[go: up one dir, main page]

WO2025032254A1 - Anthracycline derivatives and conjugates and uses thereof - Google Patents

Anthracycline derivatives and conjugates and uses thereof Download PDF

Info

Publication number
WO2025032254A1
WO2025032254A1 PCT/EP2024/072665 EP2024072665W WO2025032254A1 WO 2025032254 A1 WO2025032254 A1 WO 2025032254A1 EP 2024072665 W EP2024072665 W EP 2024072665W WO 2025032254 A1 WO2025032254 A1 WO 2025032254A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
attachment
pharmaceutically acceptable
point
acceptable salt
Prior art date
Application number
PCT/EP2024/072665
Other languages
French (fr)
Inventor
Akbar Husain KHAN
Andreas Maderna
Luke Andrew MASTERSON
Boliang DENG
Maria Elena DE ORBE IZQUIERDO
Original Assignee
Astrazeneca Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Astrazeneca Ab filed Critical Astrazeneca Ab
Publication of WO2025032254A1 publication Critical patent/WO2025032254A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/14—Ortho-condensed systems

Definitions

  • Anthracyclines are a class of potent chemotherapy drugs used in the treatment of cancer.
  • the most commonly used anthracyclines include doxorubicin, daunorubicin and idarubicin. These compounds have been used in wide range of therapeutics including breast cancer, leukaemia, lymphomas, and in other solid tumours.
  • the mechanism of action for anthracyclines primarily involves DNA intercalation, inhibition of topoisomerase II, and the generation of free radicals, ultimately leading to DNA damage and apoptosis of cancer cells.
  • anthracycline antibody-drug conjugates utilize the targeting capabilities of monoclonal antibodies (mAbs) to selectively deliver cytotoxic payloads (such as anthracyclines) to tumour cells. This selective delivery can increase the efficacy of the cytotoxic payloads while minimizing systemic toxicity.
  • mAbs monoclonal antibodies
  • anthracycline ADCs have been investigated in preclinical and clinical settings.
  • Doxorubicin and daunorubicin have been used in early examples of anthracycline ADCs. More recently, morpholino derivatives such as nemorubicin and its metabolite PNU-159682 have been developed and used in ADCs. These morpholino derivatives are reportedly highly potent while having reduced cardiotoxicity; however, off-target toxicity is still seen in ADCs incorporating these derivatives, as a result of their extremely high potency. There remains a need for anthracycline derivatives having attenuated potency and/or favourable lysosomal stability and/or favourable physicochemical properties, such as solubility and permeability, allowing for a wider therapeutic window and reduced side effects.
  • anthracycline derivatives and conjugates of the present disclosure may be used for the treatment of diseases such as cancer.
  • A is: E is: (ii) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X; (ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C 1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C 1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two
  • a compound of Formula (IIa): or a pharmaceutically acceptable salt thereof wherein A is: E 1 is: i) -NR-; ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X 1 ; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X 1 ; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which
  • a pharmaceutical composition comprising a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
  • a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.
  • a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer for use in the treatment of cancer.
  • a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.
  • a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof.
  • methods and intermediates for preparing compounds of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof are provided.
  • methods and intermediates for preparing compounds of Formula (I) or (II), or a pharmaceutically acceptable salt thereof are provided.
  • a compound of Formula (IIIa) A-E-X-Y-L 1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or , wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L 1 ; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C 1-3 alkyl)-, S, O, -S(CH 2 ) 2 NH-*, - O(CH 2 ) 2 NH-* or -NH(CH 2 ) 2 NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • a compound of Formula (III) A-E-X-Y-L 1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or , wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L 1 ; and Q is NH, S, O, -S(CH 2 ) 2 NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • a compound of Formula (IVa) A-E 1 -X 1 -Y 1 -L 1 or a salt thereof, wherein A, E 1 and X 1 are as defined herein; Y 1 is , wherein the wavy line indicates the point of attachment to X 1 and the asterisk-wavy line indicates the point of attachment to L 1 ; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C 1-3 alkyl)-, S, O, -S(CH 2 ) 2 NH-*, -O(CH 2 ) 2 NH-* or - NH(CH 2 ) 2 NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • a compound of Formula (IV) A-E 1 -X 1 -Y 1 -L 1 or a salt thereof, wherein A, E 1 and X 1 are as defined herein; Y 1 is , wherein the wavy line indicates the point of attachment to X 1 and the asterisk-wavy line indicates the point of attachment to L 1 ; and Q is NH, S, O, -S(CH 2 ) 2 NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • a pharmaceutical composition comprising a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.
  • a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer for use in the treatment of cancer.
  • a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.
  • a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof.
  • Figures 2A-2B depict results from in vivo studies described in Example 59, demonstrating effect of anti-HER2 ADCs (ADC-1 to ADC-6) and negative control ADCs (Control ADCs 1-6) on tumour growth in xenograft models.
  • ADC-1 to ADC-6 anti-HER2 ADCs
  • Control ADCs 1-6 negative control ADCs
  • Cx-y where x and y are integers, indicates the numerical range of carbon atoms that are present in a group.
  • alkyl refers to a saturated, linear or branched, hydrocarbon radical having the specified number of carbon atoms.
  • Examples of C1-3 alkyl groups include methyl (Me), ethyl (Et), n-propyl ( n Pr), and i-propyl ( i Pr).
  • Examples of C1-4 alkyl groups include methyl (Me), ethyl (Et), n-propyl ( n Pr), i-propyl ( i Pr), n-butyl ( n Bu), i-butyl ( i Bu), s-butyl ( s Bu), and t-butyl ( t Bu).
  • cycloalkyl refers to a saturated, cyclic hydrocarbon radical having the specified number of carbon atoms.
  • C3-4 cycloalkyl groups include cyclopropyl and cyclobutyl.
  • heterocycle refers to a ring structure containing the specified number of ring atoms (referred to as “members”), wherein one or more of the ring atoms is a heteroatom (i.e., oxygen, nitrogen, or sulfur, unless otherwise defined), with the remaining ring atoms being carbon.
  • a heterocycle may be monocyclic or multicyclic (e.g. bicyclic), unless otherwise specified.
  • bicyclic refers to a fused, bridged or spirocyclic bicyclic ring. Certain embodiments of this specification include a heterocycle which is said to optionally contain an oxygen atom. In further embodiments said heterocycle does not contain an oxygen atom.
  • the heteroatoms present in the heterocycle may be non- adjacent (i.e. the heteroatoms may be separated by at least one carbon atom in the heterocycle).
  • the term “linker for connection to an antibody, or antigen-binding fragment thereof” refers to a group of atoms capable of forming a covalent bond to an antibody, or antigen- binding fragment thereof, through a chemical reaction.
  • the linker in the compound of Formula (III), (IIIa), (IV) or (IVa) e.g.
  • an antibody-drug conjugate (ADC) derivatised from the compound of Formula (III), (IIIa), (IV) or (IVa) may be formed, for example a conjugate of Formula (V), (Va), (VI) or (VIa) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof may be formed.
  • conjugation group for conjugation to an antibody, or antigen-binding fragment thereof refers to an atom or group of atoms capable of forming a covalent bond to an antibody, or antigen-binding fragment thereof, through a chemical reaction.
  • the use of “ ” (referred to as a “wavy line”), “ ” (referred to as an “asterisk-wavy line”), or “ ” in formulae of this specification denotes the point of covalent attachment to a chemical moiety. indicates the point of attachment to an antibody, or antigen-binding fragment thereof.
  • Certain embodiments of this specification include a group which is said to be “optionally substituted”. In further embodiments said group is unsubstituted.
  • Certain embodiments in this specification include a group that can be “absent”. Where a group is absent, the groups on either side of this are linked by a direct covalent bond.
  • X 1 is absent; in such embodiments, Formula (II) or (IIa) (e.g. Formula (II)) could be rewritten as: A-E 1 -Y 1 -Z 1 .
  • Formula (II) or (IIa) e.g.
  • X 1 is -X a -(CH2)m-X b -, wherein X a is absent; in such embodiments, X 1 can be described as -(CH2)m-X b -.
  • SI International System of Units
  • this specification provides a compound of Formula (IIa), or a pharmaceutically acceptable salt thereof, as defined above.
  • this specification provides a compound of Formula (II), or a pharmaceutically acceptable salt thereof, as defined above.
  • the compounds of Formulae (I), (Ia), (II) or (IIa) e.g. the compounds of Formula (I) and (II)), or pharmaceutically acceptable salts thereof, are anthracycline derivatives. These compounds may be useful in the treatment of cancer. The compounds may be directly administered to a patient for treating cancer.
  • the compounds may be used to prepare ADCs, which may be administered to a patient for treating cancer (such as conjugates of Formula (V), (Va), (VI) or (VIa), e.g. conjugates of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, as disclosed herein).
  • cancer such as conjugates of Formula (V), (Va), (VI) or (VIa), e.g. conjugates of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, as disclosed herein.
  • the compounds of Formulae (I), (Ia), (II) or (IIa) e.g. the compounds of Formulae (I) or (II)
  • pharmaceutically acceptable salts thereof may be referred to as payloads.
  • the payloads may be released from the ADC at a target site, for example via enzymatic cleavage of a linker portion of the ADC.
  • Compounds of Formula (I), (Ia), (II) or (IIa) may have attenuated potency compared to known anthracyclines, such as PNU-159682.
  • Compounds of Formula (I), (Ia), (II) or (IIa) e.g.
  • compounds of Formula (I) or (II)) may exhibit advantageous physicochemical properties (for example, lower lipophilicity, higher aqueous solubility, higher permeability and/or lower plasma protein binding), and/or favourable toxicity profiles (for example a decreased activity at hERG), and/or favourable metabolic or pharmacokinetic profiles, in comparison with known anthracyclines.
  • Compounds of Formula (I), (Ia), (II) or (IIa) e.g. compounds of Formula (I) or (II)
  • a compound of Formula (I): A-E-X-Y-Z or a pharmaceutically acceptable salt thereof wherein A is: E is: i) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X; ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl;iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle
  • A is: .
  • A is:
  • E is a) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X; b) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or c) -heterocycle- NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-
  • E is selected from: wherein Ring E a is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and Ring E b is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • E is: wherein Ring E a is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • the bicyclic heterocycle of Ring E a is a bridged bicycle. In further embodiments, the bicyclic heterocycle is a 7-8 membered bridged bicycle. In embodiments, E is selected from: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In further embodiments, E is selected from: . In further embodiments, E is selected from: . In further embodiments, E is , optionally . In embodiments, the bicyclic heterocycle of Ring E a is a spirocycle. In further embodiments, the bicyclic heterocycle is an 8-9 membered spirocycle.
  • Ring E a is a spirocycle
  • the two nitrogen atoms present in the heterocycle of Ring E a are in the same ring of the spirocycle.
  • E is selected from: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • the two nitrogen atoms present in the heterocycle of Ring E a are in separate rings of the spirocycle.
  • E is selected from: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • the bicyclic heterocycle of Ring E a is a fused bicycle. In further embodiments, the bicyclic heterocycle is an 8-membered fused bicycle. In further embodiments, . I n embodiments, E is , wherein Ring E c is a monocyclic 5-8 membered saturated heterocycle having the two nitrogen atoms shown, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C 1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; and wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • the heterocycle of Ring E c is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. In further embodiments, the geminal substituent is geminal dimethyl. In embodiments, Ring E c is a piperazine geminally substituted on a carbon atom with two occurrences of C 1-3 alkyl. In further embodiments, the geminal substituent is geminal dimethyl. In embodiments, E is selected from: and the asterisk-wavy line indicates the point of attachment to X. In further embodiments E is . In embodiments, Ring E c is a monocyclic 5, 7 or 8 membered saturated heterocycle having the two nitrogen atoms shown, wherein the heterocycle is unsubstituted.
  • E is: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • E is .
  • E is -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-.
  • ring E d is a monocyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • ring E d is a piperidine.
  • E is wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • E is -heterocycle-C(O)NH-, wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -C(O)NH-.
  • ring E b is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
  • the heterocycle is a bridged bicyclic heterocycle.
  • E is: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attac
  • E is: .
  • R her Nd is eH *hment to X.
  • E is: iments, R is C3-4 cycloalkyl.
  • R is meth in * indicates the point of attachment to Y.
  • X is -C(O)CH2NH-*, wherein * indicates the point of attachment to Y.
  • X is -X a -(CH2)m-X b -, wherein X a is -C(O)- or absent; X b is -C(O)NH-*, -O- * .
  • R is H N N NHiments, R is cyclopropyl. In further e 1-4 yl.
  • X is absent. Where X is absent, Formula (I) or (Ia) (e.g.
  • Formula (I)) may be rewritten as: A-E-Y-Z.
  • X is -C(O)CHO-*, w or -NH-, wherein * indicates the point of attachment to Y; and m is 2 or 3.
  • m is 2.
  • X a is -C(O)-
  • X a is absent (i.e. X is –(CH 2 ) m -X b -).
  • X b is -C(O)NH-*, wherein * indicates the point of attachment to Y.
  • X b is -O-.
  • X b is -NH-.
  • X is selected from , wherein the wavy line indicates the point of attachment to E and the asterisk-wavy line indicates the point of attachment to Y.
  • X is .
  • E is -heterocycle-C(O)NH- and X is absent.
  • E is - heterocycle-C(O)NH-, X is -X a -(CH2)m-X b - and X a is absent (i.e. X is –(CH2)m-X b -).
  • E is -heterocycle-C(O)NH-
  • X is -X a -(CH2)m-X b -
  • X a is absent and
  • X b is -NH- (i.e. X is – (CH 2 ) m -NH-).
  • the compound is a compound of Formula (Ia)
  • E is - heterocycle-C(O)NH-
  • X is absent or –(CH 2 ) m -X b -.
  • Y is absent. Where Y is absent, Formula (I) or (Ia) (e.g. Formula (I)) may be rewritten as: A-E-X-Z.
  • Y is , for example . In further embodiments, example . In embodiments, wherein the compound is a compound of Formula (Ia), Y is: embodiments, ya or yb are each independently selected from 0, 1 and 2. In further embodiments, at least one of ya or yb is 0. In further embodiments, ya is 0 and yb is 1. In further embodiments, ya is 1 and yb is 0. In further embodiments, the total of ya and yb is no more than 3. In further embodiments, ya and yb are both 0 (i.e.
  • Y is , optionally In further embodiments, ya and yb are both independently selected from 1 and 2.
  • Q is NH, S or O.
  • Q is NH.
  • Q is S.
  • Q is O.
  • Q is -N(C1-3alkyl)-, for example -N(CH3)-.
  • Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z.
  • Q is -S-(CH 2 ) 2 NH-*.
  • Q is -O-(CH2)2NH-*. In further embodiments, Q is -NH-(CH2)2NH-*. In embodiments, X and Y are both absent. In such embodiments, Formula (I) or (Ia) (e.g. Formula (I)) may be rewritten as: A-E-Z.
  • Z is H. In embodiments, Z is C1-4 alkyl. In further embodiments, Z is methyl. In embodiments, Z is -C(O)C1-4 alkyl. In further embodiments, Z is -C(O)CH3. In embodiments, integer from 1 to 5, and each R z is independently H, C1-4 alkyl, phenyl or benzyl.
  • n is 1. In further embodiments, R Z is H. In further embodiments, n is 2, 3, 4 or 5. In further embodiments, one occurrence of R Z is C1-4 alkyl, benzyl or phenyl and the remaining occurrence or occurrences of R Z are H. In further embodiments, Z is: wherein n1 is 0, 1, 2, 3 or 4 and R z is C1-4 alkyl, benzyl, or phenyl. In further embodiments, R Z is C1-4 alkyl. In further embodiments, R Z is methyl. In further embodiments, R Z is isopropyl. In further embodiments, R Z is benzyl. In further embodiments, R Z is phenyl. In embodiments, Z is .
  • Z comprises amino acid residue(s).
  • R Z when R Z is H, Z could alternatively be defined as (Gly)1-5. When R Z is H and n is 1, Z could alternatively be defined as Gly.
  • Variable Z may comprise amino acid residues in instances where the compounds of Formula (I) or (Ia) (e.g. the compounds of Formula (I)) are payloads that have been released from an ADC, for example once a peptide linker portion has been cleaved.
  • Z is -C(O)C 1-4 alkyl, absent and X is -X a - (CH 2 ) m -X b -, wherein X b is -O- or -NH-.
  • Z is -C(O)C1-4 alkyl, absent and X is -X a - (CH2)m-X b -, wherein X b is -NH-.
  • the compound is a compound of Formula (Ia)
  • the compound of Formula (I) or (Ia) e.g. the compound of Formula (I)
  • a pharmaceutically acceptable salt thereof is selected from a compound of Table 1, or a pharmaceutically acceptable salt thereof. Table 1.
  • the compound of Formula (I) or (la) (e.g. the compound of Formula (I)), or a pharmaceutically acceptable salt thereof, is selected from: or a pharmaceutically acceptable salt thereof.
  • a compound of Formula (IIa) A-E 1 -X 1 -Y 1 -Z 1 5 or a pharmaceutically acceptable salt thereof, wherein A is: E 1 is: i) -NR-; 10 ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X 1 ; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C 1-3 alkyl; 15 iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X 1 ; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -
  • X 1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -X a -(CH2)m-X b -, wherein X b is connected to Y 1 ;
  • X a is - C(O)- or absent;
  • X b is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y 1 ;
  • m is 2 or 3;
  • the wavy line indicates the point of attachment to X 1 and 5 the asterisk-wavy line indicates the point of attachment to Z 1 ;
  • ya and yb are each independently selected from 0, 1, 2 and 3; and
  • Q is NH, -N(C1-3alkyl)-, S, O, -S-(CH2)2NH-*, -O- (CH 2 ) 2 NH-* or -NH-(CH 2 ) 2 NH-*, wherein
  • -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X 1 ; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 5 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X 1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -X a -(CH2)m-X b -, wherein X b is connected to Y 1 ; X a is - C(O)- or absent; X b
  • E 1 is -NR-.
  • E 1 is a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X 1 ; optionally wherein the heterocycle is 10 geminally substituted on a carbon atom with two occurrences of C1-3 alkyl.
  • E 1 is selected from: wherein ring E a is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and ring E b is a bicyclic 5-8 membered saturated heterocycle 15 having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X 1 .
  • E 1 is: wherein ring E a is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X 1 .
  • the bicyclic heterocycle of Ring E a is a bridged bicycle.
  • the bicyclic heterocycle is a 7-8 membered bridged bicycle.
  • E 1 is selected from: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates 10 the point of attachment to X 1 .
  • E 1 is selected from: .
  • E 1 is selected from: .
  • the bicyclic heterocycle of Ring E a is a spirocycle.
  • the 15 bicyclic heterocycle is an 8-9 membered spirocycle.
  • Ring E a is a spirocycle
  • the two nitrogen atoms present in the heterocycle of Ring E a are in the same ring of the spirocycle.
  • E 1 is selected from: 40
  • the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X 1 .
  • . 5 In embodiments in which Ring E a is a spirocycle, the two nitrogen atoms present in the heterocycle of Ring E a are in separate rings of the spirocycle.
  • E 1 is selected from: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X 1 . 10
  • the bicyclic heterocycle of Ring E a is a fused bicycle.
  • the bicyclic heterocycle is an 8-membered fused bicycle.
  • E 1 is , wherein ring E c is a monocyclic 5-8 membered saturated heterocycle having the two nitrogen atoms shown, optionally wherein the heterocycle is geminally 41
  • the heterocycle of Ring E c is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl.
  • the geminal substituent is geminal dimethyl.
  • Ring E c is a piperazine geminally substituted on a carbon atom with two occurrences of C1-3 alkyl.
  • the geminal substituent is geminal dimethyl.
  • E 1 is selected from: , wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X 1 .
  • Ring E c is a monocyclic 5 to 8 membered saturated heterocycle having the two nitrogen atoms shown, wherein the heterocycle is unsubstituted.
  • E 1 is wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X 1 .
  • E 1 is: wherein the wavy line indicates the point of attachment to A and the asterisk-wavy 15 line indicates the point of attachment to X 1 .
  • E 1 is unsubstituted piperazine.
  • E 1 is -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X 1 ; and wherein a carbon atom in the heterocycle connects to -NR-.
  • E 1 is , wherein ring E d is a monocyclic 5-8 membered saturated 20 heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of 42
  • E 1 is -heterocycle-C(O)NH-, wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -C(O)NH-.
  • ring E b is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the 10 wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X 1 .
  • the heterocycle is a bridged bicyclic heterocycle.
  • E 1 is: wherein the wavy line indicates the point of attachment t and the asterisk-wavy line indicates the point of attachment to X 1 .
  • E is: .
  • E is: . 15
  • R is H.
  • R is C3-4 cycloalkyl.
  • R is cyclopropyl.
  • R is C1-4 alkyl. In further embodiments, R is methyl.
  • X 1 is absent. Where X 1 is absent, Formula (II) or (IIa) (e.g. Formula (II)) may be rewritten as: A-E 1 -Y 1 -Z 1 .
  • X 1 is -C(O)CH2O-*, wherein * indicates the point of 20 attachment to Y 1 .
  • X 1 is -C(O)CH2NH-*, wherein * indicates the point of attachment to Y 1 . 43
  • X 1 is -X a -(CH2)m-X b -, wherein X a is -C(O)- or absent; X b is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; and m is 2 or 3. In further embodiments, m is 2. In further embodiments, X a is -C(O)-. In further embodiments, X a is absent (i.e. X 1 is -(CH2)m-X b -). In further embodiments, X b is -C(O)NH-*, wherein * indicates the point of attachment to Y 1 .
  • X b is -NH-.
  • X 1 is selected from wherein the wavy line indicates the point of attachment to E 1 and the asterisk-wavy line indicates the point of attachment to Y 1 .
  • X 1 is .
  • E 1 is -NH- and X 1 is absent.
  • E 1 is -NH-, X 1 is -X a -(CH 2 ) m -X b - 10 and X a is absent (i.e. X 1 is -(CH 2 ) m -X b -).
  • E 1 is -NH-, X 1 is -X a -(CH 2 ) m -X b -, X a is absent and X b is -NH- (i.e. X 1 is -(CH2)m-NH-).
  • E 1 is -heterocycle-C(O)NH- and X 1 is absent.
  • E 1 is - heterocycle-C(O)NH-, X 1 is -X a -(CH2)m-X b - and X a is absent (i.e. X 1 is -(CH2)m-X b -).
  • E 1 is -heterocycle-C(O)NH-
  • X 1 is -X a -(CH2)m-X b -
  • X a is absent and
  • X b is -NH- (i.e. X 1 is -15 (CH2)m-NH-).
  • the compound is a compound of Formula (IIa)
  • E 1 is - NR- or -heterocycle-C(O)NH-
  • X 1 is absent or -(CH 2 ) m -X b -.
  • the wavy line indicates the point of attachment to X 1 and the asterisk-wavy line indicates the point of attachment to Z 1 .
  • example In further embodiments, 20 . 44
  • Y 1 is: .
  • y a and y b are each independently selected from 0, 1 and 2.
  • at least one of ya or yb is 0.
  • ya is 0 and yb is 1.
  • ya is 1 and yb is 0.
  • the total of ya and yb is no more than 3.
  • ya and yb are both 0 (i.e.
  • Y 1 is In further embodiments, ya and yb are both independently selected from 1 and 2. In further embodiments of a compound of Formula (IIa), Y 1 is as described in relation to a compound of Formula (II).
  • Q is NH, S or O.
  • Q is NH.
  • Q is S. 15
  • Q is O.
  • Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z 1 .
  • Q is -S-(CH2)2NH-*.
  • Q is -O-(CH2)2NH-*.
  • Q is -NH-(CH2)2NH-*. 45
  • Q is -N(C1-3alkyl)-, for example -N(CH3)-.
  • Z 1 is H.
  • Z 1 is C 1-4 alkyl.
  • Z 1 is methyl.
  • Z 1 is -C(O)C 1- 5 4 alkyl.
  • Z 1 is -C(O)CH 3 .
  • integer from 1 to 5 and each R z is independently H, C 1-4 alkyl, phenyl or benzyl.
  • n is 1.
  • R Z is H.
  • n is 2, 3, 4 or 5.
  • R Z is C1-4 alkyl, benzyl or phenyl and the remaining occurrence or occurrences of R Z are H.
  • Z 1 is: 10 wherein n1 is 0, 1, 2, 3 or 4 and R z is C1-4 alkyl, benzyl, or phenyl.
  • R Z is C1-4 alkyl.
  • R Z is methyl.
  • R Z is isopropyl.
  • R Z is benzyl.
  • R Z is phenyl.
  • Z 1 could alternatively be defined as (Gly)1-5.
  • Z 1 15 could alternatively be defined as Gly.
  • Variable Z 1 may comprise amino acid residues in instances where the compounds of Formula (II) or (IIa) (e.g. Formula (II)) are payloads that have been released from an ADC, for example once a peptide linker portion has been cleaved.
  • the compound of Formula (IIa), or a pharmaceutically acceptable salt thereof is 20 selected from a compound of Table 2 or Table 2A, or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (II), or a pharmaceutically acceptable salt thereof is selected from a compound of Table 2, or a pharmaceutically acceptable salt thereof. 25 46 Table 2.
  • the compound of Formula (II) or (IIa) (e.g. the compound of Formula (II)), or a pharmaceutically acceptable salt thereof, is selected from:
  • a pharmaceutically acceptable salt thereof there are provided methods for preparing compounds of Formula (I), (Ia), (II) or (IIa) (e.g. compounds of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof; and intermediates in said methods.
  • the method may be any method as described herein, for example as described in General Procedures and/or reaction schemes herein.
  • the intermediate may be an Intermediate Compound disclosed herein.
  • the Intermediate Compound may be in a salt form, for example a specific salt form described herein.
  • the Intermediate Compound may be in a non-salt form (i.e. as a free base).
  • a compound of Formula (IIIa) A-E-X-Y-L 1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or , wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L 1 ; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • a compound of Formula (III) A-E-X-Y-L 1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or , wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L 1 ; and Q is NH, S, O, -S(CH 2 ) 2 NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • a compound of Formula (IVa) A-E 1 -X 1 -Y 1 -L 1 or a salt thereof, wherein A, E 1 and X 1 are as defined herein; Y 1 is , wherein the wavy line indicates the point of attachment to X 1 and the asterisk-wavy line indicates the point of attachment to L 1 ; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • this specification provides a compound of Formula (IV): A-E 1 -X 1 -Y 1 -L 1 or a salt thereof, wherein A, E 1 and X 1 are as defined herein; Y 1 is , wherein the wavy line indicates the point of attachment to X 1 and the asterisk-wavy line indicates the point of attachment to L 1 ; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L 1 ; and L 1 is a linker for connection to an antibody or antigen-binding fragment thereof.
  • the compounds of Formulae (III), (IIIa), (IV) and (IVa) (e.g. the compounds of Formulae (III) and (IV)), or salts thereof, may be referred to “linker payloads”.
  • These compounds comprise a payload portion (derived from the compounds of Formula (I), (Ia), (II) or (IIa), e.g. compounds of Formula (I) or (II)), respectively, connected to a linker portion.
  • the linker portion enables the connection of the payload(s) to an antibody, or antigen-binding fragment thereof, through covalent bond(s) to form an ADC (such as a conjugate of Formula (V), (Va), (VI) or (VIa), e.g.
  • the payload(s) may be released from the ADC at a target site, for example via enzymatic cleavage of the linker portion, to provide compounds of Formula (I), (Ia), (II) or (IIa).
  • Compounds of Formula (I), (Ia), (II) or (IIa) may exhibit advantageous properties, as described above.
  • the payload-linker may be conjugated to an anti-HER2 monoclonal antibody to obtain an anti-HER2 ADC that is effective in treating cancer, such as HER2 positive cancer.
  • the compound of Formula (III), (IIIa), (IV) or (IVa) (e.g.
  • the compound of Formula (III) or (IV)) may be in a salt form, for example a pharmaceutically acceptable salt.
  • Variable A in a compound of Formula (IIIa) or (IVa) may be as described herein, for example, as described in relation to a compound of Formula (Ia) or (IIa).
  • Variable A in a compound of Formula (III) or (IV) may be as described herein, for example, as described in relation to a compound of Formula (I) or (II).
  • Variables E and X in a compound of Formula (IIIa) may be as described herein, for example, as described in relation to a compound of Formula (Ia).
  • Variable Y in a compound of Formula (IIIa) may be as described in relation to a compound of Formula (Ia), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 1 .
  • Variables E and X in a compound of Formula (III) may be as described herein, for example, as described in relation to a compound of Formula (I).
  • Variable Y in a compound of Formula (III) may be as described in relation to a compound of Formula (I), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 1 .
  • Variables E 1 and X 1 in a compound of Formula (IVa) may be as described herein, for example, as described in relation to a compound of Formula (IIa).
  • Variable Y 1 in a compound of Formula (IVa) may be as described in relation to a compound of Formula (IIa), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 1 .
  • Variables E 1 and X 1 in a compound of Formula (IV) may be as described herein, for example, as described in relation to a compound of Formula (II).
  • Variable Y 1 in a compound of Formula (IV) may be as described in relation to a compound of Formula (II), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 1 .
  • the linker L 1 contains a reactive moiety through which the compound may be connected to an antibody or antigen-binding fragment thereof.
  • L 1 may comprise a reactive moiety which reacts with a site in the antibody or antigen-binding fragment thereof, to form a covalent bond between the linker and the antibody or antigen-binding fragment thereof.
  • the reactive moiety in the linker L 1 may be an electrophilic group, capable of reacting with a nucleophilic group within the antibody, or antigen-binding fragment thereof.
  • Nucleophilic groups on antibodies include side chain thiol groups (e.g. cysteine), side chain amine groups (e.g. lysine), N-terminal amine groups, and sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Electrophilic groups on the linker include, but are not limited to: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
  • the antibody or antigen- binding fragment thereof may contain reducible interchain disulfides, such as cysteine bridges.
  • Such antibodies or antigen fragments may be treated with a reducing agent, such as dithiothreitol, so that each disulfide forms two reactive thiol nucleotides that can react with linkers.
  • Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2- iminothiolane resulting in conversion of an amine into a thiol.
  • Reactive thiol groups may be introduced into the antibody, or antigen-binding fragment thereof, for example by preparing mutant antibodies comprising one or more non-native cysteine amino acid residues.
  • the reactive moiety in the linker may be a nucleophilic group, capable of reacting with an electrophilic group within the antibody, or antigen-binding fragment thereof.
  • Electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups.
  • Nucleophilic groups on a linker include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • the reactive moiety in the linker may be a substrate for an enzyme, where the enzyme catalyses conjugation of the reactive moiety to the antibody or antigen-binding fragment thereof.
  • the enzyme may be a transpeptidase, for example sortase A. Sortase A, originally derived from the bacterium Staphylococcus aureus, exhibits high specificity in cross-linking. Sortase A may catalyze the reaction of an amine, e.g.
  • the linker L 1 may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions.
  • the linker is cleavable by enzymatic activity, such as a protease or glucuronidase.
  • the linker may be peptide- or glucuronide-based.
  • the cleavage of the linker may be such that the released payload contains a portion of the cleaved linker. For example, if the linker is (Gly)5, then the released payload may contain 1, 2, 3, 4 or 5 glycine residues, depending on the point of enzymatic cleavage.
  • Cleavage of the linker L 1 , in a compound of Formula (III) or (IIIa) may lead to the release of a payload which is a compound of Formula (I) or (Ia), respectively (e.g. a compound of Formula (I)).
  • Cleavage of the linker L 1 , in a compound of Formula (IV) or (IVa) (e.g. a compound of Formula (IV)), may lead to the release of a payload which is a compound of Formula (II) or (IIa), respectively (e.g. a compound of Formula (II)).
  • the linker may be a non-cleavable linker. ADCs containing non-cleavable linkers may rely on degradation of the antibody, or antigen-binding fragment thereof, in order to release the payloadlinker.
  • the linker may be stable extracellularly, such that the ADC remains intact until it reaches the target cell.
  • the linker may be cleaved once inside the target cell.
  • Suitable linkers are known in the art; for example, as disclosed in Bargh et al, Chem. Soc. Rev., 2019, 48, 4391-4374 or Su et al, Acta Pharm. Sin. B, 2021, 11, 3889-3907 (incorporated herein by reference in their entirety).
  • L 1 is:
  • each R Z1 is independently H, C1.4 alkyl, phenyl or benzyl; q is 3, 4 or 5; the asterisk-wavy line indicates the point of attachment to G 1 ; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and G 1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
  • each R Z1 is independently H, CM alkyl, phenyl or benzyl; q is 3, 4 or 5; L B is selected from -(CH 2 )b- and , wherein the asterisk-wavy line indicates the point of attachment to G 1 ; b is an integer from 1 to 5; c is an integer from 5 to 15; and G 1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof; provided that, for a compound of
  • L A is absent or *-CH 2 -NH-.
  • L 1 is: . In further embodiments, q is 3. In further embodiments, q is 4. In further embodiments, q is 5. In further embodiments, b is 2. In further embodiments, c is 7. In further embodiments, R Z1 is H. In further embodiments, one occurrence of R Z1 is Ci-4 alkyl, benzyl or phenyl and the remaining occurrences of R Z1 are H. In further embodiments, L 1 is: wherein R Z1 is C M alkyl, benzyl, or phenyl and ql is
  • R Z1 is C M alkyl. In further embodiments, R Z1 is methyl. In further embodiments, R Z1 is isopropyl. In further embodiments, R Z1 is benzyl. In further embodiments, R Z1 is phenyl.
  • I_ B is -(CH 2 )b-.
  • L B is .
  • b is 2.
  • c is 7.
  • L B is .
  • d is 1. In other embodiments, d is 2.
  • L 1 is selected from: or a salt thereof.
  • LMS salt thereof.
  • L 1 is a group of Formula (L-IA): wherein Ring F 1 is a saturated bicyclic ring having 6, 7 , or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F 2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F 3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
  • R 1 is CM alkyl
  • X 2 is (CH 2 )n3, wherein n3 is 0, 1, 2 or 3,
  • Y 2 is (CH 2 )n4, wherein n4 is 0, 1, 2, 3 or 4, wherein n5 is 1, 2, 3, 4 or 5, n5A is an integer from 1 to 10, n5B is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G 1 , m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
  • G 1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
  • L 1 is a group of Formula (L-l): wherein E 2 is (CH 2 ) n2 , wherein n2 is 0, 1, 2 or 3, wherein Ring F 1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F 2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F 3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, R 1 is Ci-4 alkyl,
  • X 2 is (CH 2 ) n3 , wherein n3 is 0, 1, 2 or 3,
  • Y 2 is (CH 2 )n4, wherein n4 is 0, 1, 2, 3 or 4,
  • Z 2 is (CH 2 )n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
  • G 1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof; provided that, for a compound of Formula (III), when Y is absent, then X b is not -C(O)NH-.
  • Q 2 is
  • R 1 is CH 3 .
  • E 2 is CH 2 .
  • X 2 is CH 2 .
  • Y 2 is (CH 2 ) 2 .
  • Z 2 is (CH 2 ) 2 .
  • p is 1. In further embodiments, pl is 0. In further embodiments, pl is 1. In
  • Z 2 is n5A .
  • n5B is 2.
  • n5A is 1.
  • L 1 is selected from:
  • L 1 is a group of Formula (L-ll I) : wherein E 3 is (CH 2 )n6, wherein n6 is 0, 1, 2 or 3; R 1A is CM alkyl or H, wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or
  • Y 3 is O or NR B , wherein R B is H, Ci ⁇ 4 alkyl or C 3-4 cycloalkyl,
  • Z 3 is (CH 2 ) n io or , wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G 1 , m3 is an integer from 2 to 17; p2 is 1 or 0; q2 is 1 or 0; p3 is 0 or 1; and G 1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
  • m3 is 9, 10, 11, 12 or 13.
  • R 1A is CH 3 .
  • E 3 is CH 2 .
  • X 4 is CH 2 .
  • Y 3 is O.
  • Z 3 is (CH 2 )nio.
  • Z 3 is (CH 2 ) 2 .
  • Z 3 is absent (i.e. Z 3 is
  • nlOB is 2.
  • nlOA is 1.
  • p2 is 1.
  • q2 is 1.
  • p2 is 0.
  • p3 is 1.
  • p3 is 0.
  • L 1 is:
  • L 1 is selected from the moieties of Table 3:
  • G 1 is selected from: wherein X 3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, R K is H or CH 3 , and R L is Cv 6 alkyl.
  • G 1 is selected from: wherein X 3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, R K is H or CH 3 , and R L is Ci- 6 alkyl. In embodiments G 1 is selected from: wherein X 3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, R K is H or CH 3 , and R L is Ci- 6 alkyl. In further embodiments, G 1 is selected from: wherein X 3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, R K is H or CH 3 , and R L is Ci- 6 alkyl In further embodiments, G 1 is selected from
  • G 1 is . In further embodiments, G 1 is In further embodiments, G 1 is
  • L 1 is selected from: or a salt thereof.
  • L 1 is: salt thereof.
  • L 1 is selected from the moieties of Table 4:
  • X b is not -C(O)NH-.
  • the compound of Formula (III) or (Illa) (e.g. the compound of Formula (III)), or a salt thereof, is selected from a compound of Table 5A, or a salt thereof:
  • the compound of Formula (IVa), or a salt thereof is selected from a compound of Table 5B or Table 5C, or a salt thereof. In embodiments, the compound of Formula (IV), or a salt thereof, is selected from a compound of Table 5B, or a salt thereof.
  • a compound of Formula (III), (Illa), (IV) or (IVa) (e.g. a compound of Formula (III) or (IV)), or a salt thereof, may be prepared by reacting (i.e. conjugating) a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)) or a derivative thereof, or a salt thereof, with a precursor to
  • L 1 contains a second reactive moiety which reacts with the compound of Formula (I), (la), (II) or (Ila) (e.g. the compound of Formula (I) or (II)) or derivative thereof.
  • compound LP1 or LP2 may be prepared by reacting compound P20 or P8, respectively, or a salt thereof, with compound LI, or a salt thereof (see example section for LI structure).
  • each A, E and X is as defined herein; Y is absent , wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L 2 ; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N (Ci- 3 al kyl)-, S, O, -S(CH 2 ) 2 NH-*, - O(CH 2 ) 2 NH-* or -NH(CH 2 ) 2 NH-*, wherein * indicates the point of attachment to L 2 ; each L 2 is a linker; k is an integer from 1 to 10; and Ab is an antibody or antigen-binding fragment thereof.
  • each A, E and X is as defined herein; Y is absent , wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L 2 ; and Q is NH, S, O, -S(CH 2 ) 2 NH-*, - O(CH 2 ) 2 NH-* or -NH(CH 2 ) 2 NH-*, wherein * indicates the point of attachment to I 2 ; each L 2 is a linker; k is an integer from 1 to 10; and Ab is an antibody or antigen-binding fragment thereof.
  • the conjugates of Formula (V), (Va), (VI) and (Via) may alternatively be referred to as the compounds of Formula (V), (Va), (VI) and (Via) (e.g. compounds of Formula (V) and (VI).
  • the conjugates of Formula (V), (Va), (VI) and (Via) may also be referred to as ADCs.
  • a conjugate of Formula (V), (Va), (VI) and (Via) e.g.
  • a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof may undergo cleavage to release a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), respectively.
  • Compounds of Formula (I), (la), (II) or (Ila), and the conjugates of Formula (V), (Va), (VI) and (Via) may exhibit advantageous properties, as described above, which may result in a wider therapeutic window.
  • Variable A in a conjugate of Formula (Va) or (Via) may be as described herein, for example, as described in relation to a compound of Formula (la) or (Ila).
  • Variable A in a conjugate of Formula (V) or (VI) may be as described herein, for example, as described in relation to a compound of Formula (I) or (II).
  • Variables E and X in a conjugate of Formula (Va) may be as described herein, for example, as described in relation to a compound of Formula (la).
  • Variable Y in a conjugate of Formula (Va) may be as described in relation to a compound of Formula (la), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 2 .
  • Variables E and X in a conjugate of Formula (V) may be as described herein, for example, as described in relation to a compound of Formula (I).
  • Variable Y in a conjugate of Formula (V) may be as described in relation to a compound of Formula (I), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 2 .
  • Variables E 1 and X 1 in a conjugate of Formula (Via) may be as described herein, for example, as described in relation to a compound of Formula (Ila).
  • Variable Y 1 in a conjugate of Formula (Via) may be as described in relation to a compound of Formula (Ila), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 2 .
  • Variables E 1 and X 1 in a conjugate of Formula (VI) may be as described herein, for example, as described in relation to a compound of Formula (II).
  • Variable Y 1 in a conjugate of Formula (VI) may be as described in relation to a compound of Formula (II), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L 2 .
  • the linker L 2 may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions.
  • the linker is cleavable by enzymatic activity, such as protease or glucuronidase.
  • the linker may be peptide- or glucuronide-based. The cleavage of the linker may be such that the released payload contains a portion of the cleaved linker.
  • the released payload may contain 1, 2, 3, 4 or 5 glycine residues, depending on the point of enzymatic cleavage.
  • Cleavage of the linker L 2 , in a conjugate of Formula (Va) may lead to the release of a payload which is a compound of Formula (la).
  • Cleavage of the linker L 2 , in a compound of Formula (V) may lead to the release of a payload which is a compound of Formula (I).
  • Cleavage of the linker, L 2 , in a conjugate of Formula (Via) may lead to the release of a payload which is a compound of Formula (Ila).
  • Cleavage of the linker, L 2 , in a conjugate of Formula (VI) may lead to the release of a payload which is a compound of Formula (II).
  • the linker may be a non-cleavable linker.
  • ADCs containing non-cleavable linkers rely on degradation of the antibody, or antigen-binding fragment thereof, in order to release the payloadlinker.
  • the linker may be stable extracellularly, such that the ADC remains intact until it reaches the target cell.
  • the linker may be cleaved once inside the target cell.
  • Suitable linkers are known in the art; for example, as disclosed in Bargh et al, Chem. Soc. Rev., 2019, 48, 4391-4374 or Su et al, Acta Pharm. Sin. B, 2021, 11, 3889-3907 (incorporated herein by reference in their entirety).
  • L 2 is: , wherein L A is absent or is selected from *-CH 2 -NH-, and *-C(O)-CH 2 -L A1 -
  • L A is absent or is selected from *-CH 2 -NH-
  • each R Z1 is independently H, Ci.
  • L B is selected from — (CH 2 )b- and , wherein the asterisk-wavy line indicates the point of attachment to G 2 ; b is an integer from 1 to 5; c is an integer from 5 to 15; and G 2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof; provided that, for a conjugate of Formula (V), when Y is absent, then X b is not -C(O)NH-.
  • L A is absent or *-CH 2 -NH-.
  • L 2 is: further embodiments, q is 3. In further embodiments, q is 4. In further embodiments, q is 5. In further embodiments, b is 2. In further embodiments, c is 7. In further embodiments, R Z1 is H. In further embodiments, one occurrence of
  • R Z1 is CM alkyl, benzyl or phenyl and the remaining occurrences of R Z1 are H.
  • R Z1 is Ci. 4 alkyl.
  • R Z1 is methyl.
  • R Z1 is isopropyl.
  • R Z1 is benzyl.
  • R Z1 is phenyl.
  • L B is — (CH 2 )b-- In further embodiments, L B is
  • L B is .
  • d is 1. In other embodiments, d is 2.
  • L 2 is selected from: or a pharmaceutically acceptable salt thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
  • L 2 is: pharmaceutically acceptable salt thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
  • L 2 is a group of Formula (L-ll A): wherein Ring F 1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F 2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F 3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
  • R 1 is Ci-4 alkyl
  • X 2 is (CH 2 )n3, wherein n3 is 0, 1, 2 or 3,
  • Y 2 is (CH 2 )n4, wherein n4 is 0, 1, 2, 3 or 4, or 2, and the asterisk-wavy line indicates the point of attachment to G 2 , m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
  • G 2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
  • L 2 is a group of Formula (L-ll):
  • R 1 is Ci-4 alkyl
  • X 2 is (CH 2 )n3, wherein n3 is 0, 1, 2 or 3,
  • Y 2 is (CH 2 )n4, wherein n4 is 0, 1, 2, 3 or 4,
  • Z 2 is (CH 2 )n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
  • G 2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof; provided that, for a conjugate of Formula (V), when Y is absent, then X b is not -C(O)NH-.
  • R 1 is CH 3 .
  • E 2 is CH 2 .
  • X 2 is CH 2 .
  • Y 2 is (CH 2 ) 2 .
  • Z 2 is (CH 2 ) 2 .
  • p is 1.
  • pl is 0.
  • pl is 1.
  • n5A is 1.
  • L 2 is selected from: wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
  • X 4 is CH 2 .
  • Y 3 is O.
  • Z 3 is
  • Z 3 is (CH 2 )nio. In further embodiments, Z 3 is (CH 2 ) 2 . In further embodiments, Z 3 is absent (i.e. Z 3 is
  • nlO is 0
  • Z 3 is embodiments, nlOB is 2.
  • nlOA is 1.
  • p2 is 1.
  • q2 is 1.
  • p2 is 0.
  • p3 is 1.
  • p3 is 0.
  • L 2 is: In embodiments, L 2 is selected from a moiety of Table 6:
  • G 2 is selected from: wherein R K is H or CH 3 , R L is Cv 6 alkyl, and - x ' 1! indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
  • G 2 is selected from: wherein R K is H or CH 3 , R L is Cv 6 alkyl, and indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
  • G 2 is selected from:
  • R K is H or CH 3
  • R L is Ci_ 6 alkyl, and indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
  • G 2 is selected from:
  • G 2 is further embodiments, G 2 is In embodiments, G 2 is (i.e. G 2 a bond).
  • -L B -G 2 , -Z 2 -G 2 or -Z 3 -G 2 is:
  • L 2 is selected from: or a pharmaceutically acceptable salt thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
  • L 2 is: or a pharmaceutically acceptable salt thereof, wherein - x "' indicates the point of attachment to the antibody or antigen-binding fragment thereof.
  • L 2 is selected from a moiety of Table 7:
  • conjugate of Formula (V) or (Va) when Y is absent, then X b is not -C(O)NH-.
  • the conjugate of Formula (V) or (Va) e.g. the conjugate of Formula (V)
  • a pharmaceutically acceptable salt thereof is selected from a conjugate of Table 8A:
  • Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
  • the conjugate of Formula (VI) or (Via) (e.g. the conjugate of Formula (VI)), or a pharmaceutically acceptable salt thereof, is selected from a conjugate of Table 8B:
  • the conjugate of Formula (Via) or a pharmaceutically acceptable salt thereof is selected from a conjugate of Table 8C: or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
  • k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In further embodiments k is an integer from 2 to
  • k is an integer from 2 to 8. In further embodiments k is 4. In further embodiments k is 8.
  • isotopes will be understood to include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium.
  • isotopes of carbon include 13 C and 14 C.
  • Isotopes of nitrogen include 15 N.
  • the compounds disclosed herein may contain one or more chiral centres. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e. as individual enantiomers, diastereoisomers, or as a stereoisomerically enriched mixture. All such stereoisomer (and enriched) mixtures are included within the scope of the embodiments, unless otherwise stated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
  • the chemical structure or chemical name is intended to embrace all possible stereoisomers, diastereoisomers, conformers, rotamers and tautomers of the compound depicted.
  • a compound containing a chiral carbon atom is intended to embrace both the (R) enantiomer and the (S) enantiomer, as well as mixtures of the enantiomers, including racemic mixtures; and a compound containing two chiral carbons is intended to embrace all enantiomers and diastereoisomers including (R,R), (S,S), (R,S) and (S,R).
  • tautomeric forms e.g. keto/enol
  • the compounds disclosed herein may be prepared, used or supplied in amorphous form, crystalline form, or semicrystalline form and any given compound disclosed herein may be capable of being formed into more than one crystalline / polymorphic form, including hydrated (e.g. hemi hydrate, a mono hydrate, a di hydrate, a tri hydrate or other stoichiometry of hydrate) and/or solvated forms. It is to be understood that the present specification encompasses any and all such solid forms of the compound disclosed herein.
  • the compounds disclosed herein may be prepared as a co-crystal solid form. It is to be understood present specification encompasses any and all such co-crystals of a compound disclosed herein.
  • a suitable pharmaceutically acceptable salt of a compound disclosed herein is, for example, an acid addition salt.
  • An acid addition salt may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person.
  • An acid addition salt may for example be formed using an inorganic acid selected from hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid.
  • An acid addition salt may also be formed using an organic acid selected from trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.
  • organic acid selected from trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.
  • a suitable pharmaceutically acceptable salt of a compound disclosed herein is, for example, a base addition salt.
  • a base addition salt may be formed by bringing the compound into contact with a suitable inorganic or organic base under conditions known to the skilled person.
  • a base addition salt may for example be an alkali metal salt (such as a sodium, potassium, or lithium salt) or an alkaline earth metal salt (such as a calcium salt), which may be formed using an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g., an ethoxide or methoxide).
  • a base addition salt may also be formed using a suitably basic organic amine (e.g., a choline or meglumine salt).
  • a further suitable pharmaceutically acceptable salt of a compound of Formula (I) or (II) or a conjugate of Formula (V) or (VI) is, for example, a salt formed within a patient's body after administration of a compound of Formula (I) or (II) or a conjugate of Formula (V) or (VI) to the patient.
  • a further suitable pharmaceutically acceptable salt of a compound of Formula (la) or (Ila) or a conjugate of Formula (Va) or (Via) is, for example, a salt formed within a patient's body after administration of a compound of Formula (la) or (Ila) or a conjugate of Formula (Va) or (Via) to the patient.
  • a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof may be prepared by reacting (i.e. conjugating) a compound of Formula (III) or (IV), or a salt thereof, with an antibody or an antigen-binding fragment thereof.
  • a compound of Formula (III) or (IV) contains moiety G 1 and a conjugate of Formula (V) or (VI) contains moiety G 2 , as described above. Upon conjugation, a moiety G 1 is converted to moiety G 2 where G 2 has a point of attachment to the antibody or antigen-binding fragment thereof.
  • a conjugate of Formula (Va) or (Via), or a pharmaceutically acceptable salt thereof may be prepared by reacting (i.e. conjugating) a compound of Formula (Illa) or (IVa), or a salt thereof, with an antibody or an antigen-binding fragment thereof.
  • a compound of Formula (Illa) or (IVa) contains moiety G 1 and a conjugate of Formula (Va) or (Via) contains moiety G 2 , as described above.
  • a moiety G 1 is converted to moiety G 2 where G 2 has a point of attachment to the antibody or antigen-binding fragment thereof.
  • G 1 and G 2 include, but are not limited to, the following moieties set out in Table 9, wherein X 3 is CH or N, h is 0 or 1, R K is H or CH 3 , Hal is Cl, Br or I, R L is Ci. 6 alkyl. " indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
  • G 1 and G 2 are set out in Table 9A.
  • sortase A enzyme may be used to effect the conjugation to the antibody or antigen-binding fragment thereof.
  • the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen.
  • the antibody is isolated or recombinant.
  • isolated when used herein refers to a polypeptide, e.g., an antibody, that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated antibody will be prepared by at least one purification step. Thus, an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities.
  • the antibody comprises at least two "light chains” (LC) and two “heavy chains” (HC).
  • the light chains and heavy chains of such antibodies are polypeptides consisting of several domains.
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region (abbreviated herein as "CH”).
  • the heavy chain constant region comprises the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM).
  • Each light chain comprises a light chain variable domain (abbreviated herein as "VL”) and a light chain constant domain (abbreviated herein as "CL”).
  • the antibody is a full-length antibody.
  • An “intact” or “full-length” antibody, as used herein, refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • the variable regions VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) (also known as hypervariable regions), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region.
  • binding between an antibody and its target antigen or epitope is mediated by the CDRs.
  • epitope refers to a target protein region (e.g. polypeptide) capable of binding to (e.g. being bound by) an antibody or antigen-binding fragment of the disclosure.
  • the CDRs are the main determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al.
  • IgA immunoglobulin G
  • IgD immunoglobulin G
  • IgE immunoglobulin M
  • IgM immunoglobulin M
  • IgG molecules interact with multiple classes of cellular receptors.
  • IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII.
  • FcyR Fey receptors
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • the antibody or antigen-binding fragment thereof is an IgG isotype.
  • the antibody or antigen-binding fragment thereof can be any IgG subclass, for example IgGl, lgG2, lgG3, or lgG4 isotype.
  • the antibody or antigen-binding fragment thereof is based on an IgGl isotype.
  • Fc region refers to the portion of a native immunoglobulin that is formed by two Fc chains.
  • Each "Fc chain” comprises a constant domain CH2 and a constant domain CH3.
  • Each Fc chain may also comprise a hinge region.
  • a native Fc region is homodimeric.
  • the Fc region may be heterodimeric because it may contain modifications to enforce Fc heterodimerisation.
  • the Fc region contains the carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor), and has no antigen binding activity.
  • Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.
  • Human IgGl, lgG2, lgG3, and lgG4 heavy chain sequences can be obtained in a variety of sequence databases, including the UniProt database (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN) respectively.
  • the antibody of the disclosure is a monoclonal antibody.
  • a “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants.
  • the term “monoclonal antibody” can encompass both full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab 1 , F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site.
  • “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.
  • the antibody of the disclosure is an isolated monoclonal antibody.
  • the antibody is a fully human monoclonal antibody.
  • the antibody of the disclosure is a full-length antibody described above.
  • the antibody can be an antigen-binding fragment.
  • antigen-binding fragment as used herein incudes any naturally-occurring or artificially-constructed configuration of an antigen-binding polypeptide comprising one, two or three light chain CDRs, and/or one, two or three heavy chain CDRs, wherein the polypeptide is capable of binding to the antigen.
  • the antigen-binding fragment of the disclosure is a Fab fragment.
  • the antibody according to the disclosure can also be a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an intrabody, an IgG CH2, a minibody, a single-domain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BITE), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-lg, an (scFv)2, a mAb2 or a DARPin.
  • Fab fragment and "Fab” are used interchangeably herein and contain a single light chain (e.g. a constant domain CL and a VL) and a single heavy chain (e.g. a constant domain CHI and a VH).
  • the heavy chain of a Fab fragment is not capable of forming a disulfide bond with another heavy chain.
  • a “Fab 1 fragment” contains a single light chain and a single heavy chain but in addition to the CHI and the VH, a “Fab' fragment” contains the region of the heavy chain between the CHI and CH2 domains that is required for the formation of an inter-chain disulfide bond. Thus, two “Fab 1 fragments” can associate via the formation of a disulfide bond to form a F(ab')2 molecule.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for the formation of an inter-chain disulfide bond between two heavy chains.
  • Fv fragment contains only the variable regions of the heavy and light chain. It contains no constant regions.
  • a “single-domain antibody” is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).
  • a "single-chain Fv” (“scFv”) is antibody fragment containing the VH and VL domain of an antibody, linked together to form a single chain.
  • a polypeptide linker is commonly used to connect the VH and VL domains of the scFv.
  • a “tandem scFv”, also known as a TandAb, is a single-chain Fv molecule formed by covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.
  • a "bi-specific T cell engager” (BiTE) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumour cell antigen.
  • a “diabody” is a small bivalent and bispecific antibody fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH- VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.
  • a "DARPin” is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins.
  • a DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences.
  • a DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display, which is completely cell-free and is described in He M.
  • the antibody or antigen-binding fragment thereof can be further modified to contain additional chemical moieties not normally part of the protein.
  • Those derivatised moieties can improve the solubility, the biological half-life or absorption of the protein.
  • the moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 2nd ed., Ed. Lloyd V. Allen, Jr. (2012).
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient, and which contains no additional components which are unacceptably toxic to a patient to which the composition would be administered. Such compositions can be sterile.
  • a pharmaceutical composition according to the present specification comprises a compound of Formula (I), (la), (II) or (Ila), or a pharmaceutically acceptable salt thereof, or a conjugate of Formula (V), (Va), (VI) or (Via), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, or a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, buffer or stabilizer.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable excipient e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable excipient e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable excipient e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable excipient e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable, non-toxic, sterile carrier.
  • the carrier is a physiological saline, non-toxic buffer, or preservative.
  • Suitable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol and ethanol, as well as any combination thereof.
  • a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and one or more isotonic agents.
  • the one or more isotonic agents are selected from a sugar, a polyalcohol and sodium chloride.
  • a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, contained within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal aerosol, and a lyophilized powder which can be reconstituted to make a suspension or solution before use.
  • a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, a buffer, a surfactant and/or a stabilizer agent.
  • the buffer is an acetate, phosphate or citrate buffer.
  • the surfactant is polysorbate.
  • the stabilizer agent is human albumin.
  • composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, can be administered to a patient by any appropriate systemic or local route of administration.
  • administration may be oral, buccal, sublingual, ophthalmic, intranasal, intratracheal, pulmonary, topical, transdermal, urogenital, rectal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular or intratumoural.
  • the pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, can be formulated for administration by any appropriate means, for example by epidermal or transdermal patches, ointments, lotions, creams, or gels; by nebulizers, vaporizers, or inhalers; by injection or infusion; or in the form of capsules, tablets, liquid solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders.
  • the most suitable route for administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired properties of the formulation.
  • compositions comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • a capsule may comprise a solid carrier such a gelatin.
  • the conjugate of Formula (V), (Va), (VI) or (Via) (e.g. the conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
  • a pharmaceutical composition comprising a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
  • a pharmaceutically acceptable salt thereof may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs) or for parenteral administration (for example
  • compositions may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.
  • pharmaceutical compositions comprising a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
  • An effective amount of the compound of Formula (I), (la), (II) or (Ila) e.g. the compound of Formula (I) or (II)
  • a pharmaceutically acceptable salt thereof will normally be present in the composition.
  • a compound of Formula (I), (la), (II) or (Ila) e.g. a compound of Formula (I) or (II)
  • a pharmaceutically acceptable salt thereof for use in therapy.
  • a compound of Formula (I), (la), (II) or (Ila) e.g. a compound of Formula (I) or (II)
  • a pharmaceutically acceptable salt thereof for use in the treatment of cancer.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof for use in therapy.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof for use in the treatment of cancer.
  • cancer includes both non-metastatic cancer and also metastatic cancer, such that treating cancer involves treatment of both primary tumours and also tumour metastases.
  • the term “therapy” is intended to have its normal meaning of dealing with a disease in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology.
  • the term “therapy” also includes “prophylaxis” unless there are specific indications to the contrary.
  • the terms “therapeutic” and “therapeutically” should be interpreted in a corresponding manner.
  • prophylaxis is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease and secondary prophylaxis whereby the disease has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or the development of new symptoms associated with the disease.
  • treatment is used synonymously with “therapy”.
  • treat can be regarded as “applying therapy” where “therapy” is as defined herein.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof for use in the treatment of HER2 positive cancer.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.
  • a compound of Formula (I), (la), (II) or (Ila) e.g. a compound of Formula (I) or (II)
  • a pharmaceutically acceptable salt thereof as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof e.g. a conjugate of Formula (V) or (VI)
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof.
  • Terms such as “treating” or “treatment” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • a patient is successfully "treated” for cancer according to the methods of the present disclosure if the patient shows, e.g., total, partial, or transient remission of a certain type of cancer.
  • an effective amount means an amount of an active ingredient which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response).
  • the effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically acceptable excipient(s)/carrier(s) utilized, and like factors within the knowledge and expertise of the attending physician.
  • patient refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. In embodiments the term “patient” refers to a human subject.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, wherein the cancer is a HER2 positive cancer.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof e.g. a conjugate of Formula (V) or (VI)
  • an additional anti-tumour substance for the conjoint treatment of cancer.
  • a combination for use in the treatment of cancer comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of the Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof and an additional anti-tumour agent.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of the Formula (V) or (VI)
  • a pharmaceutically acceptable salt thereof in combination with an additional anti-tumour agent.
  • “conjoint treatment” is used in reference to a combination treatment, it is to be understood that this may refer to simultaneous, separate or sequential administration. In one aspect, “conjoint treatment” refers to simultaneous administration. In another aspect, “conjoint treatment” refers to separate administration. In a further aspect, “conjoint treatment” refers to sequential administration.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially administering at least one additional anti-tumour substance to said patient, where the amounts of the conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or pharmaceutically acceptable salt thereof, and the additional anti-tumour substance are jointly effective in producing an anti-cancer effect.
  • a conjugate of Formula (V), (Va), (VI) or (Via) e.g. a conjugate of Formula (V) or (VI)
  • the additional anti-tumour substance are jointly effective in producing an anti-cancer effect.
  • a compound of Formula (I), (la), (II) or (Ila) e.g. a compound of Formula (I) or (II)
  • a pharmaceutically acceptable salt thereof e.g. a compound of Formula (I) or (II)
  • an additional anti-tumour substance for the conjoint treatment of cancer.
  • a combination for use in the treatment of cancer comprising a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof and an additional anti-tumour agent.
  • a compound of Formula (I), (la), (II) or (Ila) e.g. a compound of Formula (I) or (II)
  • a pharmaceutically acceptable salt thereof in combination with an additional anti-tumour agent.
  • a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially administering at least one additional anti-tumour substance to said patient, where the amounts of the compound of Formula (I), (la), (II) or (Ila) (e.g. the compound of Formula (I) or (II)), or pharmaceutically acceptable salt thereof, and the additional anti-tumour substance are jointly effective in producing an anti-cancer effect.
  • a compound of Formula (I), (la), (II) or (Ila) e.g. a compound of Formula (I) or (II)
  • pharmaceutically acceptable salt thereof e.g. the compound of Formula (I) or (II)
  • Reagents and solvents (all anhydrous HPLC-grade), including PNU-159682 carboxylic acid (CAS No.: 1204819-92-0), were obtained from commercial suppliers and used without any further purification unless otherwise stated. All reagents were weighed and handled in air unless otherwise stated. Brine refers to a saturated solution of NaCI. Concentration under reduced pressure refers to the use of a rotary evaporator.
  • DMSO refers to c/6-DMSO. [ethyl cyano(hydroxyimino)acetato-O 2 ]tri-l-pyrrolidinylphosphonium
  • Method A Boc protected amine was dissolved in 1,4-dioxane, then HCI solution (4 M in dioxane) was added in one portion at 0 °C under nitrogen. The resulting mixture was stirred at 25 °C for a few hours until the reaction was completed, and then concentrated to remove the solvent. The crude product was purified by preparative HPLC using MeCN/water (containing 0.01% NH4HCO3) to afford the product.
  • Method B TFA (40 mL) was added to Boc protected amine ( ⁇ 8.0 mmol) in DCM (60 ml) under nitrogen. The resulting mixture was stirred at 25 °C for a few hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18 chromatography using elution gradient 0 to 100% MeCN in water (0.1% NH4HCO3) to afford the product.
  • HCI Fmoc tert-Butyl 3,6-diazabicyclo[3.1.1]heptane-3-carboxylate 500 mg, 2.52 mmol was added to a solution of N,N-diisopropylethylamine (978 mg, 7.57 mmol) and N-(9H-fluoren-2- ylmethoxycarbonyloxyjsuccinimide (936 mg, 2.77 mmol) in DCM (10 mL). The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water. The organic layer was separated, the aq solution was extracted with DCM. The combined organic solution was dried over Na 2 SO 4 , and concentrated to afford the crude intermediate. The crude intermediate was treated with HCI solution (4 M in dioxane) according to General Procedure II, Method A to afford
  • Step 2 Intermediate Compound 2: (9H-fluoren-9-yl)methyl 3-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((lS,3R,4aS,9S,9aR, 10aS)-9-methoxy-l-methyloctahydro-lH- pyrano[4',3':4,5]oxazolo[2,3-c][l,4]oxazin-3-yl)oxy)-6,ll-dioxo-l,2,3,4,6,ll-hexahydrotetracene-2- carbonyl)-3,6-diazabicyclo- [3.1.1]heptane-6-carboxylate
  • Step 3 Compound P1: (8S,10S)-8-(3,6-diazabicyclo[3.1.1]heptane-3-carbonyl)-6,8,11-trihydroxy-1- methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
  • Step 2 Intermediate Compound 4: (9H-fluoren-9-yl)methyl 3,6-diazabicyclo[3.1.1]heptane-3- carboxylate, TFA salt General Procedure II, Method B was followed using Intermediate Compound 3 (3.4 g, 8.09 mmol) in DCM (60 mL) to afford Intermediate Compound 4 (2.300 g, yield: 89 %) as a pale yellow solid.
  • Step 2 Intermediate Compound 7: 1-(3,6-diazabicyclo[3.1.1]heptan-3-yl)-2-hydroxyethan-1-one, HCl salt
  • Method A was followed using Intermediate Compound 6 (1g, 3.90 mmol) to afford Intermediate Compound 7 (0.600 g, 85 %) as a yellow solid.
  • Step 3 Compound P2b: (8S,10S)-6,8,11-trihydroxy-8-(3-(2-hydroxyacetyl)-3,6- diazabicyclo[3.1.1]heptane-6-carbonyl)-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':4,5] oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10- tetrahydrotetracene-5,12-dione
  • General Procedure I was followed using the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), Intermediate Compound 7 (112 mg, 0.72 mmol), PyOxim (244 mg, 0.48 mmol) and N,N-diisopropylethylamine (154 mg
  • Step 2 Intermediate Compound 9: (9H-fluoren-9-yl)methyl (1R,4R)-2,5-diazabicyclo[2.2.2]octane- 2-carboxylate, HCl salt
  • Intermediate Compound 8 (1.5 g, 3.45 mmol) to afford Intermediate Compound 9 (0.900 g, 70.3 %) as a white solid.
  • Step 2 Intermediate Compound 12: (9H-fluoren-9-yl)methyl (1S,4S)-2,5-diazabicyclo[2.2.2]octane- 2-carboxylate, HCl salt
  • Intermediate Compound 11 800 mg, 1.84 mmol
  • Intermediate Compound 12 1050 mg, 97%) as a pale yellow solid.
  • Step 2 Compound 15 (9H-fluoren-9-yl)methyl (3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrole-2(1H)- carboxylate, HCl salt
  • Intermediate Compound 14 1.25 g, 3.22 mmol
  • Step 3 Intermediate Compound 16: (9H-fluoren-9-yl)methyl (3aR,6aS)-5-((2S,4S)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl) hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate
  • General Procedure I was followed using HATU (136 mg, 0.36 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), DIEA (0.209 mL, 1.20
  • Step 3 Intermediate Compound 19: (9H-fluoren-9-yl)methyl 2-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate
  • General Procedure I was followed using the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol), PyOxim (244 mg, 0.48 mmol), DIEA (0.278 mL, 1.59 mmol) and Intermediate Compound 18 (222 mg,
  • Step 4 Compound P7 (8S,10S)-8-(3,3-dimethylpiperazine-1-carbonyl)-6,8,11-trihydroxy-1-methoxy- 10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
  • General Procedure III was followed using DCM solution of DBU (15.2 mg/mL in DCM) (0.07 mL, 0.08 mmol) and Intermediate Compound 22 (160 mg, 0.17 mmol) in DCM (4 mL)/MeCN (1.000 mL) to afford Compound P7 (21.00 mg, 17.16 %) as a red solid.
  • Step 2 Intermediate Compound 24: 1-(2,2-dimethylpiperazin-1-yl)ethan-1-one, HCl salt
  • Method A was followed using HCl in dioxane (4 M) (29.3 mL, 117.2 mmol) and Intermediate Compound 23 (5 g, 19.50 mmol) to afford Intermediate Compound 24 as a pure solid material (3 g HCl salt).
  • ES + (M+1 157).
  • Step 3 Compound P7a: (8S,10S)-8-(4-acetyl-3,3-dimethylpiperazine-1-carbonyl)-6,8,11-trihydroxy- 1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
  • General Procedure I was followed using DIEA (0.139 mL, 0.80 mmol), Intermediate Compound 24 (49.8 mg, 0.32 mmol), HATU (121 mg, 0.32 mmol) and the PNU-159682 carboxylic acid (CAS No.
  • Step 2 Intermediate Compound 26: (9H-fluoren-9-yl)methyl 2,6-diazaspiro[3.3]heptane-2- carboxylate, TFA salt
  • TFA 15 ml, 194.70 mmol
  • Intermediate Compound 25 (1.6 g, 3.80 mmol) in DCM (20 mL) to afford Intermediate Compound 26 (1.100 g, 90 %) as a pale yellow solid.
  • Step 2 Intermediate Compound 29: 1-(2,6-diazaspiro [3.3]heptan-2-yl)ethan-1-one, TFA salt
  • Method B was followed using Intermediate Compound 28 (900 mg, 3.75 mmol) and TFA (5 mL)/DCM (5.00 mL) to afford Intermediate Compound 29 (450 mg, 86 %) as a pale yellow solid.
  • NMR 400 MHz, DMSO
  • m/z (ES+), [M+H]+ 141.
  • Step 3 Compound P9a (8S,10S)-8-(6-acetyl-2,6-diazaspiro[3.3]heptane-2-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione . , . , . , . , .
  • Step 2 Intermediate Compound 31: (9H-fluoren-9-yl)methyl 3,8-diazabicyclo[3.2.1]octane-8- carboxylate, HCl salt
  • Method A was followed using HCl (5 mL, 4 M in dioxane) and Intermediate Compound 30 (1.6 g, 3.68 mmol) in 1,4-dioxane (10 mL) to afford Intermediate Compound 31 (0.950 g, 77 %) as a yellow solid.
  • Step 2 Intermediate Compound 37: (9H-fluoren-9-yl)methyl (1R,4R)-2,5- diazabicyclo[2.2.1]heptane-2-carboxylate, HCl salt
  • Method A was followed using 4 N HCL in 1,4-dioxane (10 ml, 40.00 mmol), and Intermediate Compound 36 (2 g, 4.76 mmol) in DCM (10 mL) to afford crude product Intermediate Compound 37 (2.200 g, 99 %) as a yellow solid.
  • Step 2 Intermediate Compound 40: (9H-fluoren-9-yl)methyl (1S,4S)-2,5-diazabicyclo [2.2.1]heptane-2-carboxylate, HCl salt
  • Method A was followed using 4N HCL in 1,4-dioxane (1.40 mL, 5.64 mmol), and Intermediate Compound 39 (790 mg, 1.88 mmol) in DCM (10 mL) to afford Intermediate Compound 40 (550 mg, 91 %) as a yellow solid.
  • Example 17 Synthesis of Compound P14 Step 1a and 1b - Intermediate Compound 43: (9H-fluoren-9-yl)methyl 5-oxa-2,8- diazaspiro[3.5]nonane-8-carboxylate, HCl salt tert-Butyl 5-oxa-2,8-diazaspiro[3.5]nonane-2-carboxylate (500 mg, 2.19 mmol) was added to a solution of N,N-diisopropylethylamine (849 mg, 6.57 mmol) and N-(9H-fluoren-2- ylmethoxycarbonyloxy)succinimide (813 mg, 2.41 mmol) in DCM (8 mL).
  • Step 3 Intermediate Compound 44: (9H-fluoren-9-yl)methyl 2-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl)-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate
  • General Procedure I was followed using the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol), Intermediate Compound 43 (279 mg, 0.80 mmol), N,N-diisopropylethylamine (206 mg, 1.
  • Step 4 Compound P14: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-8-(5-oxa-2,8- diazaspiro[3.5]nonane-2-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
  • General Procedure III was followed using Intermediate Compound 44 (130 mg, 0.14 mmol) and DCM solution of DBU (15.2mg/mL in DCM) (0.746 mL, ⁇ 0.07mmol) in DCM (1.5 mL) and MeCN (0.5 mL) to afford Compound P14 (20.00 mg, 20.02 %) as a red solid.
  • Step 4 Compound P15: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4’,3’:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-(5-oxa-2,8- diazaspiro[3.5]nonane-8-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
  • General Procedure III was followed using DBU (7.85 ⁇ l, 0.05 mmol) and Intermediate Compound 47 (100 mg, 0.10 mmol) in DCM (2 mL)/MeCN (0.5 mL) to afford Compound P15 (15.00 mg, 19.52 %) as a red solid.
  • ES + (M+1 738).
  • Example 19 Synthesis of Compound P15a Step 1 – Intermediate Compound 48: tert-butyl 2-acetyl-5-oxa-2,8-diazaspiro[3.5]nonane-8- carboxylate Ac2O (0.248 mL, 2.63 mmol) was added to a solution of tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane- 8-carboxylate (400 mg, 1.75 mmol) and DIEA (1.530 mL, 8.76 mmol) in DCM (15 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours.
  • Step 2 Intermediate Compound 49: 1-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)ethan-1-one, HCl salt
  • Method A was followed using Intermediate Compound 48 (410 mg, 1.52 mmol) to afford Intermediate Compound 49 (210 mg, 81 %) as a yellow solid.
  • Step 3 Compound P15a: (8S,10S)-8-(2-acetyl-5-oxa-2,8-diazaspiro[3.5]nonane-8-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
  • General Procedure I was followed using HATU (158 mg, 0.41 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 130 mg, 0.21 mmol), DIEA (0.181 mL, 1.04 mmol) and Intermediate Compound 49 (70.5 mg, 0.41 mmol) in DMA (1.5
  • Step 2 Intermediate Compound 51: (3S,3aR,6S,6aR)-3,6-diazidohexahydrofuro[3,2-b]furan Sodium azide (3.43 g, 52.80 mmol) was added to a solution of Intermediate Compound 50 (4 g, 8.80 mmol) in DMF (80 mL). The solution was stirred for 3 hours at 140 °C. The reaction mixture was diluted with water (200 mL), and extracted with EA (3 x 200 mL).
  • Step 3 Intermediate Compound 52: (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine Pd-C (0.076 g, 0.71 mmol) was added to Intermediate Compound 51 (1.4 g 7.14 mmol) in ethyl acetate (20 mL) and methanol (1 mL). The mixture was stirred for overnight hours 25 °C under H2. The reaction mixture was filtered through silica, and washed with EA (3 x 40 mL). The solvent was removed under reduced pressure to afford Intermediate Compound 52 (0.600 g, 58.3 %) as a yellow soild.
  • reaction mixture was diluted with DCM (200 mL), and washed sequentially with 0.1M HCl (2 x 200 mL) and saturated brine (2 x 200 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product.
  • Step 2 Intermediate Compound 54: (3R,3aS,6R,6aS)-6-((tert- butyldimethylsilyl)oxy)hexahydrofuro[3,2-b]furan-3-yl 4-methylbenzenesulfonate TBS-Cl (3.76 g, 24.97 mmol) was added to a solution of Intermediate Compound 53 (5 g, 16.65 mmol) and 1H-imidazole (2.267 g, 33.30 mmol) in DCM (100 mL). The solution was stirred for overnight at room temperature. The reaction mixture was diluted with DCM (100 mL), and washed with saturated water (2 x 100 mL) and brine (2 x 100 mL).
  • Step 3 Intermediate Compound 55: (((3R,3aS,6S,6aR)-6-azidohexahydrofuro[3,2-b]furan-3- yl)oxy)(tert-butyl)dimethylsilane Sodium azide (2.59 g, 39.80 mmol) was added to Intermediate Compound 54 (5.5 g, 13.27 mmol) in DMF (110 mL). The solution was stirred for 3 hours at 140°C. The reaction mixture was diluted with water (250 mL), and extracted with ethyl acetate (3 x 150 mL). The combined organic solution was washed with water (2 x 200 mL), and brine (2 x 200 mL).
  • Step 4 Intermediate Compound 56: (3S,3aR,6R,6aS)-6-((tert-butyldimethylsilyl)oxy)hexahydrofuro [3,2-b]furan-3-amine Pd/C (1.3 g) was added to Intermediate Compound 55 (700 mg, 2.45 mmol) in EtOAc (10 mL) / MeOH (0.500 mL) at 25°C under H 2 . The resulting mixture was stirred at 25 °C for 16 hours under H 2 . The reaction mixture was filtered through celite. The solvent was removed under reduced pressure to afford Intermediate Compound 56 as crude product (600 mg, 94 %) as a yellow oil.
  • Example 26 Synthesis of Compound P22 Step 1 - Intermediate Compound 53: (3R,3aS,6R,-6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl 4- methylbenzenesulfonate 4-Toluenesulfonyl chloride (137 g, 718.48 mmol) was added to a solution of pyridine (135.4 g, 1710.66 mmol), and (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol (“isomannide”; 100 g, 684.26 mmol) in DCM (1000 mL) under nitrogen.
  • Step 2 Intermediate Compound 54: (3R,3aS,6R,6aS)-6-((tert- butyldimethylsilyl)oxy)hexahydrofuro[3,2-b]furan-3-yl-4-methylbenzenesulfonate
  • TBS-Cl 38.64g, 256.38mmol
  • imidazole 31.74 g, 466.16 mmol
  • Intermediate Compound 53 70 g, 333.08 mmol
  • DMA 220 ml
  • Step 8 Intermediate Compound 63: tert-butyl 3-((3S,3aR,6S,6aR)-6- (((benzyloxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)-3,6-diazabicyclo[3.1.1]heptane-6- carboxylate tert-Butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (24.10 g, 121.55 mmol) was added to a solution of Intermediate Compound 62 (10 g, 24.31 mmol) in 1,4-dioxane (100 mL).
  • Step 9 Intermediate Compound 64: tert-butyl 3-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2- b]furan-3-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate Pd-C (1.853 g, (10% w/w)) was added to a solution of Intermediate Compound 63 (8 g, 17.41 mmol) in MeOH (160 mL) under hydrogen. The resulting mixture was stirred at 25 °C for another 2 hours. The reaction mixture was filtered through a celite pad.
  • Step 3 Intermediate Compound 70: (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine Hydrazine monohydrate (12.75 mL, 210.20 mmol) was added to a solution of Intermediate Compound 69 (17 g, 42.04 mmol) in EtOH (350 mL) under nitrogen. The resulting mixture was stirred at 80 °C for 2 hours. The reaction mixture was filtered, and wash the filter cake with EtOH (2 x 150 mL). The filtrate was evaporated to dryness to afford Intermediate Compound 70 (3.50 g, 57.7 %) as a yellow solid.
  • Step 4 Intermediate Compound 71: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)carbamate N-(9H-Fluoren-2-ylmethoxycarbonyloxy)su2Ncc ( OiSn) Him H Oide ( NS (H)1F.9m8o9c g, 5.90 mmol) was added slowly to a solution of Intermediate Compound 70 (1.7 g, 11.79 mmol) and DIEA (4.12 mL, 23.58 mmol) in THF (100 mL) under nitrogen.
  • Step 5 Intermediate Compound 72: 4-((1R,4R)-5-(tert-butoxycarbonyl)-2,5- diazabicyclo[2.2.2]octan-2-yl)-4-oxobutanoic acid Succinic anhydride (236 mg, 2.36 mmol) was added to a solution of tert-butyl (1R,4R)-2,5-diaza- bicyclo[2.2.2]octane-2-carboxylate (500 mg, 2.36 mmol) in DCM (6 mL) under nitrogen. The resulting mixture was stirred at RT for 2 hours. The solvent was removed under reduced pressure.
  • Step 2 Intermediate Compound 77: (4-(6-(tert-butoxycarbonyl)-3,6-diazabicyclo[3.1.1]heptan-3- yl)-4-oxobutanoic acid Pd-C (3.29 g, 3.09 mmol) was added to a solution of Intermediate Compound 76(6 g, 15.45 mmol) in THF (100 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 2 hours under hydrogen. The reaction mixture was filtered through a celite pad. The solvent was removed under reduced pressure. Intermediate Compound 77 was obtained as crude material (4.30 g, 93 %, white solid).
  • Example 30 Synthesis of Compound P26 Steps 1 and 2 – Intermediate Compound 86: (9H-fluoren-9-yl)methyl (2-((2-(((3S,3aR,6S,6aR)-6- ((tert-butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)amino)-2- oxoethyl)carbamate (((9H-Fluoren-9-yl)methoxy)carbonyl)glycylglycine (1 g, 2.82 mmol) was added to 1- methylimidazole (0.232 g, 2.82 mmol) and Intermediate Compound 70(0.814 g, 5.64 mmol) in DMF (8 mL) at 25°C under nitrogen.
  • Example 31 Synthesis of Compound P27 Steps 1,–2 - Intermediate Compound 90: (9H-fluoren-9-yl)methyl (2-((2-((2-(((3S,3aR,6S,6aR)-6- ((tert-butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)carbamate (((9H-Fluoren-9-yl)methoxy)carbonyl)glycylglycylglycine (1 g, 2.43 mmol) was added to 1- methylimidazole (0.200 g, 2.43 mmol) and (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine (0.701 g, 4.86
  • Step 3 Intermediate Compound 101: tert-butyl (1S,4S)-5-((3S,3aR,6S,6aR)-6-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) hexahydrofuro [3,2-b] furan-3-yl)-2,5-diazabicyclo [2.2.2] octane-2- carboxylate N-(9H-Fluoren-2-ylmethoxycarbonyloxy) succinimide (806 mg, 2.39 mmol) was added to Intermediate Compound 100 (595mg, 1.75 mmol) and DIEA (0.839 mL, 4.80 mmol) in DCM (5 mL) under hydrogen.
  • Step 3 Intermediate Compound A-111: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)(methyl)carbamate
  • Intermediate Compound A-110 (440 mg, 0.92 mmol) was added to DCM (3 mL)/HCl in dioxane (4N) (3.00 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford crude Intermediate Compound A-111 (300 mg, 86 %) as a pale yellow solid.
  • Step 4 Intermediate Compound A-112: (9H-fluoren-9-yl) methyl((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,-4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]-oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido)-hexahydrofuro[3,2-b]furan-3-yl)carbamate
  • Intermediate Compound A-111 (218 mg, 0.57 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 300 mg, 0.48 mmol), HATU
  • Example 35 Synthesis of Compound P31 Step 1 – Intermediate Compound A-113: benzyl ((3S,3aR,6S,6aR)-6-cyanohexahydrofuro[3,2- b]furan-3-yl)carbamate KCN (3.56 g, 54.70 mmol) was added to Intermediate Compound 62 (15 g, 36.47 mmol) and 18- Crown-6 (15.42 g, 58.34 mmol) in THF (100 mL) at 0°C. The resulting mixture was stirred at 0 °C for 4 hours.
  • Step 2 Intermediate Compound A-114: benzyl ((3S,3aR,6S,6aR)-6- (aminomethyl)hexahydrofuro[3,2-b]furan-3-yl)carbamate Borane-tetrahydrofuran complex (156 mL, 156.09 mmol) was added to Intermediate Compound A-113 (9 g, 31.22 mmol) in THF (100 mL) at 0°C over a period of 10 minutes under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with MeOH (100 mL) in ice bath.
  • Step 3 Intermediate Compound A-115: benzyl ((3S,3aR,6S,6aR)-6-(((((9H-fluoren-9- yl)methoxy)carbonyl)amino)methyl)hexahydrofuro-[3,2-b]furan-3-yl)carbamate N-(9H-Fluoren-2-ylmethoxycarbonyloxy)succinimide (9.23 g, 27.37 mmol) was added to Intermediate Compound A-114 (8g, 27.37 mmol) in MeCN (50 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure.
  • Example 36 Synthesis of Compound P32 Step 1 – Intermediate Compound A-118: (9H-fluoren-9-yl)methyl (((3S,3aR,6S,6aR)-6- (tritylamino)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamate
  • Intermediate Compound A-116 300 mg, 0.79 mmol was added to Triphenylmethyl chloride (264 mg, 0.95 mmol) and N,N-Diisopropylethylamine (306 mg, 2.37 mmol) in THF (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours.
  • Step 2 Intermediate Compound A-119: (3S,3aR,6S,6aR)-6-(aminomethyl)-N- tritylhexahydrofuro[3,2-b]furan-3-amine Intermediate Compound A-118 (491 mg, 0.79 mmol) was added to Diethylamine (3 mL, 0.79 mmol) in THF (5 mL) at 25°C under nitrogen.
  • Step 4 Compound P32: (2S,4S)-N-(((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3- yl)methyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro- 1H-pyrano-[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamide
  • Intermediate Compound A-120 (100 mg, 0.10 mmol) was added to 1,1,1,3,3,3-Hexafluoro-2- propanol (2 mL, 0.10 mmol) in DCM (5 mL) at 25°C under nitrogen.
  • Step 2 3 - Intermediate Compound A-122: ((3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6- diyl)dimethanamine
  • Intermediate Compound A-121 (4.6 g, 11.21 mmol) and 18-Crown-6 (6.22 g, 23.55 mmol) in THF (10 mL) at 0°C under N2.
  • KCN 1.533 g, 23.55 mmol
  • Step 6 Compound P33: (2S,4S)-N-(((3S,3aR,6S,6aR)-6-(aminomethyl)-hexahy-drofuro[3,2-b]furan- 3-yl)methyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,-9aR,-10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':4,5]oxazolo-[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamide
  • Intermediate Compound A-122 (82 mg, 0.48 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 100 mg, 0.16
  • Example 38 Synthesis of Compound P34 Step 1 - Intermediate Compound A-123: Dibenzyl (((3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6- diyl)bis(methylene))dicarbamate
  • N-(Benzyloxycarbonyloxy)succinimide 5.59 g, 22.43 mmol
  • DIEA 5.88 mL, 33.64 mmol
  • Intermediate Compound A-122 1.9 g, 11 mmol
  • THF 10 mL
  • the solvent was removed under reduced pressure.
  • Step 2 Intermediate Compound A-124: Benzyl (((3S,3aR,6S,6aR)-6- (aminomethyl)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamate
  • Intermediate Compound A-123 8 g, 18.16 mmol
  • dioxane 30 mL
  • the solvent was removed by blowing N2 gently.
  • the crude product was purified by flash C18- flash chromatography, elution gradient 0 to 100% MeCN in water (0.1%NH4HCO3).
  • Step 3 Intermediate Compound A-125: Benzyl (((3S,3aR,6S,6aR)-6- ((methylamino)methyl)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamate
  • methyl trifluoromethanesulfonate 0.321 g, 1.96 mmol
  • HFIP HFIP
  • Step 4 Intermediate Compound A-126: 1-((3S,3aR,6S,6aR)-6-(aminomethyl)hexahydrofuro[3,2- b]furan-3-yl)-N-methylmethanamine
  • Intermediate Compound A-125 200 mg, 0.62 mmol
  • dioxane 1 mL
  • HCl(12N) 5.00 mL
  • the resulting mixture was stirred at 25 °C for 5 hours.
  • Step 5 Compound P34: (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N- (((3S,3aR,6S,6aR)-6-((methylamino)methyl)hexahydrofuro[3,2-b]furan-3-yl)methyl)-6,11-dioxo- 1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
  • Intermediate Compound A-126 89 mg, 0.48 mmol
  • PNU- 159682 carboxylic acid CAS No.1204819-92-0, 150 mg, 0.24 mmol
  • HATU 182 mg
  • the reaction mixture is diluted with acetonitrile (1 mL) and purified via reverse phase purification by using acetonitrile and water (2 mmol HCOONH4) as mobile phase. Obtained fractions are lyophilized to afford the Linker-Payload. Protection and de-protection steps are utilized as necessary.
  • the linker may be loaded onto a resin prior to the coupling with the payload, followed by cleavage of the resin after coupling.
  • Example 39 Synthesis of Linker-Payload Compound LP1 Synthesis of Linker Compound L1: (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoyl)glycylglycylglycylglycylglycine Resin loading: A peptide synthesizer glass vessel (Chemglass, 250-mL) adapted with an N2 (g) flow inlet and a medium size frit filter was charged with 2-chlorotrityl chloride resin (1.4 g, 1.54 mmol, 1.1 mmol/g commercial loading capacity, Chem-Impex International).
  • the resin was swollen by agitation with a flow of N2(g) for 1.5 hours with 20 mL of anhydrous DMF and for 30 minutes with 27 mL of DMF/DCM (3:1).
  • N-Fmoc-Gly-Gly-Gly-Gly-OH (1.36 eq., 0.98 g, 2.09 mmol) was dissolved in 31 mL of anhydrous EtOAC/DMSO/DMF (20:6:5) and 1.88 mL of DIPEA (1.39 g, 7 eq, 10.78 mmol).
  • the reaction was run for 15 hours and the resin was washed with DMF (4 x 5 min x 25 mL) and DCM (4 x 5 min x 25 mL).
  • Resin capping The resin was capped with 15 mL of 17:2:1 DCM:MeOH:DIPEA solution (2 x 10 min x 15 mL) and then washed with DCM (3 x 3 min x 12 mL) followed by DMF (3 x 3 min x 15 mL).
  • Fmoc-removal The Fmoc-removal was performed using in 1:4 piperidine/DMF solution (1 x 10 min x 15 mL and 1 x 5 min x 15 mL).
  • Coupling The resin was washed with DMF (5 x 5 min x 15 mL) and coupled to N-Fmoc-Gly-OH, assuming resin loading of 1.1 mmol/g.
  • the coupling cocktail containing N-Fmoc-Gly-OH (2.29 g, 5 eq, 7.7 mmol), HOBt (1.18 g, 5 eq, 7.7 mmol), HATU (2.87 g, 4.9 eq, 7.55 mmol) and DIPEA (1.61 mL, 6 eq, 9.24 mmol) dissolved in 12 mL of DMF was pre-activated for 5 mins prior to addition to the resin.
  • the coupling reaction was run for 30 min and the completion of the reaction was monitored using Ninhydrin stain.
  • the coupling cocktail containing 3-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)propanoic acid (174 mg, 2.1 eq, 1.03 mmol), HOBt (278 mg, 3.7 eq, 1.81 mmol), HATU (373 mg, 2 eq, 0.98 mmol) and DIPEA (0.342 mL, 4 eq, 1.96 mmol) dissolved in 3.5 mL of DMF was pre-activated for 5 mins prior to addition to the resin.
  • the coupling reaction was run for 30 min and the completion of the reaction was monitored using Ninhydrin stain.
  • Linker-Payload LP1 To a 25 ml round bottom flask, Linker Compound L1 (50 mg, 1.0 eq) was added in anhydrous DMF (1 mL) and cooled to 0 ⁇ C. To the solution was added HATU (42 mg, 1.0 eq), and DIPEA (3 eq) followed by Compound P20 (80 mg, 0.95 eq). The reaction mixture was warmed to room temperature and stirred for 1 hrs. LCMS showed formation of desired product. The reaction mixture was diluted with acetonitrile (1 mL) and purified via reverse phase purification by using acetonitrile and water (2 mmol HCOONH 4 ) as mobile phase.
  • Example 42 Synthesis of Compound LP3 Step 1 - Intermediate Compound 110: tert-butyl ((3S,3aR,6S,6aR)-6-(1-(9H-fluoren-9-yl)-3,6,9- trioxo-2-oxa-4,7,10-triazadodecan-12-amido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
  • a mixture of (((9H-fluoren-9- yl)methoxy)carbonyl)glycylglycylglycine (11.2 g, 27.22 mmol) was added to tert-butyl ((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)carbamate (9.98 g, 40.83 mmol), O-(7- Azabenzotria
  • the resulting mixture was stirred at 25 °C for 2 hours.
  • the reaction mixture was diluted with DCM (30 mL) and removed under reduced pressure to give crude Intermediate Compound 115.
  • the crude Intermediate Compound 115 was added to the PNU-159682 carboxylic acid (CAS No.: 1204819-92-0; 1.1 g, 1.75 mmol), O-(7- Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoro-phosphate (1.000 g, 2.63 mmol) and N,N-diisopropylethylamine (0.680 g, 5.26 mmol) in DMA (30 mL) at 0°C under nitrogen.
  • Step 2 Intermediate Compound 111: tert-butyl ((3S,3aR,6S,6aR)-6-(14-amino-4,7,10,13-tetraoxo- 3,6,9,12-tetraazatetradecanamido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
  • diethylamine (3.89 g, 53.21 mmol) was added to Intermediate Compound 111-A (4g, 5.32 mmol) in THF (50 mL)/water (10 mL) at 25°C under nitrogen.
  • the resulting mixture was stirred at 25 °C for 1 hour.
  • the solvent was removed under reduced pressure.
  • Step 3 Intermediate Compound 116: tert-butyl ((3S,3aR,6S,6aR)-6-(1-(9H-fluoren-9-yl)- 3,30,33,36,39,42-hexaoxo-2,7,10,13,16,19,-22,25,28-nonaoxa-4,31,34,37,40,43- hexaazapentatetracontan-45-amido)hexahydrofuro-[3,2-b]furan-3-yl)carbamate
  • Intermediate Compound 111 (1 g, 1.89 mmol) was added to 1-(9H-fluoren- 9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatriacontan-30-oic acid (1.227 g, 1.89 mmol), HATU (1.436 g, 3.78 m
  • the crude product was purified by flash C18-flash chromatography, elution gradient 0 to 70% MeCN in water to afford crude product (0.4 g).
  • the crude product was further purified by preparative HPLC to afford Compound LP7 (120 mg, 6.92 %) as a red solid.
  • Step 1 Intermediate Compound 130: (9H-fluoren-9-yl)methyl (2-(((((3R,3aR,6R,6aR)-6- hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)-amino)-2-oxoethyl)carbamate
  • (2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 500g, 1357.26 mmol
  • was added to (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol (“isomannide”, CAS No.641-74-7; 7934 g, 54290.44 mmol) and pyridin-1-ium 4- methylbenzenesulfonate (51.2 g, 203.59 mmol) in D
  • Step 3 Intermediate Compound 132: benzyl (2-(((((3R,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2- b]furan-3-yl)oxy)methyl)amino)-2-oxoethyl)carbamate
  • benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (393 g, 1575.98 mmol) was added to N-ethyl-N-isopropylpropan-2-amine (339 g, 2626.64 mmol) and Intermediate Compound 131 (305 g, 1313.32 mmol) in DMF (1 L) under nitrogen.
  • Step 7 Intermediate Compound 136: benzyl (2-(((((3R,3aS,-6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)-amino)-2-oxoethyl)carbamate
  • Boc2O 57.2 mL, 246.31 mmol
  • DCM 500 mL
  • Step 1 Intermediate Compound 142: 5-(((tert-butyldimethyl-silyl)oxy)methyl)-2- hydroxybenzaldehyde
  • TBS-Cl 178 g, 1183.05 mmol
  • DCM 200 mL
  • Imidazole 134 g, 1971.75 mmol
  • DCM 500 mL
  • Step 2 Intermediate Compound 143: (2S,3S,4S,5R)-methyl 3,4,5-triacetoxy-6-(4-((tert- butyldimethylsilyloxy)methyl)-2-formylphenoxy)-tetrahydro-2H-pyran-2-carboxylate
  • 4A molecular sieves 240 g, 450.43 mmol
  • Silver(I) oxide 313 g, 1351.29 mmol
  • MeCN 2000 mL
  • Step 2 Intermediate Compound 155: 1-allyl 6-(tert-butyl) (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)hexanedioate To a 250 mL round bottom flask was added (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- (tert-butoxy)-6-oxohexanoic acid (5 g, 11.38 mmol) in dry DMF (20 mL) under nitrogen gas.
  • Step 3 Intermediate Compound 161: tert-butyl ((3S,3aR,6S,6aR)-6-(2-aminoacetamido)- hexahydrofuro[3,2-b]furan-3-yl)carbamate
  • dimethylamine 8.38 g, 114.59 mmol
  • the resulting mixture was stirred at 15 °C for 2 hours.
  • the solvent was removed under reduced pressure to afford Intermediate Compound 161 (3.00 g, 87 %) as a yellow solid.
  • Step 4 Intermediate Compound 171: N-(1-(((3R,3aS,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3- yl)oxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamide
  • Intermediate Compound 170 300 mg, 0.46 mmol
  • HFIP HFIP
  • Step 1 Intermediate Compound 178: (tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3- hydroxyazetidine-1-carboxylate
  • tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (10 g, 49.44 mmol) was added to N-ethyl-N-isopropylpropan-2-amine (19.17 g, 148.33 mmol) and N- (benzyloxycarbonyloxy)succinimide (14.79 g, 59.33 mmol) in THF (150 mL) under nitrogen.
  • Step 2 Intermediate Compound 179: tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-(2-(tert- butoxy)-2-oxoethoxy)azetidine-1-carboxylate
  • Intermediate Compound 178 13 g, 38.65 mmol
  • tert-butyl 2-bromoacetate 9.05 g, 46.38 mmol
  • sodium hydride 1.55 g, 77.29 mmol
  • Step 3 Intermediate Compound 180: tert-butyl 3-(aminomethyl)-3-(2-(tert-butoxy)-2- oxoethoxy)azetidine-1-carboxylate
  • Intermediate Compound 179 (9 g, 19.98 mmol) was added to ROYER TM Palladium Catalyst Powder (CAS 7440-05-3, 6.07 g, 19.98 mmol) in THF (120 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 6 hours. The reaction mixture was filtered through silica and washed with THF (3 x 50 mL).
  • Step 5 Intermediate Compound 182: 2-((3-((((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)- 1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetic acid
  • Intermediate Compound 181 (615 mg, 1.14 mmol) in DCM (4 mL) was added TFA (4 mL). The reaction mixture was stirred at room temperature for 1.5 h.
  • Step 6 (part 2) - Intermediate Compound 183: 2-((3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamido)methyl)-1-(2,5,8,11,14,17,20,23,26,29,-32,35-dodecaoxaoctatriacontan-38- oyl)azetidin-3-yl)oxy)acetic acid
  • 2,5-Dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoate (0.911 g, 3.42 mmol) was added to Intermediate Compound 183-A (2.5 g, 3.42 mmol) and DIEA (0.597 mL, 3.42 mmol) in DMA (30 mL) at 25°C under nitrogen.
  • Step 10 Compound LP13: In a round bottom flask, 1-Hydroxypyrrolidine-2,5-dione (147 mg, 1.27 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 400 mg, 0.64 mmol) and DIC (0.199 mL, 1.27 mmol) in DMA (3 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. Then Intermediate Compound 186 (731 mg, 0.51 mmol) and DIEA (0.668 mL, 3.82 mmol) was added into the mixture, then the obtained reaction solution was stirred at 25 °C for 2 hours.
  • Step 1 Intermediate Compound 192: (2S,3S,4S,5R)-2-((allyloxy)carbonyl)-6-bromotetrahydro-2H- pyran-3,4,5-triyl triacetate
  • 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (26.5 ml, 177.35 mmol) was added dropwise to a 1-L round bottom flask containing (2S,3S,4S,5R,6R)-3,4,5,6- tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid (31.3 g, 161.22 mmol) in DMF (100 ml) at 21 °C.
  • Step 2 Intermediate Compound 193: (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(4-(((tert- butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate
  • molecular sieves (4 ⁇ beads, 5.0 g), silver oxide (29.2 g, 125.8 mmol) and acetonitrile (150 mL), producing a black slurry.
  • CHT ceramic hydroxyapatite
  • the pH is brought back to neutral by running 5 – 10 CV of 10X PBS (pH 7.0) through the column, then equilibrated using 5 – 10 CV of 10 mM sodium phosphate buffer (pH 7.0).
  • the conductivity reading should reach a minimum before loading the conjugated antibody sample.
  • the conjugated antibody sample is diluted five-fold with water, then loaded into the column.
  • the column is washed with 10 mM sodium phosphate buffer until the 330 nm signal is at or close to 0 (this 330 nm signal corresponds to free payload in the sample).
  • the sample is eluted with 10 mM sodium phosphate buffer with 2 M NaCl (pH 7.0) and the fractions with high 280 nm signals are collected.
  • the crude reaction mixture was purified using CHT-type-II column chromatography, after which, the eluted antibody-drug conjugate (ADC) was immediately dialyzed into 20 mM histidine and 240 mM sucrose at pH 6.
  • ADC eluted antibody-drug conjugate
  • Endotoxin reading x EU/ml obtained by diluting 10-fold in biology grade water using 0.1-0.001 EU/mL sensitivity cartridge. * The free drug % was measured using reverse phase HPLC.0.1% is the LOD, and the free drug is below the limit of detection.
  • DAR drug-antibody ratio
  • SEC size exclusion chromatography
  • HIC hydrophobic interactions chromatography
  • EU endotoxin units.
  • Negative control ADCs Negative control ADCs: Negative control ADCs (Control ADC1-ADC6) were prepared in an analogous manner to that described above, using a negative control antibody NIP228 (as disclosed in WO 2015/127273 A1, the contents of which are incorporated by reference).
  • NIP228 is an IgG1 that has complementary binding to transferrin, a transmembrane protein whose expression is 5 low on the tested cell lines, therefore making it a suitable negative control.
  • Example 57 In vitro cytotoxicity assessment of Anthracycline payloads Cytotoxicity of anthracycline payloads was investigated using Multiple myeloma (MM) cell lines (MM1.s, MM1.r, NCI-H929, RPMI8826), gastric cell lines (SNU601, SNU620) and colorectral cancer (CRC) cell lines (LS411N, LS180, HT-29, LS1034).
  • MM Multiple myeloma
  • SNU601, SNU620 gastric cell lines
  • CRC colorectral cancer
  • the efficacy of the payloads was compared to PNU- 159682, with SG3924 (a Topo1 inhibitor) and/or MMAE as internal positive controls.
  • DMSO control was also included in all the studies with DMSO at 0.1%.
  • Cells were plated in 384 well plates at the density of 4000 cells /well in 30 ⁇ l of the respective media (MM) and 750 cells/well for adherent (CRC). The cells were treated with anthracycline payloads after 24hrs of incubation of the cells at 37°C in a humidified incubator at 5% CO 2 atmosphere.10 ⁇ l of 4-fold serial dilutions of each anthracycline payload and positive control in growth medium was added to the cells making the final volume to 40 ⁇ l.
  • the final concentration ranged from 100nM to 0.095fM. Each dilution was done in triplicate. After 3 days of incubation, the plates were taken out from the incubator and equilibrated to the room temperature.40 ⁇ 1Lof CellTiter-Glo® 2.0 Luminescent Solution (Promega, CatNo# G9242) was added to each well and, after shaking the plates at 45Orpm for 5min followed by a 10 min incubation without shaking, luminescence was measured on a Perkin Elmer Envision 2105 with an integration time of 0.1 second per well. The percent cell viability for the treated cells were calculated by normalizing to the untreated control.
  • MMAE and SG3924 were introduced as internal positive controls with a potency range ⁇ 1- 10 nM and ⁇ 0.1-0.4 nM IC50, respectively, against the MM cell lines. MMAE was also used as an internal positive control against the solid cell lines.
  • Table 11 IC50 values for the payloads in gastric cell lines (SNU601, SNU620) and colorectral cancer cell lines (LS411N, LS180, HT-29, LS1034).
  • Figures 1A-1C shows % cell viability against payload concentration for certain payloads in gastric and CRC cell lines, compared to PNU-159682.
  • ADC1-ADC6 Trastuzumab-based antibody-drug conjugates (ADC1-ADC6) and matched isotype controls (Control ADC1-ADC6) were tested in-vitro in cell lines with varying levels of HER2 expression. Cells were maintained in appropriate media (N87, HT-29, + 10% FBS) and passaged when 80-90% confluent. On the day of the experiment (Day 1) cells were detached from the culture flask using 3mL phenol- red free TrypLE® (Invitrogen), and neutralized using 7mL of complete media.
  • ADCs were serially diluted in media to a top concentration of 160ug/mL (4x, ⁇ 1.5uM), and lOuL of each serially diluted ADC was added to 30uL of cells will be lx at 60ug/mL final ( ⁇ 350nM). ADCs were allowed to incubate with cells for 72hrs. On day 3, CellTiterGlo 2.0 terminal reagent was used to evaluate the viability of the cells.
  • the two cell models tested have different levels of HER2 expression (N87: breast, HER2 high; HT- 29: colon, HER2 mid/low).
  • the activity of the trastuzumab ADCs strongly correlates with the level of expression of HER2.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The specification relates to anthracycline compounds and conjugates thereof. The specification further relates to the use of the compounds and conjugates in treating cancer, and to processes for preparing the compounds and conjugates.

Description

ANTHRACYCLINE DERIVATIVES AND CONJUGATES AND USES THEREOF CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This specification claims the benefit of priority to U.S. Provisional Patent Application No. 63/518,651 (filed 10 August 2023) and U.S. Provisional Patent Application No.63/631,545 (filed 9 April 2024). The entire contents of the above-referenced patent applications is incorporated by reference into this specification for all purposes. FIELD This specification relates to anthracycline derivatives and to conjugates thereof, as well as to pharmaceutical compositions containing them. This specification further relates to the use of the anthracycline derivatives and conjugates in treating diseases such as cancer. This specification further relates to processes and intermediate compounds involved in the preparation of the anthracycline derivatives and conjugates thereof. BACKGROUND Anthracyclines are a class of potent chemotherapy drugs used in the treatment of cancer. The most commonly used anthracyclines include doxorubicin, daunorubicin and idarubicin. These compounds have been used in wide range of therapeutics including breast cancer, leukaemia, lymphomas, and in other solid tumours. The mechanism of action for anthracyclines primarily involves DNA intercalation, inhibition of topoisomerase II, and the generation of free radicals, ultimately leading to DNA damage and apoptosis of cancer cells. However, the clinical use of anthracyclines is often limited by their systemic toxicity and does-dependent side effects (as a result of their high potency). To mitigate the potential toxic effects of anthracyclines, various strategies have been used. Recently, targeted therapy approach has been utilized to minimize side effects of anthracyclines. One such approach is the design of anthracycline antibody-drug conjugates (ADCs). ADCs utilize the targeting capabilities of monoclonal antibodies (mAbs) to selectively deliver cytotoxic payloads (such as anthracyclines) to tumour cells. This selective delivery can increase the efficacy of the cytotoxic payloads while minimizing systemic toxicity. Several anthracycline ADCs have been investigated in preclinical and clinical settings. Doxorubicin and daunorubicin have been used in early examples of anthracycline ADCs. More recently, morpholino derivatives such as nemorubicin and its metabolite PNU-159682 have been developed and used in ADCs. These morpholino derivatives are reportedly highly potent while having reduced cardiotoxicity; however, off-target toxicity is still seen in ADCs incorporating these derivatives, as a result of their extremely high potency.
Figure imgf000004_0001
There remains a need for anthracycline derivatives having attenuated potency and/or favourable lysosomal stability and/or favourable physicochemical properties, such as solubility and permeability, allowing for a wider therapeutic window and reduced side effects. The anthracycline derivatives and conjugates of the present disclosure may be used for the treatment of diseases such as cancer. SUMMARY According to one aspect, there is provided a compound of Formula (Ia): A-E-X-Y-Z or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000004_0002
E is: (ii) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X; (ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; (iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or (iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y; Xa is - C(O)- or absent; and Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; m is 2 or 3; Y is absent or
Figure imgf000005_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, - S(CH2)2NH-*, -O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to Z; Z is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000005_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. According to another aspect of the specification, there is provided a compound of Formula (I): A-E-X-Y-Z or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000006_0001
E is: (i) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X; (ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; (iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or (iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y; Xa is - C(O)- or absent; and Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; m is 2 or 3; Y is absent or
Figure imgf000006_0002
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z; and Q is NH, S, O, - S(CH2)2NH-*, -O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to Z; Z is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000007_0001
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl; provided that when E is -heterocycle-C(O)NH-, then X is absent or -(CH2)m-Xb-; and provided that when Z is -C(O)C1-4 alkyl,
Figure imgf000007_0002
absent, then Xb is not -C(O)NH-. According to another aspect, there is provided a compound of Formula (IIa): or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000007_0003
E1 is: i) -NR-; ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X1; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y1; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y1; m is 2 or 3;
Figure imgf000008_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S-(CH2)2NH-*, -O- (CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1; Z1 is H, C1-4 alkyl, C(O)C1-4 alkyl,
Figure imgf000008_0002
and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. According to another aspect, there is provided a compound of Formula (II): A-E1-X1-Y1-Z1 or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000009_0001
E1 is: i) -NR-; ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X1; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y1; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y1; m is 2 or 3; provided that when E1 is -NR- or -heterocycle-C(O)NH-, then X1 is absent or -(CH2)m- Xb-; Y1 is
Figure imgf000009_0002
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1; and Q is NH, S, O, -S-(CH2)2NH-*, -O- (CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1; Z1 is H, C1-4 alkyl, C(O)C1-4 alkyl,
Figure imgf000010_0001
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. In a further aspect there is provided a pharmaceutical composition comprising a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. In a further aspect there is provided a pharmaceutical composition comprising a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. In a further aspect there is provided a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof, for use in therapy. In a further aspect there is provided a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, for use in therapy. In a further aspect there is provided a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. In a further aspect there is provided a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. In a further aspect there is provided the use of a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament. In a further aspect there is provided the use of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament. In a further aspect there is provided the use of a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer. In a further aspect there is provided the use of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer. In a further aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof. In a further aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof. In further aspects, there are provided methods and intermediates for preparing compounds of Formula (Ia) or (IIa), or a pharmaceutically acceptable salt thereof. In further aspects, there are provided methods and intermediates for preparing compounds of Formula (I) or (II), or a pharmaceutically acceptable salt thereof. According to another aspect, there is provided a compound of Formula (IIIa): A-E-X-Y-L1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or
Figure imgf000011_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. According to one aspect, there is provided a compound of Formula (III): A-E-X-Y-L1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or
Figure imgf000011_0002
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L1; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. According to another aspect, there is provided a compound of Formula (IVa): A-E1-X1-Y1-L1 or a salt thereof, wherein A, E1 and X1 are as defined herein; Y1 is
Figure imgf000012_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. According to another aspect, there is provided a compound of Formula (IV): A-E1-X1-Y1-L1 or a salt thereof, wherein A, E1 and X1 are as defined herein; Y1 is
Figure imgf000012_0002
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L1; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. In further aspects, there are provided methods for preparing compounds of Formula (III) or (IV), or a salt thereof. In further aspects, there are provided methods for preparing compounds of Formula (IIIa) or (IVa), or a salt thereof. According to one aspect, there is provided a conjugate of Formula (Va): (A-E-X-Y-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E and X is as defined herein, Y is absent or
Figure imgf000012_0003
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and Ab is an antibody or antigen-binding fragment thereof. According to another aspect, there is provided a conjugate of Formula (V): (A-E-X-Y-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E and X is as defined herein, Y is absent or
Figure imgf000013_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L2; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and Ab is an antibody or antigen-binding fragment thereof. According to another aspect, there is provided a conjugate of Formula (VIa): (A-E1-X1-Y1-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E1 and X1 is as defined herein, Y1 is
Figure imgf000013_0002
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and Ab is an antibody or antigen-binding fragment thereof. According to another aspect, there is provided a conjugate of Formula (VI): (A-E1-X1-Y1-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E1 and X1 is as defined herein, Y1 is
Figure imgf000014_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L2; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and Ab is an antibody or antigen-binding fragment thereof. In a further aspect there is provided a pharmaceutical composition comprising a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In a further aspect there is provided a pharmaceutical composition comprising a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In a further aspect there is provided a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof, for use in therapy. In a further aspect there is provided a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, for use in therapy. In a further aspect there is provided a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. In a further aspect there is provided a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. In a further aspect there is provided the use of a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament. In a further aspect there is provided the use of a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament. In a further aspect there is provided the use of a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer. In a further aspect there is provided the use of a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer. In a further aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof. In a further aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof. In a further aspect there is provided a method of preparing a conjugate of Formula (Va) or (VIa), or a pharmaceutically acceptable salt thereof, by reacting a compound of Formula (IIIa) or (IVa), respectively, or a salt thereof, with an antibody or an antigen-binding fragment thereof. In a further aspect there is provided a method of preparing a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, by reacting a compound of Formula (III) or (IV), respectively, or a salt thereof, with an antibody or an antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1C depict results from in vitro studies described in Example 57, demonstrating inhibitory activity of payloads of the present disclosure against various cancer cell lines, compared to PNU-159682. Figures 2A-2B depict results from in vivo studies described in Example 59, demonstrating effect of anti-HER2 ADCs (ADC-1 to ADC-6) and negative control ADCs (Control ADCs 1-6) on tumour growth in xenograft models. DETAILED DESCRIPTION Definitions So that the present specification may be more readily understood, certain terms are explicitly defined below. In addition, definitions are set forth as appropriate throughout the detailed description. Where examples are provided for a definition, they are not limiting. The prefix Cx-y, where x and y are integers, indicates the numerical range of carbon atoms that are present in a group. As used herein the term “alkyl” refers to a saturated, linear or branched, hydrocarbon radical having the specified number of carbon atoms. Examples of C1-3 alkyl groups include methyl (Me), ethyl (Et), n-propyl (nPr), and i-propyl (iPr). Examples of C1-4 alkyl groups include methyl (Me), ethyl (Et), n-propyl (nPr), i-propyl (iPr), n-butyl (nBu), i-butyl (iBu), s-butyl (sBu), and t-butyl (tBu). Examples of C1-6 alk l i l d th l th l l i l b t l i-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl. As used herein the term “cycloalkyl” refers to a saturated, cyclic hydrocarbon radical having the specified number of carbon atoms. Examples of C3-4 cycloalkyl groups include cyclopropyl and cyclobutyl. As used herein the term “heterocycle” refers to a ring structure containing the specified number of ring atoms (referred to as “members”), wherein one or more of the ring atoms is a heteroatom (i.e., oxygen, nitrogen, or sulfur, unless otherwise defined), with the remaining ring atoms being carbon. A heterocycle may be monocyclic or multicyclic (e.g. bicyclic), unless otherwise specified. As used herein the term “bicyclic” refers to a fused, bridged or spirocyclic bicyclic ring. Certain embodiments of this specification include a heterocycle which is said to optionally contain an oxygen atom. In further embodiments said heterocycle does not contain an oxygen atom. Certain embodiments of this specification recite a saturated heterocycle that contains more than one heteroatom. In such embodiments, the heteroatoms present in the heterocycle may be non- adjacent (i.e. the heteroatoms may be separated by at least one carbon atom in the heterocycle). As used herein,
Figure imgf000016_0001
refers to a ring; for example, is ring F1. As used herein the term “linker for connection to an antibody, or antigen-binding fragment thereof” refers to a group of atoms capable of forming a covalent bond to an antibody, or antigen- binding fragment thereof, through a chemical reaction. For example, the linker in the compound of Formula (III), (IIIa), (IV) or (IVa) (e.g. the compound of Formula (III) or (IV)) enables the formation of a covalent bond between said compound and an antibody or antigen-binding fragment thereof. Accordingly, an antibody-drug conjugate (ADC) derivatised from the compound of Formula (III), (IIIa), (IV) or (IVa) (e.g. the compound of Formula (III) or (IV)) may be formed, for example a conjugate of Formula (V), (Va), (VI) or (VIa) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof may be formed. As used herein the term “conjugation group for conjugation to an antibody, or antigen-binding fragment thereof” refers to an atom or group of atoms capable of forming a covalent bond to an antibody, or antigen-binding fragment thereof, through a chemical reaction. The use of “ ” (referred to as a “wavy line”), “ ” (referred to as an “asterisk-wavy line”), or “ ” in formulae of this specification denotes the point of covalent attachment to a chemical moiety. indicates the point of attachment to an antibody, or antigen-binding fragment thereof. Certain embodiments of this specification include a group which is said to be “optionally substituted”. In further embodiments said group is unsubstituted. Certain embodiments in this specification include a group that can be “absent”. Where a group is absent, the groups on either side of this are linked by a direct covalent bond. For example, in some embodiments of Formula (II) or (IIa) (e.g. Formula (II)), X1 is absent; in such embodiments, Formula (II) or (IIa) (e.g. Formula (II)) could be rewritten as: A-E1-Y1-Z1. As another example, in some embodiments of Formula (II) or (IIa) (e.g. Formula (II)), X1 is -Xa-(CH2)m-Xb-, wherein Xa is absent; in such embodiments, X1 can be described as -(CH2)m-Xb-. Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. Compounds of Formula (I), (Ia), (II) and (IIa) In one aspect, this specification provides a compound of Formula (Ia), or a pharmaceutically acceptable salt thereof, as defined above. In another aspect, this specification provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined above. In another aspect, this specification provides a compound of Formula (IIa), or a pharmaceutically acceptable salt thereof, as defined above. In another aspect, this specification provides a compound of Formula (II), or a pharmaceutically acceptable salt thereof, as defined above. The compounds of Formulae (I), (Ia), (II) or (IIa) (e.g. the compounds of Formula (I) and (II)), or pharmaceutically acceptable salts thereof, are anthracycline derivatives. These compounds may be useful in the treatment of cancer. The compounds may be directly administered to a patient for treating cancer. Alternatively, the compounds may be used to prepare ADCs, which may be administered to a patient for treating cancer (such as conjugates of Formula (V), (Va), (VI) or (VIa), e.g. conjugates of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, as disclosed herein). In such instances, the compounds of Formulae (I), (Ia), (II) or (IIa) (e.g. the compounds of Formulae (I) or (II)), or pharmaceutically acceptable salts thereof, may be referred to as payloads. The payloads may be released from the ADC at a target site, for example via enzymatic cleavage of a linker portion of the ADC. Compounds of Formula (I), (Ia), (II) or (IIa) (e.g. compounds of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, may have attenuated potency compared to known anthracyclines, such as PNU-159682. Compounds of Formula (I), (Ia), (II) or (IIa) (e.g. compounds of Formula (I) or (II)) may exhibit advantageous physicochemical properties (for example, lower lipophilicity, higher aqueous solubility, higher permeability and/or lower plasma protein binding), and/or favourable toxicity profiles (for example a decreased activity at hERG), and/or favourable metabolic or pharmacokinetic profiles, in comparison with known anthracyclines. Compounds of Formula (I), (Ia), (II) or (IIa) (e.g. compounds of Formula (I) or (II)) may exhibit favourable lysosomal stability. Any of these properties may result in a wider therapeutic window compared to known anthracyclines and conjugates (e.g. ADCs) thereof; thus reducing side effects. As such, the compounds of Formula (I), (Ia), (II) or (IIa) (e.g. the compounds of Formula (I) or (II)) and conjugates thereof may be especially suitable as therapeutic agents, such as for the treatment of cancer. Compounds of Formula (I) and (Ia) In one aspect, provided herein is a compound of Formula (Ia): A-E-X-Y-Z or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000018_0001
E is: i) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X; (ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl;iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y; Xa is - C(O)- or absent; and Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; m is 2 or 3; Y is absent or
Figure imgf000019_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, - S(CH2)2NH-*, -O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to Z; Z is H, C1-4 alkyl, C(O)C1-4 alkyl,
Figure imgf000019_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. In another aspect, provided herein is a compound of Formula (I): A-E-X-Y-Z or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000020_0001
E is: i) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X; ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl;iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; m is 2 or 3; Y is absent or
Figure imgf000020_0002
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z; and Q is NH, S, O, - S(CH2)2NH-*, -O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to Z; Z is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000021_0001
integer from 1 to 5; and each Rz is independently H,
Figure imgf000021_0002
alkyl, phenyl or benzyl; provided that when E is -heterocycle-C(O)NH-, then X is absent or –(CH2)m-Xb-; and provided that when Z is -C(O)C1-4 alkyl,
Figure imgf000021_0003
absent, then Xb is not -C(O)NH-. Unless otherwise specified, the following embodiments relate to compounds of Formula (I) or to compounds of Formula (Ia). In embodiments, A is:
Figure imgf000021_0004
. In other embodiments, A is:
Figure imgf000022_0001
In embodiments, E is a) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X; b) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or c) -heterocycle- NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-. In embodiments, E is selected from:
Figure imgf000022_0002
wherein Ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and Ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In embodiments, E is:
Figure imgf000022_0003
wherein Ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In embodiments, the bicyclic heterocycle of Ring Ea is a bridged bicycle. In further embodiments, the bicyclic heterocycle is a 7-8 membered bridged bicycle. In embodiments, E is selected from:
Figure imgf000023_0001
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In further embodiments, E is selected from:
Figure imgf000023_0002
. In further embodiments, E is selected from: .
Figure imgf000023_0003
In further embodiments, E is , optionally . In embodiments, the bicyclic heterocycle of Ring Ea is a spirocycle. In further embodiments, the bicyclic heterocycle is an 8-9 membered spirocycle. In embodiments in which Ring Ea is a spirocycle, the two nitrogen atoms present in the heterocycle of Ring Ea are in the same ring of the spirocycle. In embodiments, E is selected from:
Figure imgf000023_0004
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In further embodiments
Figure imgf000024_0001
. In embodiments in which Ring Ea is a spirocycle, the two nitrogen atoms present in the heterocycle of Ring Ea are in separate rings of the spirocycle. In embodiments, E is selected from:
Figure imgf000024_0002
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In embodiments, the bicyclic heterocycle of Ring Ea is a fused bicycle. In further embodiments, the bicyclic heterocycle is an 8-membered fused bicycle. In further embodiments,
Figure imgf000024_0003
. I
Figure imgf000024_0004
n embodiments, E is , wherein Ring Ec is a monocyclic 5-8 membered saturated heterocycle having the two nitrogen atoms shown, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; and wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In embodiments, the heterocycle of Ring Ec is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. In further embodiments, the geminal substituent is geminal dimethyl. In embodiments, Ring Ec is a piperazine geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. In further embodiments, the geminal substituent is geminal dimethyl. In embodiments, E is selected from:
Figure imgf000025_0001
and the asterisk-wavy line indicates the point of attachment to X. In further embodiments E is
Figure imgf000025_0002
. In embodiments, Ring Ec is a monocyclic 5, 7 or 8 membered saturated heterocycle having the two nitrogen atoms shown, wherein the heterocycle is unsubstituted. In further embodiments, E is:
Figure imgf000025_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In further embodiments E is
Figure imgf000025_0004
. In embodiments, E is -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-. In further embodiments,
Figure imgf000025_0005
, wherein ring Ed is a monocyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In further embodiments, ring Ed is a piperidine.
Figure imgf000026_0001
In embodiments, E is wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In embodiments, E is -heterocycle-C(O)NH-, wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -C(O)NH-. In further embodiments,
Figure imgf000026_0002
, wherein ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. In further embodiments, the heterocycle is a bridged bicyclic heterocycle. In embodiments, E is:
Figure imgf000026_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attac In further embodiments, E is:
Figure imgf000026_0005
. In further mbodimen 2 O embo ts, R her Nd is eH *hment to X. . further embodiments, E is:
Figure imgf000026_0004
iments, R is C3-4 cycloalkyl. In further embod C alkyl. In further embodiments, R is meth in * indicates the point of attachment to Y. In embodiments, X is -C(O)CH2NH-*, wherein * indicates the point of attachment to Y. In embodiments, X is -Xa-(CH2)m-Xb-, wherein Xa is -C(O)- or absent; Xb is -C(O)NH-*, -O- * . In embodiments, R is H N N NHiments, R is cyclopropyl. In further e 1-4 yl. In embodiments, X is absent. Where X is absent, Formula (I) or (Ia) (e.g. Formula (I)) may be rewritten as: A-E-Y-Z. In embodiments, X is -C(O)CHO-*, w or -NH-, wherein * indicates the point of attachment to Y; and m is 2 or 3. In further embodiments, m is 2. In further embodiments Xa is -C(O)- In further embodiments Xa is absent (i.e. X is –(CH2)m-Xb-). In further embodiments, Xb is -C(O)NH-*, wherein * indicates the point of attachment to Y. In further embodiments, Xb is -O-. In further embodiments, Xb is -NH-. In embodiments, X is selected from
Figure imgf000027_0001
, wherein the wavy line indicates the point of attachment to E and the asterisk-wavy line indicates the point of attachment to Y. In further embodiments, X is
Figure imgf000027_0002
. In embodiments, E is -heterocycle-C(O)NH- and X is absent. In further embodiments, E is - heterocycle-C(O)NH-, X is -Xa-(CH2)m-Xb- and Xa is absent (i.e. X is –(CH2)m-Xb-). In further embodiments, E is -heterocycle-C(O)NH-, X is -Xa-(CH2)m-Xb-, Xa is absent and Xb is -NH- (i.e. X is – (CH2)m-NH-). In embodiments, wherein the compound is a compound of Formula (Ia), when E is - heterocycle-C(O)NH-, then X is absent or –(CH2)m-Xb-. In embodiments, Y is absent. Where Y is absent, Formula (I) or (Ia) (e.g. Formula (I)) may be rewritten as: A-E-X-Z.
Figure imgf000027_0003
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z.
Figure imgf000027_0004
. In embodiments, Y is
Figure imgf000028_0001
, for example
Figure imgf000028_0002
. In further embodiments,
Figure imgf000028_0004
example
Figure imgf000028_0003
. In embodiments, wherein the compound is a compound of Formula (Ia), Y is:
Figure imgf000028_0005
embodiments, ya or yb are each independently selected from 0, 1 and 2. In further embodiments, at least one of ya or yb is 0. In further embodiments, ya is 0 and yb is 1. In further embodiments, ya is 1 and yb is 0. In further embodiments, the total of ya and yb is no more than 3. In further embodiments, ya and yb are both 0 (i.e. Y is
Figure imgf000028_0006
, optionally
Figure imgf000028_0007
In further embodiments, ya and yb are both independently selected from 1 and 2. In embodiments, Q is NH, S or O. In further embodiments, Q is NH. In further embodiments, Q is S. In further embodiments, Q is O. In embodiments, wherein the compound is a compound of Formula (Ia), Q is -N(C1-3alkyl)-, for example -N(CH3)-. In embodiments, Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z. In further embodiments, Q is -S-(CH2)2NH-*. In further embodiments, Q is -O-(CH2)2NH-*. In further embodiments, Q is -NH-(CH2)2NH-*. In embodiments, X and Y are both absent. In such embodiments, Formula (I) or (Ia) (e.g. Formula (I)) may be rewritten as: A-E-Z. In embodiments, Z is H. In embodiments, Z is C1-4 alkyl. In further embodiments, Z is methyl. In embodiments, Z is -C(O)C1-4 alkyl. In further embodiments, Z is -C(O)CH3. In embodiments,
Figure imgf000029_0001
integer from 1 to 5, and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. In further embodiments, n is 1. In further embodiments, RZ is H. In further embodiments, n is 2, 3, 4 or 5. In further embodiments, one occurrence of RZ is C1-4 alkyl, benzyl or phenyl and the remaining occurrence or occurrences of RZ are H. In further embodiments, Z is:
Figure imgf000029_0002
wherein n1 is 0, 1, 2, 3 or 4 and Rz is C1-4 alkyl, benzyl, or phenyl. In further embodiments, RZ is C1-4 alkyl. In further embodiments, RZ is methyl. In further embodiments, RZ is isopropyl. In further embodiments, RZ is benzyl. In further embodiments, RZ is phenyl. In embodiments, Z is
Figure imgf000029_0003
. In these embodiments, Z comprises amino acid residue(s). For example, when RZ is H, Z could alternatively be defined as (Gly)1-5. When RZ is H and n is 1, Z could alternatively be defined as Gly. Variable Z may comprise amino acid residues in instances where the compounds of Formula (I) or (Ia) (e.g. the compounds of Formula (I)) are payloads that have been released from an ADC, for example once a peptide linker portion has been cleaved. In embodiments, Z is -C(O)C1-4 alkyl,
Figure imgf000029_0004
absent and X is -Xa- (CH2)m-Xb-, wherein Xb is -O- or -NH-. In embodiments, Z is -C(O)C1-4 alkyl,
Figure imgf000029_0005
absent and X is -Xa- (CH2)m-Xb-, wherein Xb is -NH-. In embodiments, wherein the compound is a compound of Formula (Ia), when Z is -C(O)C1-4 alkyl,
Figure imgf000029_0006
absent, then Xb is not -C(O)NH-. In embodiments, the compound of Formula (I) or (Ia) (e.g. the compound of Formula (I)), or a pharmaceutically acceptable salt thereof, is selected from a compound of Table 1, or a pharmaceutically acceptable salt thereof. Table 1.
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0002
In embodiments, the compound of Formula (I) or (la) (e.g. the compound of Formula (I)), or a pharmaceutically acceptable salt thereof, is selected from:
Figure imgf000036_0001
Figure imgf000037_0001
or a pharmaceutically acceptable salt thereof.
Compounds of Formula (II) and (IIa) In an aspect, provided herein is a compound of Formula (IIa): A-E1-X1-Y1-Z1 5 or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000038_0001
E1 is: i) -NR-; 10 ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X1; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; 15 iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and 20 wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; 36
X1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y1; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y1; m is 2 or 3;
Figure imgf000039_0001
, wherein the wavy line indicates the point of attachment to X1 and 5 the asterisk-wavy line indicates the point of attachment to Z1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S-(CH2)2NH-*, -O- (CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1; Z1 is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000039_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. 10 In another aspect, provided herein is a compound of Formula (II): A-E1-X1-Y1-Z1 or a pharmaceutically acceptable salt thereof, wherein A is: 15
Figure imgf000039_0003
iv) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X1; optionally wherein the 20 heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; 37
iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 5 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y1; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y1;10 m is 2 or 3; provided that when E1 is -NR- or -heterocycle-C(O)NH-, then X1 is absent or –(CH2)m-
Figure imgf000040_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1; and Q is NH, S, O, -S-(CH2)2NH-*, -O- (CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1; 15 Z1 is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000040_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. Unless otherwise specified, the following embodiments relate to compounds of Formula (II) or to compounds of Formula (IIa). 20 In embodiments, A is: 38
.
Figure imgf000041_0001
5 In embodiments, E1 is -NR-. In embodiments, E1 is a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X1; optionally wherein the heterocycle is 10 geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. In embodiments, E1 is selected from:
Figure imgf000041_0002
wherein ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and ring Eb is a bicyclic 5-8 membered saturated heterocycle 15 having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. 39
In embodiments, E1 is:
Figure imgf000042_0001
wherein ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. 5 In embodiments, the bicyclic heterocycle of Ring Ea is a bridged bicycle. In further embodiments, the bicyclic heterocycle is a 7-8 membered bridged bicycle. In embodiments, E1 is selected from:
Figure imgf000042_0002
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates 10 the point of attachment to X1. In further embodiments, E1 is selected from:
Figure imgf000042_0003
. further embodiments, E1 is selected from:
Figure imgf000042_0004
. In embodiments, the bicyclic heterocycle of Ring Ea is a spirocycle. In further embodiments, the 15 bicyclic heterocycle is an 8-9 membered spirocycle. In embodiments in which Ring Ea is a spirocycle, the two nitrogen atoms present in the heterocycle of Ring Ea are in the same ring of the spirocycle. In embodiments, E1 is selected from: 40
Figure imgf000043_0001
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. In further embodiments,
Figure imgf000043_0002
. 5 In embodiments in which Ring Ea is a spirocycle, the two nitrogen atoms present in the heterocycle of Ring Ea are in separate rings of the spirocycle. In embodiments, E1 is selected from:
Figure imgf000043_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. 10 In embodiments, the bicyclic heterocycle of Ring Ea is a fused bicycle. In further embodiments, the bicyclic heterocycle is an 8-membered fused bicycle. In further embodiments,
Figure imgf000043_0004
. In further embodiments,
Figure imgf000043_0005
. In embodiments, E1 is
Figure imgf000043_0006
, wherein ring Ec is a monocyclic 5-8 membered saturated heterocycle having the two nitrogen atoms shown, optionally wherein the heterocycle is geminally 41
substituted on a carbon atom with two occurrences of C1-3 alkyl; and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. In embodiments, the heterocycle of Ring Ec is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. In further embodiments, the geminal substituent is geminal dimethyl. 5 In embodiments, Ring Ec is a piperazine geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. In further embodiments, the geminal substituent is geminal dimethyl. In embodiments, E1 is selected from:
Figure imgf000044_0001
, wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. 10 In embodiments, Ring Ec is a monocyclic 5 to 8 membered saturated heterocycle having the two nitrogen atoms shown, wherein the heterocycle is unsubstituted. In further embodiments E1 is
Figure imgf000044_0002
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. In further embodiments, E1 is:
Figure imgf000044_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy 15 line indicates the point of attachment to X1. In further embodiments, E1 is unsubstituted piperazine. In embodiments, E1 is -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-. In embodiments, E1 is
Figure imgf000044_0004
, wherein ring Ed is a monocyclic 5-8 membered saturated 20 heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of 42
attachment to A and the asterisk-wavy line indicates the point of attachment to X1. In further embodiments, ring Ed is a piperidine. 1
Figure imgf000045_0001
In embodiments E is wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. 5 In embodiments, E1 is -heterocycle-C(O)NH-, wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -C(O)NH-. In further embodiments,
Figure imgf000045_0002
wherein ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the 10 wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. In further embodiments, the heterocycle is a bridged bicyclic heterocycle. In embodiments, E1 is:
Figure imgf000045_0003
wherein the wavy line indicates the point of attachment t
Figure imgf000045_0004
and the asterisk-wavy line indicates the point of attachment to X1. In further embodiments, E is:
Figure imgf000045_0005
. In further embodiments, E is:
Figure imgf000045_0006
. 15 In embodiments, R is H. In further embodiments, R is C3-4 cycloalkyl. In further embodiments, R is cyclopropyl. In further embodiments, R is C1-4 alkyl. In further embodiments, R is methyl. In embodiments, X1 is absent. Where X1 is absent, Formula (II) or (IIa) (e.g. Formula (II)) may be rewritten as: A-E1-Y1-Z1. In embodiments, X1 is -C(O)CH2O-*, wherein * indicates the point of 20 attachment to Y1. In embodiments, X1 is -C(O)CH2NH-*, wherein * indicates the point of attachment to Y1. 43
In embodiments, X1 is -Xa-(CH2)m-Xb-, wherein Xa is -C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; and m is 2 or 3. In further embodiments, m is 2. In further embodiments, Xa is -C(O)-. In further embodiments, Xa is absent (i.e. X1 is -(CH2)m-Xb-). In further embodiments, Xb is -C(O)NH-*, wherein * indicates the point of attachment to Y1. In further 5 embodiments, Xb is -NH-. In embodiments, X1 is selected from
Figure imgf000046_0001
wherein the wavy line indicates the point of attachment to E1 and the asterisk-wavy line indicates the point of attachment to Y1. In further embodiments, X1 is
Figure imgf000046_0002
. In embodiments, E1 is -NH- and X1 is absent. In further embodiments, E1 is -NH-, X1 is -Xa-(CH2)m-Xb- 10 and Xa is absent (i.e. X1 is -(CH2)m-Xb-). In further embodiments, E1 is -NH-, X1 is -Xa-(CH2)m-Xb-, Xa is absent and Xb is -NH- (i.e. X1 is -(CH2)m-NH-). In embodiments, E1 is -heterocycle-C(O)NH- and X1 is absent. In further embodiments, E1 is - heterocycle-C(O)NH-, X1 is -Xa-(CH2)m-Xb- and Xa is absent (i.e. X1 is -(CH2)m-Xb-). In further embodiments, E1 is -heterocycle-C(O)NH-, X1 is -Xa-(CH2)m-Xb-, Xa is absent and Xb is -NH- (i.e. X1 is -15 (CH2)m-NH-). In embodiments, wherein the compound is a compound of Formula (IIa), when E1 is - NR- or -heterocycle-C(O)NH-, then X1 is absent or -(CH2)m-Xb-. In compounds of Formula
Figure imgf000046_0003
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1. In embodiments,
Figure imgf000046_0005
example
Figure imgf000046_0004
. In further embodiments, 20
Figure imgf000046_0006
. 44
Figure imgf000047_0001
. 5 In compounds of Formula
Figure imgf000047_0002
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1. In further embodiments, Y1 is:
Figure imgf000047_0003
. In further embodiments, ya and yb are each independently selected from 0, 1 and 2. In further embodiments, at least one of ya or yb is 0. In further embodiments, ya is 0 and yb is 1. In further embodiments, ya is 1 and yb is 0. In further 10 embodiments, the total of ya and yb is no more than 3. In further embodiments, ya and yb are both 0 (i.e. Y1 is
Figure imgf000047_0004
In further embodiments, ya and yb are both independently selected from 1 and 2. In further embodiments of a compound of Formula (IIa), Y1 is as described in relation to a compound of Formula (II). In embodiments, Q is NH, S or O. In further embodiments, Q is NH. In further embodiments, Q is S. 15 In further embodiments, Q is O. In embodiments, Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1. In further embodiments, Q is -S-(CH2)2NH-*. In further embodiments, Q is -O-(CH2)2NH-*. In further embodiments, Q is -NH-(CH2)2NH-*. 45
In embodiments, wherein the compound is a compound of Formula (IIa), Q is -N(C1-3alkyl)-, for example -N(CH3)-. In embodiments, Z1 is H. In embodiments, Z1 is C1-4 alkyl. In further embodiments, Z1 is methyl. In embodiments, Z1 is -C(O)C1- 5 4 alkyl. In further embodiments, Z1 is -C(O)CH3. In embodiments,
Figure imgf000048_0001
integer from 1 to 5, and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. In further embodiments, n is 1. In further embodiments, RZ is H. In further embodiments, n is 2, 3, 4 or 5. In further embodiments, one occurrence of RZ is C1-4 alkyl, benzyl or phenyl and the remaining occurrence or occurrences of RZ are H. In further embodiments, Z1 is: 10
Figure imgf000048_0002
wherein n1 is 0, 1, 2, 3 or 4 and Rz is C1-4 alkyl, benzyl, or phenyl. In further embodiments, RZ is C1-4 alkyl. In further embodiments, RZ is methyl. In further embodiments, RZ is isopropyl. In further embodiments, RZ is benzyl. In further embodiments, RZ is phenyl. In embodiments,
Figure imgf000048_0003
. For example, when RZ is H, Z1 could alternatively be defined as (Gly)1-5. When RZ is H and n is 1, Z1 15 could alternatively be defined as Gly. Variable Z1 may comprise amino acid residues in instances where the compounds of Formula (II) or (IIa) (e.g. Formula (II)) are payloads that have been released from an ADC, for example once a peptide linker portion has been cleaved. In embodiments, the compound of Formula (IIa), or a pharmaceutically acceptable salt thereof, is 20 selected from a compound of Table 2 or Table 2A, or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula (II), or a pharmaceutically acceptable salt thereof, is selected from a compound of Table 2, or a pharmaceutically acceptable salt thereof. 25 46 Table 2.
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Table 2A.
Figure imgf000051_0002
Figure imgf000052_0002
In embodiments, the compound of Formula (II) or (IIa) (e.g. the compound of Formula (II)), or a pharmaceutically acceptable salt thereof, is selected from:
Figure imgf000052_0001
Figure imgf000053_0001
or a pharmaceutically acceptable salt thereof. In further aspects, there are provided methods for preparing compounds of Formula (I), (Ia), (II) or (IIa) (e.g. compounds of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof; and intermediates in said methods. The method may be any method as described herein, for example as described in General Procedures and/or reaction schemes herein. The intermediate may be an Intermediate Compound disclosed herein. The Intermediate Compound may be in a salt form, for example a specific salt form described herein. The Intermediate Compound may be in a non-salt form (i.e. as a free base). Compounds of Formula (III), (IIIa), (IV) and (IVa) In one aspect, provided herein is a compound of Formula (IIIa): A-E-X-Y-L1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or
Figure imgf000054_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. In another aspect, there is provided a compound of Formula (III): A-E-X-Y-L1 or a salt thereof, wherein A, E and X are as defined herein; and Y is absent or
Figure imgf000054_0002
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L1; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. In another aspect, there is provided a compound of Formula (IVa): A-E1-X1-Y1-L1 or a salt thereof, wherein A, E1 and X1 are as defined herein; Y1 is
Figure imgf000055_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. In another aspect, this specification provides a compound of Formula (IV): A-E1-X1-Y1-L1 or a salt thereof, wherein A, E1 and X1 are as defined herein; Y1 is
Figure imgf000055_0002
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L1; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof. The compounds of Formulae (III), (IIIa), (IV) and (IVa) (e.g. the compounds of Formulae (III) and (IV)), or salts thereof, may be referred to “linker payloads”. These compounds comprise a payload portion (derived from the compounds of Formula (I), (Ia), (II) or (IIa), e.g. compounds of Formula (I) or (II)), respectively, connected to a linker portion. The linker portion enables the connection of the payload(s) to an antibody, or antigen-binding fragment thereof, through covalent bond(s) to form an ADC (such as a conjugate of Formula (V), (Va), (VI) or (VIa), e.g. a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, as disclosed herein). The payload(s) may be released from the ADC at a target site, for example via enzymatic cleavage of the linker portion, to provide compounds of Formula (I), (Ia), (II) or (IIa). Compounds of Formula (I), (Ia), (II) or (IIa) may exhibit advantageous properties, as described above. As an example, the payload-linker may be conjugated to an anti-HER2 monoclonal antibody to obtain an anti-HER2 ADC that is effective in treating cancer, such as HER2 positive cancer. The compound of Formula (III), (IIIa), (IV) or (IVa) (e.g. the compound of Formula (III) or (IV)) may be in a salt form, for example a pharmaceutically acceptable salt. Variable A in a compound of Formula (IIIa) or (IVa) may be as described herein, for example, as described in relation to a compound of Formula (Ia) or (IIa). Variable A in a compound of Formula (III) or (IV) may be as described herein, for example, as described in relation to a compound of Formula (I) or (II). Variables E and X in a compound of Formula (IIIa) may be as described herein, for example, as described in relation to a compound of Formula (Ia). Variable Y in a compound of Formula (IIIa) may be as described in relation to a compound of Formula (Ia), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L1. Variables E and X in a compound of Formula (III) may be as described herein, for example, as described in relation to a compound of Formula (I). Variable Y in a compound of Formula (III) may be as described in relation to a compound of Formula (I), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L1. Variables E1 and X1 in a compound of Formula (IVa) may be as described herein, for example, as described in relation to a compound of Formula (IIa). Variable Y1 in a compound of Formula (IVa) may be as described in relation to a compound of Formula (IIa), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L1. Variables E1 and X1 in a compound of Formula (IV) may be as described herein, for example, as described in relation to a compound of Formula (II). Variable Y1 in a compound of Formula (IV) may be as described in relation to a compound of Formula (II), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L1. In a compound of Formula (III), (IIIa), (IV) or (IVa) (e.g. a compound of Formula (III) or (IV)), the linker L1 contains a reactive moiety through which the compound may be connected to an antibody or antigen-binding fragment thereof. For example, L1 may comprise a reactive moiety which reacts with a site in the antibody or antigen-binding fragment thereof, to form a covalent bond between the linker and the antibody or antigen-binding fragment thereof. The reactive moiety in the linker L1 may be an electrophilic group, capable of reacting with a nucleophilic group within the antibody, or antigen-binding fragment thereof. Nucleophilic groups on antibodies include side chain thiol groups (e.g. cysteine), side chain amine groups (e.g. lysine), N-terminal amine groups, and sugar hydroxyl or amino groups where the antibody is glycosylated. Electrophilic groups on the linker include, but are not limited to: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. The antibody or antigen- binding fragment thereof may contain reducible interchain disulfides, such as cysteine bridges. Such antibodies or antigen fragments may be treated with a reducing agent, such as dithiothreitol, so that each disulfide forms two reactive thiol nucleotides that can react with linkers. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2- iminothiolane resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody, or antigen-binding fragment thereof, for example by preparing mutant antibodies comprising one or more non-native cysteine amino acid residues. The reactive moiety in the linker may be a nucleophilic group, capable of reacting with an electrophilic group within the antibody, or antigen-binding fragment thereof. Electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. Nucleophilic groups on a linker include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The reactive moiety in the linker may be a substrate for an enzyme, where the enzyme catalyses conjugation of the reactive moiety to the antibody or antigen-binding fragment thereof. The enzyme may be a transpeptidase, for example sortase A. Sortase A, originally derived from the bacterium Staphylococcus aureus, exhibits high specificity in cross-linking. Sortase A may catalyze the reaction of an amine, e.g. an alkyl amine, in the linker with the antibody or antigen-binding fragment thereof. The linker L1 may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions. In embodiments the linker is cleavable by enzymatic activity, such as a protease or glucuronidase. For example, the linker may be peptide- or glucuronide-based. The cleavage of the linker may be such that the released payload contains a portion of the cleaved linker. For example, if the linker is (Gly)5, then the released payload may contain 1, 2, 3, 4 or 5 glycine residues, depending on the point of enzymatic cleavage. Cleavage of the linker L1, in a compound of Formula (III) or (IIIa) (e.g. a compound of Formula (III)), may lead to the release of a payload which is a compound of Formula (I) or (Ia), respectively (e.g. a compound of Formula (I)). Cleavage of the linker L1, in a compound of Formula (IV) or (IVa) (e.g. a compound of Formula (IV)), may lead to the release of a payload which is a compound of Formula (II) or (IIa), respectively (e.g. a compound of Formula (II)). The linker may be a non-cleavable linker. ADCs containing non-cleavable linkers may rely on degradation of the antibody, or antigen-binding fragment thereof, in order to release the payloadlinker.
The linker may be stable extracellularly, such that the ADC remains intact until it reaches the target cell. The linker may be cleaved once inside the target cell.
Suitable linkers are known in the art; for example, as disclosed in Bargh et al, Chem. Soc. Rev., 2019, 48, 4391-4374 or Su et al, Acta Pharm. Sin. B, 2021, 11, 3889-3907 (incorporated herein by reference in their entirety).
In embodiments, L1 is:
Figure imgf000058_0001
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y in a compound of Formula (III) or (Illa) or the point of attachment to Y1 in a compound of Formula (IV) or (IVa); each RZ1 is independently H, C1.4 alkyl, phenyl or benzyl; q is 3, 4 or 5;
Figure imgf000058_0002
the asterisk-wavy line indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
In embodiments, L
Figure imgf000058_0003
wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1-CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y in a compound of Formula (III) or (Illa) (e.g. a compound of Formula (III)) or the point of attachment to Y1 in a compound of Formula (IV) or (IVa) (e.g. a compound of Formula (IV)); each RZ1 is independently H, CM alkyl, phenyl or benzyl; q is 3, 4 or 5; LB is selected from -(CH2)b- and
Figure imgf000058_0004
, wherein the asterisk-wavy line indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15; and G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof; provided that, for a compound of
Formula (III), when Y is absent, then Xb is not -C(O)NH-.
In further embodiments, LA is absent or *-CH2-NH-.
In further embodiments, L1 is:
Figure imgf000059_0001
. In further embodiments, q is 3. In further embodiments, q is 4. In further embodiments, q is 5. In further embodiments, b is 2. In further embodiments, c is 7. In further embodiments, RZ1 is H. In further embodiments, one occurrence of RZ1 is Ci-4 alkyl, benzyl or phenyl and the remaining occurrences of RZ1 are H. In further embodiments, L1 is:
Figure imgf000059_0002
wherein RZ1 is CM alkyl, benzyl, or phenyl and ql is
2, 3 or 4, optionally wherein ql is 3. In further embodiments, RZ1 is CM alkyl. In further embodiments, RZ1 is methyl. In further embodiments, RZ1 is isopropyl. In further embodiments, RZ1 is benzyl. In further embodiments, RZ1 is phenyl.
In further embodiments, I_B is -(CH2)b-. In further embodiments, LB is
Figure imgf000059_0003
. In embodiments, b is 2. In embodiments, c is 7.
In further embodiments, LB is
Figure imgf000059_0004
. In embodiments, d is 1. In other embodiments, d is 2.
In further embodiments, L1 is selected from:
Figure imgf000060_0001
or a salt thereof.
In further embodiments, LMS:
Figure imgf000060_0002
salt thereof.
In embodiments, L1 is a group of Formula (L-IA):
Figure imgf000060_0003
wherein Ring F1 is a saturated bicyclic ring having 6, 7 , or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is CM alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000061_0001
wherein n5 is 1, 2, 3, 4 or 5, n5A is an integer from 1 to 10, n5B is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
In embodiments, L1 is a group of Formula (L-l):
Figure imgf000061_0002
wherein E2 is (CH2)n2, wherein n2 is 0, 1, 2 or 3,
Figure imgf000061_0003
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Z2 is (CH2)n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof; provided that, for a compound of Formula (III), when Y is absent, then Xb is not -C(O)NH-. In further embodiments of a compound of Formula (L-IA) or (L-l), Q2 is
Figure imgf000062_0001
12 or 13. In further embodiments, R1 is CH3. In further embodiments, E2 is CH2. In further embodiments, X2 is CH2. In further embodiments, Y2 is (CH2)2. In further embodiments, Z2 is (CH2)2. In further embodiments, p is 1. In further embodiments, pl is 0. In further embodiments, pl is 1. In
| ‘■J* further embodiments of a compound of Formula ( L-l A), Z2 is
Figure imgf000062_0002
n5A . In further embodiments, n5B is 2. In further embodiments, n5A is 1.
In further embodiments, L1 is selected from:
Figure imgf000062_0003
Figure imgf000063_0001
In embodiments, L1 is a group of Formula (L-ll I) :
Figure imgf000063_0002
wherein E3 is (CH2)n6, wherein n6 is 0, 1, 2 or 3; R1A is CM alkyl or H,
Figure imgf000063_0004
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or
3, and Q3 is O or NRA, wherein RA is H, CM alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety;
Y3 is O or NRB, wherein RB is H, Ci~4 alkyl or C3-4 cycloalkyl,
Z3 is (CH2)nio or
Figure imgf000063_0003
, wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m3 is an integer from 2 to 17; p2 is 1 or 0; q2 is 1 or 0; p3 is 0 or 1; and G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
In further embodiments, m3 is 9, 10, 11, 12 or 13. In further embodiments, R1A is CH3. In further
Figure imgf000064_0001
further embodiments, E3 is CH2.
In further embodiments, X4 is CH2. In further embodiments, Y3 is O. In further embodiments, Z3 is (CH2)nio. In further embodiments, Z3 is (CH2)2. In further embodiments, Z3 is absent (i.e. Z3 is
(CH2)nl0, where nlO is 0). In further embodiments, Z3 is
Figure imgf000064_0002
. In further embodiments, nlOB is 2. In further embodiments, nlOA is 1. In further embodiments, p2 is 1. In further embodiments, q2 is 1. In further embodiments, p2 is 0. In further embodiments, p3 is 1. In further embodiments, p3 is 0. In further embodiments, L1 is:
Figure imgf000064_0003
In embodiments, L1 is selected from the moieties of Table 3:
Figure imgf000064_0004
Figure imgf000065_0001
Figure imgf000066_0002
In embodiments G1 is selected from:
Figure imgf000066_0001
wherein X3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is Cv6 alkyl.
In embodiments G1 is selected from:
Figure imgf000067_0001
wherein X3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is Ci-6 alkyl. In embodiments G1 is selected from:
Figure imgf000067_0002
wherein X3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is Ci-6 alkyl In further embodiments, G1 is selected from
Figure imgf000068_0001
In further embodiments,
Figure imgf000068_0002
further embodiments, G1 is
Figure imgf000068_0003
. In further embodiments, G1 is
Figure imgf000068_0005
In further embodiments, G1 is
Figure imgf000068_0004
In some embodiments, -
Figure imgf000068_0006
In embodiments L1 is selected from:
Figure imgf000068_0007
or a salt thereof.
In further embodiments, L1 is:
Figure imgf000069_0001
salt thereof.
In embodiments, L1 is selected from the moieties of Table 4:
Figure imgf000069_0002
Figure imgf000070_0001
Figure imgf000071_0001
In embodiments, for a compound of Formula (III) or (Illa), when Y is absent, then Xb is not -C(O)NH-.
In embodiments, the compound of Formula (III) or (Illa) (e.g. the compound of Formula (III)), or a salt thereof, is selected from a compound of Table 5A, or a salt thereof:
Figure imgf000071_0002
In embodiments, the compound of Formula (IVa), or a salt thereof, is selected from a compound of Table 5B or Table 5C, or a salt thereof. In embodiments, the compound of Formula (IV), or a salt thereof, is selected from a compound of Table 5B, or a salt thereof.
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000074_0002
A compound of Formula (III), (Illa), (IV) or (IVa) (e.g. a compound of Formula (III) or (IV)), or a salt thereof, may be prepared by reacting (i.e. conjugating) a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)) or a derivative thereof, or a salt thereof, with a precursor to
L1. The precursor to L1 contains a second reactive moiety which reacts with the compound of Formula (I), (la), (II) or (Ila) (e.g. the compound of Formula (I) or (II)) or derivative thereof. For example, compound LP1 or LP2 may be prepared by reacting compound P20 or P8, respectively, or a salt thereof, with compound LI, or a salt thereof (see example section for LI structure).
Conjugates of Formula (V), (Va), (VI) and (Via)
In one aspect, provided herein is a conjugate of Formula (Va):
(A-E-X-Y-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E and X is as defined herein; Y is absent
Figure imgf000075_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N (Ci-3al kyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker; k is an integer from 1 to 10; and Ab is an antibody or antigen-binding fragment thereof.
In another aspect, provided herein is a conjugate of Formula (V):
(A-E-X-Y-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E and X is as defined herein; Y is absent
Figure imgf000075_0002
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L2; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to I2; each L2 is a linker; k is an integer from 1 to 10; and Ab is an antibody or antigen-binding fragment thereof.
In another aspect, provided herein is a conjugate of Formula (Via):
(A-E1-X1-Y1~L2)k~Ab or a pharmaceutically acceptable salt thereof, wherein each A, E1 and X1 is as defined herein; Y1 is
Figure imgf000075_0003
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(Ci_3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or -
NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker; k is an integer from 1 to 10; and Ab is an antibody or antigen-binding fragment thereof.
In another aspect, provided herein is a conjugate of Formula (VI):
(A-E1-X1-Y1-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E1 and X1 is as defined herein; Y1 is
Figure imgf000076_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk- wavy line indicates the point of attachment to L2; and Q is NH, S, 0, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker; k is an integer from 1 to 10; and Ab is an antibody or antigen-binding fragment thereof.
The conjugates of Formula (V), (Va), (VI) and (Via) (e.g. the conjugates of Formula (V) and (VI) may alternatively be referred to as the compounds of Formula (V), (Va), (VI) and (Via) (e.g. compounds of Formula (V) and (VI). The conjugates of Formula (V), (Va), (VI) and (Via) may also be referred to as ADCs. A conjugate of Formula (V), (Va), (VI) and (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, may undergo cleavage to release a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), respectively. Compounds of Formula (I), (la), (II) or (Ila), and the conjugates of Formula (V), (Va), (VI) and (Via), may exhibit advantageous properties, as described above, which may result in a wider therapeutic window.
Variable A in a conjugate of Formula (Va) or (Via) may be as described herein, for example, as described in relation to a compound of Formula (la) or (Ila). Variable A in a conjugate of Formula (V) or (VI) may be as described herein, for example, as described in relation to a compound of Formula (I) or (II).
Variables E and X in a conjugate of Formula (Va) may be as described herein, for example, as described in relation to a compound of Formula (la). Variable Y in a conjugate of Formula (Va) may be as described in relation to a compound of Formula (la), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L2.
Variables E and X in a conjugate of Formula (V) may be as described herein, for example, as described in relation to a compound of Formula (I). Variable Y in a conjugate of Formula (V) may be as described in relation to a compound of Formula (I), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L2.
Variables E1 and X1 in a conjugate of Formula (Via) may be as described herein, for example, as described in relation to a compound of Formula (Ila). Variable Y1 in a conjugate of Formula (Via) may be as described in relation to a compound of Formula (Ila), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L2. Variables E1 and X1 in a conjugate of Formula (VI) may be as described herein, for example, as described in relation to a compound of Formula (II). Variable Y1 in a conjugate of Formula (VI) may be as described in relation to a compound of Formula (II), but wherein an asterisk-wavy line or an asterisk (*) indicates the point of attachment to L2.
In a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), the linker L2 may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions. In embodiments the linker is cleavable by enzymatic activity, such as protease or glucuronidase. For example, the linker may be peptide- or glucuronide-based. The cleavage of the linker may be such that the released payload contains a portion of the cleaved linker. For example, if the linker is (Gly)5, then the released payload may contain 1, 2, 3, 4 or 5 glycine residues, depending on the point of enzymatic cleavage. Cleavage of the linker L2, in a conjugate of Formula (Va), may lead to the release of a payload which is a compound of Formula (la). Cleavage of the linker L2, in a compound of Formula (V), may lead to the release of a payload which is a compound of Formula (I). Cleavage of the linker, L2, in a conjugate of Formula (Via), may lead to the release of a payload which is a compound of Formula (Ila). Cleavage of the linker, L2, in a conjugate of Formula (VI), may lead to the release of a payload which is a compound of Formula (II).
The linker may be a non-cleavable linker. ADCs containing non-cleavable linkers rely on degradation of the antibody, or antigen-binding fragment thereof, in order to release the payloadlinker.
The linker may be stable extracellularly, such that the ADC remains intact until it reaches the target cell. The linker may be cleaved once inside the target cell.
Suitable linkers are known in the art; for example, as disclosed in Bargh et al, Chem. Soc. Rev., 2019, 48, 4391-4374 or Su et al, Acta Pharm. Sin. B, 2021, 11, 3889-3907 (incorporated herein by reference in their entirety).
In embodiments, L2 is:
Figure imgf000077_0001
, wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1-
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y in a conjugate of Formula (V) or (Va) or the point of attachment to Y1 in a conjugate of Formula (VI) or (Via); each RZ1 is independently H, C1.4 alkyl, phenyl or benzyl; q is 3, 4 or 5; LB is selected from - (CH2)b-,
Figure imgf000078_0001
, wherein the asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein
Figure imgf000078_0002
indicates the point of attachment to the antibody or antigen-binding fragment thereof.
In embodiments,
Figure imgf000078_0003
wherein LA is absent or is selected from *-CH2-NH-
, and *-C(O)-CH2-LA1-CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y in a conjugate of Formula (V) or (Va) (e.g. a compound of Formula (V)) or the point of attachment to Y1 in a conjugate of Formula (VI) or (Via) (e.g. a compound of Formula (VI)); each RZ1 is independently H, Ci.4 alkyl, phenyl or benzyl; q is 3, 4 or 5; LB is selected from — (CH2)b- and
Figure imgf000078_0004
, wherein the asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; and G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein
Figure imgf000078_0005
indicates the point of attachment to the antibody or antigen-binding fragment thereof; provided that, for a conjugate of Formula (V), when Y is absent, then Xb is not -C(O)NH-.
In further embodiments, LA is absent or *-CH2-NH-.
In further embodiments, L2 is:
Figure imgf000078_0006
further embodiments, q is 3. In further embodiments, q is 4. In further embodiments, q is 5. In further embodiments, b is 2. In further embodiments, c is 7. In further embodiments, RZ1 is H. In further embodiments, one occurrence of
RZ1 is CM alkyl, benzyl or phenyl and the remaining occurrences of RZ1 are H. In further embodiments, L
Figure imgf000078_0007
wherein RZ1 is Ci.4 alkyl, benzyl, or phenyl and ql is 2, 3 or 4, optionally wherein ql is 3. In further embodiments, RZ1 is Ci.4 alkyl. In further embodiments, RZ1 is methyl. In further embodiments, RZ1 is isopropyl. In further embodiments, RZ1 is benzyl. In further embodiments, RZ1 is phenyl.
In further embodiments, LB is — (CH2)b-- In further embodiments, LB is
Figure imgf000079_0001
In further embodiments, LB is
Figure imgf000079_0002
. In embodiments, d is 1. In other embodiments, d is 2.
In embodiments, L2 is selected from:
Figure imgf000079_0003
or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000079_0004
indicates the point of attachment to the antibody or antigen-binding fragment thereof.
In embodiments, L2 is:
Figure imgf000079_0005
pharmaceutically acceptable salt thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof. In embodiments, L2 is a group of Formula (L-ll A):
Figure imgf000080_0002
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000080_0001
or 2, and the asterisk-wavy line indicates the point of attachment to G2, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
In embodiments, L2 is a group of Formula (L-ll):
Figure imgf000081_0001
(HI), wherein E2 is (CH2)n2, wherein n2 is 0, 1, 2 or 3,
Figure imgf000081_0002
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Z2 is (CH2)n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof; provided that, for a conjugate of Formula (V), when Y is absent, then Xb is not -C(O)NH-.
In further embodiments of Formula (L-IIA) or (L-ll ), Q2 is
Figure imgf000082_0001
12 or 13. In further embodiments, R1 is CH3. In further embodiments, E2 is CH2. In further embodiments, X2 is CH2. In further embodiments, Y2 is (CH2)2. In further embodiments, Z2 is (CH2)2. In further embodiments, p is 1. In further embodiments, pl is 0. In further embodiments, pl is 1. In further embodiments of Formula
Figure imgf000082_0002
further embodiments, n5A is 1.
In further embodiments, L2 is selected from:
Figure imgf000082_0003
wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof. In embodiments, L2 is a group of Formula (L-IV):
Figure imgf000083_0001
wherein E3 is (CH2)n6, wherein n6 is 0, 1, 2 or 3, R1A is C1-4 alkyl or H;
Figure imgf000083_0002
3, and Q3 is O or NRA, wherein RA is H, C1-4 alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety; Y3 is O or NRB, wherein RB is H, C1-4 alkyl or C3-4 cycloalkyl, Z3 is (CH2)n10 or
Figure imgf000083_0003
, wherein n10 is 0, 1, 2, 3, 4 or 5, n10A is an integer from 1 to 10, n10B is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof. In further embodiments, m3 is 9, 10, 11, 12 or 13. In further embodiments, R1A is CH3. In further
Figure imgf000084_0001
further embodiments, E3 is CH2.
In further embodiments, X4 is CH2. In further embodiments, Y3 is O. In further embodiments, Z3 is
(CH2)nio. In further embodiments, Z3 is (CH2)2. In further embodiments, Z3 is absent (i.e. Z3 is
(CH2)n10, where nlO is 0). In further embodiments, Z3 is
Figure imgf000084_0002
embodiments, nlOB is 2. In further embodiments, nlOA is 1. In further embodiments, p2 is 1. In further embodiments, q2 is 1. In further embodiments, p2 is 0. In further embodiments, p3 is 1. In further embodiments, p3 is 0. In further embodiments, L2 is:
Figure imgf000084_0003
In embodiments, L2 is selected from a moiety of Table 6:
Figure imgf000084_0004
Figure imgf000085_0001
Figure imgf000086_0002
In embodiments, G2 is selected from:
Figure imgf000086_0001
wherein RK is H or CH3, RL is Cv6 alkyl, and -x'1! indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
In embodiments, G2 is selected from:
Figure imgf000087_0001
wherein RK is H or CH3, RL is Cv6 alkyl, and
Figure imgf000087_0002
indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
In embodiments, G2 is selected from:
Figure imgf000088_0001
wherein RK is H or CH3, RL is Ci_6 alkyl, and
Figure imgf000088_0002
indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
In further embodiments, G2 is selected from:
Figure imgf000088_0003
In further embodiments,
Figure imgf000088_0004
further embodiments, G2 is
Figure imgf000088_0005
Figure imgf000088_0007
Figure imgf000088_0006
further embodiments, G2 is In embodiments, G2 is (i.e. G2 a bond).
In embodiments, -LB-G2, -Z2-G2 or -Z3-G2 is:
Figure imgf000088_0008
In embodiments, L2 is selected from:
Figure imgf000089_0001
or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000089_0002
indicates the point of attachment to the antibody or antigen-binding fragment thereof.
In embodiments, L2 is:
Figure imgf000089_0003
or a pharmaceutically acceptable salt thereof, wherein -x"' indicates the point of attachment to the antibody or antigen-binding fragment thereof.
In embodiments, L2 is selected from a moiety of Table 7:
Figure imgf000089_0004
Figure imgf000090_0001
Figure imgf000091_0001
In embodiments, for a conjugate of Formula (V) or (Va), when Y is absent, then Xb is not -C(O)NH-. In embodiments, the conjugate of Formula (V) or (Va) (e.g. the conjugate of Formula (V)), or a pharmaceutically acceptable salt thereof, is selected from a conjugate of Table 8A:
Figure imgf000092_0001
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
In embodiments, the conjugate of Formula (VI) or (Via) (e.g. the conjugate of Formula (VI)), or a pharmaceutically acceptable salt thereof, is selected from a conjugate of Table 8B:
Figure imgf000092_0002
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10. In embodiments, the conjugate of Formula (Via) or a pharmaceutically acceptable salt thereof, is selected from a conjugate of Table 8C:
Figure imgf000096_0001
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
In embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In further embodiments k is an integer from 2 to
10. In further embodiments k is an integer from 2 to 8. In further embodiments k is 4. In further embodiments k is 8.
The present specification is intended to include all isotopes of atoms occurring in the present compounds and conjugates. Isotopes will be understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include 13C and 14C. Isotopes of nitrogen include 15N.
The compounds disclosed herein may contain one or more chiral centres. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e. as individual enantiomers, diastereoisomers, or as a stereoisomerically enriched mixture. All such stereoisomer (and enriched) mixtures are included within the scope of the embodiments, unless otherwise stated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the chemical structure or chemical name is intended to embrace all possible stereoisomers, diastereoisomers, conformers, rotamers and tautomers of the compound depicted. For example, a compound containing a chiral carbon atom is intended to embrace both the (R) enantiomer and the (S) enantiomer, as well as mixtures of the enantiomers, including racemic mixtures; and a compound containing two chiral carbons is intended to embrace all enantiomers and diastereoisomers including (R,R), (S,S), (R,S) and (S,R). In cases where compounds may exist in tautomeric forms (e.g. keto/enol), whether existing in equilibrium or predominantly in one form, depiction of one tautomer is intended to encompass the other tautomer.
The compounds disclosed herein may be prepared, used or supplied in amorphous form, crystalline form, or semicrystalline form and any given compound disclosed herein may be capable of being formed into more than one crystalline / polymorphic form, including hydrated (e.g. hemi hydrate, a mono hydrate, a di hydrate, a tri hydrate or other stoichiometry of hydrate) and/or solvated forms. It is to be understood that the present specification encompasses any and all such solid forms of the compound disclosed herein.
The compounds disclosed herein may be prepared as a co-crystal solid form. It is to be understood present specification encompasses any and all such co-crystals of a compound disclosed herein.
A suitable pharmaceutically acceptable salt of a compound disclosed herein (e.g. a compound of Formula (I), (II), (III) or (IV) or a conjugate of Formula (V) or (VI)) is, for example, an acid addition salt. An acid addition salt may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person. An acid addition salt may for example be formed using an inorganic acid selected from hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid. An acid addition salt may also be formed using an organic acid selected from trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.
A suitable pharmaceutically acceptable salt of a compound disclosed herein (e.g. a compound of Formula (I), (II), (III) or (IV) or a conjugate of Formula (V) or (VI)) is, for example, a base addition salt. A base addition salt may be formed by bringing the compound into contact with a suitable inorganic or organic base under conditions known to the skilled person. A base addition salt may for example be an alkali metal salt (such as a sodium, potassium, or lithium salt) or an alkaline earth metal salt (such as a calcium salt), which may be formed using an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g., an ethoxide or methoxide). A base addition salt may also be formed using a suitably basic organic amine (e.g., a choline or meglumine salt). A further suitable pharmaceutically acceptable salt of a compound of Formula (I) or (II) or a conjugate of Formula (V) or (VI) is, for example, a salt formed within a patient's body after administration of a compound of Formula (I) or (II) or a conjugate of Formula (V) or (VI) to the patient. A further suitable pharmaceutically acceptable salt of a compound of Formula (la) or (Ila) or a conjugate of Formula (Va) or (Via) is, for example, a salt formed within a patient's body after administration of a compound of Formula (la) or (Ila) or a conjugate of Formula (Va) or (Via) to the patient.
Conjugation
A conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, may be prepared by reacting (i.e. conjugating) a compound of Formula (III) or (IV), or a salt thereof, with an antibody or an antigen-binding fragment thereof. In some embodiments, a compound of Formula (III) or (IV) contains moiety G1 and a conjugate of Formula (V) or (VI) contains moiety G2 , as described above. Upon conjugation, a moiety G1 is converted to moiety G2 where G2 has a point of attachment to the antibody or antigen-binding fragment thereof.
A conjugate of Formula (Va) or (Via), or a pharmaceutically acceptable salt thereof, may be prepared by reacting (i.e. conjugating) a compound of Formula (Illa) or (IVa), or a salt thereof, with an antibody or an antigen-binding fragment thereof. In some embodiments, a compound of Formula (Illa) or (IVa) contains moiety G1 and a conjugate of Formula (Va) or (Via) contains moiety G2 , as described above. Upon conjugation, a moiety G1 is converted to moiety G2 where G2 has a point of attachment to the antibody or antigen-binding fragment thereof.
Examples of G1 and G2 include, but are not limited to, the following moieties set out in Table 9, wherein X3 is CH or N, h is 0 or 1, RK is H or CH3, Hal is Cl, Br or I, RL is Ci.6 alkyl. " indicates the point of attachment to the antibody, or antigen-binding fragment thereof.
Table 9. Exemplary G1 and G2 moieties.
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
A further example of G1 and G2 is set out in Table 9A. For the example of Table 9A, sortase A enzyme may be used to effect the conjugation to the antibody or antigen-binding fragment thereof. Table 9A:
Figure imgf000100_0002
Antibody or antigen-binding fragment thereof
As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen. In embodiments the antibody is isolated or recombinant. "Isolated", when used herein refers to a polypeptide, e.g., an antibody, that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated antibody will be prepared by at least one purification step. Thus, an "isolated antibody" refers to an antibody which is substantially free of other antibodies having different antigenic specificities.
In embodiments the antibody comprises at least two "light chains" (LC) and two "heavy chains" (HC). The light chains and heavy chains of such antibodies are polypeptides consisting of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region (abbreviated herein as "CH"). The heavy chain constant region comprises the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain (abbreviated herein as "VL") and a light chain constant domain (abbreviated herein as "CL").
In embodiments the antibody is a full-length antibody. An "intact" or "full-length" antibody, as used herein, refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.
A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) (also known as hypervariable regions), interspersed with regions that are more conserved, termed framework regions (FRs). In embodiments each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region.
Binding between an antibody and its target antigen or epitope is mediated by the CDRs. The term "epitope" refers to a target protein region (e.g. polypeptide) capable of binding to (e.g. being bound by) an antibody or antigen-binding fragment of the disclosure. The CDRs are the main determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs. The "constant domains" (or "constant regions") of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE and IgM, each with characteristic effector functions designated by isotype. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII. Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell- mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains.
In embodiments the antibody or antigen-binding fragment thereof is an IgG isotype. The antibody or antigen-binding fragment thereof can be any IgG subclass, for example IgGl, lgG2, lgG3, or lgG4 isotype. In embodiments the antibody or antigen-binding fragment thereof is based on an IgGl isotype.
The terms "Fc region", "Fc part" and "Fc" are used interchangeably herein and refer to the portion of a native immunoglobulin that is formed by two Fc chains. Each "Fc chain" comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. A native Fc region is homodimeric. In embodiments the Fc region may be heterodimeric because it may contain modifications to enforce Fc heterodimerisation. The Fc region contains the carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor), and has no antigen binding activity. Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in a number of Fc domain sites, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, resulting in minor variations between sequences. As a result, every naturally occurring IgG Fc region is referred to as a "wild type IgG Fc domain" or "WT IgG Fc domain" (i.e., any allele). Human IgGl, lgG2, lgG3, and lgG4 heavy chain sequences can be obtained in a variety of sequence databases, including the UniProt database (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN) respectively.
In embodiments the antibody of the disclosure is a monoclonal antibody. A "monoclonal antibody" (mAb) refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" can encompass both full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab1, F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals. In embodiments the antibody of the disclosure is an isolated monoclonal antibody. In further embodiments the antibody is a fully human monoclonal antibody.
In embodiments the antibody of the disclosure is a full-length antibody described above. Alternatively, the antibody can be an antigen-binding fragment. The term "antigen-binding fragment" as used herein incudes any naturally-occurring or artificially-constructed configuration of an antigen-binding polypeptide comprising one, two or three light chain CDRs, and/or one, two or three heavy chain CDRs, wherein the polypeptide is capable of binding to the antigen.
In embodiments the antigen-binding fragment of the disclosure is a Fab fragment. The antibody according to the disclosure can also be a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an intrabody, an IgG CH2, a minibody, a single-domain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BITE), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-lg, an (scFv)2, a mAb2 or a DARPin.
The terms "Fab fragment" and "Fab" are used interchangeably herein and contain a single light chain (e.g. a constant domain CL and a VL) and a single heavy chain (e.g. a constant domain CHI and a VH). The heavy chain of a Fab fragment is not capable of forming a disulfide bond with another heavy chain.
A "Fab1 fragment" contains a single light chain and a single heavy chain but in addition to the CHI and the VH, a "Fab' fragment" contains the region of the heavy chain between the CHI and CH2 domains that is required for the formation of an inter-chain disulfide bond. Thus, two "Fab1 fragments" can associate via the formation of a disulfide bond to form a F(ab')2 molecule. A "F(ab')2 fragment" contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for the formation of an inter-chain disulfide bond between two heavy chains.
An "Fv fragment" contains only the variable regions of the heavy and light chain. It contains no constant regions.
A "single-domain antibody" is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).
A "single-chain Fv" ("scFv") is antibody fragment containing the VH and VL domain of an antibody, linked together to form a single chain. A polypeptide linker is commonly used to connect the VH and VL domains of the scFv.
A "tandem scFv", also known as a TandAb, is a single-chain Fv molecule formed by covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.
A "bi-specific T cell engager" (BiTE) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumour cell antigen.
A "diabody" is a small bivalent and bispecific antibody fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH- VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.
A "DARPin" is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins. A DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences. A DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display, which is completely cell-free and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5. In embodiments the antibody or antigen-binding fragment thereof can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatised moieties can improve the solubility, the biological half-life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 2nd ed., Ed. Lloyd V. Allen, Jr. (2012).
Pharmaceutical Compositions
The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient, and which contains no additional components which are unacceptably toxic to a patient to which the composition would be administered. Such compositions can be sterile. A pharmaceutical composition according to the present specification comprises a compound of Formula (I), (la), (II) or (Ila), or a pharmaceutically acceptable salt thereof, or a conjugate of Formula (V), (Va), (VI) or (Via), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In embodiments, a pharmaceutical composition comprises a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, or a conjugate of Formula (V) or (VI), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, buffer or stabilizer. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable, non-toxic, sterile carrier. In further embodiments the carrier is a physiological saline, non-toxic buffer, or preservative. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 2nd ed., Ed. Lloyd V. Allen, Jr. (2012), the contents of which are incorporated by reference.
Examples of suitable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol and ethanol, as well as any combination thereof. In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and one or more isotonic agents. In further embodiments the one or more isotonic agents are selected from a sugar, a polyalcohol and sodium chloride.
In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, contained within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal aerosol, and a lyophilized powder which can be reconstituted to make a suspension or solution before use.
In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, a buffer, a surfactant and/or a stabilizer agent. In further embodiments the buffer is an acetate, phosphate or citrate buffer. In further embodiments the surfactant is polysorbate. In further embodiments the stabilizer agent is human albumin.
The pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, can be administered to a patient by any appropriate systemic or local route of administration. For example, administration may be oral, buccal, sublingual, ophthalmic, intranasal, intratracheal, pulmonary, topical, transdermal, urogenital, rectal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular or intratumoural.
The pharmaceutical composition comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, can be formulated for administration by any appropriate means, for example by epidermal or transdermal patches, ointments, lotions, creams, or gels; by nebulizers, vaporizers, or inhalers; by injection or infusion; or in the form of capsules, tablets, liquid solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most suitable route for administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired properties of the formulation.
Pharmaceutical compositions comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the conjugate of Formula (V), (Va), (VI) or (Via) (e.g. the conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
A pharmaceutical composition comprising a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing). Such compositions may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, pharmaceutical compositions comprising a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents. An effective amount of the compound of Formula (I), (la), (II) or (Ila) (e.g. the compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, will normally be present in the composition.
Therapy
In one aspect there is provided a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, for use in therapy.
In one aspect there is provided a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.
In one aspect there is provided a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, for use in therapy. In one aspect there is provided a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.
Where "cancer" is mentioned, this includes both non-metastatic cancer and also metastatic cancer, such that treating cancer involves treatment of both primary tumours and also tumour metastases.
The term "therapy" is intended to have its normal meaning of dealing with a disease in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology. The term "therapy" also includes "prophylaxis" unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted in a corresponding manner.
The term "prophylaxis" is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease and secondary prophylaxis whereby the disease has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or the development of new symptoms associated with the disease.
The term "treatment" is used synonymously with "therapy". Similarly, the term "treat" can be regarded as "applying therapy" where "therapy" is as defined herein.
In embodiments there is provided a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, for use in the treatment of HER2 positive cancer.
In one aspect there is provided the use of a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.
In one aspect there is provided the use of a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.
In one aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof.
In one aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof. Terms such as "treating" or "treatment" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain aspects, a patient is successfully "treated" for cancer according to the methods of the present disclosure if the patient shows, e.g., total, partial, or transient remission of a certain type of cancer.
The term "effective amount" means an amount of an active ingredient which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically acceptable excipient(s)/carrier(s) utilized, and like factors within the knowledge and expertise of the attending physician.
The term "patient" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. In embodiments the term "patient" refers to a human subject.
In embodiments there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, wherein the cancer is a HER2 positive cancer.
In embodiments there is provided a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and an additional anti-tumour substance for the conjoint treatment of cancer.
In embodiments there is provided a combination for use in the treatment of cancer comprising a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of the Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof and an additional anti-tumour agent.
In embodiments there is provided a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of the Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, in combination with an additional anti-tumour agent. Herein, where the term "conjoint treatment" is used in reference to a combination treatment, it is to be understood that this may refer to simultaneous, separate or sequential administration. In one aspect, "conjoint treatment" refers to simultaneous administration. In another aspect, "conjoint treatment" refers to separate administration. In a further aspect, "conjoint treatment" refers to sequential administration.
In embodiments there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially administering at least one additional anti-tumour substance to said patient, where the amounts of the conjugate of Formula (V), (Va), (VI) or (Via) (e.g. a conjugate of Formula (V) or (VI)), or pharmaceutically acceptable salt thereof, and the additional anti-tumour substance are jointly effective in producing an anti-cancer effect.
In embodiments there is provided a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, and an additional anti-tumour substance for the conjoint treatment of cancer.
In embodiments there is provided a combination for use in the treatment of cancer comprising a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof and an additional anti-tumour agent.
In embodiments there is provided a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, in combination with an additional anti-tumour agent.
In embodiments there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a compound of Formula (I), (la), (II) or (Ila) (e.g. a compound of Formula (I) or (II)), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially administering at least one additional anti-tumour substance to said patient, where the amounts of the compound of Formula (I), (la), (II) or (Ila) (e.g. the compound of Formula (I) or (II)), or pharmaceutically acceptable salt thereof, and the additional anti-tumour substance are jointly effective in producing an anti-cancer effect. EXAMPLES
The specification will now be illustrated by the following non-limiting Examples.
General Information
Reagents and solvents (all anhydrous HPLC-grade), including PNU-159682 carboxylic acid (CAS No.: 1204819-92-0), were obtained from commercial suppliers and used without any further purification unless otherwise stated. All reagents were weighed and handled in air unless otherwise stated. Brine refers to a saturated solution of NaCI. Concentration under reduced pressure refers to the use of a rotary evaporator.
Abbreviations
ACN acetonitrile
Boc tert-butyloxycarbonyl
DBU l,8-diazabicyclo(5.4.0)undec-7-ene
DCM dichloromethane
DEA diethylamine
DIC diisopropylcarbodiimide
DMF dimethylformamide
DMSO dimethylsulfoxide1
DMA dimethylacetamide
DIEA, DIPEA N,N-diisopropylethylamine
EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
FA formic acid
Fmoc 9-fluorenylmethyloxycarbonyl
Fmoc-OSu N-(9H-fluorenylmethoxycarbonyloxy)succinimide
HATU hexafluorophosphate azabenzotriazole tetramethyl uronium
HFIP hexafluoro-2-propanol
HOBt hydroxybenzotriazole
Int Intermediate compound mAb monoclonal antibody
MTBE methyl tert-butyl ether
NMI 1-methylimidazole
1 In the context of NMR characterisation in the below examples, "DMSO" refers to c/6-DMSO. [ethyl cyano(hydroxyimino)acetato-O2]tri-l-pyrrolidinylphosphonium
PyOxim hexafluorophosphate
RT, rt room temperature
TBAF tetra-n-butylammonium fluoride
TBS tert-butyldimethylsilyl
TCFH chloro-N,N,N',N'-tetra-methylformamidinium hexa-fluorophosphate
TEA triethylamine
TEA trifluoroacetic acid
TLC thin layer chromatography
TCEP tris(2-carboxyethyl)phosphine hydrochloride
THF tetrahydrofuran
PAYLOAD SYNTHESIS
General procedure I: Synthesis of amide by the coupling reaction of acid with amine using PyOxim or HATU
Figure imgf000112_0001
((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((lS,3R,4aS,9S,9aR,10aS)-9-methoxy-l-methyloctahydro- lH-pyrano[4',3':4,5]oxazolo[2,3-c][l,4]oxazin-3-yl)oxy)-6,ll-dioxo-l,2,3,4,6,ll- hexahydrotetracene-2-carboxylic acid (CAS No.: 1204819-92-0; "PNU-159682 carboxylic acid") was added to a solution of DIEA (5 eq) and mono-protected diamine (2-3 eq) in DMA/MeCN (v/v: 1/3) at 25 °C under nitrogen. PyOxim (2 eq)/or HATU (2 eq) was added. The resulting mixture was stirred at 25 °C for a few hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18 chromatography using 0 to 100% MeCN in water (5%NH4HCO3) to afford the product. General procedure II: deprotection of Boc-amine
Method A: Boc protected amine was dissolved in 1,4-dioxane, then HCI solution (4 M in dioxane) was added in one portion at 0 °C under nitrogen. The resulting mixture was stirred at 25 °C for a few hours until the reaction was completed, and then concentrated to remove the solvent. The crude product was purified by preparative HPLC using MeCN/water (containing 0.01% NH4HCO3) to afford the product.
Method B: TFA (40 mL) was added to Boc protected amine (~ 8.0 mmol) in DCM (60 ml) under nitrogen. The resulting mixture was stirred at 25 °C for a few hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18 chromatography using elution gradient 0 to 100% MeCN in water (0.1% NH4HCO3) to afford the product.
General procedure III: deprotection of Fmoc-amine: A DCM solution of DBU (15.2 mg/mL) (0. 45 eq) was added dropwise to Fmoc protected amine in a mixture of DCM and acetonitrile (v/v: 1/1) under nitrogen. The resulting mixture was stirred at 0 °C for a few hours until the reaction was completed. The reaction mixture was quenched with 0.1% NH4HCO3. The solvent was removed under reduced pressure. The crude product was purified by combi-flash or preparative HPLC (YMC- Actus Triant C18 Column, 20 x 250 mm, 5pm; Mobile Phase A: Water (lOmmol/L NH4HC03+0.1%NH3H20), Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 40% B to 54% B in 10 min) to afford the product.
Figure imgf000113_0001
Step 1 - Intermediate Compound 1: (9H-fluoren-9-yl)methyl 3,6-diazabicyclo[3.1.1]heptane-6- carboxylate, HCI salt
HCI
Figure imgf000114_0001
Fmoc tert-Butyl 3,6-diazabicyclo[3.1.1]heptane-3-carboxylate (500 mg, 2.52 mmol) was added to a solution of N,N-diisopropylethylamine (978 mg, 7.57 mmol) and N-(9H-fluoren-2- ylmethoxycarbonyloxyjsuccinimide (936 mg, 2.77 mmol) in DCM (10 mL). The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water. The organic layer was separated, the aq solution was extracted with DCM. The combined organic solution was dried over Na2SO4, and concentrated to afford the crude intermediate. The crude intermediate was treated with HCI solution (4 M in dioxane) according to General Procedure II, Method A to afford
Intermediate Compound 1 (800 mg, 99 %) as a white solid.
Figure imgf000114_0002
NMR (400 MHz, DMSO) 6 1.22 - 1.37 (m, 3H), 1.93 - 2.09 (m, 1H), 2.58 - 2.71 (m, 1H), 2.75 - 2.91 (m, 1H), 3.08 - 3.22 (m, 1H), 3.28 (d, J = 12.6 Hz, 1H), 4.12 - 4.47 (m, 4H), 7.35 (qd, J = 1.2, 7.3 Hz, 2H), 7.40 - 7.47 (m, 2H), 7.68 (dd, J = 7.4, 22.5 Hz, 2H), 7.86 - 7.94 (m, 2H). ES+ [M+H]+: 321.
Step 2 - Intermediate Compound 2: (9H-fluoren-9-yl)methyl 3-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((lS,3R,4aS,9S,9aR, 10aS)-9-methoxy-l-methyloctahydro-lH- pyrano[4',3':4,5]oxazolo[2,3-c][l,4]oxazin-3-yl)oxy)-6,ll-dioxo-l,2,3,4,6,ll-hexahydrotetracene-2- carbonyl)-3,6-diazabicyclo- [3.1.1]heptane-6-carboxylate
Figure imgf000114_0003
General Procedure I was followed using the PNU-159682 carboxylic acid (CAS No. 1204819-92-0; 150 mg, 0.24 mmol), DIEA (0.209 mL, 1.20 mmol) and Intermediate compound 1 (191 mg, 0.60 mmol) and PyOxim (244 mg, 0.48 mmol) to synthesize Intermediate compound 2 (110 mg, 49.5 %) as a red solid. ES+[M+1]: 930. Step 3 - Compound P1: (8S,10S)-8-(3,6-diazabicyclo[3.1.1]heptane-3-carbonyl)-6,8,11-trihydroxy-1- methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000115_0001
General procedure II was followed using DCM solution of DBU (15.2 mg/mL) (0.53 mL, ~0.05mmol) and Intermediate Compound 2 (100 mg, 0.11 mmol) to afford Compound P1 (15.00 mg, 19.71 %) as a red solid.1H NMR (300 MHz, CDCl3) δ 1.41 (t, J = 6.4 Hz, 3H), 1.62-1.71 (m, 1H),1.76-1.79 (m, 2H), 1.90-2.03 (m, 1H), 2.38 – 2.60 (m, 1H), 2.70-2.85 (m, 4H), 3.10 (t, J = 18.5 Hz, 1H), 3.39 (d, J = 6.6 Hz, 1H), 3.48 (s, 3H), 3.54 – 3.64 (m, 2H), 3.77 (dd, J = 13.7, 21.1 Hz, 2H), 3.93 (m, 3H), 3.95-4.09 (m, 5H), 4.220-4.26 (m, 1H), 4.36 (s, 1H), 4.50 (d, J = 1.9 Hz, 1H), 4.66 (d, J = 12.9 Hz, 1H), 4.73 (s, 1H), 4.91 (d, J = 7.6 Hz, 1H), 5.39 (s, 1H), 5.58 (d, J = 5.1 Hz, 1H) 7.42 (d, J = 8.5 Hz, 1H), 7.80 (t, J = 8.1 Hz, 1H), 8.06 (d, J = 7.7 Hz, 1H), 13.38 (s, 1H), 13.97 (d, J = 4.1 Hz, 1H). ES+[M+1] = 708.15. Example 2: Synthesis of Compound P2
Figure imgf000115_0002
Step 1 - Intermediate Compound 3: 3-((9H-fluoren-9-yl)methyl) 6-(tert-butyl) 3,6- diazabicyclo[3.1.1]heptane-3,6-dicarboxylate
Figure imgf000116_0001
N-(9H-Fluoren-2-ylmethoxycarbonyloxy)succinimide (3.40 g, 10.09 mmol) was added to a mixture of tert-butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (2 g, 10.09 mmol) and DIEA (3.52 mL, 20.17 mmol) in DCM (60 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with DCM (300 mL), and washed sequentially with water (3 x 250 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 3 (3.50 g, yield: 83 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.29 – 1.34 (m, 9H), 2.44 (s, 2H), 3.81 (dd, J = 2.8, 12.2 Hz, 2H), 3.99 (s, 1H), 4.06 (s, 2H), 4.23 – 4.31 (m, 2H), 4.40 (t, J = 8.7 Hz, 2H), 7.33 – 7.35 (m, 2H), 7.43 (s, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.91 (d, J = 7.5 Hz, 2H). ES+[M+23]: 443. Step 2 - Intermediate Compound 4: (9H-fluoren-9-yl)methyl 3,6-diazabicyclo[3.1.1]heptane-3- carboxylate, TFA salt
Figure imgf000116_0002
General Procedure II, Method B was followed using Intermediate Compound 3 (3.4 g, 8.09 mmol) in DCM (60 mL) to afford Intermediate Compound 4 (2.300 g, yield: 89 %) as a pale yellow solid.1H NMR (400 MHz, DMSO) δ 1.75 (dd, J = 5.7, 10.5 Hz, 1H), 2.76 – 2.87 (m, 1H), 3.68 – 3.78 (m, 3H), 3.82 (dd, J = 2.1, 12.7 Hz, 1H), 4.28 – 4.48 (m, 5H), 7.31 – 7.40 (m, 2H), 7.40 – 7.48 (m, 2H), 7.69 (dd, J = 2.2, 7.4 Hz, 2H), 7.92 (d, J = 7.5 Hz, 2H). ES+(M+1 = 321). Step 3 – Intermediate Compound 5: (9H-fluoren-9-yl)methyl 6-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4’,3’:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptane-3-carboxylate
Figure imgf000117_0001
General Procedure I was followed using PyOxim (652 mg, 1.27 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 400 mg, 0.64 mmol), Intermediate Compound 4 (511 mg, 1.59 mmol) and DIEA (0.557 mL, 3.19 mmol) in DMA (8 mL) to afford Intermediate Compound 5 (500 mg, yield: 84 %) as a red solid.
Figure imgf000117_0002
NMR (400 MHz, DMSO) δ 1.27 (dd, J = 4.0, 6.6 Hz, 3H), 1.54 (dd, J = 8.8, 17.9 Hz, 2H), 1.63 – 1.75 (m, 3H), 2.25 (d, J = 6.0 Hz, 1H), 2.30 – 2.39 (m, 2H), 2.63 – 2.69 (m, 3H), 2.85 – 3.12 (m, 4H), 3.32 (d, J = 2.4 Hz, 2H), 3.50 (d, J = 7.9 Hz, 2H), 3.61 – 3.71 (m, 3H), 3.98 (s, 3H), 4.24 – 4.37 (m, 6H), 4.89 (s, 1H), 5.23 (d, J = 5.0 Hz, 2H), 5.33 (d, J = 10.0 Hz, 1H), 7.32 – 7.37 (m, 3H), 7.39 – 7.44 (m, 3H), 7.67 (d, J = 7.7 Hz, 2H), 7.86 – 7.92 (m, 4H), 13.17 (s, 1H), 14.00 (s, 1H). ES+(M+1 = 930). Step 4 - Compound P2: (8S,10S)-8-(3,6-diazabicyclo[3.1.1]heptane-6-carbonyl)-6,8,11-trihydroxy-1- methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000117_0003
General Procedure III was followed using DCM solution of DBU（15.2 mg/mL in DCM）(0.354 mL, ~0.35 mmol) and Intermediate Compound 5 (470 mg, 0.51 mmol) to afford Compound P2 (59.0 mg, yield: 16.50 %) as a red solid.1H
Figure imgf000117_0004
NMR (400 MHz, CDCl3) δ 1.38-1.47 (m, 3H), 1.70-1.88 (m, 2H), 1.93-2.10 (m, 1H), 2.30 – 2.58 (m, 2H), 2.70 – 2.86 (m, 3H), 3.06 – 3.17 (m, 2H), 3.27 – 3.43 (m, 3H), 3.48 (s, 3H), 3.53 – 3.64 (m, 3H), 3.92 – 4.11 (m, 6H), 4.47 (dd, J = 6.3, 17.4 Hz, 2H), 4.69 (d, J = 23.4 Hz, 2H), 4.83 – 4.90 (m, 1H), 5.31 (d, J = 18.9 Hz, 1H), 5.45 – 5.55 (m, 1H), 7.39 (dd, J = 5.0, 8.6 Hz, 1H), 7.74 – 7.85 (m, 1H), 8.03 (dd, J = 2.6, 7.7 Hz, 1H), 13.91 (s, 1H). ES+(M+1 = 708). Example 3: Synthesis of Compound P2a
Figure imgf000118_0001
Compound P2a: (8S,10S)-8-(6-acetyl-3,6-diazabicyclo[3.1.1] heptane-3-carbonyl)-6,8,11-trihydroxy- 1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5] oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000118_0002
General Procedure I was followed using 1-(3,6-diazabicyclo[3.1.1]heptan-6-yl)ethan-1-one (53.6 mg, 0.38 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 120 mg, 0.19 mmol), HATU (145 mg, 0.38 mmol) and DIEA (0.100 mL, 0.57 mmol) to afford Compound P2a (23.00 mg, 16.04 %) as a red solid.1H NMR (300 MHz, DMSO) δ 1.16 – 1.30 (m, 3H), 1.54 (t, J = 10.2 Hz, 1H), 1.70 (s, 2H), 2.02 (d, J = 3.3 Hz, 3H), 2.65 (d, J = 8.5 Hz, 2H), 2.91 (dd, J = 9.9, 18.9 Hz, 3H), 2.93 – 3.02 (m, 2H), 3.05 – 3.14 (m, 3H), 3.52 (d, J = 6.1 Hz, 1H), 3.66 (d, J = 10.0 Hz, 1H),3.68 - 3.75 (m, 4H), 3.92 (dd, J = 9.2, 15.0 Hz, 1H), 4.00 (s, 3H), 4.24 (s, 1H), 4.35 (d, J = 17.8 Hz, 1H), 4.59 (s, 1H), 4.88 (s, 1H), 4.99 (s, 1H), 5.07 (s, 1H), 5.24 (s, 1H), 5.26 – 5.38 (m, 2H), 7.68 (q, J = 4.3, 4.9 Hz, 1H), 7.93 (d, J = 4.8 Hz, 2H), 13.28 (s, 1H), 14.06 (s, 1H). ES+(M+1= 750). Example 4: Synthesis of Compound P2b:
Figure imgf000119_0001
Step 1 – Intermediate Compound 6: tert-butyl 3-(2-hydroxyacetyl)-3,6-diazabicyclo[3.1.1]heptane- 6-carboxylate General Procedure I was followed using tert-butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (1 g, 5.04 mmol), 2-hydroxyacetic acid (1.918 g, 25.22 mmol), HATU (4.79 g, 12.61 mmol) and N,N- Diisopropylethylamine (3.26 g, 25.22 mmol) in DMA (20 mL) to afford Intermediate Compound 6 (1.000 g, 77 %) as a yellow solid.
Figure imgf000119_0002
NMR (400 MHz, DMSO) δ 1.36 (s, 9H), 2.42 – 2.50 (m, 1H), 3.34 (s, 1H), 3.36 (d, J = 1.2 Hz, 1H), 3.46 (d, J = 11.2 Hz, 1H), 3.75 – 3.91 (m, 1H), 3.97 – 4.20 (m, 4H), 4.57 (s, 1H). ES+(M+1 = 257). Step 2 – Intermediate Compound 7: 1-(3,6-diazabicyclo[3.1.1]heptan-3-yl)-2-hydroxyethan-1-one, HCl salt
Figure imgf000120_0001
General Procedure II, Method A was followed using Intermediate Compound 6 (1g, 3.90 mmol) to afford Intermediate Compound 7 (0.600 g, 85 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.67 – 1.82 (m, 1H), 2.84 (m, 1H), 3.72 – 3.95 (m, 2H), 4.00 – 4.10 (m, 1H), 4.19 (d, J = 15.7 Hz, 1H), 4.33 (d, J = 5.7 Hz, 1H), 4.79 (s, 3H), 10.21 (s, 1H). ES+(M+1 = 157). Step 3 – Compound P2b: (8S,10S)-6,8,11-trihydroxy-8-(3-(2-hydroxyacetyl)-3,6- diazabicyclo[3.1.1]heptane-6-carbonyl)-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':4,5] oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10- tetrahydrotetracene-5,12-dione
Figure imgf000120_0002
General Procedure I was followed using the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), Intermediate Compound 7 (112 mg, 0.72 mmol), PyOxim (244 mg, 0.48 mmol) and N,N-diisopropylethylamine (154 mg, 1.20 mmol) to afford Compound P2b (40.0 mg, 21.86 %) as a red solid.1H
Figure imgf000120_0003
NMR (400 MHz, DMSO) δ 1.15-1.25 (m, 3H), 1.53-1.58 (m, 1H), 1.70 (b, 2H), 2.01 – 2.41 (m, 3H), 2.57 – 2.78 (m, 3H), 2.85 – 2.98 (m, 1H), 3.04-3.10 (m, 1H), 3.32 (s, 3H), 3.46 – 3.76 (m, 4H), 3.79 – 4.20 (m, 9H), 4.21 – 4.26 (m, 1H), 4.36 (d, J = 24.6 Hz, 1H), 4.47 – 4.64 (m, 2H), 4.79-5.06 (m, 2H), 5.20 – 5.52 (m, 2H), 7.66 (m, 1H), 7.88 – 7.95 (m, 2H), 13.26 (s, 1H), 14.04 (s, 1H). ES+(M+1 = 766). Example 5: Synthesis of Compound P3
Figure imgf000121_0001
Step 1 – Intermediate Compound 8: 2-((9H-fluoren-9-yl)methyl) 5-(tert-butyl) (1R,4R)-2,5- diazabicyclo[2.2.2]octane-2,5-dicarboxylate
Figure imgf000121_0002
tert-Butyl (1R,4R)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate (500 mg, 2.36 mmol) was added to a mixture of N-(9H-fluoren-2-ylmethoxycarbonyloxy)succinimide (794 mg, 2.36 mmol) and DIEA (1.234 mL, 7.07 mmol) in DCM (10 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with DCM (100 mL), and washed sequentially with water ( 2 x 80 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 8 (1100 mg, 99 %) as a yellow solid.1H NMR (300 MHz, DMSO) δ 1.41 (d, J = 3.3 Hz, 9H), 1.74 (dt, J = 10.2, 20.1 Hz, 5H), 3.25 – 3.32 (m, 1H), 3.35 – 3.43 (m, 1H), 4.07 (d, J = 23.5 Hz, 2H), 4.28 (d, J = 6.0 Hz, 1H), 4.39 (dd, J = 3.7, 6.5 Hz, 2H), 5.76 (s, 1H), 7.30 – 7.47 (m, 4H), 7.64 (t, J = 6.6 Hz, 2H), 7.90 (dd, J = 1.6, 7.4 Hz, 2H). ES+(M+23 = 457.3). Step 2 – Intermediate Compound 9: (9H-fluoren-9-yl)methyl (1R,4R)-2,5-diazabicyclo[2.2.2]octane- 2-carboxylate, HCl salt
Figure imgf000121_0003
General Procedure II, Method A was followed using Intermediate Compound 8 (1.5 g, 3.45 mmol) to afford Intermediate Compound 9 (0.900 g, 70.3 %) as a white solid.1H NMR (300 MHz, DMSO) δ 1.18 – 1.33 (m, 1H), 1.74 – 1.82 (m, 3H), 2.08 (d, J = 13.6 Hz, 1H), 3.17 – 3.44 (m, 4H), 3.60 – 3.83 (m, 2H), 4.25 – 4.48 (m, 3H), 7.28 – 7.46 (m, 4H), 7.56 – 7.76 (m, 2H), 7.91 (d, J = 7.4 Hz, 2H). ES+(M+1 = 335.2). Step 3 – Intermediate Compound 10: (9H-fluoren-9-yl)methyl (1R,4R)-5-((2S,4S)-2,5,12-trihydroxy- 7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran’[4’,3’:4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-2,5- diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000122_0001
General Procedure I was followed using PyOxim (326 mg, 0.64 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol) and Intermediate Compound 9 (266 mg, 0.80 mmol) and DIEA (0.278 mL, 1.59 mmol) to afford Intermediate Compound 10 (175 mg, 58.2 %) as a red solid. NMR (400 MHz, DMSO) δ 1.21 – 1.29 (m, 3H), 1.67 (d, J = 5.8 Hz, 2H), 1.78 (s, 1H), 1.82 – 1.89 (m, 2H), 1.95 – 2.06 (m, 2H), 2.08 (s, 1H), 2.70 (s, 2H), 2.78 (d, J = 17.3 Hz, 1H), 3.37 (s, 3H), 3.41 – 3.57 (m, 4H), 3.62 – 3.72 (m, 1H), 3.79 (d, J = 10.8 Hz, 1H), 3.88 – 4.01 (m, 5H), 4.01 – 4.06 (m, 1H), 4.09 – 4.16 (m, 2H), 4.20 – 4.31 (m, 3H), 4.34 – 4.46 (m, 2H), 5.10 – 5.20 (m, 2H), 5.25 – 5.33 (m, 1H), 6.14 (s, 1H), 7.31 – 7.41 (m, 4H), 7.46 – 7.67 (m, 5H), 7.81 – 7.93 (m, 2H), 13.27 (s, 1H), 14.00 (s, 1H). ES+(M+1 = 944). Step 4 – Compound P3: (8S,10S)-8-((1R,4R)-2,5-diazabicyclo[2.2.2] octane-2-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,-9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyran’[4’,3’:4,5]oxazolo[2,3-c][1,4] oxazin-3-yl)oxy)-7,8,9, 10-tetrahydrotetracene-5,12-dione
Figure imgf000122_0002
General Procedure III was followed using DCM solution of DBU (15.2 mg/mL in DCM) (0.828 mL, 0.08 mmol) and Intermediate Compound 10 (156 mg 017 mmol) to afford Compound P3 (26.0 mg, 21.80 %) as a red solid.1H NMR (300 MHz, CDCl3) δ 0.85（s, 1H), 1.41-1.48(m, 3H), 1.70- 1.91(m, 2H), 1.92-2.05(m,3H),2.13 – 2.66 (m, 4H), 2.81 (s, 2H), 3.00-3.10 (m,1H), 3.21 – 3.74 (m, 10H), 3.74 – 4.29 (m, 7H), 4.49 (s, 1H), 4.73 (s, 1H), 4.89 (d, J = 16.5 Hz, 1H), 5.07 (s, 1H), 5.38 (s, 1H), 5.57 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.81 (t, J = 8.1 Hz, 1H), 8.06 (s, 1H), 13.96 (s, 1H). ES+(M+1 = 722). Example 6: Synthesis of Compound P4
Figure imgf000123_0001
Step 1 – Intermediate Compound 11: 2-((9H-fluoren-9-yl)methyl) 5-(tert-butyl) (1S,4S)-2,5- diazabicyclo[2.2.2]octane-2,5-dicarboxylate
Figure imgf000123_0002
tert-Butyl (1S,4S)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate (500 mg, 2.36 mmol) was added to a solution of N-(9H-fluoren-2-ylmethoxycarbonyloxy)succinimide (794 mg, 2.36 mmol) and DIEA (0.411 mL, 2.36 mmol) in DCM (15 mL) at RT. The resulting mixture was stirred at RT for 2 hours. The reaction mixture was diluted with DCM (100 mL), and washed sequentially with water (2 x 100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 11 (1100 mg, 99 %) as a yellow solid.1H
Figure imgf000123_0003
NMR (300 MHz, DMSO) δ 0.95 (d, J = 6.4 Hz, 1H), 1.41 (d, J = 3.7 Hz, 9H), 1.59 – 1.90 (m, 5H), 3.29 (d, J = 5.3 Hz, 1H), 4.07 (d, J = 23.9 Hz, 2H), 4.28 – 4.30(m, 1H), 4.34 – 4.49 (m, 2H), 5.77 (s, 1H), 7.29 – 7.47 (m, 4H), 7.64 (t, J = 6.5 Hz, 2H), 7.85 – 7.95 (m, 2H). ES+(M+23 = 457). Step 2 - Intermediate Compound 12: (9H-fluoren-9-yl)methyl (1S,4S)-2,5-diazabicyclo[2.2.2]octane- 2-carboxylate, HCl salt
Figure imgf000124_0001
General Procedure II, Method A was followed using Intermediate Compound 11 (800 mg, 1.84 mmol) to afford Intermediate Compound 12 (1050 mg, 97%) as a pale yellow solid.1H NMR (400 MHz, DMSO) δ 1.22 – 1.33 (m, 1H), 1.64 – 1.85 (m, 3H), 2.03 – 2.14 (m, 1H), 3.17 – 3.32 (m, 2H), 3.33 – 3.47 (m, 2H), 3.67 – 3.82 (m, 2H), 4.24 – 4.51 (m, 3H), 7.35 – 7.39(m, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.60 – 7.70 (m, 2H), 7.91 (d, J = 7.5 Hz, 2H). ES+(M+1 = 335). Step 3 - Intermediate Compound 13: (9H-fluoren-9-yl)methyl (1S,4S)-5-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo- [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-2,5- diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000124_0002
General Procedure I was followed using HATU (136 mg, 0.36 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), DIEA (0.209 mL, 1.20 mmol) and Intermediate Compound 12 (160 mg, 0.48 mmol) to afford Intermediate Compound 13 (160 mg, 70.9 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.18 – 1.32 (m, 3H), 1.68 (d, J = 7.8 Hz, 3H), 1.77 (s, 2H), 2.08 (s, 2H), 2.65 (d, J = 18.6 Hz, 2H), 2.77 – 2.87 (m, 1H), 3.15 – 3.32 (m, 4H), 3.35 – 3.43 (m, 3H), 3.47 – 3.59 (m, 2H), 3.65 (d, J = 7.3 Hz, 1H), 3.85-3.96 (m, 2H), 3.99 (s, 3H), 4.16 (s, 1H), 4.19 – 4.32 (m, 2H), 4.34 – 4.50 (m, 3H), 4.58 (dd, J = 2.1, 16.0 Hz, 1H), 4.95 – 5.15 (m, 2H), 5.27 (t, J = 4.4 Hz, 1H), 5.95 (d, J = 1.9 Hz, 1H), 7.28 – 7.39 (m, 3H), 7.39 – 7.46 (m, 2H), 7.63 – 7.67 (m, 2H), 7.87 – 7.94 (m, 4H), 13.32 (s, 1H), 14.02 – 14.11 (m, 1H). ES+(M+1 = 944). Step 4 - Compound P4: (8S,10S)-8-((1S,4S)-2,5-diazabicyclo[2.2.2]octane-2-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000125_0001
General Procedure III was followed using DBU (0.012 mL, 0.08 mmol) and Intermediate Compound 13 (150 mg, 0.16 mmol) to afford Compound P4 (15.00 mg, 13.08 %) as a red solid.1H NMR (300 MHz, CDCl3) δ 1.42 (d, J = 6.4 Hz, 3H), 1.72 - 2.19 (m, 9H), 2.36 - 2.49 (m, 1H), 2.62-2.68 (m, 1H), 2.74 - 2.87 (m, 2H), 3.00-3.08 (m, 1H), 3.12 - 3.25 (m, 1H), 3.30 (s, 1H), 3.33 - 3.47 (m, 3H), 3.53 - 3.66 (m, 2H), 3.70-3.75 (m, 1H), 3.87 - 4.01 (m, 1H), 4.00 - 4.19 (m, 5H), 4.24 (s, 1H), 4.49 (d, J = 1.8 Hz, 1H), 4.74 (d, J = 1.9 Hz, 1H), 4.86 (s, 1H), 4.96 (s, 1H), 5.39 (s, 1H), 5.58 (t, J = 5.3 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.80 (t, J = 8.1 Hz, 1H), 8.02 - 8.11 (m, 1H), 13.98 (s, 1H). ES+(M+1 = 722). Example 7: Synthesis of Compound P5
Figure imgf000125_0002
Step 1 - Intermediate Compound 14 ((9H-fluoren-9-yl)methyl) 5-(tert-butyl) (3aR,6aS)- tetrahydropyrrolo[3,4-c]pyrrole-2,5(1H,3H)-dicarboxylate
Figure imgf000125_0003
(9H-Fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (1.748 g, 5.18 mmol) was added to tert-butyl (3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (1g, 4.71 mmol) and DIEA (2.468 mL, 14.13 mmol) in DCM (15 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with DCM (300 mL), and washed sequentially with water (3 x 250 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 14 (1.500 g, 73.3 %) as a pale yellow oil.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.81 (s, 2H), 3.03 - 3.15 (m, 4H), 3.39 -3.54 (m, 4H), 5.76 (s, 1H), 6.28 (s, 2H), 7.30 - 7.38 (m, 2H), 7.38 - 7.46 (m, 2H), 7.81 - 7.92 (m, 4H). ES+(M+1 = 435). Step 2 - Compound 15 (9H-fluoren-9-yl)methyl (3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrole-2(1H)- carboxylate, HCl salt
Figure imgf000126_0001
General Procedure II, Method A was followed using Intermediate Compound 14 (1.4 g, 3.22 mmol) to afford Intermediate Compound 15 (1.000 g, 93 %) as a pale yellow solid.1H NMR (400 MHz, DMSO) δ 1.27 - 1.32 (m, 3H), 3.00 (s, 2H), 3.02 - 3.17 (m, 2H), 3.32 -3.43 (m, 4H), 4.24 - 4.34 (m, 3H), 7.31 - 7.39 (m, 2H), 7.39 - 7.48 (m, 2H), 7.61 - 7.70 (m, 2H), 7.89 - 7.94 (m, 2H). ES+(M+1 = 335). Step 3 - Intermediate Compound 16: (9H-fluoren-9-yl)methyl (3aR,6aS)-5-((2S,4S)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl) hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate
Figure imgf000126_0002
General Procedure I was followed using HATU (136 mg, 0.36 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), DIEA (0.209 mL, 1.20 mmol) and Intermediate Compound 15 (160 mg, 0.48 mmol) to afford Intermediate Compound 16 ((130 mg, 57.6 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.18 – 1.34 (m, 3H), 2.58 – 2.68 (m, 2H), 2.79 (s, 1H), 2.86 (s, 1H), 2.90 – 2.95 (m, 1H), 3.17 (s, 10H), 3.26 – 3.34 (m, 5H), 3.36 (d, J = 8.7 Hz, 2H), 3.69 (q, J = 9.2, 10.5 Hz, 2H), 3.91 – 4.14 (m, 7H), 4.18 – 4.30 (m, 2H), 4.55 – 4.73 (m, 1H), 5.77 (s, 1H), 6.28 (s, 2H), 7.31 – 7.45 (m, 5H), 7.81 – 7.91 (m, 6H). ES+(M+1 = 944). Step 4 – Compound P5: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-((3aR,6aS)- octahydropyrrolo[3,4-c]pyrrole-2-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000127_0001
General Procedure III was followed using Intermediate Compound 16 (120 mg, 0.13 mmol) to afford Compound P5 (13.00 mg, 14.17 %) as a red solid.
Figure imgf000127_0002
NMR (400 MHz, CDCl3) δ 1.41 (d, J = 6.3 Hz, 3H), 1.91 – 2.09 (m, 4H), 2.43 (d, J = 14.8 Hz, 1H), 2.61 (d, J = 15.3 Hz, 1H), 2.73 – 3.08 (m, 7H), 3.20 (s, 2H), 3.36 – 3.44 (m, 2H), 3.48 (s, 3H), 3.56 – 3.63 (m, 2H), 3.83 (s, 1H), 3.90 – 3.98 (m, 2H), 4.09 (d, J = 16.7 Hz, 6H), 4.49 (s, 1H), 4.73 (s, 1H), 5.36 (s, 1H), 5.57 (s, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.80 (t, J = 8.1 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 13.97 (s, 1H). ES+(M+1 = 722). Example 8: Synthesis of Compound P6
Figure imgf000127_0003
Steps 1, 2 – Intermediate Compound 18: (9H-fluoren-9-yl)methyl 2,7-diazaspiro[3.5]nonane-7- carboxylate, HCl salt
Figure imgf000128_0001
(9H-Fluoren-9-yl)methyl carbonochloridate (1.257 g, 4.86 mmol) was added to a mixture of tert- butyl 2,7-diazaspiro[3.5]nonane-2-carboxylate (1 g, 4.42 mmol) and K2CO3 (1.832 g, 13.26 mmol) in DCM (50 mL) under air. The resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered. The filtration was evaporated to afford the di-protected Intermediate Compound 17 as a crude product (1.3 g). The crude Intermediate Compound 17 was treated with HCl solution in dioxane 4M (30 mL) according to General Procedure II, Method A to afford Intermediate Compound 18 as crude product ((1.200 g, 70.6 %) as a yellow oil. ES+(M+1 = 349). Step 3 - Intermediate Compound 19: (9H-fluoren-9-yl)methyl 2-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate
Figure imgf000128_0002
General Procedure I was followed using the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol), PyOxim (244 mg, 0.48 mmol), DIEA (0.278 mL, 1.59 mmol) and Intermediate Compound 18 (222 mg, 0.64 mmol) to afford Intermediate Compound 19 (100 mg, 32.8 %) as a red solid. ES+(M+1 = 958). Step 4 - Compound P6 (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-(2,7- diazaspiro[3.5]nonane-2-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000129_0001
General Procedure III was followed u Osing O O Inte O OrHHme d OdO sOia oHl Ote id N C . Oo Nmpo NM N Ou RHnd 19 (100 mg, 0.10 mmol) to afford Compound P6 (19.00 mg, 24.74 %) as a re
Figure imgf000129_0002
O (300 MHz, CDCl3) δ 1.42 (d, J = 6.3 Hz, 3H), 1.73 – 1.87 (m, 3H), 1.95 (s, 4H), 2.05 (s, 1H), 2.36 (d, J = 13.2 Hz, 1H), 2.58 (d, J = 15.0 Hz, 1H), 2.80 (d, J = 18.3 Hz, 2H), 2.97 (s, 2H), 3.16 – 3.26 (m, 3H), 3.31 (s, 1H), 3.41 (dd, J = 6.6, 13.2 Hz, 2H), 3.49 (s, 3H), 3.60 (d, J = 11.7 Hz, 2H), 3.84 (s, 2H), 3.94 (t, J = 9.3 Hz, 2H), 4.06 (d, J = 10.5 Hz, 2H), 4.11 (s, 3H), 4.22 – 4.35 (m, 2H), 4.50 (d, J = 1.8 Hz, 1H), 4.63 (s, 1H), 4.73 (s, 1H), 5.33 (s, 1H), 5.52 (t, J = 5.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.80 (t, J = 8.1 Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 13.35 (s, 1H), 13.96 (s, 1H). ES+(M+1 = 736). Example 9: Synthesis of Compound P7
Figure imgf000129_0003
Step 1, 2 - Intermediate Compound 21: (9H-fluoren-9-yl)methyl 2,2-dimethylpiperazine-1- carboxylate, HCl salt
Figure imgf000129_0004
K2CO3 (1.935 g, 14.00 mmol) was added to a mixture of tert-butyl 3,3-dimethylpiperazine-1- carboxylate (1 g, 4.67 mmol) and (9H-fluoren-9-yl)methyl carbonochloridate (1.328 g, 5.13 mmol) in DCM (3 mL). The resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered through celite The solvent was removed under reduced pressure to afford the di-protected Intermediate Compound 20. The intermediate was treated with HCl in dioxane according to General Procedure II, Method A to afford Intermediate Compound 21 (1.300 g, 83 %) as a white solid.1H NMR (400 MHz, DMSO) δ 0.98 (s, 6H), 1.12 – 1.20 (m, 1H), 2.86 – 2.93 (m, 2H), 3.00 – 3.06 (m, 2H), 3.43 – 3.47 (m, 2H), 4.29 (s, 1H), 4.58 (d, J = 4.9 Hz, 2H), 7.33 (dd, J = 1.3, 7.5 Hz, 2H), 7.40 – 7.44 (m, 2H), 7.65 (d, J = 7.6 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H). ES+(M+1 = 337). Step 3 - Intermediate Compound 22 (9H-fluoren-9-yl)methyl 2,2-dimethyl-4-((2S,4S)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl)- piperazine-1-carboxylate
Figure imgf000130_0001
General Procedure I was followed using PyOxim (306 mg, 0.60 mmol), the PNU-159682 carboxylic acid (CAS No.: 1204819-92-0; 150 mg, 0.24 mmol), Intermediate Compound 21121 mg, 0.36 mmol) and DIEA (0.209 mL, 1.20 mmol) to afford Intermediate Compound 22 (170 mg, 75 %) as a red solid. NMR (400 MHz, DMSO) δ 0.93 (s, 3H), 0.98 – 1.05 (m, 3H), 1.19 – 1.27 (m, 3H), 1.63 – 1.72 (m, 2H), 2.05 – 2.21 (m, 2H), 2.62 – 2.70 (m, 3H), 2.85 – 2.89 (m, 3H), 3.30 (d, J = 8.5 Hz, 5H), 3.48 – 3.56 (m, 4H), 3.68 (dd, J = 8.5, 11.9 Hz, 2H), 3.83 – 3.97 (m, 5H), 4.18 – 4.30 (m, 3H), 4.60 (d, J = 2.1 Hz, 1H), 5.00 (d, J = 8.7 Hz, 1H), 5.25 (d, J = 5.5 Hz, 1H), 5.74 (s, 1H), 7.32 – 7.35 (m, 3H), 7.63 (d, J = 6.8 Hz, 3H), 7.88 (dd, J = 3.3, 7.2 Hz, 5H). ES+(M+1 = 946).
Step 4 - Compound P7 (8S,10S)-8-(3,3-dimethylpiperazine-1-carbonyl)-6,8,11-trihydroxy-1-methoxy- 10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000131_0001
General Procedure III was followed using DCM solution of DBU (15.2 mg/mL in DCM) (0.07 mL, 0.08 mmol) and Intermediate Compound 22 (160 mg, 0.17 mmol) in DCM (4 mL)/MeCN (1.000 mL) to afford Compound P7 (21.00 mg, 17.16 %) as a red solid.
Figure imgf000131_0002
NMR (400 MHz, DMSO) δ 0.80 – 1.06 (m, 7H), 1.24 (d, J = 6.3 Hz, 4H), 1.65 – 1.70 (m, 2H), 2.11 – 2.17 (m, 1H), 2.64 (d, J = 8.7 Hz, 2H), 2.84 (d, J = 17.8 Hz, 2H), 3.06 – 3.22 (m, 2H), 3.38 (d, J = 6.3 Hz, 3H), 3.47 – 3.54 (m, 2H), 3.67 (s, 1H), 3.81 (s, 1H), 3.93 (d, J = 2.0 Hz, 1H), 3.94 – 4.07 (m, 6H), 4.24 (d, J = 2.2 Hz, 1H), 4.60 (d, J = 2.1 Hz, 1H), 5.00 – 5.04 (m, 1H), 5.24 – 5.27 (m, 1H), 5.88 (s, 1H), 7.65 – 7.69 (m, 1H), 7.91 – 7.96 (m, 2H), 13.91 – 14.20 (m, 1H). ES+(M+1 = 724). Example 9A: Synthesis of Compound P7a
Figure imgf000131_0003
Step 1 - Intermediate Compound 23: tert-butyl 4-acetyl-3,3-dimethylpiperazine-1-carboxylate
Figure imgf000132_0001
Acetic anhydride (5.00 g, 48.99 mmol) was added dropwise to a solution of tert-butyl 3,3- dimethylpiperazine-1-carboxylate (5 g, 23.33 mmol) and DIEA (20.37 mL, 116.65 mmol) in DCM (110 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 4 hours. The reaction mixture was quenched with saturated brine (100 mL), extracted with DCM (2 x 75 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 23 (5.20 g, 87 %) as a white solid.
Figure imgf000132_0002
NMR (300 MHz, CDCl3) δ 1.48 (d, J = 2.6 Hz, 15H), 2.24 (s, 3H), 3.32 – 3.66 (m, 6H). ES+(M+1= 257). Step 2 - Intermediate Compound 24: 1-(2,2-dimethylpiperazin-1-yl)ethan-1-one, HCl salt
Figure imgf000132_0003
General Procedure II, Method A was followed using HCl in dioxane (4 M) (29.3 mL, 117.2 mmol) and Intermediate Compound 23 (5 g, 19.50 mmol) to afford Intermediate Compound 24 as a pure solid material (3 g HCl salt).1H
Figure imgf000132_0004
NMR (300 MHz, DMSO) δ 1.46 (s, 6H), 2.00 (s, 3H), 3.03 (t, J = 5.1 Hz, 2H), 3.07 – 3.24 (m, 2H), 3.51 – 3.69 (m, 2H). ES+(M+1 = 157). Step 3 - Compound P7a: (8S,10S)-8-(4-acetyl-3,3-dimethylpiperazine-1-carbonyl)-6,8,11-trihydroxy- 1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000132_0005
General Procedure I was followed using DIEA (0.139 mL, 0.80 mmol), Intermediate Compound 24 (49.8 mg, 0.32 mmol), HATU (121 mg, 0.32 mmol) and the PNU-159682 carboxylic acid (CAS No. 1204819-92-0; 100 mg, 0.16 mmol) to afford Compound P7a (22.01 mg, 18.04 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 1.39 (d, J = 6.4 Hz, 3H), 1.58 (d, J = 12.9 Hz, 6H), 1.70 – 1.81 (m, 1H), 1.92 – 2.03 (m, 1H), 2.13 (s, 3H), 2.47 (dd, J = 4.0, 15.3 Hz, 1H), 2.63 – 2.86 (m, 3H), 2.96 (dd, J = 10.6, 19.3 Hz, 1H), 3.38 – 3.42 (m, 1H), 3.47 (s, 4H), 3.54 – 3.63 (m, 1H), 3.66 – 3.79 (m, 4H), 3.88 – 4.13 (m, 7H), 4.27 (dd, J = 13.5, 22.8 Hz, 1H), 4.48 (d, J = 1.9 Hz, 1H), 4.72 (d, J = 1.9 Hz, 1H), 5.00 (s, 1H), 5.36 (d, J = 4.7 Hz, 1H), 5.52 – 5.59 (m, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.75 – 7.83 (m, 1H), 8.04 (d, J = 7.6 Hz, 1H), 13.33 (d, J = 10.4 Hz, 1H), 13.92 (d, J = 4.7 Hz, 1H). ES+(M+1 = 766). Example 10: Synthesis of Compound P8
Figure imgf000133_0001
Step 1 - Compound P8: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4’,3’:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-(5,8- diazaspiro[3.5]nonane-8-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000133_0002
General Procedure I was followed using HATU (273 mg, 0.72 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 300 mg, 0.48 mmol), DIEA (0.417 mL, 2.39 mmol) and 5,8- diazaspiro[3.5]nonane (121 mg, 0.96 mmol) to afford Compound P8 (50.0 mg, 14.22 %) as a red solid.1H NMR (300 MHz, CDCl3) δ 1.28 (s, 1H), 1.41 (d, J = 6.3 Hz, 3H), 1.77 (dt, J = 6.0, 15.0 Hz, 1H), 1.86 (s, 4H), 1.98 (dt, J = 5.7, 15.0 Hz, 1H), 2.47 (d, J = 15.3 Hz, 1H), 2.65 – 2.87 (m, 2H), 2.95 (d, J = 7.5 Hz, 3H), 3.03 (s, 1H), 3.39 (q, J = 6.3 Hz, 1H), 3.48 (s, 3H), 3.59 (dt, J = 3.6, 11.7 Hz, 1H), 3.87 – 4.01 (m, 1H), 4.09 (d, J = 16.5 Hz, 5H), 4.50 (d, J = 1.8 Hz, 1H), 4.74 (d, J = 1.8 Hz, 1H), 4.97 (s, 1H), 5.39 (s, 1H), 5.57 (s, 1H), 7.42 (dd, J = 1.2, 8.7 Hz, 1H), 7.81 (t, J = 8.1 Hz, 1H), 8.06 (dd, J = 1.2, 7.8 Hz, 1H), 13.36 (s, 1H), 13.97 (s, 1H). ES+(M+1 = 736). Example 11: Synthesis of Compound P9
Figure imgf000134_0001
Step 1 – Intermediate Compound 25: 2-((9H-fluoren-9-yl)methyl) 6-(tert-butyl) 2,6- diazaspiro[3.3]heptane-2,6-dicarboxylate
Figure imgf000134_0002
tert-Butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (1 g, 5.04 mmol) was added to a mixture of N- (9H-fluoren-2-ylmethoxycarbonyloxy)succinimide (1.701 g, 5.04 mmol) and DIEA (2.64 mL, 15.13 mmol) in DCM (30 mL) at RT. The resulting mixture was stirred at RT for 2 hours. The reaction mixture was diluted with DCM (20 mL), and washed with water (3 x 40 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 25 (1.700 g, 80 %) as a yellow solid. NMR (400 MHz, DMSO) δ 1.37 (s, 9H), 3.30 (s, 8H), 3.75 – 3.83 (m, 3H), 7.37 (s, 4H), 7.65 (s, 1H), 7.85 (s, 3H). ES+(M+23 = 443). Step 2 – Intermediate Compound 26: (9H-fluoren-9-yl)methyl 2,6-diazaspiro[3.3]heptane-2- carboxylate, TFA salt
Figure imgf000134_0003
General Procedure II, Method B was followed using TFA (15 ml, 194.70 mmol), Intermediate Compound 25 (1.6 g, 3.80 mmol) in DCM (20 mL) to afford Intermediate Compound 26 (1.100 g, 90 %) as a pale yellow solid.1H
Figure imgf000135_0001
NMR (400 MHz, DMSO) δ 1.19 – 1.32 (m, 2H), 4.06 – 4.14 (m, 5H), 4.19 – 4.27 (m, 1H), 4.29 (d, J = 6.1 Hz, 2H), 7.28 – 7.39 (m, 2H), 7.39 – 7.49 (m, 2H), 7.64 (d, J = 7.4 Hz, 2H), 7.91 (d, J = 7.5 Hz, 2H). ES+(M+1 = 321). Step 3 – Intermediate Compound 27: (9H-fluoren-9-yl)methyl 6-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4’,3’:4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-2,6- diazaspiro[3.3]heptane-2-carboxylate
Figure imgf000135_0002
General Procedure I was followed using HATU (136 mg, 0.36 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), DIEA (0.209 mL, 1.20 mmol) and Intermediate Compound 26 (153 mg, 0.48 mmol) to afford Intermediate Compound 27 (130 mg, 58.5 %) as a red solid.1H NMR (300 MHz, DMSO) δ 1.15 – 1.32 (m, 3H), 1.67 (t, J = 5.9 Hz, 1H), 2.18 (d, J = 14.6 Hz, 1H), 2.21 – 2.34 (m, 1H), 2.65 (d, J = 15.4 Hz, 2H), 2.91 (d, J = 18.3 Hz, 1H), 3.09 (d, J = 18.1 Hz, 1H), 3.36 (s, 1H), 3.51 (d, J = 11.6 Hz, 1H), 3.66 (d, J = 8.4 Hz, 1H), 4.02 (d, J = 19.0 Hz, 10H), 4.21 – 4.33 (m, 4H), 4.53 (d, J = 10.8 Hz, 1H), 4.56 – 4.64 (m, 2H), 4.99 (s, 1H), 5.26 (d, J = 4.1 Hz, 2H), 5.76 (s, 5H), 7.39 (dtd, J = 1.2, 7.4, 25.2 Hz, 4H), 7.64 (d, J = 7.2 Hz, 3H), 7.80 – 7.95 (m, 4H), 13.28 (s, 1H), 14.06 (s, 1H). ES+(M+1 = 930). Step 4 – Compound P9: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-(2,6- diazaspiro[3.3]heptane-2-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000135_0003
General Procedure III was followed using DBU (9.73 µl, 0.06 mmol) and Intermediate Compound 27 (120 mg, 0.13 mmol) to afford Compound P9 (11.00 mg, 12.05 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 1.43 (d, J = 6.5 Hz, 3H), 1.74 - 1.79 (m, 1H), 1.97 - 2.03 (m, 1H), 2.31 - 2.39 (m, 1H), 2.52 (d, J = 14.3 Hz, 1H), 2.73 - 2.88 (m, 2H), 3.15 (d, J = 19.0 Hz, 1H), 3.27 - 3.36 (m, 1H), 3.41 (d, J = 6.7 Hz, 1H), 3.48 (d, J = 1.4 Hz, 3H), 3.57- 3.65 (m, 1H), 3.92 - 4.16 (m, 10H), 4.23 (s, 2H), 4.50 (d, J = 1.9 Hz, 1H), 4.64 - 4.78 (m, 4H), 5.33 (s, 1H), 5.53 (t, J = 5.3 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.76 - 7.85 (m, 1H), 8.02 - 8.10 (m, 1H), 13.35 (s, 1H), 13.96 (s, 1H). ES+(M+1 = 708). Example 12: Synthesis of Compound P9a
Figure imgf000136_0001
Step 1 - Intermediate Compound 28: tert-butyl 6-acetyl-2,6-diazaspiro[3.3]heptane-2-carboxylate
Figure imgf000136_0002
Ac2O (0.714 mL, 7.57 mmol) was added to a solution of tert-butyl 2,6-diazaspiro[3.3]heptane-2- carboxylate (1g, 5.04 mmol) and DIEA (4.40 mL, 25.22 mmol) in DCM (15 mL)/DMA (1.5 mL) at 0°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water (25 mL), extracted with DCM (3 x 20 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford crude Intermediate Compound 28 (1.000 g, 83 %) as a pale yellow oil.1
Figure imgf000136_0003
H NMR (400 MHz, DMSO) δ 1.37 (s, 9H), 1.72 (s, 3H), 3.95 (d, J = 12.4 Hz, 6H), 4.21 (s, 2H). m/z (ES+), [M+H]+ = 241. Step 2 - Intermediate Compound 29: 1-(2,6-diazaspiro [3.3]heptan-2-yl)ethan-1-one, TFA salt
Figure imgf000137_0001
General Procedure II, Method B was followed using Intermediate Compound 28 (900 mg, 3.75 mmol) and TFA (5 mL)/DCM (5.00 mL) to afford Intermediate Compound 29 (450 mg, 86 %) as a pale yellow solid.
Figure imgf000137_0002
NMR (400 MHz, DMSO) δ 1.72 (s, 3H), 1.96 (s, 1H), 3.97 (d, J = 5.9 Hz, 2H), 4.10 (t, J = 6.2 Hz, 4H), 4.25 (s, 2H). m/z (ES+), [M+H]+ = 141. Step 3 - Compound P9a (8S,10S)-8-(6-acetyl-2,6-diazaspiro[3.3]heptane-2-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione .
Figure imgf000137_0003
, . , . , , . , . , . , , . , , . (dd, J = 5.3, 15.3 Hz, 1H), 2.35 (d, J = 15.0 Hz, 1H), 2.53 (d, J = 15.1 Hz, 1H), 2.68 – 2.87 (m, 2H), 3.13 (dd, J = 5.4, 19.1 Hz, 1H), 3.27 – 3.42 (m, 2H), 3.46 (s, 3H), 3.58 (d, J = 11.7 Hz, 1H), 3.87 – 4.12 (m, 6H), 4.11-4.19 (m, 2H), 4.20-4.25 (m, 2H), 4.28-4.32 (m, 2H), 4.70 – 4.82 (m, 4H), 5.32 (s, 1H), 5.51 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 7.7 Hz, 1H), 13.33 (d, J = 5.3 Hz, 1H), 13.94 (s, 1H). [M+H]+ = 750. Example 13: Synthesis of Compound P10
Figure imgf000138_0001
Step 1 - Intermediate Compound 30: 8-((9H-fluoren-9-yl)methyl) 3-(tert-butyl) 3,8- diazabicyclo[3.2.1]octane-3,8-dicarboxylate
Figure imgf000138_0002
N-(9H-Fluoren-2-ylmethoxycarbonyloxy)succinimide (1.589 g, 4.71 mmol) was added to tert-butyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (1 g, 4.71 mmol) and DIEA (1.645 mL, 9.42 mmol) in DCM (20 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was diluted with DCM (50 mL), and washed with water (2 x 50 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford afford Intermediate Compound 30 (1.700 g, 83 %) as a yellow solid. ES+(M+1 = 435). Step 2 - Intermediate Compound 31: (9H-fluoren-9-yl)methyl 3,8-diazabicyclo[3.2.1]octane-8- carboxylate, HCl salt
Figure imgf000138_0003
General Procedure II, Method A was followed using HCl (5 mL, 4 M in dioxane) and Intermediate Compound 30 (1.6 g, 3.68 mmol) in 1,4-dioxane (10 mL) to afford Intermediate Compound 31 (0.950 g, 77 %) as a yellow solid.
Figure imgf000138_0004
NMR (400 MHz, DMSO) δ 1.78 – 2.08 (m, 4H), 2.50 (s, 1H), 2.86 (s, 2H), 3.05 (d, J = 12.4 Hz, 2H), 4.17 (d, J = 43.1 Hz, 1H), 4.31 (t, J = 6.2 Hz, 1H), 4.47 (s, 2H), 7.35 (td, J = 1.2, 7.4 Hz, 2H), 7.43 (td, J = 1.2, 7.5 Hz, 2H), 7.64 (d, J = 7.5 Hz, 2H), 7.91 (d, J = 7.6 Hz, 2H). ES+(M+1 = 335). Step 3 - Intermediate Compound 32: (9H-fluoren-9-yl)methyl 3-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-3,8- diazabicyclo[3.2.1]octane-8-carboxylate
Figure imgf000139_0001
General Procedure I was followed using PyOxim (733 mg, 1.43 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 450 mg, 0.72 mmol), Intermediate Compound 31 (599 mg, 1.79 mmol) and DIEA (0.626 mL, 3.59 mmol) to afford Intermediate Compound 32 (550 mg, 81%) as a red solid. NMR (400 MHz, DMSO) δ 1.24 (s, 3H), 1.66 – 1.72 (m, 4H), 1.88 (d, J = 31.5 Hz, 2H), 2.17 (d, J = 44.7 Hz, 2H), 2.67 (dd, J = 5.9, 16.4 Hz, 4H), 3.39 (d, J = 6.6 Hz, 2H), 3.48 – 3.55 (m, 1H), 3.62 – 3.72 (m, 2H), 3.94 (d, J = 6.3 Hz, 3H), 3.99 (s, 5H), 4.01 – 4.06 (m, 2H), 4.22 – 4.31 (m, 3H), 4.50 (d, J = 24.1 Hz, 2H), 4.61 (d, J = 2.0 Hz, 2H), 5.25 – 5.29 (m, 1H), 5.85 (s, 1H), 7.32 – 7.42 (m, 4H), 7.64 (d, J = 6.0 Hz, 3H), 7.91 (d, J = 7.1 Hz, 4H), 13.32 (s, 1H), 14.04 (s, 1H). ES+(M+1 = 944). Step 4 - Compound P10: (8S,10S)-8-(3,8-diazabicyclo[3.2.1]octane-3-carbonyl)-6,8,11-trihydroxy-1- methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000139_0002
General Procedure III was followed using DCM solution of DBU (15.2 mg/mL in DCM) (3.86 mL, 0.39 mmol) and Intermediate Compound 32 (520 mg, 0.55 mmol) in DCM (24 mL)/MeCN (6.00 mL) to afford Compound P10 (130 mg, 32.7 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 1.42 (d, J = 6.4 Hz, 3H), 1.70 – 1.83 (m, 4H), 1.99 (s, 3H), 2.43 (dd, J = 4.0, 15.3 Hz, 1H), 2.63 – 3.12 (m, 5H), 3.48 (s, 6H), 3.55 – 3.68 (m, 3H), 3.90 – 4.00 (m, 1H), 4.02 – 4.13 (m, 5H), 4.28 – 4.40 (m, 1H), 4.47 – 4.61 (m, 2H), 4.74 (d, J = 1.7 Hz, 1H), 4.87 (s, 1H), 5.38 (dd, J = 2.3, 4.1 Hz, 1H), 5.56 (s, 1H), 7.42 (dd, J = 1.1, 8.6 Hz, 1H), 7.76 – 7.85 (m, 1H), 8.03 – 8.09 (m, 1H), 13.36 (s, 1H), 13.95 (s, 1H). ES+(M+1 = 722). Example 14: Synthesis of Compound P11
Figure imgf000140_0001
Steps 1, 2 - Intermediate Compound 34: (9H-fluoren-9-yl)methyl 3,8-diazabicyclo[3.2.1]octane-3- carboxylate, HCl salt K2CO3 (1758 mg, 12.72 mmol) was added to a solution of tert-butyl 3,8-diazabicyclo[3.2.1]octane-8- carboxylate (900 mg, 4.24 mmol) and (9H-fluoren-9-yl)methyl carbonochloridate (1206 mg, 4.66 mmol) in DCM (30 mL). The resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered through celite. The solution was washed with water, dried over Na2SO4, then concentrated to afford the di-protected Intermediate Compound 33 as a a yellow oil. The yellow oil was redissolved in EtOH (10 mL), then HCl in EtOH/4M (15 mL, 60.00 mmol) was added. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 34 (1200 mg, 85 %) as a yellow solid. ES+( [M+H]+ = 335. Step 3 - Intermediate Compound 35: (9H-fluoren-9-yl)methyl 8-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate
Figure imgf000141_0001
General Procedure I was followed using PyOxim (244 mg, 0.48 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), Intermediate Compound 34 (200 mg, 0.60 mmol) and DIEA (0.209 mL, 1.20 mmol) to afford Intermediate Compound 35 (140 mg, 62.1 %) as a red solid. NMR (400 MHz, DMSO) δ 1.24 (dd, J = 3.3, 6.8 Hz, 3H), 1.49 – 1.62 (m, 2H), 1.69 (d, J = 5.7 Hz, 2H), 1.74 – 1.82 (m, 2H), 1.82 – 1.93 (m, 2H), 2.05 – 2.17 (m, 2H), 2.56 (s, 1H), 2.60 – 2.71 (m, 4H), 2.88 (dd, J = 8.3, 12.8 Hz, 2H), 3.39 (d, J = 6.2 Hz, 2H), 3.47 – 3.59 (m, 2H), 3.62 – 3.72 (m, 2H), 3.93 (d, J = 6.0 Hz, 1H), 3.98 (s, 3H), 3.99 – 4.08 (m, 2H), 4.25 (dd, J = 4.7, 9.9 Hz, 2H), 4.59 (d, J = 2.1 Hz, 1H), 4.97 (d, J = 22.6 Hz, 2H), 5.26 (d, J = 7.3 Hz, 1H), 6.28 (s, 2H), 7.35 (d, J = 1.3 Hz, 2H), 7.43 (dd, J = 1.2, 7.4 Hz, 2H), 7.64 (dd, J = 4.8, 9.1 Hz, 2H), 7.89 (dd, J = 3.4, 7.7 Hz, 5H). ES+(M+1 = 945). Step 4 - Compound P11: (8S,10S)-8-(3,8-diazabicyclo[3.2.1]octane-8-carbonyl)-6,8,11-trihydroxy-1- methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000141_0002
General Procedure III was followed using DCM solution of DBU (15.2 mg/mL in DCM) (0.828 mL, 0.08 mmol) and Intermediate Compound 35 (130 mg, 0.14 mmol) in DCM (8.00 mL)/MeCN (2 mL) to afford Compound P11 (22.00 mg, 22.13 %) as a red solid.1
Figure imgf000142_0001
H NMR (400 MHz, CDCl3) δ 1.42 (d, J = 6.7 Hz, 3H), 1.72 – 1.83 (m, 1H), 1.98 (s, 6H), 2.47 (d, J = 16.8 Hz, 1H), 2.64 – 2.96 (m, 6H), 3.19 (d, J = 31.9 Hz, 2H), 3.31 – 3.44 (m, 2H), 3.48 (s, 3H), 3.57 – 3.64 (m, 1H), 3.88 – 4.00 (m, 1H), 4.11 (s, 5H), 4.50 (s, 1H), 4.80 (d, J = 53.4 Hz, 4H), 5.38 (s, 1H), 5.56 (s, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.76 – 7.84 (m, 1H), 8.06 (d, J = 7.6 Hz, 1H), 13.36 (s, 1H), 13.98 (s, 1H). ES+(M+1 = 722). Example 15: Synthesis of Compound P12
Figure imgf000142_0002
Step 1 - Intermediate Compound 36: 2-((9H-fluoren-9-yl)methyl) 5-(tert-butyl) (1R,4R)-2,5- diazabicyclo[2.2.1]heptane-2,5-dicarboxylate
Figure imgf000142_0003
tert-Butyl (1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (1g, 5.04 mmol) was added to a solution of DIEA (2.64 mL, 15.13 mmol) and N-(9H-Fluoren-2-ylmethoxycarbonyloxy)succinimide (1.701 g, 5.04 mmol) in DCM (20 mL) at RT. The resulting mixture was stirred at RT for 2 hours. The reaction mixture was diluted with DCM (200 mL), and washed with water (3 x 200 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 36 (2.300 g, 92 %) as a yellow solid.1H NMR (300 MHz, DMSO) δ 0.96 (dd, J = 6.9, 2.0 Hz, 6H), 1.40 (d, J = 4.1 Hz, 9H), 1.77 (s, 2H), 2.98 (pd, J = 6.4, 1.2 Hz, 1H), 4.27 (d, J = 22.4 Hz, 1H), 6.29 (s, 2H), 7.38 (dtd, J = 21.9, 7.4, 1.3 Hz, 4H), 7.8–7.91 (m, 3H). ES+(M+23 = 443). Step 2 - Intermediate Compound 37: (9H-fluoren-9-yl)methyl (1R,4R)-2,5- diazabicyclo[2.2.1]heptane-2-carboxylate, HCl salt
Figure imgf000143_0001
General Procedure II, Method A was followed using 4 N HCL in 1,4-dioxane (10 ml, 40.00 mmol), and Intermediate Compound 36 (2 g, 4.76 mmol) in DCM (10 mL) to afford crude product Intermediate Compound 37 (2.200 g, 99 %) as a yellow solid.
Figure imgf000143_0002
NMR (300 MHz, DMSO) δ 1.29 (dd, J = 10.6, 6.7 Hz, 4H), 1.71–2.05 (m, 1H), 2.91–3.27 (m, 2H), 3.29–3.81 (m, 3H), 4.15–4.6 (m, 3H), 5.77 (s, 1H), 7.26–7.48 (m, 2H), 7.66 (d, J = 7.4 Hz, 1H), 7.91 (d, J = 7.4 Hz, 1H), 9.30 (d, J = 60.3 Hz, 1H), 9.86 (s, 1H). ES+(M+1 = 321). Step 3 - Intermediate Compound 38 (9H-fluoren-9-yl)methyl (1R,4R)-5-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-2,5- diazabicyclo[2.2.1]heptane-2-carboxylate
Figure imgf000143_0003
General Procedure I was followed using PyOxim (293 mg, 0.57 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 180 mg, 0.29 mmol), Intermediate Compound 37 (230 mg, 0.72 mmol) and DIEA (0.250 mL, 1.43 mmol) to afford Intermediate Compound 38 (160 mg, 60.0 %) as a red solid. NMR (400 MHz, DMSO) δ 1.20 – 1.29 (m, 3H), 1.66 (d, J = 10.1 Hz, 2H), 1.77 – 1.92 (m, 2H), 2.01 (dd, J = 8.5, 12.7 Hz, 2H), 2.10 – 2.40 (m, 2H), 2.61 – 2.70 (m, 2H), 2.73 – 3.02 (m, 3H), 3.17 (d, J = 11.3 Hz, 1H), 3.38 (dd, J = 5.7, 12.7 Hz, 5H), 3.45 – 3.57 (m, 2H), 3.63 – 3.71 (m, 2H), 3.94 – 4.00 (m, 4H), 4.01 – 4.11 (m, 1H), 4.14 (d, J = 10.4 Hz, 1H), 4.22 – 4.29 (m, 2H), 4.31 – 4.37 (m, 2H), 4.61 (dd, J = 2.0, 8.6 Hz, 1H), 5.52 (s, 1H), 7.26 – 7.45 (m, 6H), 7.54 – 7.66 (m, 2H), 7.81 – 7.96 (m, 3H), 13.27 (s, 1H), 13.89 (s, 1H). ES+(M+1 = 930). Step 4 - Compound P12: (8S,10S)-8-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000144_0001
General Procedure III was followed using DCM solution of DBU (15.2 mg/mL in DCM) (1.131 mL, 0.11 mmol) and Intermediate Compound 38 (150 mg, 0.16 mmol) in DCM (8 mL)/MeCN (2.000 mL) to afford Compound P12 (42.0 mg, 36.8 %) as a red solid.
Figure imgf000144_0002
NMR (400 MHz, CDCl3) δ 1.42 (dd, J = 4.3, 6.5 Hz, 3H), 1.72 – 1.91 (m, 4H), 1.97 – 2.04 (m, 1H), 2.29 – 2.40 (m, 1H), 2.60 – 2.70 (m, 1H), 2.72 – 2.87 (m, 2H), 3.08 – 3.25 (m, 3H), 3.33 – 3.46 (m, 3H), 3.48 (s, 3H), 3.55 – 3.65 (m, 2H), 3.79 – 4.00 (m, 3H), 4.07 (d, J = 7.1 Hz, 1H), 4.11 (d, J = 4.8 Hz, 3H), 4.47 – 4.52 (m, 1H), 4.68 – 4.76 (m, 1H), 4.81 (s, 1H), 5.35 (s, 1H), 5.47 (s, 1H), 5.50 – 5.60 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.76 – 7.87 (m, 1H), 8.06 (d, J = 7.7 Hz, 1H), 13.34 (s, 1H), 13.98 (s, 1H). ES+(M+1 = 708). Example 16: Synthesis of Compound P13
Figure imgf000144_0003
Step 1 - Intermediate Compound 39: 2-((9H-fluoren-9-yl)methyl) 5-(tert-butyl) (1S,4S)-2,5- diazabicyclo[2.2.1]heptane-2,5-dicarboxylate
Figure imgf000144_0004
DIEA (0.881 mL, 5.04 mmol) was added to a solution of tert-butyl (1S,4S)-2,5- diazabicyclo[2.2.1]heptane-2-carboxylate (500 mg, 2.52 mmol) and N-(9H-Fluoren-2- ylmethoxycarbonyloxy)succinimide (851 mg, 2.52 mmol) in DCM (20 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was diluted with DCM (25 mL), and washed with water (2 x 25 mL). The bottom layer was dried over Na2SO4, filtered and evaporated to afford crude product Intermediate Compound 39 (800 mg, 75 %) as a yellow solid. NMR (400 MHz, DMSO) δ 1.40 (s, 11H), 2.61 – 3.03 (m, 4H), 3.07 – 3.22 (m, 3H), 4.12 – 4.44 (m, 2H), 7.25 – 7.47 (m, 4H), 7.59 – 7.74 (m, 1H), 7.78 – 7.95 (m, 3H). ES+(M+23 = 443). Step 2 - Intermediate Compound 40: (9H-fluoren-9-yl)methyl (1S,4S)-2,5-diazabicyclo [2.2.1]heptane-2-carboxylate, HCl salt
Figure imgf000145_0001
General Procedure II, Method A was followed using 4N HCL in 1,4-dioxane (1.40 mL, 5.64 mmol), and Intermediate Compound 39 (790 mg, 1.88 mmol) in DCM (10 mL) to afford Intermediate Compound 40 (550 mg, 91 %) as a yellow solid.
Figure imgf000145_0002
NMR (400 MHz, DMSO) δ 1.88 – 2.00 (m, 1H), 2.27 (t, J = 8.0 Hz, 2H), 2.74 – 2.81 (m, 2H), 3.26 – 3.29 (m, 2H), 3.60 (dd, J = 5.2, 17.3 Hz, 2H), 4.03 – 4.11 (m, 1H), 4.24 – 4.28 (m, 2H), 7.30 – 7.37 (m, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.75 (t, J = 7.8 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H). ES+(M+1 = 321). Step 3 - Intermediate Compound 41: (9H-fluoren-9-yl)methyl (1S,4S)-5-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-2,5- diazabicyclo[2.2.1]heptane-2-carboxylate
Figure imgf000145_0003
General Procedure I was followed using PyOxim (326 mg, 0.64 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol), Intermediate Compound 40 (255 mg, 0.80 mmol) and DIEA (0.278 mL, 1.59 mmol) to afford Intermediate Compound 41 (200 mg, 67.5 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.20 – 1.27 (m, 3H), 1.60 – 2.01 (m, 6H), 2.55 – 2.65 (m, 2H), 2.87 (d, J = 17.7 Hz, 1H), 2.98 – 3.03 (m, 1H), 3.08 – 3.21 (m, 2H), 3.31 (s, 3H), 3.89 – 4.11 (m, 6H), 4.21 – 4.31 (m, 3H), 4.35 (dd, J = 6.8, 16.4 Hz, 2H), 4.42 (s, 1H), 4.57 – 4.63 (m, 1H), 5.00 – 5.09 (m, 1H), 5.19 – 5.40 (m, 2H), 5.41 – 5.55 (m, 1H), 5.77 (s, 3H), 7.29 – 7.36 (m, 2H), 7.36 – 7.43 (m, 2H), 7.63 (dd, J = 7.2, 10.2 Hz, 3H), 7.85 – 7.93 (m, 4H), 13.21 (s, 1H), 14.05 (s, 1H). ES+(M+23 = 952). Step 4 - Compound P13: (8S,10S)-8-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000146_0001
General Procedure III was followed using DCM solution of DBU (15.2 mg/mL in DCM) (0.969 mL, 0.10 mmol) and Intermediate Compound 41 (180 mg, 0.19 mmol) in DCM (12 mL)/MeCN (3.00 mL) to afford Compound P13 (33.0 mg, 24.09 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 1.37 – 1.45 (m, 3H), 1.76 – 2.01 (m, 4H), 2.48 – 2.63 (m, 2H), 2.74 – 2.85 (m, 2H), 3.01 (d, J = 5.0 Hz, 1H), 3.13 – 3.27 (m, 2H), 3.48 (s, 6H), 3.55 – 3.67 (m, 2H), 3.90 – 3.98 (m, 3H), 3.99 – 4.14 (m, 5H), 4.49 (d, J = 1.8 Hz, 1H), 4.72 – 4.91 (m, 2H), 5.29 – 5.41 (m, 2H), 5.51 – 5.60 (m, 1H), 7.36 – 7.45 (m, 1H), 7.75 – 7.84 (m, 1H), 8.00 – 8.09 (m, 1H), 13.38 (s, 1H), 13.96 (d, J = 9.8 Hz, 1H). ES+(M+1 = 708).
Example 17: Synthesis of Compound P14
Figure imgf000147_0001
Step 1a and 1b - Intermediate Compound 43: (9H-fluoren-9-yl)methyl 5-oxa-2,8- diazaspiro[3.5]nonane-8-carboxylate, HCl salt
Figure imgf000147_0002
tert-Butyl 5-oxa-2,8-diazaspiro[3.5]nonane-2-carboxylate (500 mg, 2.19 mmol) was added to a solution of N,N-diisopropylethylamine (849 mg, 6.57 mmol) and N-(9H-fluoren-2- ylmethoxycarbonyloxy)succinimide (813 mg, 2.41 mmol) in DCM (8 mL). The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water. The organic layer was separated, dried over Na2SO4. The solvent was removed under reduced pressure. The crude di- protected product Intermediate Compound 42 was treated with 4 N HCl solution in dioxane according to General Procedure II, Method A to afford Intermediate Compound 43 (650 mg, 85 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ3.11 – 3.83 (m, 9H), 4.12-4.51 (m, 3H), 7.36 (t, J = 7.7 Hz, 2H), 7.40 – 7.49 (m, 2H), 7.65 (t, J = 6.5 Hz, 2H), 7.92 (d, J = 7.6 Hz, 2H), 9.26 (br,2H). Step 3 - Intermediate Compound 44: (9H-fluoren-9-yl)methyl 2-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl)-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate
Figure imgf000148_0001
General Procedure I was followed using the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol), Intermediate Compound 43 (279 mg, 0.80 mmol), N,N-diisopropylethylamine (206 mg, 1.59 mmol) and PyOxim (326 mg, 0.64 mmol) in DMA (2 mL) and MeCN (4 mL) to afford Intermediate Compound 44 (150 mg, 49.0 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.25 (d, J = 6.8 Hz, 2H), 1.67 (s, 2H), 2.12 – 2.37 (m, 2H), 2.57 – 3.02 (m, 4H), 3.27 – 3.32 (m, 2H), 3.36 (s, 20H), 3.61 – 3.95 (m, 4H), 4.05 – 4.65 (m, 4H), 4.95 – 5.40 (m, 2H), 6.10 (s, 1H), 7.32 – 7.68 (m, 5H), 7.79 – 7.98 (m, 4H). m/z (ES+), [M+H]+ = 960. Step 4 - Compound P14: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-8-(5-oxa-2,8- diazaspiro[3.5]nonane-2-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000148_0002
General Procedure III was followed using Intermediate Compound 44 (130 mg, 0.14 mmol) and DCM solution of DBU (15.2mg/mL in DCM) (0.746 mL, ~0.07mmol) in DCM (1.5 mL) and MeCN (0.5 mL) to afford Compound P14 (20.00 mg, 20.02 %) as a red solid.
Figure imgf000148_0003
NMR (300 MHz, DMSO) δ 1.17 (d, J = 6.7 Hz, 1H), 1.25 (d, J = 6.7 Hz, 4H), 1.67 (s, 2H), 2.28 (s, 1H), 2.65 (d, J = 15.2 Hz, 3H), 2.79 (s, 1H), 2.92 (d, J = 17.6 Hz, 1H), 3.13 (s, 1H), 3.52 (d, J = 4.6 Hz, 4H), 3.59–3.80 (m, 4H), 3.94 (d, J = 8.4 Hz, 2H), 4.00 (s, 3H), 4.07 – 4.43 (m, 5H), 4.59 (s, 1H), 4.98 (s, 1H), 5.25 (d, J = 4.4 Hz, 1H), 5.31–5.39 (m, 1H), 7.67 (t, J = 4.9 Hz, 1H), 7.93 (d, J = 4.7 Hz, 2H). m/z (ES+), [M+H]+ = 738. Example 18: Synthesis of Compound P15
Figure imgf000149_0001
Steps 1-2 - Intermediate Compound 46: (9H-fluoren-9-yl)methyl 5-oxa-2,8-diazaspiro[3.5]nonane- 2-carboxylate, HCl salt
Figure imgf000149_0002
tert-Butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (700 mg, 3.07 mmol) was added to a solution of N-(9H-fluoren-2-ylmethoxycarbonyloxy)succinimide (1138 mg, 3.37 mmol) and N,N- diisopropylethylamine (1189 mg, 9.20 mmol) in DCM (15 mL) under air. The resulting mixture was stirred at 25 °C for 2 hours. The mixture was diluted with 50 ml of DCM, and washed with water (50 mL). The organic phase was isolated and dried over Na2SO4, then evaporated under reduced pressure. The crude di-protected product Intermediate Compound 45 was treated with HCl solution (4N in 1,4-dioxane) (10 mL) according to General Procedure II, Method A to afford Intermediate Compound 46 (1000 mg, 76 %) as a yellow solid.1H NMR (300 MHz, DMSO) δ 3.09 (d, J = 19.2 Hz, 2H), 3.33 (s, 2H), 3.65 – 4.41 (m, 9H), 7.35 (td, J = 1.2, 7.4 Hz, 2H), 7.40 – 7.48 (m, 2H), 7.65 (d, J = 7.4 Hz, 2H), 7.91 (d, J = 7.4 Hz, 2H), 9.49 (s, 2H). ES+(M+1= 351). Step 3 - Intermediate Compound 47: (9H-fluoren-9-yl)methyl 8-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-5-oxa-2,8- diazaspiro[3.5]nonane-2-carboxylate
Figure imgf000150_0001
General Procedure I was followed using HATU (136 mg, 0.36 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), N,N-diisopropylethylamine (154 mg, 1.20 mmol), Intermediate Compound 46 (209 mg, 0.60 mmol) to afford Intermediate Compound 47 ( (130 mg, 56.7 %) as a red solid.
Figure imgf000150_0002
NMR (300 MHz, DMSO) δ 1.25 (d, J = 6.7 Hz, 3H), 1.66 (s, 1H), 2.17 (s, 1H), 2.59 – 2.68 (m, 2H), 2.90 (s, 1H), 3.31 (s, 5H), 3.39 - 4.06 (m, 20H), 4.24 (s, 1H), 4.59 (s, 1H), 5.07 (s, 1H), 5.26 (s, 1H), 5.77 (s, 3H), 6.09 (s, 1H), 7.39 (d, J = 7.7 Hz, 5H), 7.62 (s, 2H), 7.78 – 7.95 (m, 4H). ES+(M+1 = 960). Step 4 - Compound P15: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4’,3’:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-(5-oxa-2,8- diazaspiro[3.5]nonane-8-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000150_0003
General Procedure III was followed using DBU (7.85 µl, 0.05 mmol) and Intermediate Compound 47 (100 mg, 0.10 mmol) in DCM (2 mL)/MeCN (0.5 mL) to afford Compound P15 (15.00 mg, 19.52 %) as a red solid. ES+(M+1 = 738).
Example 19: Synthesis of Compound P15a
Figure imgf000151_0001
Step 1 – Intermediate Compound 48: tert-butyl 2-acetyl-5-oxa-2,8-diazaspiro[3.5]nonane-8- carboxylate
Figure imgf000151_0002
Ac2O (0.248 mL, 2.63 mmol) was added to a solution of tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane- 8-carboxylate (400 mg, 1.75 mmol) and DIEA (1.530 mL, 8.76 mmol) in DCM (15 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with DCM (75 mL), and washed with water (3 x 50 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 48 (420 mg, 89%) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.42 (s, 9H), 1.79 (s, 3H), 3.26 – 3.33 (m, 2H), 3.47 (d, J = 16.4 Hz, 2H), 3.52 – 3.62 (m, 2H), 3.62 – 3.71 (m, 2H), 3.96 (s, 2H). m/z (ES+), [M+H]+ = 271. Step 2 – Intermediate Compound 49: 1-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)ethan-1-one, HCl salt
Figure imgf000151_0003
General Procedure II, Method A was followed using Intermediate Compound 48 (410 mg, 1.52 mmol) to afford Intermediate Compound 49 (210 mg, 81 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.77 (s, 3H), 2.97 – 3.08 (m, 2H), 3.22 – 3.34 (m, 2H), 3.65 (d, J = 10.6 Hz, 1H), 3.79 – 3.88 (m, 2H), 3.99 (dd, J = 3.5, 10.0 Hz, 2H), 4.20 – 4.27 (m, 1H). m/z (ES+), [M+H]+ = 171. Step 3 – Compound P15a: (8S,10S)-8-(2-acetyl-5-oxa-2,8-diazaspiro[3.5]nonane-8-carbonyl)-6,8,11- trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000152_0001
General Procedure I was followed using HATU (158 mg, 0.41 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 130 mg, 0.21 mmol), DIEA (0.181 mL, 1.04 mmol) and Intermediate Compound 49 (70.5 mg, 0.41 mmol) in DMA (1.5 mL) to afford Compound P15a (46.0 mg, 28.5 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 1.42 (d, J = 6.4 Hz, 3H), 1.75 (s, 3H), 1.93 (s, 4H), 2.44 (dd, J = 4.2, 15.3 Hz, 1H), 2.65 – 3.01 (m, 4H), 3.48 (d, J = 2.3 Hz, 5H), 3.58 – 3.80 (m, 3H), 3.84 – 4.19 (m, 12H), 4.49 (d, J = 2.0 Hz, 1H), 4.69 – 4.76 (m, 1H), 5.11 (d, J = 63.0 Hz, 1H), 5.40 (s, 1H), 5.57 (d, J = 5.0 Hz, 1H), 7.43 (dd, J = 1.1, 8.6 Hz, 1H), 7.77 – 7.85 (m, 1H), 8.03 – 8.09 (m, 1H), 13.37 (s, 1H), 13.94 (d, J = 6.4 Hz, 1H). m/z (ES+), [M+H]+ = 780. Example 20: Synthesis of Compound P16
Figure imgf000152_0002
Step 1 - Compound P16: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-(2-oxa-5,8- diazaspiro[3.5]nonane-8-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000153_0001
General Procedure I was followed using HATU (91 mg, 0.24 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 100 mg, 0.16 mmol), DIEA (0.139 mL, 0.80 mmol) and 2-oxa-5,8- diazaspiro[3.5]nonane (71.5 mg, 0.56 mmol) to afford Compound P16 (30.0 mg, 25.5 %) as a red solid.1H NMR (300 MHz, CDCl3) δ 1.41 (d, J = 6.4 Hz, 3H), 1.70 - 1.81 (m, 1H), 1.92 - 2.07 (m, 1H), 2.40 - 2.50 (m, 1H), 2.67 - 2.86 (m, 3H), 2.90-3.02 (m, 3H), 3.40 (q, J = 6.4 Hz, 2H), 3.48 (s, 4H), 3.55 - 3.70 (m, 2H), 3.81 - 4.10 (m, 6H), 4.12 (s, 3H), 4.49 (d, J = 1.9 Hz, 1H), 4.51 - 4.67 (m, 3H), 4.73 (d, J = 1.9 Hz, 2H), 5.15 (s, 1H), 5.40 (s, 1H), 5.57 (t, J = 5.2 Hz, 1H), 7.38 - 7.47 (m, 1H), 7.81 (t, J = 8.1 Hz, 1H), 8.07 (dd, J = 1.1, 7.7 Hz, 1H), 13.37 (s, 1H), 13.96 (s, 1H). ES+ (M+1 = 738). Example 21: Synthesis of Compound P17
Figure imgf000153_0002
Step 1 - Compound P17: (8S,10S)-6,8,11-trihydroxy-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-8-(4,7- diazaspiro[2.5]octane-7-carbonyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000153_0003
General Procedure I was followed using HATU (91 mg, 0.24 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 100 mg, 0.16 mmol), DIEA (0.139 mL, 0.80 mmol) and 4,7- diazaspiro[2.5]octane (35.7 mg, 0.32 mmol) to afford Compound P17 (22.00 mg, 19.13 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 0.80-0.85 (m, 4H), 1.29 (d, J = 11.8 Hz, 1H), 1.42 (d, J = 6.4 Hz, 3H), 1.76 (t, J = 7.8 Hz, 1H), 1.95 – 1.99 (m, 1H), 2.40 - 2.50 (m, 1H), 2.64 – 2.87 (m, 3H), 2.98 (d, J = 18.5 Hz, 1H), 3.10 (s, 2H), 3.48 (s, 5H), 3.56 – 3.66 (m, 2H), 3.82 – 3.99 (m, 2H), 4.01 – 4.26 (m, 7H), 4.50 (d, J = 2.0 Hz, 1H), 4.74 (d, J = 1.8 Hz, 1H), 4.96 (s, 1H), 5.39 (s, 1H), 5.57 (t, J = 5.2 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.81 (t, J = 8.1 Hz, 1H), 8.03 - 8.10 (m, 1H), 13.36 (s, 1H), 13.96 (s, 1H). ES+(M+1 = 722). Example 22: Synthesis of Compound P18
Figure imgf000154_0001
Step 1 – Compound P18: (8S,10S)-8-(1,5-diazocane-1-carbonyl)-6,8,11-trihydroxy-1-methoxy-10- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3- c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000154_0002
General Procedure I was followed using HATU (145 mg, 0.38 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 120 mg, 0.19 mmol), DIEA (0.167 mL, 0.96 mmol) and 1,5-diazocane dihydrobromide (79 mg, 0.29 mmol) in DMA (3 mL) to afford Compound P18 (32.0 mg, 23.12 %) as a red solid. NMR (400 MHz, CDCl3) δ 1.28 (s, 1H), 1.41 (d, J = 6.4 Hz, 3H), 1.76 – 2.05 (m, 7H), 20 2.48 – 2.82 (m, 4H), 2.88 – 3.19 (m, 5H), 3.38 – 3.64 (m, 8H), 3.89 – 3.99 (m, 2H), 4.05-4.14 (m, 6H), 4.50 (d, J = 2.0 Hz, 1H), 4.74 (d, J = 1.6Hz, 1H), 5.37 (s, 1H), 5.52 – 5.59 (m, 1H), 7.41 (dd, J = 1.2, 8.8 Hz, 1H), 7.76 – 7.84 (m, 1H), 8.05 (dd, J = 1.2, 7.7 Hz, 1H), 13.96 (s, 1H). ES+(M+1 = 724). Example 23: Synthesis of Compound P19
Figure imgf000155_0001
Step 1 - Compound P19: (8S,10S)-8-(6,6-dimethyl-1,4-diazepane-1-carbonyl)-6,8,11-trihydroxy-1- methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000155_0002
General Procedure I was followed using HATU (145 mg, 0.38 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 120 mg, 0.19 mmol), DIEA (0.167 mL, 0.96 mmol) and 6,6-dimethyl- 1,4-diazepane dihydrochloride (77 mg, 0.38 mmol) to afford Compound P19 (10.50 mg, 7.44 %) as a red solid. NMR (400 MHz, CDCl3) δ 0.86 – 0.90 (m, 1H), 0.98 – 1.04 (m, 5H), 1.04 – 1.10 (m, 1H), 1.28 (s, 1H), 1.41 (d, J = 6.4 Hz, 3H), 1.74 – 1.81 (m, 1H), 1.98 – 2.02 (m, 1H), 2.46 – 2.54 (m, 1H), 2.62 – 2.85 (m, 5H), 2.99 (s, 1H), 3.03-3.11 (br, 2H), 3.30 (d, J = 13.6 Hz, 1H), 3.40 (d, J = 6.4 Hz, 1H), 3.48 (s, 4H), 3.53 – 3.63 (m, 2H), 3.70 – 3.86 (m, 2H), 3.91 – 3.96 (m, 1H), 4.12 (s, 6H), 4.50 (d, J = 1.9 Hz, 1H), 4.74 (d, J = 1.6 Hz, 1H), 5.00 (s, 1H), 5.36 (s, 1H), 5.54 (s, 1H), 7.39 – 7.45 (m, 1H), 7.76 – 7.85 (m, 1H), 8.06 (dd, J = 1.2, 7.6 Hz, 1H), 13.38 (s, 1H), 13.95 (s, 1H). ES+(M+1 = 738). 20 Example 24: Synthesis of Compound P20
Figure imgf000156_0001
Step 1 – Intermediate Compound 50: (3R,3aS,6R,6aS)-hexahydrofuro[3,2-b]furan-3,6-diyl bis(4- methylbenzenesulfonate)
Figure imgf000156_0002
Ts-Cl (32.6 g, 171.07 mmol) was added to a solution of (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan- 3,6-diol (“isomannide”, 10 g, 68.43 mmol) and pyridine(27 g, 5 eq ) in DCM (400 mL). The solution was stirred for overnight at room temperature. The reaction mixture was diluted with DCM (200 mL), and washed sequentially with 0.1M HCl (2 x 500 mL), saturated brine (500 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography to afford Intermediate Compound 50 (8.50 g, 27.3 %) as a white solid.
Figure imgf000156_0003
NMR (300 MHz, DMSO-d6) δ 2.42 (s, 6H), 3.51 – 3.61 (m, 2H), 3.74 – 3.84 (m, 2H), 4.34 -4.41 (m, 2H), 4.88 – 4.98 (m, 2H), 7.48 (d, 4H), 7.77- 7.85 (m, 4H). ES+(M+1 = 455). Step 2 - Intermediate Compound 51: (3S,3aR,6S,6aR)-3,6-diazidohexahydrofuro[3,2-b]furan
Figure imgf000156_0004
Sodium azide (3.43 g, 52.80 mmol) was added to a solution of Intermediate Compound 50 (4 g, 8.80 mmol) in DMF (80 mL). The solution was stirred for 3 hours at 140 °C. The reaction mixture was diluted with water (200 mL), and extracted with EA (3 x 200 mL). The combined organic solution was washed with water (2 x 300 mL), and brine (2 x 300 mL), The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product Intermediate Compound 51 (1.4 g, 81%) as a yellow oil.1
Figure imgf000157_0001
H NMR (300 MHz, DMSO-d6) δ 3.82-3.89 (m, 4H), 4.2-4.28(m, 2H), 4.58 (s, 2H). Step 3 - Intermediate Compound 52: (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine
Figure imgf000157_0002
Pd-C (0.076 g, 0.71 mmol) was added to Intermediate Compound 51 (1.4 g 7.14 mmol) in ethyl acetate (20 mL) and methanol (1 mL). The mixture was stirred for overnight hours 25 °C under H2. The reaction mixture was filtered through silica, and washed with EA (3 x 40 mL). The solvent was removed under reduced pressure to afford Intermediate Compound 52 (0.600 g, 58.3 %) as a yellow soild.1H NMR (300 MHz, DMSO-d6) δ 3.20-3.26 (m, 4H), 3.65 – 3.73 (m, 2H), 4.24 (s, 2H). ES+( M+1 = 145). Step 4 - Compound P20: (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine (161 mg, 1.12 mmol) to afford (2S,4S)-N-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3'':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamide
Figure imgf000157_0003
General Procedure I was followed using HATU (182 mg, 0.48 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol), DIEA (0.278 mL, 1.59 mmol) and Intermediate Compound 52 (161 mg, 1.12 mmol) to afford Compound P20 (30.0 mg, 12.49 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 1.40 (d, J = 6.5 Hz, 3H), 1.69-1.80 (m, 2H), 1.99-2.13 (m, 1H), 2.33-2.52 (m, 2H), 2.72-2.89 (m, 2H), 3.18-3.33 (m, 2H), 3.36-3.43 (m, 1H), 3.48 (s, 3H), 3.56-3.83 (m, 4H), 3.85- 4.03 (m, 4H), 4.12 (s, 4H), 4.38-4.56 (m, 3H), 4.70 (dd, J = 2.9, 25.8 Hz, 2H), 5.15 (s, 1H), 5.31-5.55 (m, 2H), 7.31-7.45 (m, 2H), 7.76-7.85 (m, 1H), 8.06 (dd, J = 1.1, 7.7 Hz, 1H), 13.32 (s, 1H), 13.95 (s, 1H). ES+(M+1 = 754). Example 25: Synthesis of Compound P21
Figure imgf000158_0001
Step 1 – Intermediate Compound 53: (3R,3aS,6R,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl 4- methylbenzenesulfonate
Figure imgf000158_0002
Ts-Cl (13.04 g, 68.43 mmol) was added to (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol (“isomannide”, 10 g, 68.43 mmol) and pyridine (13.77 mL, 171.07 mmol) in DCM (200 mL). The solution was stirred for overnight at room temperature. The reaction mixture was diluted with DCM (200 mL), and washed sequentially with 0.1M HCl (2 x 200 mL) and saturated brine (2 x 200 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography using 0 to 70% EtOAc in petroleum ether to afford Intermediate Compound 53 (14.50 g, 35.4 %) as a white solid.1H NMR (300 MHz, DMSO-d6) δ 2.43 (s, 3H), 3.32 (t, 1H), 3.53 - 3.61 (m, 1H), 3.73 - 3.84 (m, 2H), 4.03 - 4.14 (m, 1H), 4.23 (t, 1H), 4.39 (t, 1H), 4.80 - 5.05 (m, 2H), 7.49 (d, 2H), 7.78 -7.87 (m, 2H). m/z (ES+), [M+NH4]+ = 318. Step 2 - Intermediate Compound 54: (3R,3aS,6R,6aS)-6-((tert- butyldimethylsilyl)oxy)hexahydrofuro[3,2-b]furan-3-yl 4-methylbenzenesulfonate
Figure imgf000159_0001
TBS-Cl (3.76 g, 24.97 mmol) was added to a solution of Intermediate Compound 53 (5 g, 16.65 mmol) and 1H-imidazole (2.267 g, 33.30 mmol) in DCM (100 mL). The solution was stirred for overnight at room temperature. The reaction mixture was diluted with DCM (100 mL), and washed with saturated water (2 x 100 mL) and brine (2 x 100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 54 as crude product.1H NMR (300 MHz, DMSO-d6) δ 0.05 (s, 6H), 0.85 (s, 9H), 2.43 (s, 3H), 3.35-.341（m, 2H), 3.51 - 3.54 (m, 1H), 3.72 - 3.84 (m, 2H), 4.21 - 4.32 (m, 2H), 4.34 -4.41 (m, 1H), 4.85-4.95 (m, 1H), 7.45 - 7.52 (m, 2H), 7.77- 7.87 (m, 2H). m/z (ES+), [M+Na]+ = 437.10. Step 3 – Intermediate Compound 55: (((3R,3aS,6S,6aR)-6-azidohexahydrofuro[3,2-b]furan-3- yl)oxy)(tert-butyl)dimethylsilane
Figure imgf000159_0002
Sodium azide (2.59 g, 39.80 mmol) was added to Intermediate Compound 54 (5.5 g, 13.27 mmol) in DMF (110 mL). The solution was stirred for 3 hours at 140°C. The reaction mixture was diluted with water (250 mL), and extracted with ethyl acetate (3 x 150 mL). The combined organic solution was washed with water (2 x 200 mL), and brine (2 x 200 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 55 as crude product.1H
Figure imgf000159_0003
NMR (300 MHz, DMSO-d6) δ 0.08 (s, 6H), 0.87 (s, 9H), 3.41-3.48 (m, 1H), 3.66 -3.73 (m, 1H), 3.80-3.93 (m, 2H), 4.13 (d, 1H), 4.31 (q, 1H), 4.5- 4.42 (m, 1H), 4.47 (t, 1H). Step 4 – Intermediate Compound 56: (3S,3aR,6R,6aS)-6-((tert-butyldimethylsilyl)oxy)hexahydrofuro [3,2-b]furan-3-amine
Figure imgf000160_0001
Pd/C (1.3 g) was added to Intermediate Compound 55 (700 mg, 2.45 mmol) in EtOAc (10 mL) / MeOH (0.500 mL) at 25°C under H2. The resulting mixture was stirred at 25 °C for 16 hours under H2. The reaction mixture was filtered through celite. The solvent was removed under reduced pressure to afford Intermediate Compound 56 as crude product (600 mg, 94 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 0.07 (s, 6H), 0.87 (s, 9H), 1.66 (s, 2H), 3.22 - 3.28 (m, 1H), 3.30 – 3.40 (m, 1H), 3.48-3.56 (m, 1H), 3.62 - 3.70 (m, 1H), 3.70 - 3.78 (m, 1H), 4.12 (d, J = 4.3 Hz, 1H), 4.19 -4.35 (m, 1H), 4.39 (t, J = 4.5 Hz, 1H). m/z (ES+), [M+H]+ = 260. Step 5 – Intermediate Compound 57: (2S,4S)-N-((3S,3aR,6R,6aS)-6-((tert- butyldimethylsilyl)oxy)hexahydrofuro[3,2-b]furan-3-yl)-2,5,12-trihydroxy-7-methoxy-4- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
Figure imgf000160_0002
General Procedure I was followed using HATU (182 mg, 0.48 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol), DIEA (0.278 mL, 1.59 mmol) and Intermediate Compound 56 to afford Intermediate Compound 57 (220 mg, 79 %) as a red solid.1H
Figure imgf000160_0003
NMR (400 MHz, DMSO) δ 0.06 - 0.09 (m, 6H), 0.87 (d, J = 6.5 Hz, 12H), 1.21 (d, J = 6.5 Hz, 3H), 1.65 - 1.71 (m, 1H), 2.15 (d, J = 14.6 Hz, 1H), 2.23 - 2.29 (m, 1H), 2.63- 2.71 (m, 1H), 2.95 (d, J = 8.7 Hz, 1H), 3.32 (s, 2H), 3.34 (s, 2H), 3.34-3.38 (m, 1H), 3.38 -3.56 (m, 3H), 3.60 -3.71 (m, 2H), 3.71 - 3.82 (m, 2H), 3.89 - 3.96 (m,2H), 4.10 - 4.18 (m, 2H), 4.24 (d, J = 2.2 Hz, 1H), 4.27 - 4.31 (m, 1H), 4.37 - 4.39 (m, 1H), 4.46 (t, J = 4.9 Hz, 1H), 4.59 (d, J = 2.2 Hz, 1H), 4.94 - 5.01 (m, 1H), 5.24 (t, J = 4.8 Hz, 1H), 5.33 (s, 1H), 7.59 - 7.66 (m, 1H), 7.83-7.94 (m, 2H), 7.99 (d, J = 7.5 Hz, 1H). m/z (ES+), [M+H]+ = 869. Step 6 – Compound P21: (2S,4S)-2,5,12-trihydroxy-N-((3S,3aR,6R,6aR)-6- hydroxyhexahydrofuro[3,2-b]furan-3-yl)-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':4,5] oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamide
Figure imgf000161_0001
TBAF (57.2 mg, 0.22 mmol) was added to a solution of Intermediate Compound 57 (190 mg, 0.22 mmol) in DCM (8 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was quenched with 0.01% NH4HCO3 (2 mL). The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC to afford Compound P21 (30.0 mg, 18.18 %) as a red solid.
Figure imgf000161_0002
NMR (300 MHz, CDCl3) δ 1.40 (d, J = 6.5 Hz, 3H), 1.68 - 1.83 (m, 1H), 1.99 - 2.14 (m, 1H), 2.31-2.53 (m, 2H), 2.60 (d, J = 6.8 Hz, 1H), 2.71 - 2.91 (m, 2H), 3.24 (d, J = 7.6 Hz, 2H), 3.42 (q, J = 6.3 Hz, 1H), 3.49 (s, 3H), 3.52- 3.75 (m, 2H), 3.84- 4.06 (m, 4H), 4.06-4.16 (m, 5H), 4.29 - 4.43 (m, 1H), 4.50 (t, J = 3.0 Hz, 2H), 4.56-4.62 (m, 1H), 4.66 (t, J = 5.0 Hz, 1H), 4.73 (d, J = 1.9 Hz, 1H), 5.16 (s, 1H), 5.35 (s, 1H), 5.52 (t, J = 5.6 Hz, 1H), 7.34 -7.47 (m, 2H), 7.81 (t, J = 8.1 Hz, 1H), 8.02 - 8.10 (m, 1H), 13.31 (s, 1H), 13.95 (s, 1H). m/z (ES+), [M+H]+ = 755.
Example 26: Synthesis of Compound P22
Figure imgf000162_0001
Step 1 - Intermediate Compound 53: (3R,3aS,6R,-6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl 4- methylbenzenesulfonate
Figure imgf000163_0001
4-Toluenesulfonyl chloride (137 g, 718.48 mmol) was added to a solution of pyridine (135.4 g, 1710.66 mmol), and (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol (“isomannide”; 100 g, 684.26 mmol) in DCM (1000 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 16 hours. The reaction mixture was diluted with DCM (1000 mL), and washed sequentially with 1N HCl (1000 mL) and saturated brine (1000 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography (elution gradient 40 to 40% THF in petroleum ether) to afford Intermediate Compound 53 (70 g, 34.1 %) as a white solid.1
Figure imgf000163_0002
H NMR (400 MHz, Chloroform-d) δ 7.86 – 7.82 (m, 2H), 7.36 (d, J = 8.1 Hz, 2H), 4.94-4.84 (m, 1H), 4.49 (t, J = 4.9 Hz, 1H), 4.41 (t, J = 5.0 Hz, 1H), 4.31 – 4.25 (m, 1H), 4.01 (dd, J = 9.3, 6.6 Hz, 1H), 3.95 (dd, J = 9.2, 6.5 Hz, 1H), 3.79 (d, J = 2.0 Hz, 1H), 3.70 (d, J = 3.1 Hz, 1H), 3.57 – 3.51 (m, 1H), 2.46 (s, 3H). ES+(M+1 = 301). Step 2 - Intermediate Compound 54: (3R,3aS,6R,6aS)-6-((tert- butyldimethylsilyl)oxy)hexahydrofuro[3,2-b]furan-3-yl-4-methylbenzenesulfonate
Figure imgf000163_0003
TBS-Cl (38.64g, 256.38mmol) was added to a solution of imidazole (31.74 g, 466.16 mmol) and Intermediate Compound 53 (70 g, 333.08 mmol) in DMA (220 ml) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash silica chromatography (elution gradient 20 to 25% THF in petroleum ether) to afford Intermediate Compound 54 (76 g, 79 %) as a white solid.1H
Figure imgf000163_0004
NMR (300 MHz, DMSO) δ 3.65 (d, J = 3.1 Hz, 3H), 3.83 (dd, J = 10.5, 2.5 Hz, 1H), 3.93 (ddd, J = 15.0, 9.9, 5.2 Hz, 2H), 4.13 (s, 1H), 4.18 – 4.34 (m, 3H), 4.53 – 4.63 (m, 1H), 4.64 (d, J = 4.4 Hz, 1H), 7.34 (td, J = 7.4, 1.2 Hz, 2H), 7.43 (td, J = 7.5, 1.2 Hz, 2H), 7.50 – 7.67 (m, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.90 (d, J = 7.4 Hz, 2H), 8.33 (d, J = 6.9 Hz, 1H), 8.45 (s, 2H). ES+(M+1) = 415. Step 3 – Intermediate Compound 58: 2-((3S,3aR,6R,6aS)-6-((tert-butyldimethyl- silyl)oxy)hexahydrofuro-[3,2-b]furan-3-yl)isoindoline-1,3-dione
Figure imgf000164_0001
Phthalimide potassium (44.7 g, 241.20 mmol) was added to a solution of 18-Crown-6 (6.38 g, 24.12 mmol) and Intermediate Compound 54 (50 g, 120.60 mmol) in DMF (500 mL) under nitrogen. The resulting mixture was stirred at 140 °C for 8 hours. When the mixture was cooled to RT, water (150 ml) was added, and filtered, then washed the cake with H2O (50ml). The filter cake was dried under vacuum to afford Intermediate Compound 58 (40 g, 85%) as a white solid.1H NMR (300 MHz, DMSO-d6) δ 7.76 (p, J = 1.8 Hz, 4H), 4.81 (dd, J = 4.9, 1.9 Hz, 1H), 4.56 – 4.45 (m, 2H), 4.25-4.18 (m, 1H), 4.12-4.04 (m, 1H), 3.86 – 3.80 (m, 1H), 3.69-3.62 (m, 1H), 3.54 – 3.42 (m, 1H), 0.79 (s, 9H), 0.00 (d, J = 2.6 Hz, 6H). ES+(M+1 = 390). Step 4 – Intermediate Compound 59: (3S,3aR,6R,6aS)-6-((tert- butyldimethylsilyl)oxy)hexahydrofuro[3,2-b]furan-3-amine
Figure imgf000164_0002
Intermediate Compound 58 (40 g, 102.69 mmol) was added to a solution of hydrazine hydrate (32.1 g, 513.45 mmol) in EtOH (260 mL) under nitrogen. The resulting mixture was stirred at 80 °C for 2 hours. When the mixture was cooled, the mixture was filtrated, washed with EtOH (260 ml). The solvent was removed under reduced pressure to afford Intermediate Compound 59 (22.00 g, 83 %) as a white solid.
Figure imgf000164_0003
NMR (400 MHz, DMSO) δ 0.07 (d, J = 3.1 Hz, 6H), 0.83 – 0.90 (m, 1H), 0.87 (s, 9H), 1.94 (s, 2H), 3.26 (dt, J = 4.6, 1.5 Hz, 1H), 3.35 (dd, J = 8.3, 7.1 Hz, 1H), 3.53 (dd, J = 8.6, 1.6 Hz, 1H), 3.66 (dd, J = 8.3, 6.1 Hz, 1H), 3.75 (dd, J = 8.6, 4.4 Hz, 1H), 4.13 (d, J = 4.3 Hz, 1H), 4.21-4.30 (m, 1H), 4.39 (t, J = 4.5 Hz, 1H). ES+(M+1 = 260). Step 5 – Intermediate Compound 60: benzyl ((3S,3aR,6R,6aS)-6-((tert-butyldimethylsilyl)- oxy)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000165_0001
DIEA (40.4 mL, 231.28 mmol) was added to a solution of N-(benzyloxycarbonyloxy)succinimide (23.06 g, 92.51 mmol) and Intermediate Compound 59 (20 g, 77.09 mmol), in DCM (200 mL) under nitrogen. The resulting mixture was stirred at RT for 2 hours. The solvent was removed under reduced pressure.The crude product was purified by flash silica chromatography (elution gradient 0 to 100% THF in petroleum ether) to afford Intermediate Compound 60 (22.00 g, 72.5 %) as a white solid.1H NMR (400 MHz, DMSO) δ 0.03 – 0.11 (m, 6H), 0.86 (d, J = 7.6 Hz, 9H), 3.43 (dd, J = 8.6, 6.4 Hz, 1H), 3.65 – 3.76 (m, 2H), 3.73 – 3.87 (m, 1H), 3.87 – 3.94 (m, 1H), 4.23 – 4.35 (m, 2H), 4.40 (q, J = 6.0, 5.4 Hz, 1H), 5.04 (s, 2H), 7.28 – 7.39 (m, 5H), 7.62 (d, J = 6.4 Hz, 1H). ES+(M+1 =394). Step 6 – Intermediate Compound 61: benzyl ((3S,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan- 3-yl)carbamate
Figure imgf000165_0002
Intermediate Compound 60 (20 g, 50.82 mmol) was added to HCl solution in 1,4-dioxane (200 mL). The resulting mixture was stirred at 25 °C for 6 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 61 (13.0 g, 92 %) as a white solid.1H NMR (400 MHz, DMSO) δ 3.35 (t, J = 8.1 Hz, 1H), 3.65 – 3.85 (m, 3H), 3.81 – 3.94 (m, 2H), 4.09 (ddd, J = 7.8, 6.6, 4.6 Hz, 1H), 4.19 – 4.43 (m, 3H), 5.03 (s, 2H), 7.35 – 7.39 (m, 5H), 7.64 (d, J = 6.6 Hz, 1H). ES+(M+1 =280). Step 7 – Intermediate Compound 62: (3R,3aS,6S,6aR)-6- (((benzyloxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl trifluoromethanesulfonate
Figure imgf000165_0003
Trifluoromethanesulfonic anhydride (18.18 g, 64.45 mmol) was added to a solution of DIEA (22.51 mL, 128.90 mmol) and Intermediate Compound 61 (12 g 4297 mmol) in DCM (150 mL) at 0°C over a period of 5 minutes under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was removed under reduced pressure to afford Intermediate Compound 62 (11.00 g, 62.2 %) as a yellow oil.
Figure imgf000166_0001
NMR (400 MHz, DMSO) δ 3.73 (dd, J = 9.5, 2.6 Hz, 1H), 3.82-3.87 (m, 2H), 4.05 (d, J = 11.9 Hz, 1H), 4.54 (dd, J = 4.0, 1.6 Hz, 1H), 4.72 (d, J = 4.0 Hz, 1H), 5.04 (d, J = 3.6 Hz, 2H), 5.76 (s, 1H), 7.27 – 7.46 (m, 6H), 7.69 (d, J = 6.0 Hz, 1H). ES+(M+1 =412). Step 8 – Intermediate Compound 63: tert-butyl 3-((3S,3aR,6S,6aR)-6- (((benzyloxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)-3,6-diazabicyclo[3.1.1]heptane-6- carboxylate
Figure imgf000166_0002
tert-Butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (24.10 g, 121.55 mmol) was added to a solution of Intermediate Compound 62 (10 g, 24.31 mmol) in 1,4-dioxane (100 mL). The resulting mixture was stirred at 100 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography (elution gradient 0 to 100% THF in petroleum ether) to afford Intermediate Compound 63 (8.00 g, 71.6 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 1.47 (d, J = 8.0 Hz, 1H), 1.70 – 1.83 (m, 3H), 2.29 (q, J = 6.4 Hz, 1H), 3.04 – 3.11 (m, 1H), 3.55 – 3.75 (m, 5H), 3.85 – 3.96 (m, 4H), 4.30 – 4.43 (m, 1H), 4.51 (d, J = 5.2 Hz, 1H), 5.02 (s, 2H), 7.25-7.40 (m, 5H). ES+(M+1 =460). Step 9 – Intermediate Compound 64: tert-butyl 3-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2- b]furan-3-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate
Figure imgf000166_0003
Pd-C (1.853 g, (10% w/w)) was added to a solution of Intermediate Compound 63 (8 g, 17.41 mmol) in MeOH (160 mL) under hydrogen. The resulting mixture was stirred at 25 °C for another 2 hours. The reaction mixture was filtered through a celite pad. The solvent was removed under reduced pressure to afford Intermediate Compound 64 (5.30 g, 94 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 1.36 (s, 1H), 1.39 (s, 9H), 1.49 (d, J = 7.9 Hz, 1H), 2.22 – 2.38 (m, 1H), 2.51 (br, 3H), 2.55 – 2.91 (m 1H) 303 (td J = 5620 Hz 1H) 318 (s 1H) 323 340 (m 1H), 3.44 (dd, J = 8.8, 2.0 Hz, 1H), 3.64 (td, J = 9.4, 4.5 Hz, 1H), 3.72 (dd, J = 8.8, 4.4 Hz, 1H), 3.85 (dd, J = 9.6, 6.0 Hz, 1H), 3.95 (d, J = 6.1 Hz, 2H), 4.14 (d, J = 4.8 Hz, 1H), 4.54 (dd, J = 4.8, 2.0 Hz, 1H). ES+(M+1 =326). Step 10 – Intermediate Compound 65: tert-butyl 3-((3S,3aR,6S,6aR)-6-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)-3,6-diazabicyclo[3.1.1]heptane-6- carboxylate
Figure imgf000167_0001
A mixture of N-(9H-fluoren-2-ylmethoxycarbonyloxy)succinimide (6.74 g, 19.97 mmol) was added to a solution of DIEA (8.05 mL, 46.10 mmol) and Intermediate Compound 64 (5 g, 15.37 mmol) in CH2Cl2 (80 mL) under nitrogen was stirred at 25 °C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography (elution gradient 30 to 60% THF in petroleum ether ) to afford Intermediate Compound 65 (6.00 g, 71.3 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 1.48 (d, J = 8.0 Hz, 1H), 1.71 – 1.82 (m, 2H), 2.30 (q, J = 6.6 Hz, 1H), 2.56 – 2.86 (m, 1H), 3.09 (d, J = 6.8 Hz, 1H), 3.33 (s, 1H), 3.56 – 3.72 (m, 2H), 3.76 – 3.93 (m, 2H), 3.95 (d, J = 6.0 Hz, 2H), 4.22 (t, J = 6.8 Hz, 1H), 4.31 – 4.39 (m, 2H), 4.53 (s, 1H), 5.76 (s, 2H), 7.29 – 7.38 (m, 2H), 7.38 – 7.46 (m, 2H), 7.63 (d, J = 6.4 Hz, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H). ES+(M+1 =548). Step 11 – Intermediate Compound 66: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-(piperazin-1- yl)hexahydrofuro[3,2-b]furan-3-yl)carbamate, HCl salt
Figure imgf000167_0002
General Procedure II, Method A was followed using HCl solution (4 M in dioxane) and Intermediate Compound 65 (5.7 g, 10.41 mmol) in 1,4-dioxane (100 mL) to afford Intermediate Compound 66 (4.40 g, 97 %) as a white solid.1H NMR (300 MHz, DMSO) δ 1.96 (s, 6H), 3.57 (s, 6H), 3.88 (d, J = 8.4 Hz, 1H), 4.22 (t, J = 6.8 Hz, 1H), 4.31-4.36 (m, 5H), 4.48 (s, 1H), 7.32 – 7.46 (m, 4H), 7.71 (d, J = 7.6 Hz, 3H), 7.86 – 7.94 (m, 2H). ES+(M+1 =448). Step 12 – Intermediate Compound 67: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-(6-((2S,4S)- 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carbonyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000168_0001
General Procedure I was followed using Intermediate Compound 66 (224 mg, 0.46 mmol), DIEA (0.484 mL, 2.77 mmol), HATU (228 mg, 0.60 mmol) and the PNU-159682 carboxylic acid (CAS No. 1204819-92-0; 290 mg, 0.46 mmol) to afford Intermediate Compound 67 (160 mg, 32.8 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.20 – 1.35 (m, 5H), 1.51-1.56 (m, 1H), 1.70-1.76 (m, 9H), 1.92 (s, 1H), 3.00-3.05 (m, 11H), 3.52 (s, 1H), 3.82 – 3.93 (m, 3H), 4.00 (s, 3H), 4.10 (s, 1H), 4.23 (s, 2H), 4.32 (d, J = 7.2 Hz, 2H), 4.59 (s, 1H), 4.89 (s, 1H), 5.25 (s, 1H), 5.77 (s, 2H), 6.69 (s, 1H), 7.33 (t, J = 7.6 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.70 (d, J = 8.0 Hz, 4H), 7.87-7.92 (m, 4H), 8.17 (s, 1H), 13.26 (s, 1H), 14.06 (s, 1H). ES+(M+1 =1056.4). Step 13 - Compound P22: (8S,10S)-8-(3-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)- 3,6-diazabicyclo[3.1.1]heptane-6-carbonyl)-6,8,11-trihydroxy-1-methoxy-10- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000168_0002
General Procedure III was followed using DBU (0.021 mL, 0.14 mmol) and Intermediate Compound 67 (150 mg, 0.14 mmol) in DCM (5 mL) and MeCN (1.250 mL) to afford Compound P22 (15.00 mg, 12.66 %) as a red solid.1H NMR (300 MHz, DMSO) δ 1.15 – 1.29 (m, 3H), 1.69 (s, 2H), 1.87 (t, J = 9.2 Hz, 1H), 2.10 – 2.35 (m, 2H), 2.42 (s, 1H), 2.66 (d, J = 13.3 Hz, 2H), 2.78 – 2.94 (m, 2H), 2.98 (d, J = 5.5 Hz, 2H), 3.01 – 3.18 (m, 4H), 3.47 – 3.56 (m, 3H), 3.67 (t, J = 6.1 Hz, 3H), 3.67 – 3.93 (m, 3H), 3.93 – 4.08 (m, 6H),4.10 – 4.22 (m, 1H), 4.24 (d, J = 2.3 Hz, 3H), 4.59 (d, J = 2.3 Hz, 2H), 4.81 (s, 1H), 5.01 – 5.08 (m, 1H), 5.15 (s, 1H), 5.26 (d, J = 4.1 Hz, 1H), 7.57 – 7.72 (m, 1H), 7.92 (t, J = 6.8 Hz, 2H). ES+(M+1 =835). Example 27: Synthesis of Compound P23
Figure imgf000169_0001
Step 1 – Intermediate Compound 68: (3R,3aS,6R,6aS)hexahydrofuro[3,2-b]furan-3,6-diyl bis(4- methylbenzenesulfonate)
Figure imgf000170_0001
4-Toluenesulfonyl chloride (130 g, 684.27 mmol) was added to a solution of pyridine (110 mL, 1368.53 mmol) and (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol (“isomannide”; 20 g, 136.85 mmol) in DCM (500 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 16 hours. The reaction mixture was quenched with water (300 mL), extracted with DCM (3 x 500 mL). Combined extracts were washed with 0.1N HCl (3 x 500 ml) and then with H2O and washed with dilute NaHCO3 and saturated aqueons NaCl. The organic layer was dried over Na2SO4, filtered and evaporated to afford crude Intermediate Compound 68 (60.0 g, 96 %) as a white solid. (m,
Figure imgf000170_0003
18-Crown-6 (3.49 g, 13.20 mmol) was added to a solution of Intermediate Compound 68 (30 g, 66.01 mmol) and potassium 1,3-dioxoisoindolin-2-ide (26.9 g, 145.21 mmol) in DMF (500 mL) under nitrogen. The resulting mixture was stirred at 140 °C for 16 hours. The mixture was combined and washed with water (3 x 500 mL), dry the filter cake to afford Intermediate Compound 69 (15.00 g, 56.2 %) as a yellow solid.1
Figure imgf000170_0002
H NMR (400 MHz, DMSO) δ 3.93 (dd, J = 6.0, 9.6 Hz, 2H), 4.27 (dd, J = 7.6, 9.2 Hz, 2H), 4.69 (dd, J = 6.0, 7.6 Hz, 2H), 5.19 (s, 2H), 7.81 – 7.92 (m, 8H). ES+(M+1 = 405). Step 3 – Intermediate Compound 70: (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine
Figure imgf000171_0001
Hydrazine monohydrate (12.75 mL, 210.20 mmol) was added to a solution of Intermediate Compound 69 (17 g, 42.04 mmol) in EtOH (350 mL) under nitrogen. The resulting mixture was stirred at 80 °C for 2 hours. The reaction mixture was filtered, and wash the filter cake with EtOH (2 x 150 mL). The filtrate was evaporated to dryness to afford Intermediate Compound 70 (3.50 g, 57.7 %) as a yellow solid.1
Figure imgf000171_0002
H NMR (400 MHHz, DMSO) δ 3.25 (dd, J = 2.0, 4.4 Hz, 2H), 3.43 (dd, J = 2.0, 8.7 Hz, 2H), 3.69 (dd, J = 4.5, 8.7 Hz, 2H), 4.25 (s, 2H). ES+( M+1 = 145). Step 4 – Intermediate Compound 71: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000171_0003
N-(9H-Fluoren-2-ylmethoxycarbonyloxy)su2Ncc ( OiSn) Him H Oide ( NS (H)1F.9m8o9c g, 5.90 mmol) was added slowly to a solution of Intermediate Compound 70 (1.7 g, 11.79 mmol) and DIEA (4.12 mL, 23.58 mmol) in THF (100 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 5 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound 71 (1.200 g, 27.8 %) as a yellow solid. 1H NMR (300 MHz, DMSO) δ 3.38 – 3.48 (m, 3H), 3.55 (dd, J = 6.4, 8.4 Hz, 1H), 3.66 – 3.91 (m, 3H), 4.15 – 4.53 (m, 4H), 7.28 – 7.47 (m, 5H), 7.52 – 7.75 (m, 1H), 7.75 – 7.96 (m, 4H). ES+(M+1 = 367). Step 5 – Intermediate Compound 72: 4-((1R,4R)-5-(tert-butoxycarbonyl)-2,5- diazabicyclo[2.2.2]octan-2-yl)-4-oxobutanoic acid
Figure imgf000171_0004
Succinic anhydride (236 mg, 2.36 mmol) was added to a solution of tert-butyl (1R,4R)-2,5-diaza- bicyclo[2.2.2]octane-2-carboxylate (500 mg, 2.36 mmol) in DCM (6 mL) under nitrogen. The resulting mixture was stirred at RT for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to afford Intermediate Compound 72 (650 mg, 88%) as a white solid.1
Figure imgf000172_0001
H NMR (400 MHz, DMSO) δ 1.41 (d, J = 2.5 Hz, 9H), 1.65 – 1.93 (m, 4H), 2.32 – 2.51 (m, 4H), 3.30 – 3.47 (m, 3H), 3.47 – 3.73 (m, 2H), 4.11 – 4.19 (m, 1H), 11.56 (s, 1H). ES+(M+1 = 313). Step 6 – Intermediate Compound 73: tert-butyl (1R,4R)-5-(4-(((3S,3aR,6S,6aR)-6-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutanoyl)-2,5- diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000172_0002
General Procedure I was followed using DIEA (0.572 mL, 3.27 mmol), Intermediate Compound 72 (511 mg, 1.64 mmol), Intermediate Compound 71 (400 mg, 1.09 mmol) and HATU (830 mg, 2.18 mmol) to afford Intermediate Compound 73 (130 mg, 18.02 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.41 (d, J = 2.6 Hz, 9H), 1.68 – 1.87 (m, 4H), 2.30 – 2.50 (m, 4H), 3.34 – 3.43 (m, 3H), 3.53 – 3.62 (m, 2H), 3.62 – 3.69 (m, 1H), 3.78 – 3.92 (m, 3H), 4.07 – 4.18 (m, 2H), 4.18 – 4.26 (m, 1H), 4.30 – 4.40 (m, 3H), 4.43 (d, J = 3.8 Hz, 1H), 4.53 (d, J = 13.0 Hz, 1H), 7.30 – 7.39 (m, 2H), 7.39 – 7.47 (m, 2H), 7.71 (d, J = 7.3 Hz, 3H), 7.81 – 7.93 (m, 2H), 8.14 (d, J = 7.0 Hz, 1H). ES+(M+1 = 661). Step 7 – Intermediate Compound 74: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-(4-((1R,4R)-2,5- diazabicyclo[2.2.2]octan-2-yl)-4-oxobutanamido) hexahydrofuro[3,2-b]furan-3-yl)carbamate, HCl salt
Figure imgf000172_0003
General Procedure II, Method A was followed using 4N HCl in dioxane (3 mL, 98.74 mmol) and Intermediate Compound 73 (130 mg, 0.20 mmol) to afford Intermediate Compound 74 (100 mg, 91 %) as a yellow solid.
Figure imgf000172_0004
NMR (400 MHz, DMSO) δ 1.73 – 1.84 (m, 4H), 2.06 (s, 1H), 2.33 – 2.43 (m, 4H), 2.53 – 265 (m 2H) 318 331 (m 2H) 333 (s 1H) 337 352 (m 3H), 3.68 (d, J = 2.9 Hz, 1H), 3.76 (d, J = 5.7 Hz, 2H), 3.80 – 3.88 (m, 4H), 4.09 (s, 1H), 4.17 – 4.26 (m, 2H), 7.33 – 7.36 (m, 2H), 7.43 (s, 2H), 7.71 (d, J = 7.3 Hz, 4H), 7.90 (d, J = 7.5 Hz, 2H). ES+(M+1 = 561). Step 8 – Intermediate Compound 75: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-(4-oxo-4-((1R,4R)- 5-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro- 1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carbonyl)-2,5-diazabicyclo[2.2.2]octan-2-yl)butanamido)-hexahydrofuro[3,2- b]furan-3-yl)carbamate
Figure imgf000173_0001
General Procedure I was followed using PyOxim (212 mg, 0.41 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 130 mg, 0.21 mmol), Intermediate Compound 74 (174 mg, 0.31 mmol) and DIEA (0.181 mL, 1.04 mmol) in DMA (3 mL) to afford Intermediate Compound 75 (140 mg, 57.8 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.14 – 1.27 (m, 3H), 1.58 – 1.87 (m, 7H), 1.91 – 2.27 (m, 2H), 2.30 – 2.43 (m, 3H), 2.59 – 2.94 (m, 3H), 2.98 – 3.06 (m, 3H), 3.34 – 3.62 (m, 11H), 3.63 – 3.77 (m, 3H), 3.80 – 4.16 (m, 8H), 4.18 – 4.45 (m, 4H), 4.47 – 4.77 (m, 2H), 4.94 – 5.32 (m, 2H), 6.29 (s, 1H), 7.28 – 7.46 (m, 4H), 7.59 – 7.73 (m, 2H), 7.82 – 7.94 (m, 5H), 8.06 – 8.17 (m, 1H), 8.34 – 8.49 (m, 1H). ES+(M+1 = 1170). Step 9 – Compound P23: N-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)-4-oxo-4- ((1R,4R)-5-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':4,5]oxazolo-[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carbonyl)-2,5-diazabicyclo[2.2.2]octan-2-yl)butanamide
Figure imgf000173_0002
General Procedure III was followed using a DCM solution of DBU (15.2 mg/mL in DCM) (0.779 mL, 0.08 mmol) and Intermediate Compound 75 (130 mg, 0.11 mmol) in DCM (6 mL)/MeCN (1.5 mL) to afford Compound P23 (38.0 mg, 36.1 %) as a red solid.1H NMR (400 MHz, CDCl3) δ 1.36 – 1.47 (m, 3H), 1.73 – 1.89 (m, 3H), 1.93 – 2.05 (m, 2H), 2.19 (d, J = 12.4 Hz, 1H), 2.40 – 2.49 (m, 1H), 2.53 – 2.65 (m, 3H), 2.70 – 2.77 (m, 1H), 2.78 – 2.86 (m, 1H), 2.95 – 3.08 (m, 1H), 3.33 – 3.43 (m, 2H), 3.48 (d, J = 1.2 Hz, 3H), 3.54 – 3.86 (m, 7H), 3.94 (s, 4H), 4.11 (d, J = 2.4 Hz, 6H), 4.49 (d, J = 1.9 Hz, 2H), 4.60 (s, 1H), 4.73 (s, 1H), 4.76 – 4.86 (m, 1H), 4.90 – 5.02 (m, 1H), 5.26 (s, 1H), 5.38 (s, 1H), 5.56 (s, 1H), 6.34 – 6.38 (m, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.80 (dd, J = 6.8, 9.6 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 13.40 (s, 1H), 13.97 (s, 1H). ES+(M+1 = 948). Example 28: Synthesis of Compound P24
Figure imgf000174_0001
Step 1 – Intermediate Compound 76: tert-butyl 3-(4-(benzyloxy)-4-oxobutanoyl)-3,6- diazabicyclo[3.1.1]heptane-6-carboxylate
Figure imgf000174_0002
General Procedure I was followed using tert-butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (4.57 g, 23.05 mmol), 4-(benzyloxy)-4-oxobutanoic acid (4 g, 19.21 mmol), HATU (14.61 g, 38.42 mmol) and DIEA (10.07 mL, 57.63 mmol) in MeCN (150 mL) to afford Intermediate Compound 76 (6.00 g, 80 %) as a yellow solid.1H NMR (400 MHz, CDCl3) δ 1.44 (s, 9H), 2.49 – 2.65 (m, 1H), 2.66 – 2.76 (m, 2H), 2.78 – 2.91 (m, 1H), 3.51 (dd, J = 4.0, 11.8 Hz, 2H), 4.02 (d, J = 13.2 Hz, 1H), 4.08 – 4.28 (m, 3H), 5.08 – 5.22 (m, 2H), 7.32 – 7.46 (m, 5H). ES+(M+1= 389). Step 2 - Intermediate Compound 77: (4-(6-(tert-butoxycarbonyl)-3,6-diazabicyclo[3.1.1]heptan-3- yl)-4-oxobutanoic acid
Figure imgf000175_0001
Pd-C (3.29 g, 3.09 mmol) was added to a solution of Intermediate Compound 76(6 g, 15.45 mmol) in THF (100 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 2 hours under hydrogen. The reaction mixture was filtered through a celite pad. The solvent was removed under reduced pressure. Intermediate Compound 77 was obtained as crude material (4.30 g, 93 %, white solid).1H NMR (400 MHz, DMSO) δ 1.23-1.39 (m, 10H), 2.42 – 2.47 (m, 4H), 3.19 – 3.43 (m, 2H), 3.55 (d, J = 11.2 Hz, 1H), 3.81 (dd, J = 2.5, 13.2 Hz, 1H), 3.98 (s, 1H), 4.09 (d, J = 6.2 Hz, 2H), 12.03 (s, 1H). ES+(M+1 = 299. Step 3 - Intermediate Compound 78: tert-butyl 3-(4-(((3S,3aR,6S,6aR)-6-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutanoyl)-3,6- diazabicyclo[3.1.1]heptane-6-carboxylate
Figure imgf000175_0002
General Procedure I was followed using Intermediate Compound 71 (500 mg, 1.36 mmol), Intermediate Compound 77 (400 mg, 1.34 mmol), HATU (1020 mg, 2.68 mmol) and DIEA (0.703 mL, 4.02 mmol) in MeCN (5 mL) to afford Intermediate Compound 78 (500 mg, 57.7 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.35 (s, 10H), 2.29 – 2.40 (m, 2H), 2.48 (d, J = 8.8 Hz, 3H), 3.28 (d, J = 13.2 Hz, 1H), 3.51 – 3.70 (m, 3H), 3.77 – 3.94 (m, 4H), 4.03 – 4.12 (m, 3H), 4.22 (t, J = 6.8 Hz, 1H), 4.31 – 4.48 (m, 4H), 7.34 (td, J = 1.2, 7.6 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.71 (d, J = 7.2 Hz, 3H), 7.90 (d, J = 7.6Hz, 2H), 8.16 (d, J = 6.9 Hz, 1H). ES+(M+1 = 647). Step 4 - Intermediate Compound 79: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-(4-(3,6- diazabicyclo[3.1.1]heptan-3-yl)-4-oxobutanamido)hexahydrofuro[3,2-b]furan-3-yl)carbamate, HCl salt
Figure imgf000176_0001
General Procedure II, Method A was followed using HCl in dioxane（4M）(2 ml, 8.00 mmol) and Intermediate Compound 78 (470 mg, 0.73 mmol) in DCM (5 mL) to afford Intermediate Compound 79 (380 mg, 96 %) as a yellow solid.1
Figure imgf000176_0002
H NMR (400 MHz, DMSO) δ 1.09 – 1.30 (m, 1H), 1.64 – 1.81 (m, 1H), 2.31 – 2.45 (m, 2H), 2.58 – 2.68 (m, 1H), 2.82 (dt, J = 6.5, 12.5 Hz, 1H), 3.59 – 3.72 (m, 2H), 3.75 – 3.92 (m, 3H), 3.97 (d, J = 2.9 Hz, 2H), 4.09 (s, 1H), 4.22 (dd, J = 7.7, 11.2 Hz, 1H), 4.32-4.39 (m, 6H), 4.46 (d, J = 4.1 Hz, 1H), 7.34 (td, J = 1.2, 7.6 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.71 (d, J = 7.4 Hz, 3H), 7.90 (d, J = 7.5 Hz, 2H), 8.14 – 8.35 (m, 2H). ES+(M+1 = 547). Step 5 - Intermediate Compound 80: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-(4-oxo-4-(6- ((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro- 1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)butanamido)- hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000176_0003
General Procedure I was followed using Intermediate Compound 79 (131 mg, 0.24 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 100 mg, 0.16 mmol), PyOxim (122 mg, 0.24 mmol) and DIEA (0.083 mL, 0.48 mmol) in DMA (5 mL) to afford Intermediate Compound 80 (120 mg, 65.1 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.11 – 1.33 (m, 4H), 1.50-1.56 (m, 1H), 1.71- 1.82 (m, 20 H), 1.98 - 2.27 (m, 1H), 2.32-2.42 (m, 3H), 2.60 - 2.81 (m, 2H), 3.41-3.53 (m, 1H), 3.56 - 3.68 (m, 3H), 3.80 -3.93 (m, 2H), 3.99 (s, 3H), 4.04 - 4.15 (m, 1H), 4.23 (d, J = 5.2 Hz, 1H), 4.28 - 4.49 (m, 4H), 4.58 (s, 1H), 4.86 - 4.95 (m, 1H), 5.26 - 5.31 (m, 2H), 7.32 - 7.45 (m, 4H), 7.69 (t, J = 10.4 Hz, 4H), 7.90 (dd, J = 6.0, 12.4 Hz, 4H), 8.08 - 8.26 (m, 1H), 13.24 (s, 1H), 14.04 (s, 1H). ES+(M+1 = 1156). Step 6 – Compound P24: N-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)-4-oxo-4-(6- ((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro- 1H-pyrano[4',3':4,5] oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)butanamide
Figure imgf000177_0001
General Procedure III was followed using DBU (0.014 mL, 0.10 mmol) and Intermediate Compound 80 (110 mg, 0.10 mmol) in DCM (5 mL) and MeCN (1.25 mL) to afford Compound P24 (20.00 mg, 22.51 %) as a red solid.1H NMR (400 MHz, DMSO-d6) δ 1.19 – 1.30 (m, 4H), 1.53 (dd, J = 8.8, 16.4 Hz, 1H), 1.70 (s, 2H), 2.05 – 2.21 (m, 1H), 2.31 – 2.39 (m, 4H), 2.65 (s, 4H), 2.85 – 3.00 (m, 2H), 3.31 – 3.32 (m, 5H), 3.37 (s, 1H), 3.40 – 3.47 (m, 4H), 3.73 – 3.83 (m, 2H), 3.83 – 3.99 (m, 3H), 4.00 (s, 5H), 4.25 (dd, J = 3.6, 6.8 Hz, 2H), 4.40 (d, J = 4.0 Hz, 2H), 4.89 (s, 1H), 4.99 (s, 1H), 5.09 (s, 1H), 5.22 – 5.37 (m, 2H), 7.64 – 7.71 (m, 1H), 7.94 (dd, J = 1.6, 4.0 Hz, 2H), 8.09 – 8.18 (m, 1H). ES+(M+1 = 934). Example 29: Synthesis of Compound P25
Figure imgf000178_0001
Steps 1, 2 - Intermediate Compound 82: (9H-fluoren-9-yl) methyl (2-(((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl) amino) hexahydrofuro[3,2-b] furan-3-yl) amino)-2-oxoethyl) carbamate
Figure imgf000178_0002
2,5-Dioxopyrrolidin-1-yl (((9H-fluoren-9-yl) methoxy) carbonyl) glycinate (1 g, 2.54 mmol) was added to a mixture of Intermediate Compound 70 (0.731 g, 5.07 mmol) and DIEA (1.329 mL, 7.61 mmol) in DCM (15 mL) at RT. The resulting mixture was stirred at RT for 1 hour (to afford Intermediate Compound 81). Then (Boc)2O (2.94 mL, 12.68 mmol) was added. The resulting mixture was stirred at RT for 2 hours. The reaction mixture was quenched with water (50 mL), extracted with DCM (2 x 30 mL), and washed with water (50 mL) and 0.5N HCl (50 mL). The combined organic layer was dried over Na2SO4, filtered and evaporated to afford oil mixture. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 82 (0.527 g, 39.7 %) as a white solid.1
Figure imgf000178_0003
H NMR (300 MHz, DMSO) δ 1.39 (s, 9H), 3.54 – 3.62 (m, 3H), 3.85 (dd, J = 8.4, 4.2 Hz, 3H), 4.05 (m, J = 14.2, 7.0 Hz, 2H), 4.20 – 4.32 (m, 3H), 4.32 – 4.50 (m, 2H), 7.18 – 7.57 (m, 6H), 7.72 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.4 Hz, 2H), 8.16 (d, J = 7.1 Hz, 1H). ES+(M+1 = 524). Step 3 - Intermediate Compound 83: (9H-fluoren-9-yl)methyl (2-(((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)carbamate, HCl salt
Figure imgf000179_0001
General Procedure II, Method A was followed using Intermediate Compound 82 (520 mg, 0.99 mmol) and HCl solution (4 N, in 1,4-dioxane, 5 mL) to afford Intermediate Compound 83 (410 mg, 90 %) as a white solid.
Figure imgf000179_0002
NMR (300 MHz, DMSO) δ 3.65 (d, J = 3.1 Hz, 3H), 3.83 (dd, J = 10.5, 2.5 Hz, 1H), 3.93 (ddd, J = 15.0, 9.9, 5.2 Hz, 2H), 4.13 (s, 1H), 4.18 – 4.34 (m, 3H), 4.53 – 4.63 (m, 1H), 4.64 (d, J = 4.4 Hz, 1H), 7.34 (td, J = 7.4, 1.2 Hz, 2H), 7.43 (td, J = 7.5, 1.2 Hz, 2H), 7.50 – 7.67 (m, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.90 (d, J = 7.4 Hz, 2H), 8.33 (d, J = 6.9 Hz, 1H), 8.45 (s, 2H). ES+(M+1 = 424). Step 4 - Intermediate Compound 84: (9H-fluoren-9-yl) methyl (2-oxo-2-(((3S,3aR,6S,6aR)-6-((2S,4S)- 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano [4',3':4,5] oxazolo [2,3-c] [1,4] oxazin-3-yl) oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido) hexahydrofuro[3,2-b] furan-3-yl) amino) ethyl) carbamate
Figure imgf000179_0003
General Procedure I was followed using HATU (227 mg, 0.60 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 250 mg, 0.40 mmol), DIEA (0.348 mL, 1.99 mmol) and Intermediate Compound 83 (238 mg, 0.52 mmol) to afford Intermediate Compound 84 (280 mg, 68.0 %) as a red solid.1H NMR (300 MHz, DMSO) δ 1.07 (d, J = 6.9 Hz, 3H), 1.20 (d, J = 6.3 Hz, 5H), 1.41 (s, 3H), 1.76 (s, 1H), 1.92 (s, 2H), 3.60 (s, 5H), 4.00 (s, 5H), 4.22 – 4.34 (m, 7H), 4.60 (s, 2H), 7.31-7.42 (m, 8H), 7.72 (d, J = 7.6 Hz, 4H), 7.92 (dd, J = 12.3, 6.1 Hz, 7H), 13.28 (s, 2H), 14.08 (s, 2H). ES+(M+1 = 1033). Step 5 – Compound P25: (2S,4S)-N-((3S,3aR,6S,6aR)-6-(2-aminoacetamido) hexahydrofuro[3,2-b] furan-3-yl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1- methyloctahydro-1H-pyrano [4',3':4,5] oxazolo[2,3-c] [1,4] oxazin-3-yl) oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamide
Figure imgf000180_0001
General Procedure III was followed using a DCM solution of DBU (1.344 mL, 0.13 mmol) (15.2 mg/mL in MeCN) and Intermediate Compound 84 (250 mg, 0.24 mmol) in DCM (2mL)/MeCN (0.5 mL) to afford Compound P25 (50.0 mg, 25.5 %) as a red solid.
Figure imgf000180_0002
NMR (400 MHz, CDCl3) δ 1.34-1.46 (m, 3H), 1.75 (m, J = 15.2, 6.0 Hz, 3H), 2.04 (m, J = 15.2, 5.6 Hz, 1H), 2.36 (dd, J = 14.8, 3.6 Hz, 1H), 2.47 (d, J = 14.8 Hz, 1H), 2.66 – 2.89 (m, 2H), 3.06 – 3.34 (m, 3H), 3.41 (d, J = 5.6 Hz, 3H), 3.48 (s, 2H), 3.56 – 3.72 (m, 2H), 3.85 (d, J = 9.6 Hz, 2H), 3.89 – 4.08 (m, 4H), 4.12 (s, 4H), 4.44 – 4.57 (m, 3H), 4.63 (s, 1H), 4.73 (d, J = 2.0 Hz, 1H), 5.15 (s, 1H), 5.35 (s, 1H), 5.52 (t, J = 5.6 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 13.34 (s, 1H), 13.94 (s, 1H). ES+(M+1 = 811).
Example 30: Synthesis of Compound P26
Figure imgf000181_0001
Steps 1 and 2 – Intermediate Compound 86: (9H-fluoren-9-yl)methyl (2-((2-(((3S,3aR,6S,6aR)-6- ((tert-butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)amino)-2- oxoethyl)carbamate
Figure imgf000181_0002
(((9H-Fluoren-9-yl)methoxy)carbonyl)glycylglycine (1 g, 2.82 mmol) was added to 1- methylimidazole (0.232 g, 2.82 mmol) and Intermediate Compound 70(0.814 g, 5.64 mmol) in DMF (8 mL) at 25°C under nitrogen. The mixture was cooled to 0 °C. A solution of chloro-N,N,N',N'- tetramethylformamidinium hexafluorophosphate (0.950 g, 3.39 mmol) in DMF(2 mL) and added dropwise. The resulting mixture was then stirred at 25 °C for 4 hours (to afford Intermediate Compound 85). Di-tert-butyl dicarbonate (3.08 g, 14.11 mmol) was introduced in one portion under nitrogen. The resulting mixture was stirred at 25 °C for another 2 hours. The reaction mixture was poured into water (20 mL), extracted with EtOAc (3 x 25 mL). The combined organic solution was washed with water (2 x 50mL), dried over Na2SO4, filtered and evaporated to afford brown oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate Compound 86 (0.900 g, 54.9 %) as a yellow oil.
Figure imgf000181_0003
NMR (400 MHz, DMSO) δ 1.32-1.38 (br, 9H), 3.55 – 3.74 (m, 7H), 3.80 – 3.89 (m, 3H), 4.09 (d, J = 6.8 Hz, 1H), 4.18 – 4.29 (m, 1H), 4.31 (d, J = 8.0 Hz, 1H), 4.37 (d, J = 4.2 Hz, 1H), 4.43 (d, J = 4.0 Hz, 1H), 7.24 (d, J = 6.0 Hz, 1H), 7.30 – 7.36 (m, 2H), 7.36 – 7.43 (m, 2H), 7.44 (d, J = 1.2 Hz, 1H), 7.63 (t, J = 6.0 Hz, 1H), 7.73 (d, J = 7.6Hz, 2H), 7.82 – 7.93 (m, 2H), 8.10 (t, J = 5.6 Hz, 2H). ES+(M+1 = 581). Step 3 - Intermediate Compound 87: (9H-fluoren-9-yl)methyl (2-((2-(((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate, HCl salt
Figure imgf000182_0001
General Procedure II, Method A was followed using Intermediate Compound 86 (900 mg, 1.55 mmol) and 4 M HCl in 1.4-dioxane (10 mL) to afford Intermediate Compound 87 (750 mg, 94 %) as a pale yellow solid.1H
Figure imgf000182_0002
NMR (400 MHz, DMSO) δ 3.63 – 3.66 (m, 3H), 3.72 (d, J = 6.0 Hz, 2H), 3.86 – 3.98 (m, 3H), 4.13 (s, 1H), 4.17 – 4.34 (m, 3H), 4.59 (d, J = 4.4 Hz, 1H), 4.66 (d, J = 4.4 Hz, 1H), 7.34 (t, 2H), 7.38 – 7.47 (m, 2H), 7.60 – 7.76 (m, 3H), 7.90 (d, J = 7.6 Hz, 2H), 8.15 (t, J = 5.6 Hz, 1H), 8.27 (d, J = 6.8 Hz, 1H), 8.51 (s, 2H). ES+(M+1 = 481). Step 4 - Intermediate Compound 88: (9H-fluoren-9-yl)methyl (2-oxo-2-((2-oxo-2-(((3S,3aR,6S,6aR)- 6-((2S,4R)-2,5,12-trihydroxy-7-methoxy-4-(((1R,3S,4aR,9R,9aS,10aR)-9-methoxy-1- methyloctahydro-1H-pyrano-[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamido)hexahydro-furo[3,2-b]furan- 3yl)amino)ethyl)amino)ethyl)carbamate
Figure imgf000182_0003
General Procedure I was followed using HATU (136 mg, 0.36 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), DIEA (0.209 mL, 1.20 mmol) and Intermediate Compound 87 (161 mg, 0.31 mmol) to afford Intermediate Compound 88 (200 mg, 77 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.14 – 1.26 (m, 3H), 1.67 (s, 1H), 2.10-2.15 (m, 1H), 2.24 – 2.33 (m, 1H), 2.63 (t, J = 9.2 Hz, 1H), 2.68 (s, 1H), 2.90 (s, 1H), 2.97 (d, J = 12.0 Hz, 1H), 3.33 – 3.41 (m, 4H), 3.47 – 3.55 (m, 1H), 3.62 – 3.72 (m, 5H), 3.72 (s, 1H), 3.85 – 3.96 (m, 3H), 3.99 (s, 3H), 4.10 – 4.26 (m, 5H), 4.26 – 4.34 (m, 2H), 4.46 (d, J = 4.4 Hz, 1H), 4.50 – 4.55 (m, 1H), 4.59 (d, J = 2.2 Hz, 1H), 4.99 (d, J = 5.2 Hz, 1H), 5.24 (t, J = 4.8 Hz, 1H), 5.37 (s, 1H), 5.77 (s, 1H), 7.28 – 7.37 (m, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.59 – 7.67 (m, 2H), 7.71 (q, J = 7.6, 4.8 Hz, 2H), 7.88 (t, J = 7.2 Hz, 4H), 8.12 (q, J = 6.4, 5.6 Hz, 2H). ES+(M+1 = 1090). Step 5 – Compound P26: (2S,4S)-N-((3S,3aR,6S,6aR)-6-(2-(2- aminoacetamido)acetamido)hexahydrofuro[3,2-b]furan-3-yl)-2,5,12-trihydroxy-7-methoxy-4- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
Figure imgf000183_0001
General Procedure III was followed using a DCM solution of DBU (15.2 mg/mL in MeCN) (1.286 mL, 0.13 mmol) and Intermediate Compound 88 (200 mg, 0.18 mmol) in DCM (4 mL)/MeCN (1 mL) to afford Compound P26 (12.00 mg, 7.54 %) as a red solid.
Figure imgf000183_0002
NMR (400 MHz, CDCl3) δ 1.38 (t, J = 6.0 Hz, 3H), 1.98 – 2.09 (m, 2H), 2.28 – 2.50 (m, 2H), 2.71 – 2.88 (m, 2H), 3.24 (s, 2H), 3.38 – 3.72 (m, 7H), 3.77 – 4.25 (m, 11H), 4.49 (d, J = 2.0 Hz, 3H), 4.58 – 4.77 (m, 2H), 5.29 – 5.54 (m, 1H), 7.32 – 7.48 (m, 2H), 7.79 (t, J = 8.0 Hz, 1H), 7.94 – 8.08 (m, 1H), 13.89 (s, 1H). ES+(M+1 = 868).
Example 31: Synthesis of Compound P27
Figure imgf000184_0001
Steps 1,–2 - Intermediate Compound 90: (9H-fluoren-9-yl)methyl (2-((2-((2-(((3S,3aR,6S,6aR)-6- ((tert-butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)carbamate
Figure imgf000184_0002
(((9H-Fluoren-9-yl)methoxy)carbonyl)glycylglycylglycine (1 g, 2.43 mmol) was added to 1- methylimidazole (0.200 g, 2.43 mmol) and (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine (0.701 g, 4.86 mmol) was dissolved in DMF (15 mL) at 25°C under nitrogen. Chloro-N,N,N',N'-tetra- methylformamidinium hexafluorophosphate (0.818 g, 2.92 mmol) in DMF was added into above mixture dropwised at 0 °C. Then the resulting mixture was stirred at 25 °C for 4 hours. After the reaction finished (affording Intermediate Compound 89), then di-t-butyl dicarbonate (2.65 g, 12.15 mmol) was introduced in one portion under nitrogen. The resulting mixture was stirred at 25 °C for another 2 hours. The reaction mixture was poured into icy water (100 mL), extracted with EtOAc (3 x 50 mL), washed with water(2 x 100mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford a brown oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate Compound 90 (1.000 g, 64.5 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 2.91 (d, J = 8.7 Hz, 1H), 3.54 – 3.63 (m, 2H), 3.65 – 3.72 (m, 5H), 3.74 (d, J = 5.6 Hz, 2H), 4.07 (d, J = 7.6 Hz, 1H), 4.20 – 4.27 (m, 1H), 4.30 (d, J = 7.6 Hz, 2H), 4.37 (d, J = 4.2 Hz, 1H), 4.42 (d, J = 4.2 Hz, 1H), 7.24 (d, J = 5.9 Hz, 1H), 7.30 – 7.38 (m, 2H), 7.38 – 7.47 (m, 2H), 7.55 – 7.65 (m, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.05 – 8.15 (m, 2H), 8.20 (t, J = 5.7 Hz, 1H). ES+(M+1 = 638). Step 3 - Intermediate Compound 91: (9H-fluoren-9-yl)methyl (2-((2-((2-(((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)amino)-2-oxo-ethyl)amino)-2- oxoethyl)carbamate, HCl salt
Figure imgf000185_0001
General Procedure II, Method A was followed using Intermediate Compound 90 (1.1 g, 1.72 mmol) and HCl solution (4N in 1.4-dioxane, 12 mL) to afford Intermediate Compound 91 (0.900 g, 97 %) as a yellow solid. (HCl salt).1H
Figure imgf000185_0002
NMR (400 MHz, DMSO) δ 2.91 (d, J = 15.0 Hz, 1H), 3.65 – 3.78 (m, 7H), 3.83 – 3.98 (m, 6H), 4.08 – 4.16 (m, 1H), 4.29 (d, J = 6.3 Hz, 2H), 4.58 – 4.64 (m, 1H), 4.69 (d, J = 4.3 Hz, 1H), 7.29 – 7.38 (m, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.60 – 7.67 (m, 1H), 7.70 – 7.76 (m, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.21 (s, 1H), 8.29 (d, J = 6.3 Hz, 2H), 8.65 (s, 1H). ES+(M+1 = 538). Step 4 - Intermediate Compound 92: (9H-fluoren-9-yl)methyl (2-oxo-2-((2-oxo-2-((2-oxo-2- (((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':-4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydro-tetracene-2-carboxamido)hexahydrofuro[3,2-b]furan-3- yl)amino)ethyl)amino)ethyl)amino)ethyl)carbamate
Figure imgf000185_0003
General Procedure I was followed using HATU (136 mg, 0.36 mmol), the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 150 mg, 0.24 mmol), DIEA (0.209 mL, 1.20 mmol) and Intermediate Compound 91 (178 mg, 0.31 mmol) to afford Intermediate Compound 92 (140 mg, 51.1%) as a red solid.1H NMR (400 MHz, DMSO) δ 1.17 – 1.26 (m, 3H), 1.67 (s, 2H), 2.15 (d, J = 14.5 Hz, 1H), 2.27 (s, 1H), 2.64 (d, J = 8.3 Hz, 1H), 2.92 (s, 1H), 3.00 (d, J = 15.2 Hz, 1H), 3.30 – 3.33 (m, 6H), 3.38 (d, J = 6.4 Hz, 1H), 3.51 (d, J = 11.9 Hz, 1H), 3.59 – 3.77 (m, 8H), 3.87 (s, 1H), 3.94 (d, J = 6.4 Hz, 2H), 4.00 (s, 3H), 4.13 (d, J = 17.5 Hz, 3H), 4.18 – 4.26 (m, 2H), 4.29 (d, J = 7.0 Hz, 2H), 4.46 (d, J = 4.2 Hz, 1H), 4.52 (d, J = 4.1 Hz, 1H), 4.59 (d, J = 2.2 Hz, 1H), 5.01 (s, 1H), 5.25 (s, 1H), 5.38 (s, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.40 (q, J = 7.3 Hz, 2H), 7.59 (d, J = 6.0 Hz, 1H), 7.67-7.74 (m, 3H), 7.82 – 7.95 (m, 4H), 8.12 (t, J = 7.2 Hz, 3H), 8.19 (d, J = 6.0 Hz, 1H). ES+(M+1 = 1147). Step 5 – Compound P27: (2S,4S)-N-((3S,3aR,6S,6aR)-6-(2-(2-(2-aminoacet-amido)acetamido)- acetamido)hexahydro-furo[3,2-b]furan-3-yl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,- 9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano-[4',3':4,5]oxazolo-[2,3-c][1,4]oxazin-3-yl)oxy)- 6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
Figure imgf000186_0001
General Procedure III was followed using a DCM solution of DBU (15.2 mg/mL in MeCN) (0.856 mL, 0.13 mmol) and Intermediate Compound 92 (140 mg, 0.12 mmol) in DCM (8 mL)/MeCN (2 mL) to afford Compound P27 (26.0 mg, 23.03 %) as a red solid.
Figure imgf000186_0002
NMR (400 MHz, DMSO) δ1.09 – 1.30 (m, 4H), 1.62-1.72 (br, 2H), 2.09 – 2.37 (m, 3H), 2.61 – 2.71 (m, 2H), 3.00 (q, J = 18.3 Hz, 2H), 3.29 (s, 1H), 3.62 – 3.78 (m, 10H), 3.85 – 3.98 (m, 5H), 4.01 (d, J = 6.6 Hz, 3H), 4.07 – 4.26 (m, 5H), 4.43 – 4.65 (m, 4H), 4.95-5.06 (br, 1H), 5.25 (s, 1H), 5.40 (s, 1H), 7.68 (d, J = 4.6 Hz, 1H), 7.90 – 7.98 (m, 2H), 8.01 – 8.54 (m, 5H). ES+(M+1 = 925). Example 32: Synthesis of Compound P28
Figure imgf000187_0001
Step 1 – Intermediate Compound 94: tert-butyl 8-((3S,3aR,6S,6aR)-6- (((benzyloxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)-3,8-diazabicyclo[3.2.1]octane-3- carboxylate
Figure imgf000187_0002
tert-Butyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (3.87 g, 18.23 mmol) was added to (3R,3aS,6S,6aR)-6-(((benzyloxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl trifluoromethanesulfonate (1.5 g, 3.65 mmol) in 1,4-dioxane (25mL) at 100°C under nitrogen. The resulting mixture was stirred at 100 °C for 2 hours. The reaction mixture was quenched with water (25 mL), extracted with EtOAc (3 x 50 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford pale yellow liquid. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% THF in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 94 (1.500 g, 87 %) as a pale yellow liquid. NMR (300 MHz, DMSO) δ 1.39 (s, 9H), 1.46 (d, J = 7.4 Hz, 2H), 1.74 – 1.80 (m, 1H), 1.84 (d, J = 8.8 Hz, 1H), 2.79 – 3.01 (m, 3H), 3.06 (s, 1H), 3.36 – 3.70 (m, 5H), 3.75 – 3.89 (m, 2H), 3.99 (dd, J = 8.9, 6.2 Hz, 1H), 4.32 (d, J = 5.0 Hz, 1H), 4.45 (d, J = 3.9 Hz, 1H), 5.03 (s, 2H), 7.26 – 7.44 (m, 5H), 7.60 (d, J = 6.3 Hz, 1H). ES+(M+1 =474). Step 2 - Intermediate Compound 95: tert-butyl 8-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2- b]furan-3-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate
Figure imgf000188_0001
Pd-C (112 mg, 0.11 mmol) was added to Intermediate Compound 94 (500 mg, 1.06 mmol) in MeOH (20 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 5 hours. The reaction mixture was filtered through celite. The filtrate was evaporated to dryness to afford Intermediate Compound 95 (300 mg,83.71%).1H NMR (300 MHz, DMSO) δ 1.39 (s, 9H), 1.46 (d, J = 7.3 Hz, 2H), 1.66 (s, 1H), 1.84 (s, 1H), 2.74 – 2.91 (m, 2H), 2.99 (s, 1H), 3.06 (s, 1H), 3.21 (dd, J = 18.7, 4.3 Hz, 1H), 3.36 – 3.41 (m, 2H), 3.44 – 3.59 (m, 3H), 3.72 (dd, J = 8.8, 4.3 Hz, 1H), 3.94 (dd, J = 8.9, 6.2 Hz, 1H), 4.11 (d, J = 4.9 Hz, 1H), 4.47 (dd, J = 5.0, 2.5 Hz, 1H). ES+(M+1 = 340). Step 3 - Intermediate Compound 96: tert-butyl 8-((3S,3aR,6S,6aR)-6-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-hexahydrofuro[3,2-b]furan-3-yl)-3,8-diazabicyclo[3.2.1]octane-3- carboxylate
Figure imgf000188_0002
(9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (477 mg, 1.41 mmol) was added to DIEA (0.617 mL, 3.54 mmol) and Intermediate Compound 95 (400 mg, 1.18 mmol) in DCM (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with water (20 mL), extracted with DCM (2 x 20 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford pale yellow liquid. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% THF in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 96 (600 mg, 76.92%) as a pale yellow liquid.1H NMR (300 MHz, DMSO) δ 1.39 (s, 9H), 1.47 (d, J = 7.6 Hz, 2H), 1.84 (s, 2H), 2.76 – 3.13 (m, 4H), 3.41 (t, J = 8.2 Hz, 2H), 3.49 – 3.70 (m, 3H), 3.80 (d, J = 8.9 Hz, 2H), 3.94 – 4.01 (m, 1H), 4.23 (d, J = 6.7 Hz, 1H), 4.34 (d, J = 7.4 Hz, 2H), 4.46 (s, 1H), 7.28 – 7.48 (m, 4H), 7.61 (d, J = 6.1 Hz, 1H), 7.71 (d, J = 7.4 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H). ES+( M+1 =562), Step 4 - Intermediate Compound 97: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-(3,8- diazabicyclo[3.2.1]octan-8-yl)hexahydrofuro[3,2-b]furan-3-yl)carbamate, HCl salt
Figure imgf000189_0001
Intermediate Compound 96 (200 mg, 0.36 mmol) was added to HCl-1,4-dioxane (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The filtrate was evaporated to dryness to afford Intermediate Compound 97 (170 mg, 96 %) as a pale yellow solid.1H NMR (300 MHz, DMSO) δ 2.20 (s, 2H), 3.31 (s, 1H), 3.49 (dd, J = 8.2, 3.8 Hz, 1H), 3.57 (s, 5H), 3.71 (dt, J = 9.9, 7.0 Hz, 3H), 3.90 (d, J = 2.5 Hz, 4H), 4.23 (d, J = 7.0 Hz, 2H), 4.35 (d, J = 6.2 Hz, 2H), 4.48 (s, 1H), 7.29 – 7.48 (m, 4H), 7.71 (d, J = 8.1 Hz, 3H), 7.90 (d, J = 7.4 Hz, 2H). ES+(M+1 = 462). Step 5 - Intermediate Compound 98: (9H-fluoren-9-yl)-methyl ((3S,3aR,6S,6aR)-6-(3-((2S,4S)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]-oxazolo-[2,3-c]-[1,4]-oxazin-3-yl)-oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carbonyl)-3,8-diazabicyclo-[3.2.1]-octan-8-yl)-hexahydrofuro-[3,2-b]-furan-3- yl)-carbamate
Figure imgf000189_0002
HATU (218 mg, 0.57 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 180 mg, 0.29 mmol) in DMA (3 mL) under nitrogen. The resulting mixture 1 was stirred at RT for 10 minutes. DIEA (0.250 mL, 1.43 mmol) was added to Intermediate Compound 97 (199 mg, 0.43 mmol) in DMA (3.00 mL) under nitrogen. The resulting mixture 2 was stirred at RT for 10 minutes. After that, the mixture 1 and the mixture 2 was mixed, stirring at RT for 2 hours. The crude product was purified by C18-flash chromatography, elution gradient 0 to 100% MeCN in water (NH4HCO3). Pure fractions were evaporated to dryness to afford Intermediate Compound 98 (210 mg, 68.4 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.18 – 1.28 (m, 3H), 1.35 – 1.80 (m, 4H), 1.80 – 2.43 (m, 4H), 2.57 – 2.72 (m, 2H), 2.89 (d, J = 26.8 Hz, 3H), 3.09 (d, J = 15.3 Hz, 2H), 3.32 (s, 3H), 3.36 – 3.55 (m, 7H), 3.56 – 3.75 (m, 3H), 3.77 – 3.89 (m, 2H), 3.93 – 4.03 (m, 5H), 4.05 (d, J = 7.1 Hz, 1H), 4.13 – 4.27 (m, 2H), 4.34 (s, 2H), 4.49 (s, 1H), 4.52 – 4.75 (m, 2H), 4.75 – 5.55 (m, 1H), 6.28 (s, 1H), 7.31 – 7.37 (m, 2H), 7.38 – 7.44 (m, 2H), 7.60 (s, 1H), 7.70 (s, 1H), 7.80 – 7.95 (m, 6H). ES+(M+1 = 1071). Step 6 – Compound P28: (8S,10S)-8-(8-((3S,3aR,6S,6aR)-6-aminohexahydro-furo[3,2-b]-furan-3-yl)- 3,8-diazabicyclo[3.2.1]-octane-3-carbonyl)-6,8,11-trihydroxy-1-methoxy-10- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano-[4',3':4,5]-oxazolo-[2,3-c]- [1,4]-oxazin-3-yl)-oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000190_0001
1.066 mL of DCM solution of DBU (15.2 mg/mL in DCM) (0.11 mmol) was added to Intermediate Compound 98 (190 mg, 0.18 mmol) in DCM (8 mL)/MeCN (2.000 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The reaction mixture was quenched with 0.1% NH4HCO3 (1.5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford crude product (150 mg). The crude product was purified by preparative HPLC Column: Xselect CSH Prep C18 OBD, 30*150mm, 5um; Mobile Phase A: Water (10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min mL/min; Gradient: 30% B to 41% B in 10 min; Wavelength: 254nm/220nm; RT1(min): 8.63. Fractions containing the desired compound were evaporated to dryness to afford Compound P28 (59.0 mg, 39.2 %) as a red solid. 1H NMR (400 MHz, DMSO) δ 1.24 (d, J = 6.2 Hz, 4H), 1.39 (s, 1H), 1.63 – 1.71 (m, 2H), 1.81 (s, 3H), 2.17 (d, J = 18.5 Hz, 1H), 2.43 (s, 1H), 2.58 – 2.92 (m, 6H), 3.11 (s, 1H), 3.25 (d, J = 4.3 Hz, 3H), 3.27 – 3.31 (m, 3H), 3.46 – 3.53 (m, 2H), 3.63 – 3.75 (m, 2H), 3.80-4.10 (m, 7H), 4.13 (d, J = 5.3 Hz, 1H), 4.24 (d, J = 2.2 Hz, 1H), 4.49 (s, 2H), 4.55 – 4.67 (m, 2H), 4.86-4.95 (m, 1H), 5.25 (d, J = 11.3 Hz, 1H), 5.83 (d, J = 24.5 Hz, 1H), 7.61 – 7.69 (m, 1H), 7.91 (d, J = 5.2 Hz, 2H). ES+(M+1 = 849). Example 33: Synthesis of Compound P29
Figure imgf000191_0001
Step 1 - Intermediate Compound 99: tert-butyl (1S,4S)-5-((3S,3aR,6S,-6aR)-6- (((benzyloxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)-2,5-diazabicyclo[2.2.2]-octane-2- carboxylate
Figure imgf000191_0002
(3R,3aS,6S,6aR)-6-(((benzyloxy)carbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl trifluoromethanesulfonate (1.5 g, 3.65 mmol) and tert-butyl (1S,4S)-2,5-diazabicyclo[2.2.2]-octane- 2-carboxylate (2.322 g, 10.94 mmol) in 1,4-dioxane (20 mL) under nitrogen. The resulting mixture was stirred at 100 °C for 2 hours. The solvent was removed under reduced pressure. The reaction mixture was quenched with water (10 mL), extracted with EtOAc (2 x 50 mL), the combined organic layer was dried over Na2SO4, filtered and evaporated to afford residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate Compound 99 (1.190 g, 68.9 %) as a yellow solid.1H NMR (300 MHz, DMSO) δ 1.40 (d, J = 3.7 Hz, 8H), 1.53 (d, J = 12.5 Hz, 1H), 1.69 (s, 1H), 1.90 (d, J = 12.6 Hz, 1H), 2.78 – 2.95 (m, 3H), 3.08 (s, 1H), 3.21 (t, J = 9.3 Hz, 1H), 3.34 (s, 2H), 3.37 – 3.56 (m, 2H), 3.65 (d, J = 8.4 Hz, 1H), 3.82 (dd, J = 9.0, 4.8 Hz, 2H), 4.30 (d, J = 4.8 Hz, 1H), 4.45 (d, J = 4.8 Hz, 1H), 5.03 (s, 2H), 7.35 (d, J = 4.8 Hz, 4H), 7.39 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 6.2 Hz, 1H). ES+(M+1 =474), Step 2 - Intermediate Compound 100: tert-butyl (1S,4S)-5-((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000192_0001
Intermediate Compound 99 (850 mg, 1.79 mmol) in MeOH (8mL) under hydrogen. The resulting mixture was stirred at RT for 1 hour. The solvent was removed under reduced pressure to afford Intermediate Compound 100 (600 mg, 98%) as a yellow oil.1H NMR (300 MHz, DMSO) δ 1.40 (d, J = 3.8 Hz, 9H), 1.48 – 1.58 (m, 1H), 1.67 (d, J = 13.6 Hz, 2H), 1.91 (d, J = 8.1 Hz, 1H), 2.78 – 2.91 (m, 3H), 3.04 – 3.09 (m, 1H), 3.15 – 3.28 (m, 3H), 3.35 – 3.57 (m, 4H), 3.74 (dd, J = 8.7, 4.5 Hz, 1H), 3.79 – 3.94 (m, 2H), 4.09 (d, J = 4.8 Hz, 1H), 4.41 – 4.49 (m, 1H). ES+(M+1 = 340). Step 3 - Intermediate Compound 101: tert-butyl (1S,4S)-5-((3S,3aR,6S,6aR)-6-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) hexahydrofuro [3,2-b] furan-3-yl)-2,5-diazabicyclo [2.2.2] octane-2- carboxylate
Figure imgf000192_0002
N-(9H-Fluoren-2-ylmethoxycarbonyloxy) succinimide (806 mg, 2.39 mmol) was added to Intermediate Compound 100 (595mg, 1.75 mmol) and DIEA (0.839 mL, 4.80 mmol) in DCM (5 mL) under hydrogen. The resulting mixture was stirred at RT for 1 hour. The reaction mixture was acidified with 0.5M HCl. The reaction mixture was quenched with water (5 mL), extracted with EtOAc (2 x 5 mL), the combined organic layer was dried over Na2SO4, filtered and evaporated to afford residue.The crude product was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 101 (930 mg, 94 %) as a white solid.1H NMR (300 MHz, DMSO) δ 1.13 – 1.21 (t, J = 7.1 Hz, 1H), 1.40 (br, J = 3.6 Hz, 9H), 1.52 (s, 1H), 1.69 (s, 2H), 1.91 (d, J = 11.7 Hz, 1H), 2.00 (s, 1H), 2.80 (dd, J = 14.8, 25.0 Hz, 3H), 3.03 – 3.25 (m, 2H), 3.39 – 3.67 (m, 3H), 3.79 – 3.95 (m, 4H), 4.02 (t, J = 7.2 Hz, 1H), 4.19 – 4.37 (m, 3H), 4.44 (s, 1H), 7.29 – 7.47 (m, 4H), 7.60 (s, 1H), 7.70 (d, J = 7.4 Hz, 2H), 7.90 (d, J = 7.4 Hz, 2H). ES+(M+1 = 562). Step 4 - Intermediate Compound 102: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-((1S,4S)-2,5- diazabicyclo[2.2.2]octan-2-yl)hexahydrofuro[3,2-b]furan-3-yl)carbamate hydrochloride
Figure imgf000193_0001
Intermediate Compound 101 (920 mg, 1.64 mmol) in HCl [4 M in dioxane] (10 mL). The resulting mixture was stirred at rt for 1 hour. The solvent was removed under reduced pressure to afford Intermediate Compound 102 (800 mg, 98 %) as a yellow solid.1H NMR (300 MHz, DMSO) δ 1.82 (d, J = 12.8 Hz, 3H), 2.11 (s, 1H), 3.36 (s, 2H), 3.74 (s, 4H), 3.90 (s, 3H), 4.13 (s, 1H), 4.24 (d, J = 6.4 Hz, 1H), 4.34 (s, 2H), 4.45 (s, 1H), 5.76 (s, 4H), 7.30 – 7.48 (m, 5H), 7.70 (d, J = 7.6 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H). ES+(M+1 = 462). Step 5 - Intermediate Compound 103: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-((1S,4S)-5- ((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,-10aS)-9-methoxy-1-methyloctahydro- 1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carbonyl)-2,5-diazabicyclo[2.2.2]octan-2-yl)-hexahydrofuro[3,2-b]furan-3- yl)carbamate
Figure imgf000193_0002
The PNU-159682 carboxylic acid (CAS No.1204819-92-0; 200 mg, 0.32 mmol) was added to Intermediate Compound 102 (735 mg, 1.59 mmol), N,N-Diisopropylethylamine (124 mg, 0.96 mmol) and O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (158 mg, 0.41 mmol) in DMA (4 mL). The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound 103 (230 mg, 67.4 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.23 (d, J = 7.0 Hz, 3H), 1.45 – 2.00 (m, 3H), 2.21 – 2.37 (m, 1H), 2.50 (s, 4H), 2.52 (s, 7H), 2.61 – 2.92 (m, 3H), 3.11 (s, 2H), 3.30-3.33 (m, 9H), 3.36 – 3.53 (m, 4H), 3.54 – 3.96 (m, 3H), 4.00 (s, 3H), 4.09 – 4.63 (m, 4H), 4.69 – 5.13 (m, 2H), 5.26 (s, 1H), 5.85 (s, 1H), 7.34-7.42 (m, 4H), 7.54 – 7.74 (m, 3H), 7.81 – 7.99 (m, 4H). ES+(M+1 =1072). Step 6 – Compound P29: (8S,10S)-8-((1S,4S)-5-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan- 3-yl)-2,5-diazabicyclo[2.2.2]octane-2-carbonyl)-6,8,11-trihydroxy-1-methoxy-10- (((1S,3R,4aS,9S,9aR,-10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione
Figure imgf000194_0001
1,8-Diazabicyclo[5.4.0]undec-7-ene (1.309 mL, 0.13 mmol) (15.2mg/mL) in DCM was added to Intermediate Compound 103 (200 mg, 0.19 mmol) in DMA (1 mL), DCM (4 mL) and MeCN (1 mL). The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with 0.1% NH4HCO3 (1 mL), The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 70% MeCN in water. Pure fractions were evaporated to dryness to afford crude product (100 mg, crude). The crude product was further purified by preparative HPLC (Column: YMC-Actus Triant C18 ExRs Column, 30*150 mm, 5μm; Mobile Phase A: Water (10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 30% B to 45% B in 10 min; Wavelength: 254nm/220nm; RT1(min): 8.38). Fractions containing the desired compound were freeze dried directly to dryness to afford Compound P29 (7.30 mg, 4.61 %) as a red solid.1H NMR (400 MHz, DMSO) δ 0.85 (m, 1H) (impurity), 1.19 – 1.29 (m, 5H), 1.32 – 1.58 (m, 2H), 1.60 – 1.79 (m, 4H), 1.81 – 2.14 (m, 3H), 2.60 – 2.80 (m, 4H), 2.82 – 3.17 (m, 5H), 3.25 (d, J = 6.1 Hz, 3H), 3.45 – 3.56 (m, 3H), 3.63 – 3.79 (m, 2H), 3.81 – 3.96 (m, 4H), 4.00 (s, 3H), 4.09 (m, 1H), 4.24 (d, J = 2.0 Hz, 1H), 4.35 – 4.52 (m, 1H), 4.60 (d, J = 2.0 Hz, 1H), 4.67 – 4.88 (m, 1H), 4.93 – 5.11 (m, 1H), 5.28 (d, J = 16.4 Hz, 1H), 5.50 – 5.89 (m, 1H), 7.63 – 7.77 (m, 1H), 7.78 – 8.00 (m, 2H). ES+(M+1 =849.5). Example 34: Synthesis of Compound P30
Figure imgf000195_0001
Steps 1, 2 - Intermediate Compound A-110: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)(methyl)carbamate
Figure imgf000195_0002
Formaldehyde (340 mg, 4.30 mmol), in 150 mL round bottom flask was added to tert-butyl ((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)carbamate (CAS No.1932199-27-3; 700 mg, 2.87 mmol) in MeOH (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. Then NaBH4 (650 mg, 17.19 mmol) was added, the resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was acidified with 1M HCl to pH (7~6). Then the reaction mixture was acidified with saturated NaHCO3 to pH (9~8). N-(9H-Fluoren-2- ylmethoxycarbonyloxy)succinimide (1.45 g, 4.30 mmol) in THF (20.00 mL) was added to the mixture, the resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure. The reaction mixture was quenched with water (50 mL), extracted with DCM (3 x 100 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford pale yellow oil. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound A- 110 (590 mg, 42.8 %) as a white solid. 1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.71 (s, 3H), 3.51 - 3.62 (m, 1H), 3.63 - 3.90 (m, 4H), 4.26 – 4.42 (m, 5H), 4.47 (s, 1H), 7.18 (d, J = 6.4 Hz, 1H), 7.30 - 7.39 (m, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.62 – 7.68 (m, 2H), 7.87 – 7.93 (m, 2H). ES+(M+1 = 481), HPLC purity: 90%. Step 3: Intermediate Compound A-111: (9H-fluoren-9-yl)methyl ((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)(methyl)carbamate
Figure imgf000196_0001
Intermediate Compound A-110 (440 mg, 0.92 mmol) was added to DCM (3 mL)/HCl in dioxane (4N) (3.00 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford crude Intermediate Compound A-111 (300 mg, 86 %) as a pale yellow solid. 1H NMR (400 MHz, DMSO) δ 2.75 (s, 3H), 3.66 – 3.98 (m, 5H), 4.28 – 4.42 (m, 4H), 4.62 – 4.73 (m, 2H), 7.31 – 7.39 (m, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.65 (d, J = 7.4 Hz, 2H), 7.91 (d, J = 7.5 Hz, 2H). (2 exchangeable protons not seen). ES+(M+1 = 381), HPLC purity: 95%. Step 4 - Intermediate Compound A-112: (9H-fluoren-9-yl) methyl((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,-4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]-oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido)-hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000196_0002
Intermediate Compound A-111 (218 mg, 0.57 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 300 mg, 0.48 mmol), HATU (364 mg, 0.96 mmol) and DIEA (0.417 mL, 2.39 mmol) in DMA (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound A- 112 (430 mg, 91 %) as a red solid. 1H NMR (400 MHz, DMSO) δ 1.18 – 1.25 (m, 3H), 1.60 – 1.78 (m, 2H), 2.08 (s, 2H), 2.13 – 2.21 (m, 1H), 2.26 (t, J = 7.6 Hz, 1H), 2.59 – 2.79 (m, 5H), 2.92 – 3.03 (m, 2H), 3.39 (d, J = 6.6 Hz, 1H), 3.47 - 3.56 (m, 1H), 3.60 - 3.76 (m, 3H), 3.88 – 4.01 (m, 6H), 4.13 – 4.33 (m, 4H), 4.33 – 4.46 (m, 3H), 4.47 – 4.62 (m, 3H), 4.98 (s, 1H), 5.21 – 5.28 (m, 1H), 5.33 – 5.37 (m, 1H), 7.30 – 7.38 (m, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.59 – 7.69 (m, 3H), 7.83 – 7.93 (m, 4H), 8.05 (d, J = 7.6 Hz, 1H), 13.22 (br, 1H), 14.01 (br, 1H). ES+(M+1 = 990), HPLC purity: 90%. Step 5 – Compound P30: (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo-[2,3-c][1,4]oxazin-3-yl)oxy)-N- ((3S,3aR,6S,6aR)-6-(methylamino)hexahydrofuro[3,2-b]furan-3-yl)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamide
Figure imgf000197_0001
DBU (0.053 mL, 0.35 mmol) was added to Intermediate Compound A-112 (350 mg, 0.35 mmol) in DMA (5 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 30 minutes. The crude product was purified by preparative HPLC (Column: Xbridge Prep C18 OBD, 30*150 mm, 5μm; Mobile Phase A: Water(10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 24% B to 45% B in 8 min; Wavelength: 254nm/220nm; RT1(min): 7.35) and MeCN as eluents. Fractions containing the desired compound were freeze dried directly to dryness to afford Compound P30 (88 mg, 32.4 %) as a red solid. 1H NMR (400 MHz, DMSO) δ 1.20 (d, J = 6.5 Hz, 3H), 1.59 – 1.76 (m, 2H), 2.05 – 2.19 (m, 1H), 2.27 (s, 4H), 2.60 – 2.73 (m, 2H), 2.90 – 3.03 (m, 3H), 3.30 – 3.32 (m, 3H ), 3.37 – 3.42 (m, 2H), 3.49 – 3.69 (m, 4H), 3.77 - 3.82 (m, 1H), 3.87 – 3.96 (m, 2H), 3.99 (s, 3H), 4.09 – 4.17 (m, 2H), 4.23 (d, J = 2.2 Hz, 1H), 4.35 – 4.45 (m, 2H), 4.59 (d, J = 2.2 Hz, 1H), 4.96 – 5.03 (m, 1H), 5.24 (t, J = 4.7 Hz, 1H), 5.37 (s, 1H), 7.66 (p, J = 3.8 Hz, 1H), 7.87 – 7.97 (m, 2H), 8.04 (d, J = 7.6 Hz, 1H).2 exchangeable proton not seen. ES+(M+1 = 768), HPLC purity: 99%.
Example 35: Synthesis of Compound P31
Figure imgf000198_0001
Step 1 – Intermediate Compound A-113: benzyl ((3S,3aR,6S,6aR)-6-cyanohexahydrofuro[3,2- b]furan-3-yl)carbamate
Figure imgf000198_0002
KCN (3.56 g, 54.70 mmol) was added to Intermediate Compound 62 (15 g, 36.47 mmol) and 18- Crown-6 (15.42 g, 58.34 mmol) in THF (100 mL) at 0°C. The resulting mixture was stirred at 0 °C for 4 hours. The reaction mixture was diluted with DCM (500 mL), and washed sequentially with water (500 mL x 1) and saturated brine (500 mL x 1). The aqueous layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 60 to 60% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound A-113 (10.50 g, 100 %) as a yellow oil. 1H NMR 1H NMR (300 MHz, DMSO) δ 3.32 (s, 1H), 3.42-3.51 (m, 1H), 3.63 – 3.71 (m, 1H), 3.83 – 4.03 (m, 4H), 4.51 (d, J = 4.6 Hz, 1H), 4.88 (dd, J = 2.2, 4.6 Hz, 1H), 5.04 (s, 2H), 7.35 (d, J = 4.8 Hz, 5H), 7.69 (d, J = 6.0 Hz, 1H). ES+(M+1 = 289.111), HPLC purity: 99.1%. Step 2 - Intermediate Compound A-114: benzyl ((3S,3aR,6S,6aR)-6- (aminomethyl)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000199_0001
Borane-tetrahydrofuran complex (156 mL, 156.09 mmol) was added to Intermediate Compound A-113 (9 g, 31.22 mmol) in THF (100 mL) at 0°C over a period of 10 minutes under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with MeOH (100 mL) in ice bath. The solvent was removed under reduced pressure, which afforded Intermediate Compound A-114 (9.00 g, 99 %) as a yellow oil. 1H NMR (300 MHz, DMSO) δ 1.47 – 1.75 (m, 1H), 2.67 – 2.88 (m, 2H), 3.53 – 3.72 (m, 2H), 3.79 – 3.99 (m, 3H), 4.20 – 4.51(m, 2H), 5.03 (s, 2H), 7.36 (s, 5H), 7.67 (s, 1H), 7.90 (s, 2H). ES+( M+1 =292), HPLC purity: 93%. Step 3 - Intermediate Compound A-115: benzyl ((3S,3aR,6S,6aR)-6-(((((9H-fluoren-9- yl)methoxy)carbonyl)amino)methyl)hexahydrofuro-[3,2-b]furan-3-yl)carbamate
Figure imgf000199_0002
N-(9H-Fluoren-2-ylmethoxycarbonyloxy)succinimide (9.23 g, 27.37 mmol) was added to Intermediate Compound A-114 (8g, 27.37 mmol) in MeCN (50 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 50 to 50% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound A-115 (14.00 g, 99 %) as a white solid. 1H NMR 1H NMR (300 MHz, DMSO) δ 2.83 – 3.01 (m, J = 6.7 Hz, 2H), 3.33 (d, J = 2.0 Hz, 1H), 3.53 (td, J = 2.9, 8.4 Hz, 2H), 3.75 (dd, J = 5.9, 8.9 Hz, 1H), 3.85 (d, J = 7.9 Hz, 2H), 4.21 (t, J = 6.8 Hz, 1H), 4.29 – 4.38 (m, 4H), 5.02 (s, 2H), 7.29 – 7.37 (m, 8H), 7.41 (t, J = 7.3 Hz, 2H), 7.48 (t, J = 5.9 Hz, 1H), 7.68 (d, J = 7.4 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H). ES+( M+1 = 514), HPLC purity: 99%. Step 4 – Intermediate Compound A-116: (9H-fluoren-9-yl)methyl (((3S,3aR,6S,6aR)-6- aminohexahydrofuro-[3,2-b]furan-3-yl)methyl)carbamate, HCl salt
Figure imgf000200_0001
Pd-C (6.98 g, 13.12 mmol) was added to 0.01M HCl (3.887 mL, 0.04 mmol) and Intermediate Compound A-115 (13.5 g, 26.24 mmol) in EtOH (40 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered through celite. The solvent was removed under reduced pressure and afforded Intermediate Compound A-116 (9.50 g, 95 %) as a yellow solid. NMR (400 MHz, DMSO) δ 2.89 – 2.97 (m, 1H), 3.02 (dt, J = 6.9, 13.9 Hz, 1H), 3.54 (dd, J = 4.0, 9.0 Hz, 1H), 3.63 (dd, J = 3.1, 5.2 Hz, 1H), 3.74 (dd, J = 3.0, 10.4 Hz, 1H), 3.81 (dd, J = 6.1, 9.0 Hz, 1H), 3.92 (dd, J = 5.1, 10.4 Hz, 1H), 4.22 (t, J = 6.8 Hz, 1H), 4.31 – 4.38 (m, 2H), 4.51 (s, 2H), 7.34 (td, J = 1.2, 7.5 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.54 (t, J = 5.9 Hz, 1H), 7.68 (d, J = 7.4 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.30 (s, 3H). ES+(M+1 = 380), HPLC purity: 98%. Step 5 - Intermediate Compound A-117: (9H-fluoren-9-yl)methyl (((3S,3aR,6S,6aR)-6-((2S,4S)- 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,-9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxa-zin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido)hexahydro-furo[3,2-b]furan-3-yl)methyl)carbamate
Figure imgf000200_0002
Intermediate Compound A-116 (182 mg, 0.48 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 300 mg, 0.48 mmol), HATU (273 mg, 0.72 mmol) and DIEA (0.250 mL, 1.43 mmol) in DMA (10 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 5 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound A-117 (240 mg, 50.7 %) as a red solid. 1H NMR (400 MHz, DMSO) δ 1.17 – 1.32 (m, 5H), 1.42 – 1.56 (m, 3H), 1.63 – 1.79 (m, 3H), 2.08 (s, 3H), 2.22 – 2.37 (m, 4H), 2.86 – 3.10 (m, 3H), 3.44 – 3.71 (m, 4H), 3.78 (dd, J = 5.9, 8.9 Hz, 1H), 4.23 (s, 2H), 4.32 – 4.50 (m, 5H), 4.58 (d, J = 2.3 Hz, 1H), 5.02 (s, 2H), 5.20 – 5.43 (m, 2H), 5.77 (s, 4H), 7.29 – 7.36 (m, 3H), 7.42 (t, J = 7.5 Hz, 2H), 7.51 (t, J = 5.9 Hz, 1H), 7.61 – 7.73 (m, 4H), 7.84 – 7.97 (m, 6H), 8.07 (d, J = 7.7 Hz, 1H). ES+(M+1 = 990.45 ), HPLC purity: 89%. Step 6 - Compound P31: (2S,4S)-N-((3S,3aR,6S,6aR)-6-(aminomethyl)hexa-hydrofuro[3,2-b]furan-3- yl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamide
Figure imgf000201_0001
DBU (0.043 mL, 0.28 mmol) was added to Intermediate Compound A-117 (280 mg, 0.28 mmol) in DCM (10 mL) and MeCN (2.500 mL) under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The reaction mixture was adjusted to pH=7 with 0.1M HCl aqueous solution. Then the solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (Column: Xbridge Prep C18 OBD, 19*250mm, 5um; Mobile Phase A: Water (10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 27% B to 40% B in 10 min; Wavelength: 254nm/220nm; RT1(min): 8.72). Fractions containing the desired compound were freeze dried directly to dryness to afford Compound P31 (74.7 mg, 34.4 %) as a red solid. 1H NMR (400 MHz, DMSO) δ 1.09 – 1.25 (m, 3H), 1.67 – 1.72 (m, 2H), 2.06 – 2.16 (m, 2H), 2.21 – 2.28 (m, 1H), 2.40 – 2.46 (m, 2H), 2.66 – 2.74 (m, 2H), 3.33 – 3.43 (m, 4H), 3.46 – 3.71 (m, 5H), 3.76 – 3.82 (m, 1H), 3.86 – 3.97 (m, 3H), 3.96 (s, 3H), 3.99 (s, 2H), 4.12 (s, 1H), 4.35 – 4.45 (m, 1H), 4.48 (d, J = 4.4 Hz, 1H), 4.58 (d, J = 2.0 Hz, 1H), 4.99 (s, 1H), 5.24 (t, J = 4.8 Hz, 1H), 5.38 (s, 1H), 7.65 (t, J = 4.8 Hz, 1H), 7.91 (d, J = 4.8 Hz, 2H), 8.01 (d, J = 7.6 Hz, 1H), 8.12 – 9.01 (m, 1H). ES+(M+1 = 768.45), HPLC purity: 95.3%
Example 36: Synthesis of Compound P32
Figure imgf000202_0001
Step 1 – Intermediate Compound A-118: (9H-fluoren-9-yl)methyl (((3S,3aR,6S,6aR)-6- (tritylamino)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamate
Figure imgf000202_0002
Intermediate Compound A-116 (300 mg, 0.79 mmol) was added to Triphenylmethyl chloride (264 mg, 0.95 mmol) and N,N-Diisopropylethylamine (306 mg, 2.37 mmol) in THF (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with DCM (20 mL), and washed sequentially with water (20 mLx3), The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The product Intermediate Compound A- 118 was used in the next step directly without further purification. Step 2 - Intermediate Compound A-119: (3S,3aR,6S,6aR)-6-(aminomethyl)-N- tritylhexahydrofuro[3,2-b]furan-3-amine
Figure imgf000202_0003
Intermediate Compound A-118 (491 mg, 0.79 mmol) was added to Diethylamine (3 mL, 0.79 mmol) in THF (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed by distillation under vacuum. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% water (NH4CO3) in MeCN. Pure fractions were evaporated to dryness to afford Intermediate Compound A-119 (120 mg, 38.0 %) as a white solid. NMR (300 MHz, DMSO) δ 2.30 - 2.36 (m, 1H), 2.40 - 2.46 (m, 1H), 3.50 - 3.59 (m, 4H), 3.67 - 3.83 (m, 1H), 4.22 - 4.43 (m, 2H), 7.16 - 7.49 (m, 15H). ES+( M+23 = 423.25), HPLC purity: 82.5%. Step 3 - Intermediate Compound A-120: (2S,4S)-2,5,12-trihydroxy-7-methoxy-4- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-N-(((3S,3aR,6S,6aR)-6-(tritylamino)hexahydrofuro[3,2-b]furan-3- yl)methyl)-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
Figure imgf000203_0001
The PNU-159682 carboxylic acid (CAS No.1204819-92-0; 120 mg, 0.19 mmol) was added to Intermediate Compound A-119 (115 mg, 0.29 mmol), N,N-Diisopropylethylamine (124 mg, 0.96 mmol) and O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (145 mg, 0.38 mmol) in DMA (3 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% water (NH4CO3) in MeCN. Pure fractions were evaporated to dryness to afford Intermediate Compound A-120 (100 mg, 51.8 %) as a red solid. Step 4 - Compound P32: (2S,4S)-N-(((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3- yl)methyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro- 1H-pyrano-[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamide
Figure imgf000204_0001
Intermediate Compound A-120 (100 mg, 0.10 mmol) was added to 1,1,1,3,3,3-Hexafluoro-2- propanol (2 mL, 0.10 mmol) in DCM (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The reaction system was dried with nitrogen gas flow. The crude product was purified by preparative HPLC (Column: Xselect CSH Prep Phenyl-Hexyl OBD column, 19*250mm, 5um; Mobile Phase A: Water (10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 27% B to 37% B in 15 min; Wavelength: 254nm/220nm; RT1(min): 13.48) and MeCN as eluents. Fractions containing the desired compound were freeze dried directly to dryness to afford Compound P32 (41.8 mg, 55.0 %) as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 1.22 (d, J = 6.6 Hz, 3H), 1.63 – 1.70 (m, 2H), 2.16 (d, J = 13.8 Hz, 1H), 2.25 – 2.30 (m, 1H), 2.37 (s, 1H), 2.61 – 2.72 (m, 2H), 2.93 – 3.03 (m, 2H), 3.10 – 3.15 (m, 2H), 3.36 – 3.44 (m, 6H), 3.50 – 3.55 (m, 3H), 3.69 – 3.74 (m, 3H), 3.91 – 3.96 (m, 2H), 4.00 (s, 3H), 4.12 – 4.17 (m 2H), 4.24 (d, J = 2.1 Hz, 1H), 4.38 – 4.42 (m, 1H), 4.59 (d, J = 2.2 Hz, 1H), 5.00 – 5.10 (m, 1H), 5.23 – 5.28 (m, 1H), 5.37 (s, 1H), 7.64 – 7.69 (m, 1H), 7.93 (d, J = 4.8 Hz, 2H), 8.23 – 8.34 (m, 1H).2H were exchanged. ES+( M+1 = 768.40), HPLC purity: 90.0%. Example 37: Synthesis of Compound P33
Figure imgf000204_0002
Step 1 - Intermediate Compound A-121: (3R,3aS,6R,6aS)-hexahydrofuro[3,2-b]furan-3,6-diyl bis(trifluoromethanesulfonate)
Figure imgf000205_0001
To a 5 L flask, trifluoromethanesulfonic anhydride (93 g, 328.45 mmol) was added dropwise to (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol (“isomannide”, CAS No.641-74-7; 20 g, 136.85 mmol) and Pyridine (26.0 g, 328.45 mmol) in DCM (600 mL) at 0°C under nitrogen. The resulting mixture was stirred at rt for 16 hours. The reaction mixture was acidified with 2M HCl adjusted to pH=3. The reaction mixture was diluted with DCM (100 mL) and washed sequentially with water (500 mL x 3) and saturated brine (500 mL x 1). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound A-121 (55.0 g, 98 %) as a yellow solid.
Figure imgf000205_0002
NMR (500 MHz, CDCl3) δ 4.16 (d, J = 5.5 Hz, 4H), 4.76 – 4.80 (m, 2H), 5.19 – 5.27 (m, 2H). Step 2, 3 - Intermediate Compound A-122: ((3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6- diyl)dimethanamine
Figure imgf000205_0003
In a 150 mL round bottom flask, was added to Intermediate Compound A-121 (4.6 g, 11.21 mmol) and 18-Crown-6 (6.22 g, 23.55 mmol) in THF (10 mL) at 0°C under N2. To the reaction mixture was added KCN (1.533 g, 23.55 mmol). The resulting mixture was stirred at rt for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 70% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford crude product Intermediate Compound A-121-A, and used in the next step directly without further purification. In a 150 mL round bottom flask was added Intermediate Compound A-121-A from previous reaction and dissolved in THF (10 mL). To the reaction mixture was added borane-methyl sulfide complex in THF (22.43 mL, 224.25 mmol) at 0°C under air. The resulting mixture was stirred at rt for 16 hours. The reaction mixture was quenched with MeOH (20 mL). The solvent was removed under reduced pressure. The product was purified by using protecting group then deprotecting it. The reaction mixture was filtered through celite. The solvent was removed under reduced pressure to afford crude Intermediate Compound A-122 (120 mg, 77 %) as a pale yellow oil.1H NMR (400 MHz, MeOD) δ 2.34 – 2.46 (m, 2H), 2.67 – 2.93 (m, 4H), 3.61 – 3.69 (m, 2H), 4.01 – 4.05 (m, 2H), 4.40 – 4.50 (m, 2H). ES+(M+1) = 173. Step 6 - Compound P33: (2S,4S)-N-(((3S,3aR,6S,6aR)-6-(aminomethyl)-hexahy-drofuro[3,2-b]furan- 3-yl)methyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,-9aR,-10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':4,5]oxazolo-[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamide
Figure imgf000206_0001
To a 50 mL round bottom flask, Intermediate Compound A-122 (82 mg, 0.48 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 100 mg, 0.16 mmol), HATU (121 mg, 0.32 mmol) and DIEA (0.139 mL, 0.80 mmol) in DMA (2 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by preparative HPLC (Column: Xbridge Prep C18 OBD, 30*150 mm, 5μm; Mobile Phase A: Water(10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 22% B to 43% B in 8 min; Wavelength: 254nm/220nm; RT1(min): 6.98) and MeCN as eluents. Fractions containing the desired compound were freeze dried directly to dryness to afford Compound P33 (7.00 mg, 5.62 %) as a red solid. 1H NMR (400 MHz, CDCl3) δ 1.40 (d, J = 6.4 Hz, 3H), 1.72 – 1.79 (m, 2H), 2.00 – 2.06 (m, 1H), 2.34 – 2.55 (m, 5H), 2.72 – 2.88 (m, 4H), 3.15 – 3.28 (m, 3H), 3.35 – 3.43 (m, 3H), 3.56 – 3.72 (m, 4H), 3.91 – 4.07 (m, 5H), 4.11 (d, J = 7.5 Hz, 4H), 4.43 (s, 1H), 4.49 (d, J = 2.0 Hz, 1H), 4.59 (s, 1H), 4.73 (d, J = 2.0 Hz, 1H), 5.12 (s, 1H), 5.31 (s, 1H), 5.50 (t, J = 5.6 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.47 (s, 1H), 7.79 (t, J = 8.1 Hz, 1H), 7.95 – 8.07 (m, 1H), 13.91 (s, 1H). ES+(M+1) = 782.
Example 38: Synthesis of Compound P34
Figure imgf000207_0001
Step 1 - Intermediate Compound A-123: Dibenzyl (((3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6- diyl)bis(methylene))dicarbamate
Figure imgf000207_0002
In a round bottom flask, N-(Benzyloxycarbonyloxy)succinimide (5.59 g, 22.43 mmol) was added to DIEA (5.88 mL, 33.64 mmol) and Intermediate Compound A-122 (1.9 g, 11 mmol) in THF (10 mL) at 25°C under air. The resulting mixture was stirred at rt for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 5 to 100% water (0.1% FA) in MeCN. Pure fractions were evaporated to dryness to afford Intermediate Compound A-123 (0.400 g, 8.10 %) as a yellow solid. 1H NMR (400 MHz, DMSO) δ 2.17 – 2.31 (m, 2H), 2.81 – 3.08 (m, 4H), 3.41 – 3.51 (m, 2H), 3.68 – 3.79 (m, 2H), 4.28 (s, 2H), 5.02 (s, 4H), 7.27 – 7.42 (m, 10H), 7.46 (t, J = 5.8 Hz, 2H). ES+( M+1 = 441.15). Step 2 - Intermediate Compound A-124: Benzyl (((3S,3aR,6S,6aR)-6- (aminomethyl)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamate
Figure imgf000207_0003
In a round bottom flask, Intermediate Compound A-123 (8 g, 18.16 mmol) was added to dioxane (30 mL)/ as stirred at 25 °C for 5 hours. The solvent was removed by blowing N2 gently. The crude product was purified by flash C18- flash chromatography, elution gradient 0 to 100% MeCN in water (0.1%NH4HCO3). Pure fractions were evaporated to dryness to afford Intermediate Compound A-124 (1.800 g, 32.4 %) as a pale- yellow oil.1H NMR (400 MHz, DMSO) δ 1.71 – 1.82 (m, 2H), 2.15 (s, 1H), 2.25 (s, 1H), 2.41 - 2.49 (m, 1H), 2.54 – 2.68 (m, 1H), 2.84 - 3.04 (m, 2H), 3.43 - 3.55 (m, 2H), 3.67 – 3.90 (m, 4H), 5.02 (s, 2H), 7.27 - 7.41 (m, 5H), 7.46 (d, J = 5.9 Hz, 1H). ES+(M+1 =307) HPLC purity: 90%. Step 3 - Intermediate Compound A-125: Benzyl (((3S,3aR,6S,6aR)-6- ((methylamino)methyl)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamate
Figure imgf000208_0001
In a round bottom flask, methyl trifluoromethanesulfonate (0.321 g, 1.96 mmol) was added to Intermediate Compound A-124 (1g, 3.26 mmol) in HFIP (10 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched by a solution of 1 N HCl and then the solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.1%NH4HCO3). Pure fractions were evaporated to dryness to afford Intermediate Compound A- 125 (0.210 g, 20.08 %) as a pale yellow oil.1H NMR (400 MHz, DMSO) δ 1.74 – 1.79 (m, 1H), 2.22 (s, 1H), 2.26 (s, 3H), 2.28 – 2.37 (m, 1H), 2.38 - 2.48 (m, 1H), 2.84 - 3.04 (m, 2H), 3.43 – 3.66 (m, 3H), 3.69 - 3.83 (m, 2H), 4.21 – 4.41 (m, 2H), 5.02 (d, J = 5.2 Hz, 2H), 7.27 - 7.41 (m, 5H), 7.44 (t, J = 5.9 Hz, 1H). ES+(M+1 =321) HPLC purity: 90%. Step 4 - Intermediate Compound A-126: 1-((3S,3aR,6S,6aR)-6-(aminomethyl)hexahydrofuro[3,2- b]furan-3-yl)-N-methylmethanamine
Figure imgf000208_0002
In a round bottom flask, Intermediate Compound A-125 (200 mg, 0.62 mmol) was added to dioxane (1 mL) / HCl(12N) (5.00 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 5 hours. The solvent was removed under reduced pressure to afford crude Intermediate Compound A126 (150 mg 93 %) as a yellow solid 1H NMR (400 MHz DMSO) δ 2.45 (d, J = 6.5 Hz, 2H), 2.66 – 2.76 (m, 2H), 2.80 - 2.94 (m, 4H), 3.64 - 3.71 (m, 3H), 3.83 – 4.00 (m, 4H), 4.45 – 4.55 (m, 3H), 8.16 - 8.31 (m, 5H). ES+(M+1 =187) HPLC purity: 80%. Step 5 - Compound P34: (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9- methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N- (((3S,3aR,6S,6aR)-6-((methylamino)methyl)hexahydrofuro[3,2-b]furan-3-yl)methyl)-6,11-dioxo- 1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
Figure imgf000209_0001
In a round bottom flask, Intermediate Compound A-126 (89 mg, 0.48 mmol) was added to PNU- 159682 carboxylic acid (CAS No.1204819-92-0, 150 mg, 0.24 mmol), HATU (182 mg, 0.48 mmol) and DIEA (0.209 mL, 1.20 mmol) in DMSO (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The crude product was purified by preparative HPLC (Column: Kinetex EVO C18 Column, 25*250mm, 5um; Mobile Phase A: Water (10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 30% B to 38% B in 15 min; Wavelength: 254nm/220nm; RT1(min): 12.62) and MeCN as eluents. Fractions containing the desired compound were freeze dried directly to dryness to afford Compound P34 as a red solid (28 mg, 13%) 1H NMR (400 MHz, DMSO) δ 1.22 (s, 3H), 1.60 – 1.80 (m, 2H), 2.13 – 2.18 (m, 1H), 2.23 – 2.35 (m, 1H), 2.40 (s, 1H), 2.55 – 2.59 (m, 2H), 2.66 – 2.72 (m, 1H), 2.81 – 3.23 (m, 6H), 3.32 (s, 3H), 3.51 – 3.61 (m, 3H), 3.68- 3.78 (m, 2H), 3.80 – 3.94 (m, 2H), 3.96 (s, 1H), 4.00 (s, 3H), 4.15 (d, J = 6.8 Hz, 1H), 4.24 (s, 1H), 4.31 (d, J = 4.8 Hz, 1H), 4.44 (d, J = 5.2 Hz, 1H), 4.59 (d, J = 2.0 Hz, 1H), 5.03 (d, J = 5.2 Hz, 1H), 5.26 (d, J = 4.8 Hz, 1H), 5.40 (s, 1H), 7.68 (t, J= 4.8 Hz, 1H), 7.94 (d, J = 4.8 Hz, 2H), 8.30 (t, J = 6.0 Hz, 1H), 13.28 (s, 1H), 14.08 (s, 1H). ES+(M+1 =796) HPLC purity: 79%. LINKER PAYLOAD SYNTHESIS General procedure for Linker Payload synthesis To an appropriate size round bottom flask, a Linker (1.0 eq) is added in anhydrous DMF and cooled to 0^ C. To the solution is added HATU (1.0 eq), and DIPEA (3 eq) followed by a Payload (0.95 eq). The reaction mixture is warmed to room temperature and stirred for 1 hr. LCMS is used to confirm formation of the desired Linker-Payload. The reaction mixture is diluted with acetonitrile (1 mL) and purified via reverse phase purification by using acetonitrile and water (2 mmol HCOONH4) as mobile phase. Obtained fractions are lyophilized to afford the Linker-Payload. Protection and de-protection steps are utilized as necessary. The linker may be loaded onto a resin prior to the coupling with the payload, followed by cleavage of the resin after coupling. Example 39: Synthesis of Linker-Payload Compound LP1
Figure imgf000210_0001
Synthesis of Linker Compound L1: (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoyl)glycylglycylglycylglycylglycine Resin loading: A peptide synthesizer glass vessel (Chemglass, 250-mL) adapted with an N2 (g) flow inlet and a medium size frit filter was charged with 2-chlorotrityl chloride resin (1.4 g, 1.54 mmol, 1.1 mmol/g commercial loading capacity, Chem-Impex International). The resin was swollen by agitation with a flow of N2(g) for 1.5 hours with 20 mL of anhydrous DMF and for 30 minutes with 27 mL of DMF/DCM (3:1). N-Fmoc-Gly-Gly-Gly-Gly-OH (1.36 eq., 0.98 g, 2.09 mmol) was dissolved in 31 mL of anhydrous EtOAC/DMSO/DMF (20:6:5) and 1.88 mL of DIPEA (1.39 g, 7 eq, 10.78 mmol). The reaction was run for 15 hours and the resin was washed with DMF (4 x 5 min x 25 mL) and DCM (4 x 5 min x 25 mL). Resin capping: The resin was capped with 15 mL of 17:2:1 DCM:MeOH:DIPEA solution (2 x 10 min x 15 mL) and then washed with DCM (3 x 3 min x 12 mL) followed by DMF (3 x 3 min x 15 mL). Fmoc-removal: The Fmoc-removal was performed using in 1:4 piperidine/DMF solution (1 x 10 min x 15 mL and 1 x 5 min x 15 mL). Coupling: The resin was washed with DMF (5 x 5 min x 15 mL) and coupled to N-Fmoc-Gly-OH, assuming resin loading of 1.1 mmol/g. The coupling cocktail containing N-Fmoc-Gly-OH (2.29 g, 5 eq, 7.7 mmol), HOBt (1.18 g, 5 eq, 7.7 mmol), HATU (2.87 g, 4.9 eq, 7.55 mmol) and DIPEA (1.61 mL, 6 eq, 9.24 mmol) dissolved in 12 mL of DMF was pre-activated for 5 mins prior to addition to the resin. The coupling reaction was run for 30 min and the completion of the reaction was monitored using Ninhydrin stain. Upon completion, the coupling solution was drained and the resin-immobilized Fmoc-Gly-Gly-Gly-Gly-Gly-OH (Intermediate compound 93) was washed with DMF (5 x 5 min x 15 mL). Fmoc Removal: Resin-immobalized Fmoc-Gly-Gly-Gly-Gly-Gly-OH (Intermediate compound 93) was subjected to Fmoc-removal. The Fmoc-removal was performed using 1:4 piperidine/DMF solution (2 x 5 min 15 mL). The resin was washed with DMF (5 x 3 min x 15 mL) prior to coupling to the next carboxylic acid. Coupling and resin cleavage: The resin (0.49 mmol, assuming 1.1 mmol/g loading capacity) was coupled to 3-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)propanoic acid. The coupling cocktail containing 3-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)propanoic acid (174 mg, 2.1 eq, 1.03 mmol), HOBt (278 mg, 3.7 eq, 1.81 mmol), HATU (373 mg, 2 eq, 0.98 mmol) and DIPEA (0.342 mL, 4 eq, 1.96 mmol) dissolved in 3.5 mL of DMF was pre-activated for 5 mins prior to addition to the resin. The coupling reaction was run for 30 min and the completion of the reaction was monitored using Ninhydrin stain. Upon completion, the coupling solution was drained and the resin was washed with DMF (5 x 5 min x 8 mL) and DCM (5 x 3 min x 8 mL). Cleavage of the peptide from the resin was carried out with a 4:1 solution of DCM/HFIP (2 x 10 min x 10 mL) and the resin was washed with DCM (3 x 3 min x 5 mL). The solvent was removed in vacuo to give Linker Compound L1 as a white solid (50 mg, 0.110 mmol) in 22% yield (calculated from based on the commercial solid phase loading capacity). ES [M-H]- 453.3 m/z. Synthesis of Linker-Payload LP1: To a 25 ml round bottom flask, Linker Compound L1 (50 mg, 1.0 eq) was added in anhydrous DMF (1 mL) and cooled to 0^ C. To the solution was added HATU (42 mg, 1.0 eq), and DIPEA (3 eq) followed by Compound P20 (80 mg, 0.95 eq). The reaction mixture was warmed to room temperature and stirred for 1 hrs. LCMS showed formation of desired product. The reaction mixture was diluted with acetonitrile (1 mL) and purified via reverse phase purification by using acetonitrile and water (2 mmol HCOONH4) as mobile phase. Obtained fractions were lyophilized to afford Linker-Payload Compound LP1 as a red solid (20 mg, 15% yield) ES [M+H]+ : 1191.1 m/z. Example 40: Synthesis of Linker-Payload LP2
Figure imgf000212_0001
To a 25 ml round bottom flask, Linker Compound L1 (50 mg, 1.0 eq) was added in anhydrous DMF (1 mL) and cooled to 0^ C. To the solution was added HATU (42 mg, 1.0 eq), and DIPEA (3 eq) followed by Compound P8 (77 mg, 0.95 eq). The reaction mixture was warmed to room temperature and stirred for 1 hrs. LCMS showed formation of desired product. The reaction mixture was diluted with acetonitrile (1 mL) and purified via reverse phase purification by using acetonitrile and water (2 mmol HCOONH4) as mobile phase. Obtained fractions were lyophilized to afford Linker-Payload Compound LP2 as a red solid (31 mg, 24% yield) ES [M+H]+ : 1172.08 m/z. Example 41: Synthesis of Linker-Payload LP6
Figure imgf000213_0001
Synthesis of Intermediate compound 104: (9H-fluoren-9-yl)methyl ((S)-1-((2-(((3S,3aR,6S,6aR)-6- ((tert-butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)amino)-1-oxo-3- phenylpropan-2-yl)carbamate
Figure imgf000213_0002
To a 500 mL round bottom flask, chloro-N,N,N’,N’-tetramethylformamidinium hexafluorophosphate (15.15 g, 53.99 mmol) was added to (((9H-fluoren-9-yl)methoxy)carbonyl)-L- phenylalanylglycine (20 g, 45.00 mmol), (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine (Intermediate Compound 52, 9.73 g, 67.49 mmol) and 1-methylimidazole (7.39 g, 89.99 mmol) in DMA (250 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 3 hours. The reaction mixture was diluted with EtOAc (500 mL) and washed sequentially with water (200 mL x 2). (Boc)2O (20.89 mL, 89.99 mmol) was add to the organic layer. The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure. The reaction mixture was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 104 (12.00 g, 39.8 % for 2 steps) as a yellow solid. ES M+1 = 671.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.71 – 2.85 (m, 1H), 2.99 – 3.08 (m, 1H), 3.55 – 3.64 (m, 2H), 3.65 – 3.81 (m, 2H), 3.81 – 3.89 (m, 3H), 3.98 – 4.07 (m, 1H), 4.07 – 4.20 (m, 4H), 4.20 – 4.28 (m, 1H), 4.38 (d, J = 4.4 Hz, 1H), 4.43 (d, J = 4.4 Hz, 1H), 7.22 – 7.35 (m, 7H), 7.37 – 7.46 (m, 2H), 7.61 – 7.73 (m, 3H), 7.88 (d, J = 7.6 Hz, 2H), 8.08 (d, J = 7.0 Hz, 1H), 8.26 – 8.33 (m, 1H). Synthesis of Intermediate compound 105: tert-butyl ((3S,3aR,6S,6aR)-6-(2-((S)-2-amino-3- phenylpropanamido)acetamido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000214_0001
To a 500 mL round bottom flask, dimethylamine (35 mL, 178.90 mmol) was added to Intermediate Compound 104 (12 g, 17.89 mmol) in DCM (100 mL)/THF (50 mL) under nitrogen. The resulting mixture was stirred at RT for 1 hour. The solvent was removed under reduced pressure to afford Intermediate Compound 105 (7.00 g, 87%) as a yellow oil. No further purification was carried out as sufficiently clean for next steps. ES M+1 = 449.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.55 – 2.64 (m, 1H), 2.93 – 3.03 (m, 1H), 3.41 – 3.50 (m, 1H), 3.56 – 3.63 (m, 2H), 3.71 (d, J = 5.2 Hz, 2H), 3.80 – 3.89 (m, 3H), 4.10 (s, 1H), 4.36 (d, J = 4.0 Hz, 1H), 4.42 (d, J = 4.0 Hz, 1H), 7.23 – 7.27 (m, 3H), 7.35 (d, J = 1.2 Hz, 1H), 7.42 (d, J = 1.2 Hz, 1H), 7.82 – 7.93 (m, 4H), 8.16 – 8.20 (m, 1H). Synthesis of Intermediate compound 106: tert-butyl ((3S,3aR,6S,6aR)-6-((S)-11-benzyl-1-(9H- fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-amido)hexahydrofuro[3,2- b]furan-3-yl)carbamate
Figure imgf000214_0002
To a 500 mL round bottom flask, Intermediate Compound 105 (7 g, 15.61 mmol) was added to (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (8.30 g, 23.41 mmol), HATU (8.90 g, 23.41 mmol) and DIEA (8.18 mL, 46.82 mmol) in DMA (20 mL)/DCM (100 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate Compound 106 (9.50 g, 78 %) as a yellow solid. ES+(M+1 = 785).1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 2.99 – 3.08 (m, 1H), 3.09 – 3.20 (m, 1H), 3.58 – 3.66 (m, 6H), 3.69 – 3.77 (m, 2H), 3.81 – 3.87 (m, 3H), 4.08 (d, J = 6.6 Hz, 1H), 4.16 – 4.33 (m, 3H), 4.36 – 4.45 (m, 2H), 4.45 – 4.53 (m, 1H), 7.25 (d, J = 6.0 Hz, 4H), 7.28 – 7.37 (m, 2H), 7.38 – 7.46 (m, 2H), 7.56 – 7.64 (m, 1H), 7.72 (d, J = 7.6 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 8.00 – 8.08 (m, 2H), 8.17 (d, J = 8.0 Hz, 1H), 8.25 – 8.32 (m, 1H). Synthesis of Intermediate compound 107: tert-butyl ((3S,3aR,6S,6aR)-6-(2-((S)-2-(2-(2- aminoacetamido)acetamido)-3-phenylpropanamido)acetamido)-hexahydrofuro[3,2-b]furan-3- yl)carbamate
Figure imgf000215_0001
To a 500 mL round bottom flask, dimethylamine (40 mL, 121.04 mmol) was added to Intermediate Compound 106 (9.5 g, 12.10 mmol) in THF (40 mL)/DCM (120 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 1 hour. The solvent was removed under reduced pressure to afford Intermediate Compound 107 (6.00 g, 88 %) as a yellow solid. No further purification was carried out as sufficiently clean for next steps. ES+ M+1 = 563.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.73 – 2.83 (m, 1H), 3.08 (s, 2H), 3.30 (s, 2H), 3.58 – 3.64 (m, 3H), 3.64 – 3.69 (m, 2H), 3.81 – 3.88 (m, 4H), 4.09 (d, J = 6.8 Hz, 1H), 4.34 – 4.47 (m, 2H), 7.26 (d, J = 6.0 Hz, 5H), 7.82 – 7.92 (m, 1H), 8.07 (d, J = 7.2 Hz, 1H), 8.23 (s, 1H), 8.26 – 8.37 (m, 2H). Synthesis of Intermediate compound 108: tert-butyl ((3S,3aR,6S,6aR)-6-((S)-5-benzyl-15-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)-4,7,10,13-tetraoxo-3,6,9,12- tetraazapentadecanamido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000215_0002
To a 500 mL round bottom flask, DIEA (3.73 mL, 21.33 mmol) was added to Intermediate compound 107 (6 g, 10.66 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)propanoate (3.41 g, 12.80 mmol) in DMA (40 mL)/MeCN (40.0 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. Solvent was removed under reduced pressure.The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford Intermediate compound 108 (4.50 g, 59.1 %) as a yellow solid. ES+M+1 = 714.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.38 – 2.46 (m, 2H), 2.75 – 2.85 (m, 1H), 2.99 – 3.08 (m, 1H), 3.56 – 3.63 (m, 5H), 3.66 (d, J = 5.6 Hz, 3H), 3.70 – 3.78 (m, 2H), 3.81 – 3.87 (m, 3H), 4.09 (d, J = 7.2 Hz, 1H), 4.37 (d, J = 4.0 Hz, 1H), 4.42 (d, J = 4.0 Hz, 1H), 4.44 – 4.51 (m, 1H), 7.00 (s, 2H), 7.16 – 7.22 (m, 1H), 7.22 – 7.29 (m, 5H), 8.01 – 8.09 (m, 2H), 8.09 – 8.20 (m, 1H), 8.22 – 8.33 (m, 2H). Synthesis of Intermediate compound 109: (S)-N-(2-(((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2- b]furan-3-yl)amino)-2-oxoethyl)-2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamido)acetamido)-acetamido)-3-phenylpropanamide
Figure imgf000216_0001
To a 500 mL round bottom flask, 12M HCl (25 mL, 100.00 mmol) was added to Intermediate compound 108 (4.5 g, 6.30 mmol) in 4M HCl- EtOH (60 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.1% NH4HCO3). Pure fractions were evaporated to dryness to afford Intermediate compound 109 (3.50 g, 90 %) as a yellow solid. ES+ M+1 = 6141H NMR (400 MHz, DMSO) δ 2.36 – 2.46 (m, 2H), 2.78 – 2.90 (m, 1H), 2.99 – 3.10 (m, 1H), 3.50 – 3.71 (m, 8H), 3.71 – 3.81 (m, 2H), 3.82 – 3.98 (m, 3H), 4.12 (s, 1H), 4.37 – 4.47 (m, 1H), 4.61 (d, J = 4.4 Hz, 1H), 4.69 (d, J = 4.4 Hz, 1H), 7.13 – 7.22 (m, 1H), 7.25 (d, J = 4.0 Hz, 4H), 8.10 – 8.18 (m, 1H), 8.19 – 8.26 (m, 2H), 8.32 – 8.41 (m, 2H), 8.60 – 8.66 (m, 2H). Synthesis of Compound LP6 : (2S,4S)-N-((3S,3aR,6S,6aR)-6-((S)-5-benzyl-15-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazapentadecanamido)hexahydrofuro-[3,2-b]furan- 3-yl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4’3’:45]oxazolo[23c][14]oxazin3yl)oxy)611dioxo1234611hexahydrotetracene-2- carboxa
Figure imgf000217_0001
To a 250 mL round bottom flask, HATU (1.091 g, 2.87 mmol) was added to PNU-159682 carboxylic acid (CAS No.1204819-92-0; 1.5 g, 2.39 mmol) in DMA (40 mL) under nitrogen. The resulting mixture 1 was stirred at 15 °C for 10 minutes. DIEA (2.087 mL, 11.95 mmol) was added to Intermediate compound 109 (1.467 g, 2.39 mmol) in DMSO (20.00 mL) under nitrogen. The resulting mixture 2 was stirred at 15 °C for 10 minutes. After that, the mixture 2 was added to the mixture 1, then stirring at 15 °C for 2 hours. The reaction mixture was quenched with 10 mM NH4Cl (12 mL). The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford crude product (2.3 g, crude). The crude product was purified by preparative HPLC Column: XBridge Prep Shield RP18 OBD Column, 19*250 mm, 5μm; Mobile Phase A: Water, Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: isocratic 15% B in 2 min, 30% B to 40% B in 20 min; Wave Length: 254nm/220nm; RT1(min): 19.2. Fractions containing the desired compound were freeze dried directly to dryness to afford Linker-Payload Compound LP6 (0.640 g, 21.89 %) as a red solid. ES+(M+1 = 1223), 1H NMR (400 MHz, DMSO) δ 1.20 (d, J = 6.4 Hz, 3H), 1.62 – 1.74 (m, 2H), 2.10 – 2.17 (m, 1H), 2.24 – 2.33 (m, 1H), 2.37 – 2.46 (m, 2H), 2.59 – 2.74 (m, 2H), 2.75 – 2.85 (m, 1H), 2.92 – 3.08 (m, 3H), 3.27 – 3.33 (m, 3H), 3.47 – 3.81 (m, 13H), 3.85 – 3.97 (m, 3H), 4.00 (s, 3H), 4.10 – 4.19 (m, 3H), 4.23 (d, J = 2.0 Hz, 1H), 4.43 – 4.54 (m, 3H), 4.59 (d, J = 2.0 Hz, 1H), 4.98 – 5.04 (m, 1H), 5.22 – 5.32 (m, 1H), 5.38 (s, 1H), 6.99 (s, 2H), 7.16 – 7.21 (m, 1H), 7.26 (d, J = 5.5 Hz, 4H), 7.64 – 7.70 (m, 1H), 7.93 (d, J = 4.8 Hz, 2H), 8.03 – 8.17 (m, 4H), 8.22 – 8.33 (m, 2H), 13.26 (s, 1H), 14.07 (s, 1H).
Example 42: Synthesis of Compound LP3
Figure imgf000218_0001
Step 1 - Intermediate Compound 110: tert-butyl ((3S,3aR,6S,6aR)-6-(1-(9H-fluoren-9-yl)-3,6,9- trioxo-2-oxa-4,7,10-triazadodecan-12-amido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000218_0002
In a 500 mL round bottom flask, a mixture of (((9H-fluoren-9- yl)methoxy)carbonyl)glycylglycylglycine (11.2 g, 27.22 mmol) was added to tert-butyl ((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)carbamate (9.98 g, 40.83 mmol), O-(7- Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15.53 g, 40.83 mmol) and N,N-diisopropylethylamine (10.56 g, 81.67 mmol) in DMA (300 mL) was stirred at 25 °C for 3 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water to afford Intermediate Compound 110 (9.20 g, 53.0 %) as a white solid.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 3.54 – 3.62 (m, 2H), 3.68 (dd, J = 5.9, 10.6 Hz, 3H), 3.73 (d, J = 5.6 Hz, 2H), 3.80 – 3.88 (m, 3H), 4.08 (s, 1H), 4.19 – 4.27 (m, 1H), 4.30 (d, J = 6.9 Hz, 2H), 4.36 (d, J = 4.2 Hz, 1H), 4.42 (d, J = 4.1 Hz, 1H), 7.23 (s, 1H), 7.30 – 7.37 (m, 2H), 7.39 – 7.46 (m, 2H), 7.58 (t, J = 6.0 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.04 – 8.14 (m, 2H), 8.16 – 8.22 (m, 1H). ES+(M+1 = 638.35). Step 2 & 3 - Intermediate Compound 112: tert-butyl ((3S,3aR,6S,6aR)-6-(1-(9H-fluoren-9-yl)- 3,30,33,36-tetraoxo-2,7,10,13,16,19,22,25,28-nonaoxa-4,31,34,37-tetraazanonatriacontan-39- amido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000219_0001
In a 500 mL round bottom flask, Intermediate Compound 110 (9.2 g, 14.43 mmol) was added to diethylamine (31.7 g, 432.81 mmol) in THF (500 mL) and water (10 mL). The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure to get crude product (Int 111). The crude product was washed with MTBE (3 x 100 mL) and dried with nitrogen flow. The above crude product was added to O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.23 g, 21.64 mmol), DIEA (10.08 mL, 57.71 mmol) and 1-(9H-fluoren-9-yl)-3- oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatriacontan-30-oic acid (18.75 g, 28.85 mmol) in DMA (100 mL). The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water to afford Intermediate Compound 112 (7.30 g, 48.3 %).1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 3.38 – 3.43 (m, 2H), 3.50 (br, 24H), 3.52 (s, 4H), 3.58 (s, 2H), 3.67 – 3.74 (m, 4H), 3.77 – 3.87 (m, 4H), 3.93 (s, 2H), 4.05 – 4.14 (m, 2H), 4.21 (t, J = 6.8 Hz, 1H), 4.29 (d, J = 6.9 Hz, 3H), 4.34 – 4.43 (m, 2H), 7.23 (s, 1H), 7.42 (t, J = 7.4 Hz, 3H), 7.70 (d, J = 7.5 Hz, 3H), 7.90 (d, J = 7.3 Hz, 4H), 8.02 – 8.13 (m, 2H), 8.24 (t, J = 5.7 Hz, 1H). ES+( M+1 = 1047.65). Step 4 & 5 - Intermediate Compound 114: tert-butyl ((3S,3aR,6S,6aR)-6-(39-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-4,7,10,37-tetraoxo-12,15,18,21,24,27,30,33-octaoxa-3,6,9,36- tetraazanonatriacontanamido)hexahydrofuro-[3,2-b]furan-3-yl)carbamate
Figure imgf000219_0002
In a 500 mL round bottom flask, Intermediate Compound 112 (7.3 g, 6.97 mmol) was added to diethylamine (20 ml, 193.33 mmol) in THF (80 mL) at 0°C. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure. The reaction mixture was diluted with DCM (150 mL). The solvent was removed under reduced pressure. The above crude product (Int 113) was added to 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (2.60 g, 9.76 mmol) in THF (200 mL) at 0°C. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 70% MeCN in water(0.1%FA). Pure fractions were evaporated to dryness to afford Intermediate Compound 114 (5.00 g, 73.5 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.33 (t, J = 7.3 Hz, 2H), 3.11 – 3.17 (m, 2H), 3.47 – 3.55 (m, 22H), 3.56 – 3.65 (m, 7H), 3.67 – 3.74 (m, 4H), 3.78 – 3.87 (m, 5H), 3.94 (s, 2H), 4.08 (s, 1H), 4.34 – 4.42 (m, 2H), 5.77 (s, 1H), 7.01 (s, 1H), 7.23 (s, 1H), 7.90 (t, J = 5.9 Hz, 1H), 8.00 – 8.13 (m, 3H), 8.24 (t, J = 5.6 Hz, 1H). ES+( M+1 = 976.60). Step 6 & 7 - Compound LP3: (2S,4S)-N-((3S,3aR,6S,6aR)-6-(39-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)-4,7,10,37-tetraoxo-12,15,18,21,24,27,30,33-octaoxa-3,6,9,36- tetraazanonatriacontanamido)hexahydrofuro[3,2-b]furan-3-yl)-2,5,12-trihydroxy-7-methoxy-4- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano-[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
Figure imgf000220_0001
In a 500 mL round bottom flask, Intermediate Compound 114 (3.08 g, 3.15 mmol) was added to hydrochloric acid solution (20 ml, 80.00 mmol) (4mol/L in EtOH) at 0°C. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with DCM (30 mL) and removed under reduced pressure to give crude Intermediate Compound 115. The crude Intermediate Compound 115 was added to the PNU-159682 carboxylic acid (CAS No.: 1204819-92-0; 1.1 g, 1.75 mmol), O-(7- Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoro-phosphate (1.000 g, 2.63 mmol) and N,N-diisopropylethylamine (0.680 g, 5.26 mmol) in DMA (30 mL) at 0°C under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was quenched with saturated NH4Cl 10mM in water (8mL). The crude product was purified by flash C18-flash chromatography and preparative HPLC elution gradient 0 to 80% MeCN in water to afford Compound LP3 (0.530 g, 20.36 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.20 (d, J = 6.4 Hz, 3H), 1.68 (br, 2H), 2.15 (d, J = 14.0 Hz, 1H), 2.25 – 2.37 (m, 2H), 2.60 – 2.73 (m, 1H), 2.92 – 3.06 (m, 2H), 3.10 – 3.19 (m, 2H), 3.31 (s, 4H), 3.46 – 3.54 (m, 26H), 3.55 – 3.64 (m, 8H), 3.65 – 3.75 (m, 6H), 3.80 (d, J = 5.8 Hz, 2H), 3.85 – 3.96 (m, 5H), 4.00 (s, 3H), 4.08 – 4.19 (m, 3H), 4.23 (d, J = 2.2 Hz, 1H), 4.41 – 4.54 (m, 2H), 4.58 (d, J = 2.2 Hz, 1H), 4.98 – 5.09 (m, 1H), 5.19 – 5.30 (m, 1H), 5.37 (s, 1H), 7.00 (s, 2H), 7.62 – 7.72 (m, 1H), 7.87 – 7.95 (m, 3H), 8.01 (t, J = 5.5 Hz, 1H), 8.05 – 8.16 (m, 3H), 8.23 (t, J = 57 Hz 1H) 1325 (s 1H) 1406 (s 1H) ES+(M+1 = 148595) Example 43: Synthesis of Compound LP4
Figure imgf000221_0001
Step 1 - Intermediate Compound 111-A: tert-butyl ((3S,3aR,6S,6aR)-6-(1-(9H-fluoren-9-yl)- 3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaoctadecan-18-amido)hexahydrofuro[3,2- b]furan-3-yl)carbamate
Figure imgf000221_0002
In a 250 mL round bottom flask, tert-butyl ((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3- yl)carbamate (2.301 g, 9.42 mmol) was added to (((9H-fluoren-9- yl)methoxy)carbonyl)glycylglycylglycylglycyl-glycine CAS No. : 132742-00-8 (3.3 g, 6.28 mmol), HATU (4.78 g, 12.56 mmol) and DIEA (3.29 mL, 18.84 mmol) in DMSO (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in neutral water. Pure fractions were evaporated to dryness to afford Intermediate Compound 111-A (4.10 g, 87 %) as a pale yellow solid. ES+(M+1) = 752.
Figure imgf000222_0001
NMR (400 MHz, DMSO) δ 1.39 (d, J = 4.2 Hz, 9H), 3.55 – 3.61 (m, 2H), 3.63 – 3.88 (m, 15H), 4.08 (s, 1H), 4.19 – 4.33 (m, 3H), 7.24 (s, 1H), 7.34 (t, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.59 (t, J = 6.1 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 8.04 – 8.22 (m, 5H). Step 2 - Intermediate Compound 111: tert-butyl ((3S,3aR,6S,6aR)-6-(14-amino-4,7,10,13-tetraoxo- 3,6,9,12-tetraazatetradecanamido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000222_0002
In a 250 mL flask, diethylamine (3.89 g, 53.21 mmol) was added to Intermediate Compound 111-A (4g, 5.32 mmol) in THF (50 mL)/water (10 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure. The dried solid was titurated with MTBE (2 x 100 mL) and filtered to afford crude Intermediate Compound 111 (2.300 g, 82 %) as a pare yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (d, J = 3.1 Hz, 9H), 3.50 – 3.63 (m, 3H), 3.64 – 3.94 (m, 14H), 4.07 (d, J = 6.8 Hz, 1H), 7.25 (d, J = 5.9 Hz, 1H), 8.05 – 8.12 (m, 2H), 8.21 (t, J = 5.8 Hz, 2H). ES+(M+1 = 530), HPLC purity: 92.9%. Step 3 - Intermediate Compound 116: tert-butyl ((3S,3aR,6S,6aR)-6-(1-(9H-fluoren-9-yl)- 3,30,33,36,39,42-hexaoxo-2,7,10,13,16,19,-22,25,28-nonaoxa-4,31,34,37,40,43- hexaazapentatetracontan-45-amido)hexahydrofuro-[3,2-b]furan-3-yl)carbamate
Figure imgf000222_0003
In a 250 mL round flask, Intermediate Compound 111 (1 g, 1.89 mmol) was added to 1-(9H-fluoren- 9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatriacontan-30-oic acid (1.227 g, 1.89 mmol), HATU (1.436 g, 3.78 mmol) and DIEA (0.989 mL, 5.67 mmol) in DMSO (15 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound 116 (1.200 g, 54.7 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 3.08 (q, J = 6.0 Hz, 2H), 3.39 (t, J = 6.1 Hz, 5H), 3.48 – 3.53 (m, 23H), 3.58 (d, J = 2.9 Hz, 2H), 3.60 – 3.64 (m, 3H), 3.68 – 3.87 (m, 14H), 3.94 (s, 2H), 4.08 (d, J = 6.8 Hz, 1H), 4.34 – 4.45 (m, 2H), 7.30 – 7.46 (m, 4H), 7.81 – 7.96 (m, 5H), 8.05 – 8.16 (m, 2H), 8.16 – 8.25 (m, 3H). ES+( M+1 = 1161). Step 4 - Intermediate Compound 117: tert-butyl ((3S,3aR,6S,6aR)-6-(41-amino-4,7,10,13,16- pentaoxo-18,21,24,27,30,33,36,39-octaoxa-3,6,9,12,15- pentaazahentetracontanamido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000223_0001
In a 250 mL round flask, diethylamine (0.630 g, 8.61 mmol) was added to Intermediate Compound 116 (1 g, 0.86 mmol) in MeCN (10 mL)/water (10.00 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure. The dried solid was titurated with MTBE (2 x 50 mL) and filtered to afford crude Intermediate Compound 117 (0.530 g, 65.5 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 3.36 (s, 2H), 3.49 – 3.55 (m, 26H), 3.56 – 3.59 (m, 4H), 3.62 (d, J = 5.6 Hz, 2H), 3.68 – 3.88 (m, 15H), 3.94 (s, 2H), 4.07 (d, J = 6.8 Hz, 1H), 7.24 (d, J = 6.0Hz, 1H), 7.92 (t, J = 6.0 Hz, 1H), 8.10 – 8.25 (m, 4H). ES+(M+1 = 939), HPLC purity: 80.0%. Step 5 - Intermediate Compound 118: tert-butyl ((3S,3aR,6S,6aR)-6-(45-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-4,7,10,13,16,43-hexaoxo-18,21,24,27,30,33,36,39-octaoxa-3,6,9,12,15,42- hexaazapentatetracontanamido)-hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000223_0002
In a 250 mL round bottom flask, 2,5-Dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoate (227 mg, 0.85 mmol) was added to Intermediate Compound 117 (400 mg, 0.43 mmol) in DMSO (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water to dryness to afford Intermediate Compound 118 (400 mg, 86 %) as a pale- yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 3.10 - 3.20 (m, 2H), 3.36 (s, 2H), 3.49 (br, 23H), 3.53 - 3.65 (m, 10H), 3.66 – 3.89 (m, 14H), 3.94 (s, 2H), 4.07 (d, J = 7.1 Hz, 1H), 4.36 (d, J = 4.1 Hz, 1H), 4.42 (d, J = 4.2 Hz, 1H), 7.02 (d, J = 5.9 Hz, 2H), 7.24 (d, J = 5.9 Hz, 1H), 7.91 (t, J = 5.8 Hz, 1H), 8.00 – 8.12 (m, 3H), 8.12 – 8.22 (m, 3H). ES+(M+1 = 1091). Step 6 - Intermediate Compound 119: N-(14-(((3S,3aR,-6S,6aR)-6-aminohexahydrofuro[3,2-b]furan- 3-yl)amino)-2,5,8,11,14-pentaoxo-3,6,9,12-tetraazatetradecyl)-26-(3-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)propanamido)-3,6,9,12,-15,18,21,24-octaoxahexacosanamide
Figure imgf000224_0001
In a 250 mL round bottom flask, Intermediate Compound 118 (380 mg, 0.35 mmol) was added to HCl in EtOH(4N) (2 mL)/H2O(0.01%FA) (0.400 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure and afforded crude Intermediate Compound 119 (300 mg, 87 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 2.34 (t, J = 7.3 Hz, 2H), 2.57 (s, 1H), 3.15 (q, J = 5.6 Hz, 2H), 3.37 (t, J = 5.9 Hz, 2H), 3.49 (br, 21H), 3.56 – 3.67 (m, 9H), 3.68 – 3.88 (m, 12H), 3.90 – 3.98 (m, 4H), 4.10 – 4.16 (m, 1H), 4.59 – 4.73 (m, 2H), 7.00 (s, 2H), 7.99 (t, J = 6.0 Hz, 1H), 8.09 (d, J = 5.7 Hz, 1H), 8.19 - 8.39 (m, 5H). ES+(M+1 = 990). Step 7 - Compound LP4: (2S,4S)-N-((3S,3aR,6S,6aR)-6-(45-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- 4,7,10,13,16,43-hexaoxo-18,21,24,27,30,33,36,39-octaoxa-3,6,9,12,15,42- hexaazapentatetracontanamido)hexahydrofuro[3,2-b]furan-3-yl)-2,5,12-trihydroxy-7-methoxy-4- (((1S,3R,4aS,9S,9aR,-10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide
Figure imgf000224_0002
In a 250 mL round bottom flask, Intermediate Compound 119 (237 mg, 0.24 mmol) and DIEA (0.139 mL, 0.80 mmol) in DMA was added to the PNU-159682 carboxylic acid (CAS No.: 1204819- 92-0; 100 mg, 0.16 mmol) and HATU (121 mg, 0.32 mmol) in DMA (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18- flash chromatography, and preparative HPLC elution gradient 0 to 80% MeCN in water, to afford Compound LP4 (28.0 mg, 10.99 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.20 (d, J = 6.5 Hz, 3H), 1.62 – 1.73 (m, 2H), 2.11 – 2.19 (m, 1H), 2.26 – 2.35 (m, 3H), 2.61 – 2.72 (m, 2H), 2.91 – 3.07 (m, 2H), 3.15 (q, J = 5.8 Hz, 2H), 3.29 (s, 4H), 3.35 – 3.40 (m, 3H), 3.52 (br, 25H), 3.57 – 3.62 (m, 5H), 3.64 - 3.81 (m, 13H), 3.87 – 3.95 (m, 5H), 4.00 (s, 3H), 4.09 – 4.20 (m, 3H), 4.23 (d, J = 2.2 Hz, 1H), 4.46 (d J 43 H 1H) 452 (d J 48 H 1H) 459 (d J 22 Hz, 1H), 5.01 (d, J = 4.9 Hz, 1H), 5.22 – 5.33 (m, 1H), 5.38 (s, 1H), 7.01 (s, 2H), 7.64-7.69 (m, 1H), 7.86 – 7.96 (m, 3H), 8.02 – 8.20 (m, 7H), 13.27 (s, 1H), 14.07 (s, 1H). ES+(M+1 = 1600). Example 44: Synthesis of Compound LP5
Figure imgf000225_0001
Step 1 - Intermediate Compound 120: tert-butyl (1R,4R)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)-L- phenylalanylglycyl)-2,5-diazabicyclo-[2.2.2]octane-2-carboxylate
Figure imgf000225_0002
In a 500 mL round bottom flask, DIEA (6.91 mL, 39.57 mmol) was added to (((9H-fluoren-9- yl)methoxy)carbonyl)-L-phenylalanylglycine (7.03 g, 15.83 mmol), tert-butyl (1R,4R)-2,5- diazabicyclo[2.2.2]-octane-2-carboxylate (2.8 g, 13.19 mmol) and HATU (7.52 g, 19.78 mmol) in THF (50 mL)/DMA (10.00 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 120 (7.50 g, 89 %) as a white solid.1H NMR (400 MHz, DMSO) δ 1.37 –1.44 (m, 9H), 1.68 – 1.97 (m, 4H), 2.75 – 2.85 (m, 1H), 3.03 – 3.12 (m, 1H), 3.39 (d, J = 13.9 Hz, 3H), 3.46 – 3.64 (m, 1H), 3.67 (d, J = 7.7 H 1H) 391 (d J 51 H 1H) 397 408 ( 1H) 410 – 4.23 (m, 4H), 4.31 – 4.38 (m, 1H), 7.25 (d, J = 7.3 Hz, 2H), 7.27 – 7.37 (m, 4H), 7.37 – 7.51 (m, 3H), 7.60 – 7.69 (m, 2H), 7.74 (d, J = 8.9 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 8.12 – 8.24 (m, 1H). ES+(M+1 = 639). Step 2 - Intermediate Compound 121: tert-butyl (1R,4R)-5-(L-phenylalanylglycyl)-2,5- diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000226_0001
In a 500 mL round bottom flask, diethylamine (30 mL, 11.58 mmol) was added to Intermediate Compound 120 (7.4 g, 11.58 mmol) in DCM (30 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure. The crude was washed with MTBE (2 x 25 mL), then the mixture was filtered through filter paper, the filter residue was collected to afford Intermediate Compound 121 (4.50 g, 93 %) as a white solid.
Figure imgf000226_0002
NMR (400 MHz, DMSO) δ 1.42 (d, J = 3.3 Hz, 9H), 1.69 – 1.89 (m, 4H), 2.53 – 2.64 (m, 1H), 3.00 (d, J = 4.3 Hz, 1H), 3.02 – 3.11 (m, 1H), 3.41 – 3.71 (m, 3H), 3.86 (d, J = 4.9 Hz, 1H), 3.90 – 4.07 (m, J = 5.1 Hz, 1H), 4.11 (d, J = 13.5 Hz, 1H), 4.20 (s, 1H), 4.55 (d, J = 12.2 Hz, 1H), 7.17 – 7.33 (m, 5H), 8.13 – 8.22 (m, 1H). ES+(M+1 = 417). Step 3 - Intermediate Compound 122: tert-butyl (1R,4R)-5-((((9H-fluoren-9- yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycyl)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000226_0003
In a 500 mL round bottom flask, DIEA (5.41 mL, 30.97 mmol) was added to Intermediate Compound 121 (4.3 g, 10.32 mmol), (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (4.39 g, 12.39 mmol) and HATU (5.89 g, 15.49 mmol) in THF (50 mL)/MeCN (50.0 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water to afford Intermediate Compound 122 (7.10 g, 91 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.41 (d, J = 4.2 Hz, 9H), 1.72 (d, J = 20.4 Hz, 2H), 1.76 – 1.92 (m, 2H), 2.72 – 2.83 (m, 1H), 3.01 – 3.10 (m, 1H), 3.33 – 3.41 (m, 4H), 3.57 (d, J = 6.9 Hz, 1H), 3.63 (d, J = 6.0 Hz, 3H), 3.71 – 3.81 (m, 1H), 3.83 – 3.94 (m, 1H), 3.95 – 4.07 (m, 1H), 4.10 (d, J = 12.0 Hz, 1H), 4.18 (s, 1H), 419 427 ( 1H) 430 (d J 84 H 2H) 450 461 (m, 1H), 7.14 – 7.21 (m, 1H), 7.25 (d, J = 4.5 Hz, 4H), 7.29 – 7.36 (m, 2H), 7.39 – 7.46 (m, 2H), 7.55 – 7.62 (m, 1H), 7.71 (d, J = 7.5 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 7.99 (d, J = 7.5 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H). ES+(M+1 = 753). Step 4 - Intermediate Compound 123: tert-butyl (1R,4R)-5-(glycylglycyl-L-phenylalanylglycyl)-2,5- diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000227_0001
In a 250 mL round bottom flask, diethylamine (6 mL, 1.33 mmol) was added to Intermediate Compound 122 (1 g, 1.33 mmol) in THF (6 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford Intermediate Compound 123.1H NMR (300 MHz, DMSO) δ 1.41 (d, J = 2.4 Hz, 9H), 1.68 – 1.94 (m, 4H), 2.71 – 2.84 (m, 2H), 3.00 – 3.12 (m, 1H), 3.13 (s, 2H), 3.35 (s, 1H), 3.48 (d, J = 14.4 Hz, 1H), 3.63 (d, J = 15.9 Hz, 2H), 3.72 – 3.83 (m, 1H), 3.83 – 3.96 (m, 1H), 3.96 – 4.09 (m, 1H), 4.12 (s, 1H), 4.19 (s, 1H), 4.52 – 4.65 (m, 1H), 7.26 (d, J = 4.2 Hz, 3H), 7.32 – 7.39 (m, 2H), 7.80 – 7.93 (m, 4H), 8.21 (d, J = 8.7 Hz, 1H). ES+(M+1 = 531). Step 5 - Intermediate Compound 124: tert-butyl (1R,4R)-5-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoyl)glycylglycyl-L-phenylalanylglycyl)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000227_0002
In a 100 mL round bottom flask, 2,5-Dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoate (367 mg, 1.38 mmol) was added to Intermediate Compound 123 (610 mg, 1.15 mmol) in MeCN (15 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure.The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.1% FA ) to afford Intermediate Compound 124 (680 mg, 87 %) as a white solid.1H
Figure imgf000227_0003
NMR (300 MHz, DMSO) δ 1.41 (d, J = 2.1 Hz, 9H), 1.67 – 1.88 (m, 4H), 2.37 – 2.48 (m, 2H), 2.71 – 2.85 (m, 1H), 3.01 – 3.11 (m, 1H), 3.42 – 3.56 (m, 2H), 3.56 – 3.81 (m, 7H), 3.81 – 3.91 (m, 1H), 3.92 – 4.02 (m, 1H), 4.12 (s, 1H), 4.19 (s, 1H), 4.49 – 4.63 (m, 2H), 7.00 (s, 2H), 7.13 – 7.24 (m, 1H), 7.26 (d, J = 4.2 Hz, 4H), 7.96 – 8.21 (m, 3H), 8.22 – 8.31 (m, 1H). ES+(M+1 = 682). Step 6 - Intermediate Compound 125: (S)-N-(2-((1R,4R)-2,5-diazabicyclo[2.2.2]octan-2-yl)-2- oxoethyl)-2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)acetamido)acetamido)-3- phenylpropanamide
Figure imgf000228_0001
In a 100 mL round bottom flask, TFA (4 mL, 51.92 mmol) was added to Intermediate Compound 124 (570 mg, 0.84 mmol) in DCM (8 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford Intermediate Compound 125 (450 mg, 93 %) as a yellow oil.1H NMR (300 MHz, DMSO) δ 1.80 (d, J = 11.1 Hz, 2H), 2.03 (d, J = 24.9 Hz, 2H), 2.37 – 2.48 (m, 2H), 2.72 – 2.86 (m, 1H), 3.06 (d, J = 13.5 Hz, 1H), 3.32 – 3.56 (m, 2H), 3.56 – 3.74 (m, 6H), 3.74 – 3.90 (m, 3H), 3.91 – 4.00 (m, 1H), 4.00 – 4.18 (m, 1H), 4.53 (d, J = 10.2 Hz, 1H), 4.55 – 4.64 (m, 1H), 7.00 (s, 1H), 7.16 – 7.23 (m, 1H), 7.26 (d, J = 4.2 Hz, 3H), 7.97 – 8.14 (m, 2H), 8.18 (d, J = 4.2 Hz, 1H), 8.23 – 8.33 (m, 1H), 8.93 (s, 2H). ES+(M+1 = 582). Step 7 - Compound LP5: (S)-2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)- acetamido)acetamido) -N-(2-oxo-2-((1R,4R)-5-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4- (((1S,3R,4aS,9S,9aR,-10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]-oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonyl)-2,5-diazabicyclo- [2.2.2]-octan-2-yl)ethy
Figure imgf000228_0002
In a 100 mL round b Oott Nl) o O-3-phe m fla OsH Nnylp k, H Oropanam AT NHU (7O2 HNide 7 mOg, NH 1.91 O mO NmolN) 9-92-0; 800 mg, 1.27 mmol O N w Oas O aOH Odded O OHH to O O the O PNU-159682 carboxylic acid (CAS No.: 120481 O) in DMA (5 mL) under nitrogen. The resulting mixture 1 was stirred at 15 °C for 10 minutes. DIEA (1.113 mL, 6.37 mmol) was added to Intermediate Compound 125 (890 mg, 1.53 mmol) in DMA (5.00 mL) under nitrogen. The resulting mixture 2 was stirred at 15 °C for 10 minutes. After that, the mixture 2 was added to the mixture 1, stirring at 15 °C for 2 hours. The reaction mixture was quenched with 10 mM NH4CL (4 mL). The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, and purified again by preparative HPLC to afford Compound LP5 (202 mg, 13.30 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.17 – 1.29 (m, 3H), 1.64 – 1.71 (m, 2H), 1.72 – 2.35 (m, 6H), 2.37 – 2.46 (m, 2H), 2.61 – 2.86 (m, 4H), 3.04 (d, J = 14.3 Hz, 2H), 3.31 (s, 3H), 3.44 – 3.77 (m, 11H), 3.96 (d, J = 19.8 Hz, 8H), 4.17 (s, 1H), 4.24 (d, J = 2.2 Hz, 1H), 4.48 – 4.65 (m, 3H), 4.98 (s, 1H), 5.15 (s, 1H), 5.28 (d, J = 4.3 Hz, 1H), 5.98 (d, J = 9.2 Hz, 1H), 6.99 (s, 2H), 7.13 – 7.22 (m, 1H), 7.24 (d, J = 4.6 Hz, 4H), 7.61 – 7.68 (m, 1H), 7.85 – 7.95 (m, 2H), 7.97 – 8.04 (m, 1H), 8.05 – 8.18 (m, 2H), 8.21 – 8.30 (m, 1H), 13.30 (s, 1H), 14.08 (s, 1H). ES+ [(M+1)/2] = 1191. Example 45: Synthesis of Compound LP7
Figure imgf000229_0001
Step 1 - Intermediate Compound 126: tert-butyl (1R,4R)-5-((((9H-fluoren-9- yl)methoxy)carbonyl)glycylglycylglycylglycylglycyl)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000229_0002
In a 500 mL round bottom flask, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycylglycylglycine (10 g, 19.03 mmol) was added to tert-butyl (1R,4R)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate (4.04 g, 19.03 mmol), O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoro- phosphate (10.85 g, 28.54 mmol) and N,N-diisopropylethylamine (7.38 g, 57.09 mmol) in DMSO (150 mL). The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water to afford Intermediate Compound 126 (5.50 g, 40.2 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.41 (d, J = 2.7 Hz, 9H), 1.45 (s, 1H), 1.65 – 1.95 (m, 5H), 3.38 (s, 2H), 3.44 – 3.62 (m, 1H), 3.68 (d, J = 6.1 Hz, 2H), 3.75 (d, J = 5.8 Hz, 5H), 3.84 (d, J = 5.4 Hz, 1H), 3.89 – 3.97 (m, 1H), 4.06 – 4.32 (m, 4H), 4.53 (d, J = 12.6 Hz, 1H), 7.29 – 7.37 (m, 2H), 7.39 – 7.45 (m, 2H), 7.57 (t, J = 6.0 Hz, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 8.15 (q, J = 6.9, 7.6 Hz, 3H). ES+(M+1 = 720.50). Step 2 & 3 - Intermediate Compound 128: tert-butyl (1R,4R)-5-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)propanoyl)glycylglycylglycyl-glycyl-glycyl)-2,5-diazabicyclo[2.2.2]octane-2-carboxylate
Figure imgf000230_0001
In a 500 mL round bottom flask, Intermediate Compound 126 (4 g, 5.56 mmol) was added to diethylamine (20 mL, 193.33 mmol) in THF (30 mL) and water (3 mL) and stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure to afford crude product Intermediate Compound 127. The crude product was added to 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)propanoate (1.775 g, 6.67 mmol) in DMA (30 mL). The resulting mixture was stirred at 25 °C for 2 hours to give the crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 60% MeCN in water(0.1%FA) to afford Intermediate Compound 128 (1.600 g, 44.4 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.41 (d, J = 2.3 Hz, 9H), 1.66 – 1.97 (m, 4H), 2.39 – 2.46 (m, 2H), 3.58 – 3.64 (m, 2H), 3.70 (d, J = 5.7 Hz, 2H), 3.75 (d, J = 5.5 Hz, 6H), 3.82 – 4.24 (m, 4H), 4.49 – 4.56 (m, 1H), 7.00 (s, 2H), 7.86 – 7.98 (m, 1H), 8.09 – 8.17 (m, 4H), 8.27 (t, J = 5.7 Hz, 1H). ES+( M+1 = 649.40). Step 4 & 5 - Compound LP7: 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2,5,8,11,14-pentaoxo-14- ((1R,4R)-5-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,-4aS,9S,9aR,10aS)-9-methoxy-1- methyloctahydro-1H-pyrano[4',3':4,5]oxazolo [2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carbonyl)-2,5-diazabicyclo[2.2.2]octan-2-yl)-3,6,9,12- tetraazatetradecyl)propanamide
Figure imgf000230_0002
In 250 mL round bottom flask, Intermediate Compound 128 (972 mg, 1.50 mmol) was added to TFA (3 mL, 38.94 mmol) in DCM (12 mL) at 0°C. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 129. The crude product was added to the PNU-159682 carboxylic acid (1204819-92-0; 470 mg, 0.75 mmol), HATU (911 mg, 2.40 mmol), 1-hydroxybenzotriazole monohydrate (202 mg, 1.50 mmol) and DIEA (1.570 mL, 8.99 mmol) in DMA (15 mL). The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with saturated NH4Cl 10mM in water (2mL). The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 70% MeCN in water to afford crude product (0.4 g). The crude product was further purified by preparative HPLC to afford Compound LP7 (120 mg, 6.92 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.18 – 1.30 (m, 3H), 1.68 (t, J = 6.0 Hz, 2H), 1.73 – 1.86 (m, 3H), 1.87 – 2.08 (m, 2H), 2.09 – 2.22 (m, 1H), 2.42 (t, J = 7.5 Hz, 2H), 2.59 – 2.73 (m, 2H), 2.74 – 2.86 (m, 1H), 3.31 (s, 6H), 3.46 – 3.56 (m, 2H), 3.61 (t, J = 7.5 Hz, 2H), 3.65 – 3.78 (m, 10H), 3.85 (t, J = 5.5 Hz, 1H), 3.90 – 4.08 (m, 6H), 4.17 (s, 1H), 4.23 (d, J = 2.2 Hz, 1H), 4.59 (t, J = 4.1 Hz, 2H), 4.97 (q, J = 7.0 Hz, 1H), 5.15 (s, 1H), 5.21 – 5.35 (m, 1H), 5.98 (d, J = 5.4 Hz, 1H), 6.99 (s, 2H), 7.54 – 7.73 (m, 1H), 7.97 – 7.85 (m, 3H), 8.18 – 8.04 (m, 3H), 8.26 (t, J = 5.8 Hz, 1H), 13.30 (s, 1H), 14.08 (s, 1H). ES+( M+1 = 1158.55). Example 46: Synthesis of Compound LP8
Figure imgf000231_0001
Figure imgf000232_0002
Step 1 - Intermediate Compound 130: (9H-fluoren-9-yl)methyl (2-(((((3R,3aR,6R,6aR)-6- hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)-amino)-2-oxoethyl)carbamate
Figure imgf000232_0001
In a 250 mL round bottom flask, (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (500g, 1357.26 mmol) was added to (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol (“isomannide”, CAS No.641-74-7; 7934 g, 54290.44 mmol) and pyridin-1-ium 4- methylbenzenesulfonate (51.2 g, 203.59 mmol) in DCM (25 L) under nitrogen. The resulting mixture was stirred at 25 °C for 40 hours. The reaction mixture was diluted with water (20 L), extracted with DCM (5 x 10 L), the organic layer was dried over Na2SO4, filtered and evaporated to afford yellow solid. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether to afford Intermediate Compound 130 (610 g, 99 %) as a beige solid.1H NMR (400 MHz, DMSO) δ 3.60 – 3.68 (d, J = 6.1 Hz, 2H), 3.74 – 3.81 (t, J = 7.6 Hz, 1H), 3.84 – 3.94 (t, J = 7.7 Hz, 1H), 4.02 – 4.15 (m, 3H), 4.20 – 4.31 (m, 4H), 4.33 – 4.39 (t, J = 5.1 Hz, 1H), 4.43 – 4.52 (t, J = 4.7 Hz, 1H), 4.55 – 4.68 (m, 2H), 4.74 – 4.83 (d, J = 7.1 Hz, 1H), 7.30 – 7.37 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.55 – 7.63 (m, 1H), 7.69 – 7.76 (d, J = 7.5 Hz, 2H), 7.86 – 7.95 (d, J = 7.5 Hz, 2H), 8.64 – 8.75 (br, 1H). ES+(M+H = 455). Step 2 - Intermediate Compound 131: 2-amino-N-((((3R,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2- b]furan-3-yl)oxy)-methyl)acetamide
Figure imgf000233_0001
In a 250 mL round bottom flask, dimethylamine (4.03 L, 8053.18 mmol) was added to Intermediate Compound 130 (610 g, 1342.20 mmol) in THF (10 L) and MeOH (10 L) under nitrogen. The resulting mixture was stirred at 25 °C for 8 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 131 (310 g, 99 %) as a yellow gum.1H NMR (400 MHz, DMSO) δ 3.06 – 3.09 (s, 1H), 3.10 – 3.13 (s, 2H), 3.24 – 3.48 (m, 4H), 3.79 (t, J = 7.5 Hz, 1H), 3.88 (t, J = 7.6 Hz, 1H), 4.03 – 4.12 (m, 2H), 4.24 – 4.30 (t, J = 4.7 Hz, 1H), 4.44 – 4.50 (m, 1H), 4.55 – 4.68 (m, 2H), 8.57 – 8.77 (m, 1H). ES+(M+1 = 233). Step 3 - Intermediate Compound 132: benzyl (2-(((((3R,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2- b]furan-3-yl)oxy)methyl)amino)-2-oxoethyl)carbamate
Figure imgf000233_0002
In a 1500 mL round bottom flask, benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (393 g, 1575.98 mmol) was added to N-ethyl-N-isopropylpropan-2-amine (339 g, 2626.64 mmol) and Intermediate Compound 131 (305 g, 1313.32 mmol) in DMF (1 L) under nitrogen. The resulting mixture was stirred at 25 °C for 4 hours. The crude product was purified by flash silica chromatography, elution gradient 7 to 8% MeOH in DCM, to afford Intermediate Compound 132 (470 g, 98 %) as a white solid.1H NMR (400 MHz, DMSO) δ 3.34 – 3.37 (d, J = 6.5 Hz, 1H), 3.37 – 3.44 (d, J = 8.4 Hz, 1H), 3.55 – 3.67 (d, J = 6.2 Hz, 2H), 3.76 – 3.82 (m, 1H), 3.83 – 3.92 (t, J = 7.6 Hz, 1H), 3.99 – 4.14 (m, 2H), 4.22 – 4.32 (t, J = 4.7 Hz, 1H), 4.42 – 4.50 (t, J = 4.6 Hz, 1H), 4.55 – 4.69 (m, 2H), 4.72 – 4.81 (d, J = 6.8 Hz, 1H), 5.00 – 5.06 (s, 2H), 7.28 – 7.41 (m, 5H), 7.45 – 7.54 (t, J = 6.0 Hz, 1H), 8.64 – 8.77 (t, J = 6.6 Hz, 1H). ES+(M+1 = 367). Step 4 - Intermediate Compound 133: (3R,3aS,6R,6aR)-6-((2- (((benzyloxy)carbonyl)amino)acetamido)methoxy)- hexahydrofuro[3,2-b]furan-3-yl trifluoromethanesulfonate
Figure imgf000233_0003
In a 5 L round bottom flask, Tf2O (0.276 L, 1632.23 mmol) was added to pyridine (2.3 L, 3766.68 mmol) and Intermediate Compound 132 (460 g, 1255.56 mmol) in DCM (1L) at -78°C over a period of 2 hours under nitrogen. The resulting mixture was stirred at 0 °C for 1 hours. The reaction mixture was diluted with DCM (2 L), and washed sequentially with 2M HCl (2 L), 0.1M HCl (2 L), and water (2 L). The organic layer was dried over Na2SO4, filtered and evaporated to afford Intermediate Compound 133 (620 g, 99 %) as a yellow gum.1H NMR (400 MHz, CDCl3) δ 3.41 (dd, J = 7.3, 8.8 Hz, 1H), 3.64 (d, J = 6.1 Hz, 2H), 3.83 (dd, J = 3.0, 5.4 Hz, 1H), 3.85 (d, J = 2.9 Hz, 1H), 4.08 (s, 1H), 4.11 (s, 1H), 4.12 – 4.17 (m, 1H), 4.60 (dd, J = 6.3, 10.3 Hz, 1H), 4.65 – 4.67 (m, 2H), 5.04 (s, 2H), 5.07 (d, J = 3.1 Hz, 1H), 7.30-7.39 (m, 1H), 7.37 (d, J = 3.1 Hz, 4H), 7.50 (t, J = 6.1 Hz, 1H), 8.72 (t, J = 6.9 Hz, 1H). ES+(M+1 = 499). Step 5 - Intermediate Compound 134: benzyl (2-(((((3R,3aS,6S,6aR)-6-(1,3-dioxoisoindolin-2- yl)hexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)amino)-2-oxoethyl)carbamate
Figure imgf000234_0001
In a 5.0 L round bottom flask, phthalimide-potassium salt (272 g, 1468.62 mmol) was added to 18- Crown-6 (32.3 g, 122.39 mmol) and Intermediate Compound 133 (610g, 1223.85 mmol) in DMF (2 L) under nitrogen. The resulting mixture was stirred at 100 °C for 1.5 hours. The reaction mixture was diluted with DCM (20 L), and washed sequentially with water (20 L), and saturated brine (20 L). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 100 to 100% EtOAc in petroleum ether to Intermediate Compound 134 (598 g, 99 %) as a yellow oil.1H NMR (400 MHz, CDCl3) δ 3.57 (s, 1H), 3.66 (d, J = 6.2 Hz, 2H), 3.84 (dd, J = 6.7, 8.4 Hz, 1H), 3.92 (dd, J = 6.2, 9.3 Hz, 1H), 4.07 – 4.12 (m, 1H), 4.16 (dd, J = 7.8, 9.3 Hz, 1H), 4.50-4.60 (m, 1H), 4.62 – 4.66 (m, 1H), 4.70 (dd, J = 7.0, 10.3 Hz, 1H), 4.79 (t, J = 4.6 Hz, 1H), 4.91 (dd, J = 1.8, 4.6 Hz, 1H), 5.04 (s, 2H), 7.26-7.35 (m, 2H), 7.36 (s, 2H), 7.37 (s, 1H), 7.52 (t, J = 6.0 Hz, 1H), 7.83 – 7.89 (m, 4H), 8.76 (t, J = 6.8 Hz, 1H). ES+(M+1 = 496). Step 6 - Intermediate Compound 135: benzyl (2-(((((3R,3aS,6S,6aR)-6-aminohexahydrofuro[3,2- b]furan-3-yl)oxy)methyl)-amino)-2-oxoethyl)carbamate
Figure imgf000234_0002
In a 5.0 L round bottom flask, hydrazine (143 g, 3572.24 mmol) was Intermediate Compound 134 (590 g, 1190.75 mmol) in EtOH (10 L) under nitrogen. The resulting mixture was stirred at 80 °C for 5 hours. The reaction mixture was filtered . The solvent was removed under reduced pressure to afford Intermediate Compound 135 (420 g, 97 %) as a yellow solid.1H NMR (400 MHz, CDCl3) δ 3.51 (s, 2H), 3.56 (d, J = 8.8 Hz, 2H), 3.63 (d, J = 6.2 Hz, 2H), 3.76 (q, J = 4.5, 6.0 Hz, 2H), 4.04 (dt, J = 3.8, 7.5 Hz, 1H), 4.17 (d, J = 4.1 Hz, 1H), 4.53 – 4.61 (m, 2H), 4.63-4.68 (m, 1H), 5.03 (s, 2H), 7.32 (q, J = 4.2, 5.5 Hz, 1H), 7.37 (d, J = 5.2 Hz, 4H), 7.49 (t, J = 6.1 Hz, 1H), 8.29 (s, 1H), 8.70 (t, J = 6.8 Hz, 1H). Step 7 - Intermediate Compound 136: benzyl (2-(((((3R,3aS,-6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)oxy)methyl)-amino)-2-oxoethyl)carbamate
Figure imgf000235_0001
In a round bottom flask, Boc2O (57.2 mL, 246.31 mmol) was added to Intermediate Compound 135 (30 g, 82.10 mmol) in DCM (500 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with DCM (0.5 L), and washed sequentially with water (2 x 0.2 L) and saturated brine (0.2 L). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 50 to 60% MeCN in water, to afford Intermediate Compound 136 (38.0 g, 99 %) as a yellow solid.1H NMR (400 MHz, CDCl3) δ 1.38 (s, 9H), 3.41 – 3.49 (m, 1H), 3.64 (t, J = 6.8 Hz, 3H), 3.72-3.81 (m, 3H), 4.05-4.09 (m, 1H), 4.30 (d, J = 4.4 Hz, 1H), 4.53 (t, J = 4.6 Hz, 1H), 4.55-4.61 (m, 1H), 4.64-4.71 (m, 1H), 5.03 (s, 2H), 7.14 – 7.22 (m, 1H), 7.26-7.34 (m, 1H), 7.36 (d, J = 3.6 Hz, 4H), 7.49 (t, J = 6.1 Hz, 1H), 8.70 (t, J = 6.7 Hz, 1H). ES+(M+1 = 466). Step 8 - Intermediate Compound 137: tert-butyl ((3S,3aR,6R,6aS)-6-((2- aminoacetamido)methoxy)hexahy-drofuro-[3,2-b]furan-3-yl)carbamate
Figure imgf000235_0002
In a round bottom flask, Pd-C (8 g, 7.52 mmol) was added to ammonium hydroxide (50 mL, 64.45 mmol) and Intermediate Compound 136 (30 g, 64.45 mmol) in MeOH (100 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered through celite. The solvent was removed under reduced pressure to afford Intermediate Compound 137 (21.00 g, 98 %) as a yellow oil.1H NMR (400 MHz, CDCl3) δ 1.38 (s, 9H), 3.22 (s, 1H), 3.40 (t, J = 8.1 Hz, 1H), 3.56 – 3.73 (m, 3H), 3.76 – 3.86 (m, 3H), 4.07 (td, J = 4.8, 7.2 Hz, 1H), 4.31 (d, J = 4.3 Hz, 1H), 4.53 (t, J = 4.6 Hz, 1H), 4.57 – 4.72 (m, 2H), 7.10 – 7.24 (m, 1H), 8.78 (t, J = 6.9 Hz, 1H). ES+(M+1 = 332). Step 9 - Intermediate Compound 138: tert-butyl ((3S,3aR,6R,6aS)-6-(((S)-11-benzyl-1-(9H-fluoren-9- yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazahepta-decan-17-yl)oxy)hexahydrofuro[3,2- b]furan-3-yl)carbamate
Figure imgf000236_0001
In a round bottom flask, EDC (2.60 g, 13.58 mmol) was added to Intermediate Compound 137 (3 g, 9.05 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (5.45 g, 10.86 mmol) in DMA (60 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The reaction mixture was purified by flash C18-flash chromatography, elution gradient 5 to 100% MeCN in water, to afford Intermediate Compound 138 (4.20 g, 56.9 %) as a yellow solid.1 H NMR (400 MHz, DMSO) δ 1.39 (d, J = 3.6 Hz, 9H), 2.52 (s, 2H), 2.68 – 2.86 (m, 1H), 3.05 (d, J = 4.5 Hz, 1H), 3.39 (dd, J = 8.9, 17.0 Hz, 1H), 3.59 – 3.90 (m, 8H), 4.02 – 4.16 (m, 1H), 4.19 – 4.37 (m, 5H), 4.42 – 4.74 (m, 3H), 7.18 (dd, J = 5.5, 8.5 Hz, 1H), 7.25 (d, J = 6.4 Hz, 5H), 7.33 (t, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.58 (t, J = 6.1 Hz, 1H), 7.71 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.01 (t, J = 5.9 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.31 (dt, J = 5.3, 11.7 Hz, 1H), 8.54 (t, J = 6.6 Hz, 1H). ES+(M+1 = 815). Step 10 – Intermediate Compound 139: tert-butyl ((3S,3aR,6R,6aS)-6-(((S)-13-amino-7-benzyl- 3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)oxy)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000236_0002
In a round bottom flask, diethylamine (30 mL, 4.30 mmol) was added to Intermediate Compound 138 (3.5 g, 4.30 mmol) in DCM (30 mL). The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 139 (2.500 g, 98 %) as a yellow solid.1 H NMR (400 MHz, DMSO) δ 1.39 (d, J = 1.4 Hz, 9H), 2.08 (s, 5H), 2.72 – 2.94 (m, 3H), 3.18 (s, 2H), 3.37 – 3.41 (m,1H), 3.78 – 3.83 (m, 3H), 3.99 – 4.12 (m, 1H), 4.31 – 4.38 (m, 1H), 4.46 – 4.73 (m, 3H), 7.21 – 7.31 (m, 5H), 7.37 – 7.46 (m, 1H), 7.81 – 7.94 (m, 2H), 8.10 (d, J = 10.2 Hz, 1H), 8.18 – 8.30 (m, 1H), 8.31 – 8.43 (m, 1H), 8.58 (t, J = 6.7 Hz, 1H). ES+(M+1 = 593). Step 11 – Intermediate Compound 140: tert-butyl ((3S,3aR,6R,6aS)-6-(((S)-7-benzyl-17-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14- pentaazaheptadecyl)oxy)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000237_0001
In a round bottom flask, DIEA (1.105 mL, 6.33 mmol) was added to Intermediate Compound 139 (2.5 g, 4.22 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-propanoate (1.348 g, 5.06 mmol) in DMA (30 mL) under air. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was purified by flash C18-flash chromatography, elution gradient 5 to 100% MeCN in water, to afford Intermediate Compound 140 (2.400 g, 76 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 2.42 (dd, J = 6.6, 8.4 Hz, 2H), 2.50 (s, 2H), 2.52 (s, 2H), 2.75- 2.87 (m, 1H), 3.06 (dd, J = 4.6, 13.9 Hz, 1H), 3.40 (t, J = 8.1 Hz, 1H), 3.55 – 3.84 (m, 12H), 4.05 – 4.10 (m, 1H), 4.31 (d, J = 4.3 Hz, 1H), 4.42 – 4.72 (m, 3H), 7.00 (s, 2H), 7.15 – 7.33 (m, 6H), 8.02 – 8.05 (m, 1H), 8.12 (d, J = 8.0 Hz, 1H), 8.30 – 8.42 (m, 2H), 8.54 (t, J = 6.8 Hz, 1H). ES+(M+1 = 744). Step 12 - Intermediate Compound 141: (S)-N-(2-(((((3R,3aS,6S,6aR)-6-aminohexahydrofuro[3,2- b]furan-3-yl)oxy)methyl)-amino)-2-oxoethyl)-2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamido)acetamido)-acetamido)-3-phenylpropanamide
Figure imgf000237_0002
In a round bottom flask, 1,1,1,3,3,3-Hexafluoro-2-propanol (2 mL, 0.40 mmol) was added to Intermediate Compound 140 (300 mg, 0.40 mmol) and ammonium chloride (173 mg, 3.23 mmol) under nitrogen. The resulting mixture was stirred at 80 °C for 16 hours. The reaction mixture was filtered through celite pad. The solvent was removed by N2 flow. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 141 (120 mg, 46.2 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.11 (s, 2H), 2.27 – 2.44 (m, 3H), 2.81 (dd, J = 9.5, 13.8 Hz, 1H), 3.06 (d, J = 18.3 Hz, 2H), 3.35 – 3.41 (m, 3H), 3.46 – 3.52 (m, 1H), 3.52 – 3.66 (m, 3H), 3.70 – 3.81 (m, 2H), 4.00 – 4.10 (m, 1H), 4.19 (d, J = 4.3 Hz, 1H), 4.43 – 4.72 (m, 4H), 7.00 (d, J = 1.3 Hz, 1H), 7.14 – 7.35 (m, 7H), 7.34 – 7.51 (m, 1H), 8.09 (dd, J = 8.5, 28.9 Hz, 2H), 8.27 – 8.36 (m, 2H), 8.54 (s, 1H). ES+(M+1 = 644). Step 13 - Compound LP8: (2S,4S)-N-((3S,3aR,6R,6aS)-6-(((S)-7-benzyl-17-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaheptadecyl)oxy)hexa-hydrofuro[3,2-b]furan- 3-yl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamide
Figure imgf000238_0001
1-Hydroxypyrrolidine-2,5-dione (36.7 mg, 0.32 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 100 mg, 0.16 mmol) and DIC (0.050 mL, 0.32 mmol) in DMA (3 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. Intermediate Compound 141 (205 mg, 0.32 mmol) and DIEA (0.139 mL, 0.80 mmol) was added, the resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by preparative HPLC to afford Compound LP8 (19.00 mg, 9.51 %) as a red solid.1
Figure imgf000238_0002
H NMR (400 MHz, DMSO) δ 1.18 – 1.26 (m, 4H), 1.68 (s, 2H), 2.16 (d, J = 14.4 Hz, 1H), 2.28 (dd, J = 5.5, 14.6 Hz, 1H), 2.42 (dd, J = 6.6, 8.4 Hz, 2H), 2.69 (s, 2H), 2.81 (dd, J = 9.6, 13.8 Hz, 1H), 2.98 (s, 1H), 3.00 – 3.11 (m, 2H), 3.31 (s, 3H), 3.60 – 3.80 (m, 12H), 3.88 – 3.98 (m, 2H), 4.00 (s, 3H), 4.06 (d, J = 6.1 Hz, 1H), 4.11 – 4.17 (m, 2H), 4.23 (d, J = 2.3 Hz, 1H), 4.43 (d, J = 4.4 Hz, 1H), 4.50 (td, J = 4.6, 8.8 Hz, 1H), 4.56 – 4.65 (m, 4H), 5.02 (s, 1H), 5.25 (t, J = 4.7 Hz, 1H), 5.39 (s, 1H), 6.99 (s, 2H), 7.19 – 7.25 (m, 6H), 7.67 (s, 1H), 7.94 (d, J = 3.2, 3.9 Hz, 2H), 8.12 – 8.31 (m, 3H), 8.56 (t, J = 6.6 Hz, 1H), 13.26 (s, 1H), 14.08 (s, 1H). ES+(M+1 = 1253). Example 47: Synthesis of Compound LP9
Figure imgf000239_0001
Figure imgf000240_0001
Step 1 - Intermediate Compound 142: 5-(((tert-butyldimethyl-silyl)oxy)methyl)-2- hydroxybenzaldehyde
Figure imgf000240_0002
In a round bottom flask, TBS-Cl (178 g, 1183.05 mmol) in DCM (200 mL) was added dropwise to 2- hydroxy-5-(hydroxymethyl)-benzaldehyde (150 g, 985.88 mmol) and Imidazole (134 g, 1971.75 mmol) in DCM (500 mL) at 15°C over a period of 5 hours under nitrogen. The resulting mixture was stirred at 15 °C for 15 hours. The reaction mixture was diluted with EtOAc (500 mL), and washed sequentially with water (2 x 300 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether, to Intermediate Compound 142 (120 g, 45.7 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 0.07 (s, 6H), 0.89 (s, 9H), 4.64 (s, 2H), 6.98 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 2.4, 8.4 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 10.27 (s, 1H), 10.65 (s, 1H). ES+(M+1 =267). Step 2 - Intermediate Compound 143: (2S,3S,4S,5R)-methyl 3,4,5-triacetoxy-6-(4-((tert- butyldimethylsilyloxy)methyl)-2-formylphenoxy)-tetrahydro-2H-pyran-2-carboxylate
Figure imgf000241_0001
In a round bottom flask, 4A molecular sieves (240 g, 450.43 mmol) was added to Intermediate Compound 142 (120 g, 450.43 mmol) and Silver(I) oxide (313 g, 1351.29 mmol) in MeCN (2000 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 16 hours. The reaction mixture was filtered through a celite pad. Then the filtrate was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether, to afford Intermediate Compound 143 (150 g, 57.2 %) as a yellow solid. 1H NMR (400 MHz, DMSO) δ 0.08 (s, 6H), 0.90 (s, 9H), 2.02 (s, 9H), 3.64 (s, 3H), 4.68 – 4.81 (m, 3H), 5.11 (d, J = 9.8 Hz, 1H), 5.23 (dd, J = 7.6, 9.6 Hz, 1H), 5.52 (d, J = 9.6 Hz, 1H), 5.77 (d, J = 7.6 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.63 (dd, J = 2.4, 8.4 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 10.18 (s, 1H). ES+(M+23 =605). Step 3 - Intermediate Compound 144: (2S,3S,4S,5R)-methyl 3,4,5-triacetoxy-6-(2-((tert- butoxycarbonylamino)methyl)-4-(hydroxymethyl)phenoxy)-tetrahydro-2H-pyran-2-carboxylate
Figure imgf000241_0002
In a round bottom flask, HCl (5.53 ml, 181.92 mmol, 12 M aqueous solution) was added to tert- butyl carbamate (63.9 g, 545.76 mmol), triethylsilane (21.15 g, 181.92 mmol) and Intermediate Compound 143 (106 g, 181.92 mmol) in MeCN (1.5 L) at 0°C. The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 80 to 100% EtOAc in petroleum ether, to afford Intermedia k, Intermediate Ote Compound 144 (80 g, 77 %) as a white solid.1H NMR (300 MHz, DMSO) δ 1.40 (s, 9H), 1.93 – 2.10 (m, 10H), 3.64 (s, 3H), 4.01 (t, J = 7.2 Hz, 2H), 4.41 (s, 2H), 4.72 (s, 1H), 4.95 – 5.23 (m, 2H), 5.36 – 5.64 (m, 2H), 6.97 (br, 1H), 7.15 (br, 3H). ES+(M+1 =570). Step 4 - Intermediate Compound 145: (2S,3S,4S,5R)-methyl 3,4,5-triacetoxy-6-(2-(aminomethyl)-4- (hydroxymethyl)-phenoxy)-tetrahydro-2H-py Comp Cra oOn uO- nM2 de-carboxylate
Figure imgf000242_0001
In a round bottom flas O O O O O 144 (20 g, O 3H5.11 mmol) was added in 2M on in 1,4-dioxane (200 mL). The O resulting mixt NH HCl soluti Oure was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 145 (15.60 g, 95 %) as a white solid.1H NMR (500 MHz, DMSO) δ 1.95 – 2.06 (m, 9H), 3.64 (s, 3H), 3.83 – 3.95 (m, 2H), 4.46 (s, 2H), 4.75 (d, J = 9.5 Hz, 1H), 5.08 (t, J = 9.5Hz, 1H), 5.22 (dd, J = 8.0, 9.5 Hz, 1H), 5.51 (t, J = 9.5 Hz, 1H), 5.66 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 7.35 (dd, J = 2.1, 8.5 Hz, 1H), 7.45 – 7.52 (m, 1H), 8.43 (s, 3H) (HCl salt). ES+(M+1 =470). Step 5 - Intermediate Compound 146: (2S,3S,4S,5R)-methyl 6-(2-((3-(((9H-fluoren-9- yl)methoxy)carbonylamino)-propanamido)methyl)-4-(hydroxymethyl)phenoxy)-3,4,5-triacetoxy- tetrahydro-2H-pyran-2-carboxylate
Figure imgf000242_0002
In a round bottom flask, N,N-Diisopropylethylamine (2.75 g, 21.30 mmol) was added to Intermediate Compound 145 (10 g, 21.30mmol)and 2,3,5,6-tetrafluorophenyl3-((((9H-fluoren-9- yl)methoxy)carbo-nyl)amino)-propanoate (9.79 g, 21.30 mmol) in MeCN (100 mL). The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 90 to 100% petroleum ether in EtOAc, to afford Int Intermediate Compound 146 (8.00 g, 49.2 %) as a white solid.
Figure imgf000242_0003
NMR (400 MHz, DMSO) δ 2.02 (d, J = 4.0 Hz, 6H), 2.05 (s, 3H), 2.37 (q, J = 7.6 Hz, 2H), 3.25 (q, J = 6.4 Hz, 2H), 3.65 (s, 3H), 4.09 (dd, J = 5.6, 16.0 Hz, 1H), 4.22 (dd, J = 6.0, 12.4 Hz, 2H), 4.29 (d, J = 6.4 Hz, 2H), 4.42 (d, J = 5.6 Hz, 2H), 4.73 (d, J = 9.6 Hz, 1H), 4.99 – 5.22 (m, 3H), 5.50 (t, J = 9.6 Hz, 1H), 5.57 (d, J = 8.0 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 7.09 – 7.25 (m, 2H), 7.27 – 7.39 (m, 3H), 7.42 (t, J = 7.6 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 8.24 (t, J = 6.0 Hz, 1H). ES+(M+1 =763). Step 6 - Intermediate Compound 147: (2S,3S,4S,5R)-methyl 6-(2-((3-(((9H-fluoren-9- yl)methoxy)carbonylamino)-propanamido)methyl)-4-(((4- nitrophenoxy)carbonyloxy)methyl)phenoxy)-3,4,5-triacetoxy-tetrahydro-2H-pyran-2-carboxylate
Figure imgf000243_0001
In a round bottom flask, DIEA (3.39 mL,19.40 mmol) was added to Intermediate Compound 146 (3.7 g, 4.85 mmol) and Bis(p-nitrophenyl) carbonate (5.90 g, 19.40 mmol) in DCM (30 mL) under nitrogen. The resulting mixture was stirred at RT for 4 hours. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether, to afford Intermediate Compound 147 (2.90 g, 64.4 %) as a yellow solid.1H
Figure imgf000243_0002
NMR (400 MHz, DMSO) δ 2.00 – 2.10 (m, 9H), 2.36 – 2.44 (m, 2H), 3.22 – 3.31 (m, 2H), 3.65 (s, 3H), 4.06 – 4.16 (m, 1H), 4.17 – 4.27 (m, 3H), 4.29 (s, 1H), 4.76 (d, J = 9.6 Hz, 1H), 5.05 – 5.14 (m, 1H), 5.14 – 5.24 (m, 2H), 5.24 (s, 1H), 5.48 – 5.57 (m, 1H), 5.66 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.26 – 7.45 (m, 7H), 7.48 – 7.57 (m, 2H), 7.69 (d, J = 7.6 Hz, 2H), 7.89 (d, J = 7.6 Hz, 2H), 8.25 – 8.36 (m, 3H). ES+(M+1 =928). Step 7 - Intermediate Compound 148: (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(((((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)carbamoyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000243_0003
In a round bottom flask, DIEA (2.184 mL, 12.50 mmol) was added to Intermediate Compound 147 (2.9 g, 3.13 mmol) and tert-butyl ((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3- yl)carbamate (3.05 g, 12.50 mmol) in THF (50 mL) under nitrogen. The resulting mixture was stirred at RT for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether, to afford Intermediate Compound 148 (2.80 g, 87 %) as a white solid.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 1.99 – 2.06 (m, 9H), 2.33 – 2.45 (m, 2H), 3.20 – 3.30 (m, 2H), 3.52 – 3.61 (m, 2H), 3.65 (s, 3H), 3.78 – 3.85 (m, 3H), 3.98 – 4.13 (m, 2H), 4.16 – 4.25 (m, 2H), 4.29 (d, J = 6.3 Hz, 2H), 4.37 (s, 2H), 4.74 (d, J =9.0 Hz, 1H), 4.89 – 5.01 (m, 2H), 5.04 – 5.13 (m, 1H), 5.13 – 5.21 (m, 1H), 5.46 – 5.55 (m, 1H), 5.61 (d, J = 7.9 Hz, 1H), 6.90 – 6.98 (m, 1H), 7.05 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 2.1 Hz, 1H), 7.19 – 7.28 (m, 2H), 7.30 – 7.36 (m, 2H), 7.39 – 7.45 (m, 2H), 7.59 (d, J = 6.7 Hz, 1H), 7.70 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.22 – 8.29 (m, 1H). ES+(M+1 =1033). Step 8 & 9 - Intermediate Compound 150: (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)-hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4- (((((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)hexahydrofuro-[3,2-b]furan-3- yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000244_0001
In a round bottom flask, DBU (0.292 mL, 1.94 mmol) was added to Intermediate Compound 148 (2 g, 1.94 mmol) in THF (20 mL) at -20°C under nitrogen. The resulting mixture was stirred at -20 °C for 2 hours. The reaction mixture was adjusted to pH 6 with 1M HCl in dioxane. The solvent was removed under reduced pressure and afforded crude Intermediate Compound 149. The crude Intermediate Compound 149 was added to Intermediate Compound 158 (2.091 g, 1.94 mmol) (see below for procedure for preparing Int 158), HATU (1.472 g, 3.87 mmol) and DIEA (1.014 mL, 5.81 mmol) in DMA (20.00 mL) at 25°C under nitrogen. The resulting mixture was stirred at 20 °C for 2 hours. The reaction mixture was set onto column directly without any workup. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 150 (2.500 g, 68.9 %) as a colourless oil.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 1.54 (d, J = 28.6 Hz, 4H), 1.98 – 2.07 (m, 8H), 2.08 (s, 8H), 2.29 – 2.39 (m, 4H), 3.24 (s, 3H), 3.35 (s, 9H), 3.39 – 3.47 (m, 4H), 3.47 – 3.52 (m, 37H), 3.57 – 3.62 (m, 4H), 3.65 (s, 2H), 3.80 – 3.90 (m, 5H), 3.94 (s, 1H), 4.10 (s, 2H), 4.16 – 4.30 (m, 4H), 4.38 (d, J = 3.2 Hz, 3H), 4.73 (d, J = 9.6 Hz, 1H), 5.06 – 5.18 (m, 1H), 5.50 (t, J = 9.6 Hz, 1H), 5.60 (d, J = 8.0 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 7.14 – 7.27 (m, 3H), 7.33 (t, J = 7.6 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 6.4 Hz, 1H), 7.74 (t, J = 6.4 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 7.99 (s, 1H), 8.08 - 8.19 (m, 2H), 8.25 (s, 1H). ES+(M+1 = 1873). Step 10 - Intermediate Compound 151: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,-6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-2-amino-6-oxohexanamido)propanamido)methyl)-4-(((((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000245_0001
In a round bottom flask, K2CO3 (0.885 g, 6.41 mmol) was added to Intermediate Compound 150 (2.4 g, 1.28 mmol) in MeOH (20 mL)/water (5.00 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The reaction mixture was adjusted to pH = 7 with 0.1M HCl. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC to afford Intermediate Compound 151 (1.000 g, 51.7 %) as a colourless oil.1H NMR (300 MHz, DMSO) δ 1.38 (s, 9H), 1.50 (s, 2H), 1.62 (s, 2H), 2.28 – 2.46 (m, 4H), 3.24 (s, 7H), 3.34 (d, J = 9.3 Hz, 2H), 3.39 – 3.55 (m, 47H), 3.59 (d, J = 9.4 Hz, 8H), 3.85 (q, J = 5.7, 7.2 Hz, 7H), 4.09 (s, 2H), 4.19 (d, J = 10.4 Hz, 1H), 4.38 (s, 4H), 4.45 – 4.53 (m, 1H), 4.61 (d, J = 5.5 Hz, 1H), 4.87 – 5.03 (m, 2H), 7.09 (d, J = 8.2 Hz, 1H), 7.25 (d, J = 9.6 Hz, 2H), 7.36 (s, 1H), 7.63 (d, J = 6.4 Hz, 1H), 8.18 (s, 3H), 8.29 (d, J = 6.8 Hz, 1H), 8.53 (s, 1H), 9.27 (s, 1H). ES+(M+1 = 1511). Step 11 - Intermediate Compound 152: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6- oxohexanamido)propanamido)methyl)-4-(((((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000246_0001
In a round bottom flask, Intermediate Compound 151 (850 mg, 0.56 mmol) and DIEA (0.098 mL, 0.56 mmol) in MeCN (10 mL) was added 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-propanoate (225 mg, 0.84 mmol) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to Intermediate Compound 152 (710 mg, 76 %) as a colourless oil.1H NMR (400 MHz , DMSO) δ 1.38 (s, 13H), 2.05 (d, J = 7.2 Hz, 2H), 2.29 - 2.40 (m, 6H), 3.25 (d, J = 5.6 Hz, 9H), 3.47 – 3.53 (m, 50H), 3.54 - 3.64 (m, 10H), 3.82 (d, J = 6.4 Hz, 5H), 4.09 (s, 2H), 4.38 (d, J = 4.0 Hz, 5H), 4.95 (t, J = 9.2 Hz, 2H), 6.98 (s, 2H), 7.07 (d, J = 8.4 Hz, 1H), 7.25 (s, 1H), 7.33 (s, 1H), 7.63 (d, J = 6.6 Hz, 1H), 7.99 (s, 1H), 8.10 – 8.36 (m, 4H). ES+( M+1 = 1662). Step 12 - Intermediate Compound 153: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6- oxohexanamido)propanamido)methyl)-4-(((((3S,3aR,6S,6aR)-6-aminohe-xahydrofuro[3,2-b]furan-3- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000246_0002
In a round bottom flask, Intermediate Compound 152 (650 mg, 0.39 mmol) was added to DCM (10 mL)/TFA (2.000 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure and afforded crude Intermediate Compound 153 (530 mg, 87 %) as a pale yellow oil.1H NMR (400 MHz, DMSO) δ 1.24 (d, J = 6.8 Hz, 7H), 1.41 – 1.46 (d, 2H), 2.05 (d, J = 7.0 Hz, 2H), 2.32 (t, J = 6.4 Hz, 4H), 2.40 (t, J = 7.2 Hz, 2H), 3.24 (s, 5H), 3.42 – 3.46 (m, 3H), 3.47 – 3.53 (m, 43H), 3.55-3.62 (m, 8H), 3.79 – 3.91 (m, 6H), 4.07 – 4.15 (m, 3H), 4.21 - 4.29 (m, 1H), 4.38 (d, J = 2.8 Hz, 5H), 4.78 (s, 1H), 4.94 (s, 2H), 5.16 (s, 1H), 5.49 (s, 1H), 6.98 (s, 2H), 7.06 (d, J = 8.4 Hz, 1H), 7.18 – 7.27 (m, 3H), 7.60 (d, J = 6.8 Hz, 1H), 7.95 (t, J = 5.6 Hz, 1H), 8.10 – 8.21 (m, 3H). ES+(M+1 = 1562). Step 13 - Compound LP9: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6- oxohexanamido)propanamido)methyl)-4-(((((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido)hexahydrofuro[3,2-b]furan-3-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000247_0001
In a round bottom flask, 1-Hydroxypyrrolidine-2,5-dione (73.4 mg, 0.64 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 350 mg, 0.56 mmol) and DIC (0.099 mL, 0.64 mmol) in DMA (3 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. Intermediate Compound 153 (498 mg, 0.32 mmol) and DIEA (0.334 mL, 1.91 mmol) was added into above mixture, then the resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by preparative HPLC to afford Compound LP9 (64.0 mg, 9.25 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.16 - 1.25 (m, 7H), 1.36 – 1.57 (m, 4H), 1.62 – 1.72 (m, 2H), 2.03 (t, J = 6.8 Hz, 2H), 2.10 – 2.18 (m, 1H), 2.24 – 2.42 (m, 7H), 2.61 – 2.72 (m, 2H), 2.92 – 3.07 (m, 2H), 3.23 – 3.32 (m, 10H), 3.38 – 3.43 (m, 3H), 3.46 – 3.52 (m, 42H), 3.56 – 3.67 (m, 9H), 3.79 – 3.96 (m, 6H), 3.99 (s, 3H), 4.05 – 4.18 (m, 5H), 4.23 (t, J = 3.6 Hz, 2H), 4.33 – 4.44 (m, 3H), 4.48 (s, 2H), 4.58 (d, J = 2.0 Hz, 1H), 4.72 (d, J = 7.6 Hz, 1H), 4.88 – 5.03 (m, 3H), 5.12 (s, 1H), 5.21 – 5.31 (m, 1H), 5.37 (s, 1H), 5.46 (d, J = 3.6 Hz, 1H), 6.97 (s, 2H), 7.07 (d, J = 8.4 Hz, 1H), 7.20 – 7.32 (m, 2H), 7.60 – 7.71 (m, 2H), 7.86 – 8.02 (m, 3H), 8.08 – 8.28 (m, 4H), 8.91 (s, 1H), 13.26 (s, 1H), 14.06 (s, 1H). ES+((M+1)/2 = 1086). Synthesis of Intermediate Compound 158
Figure imgf000248_0001
Step 1 – Intermediate Compound 154: N-((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3-yl)- 2-(methoxymethyl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontanamide
Figure imgf000248_0002
To a 250 mL round bottom flask was added Intermediate Compound 70 (1.50 g, 10.40 mmol) in dry THF (25 mL) under nitrogen gas. To the solution was added sodium hydrogen carbonate (1.748 g, 20.81 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-oate (CAS No. : 2207596-93-6; 7.13 g, 10.40 mmol) in portions under nitrogen gas and stirred at 20 °C for 6 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was quenched by addition of methanol (10 mL). The reaction mixture was diluted with methanol (20 mL) filtered through celite pad. Celite pad was washed with methanol (50 mL). Filtrate was dried over magnesium sulfate. Solvent was removed under reduced pressure to get crude product. The crude product was purified via silica gel column eluting with 0-10% methanol in DCM with 3% triethylamine, to give Intermediate Compound 154 (4.00 g, 53.8 %).1H NMR (500 MHz, MeOD) δ 4.64 – 4.59 (m, 1H), 4.45 (dd, J = 4.1, 1.3 Hz, 1H), 4.29 (dt, J = 4.1, 1.9 Hz, 1H), 3.96 (ddd, J = 10.2, 9.3, 4.9 Hz, 2H), 3.81 – 3.74 (m, 3H), 3.73 – 3.62 (m, 45H), 3.61 – 3.56 (m, 2H), 3.45 (dt, J = 3.8, 1.8 Hz, 1H), 3.40 (s, 3H), 2.52 – 2.47 (m, 2H). LCMS (ESI) m/z 715.6 (M + H)+. Step 2 – Intermediate Compound 155: 1-allyl 6-(tert-butyl) (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)hexanedioate
Figure imgf000249_0001
To a 250 mL round bottom flask was added (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- (tert-butoxy)-6-oxohexanoic acid (5 g, 11.38 mmol) in dry DMF (20 mL) under nitrogen gas. To the solution was added potassium carbonate (3.14 g, 22.75 mmol) and 3-bromoprop-1-ene (1.485 mL, 17.06 mmol) in portions under nitrogen gas and stirred at 20 °C for 16 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with water (500 mL) and organic layer was extracted with ethyl acetate (2x 300 mL), washed with water (300 mL), brine (200 mL) and dried over sodium sulfate (20 g). The solvent was removed to get crude product. The crude product was purified via silica gel column by using 0 -20% ethyl acetate in hexanes to give Intermediate Compound 155 (5.10 g, 93 %).1H NMR (500 MHz, CDCl3) δ 7.79 – 7.73 (m, 2H), 7.61 (q, J = 3.9 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.32 (tt, J = 7.4, 1.2 Hz, 2H), 5.91 (ddt, J = 16.5, 10.9, 5.8 Hz, 1H), 5.42 – 5.23 (m, 3H), 4.66 (d, J = 5.8 Hz, 2H), 4.40 (q, J = 4.8 Hz, 3H), 4.23 (t, J = 7.1 Hz, 1H), 2.26 (t, J = 7.2 Hz, 2H), 1.96 – 1.82 (m, 1H), 1.72 (dq, J = 13.5, 6.1 Hz, 3H), 1.60 – 1.47 (m, 1H), 1.45 (s, 9H). LCMS (ESI) m/z 480.2 (M + H)+. Step 3 - Intermediate Compound 156: (S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- (allyloxy)-6-oxohexanoic acid
Figure imgf000249_0002
To a 100 mL round bottom flask was added Intermediate Compound 155 (5 g, 10.43 mmol) in dry THF (20 mL) under nitrogen gas. To the solution was added HCl (13.03 mL, 52.13 mmol), 4 molar in dioxane under nitrogen gas and stirred at 20 °C for 6 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with water (200 mL) and organic layer was extracted with dichloromethane (2x 300 mL), washed with brine (200 mL) and dried over sodium sulfate (20 g). The solvent was removed to afford Intermediate Compound 156 (4.20 g, 95 %) as a white solid.
Figure imgf000249_0003
NMR (500 MHz, CDCl3) δ 7.75 (dq, J = 7.6, 1.0 Hz, 2H), 7.62 – 7.52 (m, 2H), 7.42 – 7.35 (m, 2H), 7.30 (tt, J = 7.4, 1.2 Hz, 2H), 5.90 (ddt, J = 16.4, 10.8, 5.8 Hz, 1H), 5.48 (d, J = 8.4 Hz, 1H), 5.37 – 5.20 (m, 2H), 4.64 (d, J = 5.8 Hz, 2H), 4.40 (d, J = 7.2 Hz, 3H), 4.22 (t, J = 7.0 Hz, 1H), 2.45 – 2.24 (m, 2H), 1.93 (p, J = 5.6 Hz, 1H), 1.72 (td, J = 13.9, 6.8 Hz, 3H). (ESI) m/z 424.5 (M - H)-. Step 4 - Intermediate Compound 157: allyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- (((3S,3aR,6S,6aR)-6-(33-methyl-1-(l1-oxidaneyl)-3,6,9,12,15,18,21,24,27,30,33l3- undecaoxahexatriacontan-36-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-6-oxohexanoate
Figure imgf000250_0001
To a 100 mL round bottom flask was added Intermediate 156 (1.925 g, 4.55 mmol) under nitrogen gas. To the solution was added HATU (1.862 g, 4.90 mmol) followed by DIPEA (1.222 mL, 6.99 mmol). The reaction mixture was stirred at room temperature for 15 min then Intermediate Compound 154 (2.5 g, 3.50 mmol) was added and reaction mixture was stirred at 23 °C for 3 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with DCM (300 mL), washed with water (200 mL), organic layer was extracted (2 x 100 mL), washed with Brine (50 mL), dried over sodium sulfate (5 g). Solvent was removed under reduced pressure to obtain crude product. The crude product was purified via silica gel column by using 0-10% methanol in DCM to give Intermediate Compound 157 (3.40 g, 87 %) as colorless gum.1H NMR (500 MHz, MeOD) δ 7.86 (dd, J = 7.6, 1.2 Hz, 2H), 7.74 (t, J = 7.8 Hz, 2H), 7.46 (td, J = 7.5, 1.4 Hz, 2H), 7.38 (tt, J = 7.5, 1.3 Hz, 2H), 6.05 – 5.93 (m, 1H), 5.39 (dq, J = 17.2, 1.6 Hz, 1H), 5.28 (dq, J = 10.5, 1.4 Hz, 1H), 4.73 – 4.65 (m, 2H), 4.58 (qd, J = 4.1, 1.0 Hz, 2H), 4.46 (dd, J = 10.6, 7.0 Hz, 1H), 4.40 (dd, J = 10.6, 7.0 Hz, 1H), 4.36 – 4.31 (m, 2H), 4.31 – 4.24 (m, 2H), 4.01 (ddd, J = 9.6, 5.0, 1.1 Hz, 2H), 3.84 – 3.73 (m, 5H), 3.72 – 3.60 (m, 44H), 3.60 – 3.56 (m, 2H), 3.41 (s, 3H), 2.49 (td, J = 6.0, 1.9 Hz, 2H), 2.31 (hept, J = 7.2 Hz, 2H), 1.96 – 1.85 (m, 1H), 1.85 – 1.68 (m, 3H). LCMS (ESI) m/z 1121.3 (M + H)+. Step 5 - Intermediate Compound 158: (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- (((3S,3aR,6S,6aR)-6-(33-methyl-1-(l1-oxidaneyl)-3,6,9,12,15,18,21,24,27,30,33l3- undecaoxahexatriacontan-36-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-6-oxohexanoic acid
Figure imgf000250_0002
To a 50 mL round bottom flask was added Intermediate Compound 157 (4.3 g, 3.84 mmol) in dry DCM (10 mL) under nitrogen gas. To the solution was added triethylamine (0.535 mL, 3.84 mmol) followed by triphenylphosphine (0.101 g, 0.38 mmol). To the reaction mixture was added Pd(PPh3)4 (0.444 g, 0.38 mmol) then formic acid (0.147 mL, 3.84 mmol) was added and reaction mixture was stirred at 23 °C for 6 hrs. LC-MS analysis showed formation of desired product and completion of reaction. Solvent was removed under reduced pressure to get crude products. The crude product was purified via silica gel column by using 0-10% methanol in DCM with 3% formic acid to give Intermediate Compound 158 (3.50 g, 84 %) as white solid.1H NMR (500 MHz, DMSO) δ 8.12 (dd, J = 10.2, 7.0 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 7.74 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 8.1 Hz, 1H), 7.46 – 7.38 (m, 2H), 7.34 (td, J = 7.4, 1.2 Hz, 2H), 4.38 (s, 2H), 4.32 – 4.20 (m, 3H), 4.11 (ddt, J = 7.2, 4.8, 2.1 Hz, 2H), 3.93 (td, J = 8.4, 4.6 Hz, 1H), 3.85 (dd, J = 9.3, 5.1 Hz, 2H), 3.63 – 3.57 (m, 4H), 3.54 – 3.45 (m, 42H), 3.45 – 3.40 (m, 2H), 3.24 (s, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.09 (s, 3H), 1.68 (d, J = 9.1 Hz, 1H), 1.64 – 1.48 (m, 3H). LCMS (ESI) m/z 1080.6 (M + H)+. Example 48: Synthesis of Compound LP10:
Figure imgf000251_0001
Figure imgf000252_0001
Step 1 & 2 - Intermediate Compound 160: (9H-fluoren-9-yl)methyl (2-(((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)carbamate
Figure imgf000252_0002
To 500 mL round bottom flask, Intermediate Compound 70 [(3S,3aR,6S,6aR)-hexahydrofuro[3,2- b]furan-3,6-diamine] (10.97 g, 76.07 mmol) was added slowly to 2,5-dioxopyrrolidin-1-yl (((9H- fluoren-9-yl)methoxy)carbonyl)glycinate (10 g, 25.36 mmol) and DIEA (8.86 mL, 50.71 mmol) in DMA (100 mL) at 0°C over a period of 3 minutes under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The reaction mixture was diluted with EtOAc (400 mL), and washed sequentially with water (400 mL x 2) to give Intermediate Compound 159 in organic layer. The organic layer (Intermediate Compound 159) was added to (Boc)2O (11.77 mL, 50.71 mmol). The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure.The crude product was purified by flash C18-flash chromatography, elution gradient 10 to 100% MeCN in water, to afford Intermediate Compound 160 (7.00 g, 52.7 %) as a yellow solid.1 H NMR (400 MHz, DMSO) δ 1.39 (s, 10H), 3.52 – 3.70 (m, 5H), 4.02 – 4.08 (m, 2H), 4.19 – 4.32 (m, 3H), 4.35 – 4.41 (m, 2H), 7.22 (d, J = 5.6 Hz, 1H), 7.34 (d, J = 7.2 Hz, 2H), 7.40 – 7.46 (m, 2H), 7.48 (t, J = 6.0 Hz, 1H), 7.72 (d, J = 7.2 Hz, 2H), 7.90 (d, J = 7.2 Hz, 2H), 8.15 (d, J = 7.2 Hz, 1H). ES+(M+1 = 524). Step 3 - Intermediate Compound 161: tert-butyl ((3S,3aR,6S,6aR)-6-(2-aminoacetamido)- hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000253_0001
To 500 mL round bottom flask, dimethylamine (8.38 g, 114.59 mmol) was added to Intermediate Compound 160 (6 g, 11.46 mmol) in DCM (50 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 161 (3.00 g, 87 %) as a yellow solid.1H
Figure imgf000253_0002
NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.54 – 2.61 (m, 2H), 3.51 – 3.56 (m, 2H), 3.74 – 3.93 (m, 3H), 4.05 – 4.23 (m, 1H), 4.35 – 4.49 (m, 2H), 7.21 (d, J = 5.6 Hz, 1H), 7.41 – 7.53 (m, 1H), 7.80-7.89 (m, 1H), 7.98 (s, 1H). ES+(M+1 = 302). Step 4 - Intermediate Compound 162: (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)-methyl)-4-((((2-(((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2- oxoethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000253_0003
In a 500 mL round bottom flask, DIEA (1.739 mL, 9.96 mmol) was added to Intermediate Compound 147 (6.77 g, 7.30 mmol) and Intermediate Compound 161 in DMA (20 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 3 hours. The reaction mixture was purified by flash C18-flash chromatography, elution gradient 10 to 100% MeCN in water, to afford Intermediate Compound 162 (3.50 g, 48.4 %) as a yellow solid.1 H NMR (400 MHz, DMSO-d6) δ 8.12-8.25(m, 2H), 7.89 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 7.6 Hz, 2H), 7.42 – 7.48 (m, 2H), 7.39 – 7.29 (m, 3H), 7.28 – 7.12 (m, 3H), 7.05 (d, J = 8.4 Hz, 1H), 5.61 (d, J = 8.0 Hz, 1H), 5.50 (t, J = 9.6 Hz, 1H), 5.23 – 5.02 (m, 2H), 4.95 (s, 2H), 4.74 (d, J = 10.0 Hz, 1H), 4.40 (d, J = 4.0 Hz, 1H), 4.35 (d, J = 4.0 Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 4.23 (d, J = 6.4 Hz, 1H), 4.09 – 4.12 (m, 2H), 3.90 – 3.74 (m, 3H), 3.71 – 3.49 (m, 7H), 3.25 – 3.29 (m, 2H), 2.45 – 2.28 (m, 2H), 2.15 – 1.91 (m, 10H), 1.39 (s, 10H). ES+(M+1 = 1090). Step 5 - Intermediate Compound 163: (2S,3R,4S,5S,6S)-2-(2-((3-aminopropanamido)methyl)-4-((((2- (((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)-hexahydrofuro[3,2-b]furan-3-yl)amino)-2- oxoethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000254_0001
In a round bottom flask, 1,8-Diazabicyclo[5.4.0]undec-7-ene (0.209 g, 1.38 mmol) was added to Intermediate Compound 162 (1.5 g, 1.38 mmol) in 1,4-dioxane (15 mL) and THF (3.75 mL) at 0°C over a period of 3 min under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. Reaction mixture was adjusted to weak acidic solution with 0.1M HCl, then the solvent was removed under reduced pressure to Intermediate Compound 163 (1.000 g, 84 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (s, 14H), 1.97 - 2.14 (m, 9H), 2.64 - 2.76 (m, 3H), 3.03 (d, J = 6.7 Hz, 3H), 3.48 (t, J = 6.0 Hz, 3H), 3.82 - 3.91 (m, 4H), 4.32 - 4.42 (m, 3H), 4.75 (d, J = 9.6 Hz, 1H), 4.96 (d, J = 4.0 Hz, 2H), 5.03 - 5.21 (m, 2H), 5.27 - 5.34 (m, 1H), 5.50 (t, J = 9.6 Hz, 1H), 5.62 (d, J = 7.6 Hz, 1H), 6.13 - 6.22 (m, 1H), 7.06 (d, J = 8.4 Hz, 1H), 8.11 - 8.33 (m, 1H), 8.40 - 8.52 (m, 1H), 9.76 (s, 2H). ES+(M+1 = 868). Step 6 - Intermediate Compound 164: (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-((((2- (((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)-hexahydrofuro[3,2-b]furan-3-yl)amino)-2- oxoethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000254_0002
In a round bottom flask, Intermediate Compound 163 (1 g, 1.15 mmol) was added to Intermediate Compound 158 (1.369 g, 1.27 mmol), HATU (0.657 g, 1.73 mmol) and DIEA (0.604 mL, 3.46 mmol) in DMA (20 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 5 hours. The reaction mixture was purified by flash C18-flash chromatography, elution gradient 10 to 100% MeCN in water, to afford Intermediate Compound 164 (1.300 g, 58.5 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (s, 14H), 1.44 – 1.62 (m, 1H), 1.95 – 2.11 (m, 14H), 2.27 – 2.43 (m, 5H), 3.24 (s, 4H), 3.32 – 3.39 (m, 13H), 3.43 (dd, J = 3.6, 6.0 Hz, 2H), 3.48 (d, J = 9.8 Hz, 11H), 3.55 – 3.68 (m, 14H), 3.81 – 3.93 (m, 7H), 4.10 (t, J = 6.7 Hz, 6H), 4.15 – 4.30 (m, 5H), 4.31 – 4.44 (m, 6H), 4.73 (d, J = 9.6 Hz, 1H), 4.95 (s, 3H), 5.03 – 5.24 (m, 2H), 5.49 (t, J = 9.6 Hz, 1H), 5.60 (d, J = 7.9 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 7.14 – 7.28 (m, 3H), 7.28 – 7.37 (m, 4H), 7.37 – 7.50 (m, 4H), 7.73 (t, J = 6.4 Hz, 2H), 7.89 (d, J = 7.6 Hz, 3H), 7.95 (d, J = 5.7 Hz, 1H), 8.09 (d, J = 6.9 Hz, 1H), 8.14 (dd, J = 3.0, 7.2 Hz, 3H), 8.21 (t, J = 6.0 Hz, 1H). ES+(M+1 = 1930). Step 7 - Intermediate Compound 165: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro-[3,2-b]furan- 3-yl)amino)-2-amino-6-oxohexanamido)propanamido)-methyl)-4-((((2-(((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)carbamoyl)- oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000255_0001
In a round bottom flask, K2CO3 (0.931 g, 6.74 mmol) was added to Intermediate Compound 164 (1.3 g, 0.67 mmol) in MeOH (50 mL) and water (12.50 mL) under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The reaction mixture was adjusted to neutral with 0.1M HCl. The solvent was removed under reduced pressure to afford Intermediate Compound 165 (1.000 g, 95 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (s, 7H), 1.50 – 1.58 (m, 18H), 2.04 (d, J = 7.0 Hz, 4H), 2.25 – 2.47 (m, 14H), 3.25 (s, 7H), 3.39 – 3.49 (m, 17H), 3.80 – 3.86 (m, 10H), 4.05 – 4.16 (m, 9H), 4.22 – 4.48 (m, 4H), 4.48 – 4.66 (m, 2H), 4.84 – 5.12 (m, 6H), 5.34 – 5.68 (m, 1H), 7.01 – 7.10 (m, 1H), 7.11 – 7.31 (m, 5H), 7.33 – 7.45 (m, 2H), 7.80 – 7.98 (m, 1H), 8.05 – 8.27 (m, 4H), 8.31 – 8.70 (m, 1H), 9.55 (s, 1H). ES+(M+1 = 1567). Step 8 - Intermediate Compound 166: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,-6aR)-6- (2,5,8,11,14172023 26293235dodecaoxaoctatriacontan38amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6- oxohexanamido)propanamido)methyl)-4-((((2-(((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-oxoethyl)carba- moyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000256_0001
In a round bottom flask, DIEA (0.223 mL, 1.28 mmol) was added to Intermediate Compound 165 (1 g, 0.64 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (0.255 g, 0.96 mmol) in DMA (20 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The reaction mixture was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.1% FA), to afford crude product. The crude product was purified again by preparative HPLC to afford Intermediate Compound 166 (0.490 g, 44.7 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.24 (s, 1H), 1.39 (s, 10H), 1.95 – 2.10 (m, 3H), 2.22 – 2.41 (m, 6H), 3.24 (s, 4H), 3.41 – 3.65 (m, 60H), 3.76 – 3.94 (m, 6H), 4.12 (d, J = 16.4 Hz, 4H), 4.25 – 4.44 (m, 7H), 4.94 (s, 3H), 5.24 (s, 1H), 5.52 (s, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.16 – 7.38 (m, 5H), 7.93 (t, J = 5.6 Hz, 1H), 8.02 – 8.31 (m, 5H), 12.80 (s, 1H). ES+(M+1 = 1719). Step 9 - Intermediate Compound 167: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro-[3,2-b]furan- 3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6- oxohexanamido)propanamido)methyl)-4-((((2-(((3S,3aR,6S,6aR)-6-aminohexahy-drofuro[3,2- b]furan-3-yl)amino)-2-oxoethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid
Figure imgf000256_0002
In a round bottom flask, TFA (5 mL, 1.43 mmol) was added to Intermediate Compound 166 (490 mg, 0.29 mmol) in DCM (5 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure to afford Intermediate Compound 167 (400 mg, 87 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.34 – 1.61 (m, 3H), 2.02 – 2.38 (m, 9H), 2.79 (s, 6H), 2.95 (s, 5H), 3.38 – 3.68 (m, 54H), 3.73 – 4.01 (m, 7H), 4.06 – 4.16 (m, 5H), 4.19 – 4.41 (m, 4H), 4.50 – 4.66 (m, 2H), 4.91 – 5.06 (m, 3H), 7.00 (s, 2H), 7.06 (d, J = 8.4 Hz, 1H), 7.15 – 7.27 (m, 2H), 7.38 – 7.41 (m, 1H), 8.08 – 8.28 (m, 7H). ES+(M+1 = 1619). Step 10 - Compound LP10: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6- oxohexanamido)propanamido)methyl)-4-((((2-oxo-2-(((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy- 7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido)hexahydrofuro[3,2-b]furan-3-yl)amino)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000257_0001
In a round bottom flask, 1-Hydroxypyrrolidine-2,5-dione (58.7 mg, 0.51 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 160 mg, 0.25 mmol) and DIC (0.079 mL, 0.51 mmol) in DMA (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. LCMS showed the NHS ester formed. Intermediate Compound 167 (413 mg, 0.25 mmol) and DIEA (0.223 mL, 1.27 mmol) was added, the resulting mixture was stirred at 25 °C for 2 hours. The resulting mixture was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford crude product (230.0 mg) as a red solid. The crude product was purified by preparative HPLC to afford Compound LP10 (70.0 mg, 12.32 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.12 – 1.20 (m, 11H), 1.42 (s, 3H), 1.54 (s, 1H), 1.68 (d, J = 6.0 Hz, 2H), 2.04 (s, 2H), 2.10 – 2.18 (m, 1H), 2.30 – 2.39 (m, 5H), 2.40 (t, J = 7.2 Hz, 2H), 2.70 (d, J = 12.8 Hz, 1H), 2.99 – 3.03 (m, 3H), 3.24 (s, 4H), 3.27 (d, J = 5.6 Hz, 4H), 3.28 (s, 3H), 3.30 – 3.33 (m, 42H), 3.36 – 3.50 (m, 12H), 3.54 – 3.63 (m, 6H), 3.67 (s, 3H), 3.79 – 3.98 (m, 6H), 4.00 (s, 1H), 4.23 – 4.41 (m, 5H), 4.47 – 4.53 (m, 1H), 4.69 (s 1H) 502 – 510 (m 2H) 522 – 531 (m 2H) 540 (s 1H), 5.45 (s, 2H), 6.97 (s, 2H), 7.07 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.4 Hz, 3H), 7.30 (s, 1H), 7.89 – 7.98 (m, 3H), 8.15 – 8.20 (m, 5H), 13.27 (s, 1H), 14.08 (s, 1H). ES+[(M/2) + 1] = 1115. Example 49: Synthesis of Compound LP11
Figure imgf000258_0001
Step 1 - Intermediate Compound 168: (9H-fluoren-9-yl)methyl (1-(((3R,3aS,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)oxy)-3,6,9,12-tetraoxo-2,5,8,11- tetraazatridecan-13-yl)carbamate
Figure imgf000258_0002
In a round bottom flask, Intermediate Compound 137 (2.416 g, 7.29 mmol) was added to (((9H- fluoren-9-yl)methoxy)carbonyl)-glycylglycylglycine (2 g, 4.86 mmol), HATU (3.70 g, 9.72 mmol) and DIEA (2.55 mL, 14.58 mmol) in DMSO (30 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 168 (3.10 g, 88 %) as a pale-yellow solid.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 3.40 (t, J = 8.1 Hz, 1H), 3.63 – 3.87 (m, 12H), 4.00 - 4.08 (m, 1H), 4.19 – 4.27 (m, 1H), 4.29 – 4.32 (m, 2H), 4.51 – 4.68 (m, 3H), 7.18 (s, 1H), 7.29 - 7.38 (m, 2H), 7.38 - 7.46 (m, 2H), 7.57 (t, J = 6.0 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.10 - 8.20 (m, 3H), 8.61 (t, J = 6.7 Hz, 1H). ES+(M+1 = 725). Step 2 - Intermediate Compound 169: tert-butyl ((3S,3aR,6R,6aS)-6-((13-amino-3,6,9,12-tetraoxo- 2,5,8,11-tetraazatridecyl)oxy)-hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000259_0001
In a round bottom flask, diethylamine (6.05 g, 82.79 mmol) was added to Intermediate Compound 168 (3 g, 4.14 mmol) in MeCN (30 mL)/water (30 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 16 hours. The solvent was removed under reduced pressure. The dried solid was titurated with MTBE (50 ml) and filtered to afford crude Intermediate Compound 169 (2.000 g, 96 %).1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 3.17 (s, 2H), 3.38 (s, 1H), 3.66 (t, J = 5.6 Hz, 1H), 3.71 – 3.85 (m, 9H), 4.00 - 4.09 (m, 1H), 4.31 (d, J = 4.4 Hz, 1H), 4.50 – 4.69 (m, 3H), 7.16 – 7.22 (m, 1H), 8.14 - 8.25 (m, 3H), 8.61 (t, J = 6.7 Hz, 1H). ES+(M+1 = 503). Step 3 - Intermediate Compound 170: tert-butyl ((3S,3aR,6R,6aS)-6-((17-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaheptadecyl)oxy)hexahydrofuro[3,2-b]furan- 3-yl)carbamate
Figure imgf000259_0002
In a round bottom flask, 2,5-Dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoate (CAS No.55750-62-4; 1.430 g, 5.37 mmol) was added to Intermediate Compound 169 (1.8 g, 3.58 mmol) and DIEA (0.626 mL, 3.58 mmol) in DMSO (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18- flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 170 (1.600 g, 68.3 %) as a pale-yellow solid.1H NMR (400 MHz, DMSO) δ 1.38 (s, 9H), 2.40 - 2.44 (m, 2H), 3.60 – 3.83 (m, 15H), 3.99 - 4.09 (m, 1H), 4.31 (d, J = 4.3 Hz, 1H), 4.50 – 4.69 (m, 3H), 7.01 (d, J = 6.1 Hz, 2H), 7.18 (d, J = 5.7 Hz, 1H), 8.10 - 8.19 (m, 3H), 8.27 (t, J = 5.7 Hz, 1H), 8.61 (t, J = 6.7 Hz, 1H). ES+( M+1 = 654). Step 4 - Intermediate Compound 171: N-(1-(((3R,3aS,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3- yl)oxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamide
Figure imgf000259_0003
In a round bottom flask, Intermediate Compound 170 (300 mg, 0.46 mmol) was added to HFIP (6 mL) at 25°C under nitrogen. The resulting mixture was stirred at 98 °C for 5 hours. The mixture is blown dry with nitrogen and then freeze-dried with water to obtain crude Intermediate Compound 171 (110 mg, 43.3 %).1H NMR (400 MHz, DMSO) δ 3.29 – 3.44 (m, 4H), 3.71 – 3.80 (m, 13H), 4.01 – 4.06 (m, 1H), 4.59 – 4.65 (m, 2H), 5.15 – 5.20 (m, 2H), 7.01 (d, J = 5.2 Hz, 2H), 8.12 – 8.22 (m, 5H). ES+(M+1 = 554). Step 5 – Compound LP11: (2S,4S)-N-((3S,3aR,6R,6aS)-6-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- 3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaheptadecyl)oxy)hexahydrofuro[3,2-b]furan-3-yl)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4’,3’:4,5]oxazolo-[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamide
Figure imgf000260_0001
In a round bottom flask, 1-Hydroxypyrrolidine-2,5-dione (27.5 mg, 0.24 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 100 mg, 0.16 mmol) and DIC (0.037 mL, 0.24 mmol) in DMA (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. Intermediate Compound 171 (88 mg, 0.16 mmol) and DIEA (0.028 mL, 0.16 mmol) was added, the resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by preparative HPLC to afford Compound LP11 (10.58 mg, 5.71 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.20 (d, J = 6.5 Hz, 3H), 1.59 - 1.72 (m, 2H), 2.11 - 2.19 (m, 1H), 2.23 - 2.31 (m, 1H), 2.42 (t, J = 7.5 Hz, 2H), 2.62 – 2.72 (m, 2H), 2.92 – 3.07 (m, 2H), 3.38 – 3.53 (m, 4H), 3.61 (t, J = 7.5 Hz, 2H), 3.66 – 3.82 (m, 11H), 3.89 – 3.96 (m, 2H), 4.00 (s, 3H), 4.07 (t, J = 6.1 Hz, 1H), 4.14 (d, J = 6.5 Hz, 2H), 4.23 (d, J = 2.2 Hz, 1H), 4.39 – 4.45 (m, 1H), 4.55 – 4.65 (m, 3H), 4.65 - 4.72 (m, 1H), 5.01 (t, J = 4.2 Hz, 1H), 5.24 (t, J = 4.7 Hz, 1H), 5.39 (s, 1H), 7.00 (s, 2H), 7.50-7.69 (m, 1H), 7.88 – 7.96 (m, 2H), 8.05 (d, J = 7.5 Hz, 1H), 8.15 (q, J = 6.2 Hz, 3H), 8.27 (t, J = 5.8 Hz, 1H), 8.63 (t, J = 6.7 Hz, 1H), 13.26 (s, 1H), 14.07 (s, 1H). ES+(M+1 = 1163). Example 50: Synthesis of Compound LP12
Figure imgf000261_0001
Step 1 - Intermediate Compound 172: tert-butyl ((3S,3aR,6S,6aR)-6-((S)-18-benzyl-1-(9H-fluoren-9- yl)-3,10,13,16,19-pentaoxo-2,7-dioxa-4,11,14,17,20-pentaazadocosan-22- amido)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000261_0002
In a round bottom flask, Intermediate Compound 107 (1.1 g, 1.96 mmol) was added to 3-(2-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)propanoic acid (0.695 g, 1.96 mmol), HATU (1.487 g, 3.91 mmol) and DIEA (1.024 mL, 5.87 mmol) in DMA (15 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 172 (1.500 g, 85 %) as a pale yellow solid.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.40 – 2.46 (m, 2H), 2.94 – 3.08 (m, 2H), 3.12 (q, J = 5.6 Hz, 2H), 3.35 – 3.41 (m, 4H), 3.58 – 3.87 (m, 13H), 4.21 (t, J = 6.9 Hz, 1H), 4.28 - 4.31 (m, 2H), 4.37 (d, J = 4.2 Hz, 1H), 7.17 – 7.21 (m, 1H), 7.25 (d, J = 3.1 Hz, 4H), 7.31 - 7.35 (m, 4H), 7.42 (t, J = 7.4 Hz, 3H), 7.70 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.03 - 8.07 (m, 1H), 8.17 (d, J = 7.0 Hz, 1H), 8.26 (t, J = 5.8 Hz, 1H). ES+(M+1 =900). Step 2 - Intermediate Compound 173: (9H-fluoren-9-yl)methyl ((S)-1-(((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)amino)-5-benzyl-1,4,7,10,13-pentaoxo-16-oxa-3,6,9,12- tetraazaoctadecan-18-yl)carbamate
Figure imgf000262_0001
In a round bottom flask, HCl in dioxane(4N) (20 mL) was added to Intermediate Compound 172 (610 mg, 0.68 mmol) in 10 mL DCM at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford crude Intermediate Compound 172 (1.100 g, 88 %) as a pale yellow solid.1H
Figure imgf000262_0002
NMR (400 MHz, DMSO) δ 1.07 (d, J = 7.0 Hz, 2H), 1.15 – 1.31 (m, 2H), 2.41 (t, J = 7.0 Hz, 2H), 2.99 - 3.08 (m, 1H), 3.12 (d, J = 4.2 Hz, 2H), 3.36 – 3.48 (m, 4H), 3.57 – 3.98 (m, 11H), 4.27 – 4.31 (m, 2H), 4.59 – 4.68 (m, 2H), 7.15 – 7.37 (m, 7H), 7.42 (t, J = 7.4 Hz, 2H), 7.70 (d, J = 7.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.01 – 8.24 (m, 3H), 8.33 (t, J = 6.1 Hz, 1H), 8.48 – 8.59 (m, 1H). ES+(M+1 =800). Step 3 - Intermediate Compound 174: (9H-fluoren-9-yl)methyl ((S)-5-benzyl-1,4,7,10,13-pentaoxo- 1-(((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy- 1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamido)hexahydrofuro[3,2-b]furan-3-yl)amino)-16-oxa-3,6,9,12- tetraazaoctadecan-18-yl)carbamate
Figure imgf000262_0003
In a round bottom flask, Intermediate Compound 173 (828 mg, 1.04 mmol) was added to the PNU- 159682 carboxylic acid (CAS No.1204819-92-0; 500 mg, 0.80 mmol), HATU (606 mg, 1.59 mmol) and DIEA (0.696 mL, 3.98 mmol) in DMA (10 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 174 (830 mg, 73.9 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.15 – 1.26 (m, 3H), 1.59 – 1.72 (m, 1H), 2.40 (s, 2H), 2.57 – 2.74 (m, 2H), 2.76 – 2.87 (m, 1H), 2.90 – 3.15 (m, 4H), 3.29 – 3.40 (m, 16H), 3.56 – 3.82 (m, 11H), 3.84 – 4.07 (m, 7H), 4.10 – 4.32 (m, 4H), 4.44 – 4.70 (m, 4H), 7.14 – 7.29 (m, 5H), 7.30 – 7.45 (m, 5H), 7.47 – 7.71 (m, 2H), 7.82 – 7.92 (m, 4H), 8.02 – 8.22 (m, 4H), 8.24 – 8.43 (m, 2H). ES+(M+1 =1410). Step 4 - Intermediate Compound 175 : (2S,4S)-N-((3S,3aR,6S,6aR)-6-((S)-18-amino-5-benzyl- 4,7,10,13-tetraoxo-16-oxa-3,6,9,12-tetraaz-aoctadecanamido)hexahydrofuro[3,2-b]furan-3-yl)- 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo-[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamide
Figure imgf000263_0001
In a round bottom flask, DBU (0.089 mL, 0.59 mmol) was added to Intermediate Compound 174 (830 mg, 0.59 mmol) in DMA (5 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 175 (450 mg, 64.4 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.13 – 1.27 (m, 3H), 1.56 – 1.95 (m, 2H), 2.15 (d, J = 15.3 Hz, 1H), 2.39 (d, J = 6.6 Hz, 2H), 2.55 – 2.74 (m, 3H), 2.77 – 3.07 (m, 4H), 3.22 – 3.45 (m, 10H), 3.46 – 3.53 (m, 2H), 3.55 – 3.81 (m, 12H), 3.84 – 4.07 (m, 7H), 4.11 – 4.27 (m, 3H), 4.36 – 4.71 (m, 4H), 4.89 – 5.38 (m, 1H), 7.15 – 7.31 (m, 5H), 7.41 – 7.63 (m, 1H), 7.73 – 7.98 (m, 2H), 8.03 – 8.51 (m, 6H). ES+(M+1 =1187). Step 5 - Compound LP12: (2S,4S)-N-((3S,3aR,6S,6aR)-6-((S)-5-benzyl-21-bromo-4,7,10,-13,20- pentaoxo-16-oxa-3,6,9,12,19-pentaazahenicosanamido)hexahydrofuro[3,2-b]furan-3-yl)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamide
Figure imgf000264_0001
In a round bottom flask, 2,5-Dioxopyrrolidin-1-yl 2-bromoacetate (103 mg, 0.43 mmol) was added to Intermediate Compound 175 (430 mg, 0.36 mmol) in DMA (3 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The crude product was purified by preparative HPLC to afford Compound LP12 (230 mg, 48.5 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.20 (d, J = 6.5 Hz, 3H), 1.62 -1.74 (m, 2H), 2.11 - 2.19 (m, 1H), 2.29 (d, J = 9.2 Hz, 1H), 2.40 (t, J = 6.5 Hz, 2H), 2.61 –2.71 (m, 2H), 2.75 – 2.85 (m, 1H), 2.92 – 3.10 (m, 3H), 3.21 (q, J = 5.7 Hz, 2H), 3.31 (s, 2H), 3.39 (t, J = 5.6 Hz, 3H), 3.49 – 3.54 (m, 1H), 3.57 – 3.80 (m, 12H), 3.85 (s, 2H), 3.87 – 3.97 (m, 3H), 4.01 (s, 3H), 4.10 – 4.18 (m, 3H), 4.23 (d, J = 2.2 Hz, 1H), 4.44 – 4.56 (m, 3H), 4.59 (d, J = 2.2 Hz, 1H), 5.02 (s, 1H), 5.25 (s, 1H), 5.38 (s, 1H), 7.17 – 7.23 (m, 1H), 7.26 (d, J = 5.9 Hz, 4H), 7.65 – 7.71 (m, 1H), 7.89 – 7.97 (m, 2H), 8.04 – 8.10 (m, 2H), 8.14 (t, J = 7.4 Hz, 3H), 8.24 – 8.32 (m, 2H), 13.27 (s, 1H), 14.08 (s, 1H). ES-(M-1 =1305). Example 51: Synthesis of Compound LP13
Figure imgf000264_0002
Figure imgf000265_0001
Step 1 - Intermediate Compound 178: (tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3- hydroxyazetidine-1-carboxylate
Figure imgf000265_0002
In a round bottom flask, tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (10 g, 49.44 mmol) was added to N-ethyl-N-isopropylpropan-2-amine (19.17 g, 148.33 mmol) and N- (benzyloxycarbonyloxy)succinimide (14.79 g, 59.33 mmol) in THF (150 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate Compound 178 (13.00 g, 78 %) as a yellow oil. m/z (ES+), [M+H]+ = 337. Step 2 - Intermediate Compound 179: tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-(2-(tert- butoxy)-2-oxoethoxy)azetidine-1-carboxylate
Figure imgf000266_0001
In a round bottom flask, Intermediate Compound 178 (13 g, 38.65 mmol) was added to tert-butyl 2-bromoacetate (9.05 g, 46.38 mmol) and sodium hydride (1.855 g, 77.29 mmol) in THF (150 mL) under nitrogen at 0 °C. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water (50 mL), extracted with EtOAc (3 x 150 mL), the top layer was dried over Na2SO4, filtered and evaporated to afford yellow oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 179 (9.00 g, 51.7 %) as a colourless oil. m/z (ES+), [M+H]+ = 451. Step 3 - Intermediate Compound 180: tert-butyl 3-(aminomethyl)-3-(2-(tert-butoxy)-2- oxoethoxy)azetidine-1-carboxylate
Figure imgf000266_0002
In a round bottom flask, Intermediate Compound 179 (9 g, 19.98 mmol) was added to ROYERTM Palladium Catalyst Powder (CAS 7440-05-3, 6.07 g, 19.98 mmol) in THF (120 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 6 hours. The reaction mixture was filtered through silica and washed with THF (3 x 50 mL). The solvent was removed under reduced pressure to afford Intermediate Compound 180 (6.00 g, 85 %) as a yellow oil. m/z (ES+), [M+H]+ = 317. Step 4 - Intermediate Compound 181: tert-butyl 3-(((((9H-fluoren-9- yl)methoxy)carbonyl)amino)methyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-1-carboxylate
Figure imgf000266_0003
In a round bottom flask, Intermediate Compound 180 (5 g, 15.80 mmol) was added to 9- fluorenylmethyl chloroformate (6.13 g, 23.70 mmol) and N,N-diisopropylethylamine (6.13 g, 47.41 mmol) in THF (50 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 181 (3.66 g, 43.0 %) as a white solid. m/z (ES+), [M+H]+ = 539. Step 5 - Intermediate Compound 182: 2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)- 1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetic acid
Figure imgf000267_0001
In a round bottom flask, Intermediate Compound 181 (615 mg, 1.14 mmol) in DCM (4 mL) was added TFA (4 mL). The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated under reduced pressure to give a crude product 2-((3-(((((9H- fluoren-9- yl)methoxy)carbonyl)amino)methyl)azetidin-3-yl)oxy)acetic acid (182-A) which was used without further purification. A solution of 1-[(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-yl)oxy]-2,5-pyrrolidinedione (CAS No: 2207596-93-6; 708 mg, 1.03 mmol) and DIPEA (0.397 mL, 2.28 mmol) in DMF (5 mL) was added to crude 182-A (436 mg, 1.14 mmol). The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was purified by reverse phase column chromatography (5-60% MeCN in water +0.1% FA) to give Intermediate Compound 182 (654 mg, 66.5 %) as a colourless oil. m/z (ES+), [M+H]+ = 954.7 Step 6 (part 1) – Intermediate Compound 183-A: 2-((3-(amino-methyl)-1- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetic acid
Figure imgf000267_0002
In a round bottom flask, diethylamine (3.07 g, 41.97 mmol) was added to Intermediate Compound 182 (4 g, 4.20 mmol) in THF (50 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure and afforded Intermediate Compound 183-A (2.60 g, 85 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 1.14 (t, J = 7.2 Hz, 2H), 1.75 (q, J = 3.3 Hz, 2H), 2.87 (q, J = 7.2 Hz, 2H), 3.23 (s, 3H), 3.38 – 3.73 (m, 53H). ES+(M+1 =731). Step 6 (part 2) - Intermediate Compound 183: 2-((3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamido)methyl)-1-(2,5,8,11,14,17,20,23,26,29,-32,35-dodecaoxaoctatriacontan-38- oyl)azetidin-3-yl)oxy)acetic acid
Figure imgf000268_0001
In a round bottom flask, 2,5-Dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanoate (0.911 g, 3.42 mmol) was added to Intermediate Compound 183-A (2.5 g, 3.42 mmol) and DIEA (0.597 mL, 3.42 mmol) in DMA (30 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to Intermediate Compound 183 (2.300 g, 76 %) as a pale-yellow oil.1H NMR (300 MHz, DMSO) δ 2.30 (d, J = 6.3 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 3.24 (s, 5H), 3.32 – 3.47 (m, 9H), 3.50 (s, 32H), 3.55 – 3.70 (m, 9H), 3.87 (s, 2H), 3.95 (d, J = 8.3 Hz, 2H), 7.00 (s, 2H), 8.59 (s, 1H). ES+(M+1 =882). Step 7 and 8 - Intermediate Compound 185: 2S,3S,4S,5R,6S)-6-(4-(((((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahyd-rofuro[3,2-b]furan-3-yl)carbamoyl)oxy)methyl)-2-((3-(2-((3-((3-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35- dodecaox-aoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)m-ethyl)phenoxy)- 3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000269_0001
In a round botto Om N fl Oask, 2 O,3 NH,5 O,6- NTe Otr OaflOuo NrHo Oph HOe Ono O HNl O (0 O. O5H94 O O gHH, 3.5 O8 m OmHNol) H w Oa Os H ad NdHeBdo tco Intermediate Compound 183 (1.578 g, 1.79 mmol) and DIC (0.558 mL, 3.58 mmol) in MeCN (10 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours to give crude Intermediate Compound 184. Then Intermediate Compound 149 (1.2 g, 1.79 mmol) and DIEA (0.625 mL, 3.58 mmol) in DMSO (10 mL) was added into the above solution of Intermediate Compound 184, then the resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.01%FA), to afford Intermediate Compound 185 as a pale yellow oil.1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 2.29 – 2.42 (m, 6H), 3.25 (s, 4H), 3.41 - 3.45 (m, 4H), 3.50 (s, 50H), 3.57 - 3.64 (m, 5H), 3.67 - 3.75 (m, 2H), 3.81 – 3.91 (m, 6H), 3.95 (d, J = 9.5 Hz, 1H), 4.06 (d, J = 9.4 Hz, 1H), 4.31 (d, J = 8.9 Hz, 1H), 4.38 (s, 2H), 4.95 (s, 3H), 5.25 (s, 1H), 5.54 (s, 1H), 6.99 (s, 2H), 7.06 (d, J = 8.3 Hz, 1H), 7.17 - 7.24 (m, 2H), 7.57 (d, J = 6.8 Hz, 1H), 7.87 (d, J = 6.2 Hz, 1H), 8.14 (s, 2H), 8.20 - 8.30 (m, 1H). ES+(M+1 =1535). Step 9 - Intermediate Compound 186: (2S,3S,4S,5R,6S)-6-(4-(((((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)carbamoyl)oxy)methyl)-2-((3-(2-((3-((3-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)propanamido)methyl)-1-(2,5,8,11,14,-17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)-propanamido)methyl)phenoxy)- 3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000269_0002
In a round bottom flask, Intermediate Compound 185 (1g, 0.65 mmol) was added to DCM (10 mL)/TFA (5.00 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford crude Intermediate Compound 186 (0.850 g, 91 %) as a pale yellow oil.1
Figure imgf000270_0001
H NMR (400 MHz, DMSO) δ 2.28 – 2.42 (m, 6H), 3.24 (s, 4H), 3.39 - 3.46 (m, 6H), 3.50 - 3.53(m, 49H), 3.58 – 3.66 (m, 6H), 3.70 - 3.79 (m, 4H), 3.82 – 3.99 (m, 7H), 4.06 (d, J = 9.3 Hz, 1H), 4.30 - 4.39 (m, 1H), 4.57 (s, 2H), 4.96 (d, J = 5.0 Hz, 3H), 6.99 (s, 2H), 7.06 (d, J = 8.4 Hz, 1H), 7.16 – 7.26 (m, 2H), 7.67 (d, J = 6.5 Hz, 1H), 7.87 (d, J = 6.1 Hz, 1H), 8.15 (d, J = 6.0 Hz, 3H), 8.22 (d, J = 7.1 Hz, 1H). ES+(M+1 =1435). Step 10 - Compound LP13:
Figure imgf000270_0002
In a round bottom flask, 1-Hydroxypyrrolidine-2,5-dione (147 mg, 1.27 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 400 mg, 0.64 mmol) and DIC (0.199 mL, 1.27 mmol) in DMA (3 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. Then Intermediate Compound 186 (731 mg, 0.51 mmol) and DIEA (0.668 mL, 3.82 mmol) was added into the mixture, then the obtained reaction solution was stirred at 25 °C for 2 hours. The crude product was purified by preparative HPLC to afford Compound LP13 (240 mg, 18.42 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.17 – 1.24 (m, 11H), 1.59 - 1.71 (m, 2H), 2.14 (d, J = 14.0 Hz, 1H), 2.25 - 2.42 (m, 7H), 2.61 – 2.72 (m, 2H), 2.92 – 3.07 (m, 3H), 3.23 (s, 6H), 3.41 – 3.45 (m, 4H), 3.44-3.52 (m, 46H), 3.56 – 3.74 (m, 10H), 3.83 – 3.96 (m, 7H), 3.99 (s, 4H), 4.10 – 4.26 (m, 4H), 4.48 (s, 3H), 4.58 (d, J = 2.2 Hz, 1H), 4.65 (s, 1H), 4.87 – 5.03 (m, 3H), 5.09 (s, 1H), 5.22 – 5.30 (m, 1H), 5.37 (s, 1H), 5.44 (d, J = 3.9 Hz, 1H), 6.97 (s, 2H), 7.07 (d, J = 8.2 Hz, 1H), 7.22 – 7.32 (m, 2H), 7.60 - 7.71 (m, 2H), 7.84 – 7.94 (m, 3H), 8.11 (d, J = 7.7 Hz, 1H), 8.27 (d, J = 6.1 Hz, 1H), 9.14 (s, 1H), 13.26 (s, 1H), 14.06 (s, 1H). ES+(M/2+1 =1023). Example 52: Synthesis of Compound LP14
Figure imgf000271_0001
Step 1 - Intermediate Compound 188: (2S,3S,4S,5R,6S)-6-(2-((S)-12-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,10,13,17-tetraoxo-2,7-dioxa-4,11,14,18- tetraazanonadecan-19-yl)-4-(((((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)hexahydrofuro[3,2- b]furan-3-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000272_0001
In a round bottom flask, DIEA (0.994 mL, 5.69 mmol) was added to Intermediate Compound 151 (4.3 g, 2.85 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)ethoxy)propanoate (CAS No.1807521-05-6) (1.932 g, 4.27 mmol) in MeCN (80 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 5 to 100% MeCN in water, to afford Intermediate Compound 188 (3.60 g, 68.4 %) as a yellow oil.1H NMR (400 MHz, DMSO) δ 1.01 (d, J = 6.5 Hz, 11H), 1.38 (s, 13H), 2.08 (s, 7H), 2.24 – 2.49 (m, 5H), 3.13 (d, J = 6.2 Hz, 2H), 3.45 – 3.64 (m, 44H), 3.76 – 3.97 (m, 7H), 4.09 (s, 2H), 4.16 – 4.34 (m, 6H), 4.38 (d, J = 5.1 Hz, 6H), 4.66 (s, 1H), 4.96 (d, J = 13.0 Hz, 2H), 5.08 (s, 1H), 5.39 – 5.56 (m, 3H), 7.07 (d, J = 8.3 Hz, 1H), 7.15 – 7.28 (m, 2H), 7.37 (dt, J = 7.7, 34.5 Hz, 7H), 7.62-7.66 (m, 3H), 7.89 (d, J = 7.5 Hz, 3H), 7.96 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.16 (t, J = 8.1 Hz, 2H). E HS+N(M H+1 O = 1848 O). Step 2 - Intermediate Compound 189: (2S,3S,4S,5R,6S)-6-(2-((S)-12-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,10,13,17-tetraoxo-2,7-dioxa-4,11,14,18- tetraazanonadecan-19-yl)-4-(((((3S,3aR,6S,6aR)-6-aminohexahydrofuro[3,2-b]furan-3- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxy L, 12 O9H. N80 O mmo Nt l) w Oetrahydro-2H as add Ned to mixtuH N-p Int re Hyra erm wa On-2-c edia s stir Nar te re Oboxylic Comp d at 15 O acid
Figure imgf000272_0002
In a round bot2tom f Olask H, TNHFA (1 O H0O m O O OH O OHHHHN O O O O HHound 188 (3.5 g, 1.89 mmol) in DCM (60 mL) under nitrogen. The resultingHFmoc °C for 1 hour. The solvent was removed under reduced pressure to afford the crude product Intermediate Compound 189 (2.70 g, 82 %) as a yellow oil. The product was used in the next step directly without furt 5 Hz, 9H), 1.26 – 1.31 (m, 9H), 1.39 – 1.67 (m, 3H), 2.06 (s, 2H), 2.45 – 2.50 (m, 27H), 3.14 (dd, J = 3.1, 7.3 Hz, 3H), 3.20 – 3.48 (m, 9H), 3.42 – 3.53 (m, 23H), 3.56 – 3.74 (m, 6H), 3.73 – 4.02 (m, 3H), 4.10 (s, 1H), 4.16 – 4.45 (m, 6H), 4.56 (s, 1H), 4.96 (s, 2H), 7.06 (d, J = 8.4 Hz, 1H), 7.15 – 7.25 (m, 1H), 7.33 – 7.39 (m, 3H), 7.42 – 7.51 (m, 2H), 7.69 (d, J = 7.7 Hz, 2H), 7.84 – 8.00 (m, 3H), 8.05 – 8.16 (m, 3H), 8.20-8.26 (br, 2H). ES+(M+1 = 1748). Step 3 - Intermediate Compound 190: (2S,3S,4S,5R,6S)-6-(2-((S)-12-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,10,13,17-tetraoxo-2,7-dioxa-4,11,14,18- tetraazanonadecan-19-yl)-4-(((((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4- (((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3- c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido)hexahydrofuro[3,2-b]furan-3-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000273_0001
In a round bottom flask, 1-Hydroxypyrrolidine-2,5-dione (0.367 g, 3.19 mmol) was added to the PNU-159682 carboxylic acid (CAS No.1204819-92-0; 1 g, 1.59 mmol) and DIC (0.497 mL, 3.19 mmol) in DMA (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. Intermediate Compound 189 (2.79 g, 1.59 mmol) and DIEA (1.391 mL, 7.97 mmol) was added, the resulting mixture was stirred at 25 °C for 2 hours. The resulting mixture was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 190 (2.500 g, 66.6 %) as a red solid.
Figure imgf000273_0002
NMR (400 MHz, DMSO) δ 1.03 – 1.33 (m, 18H), 1.39 – 1.81 (m, 5H), 2.24 – 2.45 (m, 5H), 2.59 – 2.75 (m, 2H), 2.95 – 3.15 (m, 4H), 3.25 – 3.35 (m, 18H), 3.39 – 3.69 (m, 52H), 3.80 – 4.04 (m, 8H), 4.06 – 4.29 (m, 5H), 4.37 (s, 2H), 4.49 (s, 2H), 4.55 – 4.70 (m, 2H), 4.86 – 5.13 (m, 4H), 5.25 (s, 1H), 5.40 (d, J = 22.5 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H), 7.19 – 7.48 (m, 8H), 7.67 (t, J = 8.7 Hz, 4H), 7.82 – 8.01 (m, 5H), 8.11-8.16 (m, 2H), 14.06 (s, 1H). ES+(M+1 = 2357). Step 4 - Intermediate Compound 191: (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-15-amino-3,7,10-trioxo-13-oxa-2,6,9-triazapentadecyl)-4- (((((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy- 1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamido)hexahydrofuro[3,2-b]furan-3- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000274_0001
In a round bottom flask, DBU (0.240 mL, 1.59 mmol) was added to Intermediate Compound 190 (2.5 g, 1.06 mmol) in MeCN (30 mL) at 0°C over a period of 3 minute under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water, to afford Intermediate Compound 191 (1.780 g, 79 %) as a red solid.1H NMR (400 MHz, DMSO) δ 0.01 (s, 7H), 1.06 – 1.28 (m, 4H), 1.36 – 1.73 (m, 6H), 1.76 (s, 1H), 1.89 – 2.42 (m, 10H), 2.66 (d, J = 17.7 Hz, 2H), 2.99 (t, J = 15.5 Hz, 4H), 3.24 (s, 4H), 3.51 (br 51H), 3.62 (dd, J = 7.9, 17.4 Hz, 7H), 3.76 – 3.97 (m, 6H), 4.01 (s, 3H), 4.05 – 4.30 (m, 9H), 4.38 (s, 2H), 4.50-4.58 (m, 5H), 4.98 – 5.10 (m, 4H), 5.25 (s, 1H), 5.36-5.46 (m, 1H), 7.08 (d, J = 8.3 Hz, 1H), 7.19 – 7.48 (m, 2H), 7.60 – 7.74 (m, 2H), 7.86 – 7.98 (m, 2H), 8.00 – 8.21 (m, 4H), 9.27 (s, 1H). ES+(M+1 = 2135). Step 5 - Compound LP14: (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-18-bromo-3,7,10,17-tetraoxo-13-oxa-2,6,9,16-tetraazaoctadecyl)-4- (((((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy- 1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11- hexahydrotetracene-2-carboxamido)hexahydrofuro[3,2-b]furan-3- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000274_0002
In a round bottom flask, 2,5-Dioxopyrrolidin-1-yl 2-bromoacetate (66.3 mg, 0.28 mmol) was added to Intermediate Compound 191 (400 mg, 0.19 mmol) in DMA (5 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The crude product was purified by preparative HPLC to afford Compound LP14 (66.0 mg, 15.62 %) as a red solid.1H NMR (300 MHz, DMSO) δ 1.21 (t, J = 7.9 Hz, 3H), 1.37 – 1.79 (m, 6H), 2.11 (br, 3H), 2.23 – 2.43 (m, 8H), 2.57 – 2.76 (m, 2H), 2.89 – 3.07 (m, 2H), 3.24 – 3.31 (s, 9H), 3.36 – 3.46 (m, 6H), 3.51 (br, 46H), 3.56 – 3.69 (m, 10H), 3.79 – 3.97 (m, 8H), 4.00 (d, J = 4.1 Hz, 2H), 4.06 – 4.31 (m, 8H), 4.37 (s, 3H), 4.48 (s, 2H), 4.59 (d, J = 2.2 Hz, 1H), 4.97 (br, 4H), 5.10 – 5.19 (m, 1H), 5.20 – 5.31 (m, 1H), 5.36 (d, J = 3.1 Hz, 1H), 5.40 – 5.67 (m, 1H), 7.00 – 7.32 (m, 4H), 7.66 (br, 2H), 7.81 – 8.21 (m, 8H), 8.33 (d, J = 6.0 Hz, 1H), 13.27 (s, 1H), 14.07 (s, 1H). ES+(M+1 = 2256). Example 53: Synthesis of Compound LP15
Figure imgf000275_0001
Figure imgf000276_0001
Step 1 - Intermediate Compound 192: (2S,3S,4S,5R)-2-((allyloxy)carbonyl)-6-bromotetrahydro-2H- pyran-3,4,5-triyl triacetate
Figure imgf000276_0002
In a round bottom flask, 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (26.5 ml, 177.35 mmol) was added dropwise to a 1-L round bottom flask containing (2S,3S,4S,5R,6R)-3,4,5,6- tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid (31.3 g, 161.22 mmol) in DMF (100 ml) at 21 °C. Next, 3-bromoprop-1-ene (16.72 ml, 193.47 mmol) was added to the reaction mixture dropwise over 10 minutes and the reaction was stirred at 21 °C for 24 hours. Reaction mixture was cooled to 0 °C and treated with pyridine (104 mL, 1289.60 mmol). Acetic anhydride (244 mL, 2579.20 mmol) was next added to the reaction mixture The reaction was warmed up to room temperature and run for 2 hours at 21 °C. Reaction mixture concentrated under reduced vacuum and the remaining pyridine was azeotropically removed with toluene (1 x 100 mL). Crude material was diluted with DCM (65 mL) and cooled to 0 °C.30% Hydrobromic acid in acetic acid (175 mL, 3226.03 mmol) was next added to the reaction mixture at 0 °C. The reaction was warmed up to room temperature and run for 2 hours 30 minutes at 21 °C. Solvent was evaporated then the compound was purified by normal phase flash column chromatography using 1-10% methanol in DCM to afford Intermediate Compound 192 (33 g, 48% yield) as a beige translucent material.1H NMR (500 MHz, CDCl3) δ 6.67 (d, J = 4.0 Hz, 1H), 5.92 (ddt, J = 16.6, 10.3, 6.0 Hz, 1H), 5.64 (t, J = 9.7 Hz, 1H), 5.42 – 5.23 (m, 3H), 4.88 (dd, J = 10.0, 4.0 Hz, 1H), 4.71 – 4.58 (m, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H); LCMS (ESI) m/z 445.0 (M + Na)+. Step 2 - Intermediate Compound 193: (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(4-(((tert- butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000277_0001
To a vacuum-dried 500 mL round-bottom flask was added molecular sieves (4 Å beads, 5.0 g), silver oxide (29.2 g, 125.8 mmol) and acetonitrile (150 mL), producing a black slurry. To this slurry was added a solution of Intermediate Compound 192 (10.7 g, 25.2 mmol) in acetonitrile (50 mL) over 20 min followed by the addition of 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (13.6 g, 51.1 mmol) in acetonitrile (50 mL) in one portion. The resulting mixture was stirred vigorously at 20 °C for 16 h. After 16 h, the reaction mixture was filtered through a 5-cm pad of Celite and rinsed with dichloromethane (3 x 25 mL). Solvent was evaporated then the compound was purified by normal phase flash column chromatography by using 0-5% methanol in DCM to afford Intermediate Compound 193 (5.2 g, 34% yield).1H NMR (400 MHz, CDCl3) δ 10.34 (s, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.58 (dd, J = 8.6, 2.1 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 5.81-5.92 (m, 1H), 5.39- 5.35 (m, 4H), 5.28-5.22 (m, 2H), 4.71 (s, 2H), 4.58-4.67 (m, 2H), 4.20-4.28 (m, 1H), 2.073 (s, 3H), 2.069 (s, 3H), 2.04 (s, 3H), 0.94 (s, 9H), 0.11 (s, 6H); LCMS (ESI) m/z 626.3 (M + NH4)+. Step 3 - Intermediate Compound 194: (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-(aminomethyl)-4- (hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000278_0001
In a round bottom flask, to a solution of Intermediate Compound 193 (5.2 g, 8.6 mmol) in acetonitrile (40 mL) was added tert-butyl carbamate (3.8 g, 32.3 mmol), trifluoroacetic acid (2.0 mL, 25.9 mmol), and triethylsilane (4.1 mL, 25.8 mmol). Stirred for 2 h at 20 °C then solvent was evaporated. To the resulting colorless oil was added 1,4-dioxane (8 mL) and HCl (4.0 M in 1,4- dioxane, 50 mL, 200 mmol). The mixture was stirred at 20 °C for 30 min the solvent was evaporated. The resulting white powder was dissolved in DMSO (3 mL) then passed through cation- exchange resin pre-treated with methanol (WATERS PORAPAK CX). The desired compound was eluted off the resin with methanol to afford Intermediate Compound 194 as a white material (2.5 g, 80% over 2 steps).1H NMR (500 MHz, CDCl3) δ 7.26 (d, J = 2.2 Hz, 1H), 7.21 (dd, J = 8.3, 2.2 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 5.90-5.82 (m, 1H), 5.42 – 5.24 (m, 6H), 5.16 (d, J = 7.1 Hz, 1H), 4.64 – 4.55 (m, 4H), 4.19 (d, J = 9.3 Hz, 1H), 3.84 (d, J = 14.0 Hz, 1H), 3.67 (d, J = 14.0 Hz, 1H), 2.32 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.03 (s, 3H). LCMS (ESI) m/z 496.5 (M + H)+. Step 4 - Intermediate Compound 195: (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6- ((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000278_0002
To a stirred reactor containing Intermediate Compound 194 (2.1 kg, 90.5% w/w, 3.57 mol) and acetonitrile (19 L) was added Fmoc-β-alanine (1.11 kg, 3.57 mol). The stirred mixture was cooled to 0°C. To this was added hexafluorophosphate azabenzotriazole tetramethyl uronium (1.36 kg, 3.57 mol) and N,N-diispropylethylamine (0.92 kg, 7.14 mol), and stirred for 4 hours, maintaining the temperature at 0°C. Water (19 L) and ethyl acetate (19 L) was added to the stirred mixture. The organic phase was separated and concentrated to ~19 L under vacuum. Ethyl acetate (28.5 L) was added to the concentrated solution and stirred at 20-25°C for 18 hours. The resulting suspension was filtered , the cake washed with ethyl acetate (3.87 L), and dried under vacuum to afford Intermediate Compound 195 (1.6 kg, 99% w/w, 56%). The intermediate compound 195 was used in next step without further purification. LCMS m/z 789 [M+H]+ Step 5 - Intermediate Compound 196: (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3- aminopropanamido)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000279_0001
To a stirred reactor containing Intermediate Compound 195 (1 kg, 1.27 mol) and tetrahydrofuran (10 L) at -45°C under nitrogen was added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (385.98 g, 2.54 mol). The mixture was stirred at -45°C for four hours then diluted with acetonitrile (5 L) and quenched by the addition of hydrogen chloride in tert-butyl methyl ether solution (2.54 L, 2.0 M, 5.07 mol). The mixture was concentrated to ~5 L under vacuum, and diluted with n-heptane (5 L). The acetonitrile layer was collected containing Intermediate Compound 196 (3.88 kg of MeCN solution, 89.97% area, assumed 100%).1H NMR (500 MHz, D2O) δ 7.22 (d, J = 2.2 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 5.84 (ddt, J = 16.6, 10.5, 6.0 Hz, 1H), 5.44 (t, J = 9.2 Hz, 1H), 5.38 (dd, J = 7.6, 3.3 Hz, 1H), 5.35 – 5.23 (m, 4H), 4.62 (d, J = 9.8 Hz, 1H), 4.56 (d, J = 6.0 Hz, 2H), 4.51 (s, 2H), 4.26 (q, J = 15.3 Hz, 2H), 3.23 (t, J = 6.8 Hz, 2H), 2.68 (td, J = 6.8, 1.9 Hz, 2H), 2.06 (d, J = 10.4 Hz, 9H). LCMS (ESI) m/z 567.2 (M + H)+. LCMS m/z 566.6 [M+H]+ Step 6 - Intermediate Compound 197: (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4- (hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000279_0002
To a 100 mL round bottom flask was added Intermediate Compound 158 (0.8 g, 0.74 mmol) under nitrogen gas. To the solution was added HATU (0.366 g, 0.96 mmol) followed by DIPEA (0.388 mL, 2.22 mmol). 15 min then Intermediate Compound 196 (0.670 g, 1.11 mmol). was added and reaction mixture was stirred at 23 °C for 3 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with DCM (100 mL), washed with water (100 mL), organic layer was extracted (2 x 100 mL), washed with Brine (100 mL), dried over sodium sulfate (15 g). Solvent was removed under reduced pressure and purified via silica gel column by using 5-20% methanol in DCM to give Intermediate Compound 197 (0.640 g, 53.1 %).1H NMR (500 MHz, DMSO) δ 8.21 (t, J = 6.0 Hz, 1H), 8.11 (dd, J = 19.5, 6.9 Hz, 2H), 7.94 (t, J = 5.8 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.73 (t, J = 7.0 Hz, 2H), 7.49 – 7.39 (m, 3H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 7.17 (dd, J = 8.4, 2.2 Hz, 1H), 7.13 (s, 1H), 7.01 (d, J = 8.4 Hz, 1H), 5.89 (ddt, J = 17.3, 10.5, 5.7 Hz, 1H), 5.56 (d, J = 7.9 Hz, 1H), 5.48 (t, J = 9.6 Hz, 1H), 5.33 (dq, J = 17.2, 1.6 Hz, 1H), 5.26 (dq, J = 10.5, 1.4 Hz, 1H), 5.19 – 5.07 (m, 3H), 4.76 (d, J = 10.0 Hz, 1H), 4.62 (ddt, J = 13.3, 5.6, 1.4 Hz, 1H), 4.54 (ddt, J = 13.3, 5.8, 1.4 Hz, 1H), 4.44 – 4.36 (m, 4H), 4.31 – 4.18 (m, 4H), 4.11 (d, J = 5.9 Hz, 3H), 3.93 (d, J = 5.9 Hz, 1H), 3.88 – 3.81 (m, 2H), 3.66 – 3.56 (m, 5H), 3.56 – 3.45 (m, 42H), 3.45 – 3.39 (m, 3H), 3.29 – 3.27 (m, 1H), 3.24 (s, 3H), 3.18 (d, J = 5.0 Hz, 1H), 2.33 (ddt, J = 14.6, 10.0, 7.6 Hz, 4H), 2.10 – 2.05 (m, 2H), 2.04 (s, 3H), 1.99 (d, J = 4.6 Hz, 6H), 1.54 (dt, J = 43.7, 8.9 Hz, 4H). LCMS (ESI) m/z 1629.8 (M + H)+. Step 7 – Intermediate Compound 198: (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatria-contan-38-amido)hexahydrofuro[3,2-b]furan- 3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-((((4- nitrophenoxy)carbonyl)oxy)-methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000280_0001
To a 250 mL round bottom flask bis(p-nitrophenyl) carbonate (6.57 g, 21.61 mmol) was added to Intermediate Compound 197 (4.4 g, 2.70 mmol) and DIEA (3.77 mL, 21.61 mmol) in THF (50 mL) at 25°C under nitrogen. The resulting mixture was stirred at 60 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate Compound 198 (3.20 g, 66.0 %) as a yellow solid. 1HNMR (400 MHz, DMSO) δ 1.17 (d, J = 6.7 Hz, 6H), 1.49 (s, 1H), 1.56 (s, 1H), 1.97 - 2.05-2.09 (m, 8H), 2.28 – 2.39 (m, 3H), 3.17 (s, 19H), 3.24 (s, 3H), 3.30 (d, J = 7.6 Hz, 2H), 3.41 – 3.51 (m, 27H), 3.65 (s, 5H), 3.68 (s, 2H), 3.80 - 3.88 (m, 2H), 3.94 (s, 1H), 4.10 (s, 2H), 4.16 - 4.29 (m, 4H), 4.38 (d, J = 5.0 Hz, 2H), 4.48 – 4.67 (m, 2H), 4.74 – 4.83 (m, 1H), 5.06 – 5.21 (m, 2H), 5.22 – 5.38 (m, 2H), 5.49 (t, J = 9.7 Hz, 1H), 5.62 (d, J = 8.2 Hz, 1H), 5.83 - 5.94 (m, 1H), 6.88 – 6.97 (m, 3H), 7.04 - 7.12 (m, 1H), 7.19 (s, 1H), 7.24 – 7.38 (m, 2H), 7.38 – 7.52 (m, 2H), 7.73 (t, J = 6.1 Hz, 1H), 7.81 - 7.91 (m, 2H), 7.98 (s, 1H), 8.08 – 8.19 (m, 4H), 8.24 – 8.33 (m, 1H). ES+(M+1 =1794) HPLC purity: 80% Step 8 - Intermediate Compound 199: tert-butyl ((3S,3aR,6S,6aR)-6-(((((9H-fluoren-9- yl)methoxy)carbonyl)amino)methyl)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000281_0001
In a round bottom flask, Intermediate Compound A-116 (7 g, 18.40 mmol) was added to Di-tert- butyl dicarbonate (8.03 g, 36.80 mmol) and N,N-Diisopropylethylamine (7.13 g, 55.20 mmol) in THF (100 mL) and water (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was diluted with DCM (100 mL), and washed sequentially with water (100 mLX3) The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 25 to 100% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford Intermediate Compound 199 (4.50 g, 50.9 %) as a colourless oil.1
Figure imgf000281_0002
H NMR (300 MHz, DMSO) δ 1.39 (s, 9H), 2.86 – 2.94 (m, 2H), 3.50 – 3.59 (m, 2H), 3.70 – 3.76 (m, 1H), 3.79 – 3.85 (m, 2H), 4.20 – 4.25 (m, 2H), 4.29 – 4.36 (m, 4H), 7.17 (s, 1H), 7.28 – 7.54 (m, 5H), 7.65 – 7.71 (m, 2H), 7.90 (d, J = 7.6Hz, 2H). ES+(M+1 =503) HPLC purity: 95% Step 9 - Intermediate Compound 200: tert-butyl ((3S,3aR,6S,6aR)-6- (aminomethyl)hexahydrofuro[3,2-b]furan-3-yl)carbamate
Figure imgf000281_0003
In a 250 mL round bottom flask, Intermediate Compound 199 (4.5 g, 9.36 mmol) was added to diethylamine (6.85 g, 93.64 mmol) in THF (40 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed by distillation under vacuum to afford Intermediate Compound 200 (2.2 g, 91 %) as a colourless oil.1H NMR (300 MHz, DMSO) δ 1.39 (s, 9H), 2.10 - 2.19 (m, 1H), 2.46 - 2.54 (m, 2H), 3.46 - 3.54 (m, 1H), 3.56 - 3.62 (m, 2H), 3.79 - 3.85 (m, 2H), 4.23 - 4.46 (m, 2H). ES+(M+1 =259) HPLC purity: 80%. Step 10 - Intermediate Compound 201: (2S,3R,4S,5S,6S)-2-(2-((S)-1-(9H-fluoren-9-yl)-5-(4- (((3S,3aR,6S,6aR)-6-(33-methyl-1-(l1-oxidaneyl)-3,6,9,12,15,18,21,24,27,30,33l3- undecaoxahexatriacontan-36-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutyl)-3,6,10- trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-((((((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamoyl)oxy)methyl)phenoxy)-6- ((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000282_0001
In a 250 mL round bottom flask, Intermediate Compound 200 (1.728 g, 6.69 mmol) was added to Intermediate Compound 198 (3 g, 1.67 mmol) and DIEA (1.168 mL, 6.69 mmol) in THF (30 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate Compound 201 (3.00 g, 94 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 1.35 – 1.43 (m, 11H), 1.48 - 1.60 (m, 2H), 1.86 – 2.12 (m, 10H), 2.28 – 2.40 (m, 4H), 3.23 (s, 3H), 3.45 – 3.52 (br, 55H), 3.56 – 3.63 (m, 6H), 3.68 (s, 1H), 3.77 – 3.88 (m, 4H), 4.10 (s, 2H), 4.14 – 4.32 (m, 5H), 4.38 (s, 2H), 4.48 – 4.66 (m, 2H), 4.74 - 4.80 (m, 1H), 4.94 (s, 1H), 5.05 – 5.18 (m, 2H), 5.25 – 5.35 (m, 1H), 5.48 (t, J = 9.7 Hz, 1H), 5.60 (t, J = 8.5 Hz, 1H), 5.81 - 5.92 (m, 1H), 6.99 – 7.08 (m, 1H), 7.13 – 7.28 (m, 3H), 7.32 – 7.51 (m, 5H), 7.72 (t, J = 6.5 Hz, 1H), 7.76 – 7.91 (m, 2H), 7.98 (s, 1H), 8.10 – 8.19 (m, 2H), 8.22 – 8.31 (m, 1H). ES+(M+1 =1913) HPLC purity: 90%. Step 11 - Intermediate Compound 202: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-2-amino-6-(((3S,3aR,6S,6aR)- 6-(33-methyl-1-(l1-oxidaneyl)-3,6,9,12,15,18,21,24,27,30,33l3-undecaoxahexatriacontan-36- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-6-oxohexanamido)propanamido)methyl)-4- ((((((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3- yl)methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000283_0001
In a 250 mL round bottom flask was added K2CO3 (2.095 g, 15.16 mmol) and Intermediate Compound 201 (2.9 g, 1.52 mmol) in MeOH (30 mL)/water (7.50 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 2 hours. The crude product was purified by preparative HPLC (Column: Xselect CSH Prep Phenyl-Hexyl Column, 19*250mm, 5μm; Mobile Phase A: Water(0.1%FA), Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 18% B to 28% B in 20 min; Wavelength: 254nm/220nm; RT1(min): 12.8) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford Intermediate Compound 202 (0.740 g, 32.0 %) as a pale-yellow oil. 1H NMR (400 MHz, DMSO) δ 1.39 (s, 9H), 1.50 (s, 2H), 1.62 (d, J = 6.8 Hz, 2H), 2.08 (s, 6H), 2.11 (s, 1H), 2.26 (d, J = 7.6 Hz, 1H), 2.29 – 2.46 (m, 4H), 2.82 – 3.04 (m, 3H), 3.24 (s, 6H), 3.26 (s, 2H), 3.32 (d, J = 9.4 Hz, 5H), 3.39 – 3.45 (m, 5H), 3.48 (s, 31H), 3.55 – 3.64 (m, 7H), 3.71 - 3.78 (m, 2H), 3.78 - 3.88 (m, 4H), 4.09 (s, 2H), 4.19 (d, J = 10.7 Hz, 1H), 4.30 (d, J = 4.5 Hz, 1H), 4.32 – 4.41 (m, 3H), 4.44 – 4.55 (m, 1H), 4.60 (d, J = 5.8 Hz, 1H), 4.87 – 4.99 (m, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 5.6 Hz, 1H), 7.25 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.44 (t, J = 5.8 Hz, 1H), 8.16 (d, J = 6.0 Hz, 2H), 8.28 (d, J = 6.9 Hz, 1H), 8.51 (d, J = 6.2 Hz, 1H), 9.29 (s, 1H). ES+(M+1 =1525) HPLC purity: 90%. Step 12 - Intermediate Compound 203: (2S,3S,4S,5R,6S)-6-(4-((((((3S,3aR,6S,6aR)-6-((tert- butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamoyl)oxy)methyl)-2-((3-((S)-2- (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6-(((3S,3aR,6S,6aR)-6-(33-methyl-1-(l1- oxidaneyl)-3,6,9,12,15,18,21,24,27,30,33l3-undecaoxahexatriacontan-36- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-6-oxohexanamido)propanamido)methyl)phenoxy)- 3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000284_0001
In a 250 mL round bottom flask was added 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)propanoate (147 mg, 0.55 mmol) was added to Intermediate Compound 202 (700 mg, 0.46 mmol) and DIEA (0.080 mL, 0.46 mmol) in DMA (10 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound 203 (610 mg, 79 %) as a pale-yellow solid. 1H NMR (400 MHz, DMSO) δ 1.22 (d, J = 6.8 Hz, 11H), 1.54 (s, 2H), 2.08 (s, 8H), 2.22 – 2.31 (m, 2H), 2.33 (d, J = 6.8 Hz, 4H), 2.40 (t, J = 7.2 Hz, 3H), 2.88 (q, J = 6.8 Hz, 1H), 2.91 – 2.99 (m, 2H), 3.05 (s, 1H), 3.22 – 3.29 (m, 11H), 3.31 – 3.36 (m, 13H), 3.39 – 3.50 (m, 16H), 3.55 – 3.64 (m, 9H), 3.70 –3.77 (m, 2H), 3.77 – 3.88 (m, 5H), 4.09 (s, 2H), 4.17 – 4.25 (m, 1H), 4.29 (d, J = 4.4 Hz, 1H), 4.34 (d, J = 4.5 Hz, 2H), 4.37 – 4.47 (m, 3H), 4.68 (s, 1H), 4.93 (s, 2H), 5.12 (s, 1H), 5.46 (s, 1H), 6.98 (s, 2H), 7.06 (d, J = 8.4Hz, 1H), 7.17 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.29 (s, 1H), 7.42 (d, J = 6.3 Hz, 1H), 7.96 (s, 1H), 8.16 (d, J = 7.0 Hz, 2H), 8.22 (d, J = 8.4Hz, 1H). ES+(M+1 =1676) HPLC purity: 90% Step 13 - Intermediate Compound 204: (2S,3S,4S,5R,6S)-6-(4-((((((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)methyl)carbamoyl)oxy)methyl)-2-((3-((S)-2-(3-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)propanamido)-6-(((3S,3aR,6S,6aR)-6-(33-methyl-1-(l1-oxidaneyl)- 3,6,9,12,15,18,21,24,27,30,33l3-undecaoxahexatriacontan-36-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-6-oxohexanamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran- 2-carboxylic acid
Figure imgf000284_0002
In a 100 mL round bottom flask, Intermediate Compound 203 (600 mg, 0.36 mmol) was added to DCM (5 mL) / TFA (5.00 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford crude Intermediate Compound 204 (530 mg, 94 %) as a brown oil.1H NMR (400 MHz, DMSO) δ 1.21 – 1.28 (br, 10H), 1.43 (t, J = 11.0 Hz, 3H), 2.04 (s, 2H), 2.31 (d, J = 6.4 Hz, 4H), 2.40 (d, J = 7.6 Hz, 2H), 2.91 – 3.06 (m, 2H), 3.10 – 3.20 (m,2H), 3.24 (s, 4H), 3.31 – 3.37 (m, 3H), 3.43 (d, J = 5.2 Hz, 4H), 3.50 (br, 40H), 3.59 (d, J = 7.2 Hz, 8H), 3.79 – 3.85 (m, 2H), 3.85 – 3.96 (m, 3H), 4.10 (s, 2H), 4.29 (s, 1H), 4.37 (s, 2H), 4.49 (d, J = 4.0 Hz, 2H), 4.94 (s, 2H), 6.99 (s, 2H), 7.05 (d, J = 8.4 Hz, 1H), 7.07 – 7.24 (m, 2H), 7.45 (s, 1H), 7.94 (s, 1H), 8.12 (d, J = 7.6 Hz, 2H), 8.14 – 8.25 (m, 4H).8.14 - 8.25(m, 2H) TFA salt. ES+(M+1 =1576) HPLC purity: 90%. Step 14 - Compound LP15: (2S,3S,4S,5R,6S)-6-(2-((3-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamido)-6-(((3S,3aR,6S,6aR)-6-(33-methyl-1-(l1-oxidaneyl)- 3,6,9,12,15,18,21,24,27,30,33l3-undecaoxahexatriacontan-36-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-6-oxohexanamido)propanamido)methyl)-4-((((((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12- trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamido)hexahydrofuro[3,2-b]furan-3-yl)methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid
Figure imgf000285_0001
In a 50 mL round flask, 1-hydroxypyrrolidine-2,5-dione (92 mg, 0.80 mmol) was added to the PNU- 159682 carboxylic acid (CAS No.: 1204819-92-0; 250 mg, 0.40 mmol) and DIC (0.124 mL, 0.80 mmol) in DMA (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The LCMS showed formation of desired product. Then Intermediate Compound 204 (502 mg, 0.32 mmol) and DIEA (0.209 mL, 1.20 mmol) was added into above mixture, the resulting mixture was stirred at 25 °C for 2 hours. The crude product was purified by preparative HPLC (Column: Xselect CSH Prep Phenyl-Hexyl OBD column, 19*250mm, 5um; Mobile Phase A: Water, Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 20% B to 30% B in 15 min; Wavelength: 254nm/220nm; RT1(min): 14.98) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford Compound LP15 (102 mg, 11.72 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.17 – 1.25 (m, 5H), 1.36 – 1.75 (m, 7H), 1.98 – 2.21 (m, 4H), 2.25 – 2.41 (m, 8H), 2.62 – 2.74 (m, 3H), 2.87 – 3.03 (m, 4H), 3.23 – 3.32 (m, 12H), 3.47 – 3.60 (m, 53H), 3.77 – 3.84 (m, 3H), 3.88 – 3.96 (m, 2H), 4.00 (s, 2H), 4.04 – 4.16 (m, 5H), 4.23 (d, J = 2.2 Hz, 2H), 4.34 – 4.47 (m, 5H), 4.58 (d, J = 2.0Hz, 1H), 4.71 (s, 1H), 4.90 – 5.05 (m, 3H), 5.11 – 5.29 (m, 2H), 5.37 – 5.51 (m, 2H), 6.97 (s, 2H), 7.07 (d, J = 8.4Hz, 1H), 7.18 – 7.33 (m, 2H), 7.44 (t, J = 6.0 Hz, 1H), 7.67 (q, J = 4.4 5.2Hz, 1H), 7.88 – 8.00 (m, 3H), 8.09 – 8.25 (m, 4H), 8.92 (s, 1H), 13.26 (s, 1H), 14.06 (s, 1H). ES+[(M/2)+1] =1093.55) HPLC purity: 94.6% Example 54: Synthesis of Compound LP16
Figure imgf000286_0001
Step 1 - Intermediate Compound 205: (9H-fluoren-9-yl)methyl ((S)-5-benzyl-1-(((3S,3aR,6S,6aR)-6- ((tert-butoxycarbonyl)amino)hexahydrofuro[3,2-b]furan-3-yl)amino)-1,4,7,10,13-pentaoxo-16,19- dioxa-3,6,9,12-tetraazahenicosan-21-yl)carbamate
Figure imgf000287_0001
In a round bottom flask, Intermediate Compound 107 (8.24 g, 14.65 mmol) was added to HOBt (3.37 g, 21.98 mmol), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.62 g, 29.30 mmol) and 1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-oic acid (6.44 g, 16.12 mmol) in DMA (200 mL). The resulting mixture was stirred at 25 °C for 3 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 5 to 60% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound 205 (10.00 g, 72.3 %) as a white solid.1H NMR (300 MHz, DMSO) δ 1.39 (s, 9H), 2.40 (t, J = 6.4 Hz, 2H), 2.72 – 2.90 (m, 1H), 2.96 – 3.06 (m, 1H), 3.06 – 3.18 (m, 1H), 3.40 (d, J = 6.0 Hz, 1H), 3.48 (s, 4H), 3.60 (dd, J = 4.4, 8.0 Hz, 4H), 3.65 – 3.77 (m, 3H), 3.77 – 3.91 (m, 3H), 4.09 (s, 1H), 4.16 – 4.25 (m, 1H), 4.29 (d, J = 6.8 Hz, 2H), 4.37 (d, J = 4.0 Hz, 1H), 4.42 (d, J = 4.4 Hz, 1H), 4.47 (s, 1H), 7.16 – 7.22 (m, 1H), 7.24 (s, 3H), 7.26 (s, 1H), 7.26 (s, 1H), 7.30 – 7.38 (m, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 8.04 (d, J = 6.8 Hz, 2H), 8.10 – 8.17 (m, 1H), 8.20 (d, J = 5.6 Hz, 1H), 8.26 (t, J = 5.6 Hz, 1H). ES+( M+1 = 944.20), HPLC purity : 91.3%. Steps 2 and 3 - Intermediate Compound 207: (9H-fluoren-9-yl)methyl ((S)-5-benzyl-1,4,7,10,13- pentaoxo-1-(((3S,3aR,6S,6aR)-6-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)- 9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo- 1,2,3,4,6,11-hexahydrotetracene-2-carboxamido)hexahydrofuro[3,2-b]furan-3-yl)amino)-16,19- dioxa-3,6,9,12-tetraazahenicosan-21-yl)carbamate
Figure imgf000287_0002
In a round bottom flask, Intermediate Compound 205 (900 mg, 0.95 mmol) was added to HCl (10 mL, 40.00 mmol) in 1,4-dioxane (5 mL) and water (1 mL). The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure to give Intermediate Compound 206. The crude Intermediate Compound 206 was added to the PNU-159682 carboxylic acid (CAS No.: 1204819-92-0; 419 mg, 0.67 mmol), HATU (471 mg, 1.24 mmol) and DIEA (0.500 mL, 2.86 mmol) in DMA (15 mL). The resulting mixture was stirred at 25 °C for 90 minutes. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 70% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate Compound 207 (800 mg, 57.7 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.20 (d, J = 6.5 Hz, 4H), 1.59 – 1.74 (m, 3H), 2.39 (t, J = 6.4 Hz, 3H), 2.57 – 2.74 (m, 2H), 2.75 – 2.86 (m, 1H), 2.89 – 3.08 (m, 3H), 3.12 (d, J = 6.0 Hz, 2H), 3.31 (s, 3H), 3.38 (d, J = 5.2 Hz, 1H), 3.47 (s, 4H), 3.53 – 3.80 (m, 6H), 3.81 – 4.05 (m, 7H), 4.07 – 4.32 (m, 8H), 4.35 – 4.64 (m, 5H), 5.01 (d, J = 4.4 Hz, 1H), 5.21 – 5.31 (m, 1H), 5.37 (s, 1H), 7.13 – 7.21 (m, 1H), 7.25 (d, J = 3.6 Hz, 5H), 7.29 – 7.36 (m, 4H), 7.37 – 7.45 (m, 3H), 7.68 (t, J = 7.2 Hz, 4H), 7.84 – 7.95 (m, 6H), 8.00 – 8.10 (m, 2H), 8.11 – 8.21 (m, 3H), 8.26 (d, J = 6.0 Hz, 1H). ES+( M+1 = 1453.55), HPLC purity: 70.0%. Step 4 - Compound LP16: (2S,4S)-N-((3S,3aR,6S,6aR)-6-((S)-21-amino-5-benzyl-4,7,10,13-tetraoxo- 16,19-dioxa-3,6,9,12-tetraazahenicosanamido)hexahydrofuro[3,2-b]furan-3-yl)-2,5,12-trihydroxy-7- methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2- carboxamide
Figure imgf000288_0001
In a round bottom flask, Intermediate Compound 207 (600 mg, 0.41 mmol) was added to DBU (0.062 mL, 0.41 mmol) in DMA (15 mL). The resulting mixture was stirred at 0 °C for 2 hours. The reaction mixture was quenched with saturated NH4Cl 10mM in water (2mL). The crude product was purified by preparative HPLC (Column: Xbridge Prep C18 OBD, 19*250mm, 5um; Mobile Phase A: Water (10mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 22% B to 42% B in 15 min; Wavelength: 254nm/220nm; RT1(min): 14.6) and MeCN as eluents. Fractions containing the desired compound were freeze dried directly to afford Compound LP16 (130 mg, 25.6 %) as a red solid.1H NMR (400 MHz, DMSO) δ 1.17 (m, 4H), 1.67 (s, 2H), 2.06 – 2.32 (m, 2H), 2.40 (t, J = 6.4 Hz, 2H), 2.66 (m, 2H), 2.81 (m, 3H), 3.00 (m, 4H), 3.33 (s, 3H), 3.48 (m, 9H), 3.57 – 3.81 (m, 11H), 3.97 (m, 7H), 4.04 – 4.32 (m, 4H), 4.37 – 4.80 (m, 4H), 5.03 (m, 1H), 5.19 – 5.58 (m, 2H), 7.11 – 7.36 (m, 5H), 7.66 (s, 1H), 7.92 (d, J = 4.4 Hz, 2H), 7.98 – 8.44 (m, 6H). ES+(M+1 = 1232.75), HPLC purity: 93.4%. ANTIBODY DRUG CONJUGATE SYNTHESIS Example 55: General Procedure for ADC Generation mAb Sample Reduction The necessary molar equivalents of TCEP required to reduce the desired number of disulfide linkages in the mAb sample is added to the mAb sample. The mAb sample and TCEP mixture is incubated at 37°C for one hour with mixing. Antibody Conjugation The desired amount of the payloads (i.e. linker-payloads described herein), from a payload stock solution dissolved in DMSO, is diluted into DMSO, to result in 10% (v/v). An additional 10% DMSO (v/v) is added to the reaction mixture (corrected for payload solution to be added) to help fully dissolve the payload. Following which, the diluted stock solution is then added to the buffer containing reduced mAb. The conjugation reaction is incubated at room temperature for–1 - 2 hours with constant mixing. Progress of the conjugation is evaluated by running an aliquot of this mixture on LC/MS. Once completion of the reaction is confirmed, the crude samples are passed through a desalting spin-column and either immediately frozen (and thawed before purification) or purified using CHT-type-II column chromatography. Removal of Unreacted Linker Payload Remaining unreacted linker payload is removed using ceramic hydroxyapatite (CHT) chromatography in accordance with the manufacturer’s instructions. To briefly summarize this protocol, an appropriately sized column is packed with CHT type II resin and connected to an AKTA purification system equipped with a pH meter, electrical conductivity meter, and UV-Vis detector set to 280 nm and 330 nm.5 – 10 column volumes (CV) 0.1 M NaOH is run through the column to strip off anything bound to the resin and the increase in ph is monitored. The pH is brought back to neutral by running 5 – 10 CV of 10X PBS (pH 7.0) through the column, then equilibrated using 5 – 10 CV of 10 mM sodium phosphate buffer (pH 7.0). The conductivity reading should reach a minimum before loading the conjugated antibody sample. The conjugated antibody sample is diluted five-fold with water, then loaded into the column. The column is washed with 10 mM sodium phosphate buffer until the 330 nm signal is at or close to 0 (this 330 nm signal corresponds to free payload in the sample). The sample is eluted with 10 mM sodium phosphate buffer with 2 M NaCl (pH 7.0) and the fractions with high 280 nm signals are collected. The fractions are combined, then the conjugated antibody sample is dialyzed in formulation buffer (1x PBS, pH 7.2) using slide-a-lyzer 10 MWCO dialysis cassettes. Following dialysis, the resulting sample is passed through a 0.2-micron filter and packed for delivery. Example 56: Preparation of ADCs Trastuzumab ADCs: To a 10 mg/mL solution of the antibody trastuzumab (HerceptinTM) suspended in 1x PBS (pH = 7.2) with 1 mM EDTA, 2-2.5 equivalents of TCEP was added and incubated at 37°C for 2 h with mixing. To the partially reduced antibody solution, 4-4.2 equivalents of the linker-payload (LP1, LP6-LP10) was added in DMSO to result in a 10% v/v of the co-solvent. (Note: the addition of linker-payload in smaller batches over 10-minute intervals resulted in a normal distribution of different DAR species.) Upon incubating the conjugation reaction at room temperature for 1 – 2 hours with constant mixing, the progress was evaluated by reduced LC/MS-QTOF. This process was repeated until a desired DAR in the range of 3.6 – 4.4 was obtained. Once the conjugation reaction proceeded to completion, the excess of maleimide was quenched with 5 x N-acetyl-L-cysteine. The crude reaction mixture was purified using CHT-type-II column chromatography, after which, the eluted antibody-drug conjugate (ADC) was immediately dialyzed into 20 mM histidine and 240 mM sucrose at pH 6. Summarized below is the characterization data of the antibody-drug conjugates that were synthesized:
Figure imgf000290_0001
€ ¥DAR calculated using LC-MS-QTOF and Agilent DAR Calculator - Build 2.2.0.0 software. The ADC concentration was measured using nanodrop (ƐHER2 = 1.42). An assumption is made that the TM mutation doesn’t i £mpact the Ɛ. Endotoxin reading = x EU/ml obtained by diluting 10-fold in biology grade water using 0.1-0.001 EU/mL sensitivity cartridge. * The free drug % was measured using reverse phase HPLC.0.1% is the LOD, and the free drug is below the limit of detection. DAR = drug-antibody ratio; SEC = size exclusion chromatography; HIC = hydrophobic interactions chromatography; EU = endotoxin units. Negative control ADCs: Negative control ADCs (Control ADC1-ADC6) were prepared in an analogous manner to that described above, using a negative control antibody NIP228 (as disclosed in WO 2015/127273 A1, the contents of which are incorporated by reference). NIP228 is an IgG1 that has complementary binding to transferrin, a transmembrane protein whose expression is 5 low on the tested cell lines, therefore making it a suitable negative control. Example 57: In vitro cytotoxicity assessment of Anthracycline payloads Cytotoxicity of anthracycline payloads was investigated using Multiple myeloma (MM) cell lines (MM1.s, MM1.r, NCI-H929, RPMI8826), gastric cell lines (SNU601, SNU620) and colorectral cancer (CRC) cell lines (LS411N, LS180, HT-29, LS1034). The efficacy of the payloads was compared to PNU- 159682, with SG3924 (a Topo1 inhibitor) and/or MMAE as internal positive controls. DMSO control was also included in all the studies with DMSO at 0.1%. Cells were plated in 384 well plates at the density of 4000 cells /well in 30µl of the respective media (MM) and 750 cells/well for adherent (CRC). The cells were treated with anthracycline payloads after 24hrs of incubation of the cells at 37°C in a humidified incubator at 5% CO2 atmosphere.10 µl of 4-fold serial dilutions of each anthracycline payload and positive control in growth medium was added to the cells making the final volume to 40μl. For all test articles, the final concentration ranged from 100nM to 0.095fM. Each dilution was done in triplicate. After 3 days of incubation, the plates were taken out from the incubator and equilibrated to the room temperature.40μ1Lof CellTiter-Glo® 2.0 Luminescent Solution (Promega, CatNo# G9242) was added to each well and, after shaking the plates at 45Orpm for 5min followed by a 10 min incubation without shaking, luminescence was measured on a Perkin Elmer Envision 2105 with an integration time of 0.1 second per well. The percent cell viability for the treated cells were calculated by normalizing to the untreated control. MMAE and SG3924 were introduced as internal positive controls with a potency range ~1- 10 nM and ~0.1-0.4 nM IC50, respectively, against the MM cell lines. MMAE was also used as an internal positive control against the solid cell lines. Table 10: IC50 values for the payloads in Multiple Myeloma cell lines (MM1.s, MM1.r, H929, RPMI8826)
Figure imgf000292_0001
NC = not calculable PNU-159682 was tested against MM. IS and MM.1R cell lines. Within the range of concentrations tested (100 nM to 0.09 pM) for the serially diluted PNU-159682, the % viability of the cells remained below 10%. Based on this, and when compared to the other positive controls, the calculable IC50 value for PNU-159682 is below 0.09 pM for the MM. IS and MM.1R cell lines.
Table 11: IC50 values for the payloads in gastric cell lines (SNU601, SNU620) and colorectral cancer cell lines (LS411N, LS180, HT-29, LS1034).
Figure imgf000293_0001
Figure imgf000294_0001
Figures 1A-1C shows % cell viability against payload concentration for certain payloads in gastric and CRC cell lines, compared to PNU-159682.
Example 58: In vitro toxicity of ADCs
Trastuzumab-based antibody-drug conjugates (ADC1-ADC6) and matched isotype controls (Control ADC1-ADC6) were tested in-vitro in cell lines with varying levels of HER2 expression. Cells were maintained in appropriate media (N87, HT-29, + 10% FBS) and passaged when 80-90% confluent. On the day of the experiment (Day 1) cells were detached from the culture flask using 3mL phenol- red free TrypLE® (Invitrogen), and neutralized using 7mL of complete media. Cells were counted on a ViCell counter, resuspended to 25e4 cells/mL, after which 30uL of cells (750cells/well) was dispensed into the inner 240 wells of a clear bottom, 384-well white plate (Corning, 3903). ADCs were serially diluted in media to a top concentration of 160ug/mL (4x, ~1.5uM), and lOuL of each serially diluted ADC was added to 30uL of cells will be lx at 60ug/mL final (~350nM). ADCs were allowed to incubate with cells for 72hrs. On day 3, CellTiterGlo 2.0 terminal reagent was used to evaluate the viability of the cells. Reagent and plates were allowed to equilibrate to room temperature, after which 30uL of reagent was added to each treatment well and placed on a plate shaker for 15 minutes. Luminescence was measured using a Perkin Elmer Envision plate reader, and IC50 values were calculated from raw data transformation using GraphPad Prism analysis software. Table 12: IC5o values for the ADCs in a breast cell line (N87) and a colorectral cancer cell line (HT-29).
Figure imgf000295_0001
NC = not calculated
The two cell models tested have different levels of HER2 expression (N87: breast, HER2 high; HT- 29: colon, HER2 mid/low). The activity of the trastuzumab ADCs strongly correlates with the level of expression of HER2.
Non-targeting control ADCs were tested against the N87 cell line. These had IC50 values over 1000- fold higher than the targeting (trastuzumab) ADCs, demonstrating the target mediated activity of the trastuzumab ADCs. Example 59: In vivo toxicity of ADCs
Human cell line xenograft models (HCC1954, HT29 and N87) were established in female BALB/c Nude mice (GemPharmatech Co. Ltd). The cells were implanted subcutaneously in the right flank of each mouse. Tumor growth was monitored over the study, and tumor volume was calculated using the formula [length (mm) x width (mm)2] / 2. Once the tumors reached an average volume of 150- 200mm3, the mice were randomly assigned into groups using the Matched Distribution method in the StudyLog software package (StudyLog Systems, Inc., South San Francisco, CA).
Following randomization, a single intravenous dose of each ADC was administered in ADC buffer (20mM Histidine, 240mM Sucrose, 0.02% PS-80, pH6) based on the body weight of the mice.
Mouse body weight and tumor measurements were collected twice weekly for the duration of the study. Mice were euthanized if they showed evidence of significant disease burden (such as weight loss of more than 20%, a large tumor mass greater than 2000mm3, tumor ulceration, hypothermia, etc.). Data are presented in growth curves showing tumor growth for each cell line - see Figures 2A-2B.
All animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.
Example 60: Lysosomal stability
The stability of payloads in lysosomal extract was evaluated over a 24h period. Compounds P20, P30 and P31 all had a half-life of over 3600 minutes.
Procedure for sample preparation:
Test compounds were incubated with lysosomal extract (Sekisui Xenotech LLC, K5200) in a CO2 incubator at 37°C and 300 rpm. At multiple time points up to 24 hours, aliquots were sampled into a plate containing acetonitrile and an internal standard, then stored in the refrigerator. After the final time point were collected, the plate was centrifuged at 4000 rpm for 30 minutes. Samples were then analysed by quantitative liquid chromatography-mass spectrometry to determine the amount of compound remaining with respect to time point 0. The peak areas of the test compound at different time points were used to calculate its half-life. . NUMBERED EMBODIMENTS A1. A compound of Formula (Ia): A-E-X-Y-Z or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000297_0001
(i) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X; (ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; (iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or (iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y; Xa is - C(O)- or absent; and Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; m is 2 or 3; Y is absent or
Figure imgf000298_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to Z; Z is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000298_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. A2. A compound of Formula (I): A-E-X-Y-Z or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000298_0003
E is: (i) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X; (ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; (iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or (iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y; Xa is - C(O)- or absent; and Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; m is 2 or 3; Y is absent or
Figure imgf000299_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z; and Q is NH, S, O, - S(CH2)2NH-*, -O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to Z; Z is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000299_0002
integer from 1 to 5; and each Rz is independently H, C
Figure imgf000299_0003
4 alkyl, phenyl or benzyl; provided that when E is -heterocycle-C(O)NH-, then X is absent or -(CH2)m-Xb; and provided that when Z is -C(O)C1-4 alkyl,
Figure imgf000300_0001
absent, then Xb is not -C(O)NH-. A3. The compound of embodiment A1 or A2, or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000300_0002
. A3a. The compound of any of embodiments A1 to A3, or a pharmaceutically acceptable salt thereof, wherein when Z is -C(O)C1-4 alkyl,
Figure imgf000300_0003
absent, then Xb is not -C(O)NH-. A3b. The compound of any of embodiments A1 to A3 and A3a, or a pharmaceutically acceptable salt thereof, wherein when E is -heterocycle-C(O)NH-, then X is absent or -(CH2)m- Xb-. A4. The compound of any of embodiments A1 to A3, A3a and A3b, or a pharmaceutically acceptable salt thereof, wherein E is: a) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X; b) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or c) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-. A5. The compound of embodiment A4, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000301_0001
wherein Ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and Ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A6. The compound of any of embodiments A1-A5, or a pharmaceutically acceptable salt thereof, wherein E is:
Figure imgf000301_0002
wherein Ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A7. The compound of embodiment A6, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a bridged bicycle. A8. The compound of embodiment A7, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a 7-8 membered bridged bicycle. A9. The compound of embodiment A8, or a pharmaceutically acceptable salt thereof, wherein E is selected from: ;
Figure imgf000302_0001
optionally wherein E is ; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A10. The compound of embodiment A9, or a pharmaceutically acceptable salt thereof, wherein E is selected from: ;
Figure imgf000302_0002
optionally wherein E is . A11. The compound of embodiment A9, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000302_0003
. A12. The compound of embodiment A6, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a spirocycle. A13. The compound of embodiment A12, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is an 8-9 membered spirocycle. A14. The compound of embodiment A12 or A13, or a pharmaceutically acceptable salt thereof, wherein the two nitrogen atoms present in the heterocycle are in the same ring of the spirocycle. A15. The compound of embodiment A14, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000303_0001
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A16. The compound of embodiment A15, or a pharmaceutically acceptable salt thereof, wherein E is:
Figure imgf000303_0002
. A17. The compound of embodiment A12 or A13, or a pharmaceutically acceptable salt thereof, wherein the two nitrogen atoms present in the heterocycle are in separate rings of the spirocycle. A18. The compound of embodiment A17, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000303_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A19. The compound of embodiment A6, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a fused bicycle. A20. The compound of embodiment A19, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is an 8-membered fused bicycle. A21. The compound of embodiment A20, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000304_0001
. A22. The compound of embodiment A21, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000304_0002
. A23. The compound of any of embodiments A1 to A3 and A3a, or a pharmaceutically acceptable salt thereof, wherein E is
Figure imgf000304_0003
, wherein Ring Ec is a monocyclic 5-8 membered saturated heterocycle having the two nitrogen atoms shown, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when ring Ec is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A24. The compound of embodiment A23, or a pharmaceutically acceptable salt thereof, wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. A25. The compound of embodiment A23 or A24, or a pharmaceutically acceptable salt thereof, wherein ring EC is a piperazine geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. A26. The compound of any of embodiments A23 to A25, or a pharmaceutically acceptable salt thereof, wherein the geminal substituent is geminal dimethyl. A27. The compound of any of embodiments A23 to A26, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000304_0004
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A28. The compound of embodiment A23, or a pharmaceutically acceptable salt thereof, wherein Ring Ec is a monocyclic 5, 7 or 8 membered saturated heterocycle having the two nitrogen atoms shown, wherein the heterocycle is unsubstituted. A29. The compound of embodiment A23, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000305_0001
. A30. The compound of any of embodiments A1 to A4, or a pharmaceutically acceptable salt thereof, wherein E is -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-. A31. The compound of embodiment A30, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000305_0002
wherein ring Ed is a monocyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A32. The compound of embodiment A31, or a pharmaceutically acceptable salt thereof, wherein ring Ed is a piperidine. A33. The compound of embodiment A30, or a pharmaceutically acceptable salt thereof, wherein E is:
Figure imgf000306_0001
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A34. The compound o *f any of embodiments A1 to A4, or a pharmaceutically acceptable salt thereof, wherein E is -heterocycle-C(O)NH-, wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -C(O)NH-. A35. The compound of embodiment A34, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000306_0002
wherein ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A36. The compound of embodiment A35, or a pharmaceutically acceptable salt thereof, wherein the heterocycle is a bridged bicyclic heterocycle. A37. The compound of embodiment A36, or a pharmaceutically acceptable salt thereof, wherein E is:
Figure imgf000306_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X. A38. The compound of embodiment A37, or a pharmaceutically acceptable salt thereof, wherein E is:
Figure imgf000306_0004
. A39. The compound of any of embodiments A1 to A5 and A30 to A33, or a pharmaceutically acceptable salt thereof, wherein R is H. A40. The compound of any of embodiments A1 to A5 and A30 to A33, or a pharmaceutically acceptable salt thereof, wherein R is C3-4 cycloalkyl. A41. The compound of embodiment A40, or a pharmaceutically acceptable salt thereof, wherein R is cyclopropyl. A42. The compound of any of embodiments A1 to A5 and A30 to A33, or a pharmaceutically acceptable salt thereof, wherein R is C1-4 alkyl, optionally methyl. A43. The compound of any of embodiments A1 to A42, or a pharmaceutically acceptable salt thereof, wherein X is absent. A44. The compound of any of embodiments A1 to A42, or a pharmaceutically acceptable salt thereof, wherein X is -C(O)CH2O-*, wherein * indicates the point of attachment to Y. A45. The compound of any of embodiments A1 to A42, or a pharmaceutically acceptable salt thereof, wherein X is -C(O)CH2NH-*, wherein * indicates the point of attachment to Y. A46. The compound of any of embodiments A1 to A42, or a pharmaceutically acceptable salt thereof, wherein X is -Xa-(CH2)m-Xb-. A47. The compound of embodiment A46, or a pharmaceutically acceptable salt thereof, wherein m is 2. A48. The compound of embodiment A46 or A47, or a pharmaceutically acceptable salt thereof, wherein Xa is -C(O)-. A49. The compound of embodiment A46 or A47, or a pharmaceutically acceptable salt thereof, wherein Xa is absent (i.e. X is -(CH2)m-Xb). A50. The compound of any of embodiments A46 to A49, or a pharmaceutically acceptable salt thereof, wherein Xb is -C(O)NH-*, wherein * indicates the point of attachment to Y. A51. The compound of any of embodiments A46 to A49, or a pharmaceutically acceptable salt thereof, wherein Xb is -O-. A52. The compound of any of embodiments A46 to A49, or a pharmaceutically acceptable salt thereof, wherein Xb is -NH-. A53. The compound of any of embodiments Al to A42, or a pharmaceutically acceptable salt thereof, wherein X is selected from:
Figure imgf000308_0001
wherein the wavy line indicates the point of attachment to E and the asterisk-wavy line indicates the point of attachment to Y.
A54. The compound of any of embodiments Al to A53, or a pharmaceutically acceptable salt thereof, wherein Y is absent.
A55. The compound of any of embodiments Al or A3 to A53, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of Formula (la) or a pharmaceutically acceptable salt thereof, and wherein
Figure imgf000308_0002
optionally
Figure imgf000308_0003
A56. The compound of embodiment A55, or a pharmaceutically acceptable salt thereof, wherein ya and yb are each independently selected from 0, 1 and 2.
A57. The compound of embodiment A55 or A56, or a pharmaceutically acceptable salt thereof, wherein at least one of ya or yb is 0.
A58. The compound of embodiment A55, or a pharmaceutically acceptable salt thereof, wherein ya is 0 and yb is 1.
A59. The compound of embodiment A55, or a pharmaceutically acceptable salt thereof, wherein ya is 1 and yb is 0.
A60. The compound of embodiment A55 or A56, or a pharmaceutically acceptable salt thereof, wherein the total of ya and yb is no more than 3.
A61. The compound of embodiment A55, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both 0. A62. The compound of embodiment A55, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both independently selected from 1 and 2.
A63. The compound of any of embodiments Al to A53, or a pharmaceutically acceptable salt thereof, wherein Y is
Figure imgf000309_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z.
A64. The compound of embodiment A63, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000309_0002
A65. The compound of any of embodiments A55 to A64, or a pharmaceutically acceptable salt thereof, wherein Q is NH, S or O.
A66. The compound of embodiment A65, or a pharmaceutically acceptable salt thereof, wherein Q is NH.
A67. The compound of embodiment A65, or a pharmaceutically acceptable salt thereof, wherein Q is S.
A68. The compound of embodiment A65, or a pharmaceutically acceptable salt thereof, wherein Q is O.
A69. The compound of any of embodiments A55 to A64, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of Formula (la) or a pharmaceutically acceptable salt thereof, and wherein Q is -N(Ci.3alkyl )-, for example -N(CH3)-.
A70. The compound of any of embodiments A55 to A64, or a pharmaceutically acceptable salt thereof, wherein Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z.
A71. The compound of embodiment A70, or a pharmaceutically acceptable salt thereof, wherein
Q is -S-(CH2)2NH-*.
A72. The compound of embodiment A70, or a pharmaceutically acceptable salt thereof, wherein
Q is -O-(CH2)2NH~*. A73. The compound of embodiment A70, or a pharmaceutically acceptable salt thereof, wherein Q is -NH-(CH2)2NH-*. A74. The compound of any of embodiments A1 to A73, or a pharmaceutically acceptable salt thereof, wherein Z is H. A75. The compound of any of embodiments A1 to A73, or a pharmaceutically acceptable salt thereof, wherein Z is C1-4 alkyl, optionally methyl. A76. The compound of any of embodiments A1 to A73, or a pharmaceutically acceptable salt thereof, wherein Z is -C(O)C1-4 alkyl, optionally -C(O)CH3. A77. The compound of any of embodiments A1 to A73, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000310_0001
integer from 1 to 5, and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. A78. The compound of embodiment A77, or a pharmaceutically acceptable salt thereof, wherein n is 1. A79. The compound of embodiment A76 or A77, or a pharmaceutically acceptable salt thereof, wherein RZ is H. A80. The compound of embodiment A77, or a pharmaceutically acceptable salt thereof, wherein n is 2, 3, 4 or 5. A81. The compound of embodiment A80, or a pharmaceutically acceptable salt thereof, wherein one occurrence of RZ is C1-4 alkyl, benzyl or phenyl and the remaining occurrence or occurrences of RZ are H. A82. The compound of embodiment A77, or a pharmaceutically acceptable salt thereof, wherein Z is:
Figure imgf000310_0002
wherein n1 is 0, 1, 2, 3 or 4 and Rz is C1-4 alkyl, benzyl, or phenyl. A83. The compound of any of embodiments A77, A78, A80 or A82, or a pharmaceutically acceptable salt thereof, wherein RZ is C1-4 alkyl. A84. The compound of embodiment A83, or a pharmaceutically acceptable salt thereof, wherein RZ is methyl. A85. The compound of embodiment A83, or a pharmaceutically acceptable salt thereof, wherein RZ is isopropyl. A86. The compound of any of embodiments A77, A78, A80 or A82, or a pharmaceutically acceptable salt thereof, wherein RZ is benzyl. A87. The compound of any of embodiments A77, A78, A80 or A82, or a pharmaceutically acceptable salt thereof, wherein RZ is phenyl. A88. The compound of any of embodiments A1 to A73, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000311_0001
. A89. The compound of embodiment A1 or A2, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from a compound of Table 1, or a pharmaceutically acceptable salt thereof. A90. A pharmaceutical composition comprising a compound of any of embodiments A1 to A89, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. A91. A compound according to any of embodiments A1 to A89, or a pharmaceutically acceptable salt thereof, for use in therapy. A92. A compound according to any of embodiments A1 to A89, or a pharmaceutically acceptable salt thereof, for use in treating cancer. A93. Use of a compound according to any of embodiments A1 to A89, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer. A94. A method of treating cancer in a patient comprising administering to the patient a compound according to any of embodiments A1 to A89, or a pharmaceutically acceptable salt thereof. A95. A pharmaceutical composition according to embodiment A90 for use in therapy. A96. A pharmaceutical composition according to embodiment A90 for use in treating cancer. A97. A method of treating cancer in a patient comprising administering to the patient a pharmaceutical composition according to embodiment A90, or a pharmaceutically acceptable salt thereof. B1. A compound of Formula (IIa): A-E1-X1-Y1-Z1 or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000312_0001
E1 is: i) -NR-; ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X1; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y1; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y1; m is 2 or 3;
Figure imgf000313_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S-(CH2)2NH-*, -O- (CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1; Z1 is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000313_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. B2. A compound of Formula (II): A-E1-X1-Y1-Z1 or a pharmaceutically acceptable salt thereof,
Figure imgf000313_0003
E1 is: iv) -NR-; ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X1; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; iv) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-; R is H, C1-4 alkyl or C3-4 cycloalkyl; X1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y1; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y1; m is 2 or 3; provided that when E1 is -NR- or -heterocycle-C(O)NH-, then X1 is absent or –(CH2)m-
Figure imgf000314_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1; and Q is NH, S, O, -S-(CH2)2NH-*, -O- (CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1; Z1 is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000314_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. B3. The compound of embodiment B1 or B2, or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000314_0003
. B3a. The compound of any of embodiments B1 to B3, or a pharmaceutically acceptable salt thereof, wherein when E1 is -NR- or -heterocycle-C(O)NH-, then X1 is absent or -(CH2)m-Xb-. B4. The compound of any of embodiments B1 to B3 and B3a, or a pharmaceutically acceptable salt thereof, wherein E1 is -NR-. B5. The compound of any of embodiments B1 to B3, or a pharmaceutically acceptable salt thereof, wherein E1 is a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen in the heterocycle is connected to X1; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. B6. The compound of any of embodiments B1 to B3 and B3a, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000315_0001
wherein ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B7. The compound of embodiment B6, or a pharmaceutically acceptable salt thereof, wherein E1 is:
Figure imgf000315_0002
wherein ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B8. The compound of embodiment B6 or B7, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a bridged bicycle. B9. The compound of embodiment B8, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a 7-8 membered bridged bicycle. B10. The compound of embodiment B7, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000316_0001
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B11. The compound of embodiment B10, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000316_0002
. B12. The compound of embodiment B10, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000316_0003
. B13. The compound of embodiment B6 or B7, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a spirocycle. B14. The compound of embodiment B13, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is an 8-9 membered spirocycle. B15. The compound of embodiment B13 or B14, or a pharmaceutically acceptable salt thereof, wherein the two nitrogen atoms present in the heterocycle are in the same ring of the spirocycle. B16. The compound of embodiment B15, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000316_0004
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B17. The compound of embodiment B16, or a pharmaceutically acceptable salt thereof, wherein E1 is:
Figure imgf000317_0001
. B18. The compound of embodiment B13 or B14, or a pharmaceutically acceptable salt thereof, wherein the two nitrogen atoms present in the heterocycle are in separate rings of the spirocycle. B19. The compound of embodiment B18, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000317_0002
or a pharmaceutically acceptable salt thereof, wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B20. The compound of embodiment B6 or B7, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a fused bicycle. B21. The compound of embodiment B20, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is an 8-membered fused bicycle. B22. The compound of embodiment B21, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000317_0003
. B23. The compound of embodiment B22, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000317_0004
. B24. The compound of any of embodiments B1 to B3, or a pharmaceutically acceptable salt thereof, wherein E1 is
Figure imgf000318_0001
wherein ring Ec is a monocyclic 5-8 membered saturated heterocycle having the two nitrogen atoms shown, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; and the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B25. The compound of embodiment B24, or a pharmaceutically acceptable salt thereof, wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. B26. The compound of embodiment B24 or B25, or a pharmaceutically acceptable salt thereof, wherein ring Ec is a piperazine geminally substituted on a carbon atom with two occurrences of C1-3 alkyl. B27. The compound of any of embodiments B24 to B26, or a pharmaceutically acceptable salt thereof, wherein the geminal substituent is geminal dimethyl. B28. The compound of any of embodiments B24 to B27, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000318_0002
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B29. The compound of embodiment B24, or a pharmaceutically acceptable salt thereof, wherein ring Ec is a monocyclic 5 to 8 membered saturated heterocycle having the two nitrogen atoms shown, wherein the heterocycle is unsubstituted. B30. The compound of embodiment B24, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000319_0001
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B31. The compound of any of embodiments B1 to B3, or a pharmaceutically acceptable salt thereof, wherein E1 is -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-. B32. The compound of embodiment B31, or a pharmaceutically acceptable salt thereof, wherein E1 is:
Figure imgf000319_0002
, wherein ring Ed is a monocyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B33. The compound of embodiment B32, or a pharmaceutically acceptable salt thereof, wherein ring Ed is a piperidine. B34. The compound of embodiment B32 or B33, or a pharmaceutically acceptable salt thereof, wherein E1 is:
Figure imgf000319_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B35. The compound of any of embodiments B1 to B3 and B3a, or a pharmaceutically acceptable salt thereof, wherein E1 is -heterocycle-C(O)NH-, wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -C(O)NH-. B36. The compound of em *bodiment B35, or a pharmaceutically acceptable salt thereof, wherein E1 is
Figure imgf000320_0001
wherein ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B37. The compound of embodiment B36, or a pharmaceutically acceptable salt thereof, wherein the heterocycle is a bridged bicyclic heterocycle. B38. The compound of embodiment B37, or a pharmaceutically acceptable salt thereof, wherein E1 is:
Figure imgf000320_0002
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1. B39. The compound of embodiment B38, or a pharmaceutically acceptable salt thereof, wherein E1 is:
Figure imgf000320_0003
. B40. The compound of any of embodiments B1-B4, B6 and B32-B34, or a pharmaceutically acceptable salt thereof, wherein R is H. B41. The compound of any of embodiments B1-B4, B6 and B32-B34, or a pharmaceutically acceptable salt thereof, wherein R is C3-4 cycloalkyl. B42. The compound of embodiment B41, or a pharmaceutically acceptable salt thereof, wherein R is cyclopropyl. B43. The compound of any of embodiments B1-B4, B6 and B32-B34, or a pharmaceutically acceptable salt thereof, wherein R is C1-4 alkyl, optionally methyl. B44. The compound of any of embodiments B1 to B43, or a pharmaceutically acceptable salt thereof, wherein X1 is absent. B45. The compound of any of embodiments Bl to B43, or a pharmaceutically acceptable salt thereof, wherein X1 is -C(O)CH2O-*, wherein * indicates the point of attachment to Y1.
B46. The compound of any of embodiments Bl to B43, or a pharmaceutically acceptable salt thereof, wherein X1 is -C(O)CH2NH-*, wherein * indicates the point of attachment to Y1.
B47. The compound of any of embodiments Bl to B43, or a pharmaceutically acceptable salt thereof, wherein X1 is -Xa-(CH2)m-Xb-.
B48. The compound of embodiment B47, or a pharmaceutically acceptable salt thereof, wherein m is 2.
B49. The compound of embodiment B47 or B48, or a pharmaceutically acceptable salt thereof, wherein Xa is -C(O)-.
B50. The compound of embodiment B47 or B48, or a pharmaceutically acceptable salt thereof, wherein Xa is absent (i.e. X1 is -(CH2)m-Xb).
B51. The compound of any of embodiments B47 to B50, or a pharmaceutically acceptable salt thereof, wherein Xb is -C(O)NH-*, wherein * indicates the point of attachment to Y1.
B52. The compound of any of embodiments B47 to B50, or a pharmaceutically acceptable salt thereof, wherein Xb is -O-.
B53. The compound of any of embodiments B47 to B50, or a pharmaceutically acceptable salt thereof, wherein Xb is -NH-.
B54. The compound of any of embodiments Bl to B43, or a pharmaceutically acceptable salt thereof, wherein X1 is selected from:
Figure imgf000321_0001
wherein the wavy line indicates the point of attachment to E1 and the asterisk-wavy line indicates the point of attachment to Y1. B54a. The compound of any of embodiments Bl or B3 to B54, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of Formula (Ila) or a pharmaceutically acceptable salt thereof, and wherein Y1 is
Figure imgf000322_0001
B55. The compound of any of embodiments Bl, B3 to B54, or B54a, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of Formula (Ila) or a pharmaceutically acceptable salt thereof, and wherein ya and yb are each independently selected from 0, 1 and 2.
B56. The compound of embodiment B54a or B55, or a pharmaceutically acceptable salt thereof, wherein at least one of ya or yb is 0.
B57. The compound of embodiment B54a or B55, or a pharmaceutically acceptable salt thereof, wherein ya is 0 and yb is 1.
B58. The compound of embodiment B54a or B55, or a pharmaceutically acceptable salt thereof, wherein ya is 1 and yb is 0.
B59. The compound of embodiment B54a or B55, or a pharmaceutically acceptable salt thereof, wherein the total of ya and yb is no more than 3.
B60. The compound of embodiment B54a or B55, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both 0.
B61. The compound of embodiment B54a or B55, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both independently selected from 1 and 2.
B62. The compound of any of embodiments Bl to B54, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000322_0002
B63. The compound of any of embodiments Bl to B62, or a pharmaceutically acceptable salt thereof, wherein Q is NH, S or O.
B64. The compound of embodiment B63, or a pharmaceutically acceptable salt thereof, wherein Q is NH. B65. The compound of embodiment B63, or a pharmaceutically acceptable salt thereof, wherein Q is S.
B66. The compound of embodiment B63, or a pharmaceutically acceptable salt thereof, wherein Q is O.
B67. The compound of any of embodiments Bl or B3 to B62, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of Formula (Ila) or a pharmaceutically acceptable salt thereof, and wherein Q is -N(Ci 3alkyl )-, for example -N(CH3)~.
B68. The compound of any of embodiments Bl to B62, or a pharmaceutically acceptable salt thereof, wherein Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to Z1.
B69. The compound of embodiment B68, or a pharmaceutically acceptable salt thereof, wherein Q is -S-(CH2)2NH-*.
B70. The compound of embodiment B68, or a pharmaceutically acceptable salt thereof, wherein Q is -O-(CH2)2NH-*.
B71. The compound of embodiment B68, or a pharmaceutically acceptable salt thereof, wherein Q, is -NH-(CH2)2NH-*.
B72. The compound of any of embodiments Bl to B71, or a pharmaceutically acceptable salt thereof, wherein Z1 is H.
B73. The compound of any of embodiments Bl to B71, or a pharmaceutically acceptable salt thereof, wherein Z1 is CM alkyl, optionally methyl.
B74. The compound of any of embodiments Bl to B71, or a pharmaceutically acceptable salt thereof, wherein Z1 is -C(O)Ci-4 alkyl, optionally -C(O)CH3.
B75. The compound of any of embodiments Bl to B71, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000323_0001
integer from 1 to 5, and each Rz is independently H, Cw alkyl, phenyl or benzyl.
B76. The compound of embodiment B75, or a pharmaceutically acceptable salt thereof, wherein n is 1. B77. The compound of embodiment B75 or B76, or a pharmaceutically acceptable salt thereof, wherein Rz is H.
B78. The compound of embodiment B75, or a pharmaceutically acceptable salt thereof, wherein n is 2, 3, 4 or 5.
B79. The compound of embodiment B78, or a pharmaceutically acceptable salt thereof, wherein one occurrence of Rz is CM alkyl, benzyl or phenyl and the remaining occurrence or occurrences of Rz are H.
B80. The compound of embodiment B75, or a pharmaceutically acceptable salt thereof, wherein Z1 is:
Figure imgf000324_0001
wherein nl is 0, 1, 2, 3 or 4 and Rz is Cw alkyl, benzyl, or phenyl.
B81. The compound of any of embodiments B75, B76, B78 or 80, or a pharmaceutically acceptable salt thereof, wherein Rz is CM alkyl.
B82. The compound of embodiment B81, or a pharmaceutically acceptable salt thereof, wherein Rz is methyl.
B83. The compound of embodiment B81, or a pharmaceutically acceptable salt thereof, wherein Rz is isopropyl.
B84. The compound of any of embodiments B75, B76, B78 or 80, or a pharmaceutically acceptable salt thereof, wherein Rz is benzyl.
B85. The compound of any of embodiments B75, B76, B78 or 80, or a pharmaceutically acceptable salt thereof, wherein Rz is phenyl.
B86. The compound of any of embodiments Bl to B71, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000324_0002
B87. The compound of embodiment Bl, or a pharmaceutically acceptable salt thereof, which is selected from a compound of Table 2 or Table 2A, or a pharmaceutically acceptable salt thereof. B88. The compound of embodiment Bl or B2, or a pharmaceutically acceptable salt thereof, which is selected from a compound of Table 2, or a pharmaceutically acceptable salt thereof.
B89. A pharmaceutical composition comprising a compound of any of embodiments Bl to B88, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
B90. A compound according to any of embodiments Bl to B88, or a pharmaceutically acceptable salt thereof, for use in therapy.
B91. A compound according to any of embodiments Bl to B88, or a pharmaceutically acceptable salt thereof, for use in treating cancer.
B92. Use of a compound according to any of embodiments Bl to B88, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer.
B93. A method of treating cancer in a patient comprising administering to the patient a compound according to any of embodiments Bl to B88, or a pharmaceutically acceptable salt thereof.
B94. A pharmaceutical composition according to embodiment B89 for use in therapy.
B95. A pharmaceutical composition according to embodiment B89 for use in treating cancer.
B96. A method of treating cancer in a patient comprising administering to the patient a pharmaceutical composition according to embodiment B89, or a pharmaceutically acceptable salt thereof.
Cl. A compound of Formula (Illa):
A-E-X-Y-L1 or a salt thereof, wherein A, E and X are as defined in any of embodiments Al to A53; and Y is absent or
Figure imgf000326_0001
, wherein the wavy line indicates the point of attachment to
X and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N (Ci_3al kyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof.
C2. A compound of Formula (III):
A-E-X-Y-L1 or a salt thereof, wherein A, E and X are as defined in any of embodiments Al to A53; and
/ °X
Y is absent or r OyV , wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L1; and Q, is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof.
C3. The compound of embodiment Cl or C2, or a salt thereof, wherein Y is absent.
C4. The compound of embodiment Cl, or a pharmaceutically acceptable salt thereof, wherein Y is
Figure imgf000326_0002
' p y
C5. The compound of embodiment C4, or a pharmaceutically acceptable salt thereof, wherein ya and yb are each independently selected from 0, 1 and 2.
C6. The compound of embodiment C4 or C5, or a pharmaceutically acceptable salt thereof, wherein at least one of ya or yb is 0.
C7. The compound of embodiment C4, or a pharmaceutically acceptable salt thereof, wherein ya is 0 and yb is 1. C8. The compound of embodiment C4, or a pharmaceutically acceptable salt thereof, wherein ya is 1 and yb is 0.
C9. The compound of embodiment C4 or C5, or a pharmaceutically acceptable salt thereof, wherein the total of ya and yb is no more than 3.
CIO. The compound of embodiment C4, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both 0.
Cll. The compound of embodiment C4, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both independently selected from 1 and 2.
C12. The compound of embodiment Cl or C2, or a salt thereof, wherein
Figure imgf000327_0001
wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L1.
C13. The compound of embodiment C12, or a salt thereof, wherein Y is
Figure imgf000327_0002
C14. The compound of any of embodiments C4 to C13, or a salt thereof, wherein Q is NH, S or O.
C15. The compound of embodiment C14, or a salt thereof, wherein Q is NH.
C16. The compound of embodiment C14, or a salt thereof, wherein Q is S.
C17. The compound of embodiment C14, or a salt thereof, wherein Q is O.
C18. The compound of any of embodiments C4 to C13, or a salt thereof, wherein the compound is a compound of Formula (Illa) or a salt thereof, and wherein Q, is -N(Cv3alkyl)-, for example - N(CH3)-.
C19. The compound of any of embodiments C4 to C13, or a salt thereof, wherein Q is -S- (CHzhNH-*, -O-(CH2)2NH-* or -NH-fCHzhNH-*, wherein * indicates the point of attachment to L1.
C20. The compound of embodiment C19, or a salt thereof, wherein Q, is -S-(CH2)2NH-*.
C21. The compound of embodiment C19, or a salt thereof, wherein Q, is -O-(CH2)2NH-*. C22. The compound of embodiment C19, or a salt thereof, wherein Q is -NH-(CH2)2NH-*.
C23. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is:
Figure imgf000328_0001
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y; each RZ1 is independently H, CM alkyl, phenyl or benzyl; q is 3, 4 or 5;
Figure imgf000328_0002
asterisk-wavy line indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
C24. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is:
Figure imgf000328_0003
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y; each RZ1 is independently H, C1.4 alkyl, phenyl or benzyl; q is 3, 4 or 5;
Figure imgf000328_0004
indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15; and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof; provided that when Y is absent, then Xb is not -C(O)NH-.
C25. The compound of embodiment C23 or C24, or a salt thereof, wherein LA is absent or *-CH2- NH-, wherein * indicates the point of attachment to Y.
C26. The compound of embodiment C23 or C24, or a salt thereof, wherein L1 is:
Figure imgf000329_0001
C27. The compound of any of embodiments C23 to C26, or a salt thereof, wherein q is 3.
C28. The compound of any of embodiments C23 to C26, or a salt thereof, wherein q is 4.
C29. The compound of any of embodiments C23 to C26, or a salt thereof, wherein q is 5.
C30. The compound of any of embodiments C23 to C29, or a salt thereof, wherein LB is — (CH2)b-
C31. The compound of any of embodiments C23 to C29, or a salt thereof, wherein LB is
Figure imgf000329_0002
C32. The compound of any of embodiments C23 to C29 or C31, or a salt thereof, wherein c is 7.
C33. The compound of any of embodiments C23 to C32, or a salt thereof, wherein b is 2.
C34. The compound of embodiment C23 or of any of embodiments C25 to C29 (where dependent on C23), or a salt thereof, wherein LB is
Figure imgf000329_0003
C35. The compound of embodiment C34, or a salt thereof, wherein d is 1.
C36. The compound of embodiment C34, or a salt thereof, wherein d is 2.
C37. The compound of any of embodiments C23 to C36, or a salt thereof, wherein RZ1 is H.
C38. The compound of any of embodiments C23 to C36, or a salt thereof, wherein one occurrence of RZ1 is C1.4 alkyl, benzyl or phenyl and the remaining occurrences of RZ1 are H. C39. The compound of embodiment C23 or C24, or a salt thereof, wherein L1 is:
Figure imgf000330_0001
wherein RZ1 is CM alkyl, benzyl, or phenyl and ql is 2, 3 or 4, optionally wherein ql is 3.
C40. The compound of embodiment C39, or a salt thereof, wherein LB is as defined in any of embodiments C30 to C36.
C41. The compound of any of embodiments C23 to C36, C39 or C40, or a salt thereof, wherein RZ1 is CM alkyl.
C42. The compound of embodiment C41, or a salt thereof, wherein RZ1 is methyl.
C43. The compound of embodiment C41, or a salt thereof, wherein RZ1 is isopropyl.
C44. The compound of any of embodiments C23 to C36, C39 or C40, or a salt thereof, wherein RZ1 is benzyl.
C45. The compound of any of embodiments C23 to C36, C39 or C40, or a salt thereof, wherein
RZ1 is phenyl.
C46. The compound of embodiment C23 or C24, or a salt thereof, wherein L1 is selected from:
Figure imgf000330_0002
or a salt thereof.
C47. The compound of embodiment C23 or C24, or a salt thereof, wherein L1 is:
Figure imgf000331_0001
or a salt thereof.
C48. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is a group of
Formula (L-IA):
Figure imgf000331_0002
(HA), wherein E2 is (CH2)n2, wherein n2 is 0, 1, 2 or 3,
Figure imgf000331_0003
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000331_0004
1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
C49. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is a group of Formula (L-l):
Figure imgf000332_0001
wherein E2 is (CH2)n2, wherein n2 is 0, 1, 2 or 3,
Figure imgf000332_0002
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is CM alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Z2 is (CH2)n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof; provided that when Y is absent, then Xb is not -C(O)NH-.
C50. The compound of embodiment C48 or C49, or a salt thereof, wherein Q2 is
Figure imgf000333_0001
C51. The compound of embodiment C50, or a salt thereof, wherein Q2 is
Figure imgf000333_0002
C52. The compound of any of embodiments C48 to C51, or a salt thereof, wherein m2 is 9, 10, 11, 12 or 13.
C53. The compound of any of embodiments C48 to C52, or a salt thereof, wherein R1 is CH3.
C54. The compound of any of embodiments C48 to C53, or a salt thereof, wherein E2 is CH2.
C55. The compound of any of embodiments C48 to C54, or a salt thereof, wherein X2 is CH2.
C56. The compound of any of embodiments C48 to C55, or a salt thereof, wherein Y2 is (CH2)2-
C57. The compound of any of embodiments C48 to C56, or a salt thereof, wherein Z2 is (CH2)2.
C58. The compound of any of embodiments C48 or C50 to C56, or a salt thereof, wherein L1 is a group of Formula (L-l A), and Z2 is
Figure imgf000333_0003
; optionally wherein n5B is 2 and/or n5A is 1.
C59. The compound of any of embodiments C48 to C58, or a salt thereof, wherein p is 1.
C60. The compound of any of embodiments C48 to C59, or a salt thereof, wherein pl is 0.
C61. The compound of any of embodiments C48 to C59, or a salt thereof, wherein pl is 1.
C62. The compound of embodiment C48 or C49, or a salt thereof, wherein L1 is selected from:
Figure imgf000334_0001
C63. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is a group of
Formula (L-lll):
Figure imgf000334_0002
wherein E3 is (CH2)ne, wherein n6 is 0, 1, 2 or 3,
R1A is Ci-4 alkyl or H,
Figure imgf000334_0003
wherein R3 is CO2H or CH2OH,
Figure imgf000335_0001
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, Cw alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety;
Y3 is O or NRB, wherein
Figure imgf000335_0002
cycloalkyl,
Z3 is (CH2)nio or
Figure imgf000335_0003
, wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
C64. The compound of embodiment C63, or a salt thereof, wherein m3 is 9, 10, 11, 12 or 13.
C65. The compound of embodiment C63 or C64, or a salt thereof, wherein R1A is CH3.
C66. The compound of any of embodiments C63 to C65, or a salt thereof, wherein R2 is
Figure imgf000335_0004
C67. The compound of any of embodiments C63 to C66, or a salt thereof, wherein E3 is CH2.
C68. The compound of any of embodiments C63 to C67, or a salt thereof, wherein X4 is CH2.
C69. The compound of any of embodiments C63 to C68, or a salt thereof, wherein Y3 is O.
C70. The compound of any of embodiments C63 to C69, or a salt thereof, wherein Z3 is (CH2)nio, optionally (CH2)2.
C71. The compound of any of embodiments C63 to C70, or a salt thereof, wherein p2 is 1.
C72. The compound of any of embodiments C63 to C70, or a salt thereof, wherein p2 is 0.
C73. The compound of any of embodiments C63 to C72, or a salt thereof, wherein q2 is 1. C74. The compound of any of embodiments C63 to C73, or a salt thereof, wherein p3 is 1.
C75. The compound of any of embodiments C63 to C73, or a salt thereof, wherein p3 is 0.
C76. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is selected from the moieties of Table 3. C77. The compound of any of embodiments C23 to C76, or a salt thereof, wherein G1 is selected
Figure imgf000336_0001
optionally wherein G1 is selected from:
Figure imgf000337_0001
wherein X3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is Ci_6 alkyl
C78. The compound of embodiment C77, or a salt thereof, wherein G1 is selected from:
Figure imgf000337_0002
C79. The compound of embodiment C78, or a salt thereof, wherein G1 is
Figure imgf000337_0003
C80. The compound of embodiment C78, or a salt thereof, wherein G1 is
Figure imgf000337_0004
C81. The compound of embodiment C78, or a salt thereof, wherein G1 is
Figure imgf000338_0001
C82. The compound of any of embodiments C23 to C78, or a salt thereof, wherein -P-G1, -Z^G1 or -Z3-G1 is:
Figure imgf000338_0002
C83. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is selected from:
Figure imgf000338_0003
or a salt thereof.
C84. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is:
Figure imgf000338_0004
salt thereof.
C85. The compound of any of embodiments Cl to C22, or a salt thereof, wherein L1 is selected from the moieties of Table 4.
C85a. The compound of any of embodiments C23 to C85, or a salt thereof, wherein when Y is absent, then Xb is not -C(O)NH-.
C86. The compound of embodiment Cl or C2, or a salt thereof, wherein the compound is:
Figure imgf000339_0001
or a salt thereof.
C87. The compound of embodiment Cl or C2, or a salt thereof, wherein the compound is selected from:
Figure imgf000339_0002
or a salt thereof. C88. The compound of embodiment Cl or C2, or a salt thereof, which is selected from a compound of Table 5A, or a salt thereof.
DI. A compound of Formula (IVa): or a salt thereof, wherein A, E1 and X1 are as defined in any of embodiments Bl to B54; Y1 is
Figure imgf000340_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(Cwalkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof.
D2. A compound of Formula (IV): or a salt thereof, wherein A, E1 and X1 are as defined in any of embodiments Bl to B54;
Figure imgf000340_0002
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L1; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof.
D3. The compound of embodiment DI, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000340_0003
D4. The compound of embodiment DI or D3, or a pharmaceutically acceptable salt thereof, wherein ya and yb are each independently selected from 0, 1 and 2.
D4a. The compound of embodiment DI, D3 or D4, or a pharmaceutically acceptable salt thereof, wherein at least one of ya or yb is 0.
D5. The compound of embodiment DI or D3, or a pharmaceutically acceptable salt thereof, wherein ya is 0 and yb is 1.
D6. The compound of embodiment DI or D3, or a pharmaceutically acceptable salt thereof, wherein ya is 1 and yb is 0. D7. The compound of embodiment DI, D3 or D4, or a pharmaceutically acceptable salt thereof, wherein the total of ya and yb is no more than 3.
D8. The compound of embodiment DI or D3, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both 0.
D9. The compound of embodiment DI or D3, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both independently selected from 1 and 2.
DIO. The compound of embodiment DI or D2, or a salt thereof, wherein Y1 is
Figure imgf000341_0001
Dll. The compound of any of embodiments DI to DIO, or a salt thereof, wherein Q is NH, S or O.
D12. The compound of embodiment Dll, or a salt thereof, wherein Q is NH.
D13. The compound of embodiment Dll, or a salt thereof, wherein Q is S.
D14. The compound of embodiment Dll, or a salt thereof, wherein Q is O.
D15. The compound of any of embodiments DI or D3 to DIO, or a salt thereof, wherein the compound is a compound of Formula (IVa) or a salt thereof, and wherein Q is -N(Ci.3al kyl)-, for example -N(CH3)-.
D16. The compound of any of embodiments DI to DIO, or a salt thereof, wherein Q is -S- (CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*.
D17. The compound of embodiment D16, or a salt thereof, wherein Q, is -S-(CH2)2NH-*.
D18. The compound of embodiment D16, or a salt thereof, wherein Q is -O-(CH2)2NH-*.
D19. The compound of embodiment D16, or a salt thereof, wherein Q, is -NH-(CH2)2NH-*.
D20. The compound of any of embodiments DI to D19, or a salt thereof, wherein L1 is:
Figure imgf000341_0002
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y1; each RZ1 is independently H, Cw alkyl, phenyl or benzyl; q is 3, 4 or 5;
LB is selected from — (CH2)t>-7
Figure imgf000342_0001
, wherein the asterisk-wavy line indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
D21. The compound of any of embodiments Dl to D19, or a salt thereof, wherein L1 is:
Figure imgf000342_0002
wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1-
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y1; each RZ1 is independently H, C1.4 alkyl, phenyl or benzyl; q is 3, 4 or 5;
LB is selected from - (CH2)t>- and
Figure imgf000342_0003
, wherein the asterisk-wavy line indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15;
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
D22. The compound of embodiment D20 or D21, or a salt thereof, wherein LA is absent or *-CH2- NH-, wherein * indicates the point of attachment to Y1. D23. The compound of embodiment D20 or D21, or a salt thereof, wherein L1 is:
Figure imgf000343_0001
D24. The compound of any of embodiments D20 to D23, or a salt thereof, wherein q is 3.
D25. The compound of any of embodiments D20 to D23, or a salt thereof, wherein q is 4.
D26. The compound of any of embodiments D20 to D23, or a salt thereof, wherein q is 5.
D27. The compound of any of embodiments D20 to D26, or a salt thereof, wherein LB is — (CH2)b-
D28. The compound of any of embodiments D20 to D26, or a salt thereof, wherein LB is
Figure imgf000343_0002
D29. The compound of any of embodiments D20 to D26 or D28, or a salt thereof, wherein c is 7.
D30. The compound of any of embodiments D20 to D29, or a salt thereof, wherein b is 2.
D31. The compound of embodiment D20 or any of embodiments D22 to D26 (when dependent
Figure imgf000343_0003
D32. The compound of embodiment D31, or a salt thereof, wherein d is 1.
D33. The compound of embodiment D31, or a salt thereof, wherein d is 2.
D34. The compound of any of embodiments D20 to D33, or a salt thereof, wherein RZ1 is H.
D35. The compound of any of embodiments D20 to D33, or a salt thereof, wherein one occurrence of RZ1 is CM alkyl, benzyl or phenyl and the remaining occurrences of RZ1 are H.
D36. The compound of embodiment D20 or D21, or a salt thereof, wherein L1 is:
Figure imgf000343_0004
wherein RZ1 is C1.4 alkyl, benzyl, or phenyl and ql is 2, 3 or 4, optionally wherein ql is 3. D37. The compound of embodiment D36, or a salt thereof, wherein LB is as defined in any of embodiments D27 to D33.
D38. The compound of any of embodiments D20 to D33, D36 or D37, or a salt thereof, wherein RZ1 is CM alkyl. D39. The compound of embodiment D38, or a salt thereof, wherein RZ1 is methyl.
D40. The compound of embodiment D38, or a salt thereof, wherein RZ1 is isopropyl.
D41. The compound of any of embodiments D20 to D33, D36 or D37, or a salt thereof, wherein RZ1 is benzyl.
D42. The compound of any of embodiments D20 to D33, D36 or D37, or a salt thereof, wherein RZ1 is phenyl.
D43. The compound of embodiment D20 or D21, or a salt thereof, wherein L1 is selected from:
Figure imgf000344_0001
or a salt thereof.
D44. The compound of embodiment D20 or D21, or a salt thereof, wherein L1 is:
Figure imgf000344_0002
salt thereof.
D45. The compound of any of embodiments DI to D19, or a salt thereof, wherein L1 is a group of
Formula (L-IA):
Figure imgf000345_0001
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is CM alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000345_0002
1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
D46. The compound of any of embodiments DI to D19, or a salt thereof, wherein L1 is a group of
Formula (L-l):
Figure imgf000346_0001
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is CM alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Z2 is (CH2)n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
D47. The compound of embodiment D45 or D46, or a salt thereof, wherein Q2 is
Figure imgf000346_0002
D48. The compound of embodiment D47, or a salt thereof, wherein Q2 is
Figure imgf000347_0001
p y
D49. The compound of any of embodiments D45 to D48, or a salt thereof, wherein m2 is 9, 10,
11, 12 or 13.
D50. The compound of any of embodiments D45 to D49, or a salt thereof, wherein R1 is CH3.
D51. The compound of any of embodiments D45 to D50 or a salt thereof, wherein E2 is CH2.
D52. The compound of any of embodiments D45 to D51, or a salt thereof, wherein X2 is CH2.
D53. The compound of any of embodiments D45 to D52, or a salt thereof, wherein Y2 is (CH2)2.
D54. The compound of any of embodiments D45 to D53, or a salt thereof, wherein Z2 is (CH2)2.
D55. The compound of any of embodiments D45 or D47 to D53, or a salt thereof, wherein L1 is a group of Formula (L-IA), and Z2 is
Figure imgf000347_0002
; optionally wherein n5B is 2 and/or n5A is 1.
D56. The compound of any of embodiments D45 to D55, or a salt thereof, wherein p is 1.
D57. The compound of any of embodiments D45 to D56, or a salt thereof, wherein pl is 0.
D58. The compound of any of embodiments D45 to D56, or a salt thereof, wherein pl is 1.
D59. The compound of embodiment D45 or D46, or a salt thereof, wherein L1 is selected from:
Figure imgf000347_0003
Figure imgf000348_0001
. D60. The compound of any of embodiments D1 to D19, or a salt thereof, wherein L1 is a group of Formula (L-III):
Figure imgf000348_0002
(L-III), wherein E3 is (CH2)n6, wherein n6 is 0, 1, 2 or 3, R1A is C1-4 alkyl or H,
Figure imgf000348_0003
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, C1-4 alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety; Y3 is O or NRB, wherein RB is H, C1-4 alkyl or C3-4 cycloalkyl, Z3 is (CH2)nio or
Figure imgf000349_0001
; wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
D61. The compound of embodiment D60, or a salt thereof, wherein m3 is 9, 10, 11, 12 or 13.
D62. The compound of embodiment D60 or D61, or a salt thereof, wherein R1A is CH3.
D63. The compound of any of embodiments D60 to D62, or a salt thereof, wherein R2 is
Figure imgf000349_0002
D64. The compound of any of embodiments D60 to D63, or a salt thereof, wherein E3 is CH2.
D65. The compound of any of embodiments D60 to D64, or a salt thereof, wherein X4 is CH2.
D66. The compound of any of embodiments D60 to D65, or a salt thereof, wherein Y3 is O.
D67. The compound of any of embodiments D60 to D66, or a salt thereof, wherein Z3 is (CH2)nio, optionally (CH2)2.
D68. The compound of any of embodiments D60 to D67, or a salt thereof, wherein p2 is 1.
D69. The compound of any of embodiments D60 to D67, or a salt thereof, wherein p2 is 0.
D70. The compound of any of embodiments D60 to D69, or a salt thereof, wherein q2 is 1.
D71. The compound of any of embodiments D60 to D70, or a salt thereof, wherein p3 is 1.
D72. The compound of any of embodiments D60 to D70, or a salt thereof, wherein p3 is 0.
D73. The compound of any of embodiments DI to D19, or a salt thereof, wherein L1 is selected from the moieties of Table 3.
D74. The compound of any of embodiments D20 to D73, or a salt thereof, wherein G1 is selected from:
Figure imgf000350_0001
optionally wherein G1 is selected from:
Figure imgf000350_0002
wherein X3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is Ci_6 alkyl.
D75. The compound of embodiment D74, or a salt thereof, wherein G1 is selected from:
Figure imgf000351_0001
D76. The compound of embodiment D75, or a salt thereof, wherein G1 is
Figure imgf000351_0002
D77. The compound of embodiment D75, or a salt thereof, wherein G1 is
Figure imgf000351_0003
D78. The compound of embodiment D75, or a salt thereof, wherein G1 is
BrX^A
D79. The compound of any of embodiments D20 to D74, or a salt thereof, wherein -P-G1, -Z^G1 or -Z^G1 is:
Figure imgf000351_0004
D80. The compound of any of embodiments DI to D19, or a salt thereof, wherein L1 is selected from:
Figure imgf000352_0001
or a salt thereof.
D81. The compound of any of embodiments DI to D19, or a salt thereof, wherein L1 is:
Figure imgf000352_0002
salt thereof.
D82. The compound of any of embodiments DI to D19, or a salt thereof, wherein L1 is selected from the moieties of Table 4.
D83. The compound of embodiment DI or D2, or a salt thereof, wherein the compound is selected from:
Figure imgf000352_0003
Figure imgf000353_0001
or a salt thereof. D84. The compound of embodiment DI or D2, or a salt thereof, wherein the compound is selected from:
Figure imgf000353_0002
and
Figure imgf000354_0001
or a salt thereof.
D85. The compound of embodiment DI or D2, or a salt thereof, which is selected from a compound of Table 5B, or a salt thereof.
D86. The compound of embodiment DI, or a salt thereof, which is selected from a compound of Table 5B or Table 5C, or a salt thereof.
El. A conjugate of Formula (Va):
(A-E-X-Y-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E and X is as defined in any of embodiments Al to A53,
Y is absent
Figure imgf000354_0002
, wherein the wavy line indicates the point of attachment to
X and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N (Ci.3al kyl)-, S, O, -SfCFbhNH-*, - O(CH2)2NH-* or -NHjCHzhNH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and
Ab is an antibody or antigen-binding fragment thereof.
E2. A conjugate of Formula (V)
(A-E-X-Y-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E and X is as defined in any of embodiments Al to A53, Y is absent or
Figure imgf000355_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to L2; and Q. is NH, S, O, -SjCHzhNH-*, - O(CH2hNH-* or -NHjCHzjzNH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and
Ab is an antibody or antigen-binding fragment thereof.
E3. The conjugate of embodiment El or E2, or a pharmaceutically acceptable salt thereof, wherein Y is absent.
E4. The conjugate of embodiment El, or a pharmaceutically acceptable salt thereof, wherein Y is
Figure imgf000355_0002
E5. The conjugate of embodiment E4, or a pharmaceutically acceptable salt thereof, wherein ya and yb are each independently selected from 0, 1 and 2.
E6. The conjugate of embodiment E4 or 45, or a pharmaceutically acceptable salt thereof, wherein at least one of ya or yb is 0.
E7. The conjugate of embodiment E4, or a pharmaceutically acceptable salt thereof, wherein ya is 0 and yb is 1.
E8. The conjugate of embodiment E4, or a pharmaceutically acceptable salt thereof, wherein ya is 1 and yb is 0.
E9. The conjugate of embodiment E4 or E5, or a pharmaceutically acceptable salt thereof, wherein the total of ya and yb is no more than 3.
E10. The conjugate of embodiment E4, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both 0.
Ell. The conjugate of embodiment E4, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both independently selected from 1 and 2. E12. The conjugate of embodiment El or E2, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000356_0001
wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to I2.
E13. The conjugate of embodiment E12, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000356_0002
E14. The conjugate of any of embodiments E4 to E13, or a pharmaceutically acceptable salt thereof, wherein Q is NH, S or O.
E15. The conjugate of embodiment E14, or a pharmaceutically acceptable salt thereof, wherein Q is NH.
E16. The conjugate of embodiment E14, or a pharmaceutically acceptable salt thereof, wherein Q is S.
E17. The conjugate of embodiment E14, or a pharmaceutically acceptable salt thereof, wherein Q is O.
E18. The conjugate of any of embodiments E4 to E13, or a pharmaceutically acceptable salt thereof, wherein the conjugate is a conjugate of Formula (Va) or a pharmaceutically acceptable salt thereof, and wherein Q is -N(Ci.3alkyl )-, for example -N(CH3)-.
E19. The conjugate of any of embodiments E4 to E13, or a pharmaceutically acceptable salt thereof, wherein Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*, wherein * indicates the point of attachment to L2.
E20. The conjugate of embodiment E19, or a pharmaceutically acceptable salt thereof, wherein Q is -S-(CH2)2NH-*.
E21. The conjugate of embodiment E19, or a pharmaceutically acceptable salt thereof, wherein Q is -O-(CH2)2NH-*.
E22. The conjugate of embodiment E19, or a pharmaceutically acceptable salt thereof, wherein
Q is -NH-(CH2)2NH-*. E23. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000357_0001
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y; each RZ1 is independently H, CM alkyl, phenyl or benzyl; q is 3, 4 or 5;
Figure imgf000357_0002
asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
E24. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000357_0003
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y; each RZ1 is independently H, CM alkyl, phenyl or benzyl; q is 3, 4 or 5; LB is selected from -(CH2)b- and
Figure imgf000358_0001
, wherein the asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof; provided that when Y is absent, then Xb is not -C(O)NH-.
E25. The conjugate of embodiment E23 or E24, or a pharmaceutically acceptable salt thereof, wherein LA is absent or *-CH2-NH-, wherein * indicates the point of attachment to Y.
E26. The conjugate of embodiment E23 or E24, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000358_0002
E27. The conjugate of any of embodiments E23 to E26, or a pharmaceutically acceptable salt thereof, wherein q is 3.
E28. The conjugate of any of embodiments E23 to E26, or a pharmaceutically acceptable salt thereof, wherein q is 4.
E29. The conjugate of any of embodiments E23 to E26, or a pharmaceutically acceptable salt thereof, wherein q is 5.
E30. The conjugate of any of embodiments E23 to E29, or a pharmaceutically acceptable salt thereof, wherein LB is -(CH2)b.
E31. The conjugate of any of embodiments E23 to E29, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000358_0003
E32. The conjugate of any of embodiments E23 to E29 or E31, or a pharmaceutically acceptable salt thereof, wherein c is 7. E33. The conjugate of any of embodiments E23 to E32, or a pharmaceutically acceptable salt thereof, wherein b is 2.
E34. The conjugate of embodiment E23 or any of embodiments E25 to E29 (when dependent on
E23), or a pharmaceutically acceptable salt thereof, wherein LB is
Figure imgf000359_0001
E35. The conjugate of embodiment E34, or a pharmaceutically acceptable salt thereof, wherein d is 1.
E36. The conjugate of embodiment E34, or a pharmaceutically acceptable salt thereof, wherein d is 2.
E37. The conjugate of any of embodiments E23 to E36, or a pharmaceutically acceptable salt thereof, wherein RZ1 is H.
E38. The conjugate of any of embodiments E23 to E36, or a pharmaceutically acceptable salt thereof, wherein one occurrence of RZ1 is
Figure imgf000359_0002
alkyl, benzyl or phenyl and the remaining occurrences of RZ1 are H.
E39. The conjugate of embodiment E23 or E24, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000359_0003
wherein RZ1 is C1.4 alkyl, benzyl, or phenyl and ql is 2, 3 or 4, optionally wherein ql is 3.
E40. The conjugate of embodiment E39, or a pharmaceutically acceptable salt thereof, wherein LB is as defined in any of embodiments E30 to E36.
E41. The conjugate of any of embodiments E23 to E36, E39 or E40, or a pharmaceutically acceptable salt thereof, wherein RZ1 is CM alkyl.
E42. The conjugate of embodiment E41, or a pharmaceutically acceptable salt thereof, wherein RZ1 is methyl.
E43. The conjugate of embodiment E41, or a pharmaceutically acceptable salt thereof, wherein RZ1 is isopropyl.
E44. The conjugate of any of embodiments E23 to E36, E39 or E40, or a pharmaceutically acceptable salt thereof, wherein RZ1 is benzyl. E45. The conjugate of any of embodiments E23 to E36, E39 or E40, or a pharmaceutically acceptable salt thereof, wherein RZ1 is phenyl.
E46. The conjugate of embodiment E23 or E24, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from:
Figure imgf000360_0001
or a pharmaceutically acceptable salt thereof.
E47. The conjugate of embodiment E23 or E24, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000360_0002
pharmaceutically acceptable salt thereof.
E48. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IIA):
Figure imgf000360_0003
(L-IIA), wherein E2 is (CH2)n2, wherein n2 is 0, 1, 2 or 3,
Figure imgf000361_0001
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000361_0002
, wherein n5 is 1, 2, 3, 4 or 5, n5A is an integer from 1 to 10, n5B is
1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
E49. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-l I) :
Figure imgf000361_0003
(HI), wherein E2 is (CH2)n2, wherein n2 is 0, 1, 2 or 3,
Figure imgf000362_0001
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Z2 is (CH2)n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof; provided that when Y is absent, then Xb is not -C(O)NH-.
E50. The conjugate of embodiment E48 or E49, or a pharmaceutically acceptable salt thereof, wherein Q2 is
Figure imgf000362_0002
E51. The conjugate of embodiment E50, or a pharmaceutically acceptable salt thereof, wherein
Q2 is
Figure imgf000362_0003
E52. The conjugate of any of embodiments E48 to E51, or a pharmaceutically acceptable salt thereof, wherein m2 is 9, 10, 11, 12 or 13. E53. The conjugate of any of embodiments E48 to E52, or a pharmaceutically acceptable salt thereof, wherein R1 is CH3.
E54. The conjugate of any of embodiments E48 to E53, or a pharmaceutically acceptable salt thereof, wherein E2 is CH2.
E55. The conjugate of any of embodiments E48 to E54, or a pharmaceutically acceptable salt thereof, wherein X2 is CH2.
E56. The conjugate of any of embodiments E48 to E55, or a pharmaceutically acceptable salt thereof, wherein Y2 is (CH2)2.
E57. The conjugate of any of embodiments E48 to E56, or a pharmaceutically acceptable salt thereof, wherein Z2 is (CH2)2.
E58. The conjugate of any of embodiments E48 or E50 to E56, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IIA), and Z2 is
Figure imgf000363_0001
; optionally wherein n5B is 2 and/or n5A is 1.
E59. The conjugate of any of embodiments E48 to E58, or a pharmaceutically acceptable salt thereof, wherein p is 1.
E60. The conjugate of any of embodiments E48 to E59, or a pharmaceutically acceptable salt thereof, wherein pl is 0.
E61. The conjugate of any of embodiments E48 to E59, or a pharmaceutically acceptable salt thereof, wherein pl is 1.
E62. The conjugate of embodiment E48 or E49, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from:
Figure imgf000363_0002
and
Figure imgf000364_0001
E63. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IV):
Figure imgf000364_0003
, ,
Figure imgf000364_0002
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, Cw alkyl or C3~4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety;
Y3 is O or NRB, wherein RB is H, CM alkyl or C3-4 cycloalkyl, Z3 is (CH2)nio or
Figure imgf000365_0001
, wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
E64. The conjugate of embodiment E63, or a pharmaceutically acceptable salt thereof, wherein m3 is 9, 10, 11, 12 or 13.
E65. The conjugate of embodiment E63 or E64, or a pharmaceutically acceptable salt thereof, wherein R1A is CH3.
E66. The conjugate of any of embodiments E63 to E65, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000365_0002
E67. The conjugate of any of embodiments E63 to E66, or a pharmaceutically acceptable salt thereof, wherein E3 is CH2.
E68. The conjugate of any of embodiments E63 to E67, or a pharmaceutically acceptable salt thereof, wherein X4 is CH2.
E69. The conjugate of any of embodiments E63 to E68, or a pharmaceutically acceptable salt thereof, wherein Y3 is O.
E70. The conjugate of any of embodiments E63 to E69, or a pharmaceutically acceptable salt thereof, wherein Z3 is (CH2)nio, optionally (CH2)2.
E71. The conjugate of any of embodiments E63 to E70, or a pharmaceutically acceptable salt thereof, wherein p2 is 1.
E72. The conjugate of any of embodiments E63 to E70, or a pharmaceutically acceptable salt thereof, wherein p2 is 0. E73. The conjugate of any of embodiments E63 to E72, or a pharmaceutically acceptable salt thereof, wherein q2 is 1.
E74. The conjugate of any of embodiments E63 to E73, or a pharmaceutically acceptable salt thereof, wherein p3 is 1. E75. The conjugate of any of embodiments E63 to E73, or a pharmaceutically acceptable salt thereof, wherein p3 is 0.
E76. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from the moieties of Table 6.
E77. The conjugate of any of embodiments E23 to E76, or a pharmaceutically acceptable salt thereof, wherein G2 is selected from:
Figure imgf000366_0001
optionally wherein G2 is selected from:
Figure imgf000367_0001
wherein RK is H or CH3, RL is Ci-6 alkyl, and
Figure imgf000367_0002
indicates the point of attachment to the antibody or antigen-binding fragment thereof.
E78. The conjugate of embodiment E77, or a pharmaceutically acceptable salt thereof, wherein
G2 is selected from:
Figure imgf000367_0003
E79. The conjugate of embodiment E78, or a pharmaceutically acceptable salt thereof, wherein
G2 is
Figure imgf000368_0001
E80. The conjugate of embodiment E78, or a pharmaceutically acceptable salt thereof, wherein
G2 is
Figure imgf000368_0002
E81. The conjugate of embodiment E78, or a pharmaceutically acceptable salt thereof, wherein
G2 is
Figure imgf000368_0003
E82. The conjugate of any of embodiments E23 to E78, or a pharmaceutically acceptable salt thereof, wherein -LB-G2, -Z2-G2 or -Z3-G2 is:
Figure imgf000368_0004
E83. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from:
Figure imgf000368_0005
or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000368_0006
indicates the point of attachment to the antibody or antigen-binding fragment thereof.
E84. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein I2 is:
Figure imgf000369_0001
pharmaceutically acceptable salt thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
E85. The conjugate of any of embodiments El to E22, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from a moiety of Table 7.
E85a. The conjugate of any of embodiments E23 to E85, or a pharmaceutically acceptable salt thereof, wherein when Y is absent, then Xb is not -C(O)NH-.
E86. The conjugate of embodiment El or E2, or a pharmaceutically acceptable salt thereof, wherein the conjugate is:
Figure imgf000369_0002
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
E87. The conjugate of embodiment El or E2, or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from:
Figure imgf000369_0003
Figure imgf000370_0001
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
E87a. The conjugate of embodiment El or E2, or a pharmaceutically acceptable salt thereof, which is selected from a conjugate of Table 8A, or a pharmaceutically acceptable salt thereof.
E88. The conjugate of any of embodiments El to E87 and E87a, or a pharmaceutically acceptable salt thereof, wherein k is an integer from 2 to 10.
E89. The conjugate of embodiment E88, wherein k is an integer from 2 to 8.
E90. The conjugate of embodiment E88, wherein k is 4.
E91. The conjugate of embodiment E88, wherein k is 8.
E92. A pharmaceutical composition comprising a conjugate of any of embodiments El to E91, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
E93. A conjugate of any of embodiments El to E91, or a pharmaceutically acceptable salt thereof, for use in therapy.
E94. A conjugate of any of embodiments El to E91, or a pharmaceutically acceptable salt thereof, for use in treating cancer.
E95. Use of a conjugate of any of embodiments El to E91, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer.
E96. A method of treating cancer in a patient comprising administering to the patient a conjugate of any of embodiments El to E91, or a pharmaceutically acceptable salt thereof.
E97. A pharmaceutical composition according to embodiment E92 for use in therapy.
E98. A pharmaceutical composition according to embodiment E92 for use in treating cancer. E99. A method of treating cancer in a patient comprising administering to the patient a pharmaceutical composition according to embodiment E92, or a pharmaceutically acceptable salt thereof.
Fl. A conjugate of Formula (Via):
(A-E1-X1-Y1-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E1 and X1 is as defined in any of embodiments Bl to B54,
Y1 is AA 0 V yb V. , wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(Ci 3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and
Ab is an antibody or antigen-binding fragment thereof.
F2. A conjugate of Formula (VI)
(A-E1-X1-Y1-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E1 and X1 is as defined in any of embodiments Bl to B54,
Figure imgf000371_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L2; and Q is NH, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and
Ab is an antibody or antigen-binding fragment thereof. F3. The compound of embodiment Fl, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000372_0001
F4. The conjugate of embodiment Fl or F3, or a pharmaceutically acceptable salt thereof, wherein ya and yb are each independently selected from 0, 1 and 2.
F4a. The conjugate of embodiment Fl, F3 or F4, or a pharmaceutically acceptable salt thereof, wherein at least one of ya or yb is 0.
F5.The conjugate of embodiment Fl or F3, or a pharmaceutically acceptable salt thereof, wherein ya is 0 and yb is 1.
F6.The conjugate of embodiment Fl or F3, or a pharmaceutically acceptable salt thereof, wherein ya is 1 and yb is 0.
F7.The conjugate of embodiment Fl, F3 or F4, or a pharmaceutically acceptable salt thereof, wherein the total of ya and yb is no more than 3.
F8.The conjugate of embodiment Fl or F3, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both 0.
F9.The conjugate of embodiment Fl or F3, or a pharmaceutically acceptable salt thereof, wherein ya and yb are both independently selected from 1 and 2.
F10. The conjugate of embodiment Fl or F2, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000372_0002
Fll. The conjugate of any of embodiments Fl to F10, or a pharmaceutically acceptable salt thereof, wherein Q is NH, S or O.
F12. The conjugate of embodiment Fll, or a pharmaceutically acceptable salt thereof, wherein Q is NH.
F13. The conjugate of embodiment Fll, or a pharmaceutically acceptable salt thereof, wherein Q is S.
F14. The conjugate of embodiment Fll, or a pharmaceutically acceptable salt thereof, wherein Q is O. F15. The conjugate of any of embodiments Fl or F3 to F10, or a pharmaceutically acceptable salt thereof, wherein the conjugate is a conjugate of Formula (Via) or a pharmaceutically acceptable salt thereof, and wherein Q is -N(Ci.3alkyl )-, for example -N(CH3)-.
F16. The conjugate of any of embodiments Fl to F10, or a pharmaceutically acceptable salt thereof, wherein Q is -S-(CH2)2NH-*, -O-(CH2)2NH-* or -NH-(CH2)2NH-*.
F17. The conjugate of embodiment F16, or a pharmaceutically acceptable salt thereof, wherein Q is -S-(CH2)2NH-*.
F18. The conjugate of embodiment F16, or a pharmaceutically acceptable salt thereof, wherein Q is -O-(CH2)2NH~*.
F19. The conjugate of embodiment F16, or a pharmaceutically acceptable salt thereof, wherein Q is -NH-(CH2)2NH-*.
F20. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000373_0001
, wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1-
CH2-NH-, wherein lA1 is O, S or NH, and * indicates the point of attachment to Y1; each RZ1 is independently H, CM alkyl, phenyl or benzyl; q is 3, 4 or 5;
Figure imgf000373_0002
asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof. F21. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000374_0001
, wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1-
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y1; each RZ1 is independently H, CM alkyl, phenyl or benzyl; q is 3, 4 or 5;
LB is selected from - (CH2)b- and
Figure imgf000374_0002
, wherein the asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
F22. The conjugate of embodiment F20 or F21, or a pharmaceutically acceptable salt thereof, wherein LA is absent or *-CH2-NH-, wherein * indicates the point of attachment to Y1.
F23. The conjugate of embodiment F20 or F21, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000374_0003
F24. The conjugate of any of embodiments F20 to F23, or a pharmaceutically acceptable salt thereof, wherein q is 3.
F25. The conjugate of any of embodiments F20 to F23, or a pharmaceutically acceptable salt thereof, wherein q is 4.
F26. The conjugate of any of embodiments F20 to F23, or a pharmaceutically acceptable salt thereof, wherein q is 5. F27. The conjugate of any of embodiments F20 to F26, or a pharmaceutically acceptable salt thereof, wherein LB is -(CH2)b.
F28. The conjugate of any of embodiments F20 to F26, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000375_0001
F29. The conjugate of any of embodiments F20 to F26 or F28, or a pharmaceutically acceptable salt thereof, wherein c is 7.
F30. The conjugate of any of embodiments F20 to F29, or a pharmaceutically acceptable salt thereof, wherein b is 2.
F31. The conjugate of embodiment F20 or of any of embodiments F22 to F26 (when dependent on F20), or a pharmaceutically acceptable salt thereof, wherein LB is
Figure imgf000375_0002
F32. The conjugate of embodiment F31, or a pharmaceutically acceptable salt thereof, wherein d is 1.
F33. The conjugate of embodiment F31, or a pharmaceutically acceptable salt thereof, wherein d is 2.
F34. The conjugate of any of embodiments F20 to F33, or a pharmaceutically acceptable salt thereof, wherein RZ1 is H.
F35. The conjugate of any of embodiments F20 to F33, or a pharmaceutically acceptable salt thereof, wherein one occurrence of RZ1 is Cw alkyl, benzyl or phenyl and the remaining occurrences of RZ1 are H.
F36. The conjugate of embodiment F20 or F21, or a pharmaceutically acceptable salt thereof, wherein I2 is:
Figure imgf000375_0003
wherein RZ1 is CM alkyl, benzyl, or phenyl and ql is 2, 3 or 4, optionally wherein ql is 3.
F37. The conjugate of embodiment F36, or a pharmaceutically acceptable salt thereof, wherein LB is as defined in any of embodiments F27 to F33. F38. The conjugate of any of embodiments F20 to F33, F36 or F37, or a pharmaceutically acceptable salt thereof, wherein RZ1 is alkyl.
F39. The conjugate of embodiment F38, or a pharmaceutically acceptable salt thereof, wherein RZ1 is methyl. F40. The conjugate of embodiment F38, or a pharmaceutically acceptable salt thereof, wherein
RZ1 is isopropyl.
F41. The conjugate of any of embodiments F20 to F33, F36 or F37, or a pharmaceutically acceptable salt thereof, wherein RZ1 is benzyl.
F42. The conjugate of any of embodiments F20 to F33, F36 or F37, or a pharmaceutically acceptable salt thereof, wherein RZ1 is phenyl.
F53. The conjugate of embodiment F20 or F21, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from:
Figure imgf000376_0001
or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000376_0002
indicates the point of attachment to the antibody or antigen-binding fragment thereof.
F44. The conjugate of embodiment F20 or F21, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000376_0003
thereof. F45. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IIA):
Figure imgf000377_0001
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,
R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000377_0002
wherein n5 is 1, 2, 3, 4 or 5, n5A is an integer from 1 to 10, n5B is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
F46. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-l I) :
Figure imgf000378_0001
wherein Ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, Ring F2 is a saturated bicyclic ring having the 2 nitrogen atoms shown, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and Ring F3 is a saturated bicyclic ring having the 1 nitrogen atom shown, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, R1 is CM alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Z2 is (CH2)n5, wherein n5 is 1, 2, 3, 4 or 5, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
F47. The conjugate of embodiment F45 or F46, or a pharmaceutically acceptable salt thereof, wherein Q2 is
Figure imgf000378_0002
F48. The conjugate of embodiment F47, or a pharmaceutically acceptable salt thereof, wherein
Q2 is
Figure imgf000379_0001
F49. The conjugate of any of embodiments F45 to F48, or a pharmaceutically acceptable salt thereof, wherein m2 is 9, 10, 11, 12 or 13.
F50. The conjugate of any of embodiments F45 to F49, or a pharmaceutically acceptable salt thereof, wherein R1 is CH3.
F51. The conjugate of any of embodiments F45 to F50, or a pharmaceutically acceptable salt thereof, wherein E2 is CH2.
F52. The conjugate of any of embodiments F45 to F51, or a pharmaceutically acceptable salt thereof, wherein X2 is CH2.
F53. The conjugate of any of embodiments F45 to F52, or a pharmaceutically acceptable salt thereof, wherein Y2 is (CH2)2.
F54. The conjugate of any of embodiments F45 to F53, or a pharmaceutically acceptable salt thereof, wherein Z2 is (CH2)2.
F55. The conjugate of any of embodiments F45 or F47 to F53, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IIA), and Z2 is
Figure imgf000379_0002
; optionally wherein n5B is 2 and/or n5A is 1.
F56. The conjugate of any of embodiments F45 to F55, or a pharmaceutically acceptable salt thereof, wherein p is 1.
F57. The conjugate of any of embodiments F45 to F56, or a pharmaceutically acceptable salt thereof, wherein pl is 0.
F58. The conjugate of any of embodiments F45 to F56, or a pharmaceutically acceptable salt thereof, wherein pl is 1.
F59. The conjugate of embodiment F45 or F46, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from:
Figure imgf000380_0001
F60. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IV):
Figure imgf000380_0002
, ,
Figure imgf000381_0001
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, Cw alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety;
Y3 is O or NRB, wherein
Figure imgf000381_0002
cycloalkyl,
Z3 is (CH2)nio or
Figure imgf000381_0003
, wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
F61. The conjugate of embodiment F60, or a pharmaceutically acceptable salt thereof, wherein m3 is 9, 10, 11, 12 or 13.
F62. The conjugate of embodiment F60 or F61, or a pharmaceutically acceptable salt thereof, wherein R1A is CH3.
F63. The conjugate of any of embodiments F60 to F62, or a pharmaceutically acceptable salt
Figure imgf000381_0004
F64. The conjugate of any of embodiments F60 to F63, or a pharmaceutically acceptable salt thereof, wherein E3 is CH2.
F65. The conjugate of any of embodiments F60 to F64, or a pharmaceutically acceptable salt thereof, wherein X4 is CH2.
F66. The conjugate of any of embodiments F60 to F65, or a pharmaceutically acceptable salt thereof, wherein Y3 is O. F67. The conjugate of any of embodiments F60 to F66, or a pharmaceutically acceptable salt thereof, wherein Z3 is (CH2)nio, optionally (CH2)2.
F68. The conjugate of any of embodiments F60 to F67, or a pharmaceutically acceptable salt thereof, wherein p2 is 1. F69. The conjugate of any of embodiments F60 to F67, or a pharmaceutically acceptable salt thereof, wherein p2 is 0.
F70. The conjugate of any of embodiments F60 to F69, or a pharmaceutically acceptable salt thereof, wherein q2 is 1.
F71. The conjugate of any of embodiments F60 to F70, or a pharmaceutically acceptable salt thereof, wherein p3 is 1.
F72. The conjugate of any of embodiments F60 to F70, or a pharmaceutically acceptable salt thereof, wherein p3 is 0.
F73. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from the moieties of Table 6. F74. The conjugate of any of embodiments F20 to F73, or a pharmaceutically acceptable salt thereof, wherein G2 is selected from:
Figure imgf000382_0001
optionally wherein G2 is selected from:
Figure imgf000383_0001
wherein RK is H or CH3, RL is Ci-6 alkyl, and
Figure imgf000383_0002
indicates the point of attachment to the antibody or antigen-binding fragment thereof. F75. The conjugate of embodiment F74, or a pharmaceutically acceptable salt thereof, wherein
G2 is selected from:
Figure imgf000383_0003
F76. The conjugate of embodiment F75, or a pharmaceutically acceptable salt thereof, wherein G2 is
Figure imgf000383_0004
F77. The conjugate of embodiment F75, or a pharmaceutically acceptable salt thereof, wherein
G2 is
Figure imgf000384_0001
F78. The conjugate of embodiment F75, or a pharmaceutically acceptable salt thereof, wherein
G2 is
Figure imgf000384_0002
F79. The conjugate of any of embodiments F20 to F74, or a pharmaceutically acceptable salt thereof, wherein -LB-G2, -Z2-G2 or -Z3-G2 is:
Figure imgf000384_0003
F80. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from:
Figure imgf000384_0004
or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000384_0005
indicates the point of attachment to the antibody or antigen-binding fragment thereof.
F81. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000385_0001
pharmaceutically acceptable salt thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
F82. The conjugate of any of embodiments Fl to F19, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from a moiety of Table 7.
F83. The conjugate of embodiment Fl or F2, or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from:
Figure imgf000385_0002
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
F84. The conjugate of embodiment Fl or F2, or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from:
Figure imgf000386_0001
Figure imgf000387_0001
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof and k is an integer from 1 to 10.
F85. The conjugate of embodiment Fl or F2, or a pharmaceutically acceptable salt thereof, which is selected from a conjugate of Table 8B or a pharmaceutically acceptable salt thereof.
F86. The conjugate of embodiment Fl, or a pharmaceutically acceptable salt thereof, which is selected from a conjugate of Table SB or Table 8C, or a pharmaceutically acceptable salt thereof.
F87. The conjugate of any of embodiments Fl to F86, or a pharmaceutically acceptable salt thereof, wherein k is an integer from 2 to 10.
F88. The conjugate of embodiment F87, wherein k is an integer from 2 to 8.
F89. The conjugate of embodiment F87, wherein k is 4.
F90. The conjugate of embodiment F87, wherein k is 8.
F91. A pharmaceutical composition comprising a conjugate of any of embodiments Fl to F90, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
F92. A conjugate of any of embodiments Fl to F90, or a pharmaceutically acceptable salt thereof, for use in therapy.
F93. A conjugate of any of embodiments Fl to F90, or a pharmaceutically acceptable salt thereof, for use in treating cancer. F94. Use of a conjugate of any of embodiments Fl to F90, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer.
F95. A method of treating cancer in a patient comprising administering to the patient a conjugate of any of embodiments Fl to F90, or a pharmaceutically acceptable salt thereof.
F96. A pharmaceutical composition according to embodiment F91 for use in therapy.
F97. A pharmaceutical composition according to embodiment F91 for use in treating cancer.
F98. A method of treating cancer in a patient comprising administering to the patient a pharmaceutical composition according to embodiment F91, or a pharmaceutically acceptable salt thereof.
The above description of illustrative embodiments is intended only to acquaint others skilled in the art with the specification, its principles, and its practical application so that others skilled in the art may readily adapt and apply the specification in its numerous forms, as they may be best suited to the requirements of a particular use. This description and its specific examples, while indicating embodiments of this specification, are intended for purposes of illustration only. This specification, therefore, is not limited to the illustrative embodiments described in this specification, and may be variously modified. In addition, it is to be appreciated that various features of the specification that are, for clarity reasons, described in the context of separate embodiments, also may be combined to form a single embodiment. Conversely, various features of the specification that are, for brevity reasons, described in the context of a single embodiment, also may be combined to form subcombinations thereof.

Claims

1. A compound of Formula (la):
A-E-X-Y-Z or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000389_0001
E is:
(i) a bicyclic 7-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X;
(ii) a monocyclic 5-8 membered saturated heterocycle containing two nitrogen atoms, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X, optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; provided that when E is a piperazine, the piperazine is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl;
(iii) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X; and wherein a carbon atom in the heterocycle connects to -NR-; or
(iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or - C(O)NH-;
R is H, C1-4 alkyl or C3-4 cycloalkyl;
X is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y; Xa is - C(O)- or absent; and Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y; m is 2 or 3; Y is absent or
Figure imgf000390_0001
, wherein the wavy line indicates the point of attachment to X and the asterisk-wavy line indicates the point of attachment to Z; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, - O(CH2)2NH-* or -NH(CH2)2NH-*, wherein * indicates the point of attachment to Z; Z is H, C1-4 alkyl, -C(O)C1-4 alkyl,
Figure imgf000390_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000390_0003
. 3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000390_0004
wherein ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
4. The compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a bridged bicycle; optionally a 7-8 membered bridged bicycle.
5. The compound of claim 3 or 4, or a pharmaceutically acceptable salt thereof, wherein E is:
Figure imgf000391_0001
6. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein E is selected from:
Figure imgf000391_0002
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X.
7. The compound of any of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R is H.
8. The compound of any of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein X is absent.
9. The compound of any of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein X is -Xa-(CH2)m-Xb-.
10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein m is 2 and/or wherein Xb is -NH- or -C(O)NH-*, wherein * indicates the point of attachment to Y.
11. The compound of any of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein X is selected from:
Figure imgf000391_0003
wherein the wavy line indicates the point of attachment to E and the asterisk-wavy line indicates the point of attachment to Y.
12. The compound of any of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein
Y is absent.
13. The compound of any of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000392_0001
14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein Y is
Figure imgf000392_0002
15. The compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein Y is
Figure imgf000392_0003
16. The compound of any of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein Q is NH.
17. The compound of any of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein Z is H.
18. The compound of any of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000392_0004
integer from 1 to 5, and each Rz is independently H, Ci-4 alkyl, phenyl or benzyl.
19. The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein Rz is H.
20. The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein Z is:
Figure imgf000393_0001
wherein nl is 0, 1, 2, 3 or 4 and Rz is C1-4 alkyl, benzyl, or phenyl.
21. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from a compound of Table 1, or a pharmaceutically acceptable salt thereof.
22. A compound of Formula (Ila):
A-EW-Z1 or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000393_0002
iv) -NR-; ii) a 5-10 membered saturated heterocycle containing two nitrogen atoms and optionally an oxygen atom, wherein one nitrogen atom in the heterocycle is connected to A and the second nitrogen atom in the heterocycle is connected to X1; optionally wherein the heterocycle is geminally substituted on a carbon atom with two occurrences of C1-3 alkyl; iv) -NR-heterocycle- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to X1; and wherein a carbon atom in the heterocycle connects to -NR-; or iv) -heterocycle-NR- or -heterocycle-C(O)NH- wherein the heterocycle is a saturated 5-8 membered heterocycle containing a nitrogen atom which is connected to A; and wherein a carbon atom in the heterocycle connects to -NR- or -C(O)NH-;
R is H, Ci-4 alkyl or C3-4 cycloalkyl;
X1 is absent, -C(O)CH2O-*, -C(O)CH2NH-*, or -Xa-(CH2)m-Xb-, wherein Xb is connected to Y1; Xa is - C(O)- or absent; Xb is -C(O)NH-*, -O- or -NH-, wherein * indicates the point of attachment to Y1; m is 2 or 3;
Figure imgf000394_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to Z1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q. is NH, -N(Ci-3alkyl)-, S, O, -S-fCHzhNH-*, -O- (CHzhNH-* or -NH-fCI-bhNH-*, wherein * indicates the point of attachment to Z1;
Z1 is H, C 1-4 alkyl, -C(O)Ci-4 alkyl,
Figure imgf000394_0002
integer from 1 to 5; and each Rz is independently H, C1-4 alkyl, phenyl or benzyl.
23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein A is:
Figure imgf000394_0003
24. The compound of claim 22 or 23, or a pharmaceutically acceptable salt thereof, wherein E1 is - NR-.
25. The compound of claim 22 or 23, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000395_0001
wherein ring Ea is a bicyclic 7-10 membered saturated heterocycle having the two nitrogen atoms shown and optionally an oxygen atom, and ring Eb is a bicyclic 5-8 membered saturated heterocycle having the nitrogen atom shown; wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1; optionally wherein E1 is:
Figure imgf000395_0002
26. The compound of claim 25, or a pharmaceutically acceptable salt thereof, wherein the bicyclic heterocycle is a bridged bicycle; optionally a 7-8 membered bridged bicycle.
27. The compound of claim 26, or a pharmaceutically acceptable salt thereof, wherein E1 is selected from:
Figure imgf000395_0003
wherein the wavy line indicates the point of attachment to A and the asterisk-wavy line indicates the point of attachment to X1.
28. The compound of any of claims 22 to 26, or a pharmaceutically acceptable salt thereof, wherein R is H.
29. The compound of any of claims 22 to 28, or a pharmaceutically acceptable salt thereof, wherein X1 is absent.
30. The compound of any of claims 22 to 28, or a pharmaceutically acceptable salt thereof, wherein X1 is -Xa-(CH2)m-Xb-.
31. The compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein m is 2 and/or wherein Xb is -NH- or -C(O)NH-*, wherein * indicates the point of attachment to Y1.
32. The compound of any of claims 22 to 28, or a pharmaceutically acceptable salt thereof, wherein X1 is selected from:
Figure imgf000396_0001
wherein the wavy line indicates the point of attachment to E1 and the asterisk-wavy line indicates the point of attachment to Y1.
33. The compound of any of claims 22 to 32, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000396_0002
34. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein Y1 is
Figure imgf000396_0003
35. The compound of any of claims 22 to 34, or a pharmaceutically acceptable salt thereof, wherein Q. is NH.
36. The compound of any of claims 22 to 35, or a pharmaceutically acceptable salt thereof, wherein Z1 is H.
37. The compound of any of claims 22 to 35, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000396_0004
integer from 1 to 5, and each Rz is independently H, Ci-4 alkyl, phenyl or benzyl.
38. The compound of claim 37, or a pharmaceutically acceptable salt thereof, wherein Rz is H.
39. The compound of claim 37, or a pharmaceutically acceptable salt thereof, wherein Z1 is:
Figure imgf000397_0001
wherein nl is 0, 1, 2, 3 or 4 and Rz is C1-4 alkyl, benzyl, or phenyl.
40. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from a compound of Table 2 or Table 2A, or a pharmaceutically acceptable salt thereof.
41. A compound of Formula (Illa):
A-E-X-Y-L1 or a salt thereof, wherein A, E and X are as defined in any of claims 1 to 11; and
Y is absent or
Figure imgf000397_0002
, wherein the wavy line indicates the point of attachment to
X and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q. is NH, -N(Ci-3alkyl)-, S, O, -SfCHzhNH-*, - OfCHzhNH-* or -NH(CH2hNH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof.
The compound of claim 41, or a salt thereof, wherein Y is absent.
43. The compound of claim 41, or a salt thereof, wherein Y is
Figure imgf000397_0003
44. The compound of claim 41 or 43, or a salt thereof, wherein Q. is NH.
45. The compound of any of claims 41 to 44, or a salt thereof, wherein L1 is:
Figure imgf000397_0004
> wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1-
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y; each RZ1 is independently H, C1-4 alkyl, phenyl or benzyl; q is 3, 4 or 5;
LB is selected from -(CHz)b-,
Figure imgf000398_0001
, wherein the asterisk-wavy line indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
46. The compound of any of claims 41 to 44, or a salt thereof, wherein L1 is a group of Formula
(L-IA):
Figure imgf000398_0003
R1 is Ci-4 alkyl,
X2 is (CHjjna, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Z2 is (CHjjns or
Figure imgf000398_0002
, wherein n5 is 1, 2, 3, 4 or 5, n5A is an integer from 1 to 10, n5B is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
47. The compound of any of claims 41 to 44, or a salt thereof, wherein L1 is a group of Formula
(L-lll):
Figure imgf000399_0001
wherein E3 is (CH2)n6, wherein n6 is 0, 1, 2 or 3,
R1A is Ci-4 alkyl or H,
Figure imgf000399_0003
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, Ci-4 alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety;
Y3 is O or NRB, wherein RB is H, C1-4 alkyl or C3-4 cycloalkyl,
Z3 is (CH2)nio or
Figure imgf000399_0002
, wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
48. A compound of Formula (IVa):
A-EW-L1 or a salt thereof, wherein A, E1 and X1 are as defined in any of claims 22 to 32;
Y1 is
Figure imgf000400_0001
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L1; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(Ci.3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L1; and L1 is a linker for connection to an antibody or antigen-binding fragment thereof.
49. The compound of claim 48, or a salt thereof, wherein Y1 is
Figure imgf000400_0002
50. The compound of claim 48 or 49, or a salt thereof, wherein Q. is NH.
51. The compound of any of claims 48 to 50, or a salt thereof, wherein L1 is:
Figure imgf000400_0003
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y1; each RZ1 is independently H, Ci-4 alkyl, phenyl or benzyl; q is 3, 4 or 5;
LB is selected from -(CH2)b-,
Figure imgf000400_0004
, wherein the asterisk-wavy line indicates the point of attachment to G1; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
52. The compound of any of claims 48 to 50, or a salt thereof, wherein L1 is a group of Formula (L-IA):
Figure imgf000401_0001
R1 is Ci-4 alkyl,
X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3,
Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000401_0002
, wherein n5 is 1, 2, 3, 4 or 5, n5A is an integer from 1 to 10, n5B is
1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof.
53. The compound of any of claims 48 to 50, or a salt thereof, wherein L1 is a group of Formula
Figure imgf000401_0003
(L-lll), wherein E3 is (CH2)n6, wherein n6 is 0, 1, 2 or 3, R1A is C1-4 alkyl or H,
Figure imgf000402_0002
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, C1-4 alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety;
Y3 is O or NRB, wherein RB is H, Ci-4 alkyl or C3-4 cycloalkyl,
Z3 is (CH2)nio or
Figure imgf000402_0001
; wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G1, m3 is an integer from 2 to 17, p2 is l or O, q2 is 1 or 0, p3 is 0 or 1, and
G1 is a conjugation group for conjugation to an antibody or antigen-binding fragment thereof. 54. The compound of any of claims 41 to 44 and 48 to 50, or a salt thereof, wherein L1 is selected from the moieties of Table 3 or Table 4.
55. The compound of any of claims 45 to 47 and 51 to 54, or a salt thereof, wherein G1 is selected from:
Figure imgf000403_0001
wherein X3 is CH or N, h is 0 or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is Ci.g alkyl.
56. The compound of claim 41, or a salt thereof, wherein the compound is selected from a compound of Table 5A, or a salt thereof. 57. The compound of claim 48, or a salt thereof, wherein the compound is selected from a compound of Table 5B or Table 5C, or a salt thereof.
58. A conjugate of Formula (Va): (A-E-X-Y-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E and X is as defined in any of claims 1 to 11,
Y is absent or
Figure imgf000403_0002
, wherein the wavy line indicates the point of attachment to
X and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q. is NH, -N(Ci-3alkyl)-, S, O, -S(CH2)?NH-*, - OfCHzhNH-* or -NHfCHzhNH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and
Ab is an antibody or antigen-binding fragment thereof.
59. The conjugate of claim 58, or a pharmaceutically acceptable salt thereof, wherein Y is absent.
60. The conjugate of claim 58, or a pharmaceutically acceptable salt thereof, wherein Y is
Figure imgf000404_0001
61. The conjugate of claim 58 or 60, or a pharmaceutically acceptable salt thereof, wherein Q. is NH.
62. The conjugate of any of claims 58 to 61, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000404_0002
, wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1-
CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y; each RZ1 is independently H, C1-4 alkyl, phenyl or benzyl; q is 3, 4 or 5;
LB is selected from -(Cl-bjb-,
Figure imgf000404_0003
, wherein the asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; dl and cl are each independently 1 or 2; and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein s'* indicates the point of attachment to the antibody or antigen-binding fragment thereof.
63. The conjugate of any of claims 58 to 61, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IIA):
Figure imgf000405_0001
1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m2 is an integer from 5 to 17, p is 1 or 0, pl is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
64. The conjugate of any of claims 58 to 61, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IV):
Figure imgf000405_0002
(L-IV) wherein E3 is (CH2)n6, wherein n6 is 0, 1, 2 or 3,
R1A is Ci-4 alkyl or H,
Figure imgf000406_0003
wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, Ci-4 alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety;
Y3 is O or NRB, wherein RB is H, C1-4 alkyl or C3-4 cycloalkyl,
Z3 is (CH2)nio or
Figure imgf000406_0001
; wherein nlO is 0, 1, 2, 3, 4 or 5, nlOA is an integer from 1 to 10, nlOB is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and
G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
A conjugate of Formula (Via)
(A-E1-X1-Y1-L2)k-Ab or a pharmaceutically acceptable salt thereof, wherein each A, E1 and X1 is as defined in any of claims 22 to 32,
Y1 is
Figure imgf000406_0002
, wherein the wavy line indicates the point of attachment to X1 and the asterisk-wavy line indicates the point of attachment to L2; ya and yb are each independently selected from 0, 1, 2 and 3; and Q is NH, -N(C1-3alkyl)-, S, O, -S(CH2)2NH-*, -O(CH2)2NH-* or - NH(CH2)2NH-*, wherein * indicates the point of attachment to L2; each L2 is a linker, k is an integer from 1 to 10, and Ab is an antibody or antigen-binding fragment thereof. 66. The conjugate of claim 65, or a pharmaceutically acceptable salt thereof, wherein Y1 is
Figure imgf000407_0001
. 67. The conjugate of claim 65 or 66, or a pharmaceutically acceptable salt thereof, wherein Q is NH. 68. The conjugate of any of claims 65 to 67, or a pharmaceutically acceptable salt thereof, wherein L2 is:
Figure imgf000407_0002
, wherein LA is absent or is selected from *-CH2-NH-, and *-C(O)-CH2-LA1- CH2-NH-, wherein LA1 is O, S or NH, and * indicates the point of attachment to Y1; each Rz1 is independently H, C1-4 alkyl, phenyl or benzyl; q is 3, 4 or 5; LB is selected from –(CH2)b-,
Figure imgf000407_0003
, wherein the asterisk-wavy line indicates the point of attachment to G2; b is an integer from 1 to 5; c is an integer from 5 to 15; d is an integer from 1 to 10; d1 and c1 are each independently 1 or 2; and G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof.
69. The conjugate of any of claims 65 to 67, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IIA):
Figure imgf000408_0002
R1 is C1-4 alkyl, X2 is (CH2)n3, wherein n3 is 0, 1, 2 or 3, Y2 is (CH2)n4, wherein n4 is 0, 1, 2, 3 or 4,
Figure imgf000408_0001
wherein n5 is 1, 2, 3, 4 or 5, n5A is an integer from 1 to 10, n5B is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m2 is an integer from 5 to 17, p is 1 or 0, p1 is 0 or 1, and G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof. 70. The conjugate of any of claims 65 to 67, or a pharmaceutically acceptable salt thereof, wherein L2 is a group of Formula (L-IV):
Figure imgf000409_0003
, ,
Figure imgf000409_0001
, wherein n7 and n9 are 0, 1, 2 or 3, n8 is 1, 2 or 3, and Q3 is O or NRA, wherein RA is H, C1-4 alkyl or C3-4 cycloalkyl, and wherein the asterisk-wavy line indicates the point of attachment to the (C=O) moiety; Y3 is O or NRB, wherein RB is H, C1-4 alkyl or C3-4 cycloalkyl, Z3 is (CH2)n10 or
Figure imgf000409_0002
, wherein n10 is 0, 1, 2, 3, 4 or 5, n10A is an integer from 1 to 10, n10B is 1 or 2, and the asterisk-wavy line indicates the point of attachment to G2, m3 is an integer from 2 to 17, p2 is 1 or 0, q2 is 1 or 0, p3 is 0 or 1, and G2 is a conjugation group conjugated to the antibody or antigen-binding fragment thereof, wherein indicates the point of attachment to the antibody or antigen-binding fragment thereof. 71. The conjugate of any of any of claims 58 to 61 or 65 to 67, or a pharmaceutically acceptable salt thereof, wherein L2 is selected from a moiety of Table 6 or Table 7.
72. The conjugate of any of claims 62 to 64 and 68 to 71, or a pharmaceutically acceptable salt thereof, wherein G2 is selected from:
Figure imgf000410_0001
wherein RK is H or CH3, RL is C1-6 alkyl, and
Figure imgf000410_0002
indicates the point of attachment to the antibody or antigen-binding fragment thereof. 73. The conjugate of claim 58, or a pharmaceutically acceptable salt thereof, which is selected from a conjugate of Table 8A, or a pharmaceutically acceptable salt thereof. 74. The conjugate of claim 65, or a pharmaceutically acceptable salt thereof, which is selected from a conjugate of Table 8B or Table 8C, or a pharmaceutically acceptable salt thereof. 75. A pharmaceutical composition comprising a compound according to any of claims 1 to 40 or a conjugate of any of claims 58 to 74, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 76. A compound according to any of claims 1 to 40 or a conjugate according to any of claims 58 to 74, or a pharmaceutically acceptable salt thereof, for use in therapy. 77. A compound according to any of claims 1 to 40 or a conjugate according to any of claims 58 to 74, or a pharmaceutically acceptable salt thereof for use in treating cancer.
78. Use of a compound according to any of claims 1 to 40 or a conjugate according to any of claims 58 to 74, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer. 5 79. A method of treating cancer in a patient comprising administering to the patient a compound according to any of claims 1 to 40 or a conjugate according to any of claims 58 to 74, or a pharmaceutically acceptable salt thereof. 10 80. A pharmaceutical composition according to claim 75 for use in therapy. 81. A pharmaceutical composition according to claim 75 for use in treating cancer. 82. A method of treating cancer in a patient comprising administering to the patient a composition according to claim 75, or a pharmaceutically acceptable salt thereof.
PCT/EP2024/072665 2023-08-10 2024-08-09 Anthracycline derivatives and conjugates and uses thereof WO2025032254A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363518651P 2023-08-10 2023-08-10
US63/518,651 2023-08-10
US202463631545P 2024-04-09 2024-04-09
US63/631,545 2024-04-09

Publications (1)

Publication Number Publication Date
WO2025032254A1 true WO2025032254A1 (en) 2025-02-13

Family

ID=92424190

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/072665 WO2025032254A1 (en) 2023-08-10 2024-08-09 Anthracycline derivatives and conjugates and uses thereof

Country Status (1)

Country Link
WO (1) WO2025032254A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010009124A2 (en) * 2008-07-15 2010-01-21 Genentech, Inc. Anthracycline derivative conjugates, process for their preparation and their use as antitumor compounds
WO2014210064A1 (en) * 2013-06-24 2014-12-31 Genentech, Inc. Anti-fcrh5 antibodies
WO2015127273A1 (en) 2014-02-21 2015-08-27 Medimmune, Llc Anti-pcsk9~glp-1 fusions and methods for use

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010009124A2 (en) * 2008-07-15 2010-01-21 Genentech, Inc. Anthracycline derivative conjugates, process for their preparation and their use as antitumor compounds
WO2014210064A1 (en) * 2013-06-24 2014-12-31 Genentech, Inc. Anti-fcrh5 antibodies
WO2015127273A1 (en) 2014-02-21 2015-08-27 Medimmune, Llc Anti-pcsk9~glp-1 fusions and methods for use

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"Concise Dictionary of Biomedicine and Molecular Biology", 2002, CRC PRESS
"Remington's Pharmaceutical Sciences", 2012
"The Dictionary of Cell and Molecular Biology", 1999, ACADEMIC PRESS
AL-LAZIKANI ET AL., J. MOLEC. BIOL., vol. 273, 1997, pages 927 - 948
BARGH ET AL., CHEM. SOC. REV., vol. 48, 2019, pages 4391 - 4374
HE M.TAUSSIG MJ., BIOCHEM SOC TRANS, vol. 35, November 2007 (2007-11-01), pages 962 - 5
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
KIPRIYANOV, INT. J. CANCER, vol. 77, 1998, pages 763 - 772
no. 1807521-05-6
SU ET AL., ACTA PHARM. SIN. B, vol. 11, 2021, pages 3889 - 3907

Similar Documents

Publication Publication Date Title
US20250114647A1 (en) ANTIBODY-DRUG CONJUGATES COMPRISING SUBSTITUTED BENZO[e]PYRROLO[1,2-a][1,4]DIAZEPINES
JP2023105175A (en) Process for preparation of glucuronide drug-linkers and intermediates thereof
AU2023202358B2 (en) Pladienolide compounds and their use
EP0793497B1 (en) Novel enediyne quinone imines and methods of preparation and use thereof
CA2948082A1 (en) Method for targeted conjugation of peptides and proteins by paired c2 bridging of cysteine amino acids
WO2025032254A1 (en) Anthracycline derivatives and conjugates and uses thereof
WO2024158996A2 (en) Immunoconjugates and methods
US12178876B2 (en) Conjugates comprising cleavable linkers
KR102728434B1 (en) Improved method for the synthesis of linker-drug vc-seco-DUBA
TW202506191A (en) Conjugates comprising cleavable linkers
US20250057969A1 (en) Conjugates comprising cleavable linkers
TW202504639A (en) Conjugates comprising cleavable linkers
KR20250039375A (en) Antibody-drug conjugate and method for producing and using the same
EP4527415A1 (en) Antibody-drug conjugate containing protein degradation agent bioactive compound, method for preparing same, and use thereof
RU2777160C2 (en) Improved method for synthesis of linker-drug vc-seco-duba
WO2023096904A2 (en) C-16 modified trioxacarcins, antibody drug conjugates, and uses thereof
WO2024218361A1 (en) Bispecific cytotoxicity targeting chimeras

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24755537

Country of ref document: EP

Kind code of ref document: A1