heterocyclyl.
The compound of claim 47, wherein R^b is C₁-C₆ alkyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂.
The compound of claim 47, wherein R^b is hydrogen, methyl, ethyl, 2-hydroxyethyl, 2-dimethylaminoethyl, 2-methoxyethyl, ethenyl, or oxetan-3-yl.
The compound of any one of claims 1 to 49, wherein R^c is C₁-C₆ alkyl, C₂-C₆ alkenyl, or 3 to 6-membered heterocyclyl.
The compound of claim 50, wherein R^c is C₁-C₆ alkyl terminally substituted with hydroxy, C₁-C₆ alkoxy, -NH₂, -NH (C₁-C₆ alkyl) , or -N (C₁-C₆ alkyl) ₂.
The compound of claim 50, wherein R^c is methyl, ethyl, 2-hydroxyethyl, 2-dimethylaminoethyl, 2-methoxyethyl, ethenyl, or oxetan-3-yl.
The compound of any one of claims 1 to 52, wherein R^a1 is C₁-C₃ alkyl or C₁-C₃ alkoxy.
The compound of claim 53, wherein R^a1 is methyl or methoxy.
The compound of any one of claims 1 to 54, wherein R^a3 is hydrogen, halogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy.
The compound of claim 55, wherein R^a3 is hydrogen, fluorine, methyl, or methoxy.
The compound of claim any one of claims 1 to 56, wherein R^a5 is hydrogen, halogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy.
The compound of claim 57, wherein R^a5 is fluorine.
The compound of any one of claims 1 to 58, wherein R is C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or 3 to 6-membered heterocyclyl.
The compound of claim 59, wherein R is C₁-C₆ alkyl substituted with one or more halogens.
The compound of claim 59, wherein R is trifluoromethyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxiran-2-yl, oxetan-2-yl, oxetan-3-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, or tetrahydro-2H-pyran-4-yl.
The compound of any one of claims 1 to 61, wherein the carbon connected to R has S-configuration.
The compound of any one of claims 1 to 61, wherein the carbon connected to R has R-configuration.
A compound in Table 1, or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition, comprising a compound of any one of claims 1 to 64 and a pharmaceutically acceptable excipient.
A method of treating a cancer, comprising administering to a subject having the cancer a therapeutically effective amount of a compound of any one of claims 1 to 64 or a pharmaceutical composition of claim 65.
The method of claim 66, wherein the cancer is a PI3Kα-associated cancer.
The method of claim 66 or 67, wherein the cancer is head and neck cancer, brain cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, liver cancer, stomach cancer, hematological cancer, thyroid cancer, colon cancer, or gastric cancer.
The method of claim 68, wherein the cancer is breast cancer.
The method of any one of claims 66 to 69, wherein the subject has one or more mutations in the PIK3CA gene, or in the amino acid sequence of PI3Kα protein.
The method of claim 70, wherein the mutation is in Exon 7, Exon 9, or Exon 20 of the PIK3CA gene.
The method of claim 70, wherein the mutation is C420R, E542K, E545A, E545D, E545G, E545K, Q546E, Q546R, H1047L, H1047R, or H1047Y.
The method of any one of claims 66 to 72, further comprising a step of diagnosing the subject as having a PI3Kα-associated cancer.