Mcl-1 inhibitor compounds and use in antibody drug conjugates

Abstract

Compounds of Formula (I) and pharmaceutically acceptable salts thereof are described herein. Further described herein are antibody drug conjugates (ADCs) comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as a payload. Methods of treating conditions are also described.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Publication Number (43) Publication Date (21) Application Number 202480072657.0 (22) Application Date 2024.11.14 (30) Priority Data 63 / 600,479 2023.11.17 US (85) PCT International Application Entering National Phase Date 2026.05.14 (86) PCT International Application Application Data PCT / US2024 / 055864 2024.11.14 (87) PCT International Application Publication Data WO2025 / 106634 EN 2025.05.22 (71) Applicant Genentech Corporation Address California, USA (72) Inventors T.H. Pilo, G.D. Levi, S.M. Monsen, M.L. Landry (74) Patent Agency King & Wood Mallesons, Beijing 11256 Patent Attorney Tai Hong (51) Int.Cl. C07D 515 / 16 (2006.01) A61K 47 / 54 (2006.01) A61P 35 / 00 (2006.01) A61K 31 / 519 (2006.01) (54) Invention Title MCL-1 Inhibitor Compound and Use in Antibody-Drug Conjugates (57) Abstract This document describes compounds of formula (I) and pharmaceutically acceptable salts thereof. This document further describes antibody-drug conjugates (ADCs) comprising compounds of formula (I) or pharmaceutically acceptable salts thereof as payloads. Methods of treating the condition are also described.Claims 9 pages, Description 90 pages, Drawings 15 pages, CN 122295346 A 2026.06.26 CN 1 22 29 53 46 A 1. A compound of formula (X): (X), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; Ring A is phenyl or 6-membered heteroaryl; Ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more independently selected halogens, alkyl, -OH, -OR2b and -O(R2bO)qR2c The substituents of the group are substituted, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b are independently alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy, and haloalkoxy.2. The compound according to claim 1, wherein the compound is a compound of formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or 6-membered heteroaryl; ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of halogens, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; each R2a and each R2b R1 is independently alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently alkyl, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen and –OH. 3. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein ring A and ring B are independently phenyl or heteroaryl containing one or two ring N atoms. 4. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein ring A is pyrimidine and ring B is phenyl, pyrimidine, or phenyl, wherein the point of connection with ring A is indicated. 5. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein R1 is a phenyl substituted with one fluorine. 6. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R2 is a C1-C6 alkyl group that is unsubstituted or substituted by one or more substituents independently selected from the group consisting of –OH and halogen. 7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Z is N-CH3 or N-CH2CH3. 8. The compound of any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein R3 is –NH2 or –NHCH3. 9. The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein X is O.10. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated; R1 is a fluorinated phenyl; R2 is an unsubstituted or fluorinated C1-C6 alkyl; R3 is –NH2 or –NH (C1-6 alkyl); and X is O. 11. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein X is O, R1 is a fluorinated phenyl, R2 is an alkyl, and R3 is –NH2. 12. The compound of claim 2, wherein the compound is a compound of formula (II): [Claims 2 / 9, page 3, CN 122295346 A (II)], or a pharmaceutically acceptable salt thereof, wherein: X, R1, R2, and R3 are as defined with respect to formula (X). 13. The compound of claim 12 or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or fluorinated C1-C6 alkyl group; and R3 is –NH2 or –NH(C1-6 alkyl). 14. The compound of claim 1, wherein the compound is: […], […], […], […], […], […], […], […], or a pharmaceutically acceptable salt thereof. (Claims 3 / 9, page 4, CN 122295346 A)15. A conjugate of formula (A): Ab-(L-(DP)r)m, or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody; L is a linker; DP is a drug payload; r is an integer from 1 to 8; and m is an integer from 1 to 10; wherein the drug payload is a compound of formula (X): (X), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or a 6-membered heteroaryl; ring B is phenyl or a 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted with one or more independently selected halogens, alkyl groups, -OH, -OR2b The substituents of the group consisting of -O(R2bO)qR2c are substituted, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b are independently alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy, and haloalkoxy. 16. The conjugate of claim 15, wherein the conjugate of formula (A) is a conjugate of formula (B): (B), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a–, or –N+(R3a)2–; m is an integer from 1 to 10; and X, R1, R2, R3a, Z, ring A, and ring B are as defined for formula (X). 17. The conjugate of claim 15 or 16, or a pharmaceutically acceptable salt thereof, wherein the linker is a peptide linker or a peptide mimic linker.18. The conjugate according to any one of claims 15 to 17, wherein the conjugate is a conjugate of formula (B-1): R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; X, R1, R2, R3a, Z, ring A and ring B are as defined with respect to formula (X); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; and RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 together can form C3-C7 cycloalkyl. 19. The conjugate according to any one of claims 15 to 18, wherein: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated; R1 is a fluorinated phenyl; R2 is an unsubstituted or fluorinated C1-C6 alkyl; R3 is –NH2 or –NH(C1-6 alkyl); and X is O. 20. The conjugate according to claim 16, wherein the conjugate of formula (A) is a conjugate of formula (C): (C), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH, –NR3a, or –N+(R3a)2; m is an integer from 1 to 10; and X, R1, R2, and R3a are as defined with respect to formula (X). 21. The conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or fluorinated C1-C6 alkyl group; and R3 is –NH2 or –NH(C1-6 alkyl). 22. The conjugate of any one of claims 15 to 18 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical loading is: […]. 23. The conjugate of claim 15, wherein the conjugate has the following structure: […].24. The conjugate according to any one of claims 15 to 23, wherein the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from the group consisting of: CLL1, CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, STEAP1, STEAP2, TrpM4, CD21, CD79a, CD72, MUC16, HER2, CD33, CD22, CD79b, LIV1, CD123, CD74, BCMA, and FcRH5. 25. A pharmaceutical composition comprising: the conjugate according to any one of claims 15 to 24, and a pharmaceutically acceptable excipient. 26. A method of treating a condition in a subject with this need, the method comprising administering to the subject a therapeutically effective amount of the conjugate according to any one of claims 15 to 24. 27. The method according to claim 26, wherein the condition is cancer, a tumor, or other malignancy. 28. The method of claim 27, wherein the condition is selected from the group consisting of: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse macrohematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), and myeloid cellular leukemia (MCL). 29. A method of using a compound to treat a condition in a subject with this need, wherein the compound is a conjugate according to any one of claims 15 to 24. 30. The compound used according to claim 29, wherein the condition is cancer, tumor or other malignant tumor.31. The compound used according to claim 30, wherein the disease is selected from the group consisting of: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), and myeloid cell leukemia (MCL). Claims 7 / 9, page 8, CN 122295346 A 32. A pharmaceutical payload-connector conjugate, wherein the pharmaceutical payload-connector conjugate is of formula (B-L1): (B-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a–, or –N+(R3a)2–; wherein each R3a is independently alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy, and haloalkoxy; each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2, or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl, or heteroarylalkyl, or RL3 and RL2 Together they can form a C3-C7 cycloalkyl; X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or a 6-membered heteroaryl; ring B is phenyl or a 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of halogen, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or the two substituents together with the atoms they are attached to form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; and p and q are independently 1 to 8 Integers.33. The drug payload-connector conjugate according to claim 32, wherein the drug payload-connector intermediate is of formula (C-L1): (C-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy, and haloalkoxy; each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl, or heteroarylalkyl, or RL3 and RL2 together can form a C3-C7 cycloalkyl group; X R1 is O or NH; R2 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R3 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-, wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of halogens, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; and p and q are independently integers from 1 to 8. 34. The drug payload-connector conjugate according to claim 32, wherein the drug payload-connector intermediate has the following structure: Claims 9 / 9 Page 10 CN 122295346 A MCL-1 Inhibitor Compound and Use in Antibody-Drug Conjugates Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 600,479, filed November 17, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to inhibitors of inducible myeloid leukemia cell differentiation protein (MCL-1), compositions comprising such compounds, antibody-drug conjugates (ADCs) comprising such compounds, and methods of using such compounds, compositions, and ADCs. Background Art

[0003] Myeloid leukemia-1, or MCL1, is a member of the Bcl2 family of anti-apoptotic proteins and is an attractive target for cancer therapy.MCL1 is found in the outer membrane of mitochondria and participates in a series of protein interactions with pro-apoptotic BH3 proteins (BIM, PUMA, NOXA) to regulate apoptosis. Inhibition of MCL1 by BH3 mimicry therapeutics ultimately leads to the release of BCL effectors BAK and BAX, which oligomerize to form pores in the mitochondrial membrane, resulting in the release of cytochrome c into the cytosol, which subsequently leads to apoptosis. MCL1 is amplified in approximately 10% of human cancers, is widely expressed in both hematologic malignancies and solid tumors, and is upregulated as a mechanism of resistance to chemotherapy and targeted therapies. Therefore, MCL1 is a highly promising target for the treatment of various cancers, including AML.

[0004] Acute myeloid leukemia (or AML) is a type of blood cell carcinoma that originates in the bone marrow and spreads to the blood and potentially other organs, ultimately interfering with the body's ability to produce blood cells. If left untreated, AML can progress rapidly and eventually lead to death. Recent advances in targeted therapy have allowed subsets of patients to receive newer treatments, such as the BCL2 inhibitor venclexta or the antibody-drug conjugate Mylotarg. However, treatment for most AML patients has not changed significantly over the past 40 years, and long-term survival relative to the disease remains low.

[0005] One of the biggest challenges to the successful application of antibody-drug conjugates (ADCs) for the treatment of cancer patients has been the presence of toxicity at effective doses (or even below effective doses). The resulting narrow therapeutic index, or the absence of a therapeutic index, can be the result of a variety of factors. The therapeutic use of first-generation antibody-drug conjugates, such as Mylotarg, has been limited because the linker between the drug or payload and the antibody is unstable. Premature release of the cytotoxic payload reduces efficacy and increases off-target toxicity. However, even ADCs with stable linkers still have a narrow therapeutic index, which may be caused by non-specific uptake of the ADC in normal tissues (e.g., pinocytosis). Despite decades of effort in the field of targeted therapies (including ADCs) for different types of cancer, including AML, there remains a significant unmet need that directly impacts patients' lives. One approach to improve the safety profile of ADCs is to utilize novel payloads that exhibit improved selectivity between tumor cells and normal tissues.

[0006] In some aspects, this invention provides a compound of formula (I): (See specification 1 / 90 page 11 CN 122295346 A (I), or a pharmaceutically acceptable salt thereof), wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or 6-membered heteroaryl; ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R2a and each R2b is independently alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently alkyl, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogens and –OH.

[0007] In other aspects, sub-formulas of formula (I) and exemplary compounds of formula (I) are provided herein. Further provided are conjugates comprising compounds of formula (I), and methods of using said compounds and conjugates.Therefore, in another aspect, this document provides a conjugate of formula (A): Ab-(L-(DP)r)m, or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody; L is a linker; DP is a drug payload; r is an integer from 1 to 8; and m is an integer from 1 to 10; wherein the drug payload is a compound of formula (I): (See specification 2 / 90 page 12 CN 122295346 A (I), or a pharmaceutically acceptable salt thereof), wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or a 6-membered heteroaryl; ring B is phenyl or a 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, -NHR3a, -N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen and –OH.

[0008] In some aspects of the ADCs provided herein, the linker is a peptide linker or a peptide mimic linker. In some aspects, the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from the group consisting of: CLL1, CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, STEAP1, STEAP2, TrpM4, CD21, CD79a, CD72, MUC16, HER2, CD33, CD22, CD79b, LIV1, CD123, CD74, BCMA, and FcRH5.

[0009] In additional aspects, a pharmaceutical composition is provided herein comprising any of the conjugates provided herein, and a pharmaceutically acceptable excipient. Further provided is a method of treating a subject with a condition by administering to a subject a therapeutically effective amount of a conjugate or pharmaceutical composition as provided herein. In some aspects, the condition is cancer, tumor, or other malignancy.

[0010] Additionally, this document provides a pharmaceutical payload-connector conjugate of formula (B-L1): (B-L1), specification 3 / 90 page 13 CN 122295346 A or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen and –OH; each RL1 is independently a C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form a C3-C7 cycloalkyl group; X R1 is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or 6-membered heteroaryl; ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl or haloalkyl; and p and q are independently integers from 1 to 8.

[0011] Additional aspects of these compounds, conjugates, compositions, and methods are described in more detail herein. It should be understood that one, some, or all of the features of the various aspects and embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. Brief Description of the Drawings

[0012] Figure 1 provides a reaction scheme for the synthesis of 1 g of a macrocyclic intermediate, as described in Example 2.

[0013] Figure 2 provides a comparative structure of compounds A, B, and C, which are evaluated in some of the biological examples provided herein.

[0014] Figures 3A and 3B are graphs showing the cytotoxic efficacy of different concentrations of compounds A, B, and C as free drugs against SK-BR-3 adenocarcinoma cells (Figure 3A) and CAMA-1 human breast cancer cells (Figure 3B), as described in Biological Example 1.

[0015] Figures 4A and 4B are graphs showing the cytotoxic efficacy of different concentrations of compounds A, B, and C as free drugs against SU-DHL5 (Figure 4A) and SU-DHL10 (Figure 4B) acute myeloid leukemia (AML) cell lines, as described in Biological Example 3.

[0016] Figures 5A, 5B, and 5C are graphs showing the cytotoxic efficacy of different concentrations of compounds A, B, and 100 against MOLM-16 (Figure 5A), HL-60 (Figure 5B), and OCI-AML3 (Figure 5C) AML cell lines in vitro.

[0017] Figures 6A, 6B, and 6C are graphs showing the cytotoxic efficacy of compounds A, B, and 100 as ADCs conjugated to anti-CD33 antibody 15G15 via the sq-Cit linker against AML cell lines EOL-1 (Figure 6A), MV-4-11 (Figure 6B), and NOMO-1 (Figure 6C), as described in Biological Example 5.

[0018] Figure 7 is a graph showing the cytotoxic efficacy of compounds B, C, and 100 conjugated to 7C2 antibody via the sq-Cit linker against SK-BR-2 adenocarcinoma cells in vitro.

[0019] Figure 8A is a particle size sieving chromatography (SEC) trace of the exemplary anti-Her2 7C2-sqCit linker-compound 100 ADC prepared according to Example 11.

[0020] Figure 8B is an LC-MS analysis of the light chain of the exemplary anti-Her2 7C2-sqCit linker-compound 100 ADC prepared according to Example 11.

[0021] Figure 8C is an LC-MS analysis of the heavy chain of the exemplary anti-Her2 7C2-sqCit linker-compound 100 ADC prepared according to Example 11.

[0022] Figure 9 is a graph showing the cytotoxicity of the exemplary anti-CD33-sqCit ADC containing the payload described herein against MV-4-11 cells in vitro, as described in Biological Example 7.

[0023] Figure 10 is a graph showing the cytotoxicity of an anti-CD33-sqCit ADC containing an exemplary or comparative payload; a non-target ADC containing the same payload; and the cytotoxicity of the cytotoxic agent in NSG mice in an in vivo MV4-11 assay, as described in Biological Example 8. The graph presents the percentage of tumor burden assessed 9 days after ADC administration.

[0024] Figure 11 is a graph showing the dose-response of an exemplary DAR6 ADC using anti-CD33 antibody 15G15, a sq-Cit linker, and payload compound 100 assessed in NSG mice in an in vivo MV4-11 assay, as described in Biological Example 9. The graph presents the bone marrow tumor burden (expressed as live tumor cells / 50kJ) on day 7.

[0025] Figure 12 is a graph showing the cytotoxicity of an anti-CD33-sqCit ADC containing an exemplary or comparative payload in an in vivo subcutaneous MV4-11 assay in SCID mice, as described in Biological Example 10. The graph presents tumor volume over time. Detailed Description

[0026] This document discloses compounds of formulas (X) and (I), or pharmaceutically acceptable salts thereof, and antibody-drug conjugates (ADCs) containing said compounds. The ADCs provided herein can be used to treat cancers, such as liquid cancers. The compounds provided herein, as payloads in ADCs, exhibit surprisingly improved cytotoxic potency compared to previously known cytotoxic macrocyclic compounds. In particular, in some cases, the compounds provided herein exhibit poor cytotoxicity as free drugs but high cytotoxicity as ADCs. In some cases, the compounds provided herein, as free drugs, perform poorly in terms of cell killing compared to other previously known macrocyclic compounds, but are comparable or better in terms of cell killing as ADCs. The reversal of the cytotoxic activity of the free drug and the ADC was surprising and unexpected.

[0027] The compounds (including conjugates) (e.g., compounds of formula (X), (I), or (A)) or their pharmaceutically acceptable salts are defined as existing in one or more stereoisomeric forms (e.g., containing one or more asymmetric carbon atoms). Various stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the subject matter disclosed herein. Likewise, it should be understood that compounds or salts may exist in tautomeric forms other than those shown in the formula and are also covered within the scope of the subject matter disclosed herein. It should be understood that the subject matter disclosed herein includes combinations and subsets of the specific groups described herein. The scope of the subject matter disclosed herein includes mixtures of stereoisomers as well as purified enantiomers or enantiomer / diastereomer-enriched mixtures.It should be understood that the subject matter disclosed herein includes combinations and subsets of the specific groups defined herein. Specification 5 / 90 pages 15 CN 122295346 A

[0028] The subject matter disclosed herein also includes isotopic notation forms of the compounds described herein, such as one or more atoms being replaced by atoms with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that can be incorporated into the compounds described herein (and their pharmaceutically acceptable salts) include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl, 123I, and 125I.

[0029] As described herein, the compounds (including conjugates) of this disclosure may optionally be substituted with one or more substituents, such as those generally exemplified herein or illustrated as specific categories, subclasses, and types of this disclosure. Generally, the term “substituted” means that a hydrogen atom in a given structure is substituted with a specified substituent. In some embodiments, more than one hydrogen atom is substituted with a specified substituent (e.g., when two hydrogen atoms are substituted with an oxygen substituent). Combinations of substituents contemplated by this disclosure are generally those that result in the formation of stable or chemically viable compounds.

[0030] As used herein, the terms “comprising,” “containing,” and “including” are used in their open, non-limiting sense.

[0031] The article “a / an” as used in this disclosure means one or more of the grammatical objects of the article (e.g., at least one). By way of example, “an element” may mean one element or more elements.

[0032] The term “antibody” is used in the broadest sense herein and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific, sex antibodies), and antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or antibodies derived from other species. Antibodies are proteins produced by the immune system that recognize and bind to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th ed., Garland Publishing, New York). Target antigens typically have multiple binding sites, also known as epitopes, which are recognized by CDRs (complementarity-determining regions) on multiple antibodies. Each antibody that specifically binds to different epitopes has a different structure.Therefore, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunoactive portions of full-length immunoglobulin molecules, i.e., molecules containing an antigen-binding site that specifically binds to an antigen or a portion thereof to a target, including but not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. Immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may be derived from any species. However, in some aspects, immunoglobulins are immunoglobulins derived from humans, mice, or rabbits.

[0033] As used herein, the term "antibody fragment" includes a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bisomatic antibodies; linear antibodies; microantibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315-323), fragments generated from Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions) and epitope-binding fragments of any of the above (which immune-specifically bind to cancer cell antigens, viral antigens, or microbial antigens), single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0034] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies contained in the group are identical except for a small number of naturally occurring mutations that may be present. Monoclonal antibodies have high specificity for a single antigenic site. Furthermore, unlike polyclonal antibody formulations which include different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier “monoclonal” indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, based on the specification 6 / 90 page 16 CN 122295346 A, the monoclonal antibodies used in the subject matter described herein can be prepared by a hybridoma method first described by Kohler et al. (1975) Nature, 256: 495, or by a recombinant DNA method (see, for example: US 4816567, US 5807715).Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described in, for example, the following literature: Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597.

[0035] Monoclonal antibodies in this document specifically include “chimeric” antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). The target chimeric antibodies in this article include “primate-like” antibodies, which contain a variable domain antigen-binding sequence derived from non-human primates (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0036] The term “chimeric” antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0037] The “class” of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

[0038] As used herein, the term "intact antibody" is an antibody comprising VL and VH domains and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effective functions," which refer to those biological activities attributable to the antibody's Fc constant region (native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors, such as B cell receptors and BCRs.

[0039] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region.The term includes both native Fc regions and variant Fc regions.

[0040] A “human antibody” is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or to an amino acid sequence derived from a non-human antibody using a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0041] A “humanized” antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will substantially contain at least one of all, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all FRs correspond to the FRs of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. An antibody in a “humanized form,” such as a non-human antibody, refers to an antibody that has been humanized.

[0042] As used herein, the term "free cysteine ​​amino acid" refers to a cysteine ​​amino acid residue that has been modified into a parent antibody, has a thiol functional group (-SH), and is not paired into an intramolecular or intermolecular disulfide bond.

[0043] As used herein, the term "amino acid" refers to glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, or citrulline.

[0044] As used herein, "alkyl" refers to a straight-chain or branched saturated hydrocarbon chain. In some embodiments, unless otherwise stated, an alkyl group comprises 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Examples of alkyl groups may include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, isopentyl, neopentyl, n-hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When referring to an alkyl residue having a specific number of carbons, all geometric isomers having that number of carbons may be covered. Thus, for example, "butyl" may include n-butyl, sec-butyl, isobutyl, and tert-butyl, and "propyl" may include n-propyl and isopropyl.

[0045] As used herein, "alkoxy" refers to the group –OR, where R is an alkyl group as used herein.In some embodiments, unless otherwise stated, an alkoxy group comprises 1 to 12 carbon atoms (C1-C12 alkoxy), 1 to 8 carbon atoms (C1-C8 alkoxy), 1 to 6 carbon atoms (C1-C6 alkoxy), or 1 to 4 carbon atoms (C1-C4 alkoxy). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and butoxy.

[0046] "Alkyne" means an unsaturated straight-chain or branched monovalent hydrocarbon chain or combination thereof having at least one alkyne unsaturated site (i.e., having at least one part of the formula C≡C) having a specified number of carbon atoms (i.e., C2-10 means having two to ten carbon atoms). Specific alkynyl groups include those alkynyl groups having 2 to 8 carbon atoms ("C2-C8 alkynyl"), 2 to 6 carbon atoms ("C2-C6 alkynyl"), and 2 to 4 carbon atoms ("C2-C4 alkynyl"). Examples of alkyne groups include, but are not limited to, groups such as ethynyl (or acetylenyl), propynyl-1-ynyl, propynyl-2-ynyl (or propynyl), butynyl-1-ynyl, butynyl-2-ynyl, and butynyl-3-ynyl. In particular, alkynes include propynyl-1-ynyl.

[0047] As used herein, “cycloalkyl” refers to a monocyclic or polycyclic saturated or partially unsaturated non-aromatic hydrocarbon. In some embodiments, unless otherwise stated, a cycloalkyl group comprises 3 to 12 carbon atoms (C3-C12 cycloalkyl), 3 to 8 carbon atoms (C3-C8 cycloalkyl), 3 to 6 carbon atoms (C3-C6 cycloalkyl), or 3 to 5 carbon atoms (C3-C5 cycloalkyl). In some embodiments, the cycloalkyl group is a saturated monocyclic or polycyclic hydrocarbon. In other embodiments, the cycloalkyl group comprises one or more double bonds (e.g., a monocyclic non-aromatic hydrocarbon comprising one or two double bonds). Polycyclic cycloalkyl groups may comprise spirocyclic, fused, or bridged polycyclic moieties, wherein each ring is a saturated or partially unsaturated non-aromatic hydrocarbon. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, octahydrocyclopentadienyl, spiro[3.3]heptyl, etc.

[0048] As used herein, “halocycloalkyl” refers to a cycloalkyl group in which one or more hydrogen atoms are halogenated, wherein each halogen is independently selected. In some embodiments, one to four hydrogen atoms are halogenated, wherein each halogen is independently selected. Thus, halocycloalkyl groups include, for example, C3-C6 membered cycloalkyl groups in which one or more hydrogen atoms are independently substituted with fluorine, chlorine, iodine, or bromine.

[0049] “halogenated” or “halogen” includes fluorine, chlorine, bromine, and iodine.When a part is substituted with more than one halogen, it can be indicated by using a prefix corresponding to the number of halogen moieties attached, such as dihaloaryl, dihaloaryl, trihaloaryl, etc., which refer to aryl and alkyl groups substituted with two (“di”) or three (“tri”) halogen groups, which may be, but do not have to be, the same halogen; thus, 4-chloro-3-fluorophenyl is within the range of dihaloaryl.

[0050] “Haloalkyl” means an alkyl group in which one or more hydrogen atoms are substituted with a halogen, wherein each halogen is independently selected. Thus, haloalkyl includes, for example, C1-C6 alkyl groups in which one or more hydrogen atoms are independently substituted with fluorine, chlorine, iodine, or bromine.

[0051] As used herein, “haloalkoxy” means an alkoxy group in which one or more hydrogen atoms are substituted with a halogen, wherein each halogen is independently selected. Therefore, haloalkoxy groups include, for example, C1-C6 alkoxy groups in which one or more hydrogen atoms are independently substituted with fluorine, chlorine, iodine, or bromine. Haloalkoxy groups may include, for example, C1-C12 haloalkoxy groups, C1-C8 haloalkoxy groups, C1-C6 haloalkoxy groups, or C1-C4 haloalkoxy groups. In some embodiments, the haloalkoxy group is a fluoromethoxy or fluoroethoxy group, such as a trifluoromethoxy or trifluoroethoxy group.

[0052] As used herein, “heteroaryl” refers to a monocyclic or polycyclic group comprising at least one aromatic ring, wherein the aromatic ring comprises at least one cyclic heteroatom. In some embodiments, the heteroatom is independently selected from the group consisting of N, O, and S. Unless otherwise specified, a heteroaryl group may comprise 5, 6, 7, 8, 9, 10, 11, 12 or more ring atoms, wherein a ring atom refers to the sum of carbon atoms and heteroatoms in one or more rings (e.g., 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered or 12-membered heteroaryls). A heteroaryl group may also comprise a polycyclic group having at least one aromatic ring comprising at least one cyclic heteroatom fused to a non-aromatic ring (e.g., 5,6,7,8-tetrahydroquinolinyl; 4,5,6,7-tetrahydroisobenzofuranyl). A heteroaryl group may also comprise a polycyclic group having at least one aromatic ring comprising at least one cyclic heteroatom fused to an aromatic ring (e.g., quinolinyl, quinoxalinyl, benzothiazolyl). A heteroaryl group may comprise a polycyclic group having two fused aromatic rings, wherein each ring comprises at least one cyclic heteroatom (e.g., naphthidyl). The heteroaryl group is a polycyclic group, and the connection point with another part (e.g., with the rest of the formula) can appear on any ring. Examples of heteroaryl groups include, but are not limited to, pyrrole, imidazolyl, triazolyl, furanyl, acezolyl, phenylthio, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, and indole.

[0053] As used herein, “heterocyclic alkyl” refers to a saturated or unsaturated non-aromatic cyclic group having a monocyclic or multiple condensed rings and having 1 to 14 cyclic (i.e., cyclic) carbon atoms and 1 to 6 cyclic (i.e., cyclic) heteroatoms such as nitrogen, phosphorus, sulfur, or oxygen. Heterocyclic rings comprising more than one ring can be fused, spirocyclic, or bridged, or any combination thereof. In fused-ring systems, one or more fused rings can be cycloalkyl. The specific heterocyclic groups are: 3- to 14-membered rings having 1 to 13 cyclic carbon atoms and 1 to 6 cyclic heteroatoms independently selected from nitrogen, phosphorus, oxygen, and sulfur; 3- to 12-membered rings having 1 to 11 cyclic carbon atoms and 1 to 6 cyclic heteroatoms independently selected from nitrogen, phosphorus, oxygen, and sulfur; 3- to 10-membered rings having 1 to 9 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, phosphorus, oxygen, and sulfur; 3- to 8-membered rings having 1 to 7 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, phosphorus, oxygen, and sulfur; and 3- to 6-membered rings having 1 to 5 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, phosphorus, oxygen, and sulfur. In one variant, the heterocyclic alkyl group comprises a monocyclic 3-, 4-, 5-, 6-, or 7-membered ring having 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6 cyclic carbon atoms and 1 to 2, 1 to 3, or 1 to 4 cyclic heteroatoms independently selected from nitrogen, phosphorus, oxygen, and sulfur. In a particular embodiment, the heterocyclic alkyl group is a 3- to 8-membered saturated monocyclic ring comprising one or two cyclic heteroatoms selected from S, O, and N. In some embodiments, the heterocyclic alkyl group is a 3- to 6-membered saturated monocyclic ring comprising one or two cyclic heteroatoms selected from S, O, and N.

[0054] As used herein, “halogenated heterocyclic alkyl” refers to a heterocyclic alkyl group in which one or more hydrogen atoms are halogenated, wherein each halogen is independently selected. In some embodiments, one to four hydrogen atoms are halogenated, wherein each halogen is independently selected. Therefore, halogenated heterocyclic alkyl groups include, for example, 3- to 8-membered heterocyclic alkyl groups in which one or more hydrogen atoms are independently substituted with fluorine, chlorine, iodine, or bromine.

[0055] “Patient” or “subject” may encompass both mammals and non-mammals. Examples of mammals may include, but are not limited to, any member of the class Mammalia: humans; non-human primates such as chimpanzees, monkeys, baboons, or rhesus monkeys and other apes and monkeys; agricultural animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs and the like.Examples of non-mammals include, but are not limited to, birds, fish, etc. In some embodiments, the patient or subject is a human. Specification 9 / 90 pages 19 CN 122295346 A

[0056] The term “effective amount” or “therapeutic effective amount” refers to an amount of a compound, its pharmaceutically acceptable salt, or a pharmaceutical composition sufficient to produce a desired therapeutic outcome (such as reducing the severity of the condition during its duration, stabilizing its severity, or eliminating one or more signs, symptoms, or causes). For therapeutic use, beneficial or desired outcomes may include, for example: reducing one or more symptoms (biochemical, histological, and / or behavioral) caused by the condition, including its complications and intermediate pathological phenotypes that occur during the course of the condition; improving the quality of life of patients suffering from the condition; reducing the dosage of other drugs required to treat the condition; enhancing the efficacy of another drug; delaying disease progression; and / or prolonging patient survival. In some embodiments, the compound is a conjugate as provided herein, such as an ADC.

[0057] As used herein, the term “excipient” refers to an inert or inactive substance that can be used to produce a pharmaceutical or pharmaceutical composition, such as tablets containing a compound (including conjugates, such as ADCs) (or pharmaceutically acceptable salts) as an active ingredient. The term “excipient” can cover a wide range of substances, including but not limited to any substance used as a diluent, filler or spreader, binder, disintegrant, humectant, coating, emulsifier or dispersant, compression / encapsulation aid, cream or lotion, lubricant, parenteral solution, material for chewable tablets, sweetener or flavoring agent, suspending / gelling agent, or wet granulation agent. In some cases, the term “excipient” covers pharmaceutically acceptable carriers.

[0058] “Pharmaceutically acceptable salts” include salts that are generally safe and are neither biologically undesirable nor otherwise undesirable, and include those salts acceptable for veterinary and human pharmaceutical use. Such salts can be prepared by any suitable method, for example, by treating a free acid with an inorganic or organic base (e.g., if the compound is a free acid) or a free base with an inorganic or organic acid (e.g., if the compound is a free base). Suitable pharmaceutically acceptable salts may include, for example, those derived from inorganic acids, organic acids, pyranoside esters, amino acids, aromatic acids, sulfonic acids, etc. Suitable pharmaceutically acceptable salts may also include, for example, those derived from organic bases (such as amines, e.g., primary, secondary, or tertiary amines), alkali metal hydroxides, or alkaline earth metal hydroxides, etc. Exemplary examples of suitable salts include, but are not limited to, organic and inorganic salts, wherein the organic salt is derived from: amino acids (such as glycine or arginine); ammonia; primary, secondary, and tertiary amines; cyclic amines (such as piperidine, morpholine, and piperazine). In some embodiments, the compound is a conjugate as provided herein, such as an ADC or a drug payload-connector conjugate.

[0059] As used herein, the term “peptide mimic” refers to a non-peptide chemical portion. A peptide is a short chain of amino acid monomers linked by a peptide (amide) bond (a covalent chemical bond formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid). The shortest peptide is a dipeptide (consisting of two amino acids linked by a single peptide bond), followed by tripeptides, tetrapeptides, and so on. A peptide mimic chemical portion includes a non-amino acid chemical portion. A peptide mimic chemical portion may also contain one or more amino acids separated by one or more non-amino acid chemical units. A peptide mimic chemical portion may not contain two or more adjacent amino acids linked by peptide bonds at any part of its chemical structure.

[0060] Numerical ranges as used herein may include consecutive integers. For example, a range expressed as “0 to 5” would include 0, 1, 2, 3, 4, and 5.

[0061] As used herein, the term “unsubstituted” may mean that the portion is free of substituents (e.g., where hydrogen satisfies its valence).

[0062] As used herein, the term “treat” means to delay the onset of one or more conditions; to prevent the onset of one or more conditions; and / or to reduce the severity of one or more symptoms of a condition that will occur or is expected to occur. Therefore, these terms may include improving symptoms of one or more existing conditions; preventing one or more other symptoms; improving or preventing the underlying cause of one or more symptoms; suppressing a condition, e.g., blocking the onset of a condition; alleviating a condition; causing a condition to subside; reducing symptoms caused by a condition; or terminating or reducing symptoms of a condition.

[0063] As used herein, the term “about” when referring to a numerical value is intended to cover various variations of the specified amount, for example, in some embodiments of the specification (pages 10 / 90 of 20 CN 122295346 A), covering a specified amount ± 20%; in some embodiments, covering a specified amount ± 10%; in some embodiments, covering a specified amount ± 5%; in some embodiments, covering a specified amount ± 1%; in some embodiments, covering a specified amount ± 0.5%; in some embodiments, covering a specified amount ± 0.1%; because these variations are suitable for performing the disclosed methods or employing the disclosed compositions.

[0064] Where a numerical range is provided, unless the context explicitly specifies otherwise, it should be understood that every tenth of each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range, is covered by the invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered by the invention, subject to any explicitly excluded limitations within the specified range. When the range includes one or both limits, the range excluding one or both of those included limits is also included in the invention.

[0065] Chemical names can be generated based on the compound structures provided herein, according to naming conventions known to those skilled in the art, such as those provided by the International Union of Pure and Applied Chemistry (IUPAC). Chemical names can also be generated using ChemDraw® software, such as ChemDraw® version 19.1. In case of any discrepancy between structure and name, the structure shall prevail.

[0066] The compounds described herein (including conjugates) or pharmaceutically acceptable salts thereof may exist in one or more stereoisomeric forms (e.g., containing one or more asymmetric carbon atoms). Various stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the subject matter disclosed herein. Likewise, it should be understood that compounds or salts may exist in tautomeric forms other than those shown in the formula and are also covered within the scope of the subject matter disclosed herein. It should be understood that the subject matter disclosed herein includes combinations and subsets of the specific groups described herein. Unless otherwise specified, the scope of the subject matter disclosed herein includes mixtures of stereoisomers and purified mirror isomers or mixtures of enantiomers / diastereomeric enrichments. It should be understood that the topics disclosed herein include combinations and subsets of the specific groups defined herein.

[0067] 1. Compound of Formula (X) This document provides a compound of formula (X): (X), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; Ring A is phenyl or 6-membered heteroaryl; Ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy, and haloalkoxy.

[0068] In some embodiments, the compound of formula (X) is the compound of formula (I) or a pharmaceutically acceptable salt thereof. This document provides compounds of formula (I): (I), or pharmaceutically acceptable salts thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or 6-membered heteroaryl; ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q Independently an integer from 1 to 8; R3 is –NH2, –NHR3a, –N(R3a)2 or –N+(R3a)3, wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogens and –OH.

[0069] In some embodiments of compounds of formula (X) or (I): X is O or NH; Z is N-CH3, N-CH2CH3 or NH; ring A is phenyl or a 6-membered heteroaryl containing one to three cyclic N atoms; ring B is phenyl or a 6-membered heteroaryl containing one to three cyclic N atoms; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted by one to three independently selected halogens; R2 is a C1-C6 alkyl, C3-C8 cycloalkyl, or a 3- to 8-membered heteroaryl containing one to three cyclic heteroatoms independently selected from O, N and S. A heterocyclic alkyl group, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocyclic alkyl group is unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OH, -OR2b, and -O (R2bO)qR2c, or two substituents together with the atoms to which they are attached form a C3-C8 cycloalkyl, a C3-C8 halocycloalkyl, a 3- to 8-membered heterocyclic alkyl group containing one to three cyclic heteroatoms independently selected from O, N, and S, or a 3- to 8-membered halocycloalkyl group containing one to three cyclic heteroatoms independently selected from O, N, and S; each R2a and each R2b is independently a C1-C6 alkyl or a C1-C6 haloalkyl; each R2c is independently hydrogen, a C1-C6 alkyl, or a C1-C6 haloalkyl; p and q are independently integers from 1 to 8; R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently a C1-C6 alkyl group, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogens and –OH.

[0070] In some embodiments of compounds of formula (X) or (I), or pharmaceutically acceptable salts thereof, ring A and ring B are independently phenyl or heteroaryl containing one or two ring N atoms. In some embodiments, both ring A and ring B are independently heteroaryl containing one or two ring N atoms. In some embodiments, ring A is pyrimidine. In some embodiments, ring B is phenyl or pyridine. In some embodiments, ring B is phenyl or pyridine, wherein the connection point with ring A is indicated. In some embodiments, ring A is pyrimidine and ring B is phenyl, pyrimidine, or pyridine. In some embodiments, ring A is pyrimidine and ring B is phenyl or pyridine. In some embodiments, ring A is pyrimidine and ring B is phenyl, pyrimidine, or phenyl, wherein the connection point with ring A is indicated.In some other embodiments, ring A is pyrimidine and ring B is phenyl or , wherein the connection point with ring A is indicated. In still other embodiments, ring A is pyrimidine and ring B is , wherein the connection point with ring A is indicated. In some embodiments, Z is N-H. In other embodiments, Z is N-CH3. In still other embodiments, Z is N-CH2CH3.

[0071] In some embodiments, the compound of formula (X) or (I) is a compound of formula (II): (II), or a pharmaceutically acceptable salt thereof, wherein: X, R1, R2 and R3 are as defined for formula (I). Instructions for Use, 13 / 90 pages, 23 CN 122295346 A

[0072] In some embodiments of formula (II) or its pharmaceutically acceptable salts: X is O; Z is N-CH3, N-CH2CH3 or NH; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is a C1-C6 alkyl, C3-C8 cycloalkyl, a 3- to 8-membered heterocyclic alkyl comprising one to three cyclic heteroatoms independently selected from O, N and S, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocyclic alkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OH, -OR2b and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a C3-C8 cycloalkyl, C3-C8 halocycloalkyl, The alkyl group comprises one to three cyclic heteroatoms independently selected from O, N, and S, consisting of 3 to 8-membered heterocyclic alkyl groups, or comprising one to three cyclic heteroatoms independently selected from O, N, and S, consisting of 3 to 8-membered halocyclic alkyl groups; each R2a and each R2b is independently C1-C6 alkyl or C1-C6 haloalkyl; each R2c is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; p and q are independently integers from 1 to 8; R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently C1-C6 alkyl, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen and –OH.

[0073] In some embodiments of compounds of formula (X) or (I), or compounds of formula (II), or pharmaceutically acceptable salts thereof, R1 is a halogen-substituted phenyl. In some embodiments, R1 is a fluorine-substituted phenyl (such as one fluorine-substituted). In some embodiments, X is O.

[0074] In some embodiments of compounds of formula (X) or (I), or compounds of formula (II), or pharmaceutically acceptable salts thereof, R2 is an unsubstituted C1-C6 alkyl group or substituted with one or more substituents independently selected from the group consisting of –OH and a halogen. In some embodiments, R2 is an unsubstituted C2-C4 alkyl group or substituted with one or more substituents independently selected from the group consisting of –OH and a halogen. In some embodiments, R2 is an unsubstituted C2-C4 alkyl group or substituted with one or more fluorines. In other embodiments, R2 is an unsubstituted C2-C4 alkyl group, such as –CH2CH2–, –CH2CH2CH2–, or –CH2CH2CH2CH2–. In still other embodiments, R2 is an unsubstituted ethyl group or substituted with one or more substituents independently selected from the group consisting of –OH and a halogen (such as fluorine). In some embodiments, R2 is –CH2CH2–.

[0075] In some embodiments of the compound of formula (X) or a pharmaceutically acceptable salt thereof, R3 is –NH2, –NHR3a, –N(R3a)2 or –N+(R3a)3, wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of halogens, –OH, alkoxy groups and haloalkoxy groups. In some embodiments, each R3a is independently a C1-6 alkyl group, wherein each C1-6 alkyl group is independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of halogens, –OH, C1-6 alkoxy groups and C1-6 haloalkoxy groups. In some embodiments, each R3a is independently a C1-6 alkyl group, wherein each alkyl group is unsubstituted or substituted with halogens, –OH or –OCH3. In some embodiments of compounds of formula (X) or (I), or compounds of formula (II), or pharmaceutically acceptable salts thereof, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogens and –OH. In some embodiments, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an unsubstituted C1-C6 alkyl group. In some embodiments, R3 is –NH2 or –NH(C1-C6 alkyl), such as –NH2 or –NH(CH3).Specification 14 / 90 pages 24 CN 122295346 A

[0076] In some embodiments of the compound of formula (I) or a pharmaceutically acceptable salt thereof: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine or pyridine; R1 is a fluoro-substituted phenyl; R2 is an unsubstituted or fluoro-substituted C1-C6 alkyl; R3 is –NH2 or –NH(C1-6 alkyl); and X is O.

[0077] In some embodiments of the compound of formula (I) or a pharmaceutically acceptable salt thereof: Z is N-C1-6 alkyl; ring A is pyrimidine; ring B is phenyl, pyrimidine or, wherein the connection point with ring A is indicated; R1 is a fluoro-substituted phenyl; R2 is an unsubstituted or fluoro-substituted C1-C6 alkyl; R3 is –NH2 or –NH(C1-6 alkyl); and X is O.

[0078] In some embodiments of the compound of formula (I) or a pharmaceutically acceptable salt thereof: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated; R1 is a fluoro-substituted phenyl; R2 is an unsubstituted or fluoro-substituted C1-C6 alkyl; R3 is –NH2 or –NH (C1-6 alkyl); and X is O.

[0079] In some embodiments of the compound of formula (I) or a pharmaceutically acceptable salt thereof: Z is N-CH3; ring A is pyrimidine; ring B is pyridine; R1 is a fluoro-substituted phenyl; R2 is an unsubstituted C1-C6 alkyl; R3 is –NH2; and X is O.

[0080] In some embodiments of the compound of formula (II) or a pharmaceutically acceptable salt thereof: R1 is a fluorinated phenyl; R2 is an unsubstituted C1-C6 alkyl; R3 is –NH2; and AX is O.

[0081] In some embodiments of the compound of formula (X) or (I) or a pharmaceutically acceptable salt thereof: X is O, Y is N, R1 is a fluorinated phenyl, R2 is an alkyl, and R3 is –NH2.

[0082] In some embodiments, the compound of formula (X) or (I) is selected from the group consisting of: , , , , , , , and, or a pharmaceutically acceptable salt thereof.

[0083] In some embodiments, the compound of formula (X) is selected from the group consisting of: , ... In some embodiments, the payload is a compound of formula (II). In some embodiments, the conjugate is an antibody-drug conjugate (ADC).

[0087] In some embodiments, this document provides a conjugate of formula (A): Ab-(L-(DP)r)m, 28 CN 122295346 A or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody; L is a linker; DP is a drug payload; r is an integer from 1 to 8; and m is an integer from 1 to 10; wherein DP is a compound of formula (X) or a pharmaceutically acceptable salt thereof.

[0088] In some embodiments, the compound of formula (X) is a compound of formula (I). Therefore, in some embodiments, this document provides a conjugate of formula (A): Ab-(L-(DP)r)m, or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody; L is a linker; DP is a drug payload; r is an integer from 1 to 8; and m is an integer from 1 to 10; wherein DP is a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0089] The drug payload may be a compound of formula (X) or (I) as provided herein, or any of the embodiments therein. For example, in some embodiments, the drug payload is a compound of formula (X) or (I), wherein ring A and ring B are independently phenyl or heteroaryl containing one or two ring N atoms. In some embodiments, both ring A and ring B are independently heteroaryl containing one or two ring N atoms. In some embodiments, ring A is pyrimidine. In some embodiments, ring B is pyridine. In some embodiments, ring A is pyrimidine and ring B is pyridine.In some embodiments, ring A is pyrimidine and ring B is phenyl, pyrimidine, or pyridine. In some embodiments, Z is N-H. In other embodiments, Z is an N-C1-6 alkyl group, such as, for example, N-CH3 or N-CH2CH3. In still some embodiments, Z is N-CH3. In some embodiments, the pharmaceutical payload is a compound of formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments of the pharmaceutical payload, R2 is an unsubstituted or C1-C6 alkyl group substituted with one or more substituents independently selected from the group consisting of –OH and a halogen. In some embodiments, R2 is an unsubstituted or C2-C4 alkyl group substituted with one or more substituents independently selected from the group consisting of –OH and a halogen. In some embodiments, R2 is an unsubstituted or C2-C4 alkyl group substituted with one or more fluorine groups. In other embodiments, R2 is an unsubstituted C2-C4 alkyl group, such as –CH2CH2–, –CH2CH2CH2–, or –CH2CH2CH2CH2–. In some embodiments, R2 is an unsubstituted or substituted ethyl group, or a halogen, substituted with one or more substituents independently selected from the group consisting of: –OH and halogen. In some embodiments, R2 is –CH2CH2–. In some embodiments, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, –OH, alkoxy, and haloalkoxy. In some embodiments, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen and –OH. In some embodiments, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently unsubstituted or substituted with one to four halogens selected from the group consisting of halogens or C1-C6 alkyl groups. In some embodiments, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently unsubstituted or substituted with one to four halogens. In some examples described on pages 19 / 90 of CN 122295346 A, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently unsubstituted or substituted with one to four halogens.In some embodiments, R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an unsubstituted or fluorinated C1-C4 alkyl group. In still other embodiments, R3 is –NHR3a, wherein each R3a is independently an unsubstituted or fluorinated C1-C4 alkyl group. In some embodiments, R3 is –NH2 or –NH(C1-C6 alkyl), such as –NH2 or –NH(CH3). In some embodiments, r is an integer from 1 to 2; and m is an integer from 1 to 10. In some embodiments, r is 1, and m is an integer from 2 to 8, such as 2, 3, 4, 5, 6, 7, or 8.

[0090] The one or more drug payloads in the ADC of formula (A) can be linked to the antibody via one or more adapters, wherein the adapters are linked to the drug payload at any chemically feasible site. In embodiments of an ADC comprising any of the compounds of formula (X), formula (I), or their sub-formulas or compounds, the connection of the connector to the compound requires the formation of one or more covalent bonds with the compound, and the structure of the compound is tuned accordingly, such as by substitution of hydrogen atoms via bond bonding to the connector, or by adding bonds to the connector and adding charges at chemically feasible sites. In some embodiments, the connector site is an N atom. In some embodiments, the connector is connected via a single covalent bond through a variable Z, and the conjugate comprises a compound wherein the variable Z is N (substitution of hydrogen atoms via bond bonding to the connector) or N+C1-C6 alkyl (where the bond is formed by a tertiary amine, converting it to a quaternary amine). In other embodiments, this is done via a single covalent bond to a variable R3, and the variable R3 in the conjugate is –NH–, –NR3a–, or –N+(R3a)2–. Those skilled in the art will readily recognize how the structure of the compound is tuned when the compound is covalently connected to the connector through different sites.

[0091] Therefore, this document provides an ADC of formula (A), wherein the ADC of formula (A) is the ADC of formula (B): (B), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; and X, R1, R2, R3a, Z, ring A and ring B are as defined for formula (X).

[0092] In some embodiments, the compound of formula (X) is the compound of formula (I), and the ADC of formula (B) is: (B), page 20 / 90 of the specification 30 CN 122295346 A or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; and X, R1, R2, R3a, Z, ring A and ring B are as defined with respect to formula (I).

[0093] The present document further provides an ADC of formula (A), wherein the ADC of formula (A) is the ADC of formula (C): (C), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; and X, R1, R2 and R3a are as defined with respect to formula (X).

[0094] In some embodiments, the compound of formula (X) is a compound of formula (I), and the ADC of formula (C) is: (C), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; and X, R1, R2 and R3a are as defined with respect to formula (I).

[0095] In still some embodiments, an ADC of formula (A) is provided herein, wherein the ADC of formula (A) is an ADC of formula (D): (D), or a pharmaceutically acceptable salt thereof, wherein: m is an integer from 1 to 10; Z is N+-C1-6 alkyl or N; and X, R1, R2 and R3 are as defined with respect to formula (X).

[0096] In some embodiments, the compound of formula (X) is a compound of formula (I), and the ADC of formula (D) is: (D), or a pharmaceutically acceptable salt thereof, wherein: m is an integer from 1 to 10; Z is an N+-C1-6 alkyl or N; and X, R1, R2 and R3 are as defined for formula (I).

[0097] In other embodiments, an ADC of formula (A) is provided herein, wherein the ADC of formula (A) is an ADC of formula (E): (E), or a pharmaceutically acceptable salt thereof, wherein: m is an integer from 1 to 10; and X, R1, R2 and R3 are as defined for formula (X).Specification 22 / 90 pages 32 CN 122295346 A

[0098] In some embodiments, the compound of formula (X) is a compound of formula (I), and the ADC of formula (E) is: (E), or a pharmaceutically acceptable salt thereof, wherein: m is an integer from 1 to 10; and X, R1, R2, and R3 are as defined with respect to formula (I).

[0099] Each of the examples or combinations of examples described herein with respect to compounds of formula (X), (I), or (II) is applicable to ADCs comprising compounds of formula (X), (I), or (II), including ADCs of formulas (A), (B), (C), (D), and (E). Antibodies and adapter components of said antibodies are described in further detail below.

[0100] a. Antibody (Ab) As described herein, compounds of formula (I) (including compounds of formula (II)) may be used in combination with antibodies (Abs) and adapters to form antibody-drug conjugates (ADCs), such as ADCs of formula (A), (B), (C), (D), or (E). In such embodiments, one or more compounds of formula (I) are covalently linked to Abs via adapters to form an ADC. In the case of using multiple compounds of formula (X) or (I), each compound may be linked to an antibody via a separate adapter. In an ADC, non-antibody, non-adaptor chemical groups (such as compounds of formula (X) or (I), which include compounds of formula (II)) are generally referred to as “payload,” “drug,” or “drug payload.”

[0101] As described herein, antibodies (such as monoclonal antibodies (mABs)) are used to deliver payloads to target cells, such as cells expressing specific proteins targeted by the antibody. The antibody portion of an ADC may target cells expressing antigens, such as cell surface antigens.

[0102] In certain embodiments, the antibody may be mutated to reduce effector function. Examples of mutations that regulate Fc effector function include the LALAPG mutation and the NG2LH mutation.

[0103] In certain embodiments, the antibody is THIOMAB™, such as the THIOMAB™ antibody previously described in WO2016 / 04856. In some embodiments, various combinations are contemplated such that any antibody target can be combined with any suitable THIOMAB™ mutation (including the LALAPG or NG2LH mutation) with or without any Fc effector regulation.

[0104] In some embodiments, the antibody is a human antibody.Human antibodies generally include those described, for example, in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368–74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450–459 (2008). In some embodiments, the antibody is a library-derived antibody. Various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding properties. Such methods are reviewed and further described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. .338(2): 299-310 (2004); Lee et al., J . Mol . Biol .340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA101(34): 12467-12472 (2004); and Lee et al., J . Immunol. Methods284(1-2): 119-132 (2004).

[0105] In some embodiments, the antibody is a chimeric antibody and a humanized antibody.Humanized antibodies and their manufacturing methods are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) transplantation); Padlan, Mol. Immunol. 28:489-498. (1991) (description of “surface rework”); Dall'Acqua et al., Methods36:43-60 (2005) (description of “FR reorganization”); and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (description of “guided selection” approach for FR reorganization).

[0106] In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody. As used herein, the term “multispecific antibody” refers to an antibody comprising an antigen-binding domain having multi-antigen determinant specificity (i.e., capable of binding to two or more different epitope determinants on one molecule or capable of binding to epitope determinants on two or more different molecules). As used herein, the term “bispecific antibody” is a multispecific antibody comprising an antigen-binding domain capable of binding to two different epitopes on one biomolecule or capable of binding to epitopes on two different biomolecules. Bispecific antibodies may also be referred to herein as having “dual specificity” or “dual-specific.” Exemplary bispecific antibodies can bind to both proteins and any other antigens.Techniques for preparing multispecific antibodies include, but are not limited to: recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829 and Traunecker et al., EMBO J.10: 3655 (1991)), and “mortar and pestle structure” modification (see, for example, US Patent No. 5,731,168, WO2009 / 089004, US2009 / 0182127, US2011 / 0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26 (6):649-658, and Kontermann (2005) Acta Pharmacol. Sin., 26:1-9). Multispecific antibodies can also be prepared using the following techniques: engineered electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980; and Brennan et al., Science 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148(5): 1547–1553 (1992)); using “dimeric antibody” techniques to prepare bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444–6448 (1993)); and using single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol. 152:5368). (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991). This document also includes engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies” or “dual variable domain immunoglobulins” (DVD) (see, for example, US 2006 / 0025576A1; and Wu et al. Nature Biotechnology (2007)). Antibodies or fragments described herein also include “dual-acting FAbs” or “DAFs” that contain antigen-binding sites that bind to the target protein and another different antigen (see, for example, US 2008 / 0069820).

[0107] In some embodiments, the antibody is an antibody fragment.Antibody fragments may include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see the specification, page 24 / 90, 34 CN 122295346 A Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and US Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding antigen-determining residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. Biantibodies are antibody fragments having two antigen-binding sites (which may be bivalent or bispecific). See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129–134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA90: 6444–6448 (1993). Trisomic and tetrasomic antibodies are also described in Hudson et al., Nat. Med. 9:129–134 (2003). Single-domain antibodies are antibody fragments containing all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1). Antibody fragments can be prepared using various techniques, including but not limited to proteolytic digestion of intact antibodies and production via recombinant host cells.

[0108] In some embodiments, the antibody is an antibody variant. In some embodiments, amino acid sequence variants of the antibodies provided herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the antibody amino acid sequence.Any combination of deletions, insertions, and substitutions can be implemented to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0109] In some embodiments, antibodies used in ADCs as provided herein can be generated using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. Referring to antibody affinity, in some embodiments, the antibody binds to one or more tumor-associated antigens or cell surface receptors.

[0110] In some embodiments, the tumor-associated antigen or cell surface receptor is selected from the group consisting of: CLL1, CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, STEAP1, STEAP2, TrpM4, CD21, CD79a, CD72, MUC16, HER2, CD33, CD22, CD79b, LIV1, CD123, CD74, BCMA, and FcRH5. Therefore, in some embodiments, the ADC provided herein may contain antibodies such as: anti-CLL1, anti-CD71, anti-Trop2, anti-MSLN, anti-NaPi2b, anti-Ly6E, anti-EpCAM, anti-STEAP1, anti-STEAP2, anti-TrpM4, anti-CD21, anti-CD79a, anti-CD72, anti-MUC16, anti-HER2, anti-CD33, anti-CD22 antibody, anti-CD79b antibody, anti-LIV1 antibody, anti-CD123 antibody, anti-CD74 antibody, anti-BCMA antibody, or anti-FcRH5 antibody.

[0111] In some embodiments, the ADC contains an anti-Trop2 antibody. Trop2 (trophoblast antigen 2) is a transmembrane glycoprotein and an intracellular calcium signaling transducer expressed differentially in various cancers. It signals cells to self-renewal, proliferation, invasion, and survival. Trop 2 is also known as cell surface glycoprotein Trop-2 / Trop2, gastrointestinal tumor-associated antigen GA7331, pancreatic cancer marker protein GA733-1 / GA733, membrane component chromosome 1 surface marker 1 M1S1, epithelial glycoprotein-1, EGP-1, CAA1, droplet corneal dystrophy GDLD, and TTD2. In some embodiments of ADCs containing anti-Trop2 antibodies, the anti-Trop2 antibody is humanized. In one embodiment, the anti-Trop2 antibody is an antibody described in US-2014 / 0377287 or US-2015 / 0366988. In some embodiments, the antibody is a cysteine-engineered variant of the disclosed antibody.

[0112] In some embodiments, the ADC contains an anti-Her2 antibody.Anti-Her2 antibodies are described in more detail in PCT / US1997 / 018385 and US7862817. In some embodiments, the ADC comprises anti-Her2 antibody 7C2, which is described in more detail in PCT / US1997 / 018385, 25 / 90 pages, CN 122295346 A, which is incorporated herein by reference in its entirety. In some embodiments of the ADC comprising anti-Her2 antibodies, the anti-Her2 antibodies are humanized. In some embodiments, the antibody is a cysteine-engineered variant of the disclosed antibody.

[0113] In some embodiments, the ADC comprises anti-CD33 antibody. CD33 is a sialic acid-binding, immunoglobulin-like lectin family member and a 67 kDa glycosylated transmembrane protein. CD33 is expressed on most myeloid and monocytic leukemia cells, except for directed bone marrow mononuclear globulin and erythrocytic precursor cells. It is not found in the earliest pluripotent stem cells, mature granulocytes, lymphoid cells, or non-hematopoietic cells (Sabbath et al., (1985). Clin. Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33 contains two tyrosine residues at its cytoplasmic tail, each followed by a hydrophobic residue, similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors. In some embodiments, the CD33 antibody is an antibody described in PCT / US2014 / 069874, such as the 15G15 antibody described in PCT / US2014 / 069874, which is incorporated herein by reference in its entirety. In some embodiments, the antibody is a cysteine-engineered variant of the disclosed antibody.

[0114] In yet other embodiments, the ADC comprises an anti-CD22 antibody. CD22 (B cell receptor CD22-B isotype, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number AK026467); Wilson et al. (1991) J. Exp. Med. 173:137-146; WO2003072036 (claim 1; Figure 1); Cross-reference: MIM:107266; ​​NP_001762.1; NM_001771_1. In some embodiments, the ADC comprises an anti-CD22 antibody 10F4v3, which is described in more detail in PCT / US2007 / 069889, which is incorporated herein by reference in its entirety.In some embodiments of the ADC containing an anti-CD22 antibody, the anti-CD22 antibody is humanized. In some embodiments, the antibody is a cysteine-engineered variant of the disclosed antibody.

[0115] b. Connector As described herein, compounds of formula (X) or (I) (including compounds of formula (II)) may be used in combination with antibodies to form antibody-drug conjugates (ADCs), such as those of formula (A), (B), or (C); or (D) or (E). In such embodiments, one or more compounds of formula (X) or (I) are covalently linked to the Ab via a connector to form the ADC. In the case of using multiple compounds of formula (X) or (I), each compound may be linked to the antibody via a separate connector.

[0116] A “connector” (L) is a bifunctional or multifunctional part that can be used to connect one or more pharmaceutical parts (D) to an antibody (Ab) to form an ADC, wherein the pharmaceutical part is a compound of formula (X) or (I) (and may also be referred to as a payload or pharmaceutical payload, such as “DP”). In some embodiments, an ADC may be prepared using a connector having a reactive functional group for covalently connecting to a drug and covalently connecting to an antibody. For example, in some embodiments, the cysteine ​​thiol of the Ab may form a bond with a reactive functional group of the connector or drug-connector intermediate to prepare an ADC.

[0117] Connectors used in the methods and compositions provided herein may include cleavable connectors (such as peptides, hydrazones, disulfides, peptide mimics, and glucuronides) and non-cleavable connectors (such as thioethers).

[0118] Methods of connecting a connector to an antibody are well known in the art and include using a reactive functional group, such as NHS esters, isothiocyanates, haloacetamides, mixed disulfides, and maleimides, on the connector. Therefore, the ADCs provided herein include those in which the Ab is covalently linked to the linker via a thio-succinimide, disulfide, ester, amide, or triazole functional group.

[0119] In some embodiments, the linker has a functional group capable of reacting with free cysteine ​​present on the antibody to form a covalent bond. Such reactive functional groups, non-limiting and exemplary, include maleimide, haloacetamide, α-haloacetyl, activated esters such as succinimide, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydride, acyl chloride, sulfonyl chloride, isocyanate, and isothiocyanate. See, for example, Klussman, et al. (2004), Bioconjugate Chemistry 15 (4): 765-773, page 766, for conjugation methods, and examples herein.Therefore, in some embodiments, the Ab includes a reactive cysteine ​​thiol, and the connector forms a bond with the reactive cysteine ​​thiol to attach the payload to the Ab.

[0120] In some embodiments, the connector has a functional group capable of reacting with an electrophilic group present on the antibody. Exemplary electrophilic groups include, but are not limited to, aldehydes and ketone carbonyl groups. In some embodiments, the heteroatoms of the reactive functional group of the connector may react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting examples of such reactive functional groups include, but are not limited to, acylhydrazides, oximes, amino groups, hydrazides, thiohexahydrazones, carboxylic acid hydrazides, and aryl acylhydrazides.

[0121] The connector may comprise one or more connector components. Exemplary connector components include 6-maleiminohexanoyl (“MC”), maleiminopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimino-4-(2-pyridinylthio)valerate (“SPP”), 4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid ester (“MCC”), 1-(5-aminopentyl)-1H-pyrrole-2,5-dione, cyclobutane-1,1-dicarboxaldehyde (“sq”), or cyclobutane-1,1-dicarboxaldehyde-citrulline (“sq-cit”). Various connector components are known in the art, some of which are described below.

[0122] The connector may be a “cuttable connector” that facilitates the release of a drug. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, light-labile linkers, disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US 5208020), and β-glucuronide linkers (e.g., cleavable by β-glucuronidase). Cleavable linkers include peptide linkers that can be hydrolyzed by lysosomal enzymes such as lysosomal cysteine ​​proteases and lysosomal thiol reductases. Such cleavable linkers may include those containing a valine-citrulline (Val-Cit) dipeptide, which can be cleaved by cathepsin B (see, for example, US 6,214,345). Cleavable linkers further include peptide mimic linkers and non-peptide linkers having certain properties of peptides. Cleavable linkers include those containing disulfide bonds and may be referred to as disulfide linkers. Disulfide bonds (which can be alternatively called disulfide bridges) can appear anywhere in the linker, including at the junction of the linker with another component of the ADC, such as the linker to the antibody, the linker to the payload, or the linker to the masking portion, depending on the ADC's construction.Disulfide linkers can be cleaved via reduction, thiol-disulfide exchange, or enzymatic cleavage. Intracellular enzymatic cleavage can occur, for example, by the action of enzymes of the thioredoxin family. Other types of enzymatically cleavable linkers exist, including β-glucuronide linkers, i.e., those linkers having β-glucuronide glycosidic bonds that can be cleaved by the lysosomal enzyme β-glucuronidase.

[0123] In some embodiments, the linkers of the ADCs provided herein are cleavable linkers, wherein the cleavable linkers comprise cleavable peptide bonds, disulfide bonds, or β-glucuronide glycosidic bonds. In some embodiments, the cleavable linkers are peptide mimic linkers.

[0124] In some embodiments, the payload is linked to an antibody via a non-peptide mimic linker that can be cleaved by a lysosomal enzyme. For example, the amide bond in the middle of the dipeptide (e.g., Val-Cit) can be replaced with an amide mimic; and / or the entire amino acid (e.g., valine in the Val-Cit dipeptide) can be replaced by a non-amino acid portion (e.g., a cycloalkyl dicarbonyl structure (e.g., ring size = 4 or 5)).

[0125] In other embodiments, the antibody is linked to the remainder of the ADC via an uncleavable linker. The uncleavable linker includes a linker containing a peptide that is not cleavable by lysosomal proteases. The uncleavable linker also includes a linker that does not contain a peptide and is not cleavable by lysosomal proteases.

[0126] In still some embodiments, the linker includes a functional group covalently linked thereto to the Ab; a spacer assembly; and a functional group covalently linked thereto to the remainder of the ADC (e.g., to the payload, such as a compound of formula (I)). Example: 27 / 90 pages, 37 CN 122295346 A. For example, the connector may be covalently linked to Ab via a thio-succinimide, disulfide, ester, amide, or triazole functional group; includes a spacer assembly; and is then linked to the compound of formula (I) via a thio-succinimide, disulfide, ester, amide, or triazole functional group. In some embodiments, the spacer assembly comprises an alkyl chain or an ether. In some embodiments, the spacer assembly comprises a C1-C10 alkyl group or comprises polyethylene glycol (PEG). In some embodiments, the spacer assembly comprises [-O-CH2CH2-]1-10. Connectors used in the methods and compositions provided herein include, for example, maleimide-PEGn-succinimide, where n is an integer from 1 to 20 (such as 1 to 10 or 3 to 6).

[0127] In some embodiments, the adapter has the following formula X: Formula X where A is a “stretching unit” and a is an integer from 0 to 1; W is an “amino acid unit” and w is an integer from 0 to 12; Y is a “spacer base unit” and y is 0, 1, or 2; and Ab, D, and p are defined above with respect to Formula X. Exemplary embodiments of such adapters are described in U.S. Patent No. 7,498,298, which is expressly incorporated herein by reference.

[0128] In some embodiments, the adapter component includes a “stretching unit” that links an antibody to another adapter component or a portion of a drug. Non-limiting exemplary stretching units are shown below (where wavy lines indicate sites covalently linked to an antibody, drug, or additional adapter component; and in some embodiments, indicate sites of connection to an antibody): MC; MP; mPEG; and in some embodiments, the adapter component includes an “amino acid unit”. In some of these embodiments, the amino acid unit allows protease cleavage of the linker, thereby promoting drug release from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit may contain naturally occurring and / or minor amino acids and / or non-naturally occurring amino acid analogs such as citrulline amino acid residues. The amino acid unit may be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasminase proteases.

[0129] In some embodiments, the linker component includes a "spacer" unit that directly or indirectly links the antibody to the drug moiety via stretching units and / or amino acid units. The spacer unit may be "self-eliminating" or "non-self-eliminating." A "non-self-eliminating" spacer unit is one in which a portion or all of the spacer unit remains bound to the drug moiety after ADC cleavage. Examples of non-self-eliminating spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units.In some embodiments, the ADC containing glycine-glycine spacer units is cleaved by tumor cell-associated proteases, resulting in the release of the glycine-glycine-drug moiety from the remainder of the ADC. In some such embodiments, the glycine-glycine-drug moiety undergoes a hydrolysis step in tumor cells, thereby cleaving the glycine-glycine spacer units from the drug moiety.

[0130] “Self-eliminating” spacer units can release the drug moiety. In some embodiments, the spacer units of the connector contain p-aminobenzyl units. In some of these embodiments, p-aminobenzyl alcohol is linked to the amino acid unit via an amide bond, and a carbamate, methyl carbamate, or carbonate is prepared between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103). In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising a self-consuming linker has the following structure: wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4; p is in the range of 1 to about 20; and D is the drug payload (DP). In some embodiments, p is in the range of 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

[0131] Other examples of self-consuming spacer groups include, but are not limited to, aromatic compounds that are electronically similar to a PAB group, such as 2-aminoimidazolium-5-methanol derivatives (US Patent No. 7,375,078; Hay et al., (1999) Bioorg. Med. Chem. Lett. 9:2237) and o-aminobenzyl acetal or p-aminobenzyl acetal. In some embodiments, spacer groups that undergo cyclization upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclic [2.2.1] and bicyclic [2.2.2] ring systems (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al., (1990) J. Org. Chem. 55:5867). The linking of the drug to the α-carbon of a glycine residue is another example of a self-consuming spacer group that may be useful in ADCs (Kingsbury et al., (1984) J. Med. Chem. 27:1447).

[0132] In some embodiments, the linker is a peptide mimic linker, such as the peptide mimic linker described in WO2015 / 095227 A2, which is incorporated herein by reference in its entirety. In some embodiments, the linker is referred to as the MC-sq-Ala linker or the MC-sq-Cit-PAB linker. In some embodiments and examples provided herein, it is simply described as the “sq-Cit” linker. In case of any confusion or difference between nomenclature and structure, the structure provided herein shall prevail.

[0133] In some embodiments, the connector has the following formula (T): — Str— (PM)— Sp—, (T) Specification 29 / 90 pages 39 CN 122295346 A Wherein Str is a stretching unit covalently linked to Ab; Sp is a spacer unit bonded or covalently linked to the drug portion; PM is a non-peptide chemical portion selected from the group consisting of: , and, W is -NH-heterocyclic alkyl- or heterocyclic alkyl; Y is heteroaryl, aryl, -C(O)C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkyl or –C1-C6 alkyl-NH-; Each R1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; R3 and R2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl, or heteroarylalkyl, or R3 and R2 together can form a C3-C7 cycloalkyl; and R4 and R5 are each independently C1-C10 alkyl, C1-C10 alkenyl, arylalkyl, heteroarylalkyl, (C1-C10 alkyl)OCH2-, or R4 and R5 together can form a C3-C7 cycloalkyl ring.

[0134] In some such embodiments, PM is covalently linked to the Str unit via the left-hand side of the chemical portion as shown in the figure, i.e., via a bond to the carbon of the C(O) portion of PM in the first two cases, or via a bond to the nitrogen of the NHC(O)(CR4R3)- portion of PM in the last case.

[0135] In some embodiments of the ADC comprising the connector of formula (T), PM is, wherein the Str unit is bound to PM via a covalent bond (which is bonded to nitrogen on the left-hand side of the portion shown in the figure). In some embodiments of the connector of formula (T), Sp is a spacer base of the formula, wherein each n is independently an integer from 1 to 6 and indicates the connection point with the PM unit of the connector. In some embodiments of the connector of formula (T), Sp is a spacer base of the formula, wherein n is 0 or 1 and indicates the connection point with the PM unit of the connector.

[0136] In some embodiments of the ADC (such as the ADC of formula (A), (B) or (C)) provided herein, the connector has formula (T-1): Specification 30 / 90 pages 40 CN 122295346 A (T-1), wherein: each R1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; R3 and R2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or R3 and R2 may together form C3-C7 cycloalkyl; and indicate the connection point with Ab; and Sp is a spacer group of the following formula where n is 0 or 1, and indicates the connection point with the rest of the connector.

[0137] Therefore, in some embodiments, an ADC of formula (B-1) is provided herein: or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; X, R1, R2, R3a, Z, ring A and ring B are as defined with respect to formula (X); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl; and Ab is an antibody.

[0138] In some embodiments, the compound of formula (X) is a compound of formula (I), and an ADC of formula (B-1) is provided herein: Specification 31 / 90 pages 41 CN 122295346 A or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; X, R1, R2, R3a, Z, ring A and ring B are as defined for formula (I); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl; and Ab It is an antibody.

[0139] This document further provides an ADC of formula (C-1): or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; X, R1, R2 and R3a are as defined with respect to formula (X); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl; and Ab is an antibody.

[0140] In some embodiments, the compound of formula (X) is a compound of formula (I), and an ADC of formula (C-1) is provided herein: Specification 32 / 90 pages 42 CN 122295346 A or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; X, R1, R2 and R3a are as defined for formula (I); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl; and Ab is an antibody.

[0141] In some embodiments of the ADCs of formulas (B-1) and (C-1), RL1 is a C1-C10 alkylNHC(NH)NH2, and RL3 and RL2 together form a C3-C7 cycloalkyl group. In some embodiments, RL1 is a C1-C10 alkylNHC(NH)NH2, and RL3 and RL2 together form a C4 cycloalkyl group.

[0142] In some embodiments, the linker has the following structure: , wherein the site for covalently linking with the antibody is indicated.

[0143] In some embodiments, the ADC has the following structure: where m is an integer from 1 to 10, and Ab is an antibody as described herein.

[0144] In some embodiments of the various ADCs provided herein, the connector L may be a dendritic connector for covalently linking more than one drug moiety to an antibody via branched, multifunctional connector portions (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic connectors can increase the molar ratio of drug to antibody, i.e., the loading, which is related to the efficacy of the ADC. Therefore, even when the antibody carries only one reactive cysteine ​​thiol group, a large number of drug moieties can be linked via a dendritic connector.

[0145] Non-limiting exemplary adapters are shown in the context of an ADC of formula (A) as provided herein: val-cit; MC-val-cit; MC-val-cit-PAB; MC-sq-Ala; and MC-sq-Cit-PAB.

[0146] Other non-limiting exemplary ADCs include the following structures, wherein -S- is a portion of an antibody: , , , , , where X is: ; Y is: ; each R is independently H or C1-C6 alkyl; n is 1 to 12; and D is the drug payload (DP) of formula (A).

[0147] In some embodiments, the adapter is substituted with groups that modulate solubility and / or reactivity. As a non-limiting example, charged substituents such as sulfonate (-SO3-) or ammonium can increase the water solubility of the linker reagent and promote the coupling reaction of the linker reagent with the antibody and / or drug moiety, or promote the coupling reaction of Ab-L (antibody-linker intermediate) with DP or DP-L (drug payload-linker intermediate) with Ab, depending on the synthetic route used to prepare the ADC. In some embodiments, a portion of the linker is coupled with the antibody and a portion of the linker is coupled with the drug, and then Ab- (linker moiety) a is coupled with the drug payload- (linker moiety) b to form an ADC of formula (A). In some such embodiments, the antibody contains more than one (linker moiety) a substituent, such that more than one drug is coupled with the antibody in the ADC of formula (A). In other embodiments, the linker is first coupled with the drug payload, and then the drug payload-linker (DP-L) is coupled with Ab. In some embodiments, multiple DP-Ls are coupled with Ab.

[0148] In some embodiments, a pharmaceutical payload-connector conjugate of formula (B-L1) is provided: Specification 35 / 90 pages 45 CN 122295346 A (B-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; X, R1, R2, R3a, Z, ring A and ring B are as defined for formula (X); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; and RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl.

[0149] In some embodiments, a pharmaceutical payload-connector conjugate of formula (B-L1) is also provided: (B-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; X, R1, R2, R3a, Z, ring A and ring B are as defined with respect to formula (I); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; and RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form a C3-C7 cycloalkyl group.

[0150] This document further provides a pharmaceutical payload-connector conjugate of formula (C-L1): (C-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; X, R1, R2 and R3a are as defined with respect to formula (X); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; and RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form a C3-C7 cycloalkyl group.

[0151] This document also provides a pharmaceutical payload-connector conjugate of formula (C-L1): Specification 36 / 90 pages 46 CN 122295346 A (C-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; X, R1, R2 and R3a are as defined with respect to formula (I); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; and RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl.

[0152] In some embodiments of the drug payload-connector conjugates of formula (B-L1) and (C-L1), X is 0. In still other embodiments of the drug payload-connector conjugates of formula (B-L1) and (C-L1), X is 0, RL1 is a C1-C10 alkylNHC(NH)NH2, and RL3 and RL2 together form a C3-C7 cycloalkyl group. In some embodiments, RL1 is a C1-C10 alkylNHC(NH)NH2, and RL3 and RL2 together form a C4 cycloalkyl group.

[0153] The following drug payload-connector conjugates with the following structures are provided herein:

[0154] The compounds of the present invention are specifically envisioned, but not limited to, ADCs prepared using the following linker reagents: bis-maleimino-trioxyethylene glycol (BMPEO), N-(β-maleiminopropyloxy)-N-hydroxysuccinimide (BMPS), N-(ε-maleiminohexanoyloxy)succinimide (EMCS), N-[γ-maleiminobutyryloxy]succinimide (GMBS), 1,6-hexane-bis-vinyl sulfone (HBVS), 4-(N-maleiminomethyl)cyclohexane-1-carboxyl-(6-acylaminohexanoic acid)succinimide (LC-SMCC), m-maleiminobenzoyl-N- Hydroxysuccinimide (MBS), hydrazine 4-(4-N-maleiminophenyl)butyrate (MPBH), succinimide 3-(bromoacetamido)propionate (SBAP), iodoacetic acid succinimide (SIA), (4-iodoacetyl)aminobenzoic acid succinimide (SIAB), N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridylthio)valerate (SPP), 4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), 4-(p-maleiminophenyl)butyrate succinimide (SMPB), 6-[(β-maleiminopropionylamino)hexanoic acid]succinimide (SMPH), iminothiacyclopentane (IT), sulfonyl-EMCS, sulfonyl-GMBS, sulfonyl- KMUS, sulfonyl-MBS, sulfonyl-SIAB, sulfonyl-SMCC, and sulfonyl-SMPB, and succinimino-(4-vinyl sulfone)benzoate (SVSB), and including bis-maleimide reagents: dithiobismaleiminoethane (DTME), 1,4-bismaleiminobutane (BMB), 1,4-bismaleimino-2,3-dihydroxybutane (BMDB), bismaleiminohexane (BMH), bismaleiminoethane (BMOE), BM(PEG)2 (as shown below), and BM(PEG)3 (as shown below); bifunctional derivatives of imide esters (such as dimethyl diimino-adipate HCl), active esters (such as octanoic acid, 37 / 90 pages, 47 CN 122295346 A) Disuccinimidyl esters, aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazo derivatives (such as bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).In some embodiments, the bis-maleimide reagent allows the thiol group of a cysteine ​​residue in an antibody to be linked to a thiol-containing pharmaceutical moiety, a linker, or a linker-pharmaceutical intermediate. Other functional groups that react with the thiol group include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

[0155] Certain useful adapter reagents are available from a variety of commercial sources, such as Pierce Biotechnology, Inc. (Rockford, IL), Molecular Biosciences Inc. (Boulder, CO), or synthesized according to procedures described in the art, for example, Toki et al., (2002) J. Org. Chem. 67:1866-1872; Dubowchik et al., (1997) Tetrahedron Letters, 38:5257-60; Walker, M.A. (1995) J. Org. Chem. 60:5352-5355; Frisch et al., (1996) Bioconjugate Chem. 7:180-186; US 6214345; WO 02 / 088172; US As described in 2003130189; US2003096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0156] c. Drug Loading Capacity of ADC The drug loading capacity is the average number of drug moieties per antibody. ADCs provided herein (such as those of formula (A), (B), (B-1), (C), (C-1), (D), or (E)) may include a drug loading capacity in the range of 1 to 10 drug molecules (D) per antibody (Ab). Thus, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug moieties may be covalently linked to an antibody, wherein the drug moieties are drug loadings of formula (I) (including compounds of formula (II)). Compositions of ADCs may include antibodies conjugated to a series of drugs (1 to 10) to form compositions having a series of drug loadings. In ADC compositions (including those pharmaceutical compositions such as those described herein), the average drug load per antibody is commonly referred to as the drug / antibody ratio or "DAR". In ADC preparation from a conjugation reaction, the average amount of drug per antibody can be characterized using conventional methods such as mass spectrometry (including LCMS), ELISA, electrophoresis, or HPLC. The quantitative distribution of the ADC can also be determined with respect to m.In some cases, homogeneous ADCs, where m is a certain value, can be separated, purified, and characterized from ADCs with other drug loadings by methods such as reversed-phase HPLC, electrophoresis, or LC-MS.

[0157] For some antibody-drug conjugates, the number of drug moieties per antibody, "m", may be limited by the number of linker sites on the antibody. For example, the antibody may have only one or a few cysteine ​​thiols, or only one or a few thiol groups with sufficiently high reactivity that can be linked to a linker. Higher drug loadings, such as m > 5, may result in aggregation, insolubility, toxicity, or loss of cell permeability in some antibody-drug conjugates.

[0158] Typically, less than the theoretical maximum drug moieties are conjugated to the antibody during the conjugation reaction. The antibody may contain, for example, many lysine residues that do not react with linker-drug intermediates or linker reagents. Additionally, in some embodiments, only the most reactive cysteine ​​thiols can react with thiol-reactive linker reagents or linker-drug intermediates. Generally, antibodies do not contain numerous (if any) free reactive cysteine ​​thiols that can be linked to the drug moiety. Most cysteine ​​thiols in an antibody compound are present as disulfide bonds and must be reduced with a reducing agent (such as dithiothreitol (DTT) or TCEP) under partial or complete reduction conditions. Specification 38 / 90 pages 48 CN 122295346 A

[0159] The DAR (drug loading as drug / antibody ratio) of an ADC can be controlled in several different ways, including: (i) limiting the molar excess of the linker-drug intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partially or limiting the reduction conditions for cysteine ​​thiols modification. When more than one nucleophilic or electrophilic group of an antibody reacts with a linker-drug intermediate or linker reagent, and subsequently with a drug moiety reagent, the resulting product can be a mixture of ADC molecules in which drug moieties linked to the antibody are distributed, for example, 1, 2, 3, etc. Therefore, the DAR of an ADC composition may not be an integer, but rather an average of the existing molecules in the composition. Liquid chromatography methods (such as polymeric reversed-phase (PLRP) and hydrophobic interaction (HIC)) can separate compounds in a mixture by drug loading value. Formulations of ADCs with a single drug loading value (m) can be separated; however, these single-loading-value ADCs may still be heterogeneous mixtures because the drug moieties may be linked to different sites on the antibody via linkers. Therefore, the antibody-drug conjugate compositions of the present invention comprise mixtures of antibody-drug conjugate compounds, wherein the antibody has one or more drug moieties and wherein the drug moieties may be linked to the antibody at various amino acid residues.

[0160] 3. Methods for Preparing the Compounds The compounds disclosed herein can be prepared by methods known in the field of organic synthesis, which are described in part by the following synthetic schemes. In the schemes described herein, it should be understood that, in accordance with general principles or chemical methods, protecting groups are employed where necessary for sensitive or reactive groups. The operation of protecting groups is carried out according to standard methods of organic synthesis (TW Greene and PGM Wuts, “Protective Groups in Organic Synthesis”, 3rd edition, Wiley, New York, 1999). At convenient stages in the synthesis of the compounds, these groups are removed using methods obvious to those skilled in the art. The selection of processes and reaction conditions and the order in which they are performed should be consistent with the preparation of the compounds disclosed herein. The compounds described herein can be prepared from commercially available starting materials or synthesized using known organic, inorganic and / or enzymatic methods.

[0161] Those skilled in the art will recognize the presence or absence of stereocenters in the compounds disclosed herein. In some embodiments, the compounds disclosed herein may exist in the form of enantiomeric or diastereomeric forms. Therefore, this disclosure includes two possible stereoisomers (unless otherwise stated in the synthesis) and includes not only racemic compounds but also individual enantiomers and / or diastereomers. When the desired compound is a single enantiomer or diastereomer, it can be obtained by stereooriented synthesis or by resolving the final product or any convenient intermediate. For example, the enantiomerically pure compounds of this disclosure can be prepared using enantiomerically pure chiral structural units. Alternatively, a racemic mixture of the final compound or a racemic mixture of advanced intermediates can be subjected to the chiral purification described herein to provide the desired enantiomerically pure intermediate or final compound. In the case where an advanced intermediate is purified to its individual enantiomers, each individual enantiomer can continue to participate in the reaction individually to provide the final enantiomerically pure compound of this disclosure. The resolution of the final product, intermediate, or starting material can be carried out by any suitable method known in the art. See, for example, “Stereochemistry of Organic Compounds” (E. L. Eliel, S. H. Wilen, and L. N. Mander, Wiley Interscience, 1994).

[0162] 4. Pharmaceutical Composition This document provides a pharmaceutical composition comprising an ADC of formula (A) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an ADC of formula (B) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.In some embodiments, the pharmaceutical composition comprises an ADC of formula (B-1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an ADC of formula (C) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In still other embodiments, the pharmaceutical composition comprises an ADC of formula (C-1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In still other embodiments, the ADC has formula (D) or (E).

[0163] Pharmaceutical compositions of ADCs are generally prepared for parenteral administration, such as by bolus, intravenous, or intratumoral injection. Such pharmaceutical compositions may be prepared in an aqueous solution or prepared in a lyophilized form for reconstitution into an aqueous solution for administration. In some embodiments, the pharmaceutical composition is a liquid for intravenous administration. Further information regarding the preparation of pharmaceutical compositions (including those for parenteral administration, aqueous solutions, or as lyophilized forms for reconstitution into aqueous solutions for administration) can be found, for example, in Remington's Pharmaceutical Sciences (2020), 23rd edition, edited by Adejare, A. Such pharmaceutically acceptable excipients may include, for example, buffers; pyrogen-free water; isotonic saline; Ringer's solution; and phosphate buffer solutions. Preservatives and antioxidants may also be present in the pharmaceutical composition at the discretion of the formulator.

[0164] 5. Methods of Use Methods of treating a condition in a subject of need are provided herein, comprising administering to the subject a therapeutically effective amount of an ADC as described herein. Further methods of treating a condition in a subject of need are provided, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an ADC as described herein and pharmaceutically acceptable excipients. The ADC of the methods described herein (including administration of an ADC or administration of a pharmaceutical composition comprising an ADC and a pharmaceutically acceptable excipient) may be an ADC of formula (A), (B), (B-1), (C), or (C-1). For example, in some embodiments of the methods described herein, the ADC has formula (B) or formula (B-1). In other embodiments, the ADC has formula (C) or formula (C-1). In still other embodiments, the ADC has formula (D) or (E). In some embodiments, the subject is a human.

[0165] A compound for treating a condition in a subject in need is also provided, wherein the compound is an ADC as described herein (e.g., an ADC of formula (A), (B), (B-1), (C), (C-1), (D), or (E)). Further, use of the compound as described herein in treating a condition in a subject in need is provided, wherein the compound is an ADC as described herein (e.g., an ADC of formula (A), (B), (B-1), (C), (C-1), (D), or (E)). Also provided is use of the compound as described herein in manufacturing a medicament for treating a condition in a subject in need, wherein the compound is an ADC as described herein (e.g., an ADC of formula (A), (B), (B-1), (C), (C-1), (D), or (E)). In some embodiments, the subject is a human.

[0166] In some embodiments, the condition is cancer, a tumor, or other malignancy. As used herein, cancer, tumor, and malignancy refer to a condition or cells or tissue associated with a condition characterized by abnormal or abnormal cell proliferation, differentiation, and / or migration, and often accompanied by abnormal or abnormal molecular phenotypes, including one or more gene mutations or other genetic changes associated with tumorigenesis, expression of tumor markers, loss of expression or activity of tumor suppressors, and / or abnormal or abnormal expression of cell surface markers. In some embodiments, the condition is selected from benign or malignant solid tumors and hematologic disorders such as leukemia and lymphomas. In some embodiments, cancer, tumor, and malignancy may include, but are not limited to, sarcomas, lymphomas, leukemias, solid tumors, germ cell tumors, gliomas, carcinomas, melanomas, and metastatic cancers.

[0167] More generally, this document provides a method of treating a subject with a hyperproliferative disease (such as cancer) in need, the method comprising administering to the subject a therapeutically effective amount of an ADC as provided herein. In some embodiments, the ADC has formula (A), such as having formula (B), formula (B-1), formula (C), formula (C-1), formula (D), or formula (E). Examples of cancers treated in this article include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.More specific examples of this type of cancer include: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML) or myeloid cell leukemia (MCL). Also provided are compounds for treating conditions, uses of compounds for treating conditions, and uses of compounds as described herein for manufacturing medicaments for treating conditions, wherein the compound is an ADC as described herein (e.g., an ADC of formula (A), (B), (B-1), (C), (C-1), (D), or (E)), and the condition is as described herein. In some embodiments, the subject is a human.

[0168] In some embodiments, a method of using an ADC as described herein, comprising an anti-CD33 antibody, for treating hematologic malignancies such as non-Hodgkin lymphoma (NHL), diffuse macrohematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), or myeloid cell leukemia (MCL), including B-cell-related cancers and proliferative conditions. In some embodiments, a method of using an ADC as described herein, comprising an anti-HER2 antibody, for treating HER2+ cancers such as HER2+ breast cancer or gastric cancer.

[0169] For the prevention or treatment of a disease, the appropriate dose of the ADC will depend on the type of disease to be treated, the severity and duration of the disease, the administration of the molecule for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The molecule is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, an initial candidate dose of about 1 µg / kg to 15 mg / kg (e.g., 0.1–20 mg / kg) of the molecule is given to the patient, for example, by a single or multiple separate administrations or by continuous infusion. Depending on the factors described above, a typical daily dose may range from about 1 μg / kg to 100 mg / kg or more.

[0170] Examples Example 1. A compound having formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; Ring A is phenyl or 6-membered heteroaryl; Ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; Specification 41 / 90 pages 51 CN 122295346 A Each R2c is independently hydrogen, alkyl or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, –NHR3a, –N(R3a)2 or –N+(R3a)3, wherein each R3a is independently alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen and –OH.

[0171] Example 2. The compound according to Example 1, or a pharmaceutically acceptable salt thereof, wherein ring A and ring B are independently phenyl or heteroaryl containing one or two ring N atoms.

[0172] Example 3. The compound according to Example 1, or a pharmaceutically acceptable salt thereof, wherein ring A is pyrimidine and ring B is phenyl, pyrimidine or, wherein the connection point with ring A is indicated.

[0173] Example 4. A compound according to any one of Examples 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R1 is a fluoro-substituted phenyl group.

[0174] Example 5. A compound according to any one of Examples 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or C1-C6 alkyl group substituted with one or more substituents independently selected from the group consisting of –OH and a halogen.

[0175] Example 6. A compound according to any one of Examples 1 to 5, or a pharmaceutically acceptable salt thereof, wherein Z is N-CH3 or N-CH2CH3.

[0176] Example 7. A compound according to any one of Examples 1 to 6, or a pharmaceutically acceptable salt thereof, wherein R3 is –NH2 or –NHCH3.

[0177] Example 8. The compound according to any one of Examples 1 to 7, or a pharmaceutically acceptable salt thereof, wherein X is O.

[0178] Example 9. The compound according to Example 1, or a pharmaceutically acceptable salt thereof, wherein: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated; R1 is a fluorinated phenyl; R2 is an unsubstituted or fluorinated C1-C6 alkyl; R3 is –NH2 or –NH (C1-6 alkyl); and X is O.

[0179] Example 10. The compound according to Example 1, or a pharmaceutically acceptable salt thereof, wherein X is O, R1 is a fluorinated phenyl, R2 is an alkyl, and R3 is –NH2.

[0180] Example 11. The compound according to Example 1, wherein the compound has formula (II): (See page 42 / 90 of specification, CN 122295346 A (II)), or a pharmaceutically acceptable salt thereof, wherein: X, R1, R2 and R3 are as defined with respect to formula (I).

[0181] Example 12. The compound according to Example 11, or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or fluorinated C1-C6 alkyl; and R3 is –NH2 or –NH (C1-6 alkyl).

[0182] Example 13. The compound according to Example 1, or a pharmaceutically acceptable salt thereof, wherein the compound is: , , , , , , , or, or a pharmaceutically acceptable salt thereof.

[0183] Example 13a. The compound according to Example 1, or a pharmaceutically acceptable salt thereof, wherein the compound is: (See page 43 / 90 of the specification, CN 122295346 A, , , , , , , , , , , , , or, or a pharmaceutically acceptable salt thereof.)

[0184] Example 14. A conjugate of formula (A): Ab-(L-(DP)r)m, or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody; L is a linker; A DP is a drug payload; r is an integer from 1 to 8; and m is an integer from 1 to 10; wherein the drug payload is a compound of formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or a 6-membered heteroaryl; ring B is phenyl or a 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, -NHR3a, -N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen and –OH.

[0185] Example 15. The conjugate according to Example 14, wherein the conjugate of formula (A) is the conjugate of formula (B): (B), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; and X, R1, R2, R3a, Z, ring A and ring B are as defined for formula (I).

[0186] Example 16. The conjugate according to Example 14 or 15, or a pharmaceutically acceptable salt thereof, wherein the linker is a peptide linker or a peptide mimic linker.

[0187] Example 17. A conjugate according to any one of Examples 14 to 16, wherein the conjugate has the formula (B-1): R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; X, R1, R2, R3a, Z, ring A and ring B are as defined with respect to formula (I); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; and RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form a C3-C7 cycloalkyl.

[0188] Example 18. A conjugate according to any one of Examples 14 to 17, wherein: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated; R1 is a fluoro-substituted phenyl; R2 is an unsubstituted or fluoro-substituted C1-C6 alkyl; R3 is –NH2 or –NH (C1-6 alkyl); and X is O.

[0189] Example 19. The conjugate according to Example 15, wherein the conjugate of formula (A) is the conjugate of formula (C): (C) , CN 122295346 A (C), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH, –NR3a or –N+(R3a)2; m is an integer from 1 to 10; and X, R1, R2 and R3a are as defined with respect to formula (I).

[0190] Example 20. The conjugate according to Example 19, or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or fluorinated C1-C6 alkyl; and R3 is –NH2 or –NH(C1-6 alkyl).

[0191] Example 21. The conjugate according to any one of Examples 14 to 17, or a pharmaceutically acceptable salt thereof, wherein the drug loading is: , , , , , , , or.

[0192] Example 21a. The conjugate or a pharmaceutically acceptable salt thereof according to any one of Examples 14 to 17, wherein the drug payload is: (Instructions for Use, Page 47 / 90, 57 CN 122295346 A, , , , , , , , , , , , or).

[0193] Example 22. The conjugate according to Example 14, wherein the conjugate has the following structure: Specification 48 / 90 pages 58 CN 122295346 A.

[0194] Example 23. The conjugate according to any one of Examples 14 to 22, wherein the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from the group consisting of: CLL1, CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, STEAP1, STEAP2, TrpM4, CD21, CD79a, CD72, MUC16, HER2, CD33, CD22, CD79b, LIV1, CD123, CD74, BCMA, and FcRH5.

[0195] Example 24. A pharmaceutical composition comprising the conjugate according to any one of Examples 14 to 23, and a pharmaceutically acceptable excipient.

[0196] Example 25. A method of treating a condition in a subject in need, comprising administering to the subject a therapeutically effective amount of the conjugate according to any one of Examples 14 to 23.

[0197] Example 26. The method according to Example 25, wherein the condition is cancer, tumor, or other malignancy.

[0198] Example 27. The method according to Example 26, wherein the disease is selected from the group consisting of: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), and myeloid cell leukemia (MCL).

[0199] Example 28. A compound used in a method of treating a condition in a subject in need, wherein the compound is a conjugate according to any one of Examples 14 to 23.

[0200] Example 29. The compound used according to Example 28, wherein the condition is cancer, tumor or other malignant tumor.

[0201] Example 30. The compound used according to Example 29, wherein the disease is selected from the group consisting of: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), and myeloid cell leukemia (MCL).

[0202] Example 31. A drug payload-connector conjugate, wherein the drug payload-connector conjugate is of formula (B-L1): Specification 49 / 90 pages 59 CN 122295346 A (B-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; wherein each R3a is independently alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen and –OH; each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl, or heteroarylalkyl, or RL3 and RL2 together may form C3-C7 cycloalkyl; X is O or NH; Z is N-C1-6 alkyl or NH; Ring A is phenyl or 6-membered heteroaryl; Ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; Each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl or haloalkyl; and p and q are independently integers from 1 to 8.

[0203] Example 32. The drug payload-connector conjugate according to Example 31, wherein the drug payload-connector intermediate is of formula (C-L1): (C-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl; X is O or NH; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted by 1 to 3 R2 is an independently selected halogen substituted compound; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; and p and q are independently integers from 1 to 8.

[0204] Example 33. The drug payload-connector conjugate according to Example 31, wherein the drug payload-connector intermediate has the following structure:

[0205] Example 34. A compound of formula (X): (X), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or 6-membered heteroaryl; ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; Each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; Specification 51 / 90 pages 61 CN 122295346 A R3 is –NH2, –NHR 3a, –N(R3a)2 or –N+(R3a)3, wherein each R3a is independently alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, –OH, alkoxy and haloalkoxy.

[0206] Example 35. The compound of claim 34, wherein the compound of formula (X) is the compound of formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or 6-membered heteroaryl; ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R2a and each R2b is independently alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen and –OH.

[0207] Example 36. The compound according to Example 34 or 35, or a pharmaceutically acceptable salt thereof, wherein ring A and ring B are independently phenyl or heteroaryl containing one or two ring N atoms.

[0208] Example 37. The compound according to Example 34 or 35, or a pharmaceutically acceptable salt thereof, wherein ring A is pyrimidine and ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated.

[0209] Example 38. A compound according to any one of Examples 34 to 37, or a pharmaceutically acceptable salt thereof, wherein R1 is a fluoro-substituted phenyl group.

[0210] Example 39. A compound according to any one of Examples 34 to 38, or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or substituted C1-C6 alkyl group: –OH and a halogen, substituted with one or more substituents independently selected from the group consisting of 1, 2, 3, 4, 52 / 90 pages, 62 CN 122295346 A

[0211] Example 40. A compound according to any one of Examples 34 to 39, or a pharmaceutically acceptable salt thereof, wherein Z is N-CH3 or N-CH2CH3.

[0212] Example 41. A compound according to any one of Examples 34 to 40, or a pharmaceutically acceptable salt thereof, wherein R3 is –NH2 or –NHCH3.

[0213] Example 42. A compound according to any one of Examples 34 to 41, or a pharmaceutically acceptable salt thereof, wherein X is O.

[0214] Example 43. A compound according to Example 34 or 35, or a pharmaceutically acceptable salt thereof, wherein: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated; R1 is a fluorinated phenyl; R2 is an unsubstituted or fluorinated C1-C6 alkyl; R3 is –NH2 or –NH(C1-6 alkyl); and X is O.

[0215] Example 44. The compound of Example 34 or 35, or a pharmaceutically acceptable salt thereof, wherein X is O, R1 is a fluorinated phenyl, R2 is an alkyl, and R3 is –NH2.

[0216] Example 45. The compound of Example 34 or 35, wherein the compound has formula (II): (II), or a pharmaceutically acceptable salt thereof, wherein: X, R1, R2 and R3 are as defined with respect to formula (I).

[0217] Example 46. The compound of Example 45, or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or fluorinated C1-C6 alkyl; and R3 is –NH2 or –NH (C1-6 alkyl).

[0218] Example 47. The compound according to Example 34, or a pharmaceutically acceptable salt thereof, wherein the compound is: , , , , , , , , , , , , , , , or a pharmaceutically acceptable salt thereof. (See page 53 / 90 of the specification, CN 122295346 A, ...

[0219] Example 48. A conjugate of formula (A): Ab-(L-(DP)r)m, or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody; L is a linker; DP is a drug payload; r is an integer from 1 to 8; and m is an integer from 1 to 10; wherein the drug payload is a compound of formula (X): Specification 54 / 90 pages 64 CN 122295346 A (I), or a pharmaceutically acceptable salt thereof, wherein: X is O or NH; Z is N-C1-6 alkyl or NH; ring A is phenyl or 6-membered heteroaryl; ring B is phenyl or 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl, or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl, or heterocycloalkyl is unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b, and -O(R2bO)qR2c, or the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; p and q are independently integers from 1 to 8; and R3 is –NH2, –NHR3a, –N(R3a)2, or –N+(R3a)3, wherein each R3a is independently an alkyl, wherein each alkyl is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, –OH, alkoxy, and haloalkoxy.

[0220] Example 49. The conjugate according to Example 48, wherein the conjugate of formula (A) is the conjugate of formula (B): (B), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; and X, R1, R2, R3a, Z, ring A and ring B are as defined for formula (X).

[0221] Example 50. The conjugate according to Example 48 or 49, or a pharmaceutically acceptable salt thereof, wherein the linker is a peptide linker or a peptide mimic linker.

[0222] Example 51. A conjugate according to any one of Examples 48 to 50, wherein the conjugate has the formula (B-1): R3 is –NH–, –NR3a– or –N+(R3a)2–; m is an integer from 1 to 10; X, R1, R2, R3a, Z, ring A and ring B are as defined with respect to formula (X); each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; and RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form a C3-C7 cycloalkyl.

[0223] Example 52. A conjugate according to any one of Examples 48 to 51, wherein: Z is N-CH2CH3 or N-CH3; ring A is pyrimidine; ring B is phenyl, pyrimidine, or, wherein the connection point with ring A is indicated; R1 is a fluoro-substituted phenyl; R2 is an unsubstituted or fluoro-substituted C1-C6 alkyl; R3 is –NH2 or –NH (C1-6 alkyl); and X is O.

[0224] Example 53. The conjugate according to Example 48, wherein the conjugate of formula (A) is a conjugate of formula (C): (C), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH, –NR3a or –N+(R3a)2; m is an integer from 1 to 10; and X, R1, R2 and R3a are as defined with respect to formula (X).

[0225] Example 54. The conjugate according to Example 53, or a pharmaceutically acceptable salt thereof, wherein R2 is an unsubstituted or fluorinated C1-C6 alkyl; and R3 is –NH2 or –NH(C1-6 alkyl).

[0226] Example 55. The conjugate according to any one of Examples 48 to 54, or a pharmaceutically acceptable salt thereof, wherein the drug payload is: , ...

[0228] Example 57. A conjugate according to any one of Examples 48 to 56, wherein the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from the group consisting of: CLL1, CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, STEAP1, STEAP2, TrpM4, CD21, CD79a, CD72, MUC16, HER2, CD33, CD22, CD79b, LIV1, CD123, CD74, BCMA, and FcRH5.

[0229] Example 58. A pharmaceutical composition comprising a conjugate according to any one of Examples 48 to 57, and a pharmaceutically acceptable excipient.

[0230] Example 59. A method of treating a condition in a subject in need, comprising administering to the subject a therapeutically effective amount of a conjugate according to any one of Examples 48 to 57.

[0231] Example 60. The method according to Example 59, wherein the disease is cancer, tumor or other malignant tumor.

[0232] Example 61. The method according to Example 59, wherein the disease is selected from the group consisting of: squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML) and myeloid cell leukemia (MCL).

[0233] Example 62. A compound used in a method of treating a condition in a subject in need, wherein the compound is a conjugate according to any one of Examples 48 to 57.

[0234] Example 63. The compound used according to Example 62, wherein the condition is cancer, tumor or other malignancy.

[0235] Example 64. The compound used according to Example 62, wherein the disease is selected from the group consisting of: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML) and myeloid cell leukemia (MCL).

[0236] Example 65. A drug payload-connector conjugate, wherein the drug payload-connector conjugate is of formula (B-L1): (B-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, –OH, alkoxy and haloalkoxy; each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form a C3-C7 cycloalkyl group; X is O or NH; Z R1 is an N-C1-6 alkyl or NH; ring A is phenyl or a 6-membered heteroaryl; ring B is phenyl or a 6-membered heteroaryl; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c, or two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; each R2a and each R2b is independently an alkyl or haloalkyl; each R2c is independently hydrogen, alkyl or haloalkyl; and p and q are independently integers from 1 to 8.

[0237] Example 66. The drug payload-connector conjugate according to Example 65, wherein the drug payload-connector intermediate is of formula (C-L1): (C-L1), or a pharmaceutically acceptable salt thereof, wherein: R3 is –NH–, –NR3a– or –N+(R3a)2–; wherein each R3a is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, –OH, alkoxy group and haloalkoxy group.

[0238] Each RL1 is independently C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkylNHC(NH)NH2 or C1-C10 alkylNHC(O)NH2; RL3 and RL2 are each independently H, C1-C10 alkyl, C1-C10 alkenyl, arylalkyl or heteroarylalkyl, or RL3 and RL2 may together form C3-C7 cycloalkyl; X is O or NH; R1 is an alkyne or phenyl, wherein the phenyl is unsubstituted or substituted with 1 to 3 independently selected halogens; R2 is an alkyl, cycloalkyl, heterocycloalkyl or -R2a(OR2a)p-; wherein the alkyl, cycloalkyl or heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R2bO)qR2c Or, the two substituents together with the atoms to which they are attached form a cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl; each R2a and each R2b are independently alkyl or haloalkyl; each R2c is independently hydrogen, alkyl, or haloalkyl; and p and q are independently integers from 1 to 8.

[0239] Example 67. The drug payload-connector conjugate according to Example 65, wherein the drug payload-connector intermediate has the following structure:

[0240] Examples The abbreviations used in the following examples may include: Specification 60 / 90 pages 70 CN 122295346 A Synthesis Example 1: Synthesis of Compound 100 Specification 61 / 90 pages 71 CN 122295346 A Step 1: Preparation of Intermediate 1b At 0°C, isobutyl chloroformate (9.0 mL, 69.4 mmol) was slowly added to a solution of 2-chloropyrimidine-4-carboxylic acid (10 g, 63.1 mmol) and triethylamine (9.7 mL, 69.4 mmol) in tetrahydrofuran (120 mL). The mixture was stirred at 0°C for 1 h. The reaction mixture was filtered, and the filtrate was used directly in the next step.

[0241] Step 2: Preparation of intermediate 1c Sodium borohydride (4.7 g, 124.8 mmol) was added dropwise to a solution of 2-chloropyrimidin-4-carboxylic acid (isobutyl carbonate) anhydride (16.3 g, 63.0 mmol) in tetrahydrofuran (200 mL) and water (20 mL) at 0 °C. The mixture was then stirred at 0 °C for 1 hour. The reaction mixture was quenched with NH4Cl aqueous solution (20 mL) and diluted with water (100 mL), extracted with ethyl acetate (100 mL x 2), washed with brine (50 mL x 2), dried over Na2SO4, filtered, and concentrated to the residue. The residue was purified by silica gel chromatography (solvent gradient: 0% to 50% ethyl acetate in petroleum ether) to give a yellow solid (2-chloropyrimidin-4-yl)methanol (3 g, yield 33%).

[0242] Step 3: Preparation of Intermediate 1d (Instructions for use, pages 62 / 90, CN 122295346 A) Pd(dppf)Cl2 (2.3 g, 3.1 mmol) was added to a solution of (2-chloropyrimidin-4-yl)methanol (4.5 g, 31.1 mmol), pinacol ester of 2-fluoropyridine-5-boronate (8.3 g, 37.4 mmol), and K2CO3 (12.9 g, 93.4 mmol) in dimethyl ether (120 mL) and water (12 mL). The reaction mixture was degassed three times with N2. The resulting mixture was stirred at 100 °C for 12 hours. The mixture was filtered and concentrated to the residue, which was then purified by silica gel chromatography (solvent gradient: 0% to 50% ethyl acetate in petroleum ether) to give a white solid (2-(6-fluoropyridin-3-yl)pyrimidin-4-yl)methanol (4.7 g, 74% yield). 1H NMR (400MHz, DMSO‑d6) δ 9.15 (d, J = 2.4 Hz, 1H) , 8.92 (d, J = 5.2 Hz, 1H) , 8.85‑8.80 (m, 1H) , 7.56 (d, J = 5.2 Hz, 1H) , 7.36‑ 7.33 (m, 1H), 5.73 (t, J = 6.0 Hz, 1H), 4.67 - 4.63 (m, 2H).

[0243] Step 4: Preparation of intermediate 1e: At 25°C, N,N-diisopropylethylamine (12.7 mL, 73.1 mmol) was added to a solution of [2-(6-fluoro-3-pyridyl)pyrimidin-4-yl]methanol (3.0 g, 14.6 mmol) and tert-butyl-N-(2-aminoethyl)carbamate (6.9 mL, 43.8 mmol) in DMSO (70 mL). The mixture was heated to 110°C and stirred for 16 hours. The reaction mixture was diluted with water (30 mL), extracted with ethyl acetate (50 mL x 2), washed with brine (50 mL x 2), dried over Na2SO4, filtered, and concentrated to the residue. The residue was purified by silica gel chromatography (solvent gradient: 0% to 5% methanol in dichloromethane) to give a yellow solid (4.3 g, 85% yield) of tert-butyl 2-(2-(5-(4-(hydroxymethyl)pyrimidin-2-yl)pyridin-2-yl)amino)ethyl)carbamate. 1H NMR (400 MHz, DMSO‑d6): δ 8.98 (d, J = 2.0 Hz, 1H), 8.74 (d, J = 5.2 Hz, 1H), 8.26 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 5.2 Hz, 1H), 7.09 (t, J = 5.2 Hz, 1H) , 6.89 (t, J = 5.2 Hz, 1H) , 6.54 (d, J = 8.8 Hz, 1H) , 5.60 (t, J = 6.0 Hz, 1H) , 4.57 (d, J = 6.0 Hz, 2H) , 3.44 (d, J = 7.2 Hz, 2H), 3.11 (d, J = 6.4 Hz, 2H), 1.38 (s, 9H).

[0244] Step 5: Preparation of intermediate 1f TsCl (1.1 g, 5.8 mmol) was added to a solution of N-[2-[[5-[4-(hydroxymethyl)pyrimidin-2-yl]-2-pyridyl]amino]ethyl]carbamate tert-butyl ester (1 g, 2.9 mmol) and N,N-diisopropylethylamine (2.02 mL, 11.6 mmol) in dichloromethane (10 mL), and the mixture was stirred at 25 °C for 16 hours.The mixture was concentrated under vacuum and purified by silica gel chromatography (solvent gradient: 0% to 2% methanol in dichloromethane) to give a white solid methyl 4-methylbenzenesulfonic acid (2-(6-((2-((tert-butoxycarbonyl)amino)ethyl)amino)pyridin-3-yl)pyrimidin-4-yl) methyl ester (660 mg, 46% yield). LCMS (5-95AB, 1.5 min): Rt = 0.823 min, m / z = 500.2 [M+H]+.

[0245] Step 6: Preparation of intermediate 1h Instructions 63 / 90 pages 73 CN 122295346 A Racemic-(11R,20R)-23,26-dichloro-3-(4-fluorophenyl)-14-hydroxy-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113, 17.09,28]octadecane 1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaen-11-carboxylic acid tert-butyl ester (300 mg, 0.37 A solution of methyl 4-methylbenzenesulfonic acid [2-[6-[2-(tert-butoxycarbonylamino)ethylamino]-3-pyridyl]pyrimidin-4-yl]methyl ester (204 mg, 0.41 mmol) and Cs₂CO₃ (302 mg, 0.93 mmol) in DMF (4 mL) was stirred at 25 °C for 1 h. The mixture was poured into water (20 mL), extracted with ethyl acetate (20 mL × 2) and washed with brine (10 mL × 3), then concentrated and purified by rapid chromatography (0% to 6% methanol in dichloromethane) to give a white solid intermediate (320 mg, 76% yield) for 1 h. LCMS (5–95 AB, 1.5 min): Rt = 0.930 min, m / z = 1136.5 [M+H]⁺.

[0246] Step 7: Preparation of compound 100 (11R,20R)-14-[[2-[6-[2-(tert-butoxycarbonylamino)ethylamino]-3-pyridyl]pyrimidin-4-yl]methoxy]-23,26-dichloro-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octadecane-1 (25),2,5 (28),6,8,13,15,17 (27),22 (26),23-decaen-11-carboxylic acid tert-butyl ester (320) TFA (3 mL) was added to a solution of dichloromethane (2 mL) at 0.28 mg (mg, 0.28 mmol) and the mixture was stirred at 25 °C for 16 hours. The mixture was concentrated to give a crude product. It was purified by preparative HPLC (15% to 45% / water (TFA)-ACN) to give compound 100 (320 mg, 94% yield) as a yellow solid. 1H NMR (400MHz, DMSO‑d6) δ 9.57 (s, 1H), 9.03 (d, J = 2.0 Hz, 1H), 8.80 (d, J = 5.2 Hz, 1H), 8.76 (s, 1H), 8.39‑8.34 (m, 1H), 7.86 (s, 3H), 7.42 (d, J = 5.2 Hz, 1H), 7.24-7.11 (m, 4H), 6.93-6.89 (m, 1H), 6.86-6.80 (m, 1H), 6.69 (d, J = 8.8 Hz, 1H) , 6.28 ‑ 6.23 (m, 1H) , 5.80 (d , J = 2.4 Hz, 1H) , 5.24 ‑ 5.09 (m, 2H) , 4.96 ‑ 4.94 (m, 1H) , 4.52‑ 4.41 (m , 2H) , 3.63 ‑ 3.58 (m , 5H) , 3.41 ‑ 3.40 (m , 2H) , 3.23 ‑ 3.21 (m , 1H) , 3.13 ‑ 2.98 (m , 6H) , 2.86 ‑ 2.84 (m , 2H) , 2.80 (s , 3H) , 1.97 (s, 6H).LCMS (5-95 AB, 1.5 min): Rt = 0.797 min, m / z = 980.4 [M+H]+.

[0247] Example 2: Synthesis of 1 g of macrocyclic intermediate 64 / 90 pages 74 CN 122295346 A Figure 1 shows the synthetic scheme for producing 1 g of macrocyclic intermediate. The intermediate compounds in this example are numbered according to the numbers in Figure 1.

[0248] Step 1, Preparation of 5,6-diiodo-3H-thieno[2,3-d]pyrimidin-4-one (intermediate 2): Under N2, a mixture of 3H-thieno[2,3-d]pyrimidin-4-one (10 g, 65.7 mmol), periodic acid (12.71 g, 65.7 mmol), iodine (38.41 g, 151.1 mmol), and sulfuric acid (1.0 mL, 65.7 mmol) in acetic acid (150 mL) and water (35 mL) was stirred at 110 °C for 3 hours. TLC (in 50% ethyl acetate in petroleum ether, Rf = 0.4) showed new spots. The mixture was cooled to room temperature, and then MTBE (100 mL) was added and stirred further at 10 °C for 30 minutes. The precipitate was filtered off and washed with a mixture of MTBE and ethanol (100 mL, 2:1), then washed with MTBE (50 mL x 3) and dried under vacuum to give a yellow solid, 5,6-diiodo-3H-thieno[2,3-d]pyrimidin-4-one (18.92 g, 71% yield). ¹H NMR (400 MHz, DMSO-d6): δ 11.67 (br, ¹H), 8.15 (s, ¹H).

[0249] Step 2, Preparation of 4-chloro-5,6-diiodo-thieno[2,3-d]pyrimidine (intermediate 3): N,N-dimethylaniline (1.2 mL, 9.3 mmol) was added to a mixture of 5,6-diiodo-3H-thieno[2,3-d]pyrimidine-4-one (10.00 g, 24.8 mmol) and phosphorus oxychloride (50 mL), and the mixture was stirred at 105 °C for 2 h. The resulting suspension was cooled to room temperature and 100 mL of hexane was added, and the mixture was stirred for 20 min. The precipitate was filtered off, washed with hexane (50 mL x 3) and water (50 mL x 3), and dried under vacuum to give a gray solid 4-chloro-5,6-diiodo-thieno[2,3-d]pyrimidine (9.23 g, yield 88%). 1H NMR (400 MHz, DMSO‑d6): δ 8.88 (s, 1H).

[0250] Step 3, Preparation of 4-chloro-5-iodo-thieno[2,3-d]pyrimidine (intermediate 4): Tert-butylmagnesium chloride (20.70 mL, 20.7 mmol) was added to a solution of 4-chloro-5,6-diiodo-thieno[2,3-d]pyrimidine (8.80 g, 20.7 mmol) in tetrahydrofuran (100 mL) over 20 minutes at -20°C, and the mixture was stirred at 0°C for 2 h. The reaction was quenched with water (7 mL) and concentrated under reduced pressure. The crude product was sonicated in a mixture of acetonitrile and water (100 mL, 3 / 1) and the solid was collected by filtration to obtain a yellow solid of 4-chloro-5-iodo-thieno[2,3-d]pyrimidine (5.19 g, 84%). 1H NMR (400 MHz, DMSO‑d6): δ 8.96 (s, 1H) , 8.46 (s, 1H).

[0251] Step 4, Preparation of 4-chloro-5-(4-methoxy-2,6-dimethyl-phenyl)thieno[2,3-d]pyrimidine (intermediate 5): Under N2, a mixture of 4-chloro-5-iodo-thieno[2,3-d]pyrimidine (5.00 g, 16.9 mmol), (2,6-dimethyl-4-methoxyphenyl)boronic acid (6.07 g, 33.7 mmol), [2-(2-aminophenyl)phenyl]-chloro-palladium; bis(1-adamantyl)-butylphosphine (2.39 g, 3.37 mmol), cesium carbonate (10.99 g, 33.7 mmol) in toluene (30 mL) and water (6 mL) was stirred at 100 °C for 16 hours. The mixture was diluted with ethyl acetate (160 mL), washed with brine (50 mL x 2), and concentrated to give a crude product. This crude product was purified by column chromatography (solvent gradient: 0% to 4% ethyl acetate in petroleum ether) to give a yellow solid, 4-chloro-5-(4-methoxy-2,6-dimethyl-phenyl)thieno[2,3-d]pyrimidine (1.47 g, 29% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.89 (s, 1H), 7.35 (s, 1H), 6.70 (s, 2H), 3.86 (s, 3H), 1.99 (s, 6H).Instructions for Use, pages 65 / 90, CN 122295346 A

[0252] Step 5, Preparation of 4-chloro-6-iodo-5-(4-methoxy-2,6-dimethyl-phenyl)thieno[2,3-d]pyrimidine (intermediate 6): Lithium (6.50 mL, 13.0 mmol) was added dropwise to a solution of 4-chloro-5-(4-methoxy-2,6-dimethyl-phenyl)thieno[2,3-d]pyrimidine (3.60 g, 11.8 mmol) in tetrahydrofuran (40 mL) at -78 °C under an argon atmosphere, and the mixture was stirred for 15 minutes. Iodine (5.99 g, 23.6 mmol) was added dropwise to a solution of tetrahydrofuran (20 mL), and then the cooling bath was removed and the mixture was stirred at 20 °C for 3 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.3) indicated that the reaction was complete. The reaction was quenched with 10 mL of aqueous NH4Cl solution and extracted with ethyl acetate (30 mL x 3). The extract was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (solvent gradient: 0% to 10% ethyl acetate in petroleum ether) to give a yellow solid, 4-chloro-6-iodo-5-(4-methoxy-2,6-dimethyl-phenyl)thieno[2,3-d]pyrimidine (2.90 g, 52% yield). ¹H NMR (400 MHz, CDCl3): δ 8.82 (s, 1H), 6.72 (s, 2H), 3.87 (s, 3H), 1.93 (s, 6H).

[0253] Step 6, Preparation of 4-chloro-3-(3,5-dichloro-4-methoxy-2,6-dimethylphenyl)-2-iodothieno[3,2-c]pyridine (intermediate 7): NCS (3.66 g, 27.4 mmol) was added to a solution of 4-chloro-6-iodo-5-(4-methoxy-2,6-dimethylphenyl)thieno[2,3-d]pyrimidine (2.95 g, 6.8 mmol) in acetonitrile (50 mL) at 26 °C. The reaction mixture was stirred at 90 °C for 3 h. New spots were observed by TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.4). The reaction was quenched with an aqueous solution of Na2SO3 (30 mL) and extracted with ethyl acetate (50 mL x 3).The organic layer was washed with brine (50 mL) and concentrated to the residue, which was then purified by column chromatography (solvent gradient: 0% to 5% ethyl acetate in petroleum ether) to give a yellow solid 4-chloro-3-(3,5-dichloro-4-methoxy-2,6-dimethylphenyl)-2-iodothieno[3,2-c]pyridine (2.1 g, 59% yield).

[0254] Step 7, Preparation of 2,6-dichloro-4-(4-chloro-6-iodothieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenol (intermediate 8): A mixture of 4-chloro-5-(3,5-dichloro-4-methoxy-2,6-dimethyl-phenyl)-6-iodothieno[2,3-d]pyrimidin (4.20 g, 8.4 mmol) and aluminum chloride (3.36 g, 25.2 mmol) in 1,2-dichloroethane (30 mL) was stirred at 70 °C for 12 h under N2. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.3) showed that the reaction was complete. The reaction was quenched with saturated NaHCO3 (3 mL) and the mixture was stirred for 2 min. Then saturated NH4Cl (15 mL) was added. The extract was obtained by extraction with ethyl acetate (50 mL), washed with brine (50 mL), and concentrated to give a crude product. This crude product was purified by column chromatography (solvent gradient: 0% to 10% ethyl acetate in petroleum ether) to give a yellow solid, 2,6-dichloro-4-(4-chloro-6-iodothieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenol (2.4 g, 59% yield). LCMS (AB_5-95_1.5 min): RT = 1.038 min, M / Z = 484.8 [M+H]+.

[0255] Step 8, Preparation of (R)-3-(allyloxy)-2-(2,6-dichloro-4-(4-chloro-6-iodothieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenoxy)propyl acetate (intermediate 10): Di-tert-butyl azodicarboxylate (0.84 mg, 3.6 mmol) was slowly added to a solution of triphenylphosphine (3.50 g, 13.2 mmol), (S)-3-(allyloxy)-2-hydroxypropyl acetate (2.31 g, 13.2 mmol), and 2,6-dichloro-4-(4-chloro-6-iodothieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenol (3.25 g, 6.6 mmol) in toluene (50 mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred at 50°C for 16 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.2) indicated that the reaction was complete.The mixture was diluted with ethyl acetate (150 mL) and washed with brine (30 mL x 2). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a crude product. The crude product was purified by column chromatography (solvent gradient: 0% to 6% ethyl acetate in petroleum ether) to give a white solid (R)-3-(allyloxy)-2-(2,6-dichloro-4-(4-chloro-6-iodothieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenoxy)propyl acetate (4.1 g, 95% yield). LCMS (AB, 5–95, 1.5 min): RT = 1.172 min, m / z = 642.8 [M+H]+. Instructions for Use, Pages 66 / 90, CN 122295346 A

[0256] Step 9, Preparation of (R)-3-(allyloxy)-2-(2,6-dichloro-4-(4-chloro-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenoxy)propyl acetate (intermediate 12): 4-chloro-6-iodo-5-(4-methoxy-2,6-dimethyl-phenyl)thieno[2,3-d]pyrimidinyl (5 g, 7.8 mmol), 4-fluorophenylboronic acid (2.2 g, 15.6 mmol), tris(dibenzylacetone)dipalladium(O) (713 mg, 0.78 mmol), and 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (331 mg, 0.78 mmol) were prepared under N2. A mixture of K3PO4 (3.3 g, 15.6 mmol) and K2PO4 (3.3 g, 15.6 mmol) in tetrahydrofuran (40 mL) and water (10 mL) was stirred at 65 °C for 16 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.4) showed that the reaction was complete. The mixture was diluted with ethyl acetate (160 mL), washed with brine (50 mL x 2) and concentrated to give a crude product, which was purified by column chromatography (solvent gradient: 0% to 8% ethyl acetate in petroleum ether) to give a yellow solid (R)-3-(allyloxy)-2-(2,6-dichloro-4-(4-chloro-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenoxy)propyl acetate (4.0 g, 84% yield). LCMS (AB_ 5‑95_1.5min): RT= 1.189 min, M / Z=610.8 [M+H]+.

[0257] Step 10, Preparation of (R)-tert-butyl 2-((5-(4-(((R)-1-acetoxy-3-(allyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thiopheno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (intermediate 14): At 25°C, (R)- tert-butyl 3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)-2-hydroxypropionate (2.92 g, 6.36 mmol) and cesium carbonate (4.15 g, 12.7 mmol) in anhydrous tert-butanol (40 mL) were added. 3-(allyloxy)-2-(2,6-dichloro-4-(4-chloro-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-3,5-dimethylphenoxy)propyl acetate (3.88 g, 6.36 mmol) was mixed and stirred at 65 °C for 12 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.3) indicated that the reaction was complete. The resulting homogeneous mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over anhydrous Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography (solvent gradient: 0% to 10% ethyl acetate in petroleum ether) to give a colorless oily (R)-tert-butyl 2-((5-(4-(((R)-1-acetoxy-3-(allyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (2.4 g, yield 37%). LCMS (R_10‑80_1 .5min): RT= 1.396 min, M / Z=1031.3 [M+H]+.

[0258] Step 11, Preparation of (R)-tert-butyl 2-((5-(4-(((S)-1-(allyloxy)-3-hydroxypropane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thiopheno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (intermediate 15): at 25°C Sodium ethoxide (31.6 mg, 0.46 mmol) was added to a solution of (R)-tert-butyl-2-((5-(4-(((R)-1-acetoxy-3-(allyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (2.4 g, 2.32 mmol) in ethanol (20 mL), and the mixture was stirred for 12 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.2) indicated that the reaction was complete. The resulting homogeneous mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over anhydrous Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography (solvent gradient: 0% to 8% ethyl acetate in petroleum ether) to give a white solid (R)-tert-butyl 2-((5-(4-(((S)-1-(allyloxy)-3-hydroxypropane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thiopheno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (1.9 g, yield 83%). LCMS (10‑80, R, 1.5min): RT= 1.363 min, m / z=989.4 [M +H]+.Instruction manual, pages 67 / 90, CN 122295346 A

[0259] Step 12, Preparation of (R)-tert-butyl 2-((5-(4-(((R)-1-(allyloxy)-3-(toluenesulfonyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thiophene[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (intermediate 16): at 0°C, to (R)-tert-butyl 2-((5-(4-(((S)-1-(allyloxy)-3-hydroxypropane-2-yl)oxy)-3,5-dichloro-2,6- A solution of dimethylphenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (5.5 g, 5.5 mmol) in anhydrous dichloromethane (50 mL) was mixed with 1,4-diazabicyclo[2.2.2]octane (DABCO; 3.10 mL, 27.8 mmol) and p-toluenesulfonyl chloride (4.24 g, 22.2 mmol). The reaction was stirred at 25 °C for 12 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.3) indicated that the reaction was complete. The resulting homogeneous mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over anhydrous Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography (solvent gradient: 0% to 8% ethyl acetate in petroleum ether) to give a white solid (R)-tert-butyl 2-((5-(4-(((R)-1-(allyloxy)-3-(toluenesulfonyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thiopheno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (5.5 g, yield 86%). LCMS (85‑100, AB, 2min): RT= 1.357 min, m / z=1143.2 [M+H]+.

[0260] Step 13, Preparation of (R)-tert-butyl 2-((5-(4-(((R)-1-(allyloxy)-3-(toluenesulfonyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thiopheno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-hydroxyphenyl)propionate (intermediate 17): at 0°C, to (R)-tert-butyl 2-((5- (4-(((R)-1-(allyloxy)-3-(toluenesulfonyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (5.5 g, 4.8 mmol) was slowly added to a solution in anhydrous tetrahydrofuran (50 mL), and the mixture was stirred at 25 °C for 2 h. TLC (in 25% ethyl acetate in petroleum ether, Rf = 0.4) indicated that the reaction was complete. The resulting homogeneous mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over anhydrous Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography (solvent gradient: 0% to 15% ethyl acetate in petroleum ether) to give a colorless oily (R)-tert-butyl 2-((5-(4-(((R)-1-(allyloxy)-3-(toluenesulfonyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-hydroxyphenyl)propionate (4.8 g, 97% yield).

[0261] Step 14, Preparation of intermediate 18: Cesium carbonate (7.59 g, 23.3 mmol) was added to a solution of (R)-tert-butyl-2-((5-(4-(((R)-1-(allyloxy)-3-(toluenesulfonyloxy)propane-2-yl)oxy)-3,5-dichloro-2,6-dimethylphenyl)-6-(4-fluorophenyl)thiopheno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-hydroxyphenyl)propionate (4.8 g, 4.7 mmol) in anhydrous N,N-dimethylformamide (50 mL) at 25 °C, and the mixture was stirred for 2 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.2) indicated that the reaction was complete.The resulting homogeneous mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over anhydrous Na₂SO₄, filtered, and concentrated to give a crude product. This crude product was purified by column chromatography (solvent gradient: 0% to 10% ethyl acetate in petroleum ether) to give the title compound (3.2 g, 80% yield) as a white solid. LCMS (AB, 5–95, 1.5 min): RT = 1.367 min, m / z = 858.6 [M+H]⁺.

[0262] Step 15, Preparation of intermediate 19: At 25°C, 1,3-dimethylbarbituric acid (3.64 g, 23.3 mmol) and Pd(Ph3P)4 (1.35 g, 1.2 mmol) were added to a mixture of intermediate 18 (2.50 g, 2.9 mmol) from step 14 in dichloromethane (20 mL) and methanol (10 mL). The reaction mixture was degassed three times with N2 and then stirred for 16 h. The mixture was concentrated and purified by column chromatography (solvent gradient: 0% to 30% ethyl acetate in petroleum ether) to give intermediate 19 (3.8 g, 96% yield) as a white solid. LCMS (5–95, AB, 1.5 min): RT = 1.235 min, m / z = 819.1 [M+H]+.

[0263] Step 16, Preparation of intermediate 20: p-Toluenesulfonyl chloride (2.66 g, 13.9 mmol) was added to a mixture of intermediate 19 from step 15 (3.80 g, 2.8 mmol) and 1,4-diazabicyclo[2.2.2]octane (DABCO; 2.45 mL, 22.3 mmol) in dichloromethane (30 mL) at 25 °C, and the mixture was stirred for 16 h. TLC (in 30% ethyl acetate in petroleum ether, Rf = 0.5) showed the desired product. The resulting mixture was concentrated to obtain a crude product, which was purified by column chromatography (solvent gradient: 0% to 15% ethyl acetate in petroleum ether) to give intermediate 20 (2.2 g, 81% yield) as a white solid. LCMS (5–95, AB, 1.5 min 220 & 254): RT = 1.303 min, m / z = 972.8 [M+H]+.

[0264] Step 17, Preparation of intermediate 22: KI (0.75 g, 4.53 mmol), 1-methylpiperazine (100 mL, 905.4 mmol), and cesium carbonate (1.47 g, 4.52 mmol) were added to a mixture of intermediate 20 (2.20 g, 2.26 mmol) from step 17 in anhydrous acetonitrile (30 mL). The mixture was stirred at 60 °C for 16 h. The solvent was concentrated under vacuum. The residue was purified by column chromatography (solvent gradient: 0% to 10% methanol in dichloromethane) to give intermediate 22 (2.01 g, 98% yield) as a yellow solid. LCMS (5-95AB, 1.5 min 220 & 254): RT = 0.970 min, m / z = 899.3 [M+H]+.

[0265] Step 18, Preparation of 1 g of intermediate: 10% palladium on carbon (2.36 g, 2.22 mmol) was added to a mixture of intermediate 22 (2 g, 2.22 mmol) from step 17 in tetrahydrofuran (30 mL) at 25 °C, and the mixture was stirred for 16 h at H2 (15 psi). TLC (10% MeOH in DCM, Rf = 0.4) showed the desired product. The mixture was filtered and concentrated to give 1 g (1.50 g, 83% yield) of a white solid macrocyclic intermediate. LCMS (5-95AB, 1.5 min -220 & 254): RT = 0.962 min, m / z = 809.4 [M+H]+.

[0266] Example 3: Synthesis of alcohol intermediate 13, step 1, preparation of intermediate 13b: TBSCl (21.82 g, 144.8 mmol) was added to a solution of 2,5-dihydroxybenzaldehyde (20 g, 144.8 mmol) and imidazole (19.72 g, 289.6 mmol) in dichloromethane (300 mL), and the mixture was stirred at 25 °C for 16 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.3) showed that the starting materials had been consumed. The mixture was diluted with water (150 mL), washed with brine (200 mL x 3), and the organic solution was concentrated to give a crude product. The crude product was purified by column chromatography (solvent gradient: 0% to 10% ethyl acetate in petroleum ether) to give a yellow oily 5-((tert-butyldimethylsilyl)oxy)-2-hydroxybenzaldehyde (30.5 g, yield 72%).

[0267] Step 2, Preparation of intermediate 13c: Benzyl bromide (7.76 mL, 65.2 mmol) was added to a solution of 5-[tert-butyl(dimethyl)silyl]oxy-2-hydroxy-benzaldehyde (22 g, 87.2 mmol) and K₂CO₃ (14.46 g, 104.6 mmol) in acetonitrile (200 mL). The mixture was stirred at 60 °C for 16 h. TLC (in 5% ethyl acetate in petroleum ether, Rf = 0.5) showed that the reaction was complete. The mixture was partitioned between ethyl acetate (200 mL) and H2O (100 mL), the organic solution was concentrated and purified by column chromatography (solvent gradient: 0% to 3% ethyl acetate in petroleum ether) to give a yellow oily 2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)benzaldehyde (28.2 g, 94% yield).

[0268] Step 3, preparation of intermediate 13d: (tert-butoxycarbonylmethylene)triphenylphosphine (35.8 g, 3.21 mmol) was added to a solution of 2-benzyloxy-5-[tert-butyl(dimethyl)silyl]oxy-benzaldehyde (35.8 g, 104.5 mmol) in tetrahydrofuran (300 mL) at 0 °C, and the mixture was stirred at 25 °C for 16 h. TLC (in 30% ethyl acetate in petroleum ether, Rf = 0.4) showed that the starting material had been consumed and new spots were found. The mixture was partitioned between ethyl acetate (300 mL) and H₂O (150 mL), the organic solution was concentrated, and purified by column chromatography (solvent gradient: 0% to 2% ethyl acetate in petroleum ether) to give a yellow oily (E)-tert-butyl 3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)acrylate (35.1 g, 76% yield). LCMS (5–95 , AB , 1.5 min): RT = 1.3 min, m / z = 881.0 [2M+H]+.

[0269] Step 4, Preparation of intermediate 13e: 10% palladium on carbon (12.68 g) was added to a solution of (E)-tert-butyl 3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)acrylate (35 g, 79.4 mmol) in tetrahydrofuran (300 mL), and the mixture was stirred at 25 °C for 16 h under H2 (30 psi). TLC (in 20% ethyl acetate in petroleum ether, Rf = 0.4) showed that the starting material had been consumed.The mixture was filtered and concentrated to give tert-butyl 3-(5-((tert-butyldimethylsilyl)oxy)-2-hydroxyphenyl)propionate (23.2 g, 82% yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃): δ 6.95 (br s, 1H), 6.59 (d, J = 8.4 Hz, 1H), 6.47–6.39 (m, 2H), 2.65–2.61 (m, 2H), 2.46–2.43 (m, 2H), 1.25 (s, 9H), 0.81 (s, 9H), 0.01 (m, 6H).

[0270] Step 5, Preparation of intermediate 13f: Potassium carbonate (9.02 g, 65.2 mmol) and benzyl bromide (7.76 mL, 65.2 mmol) were added to a solution of tert-butyl 3-(5-((tert-butyldimethylsilyl)oxy)-2-hydroxyphenyl)propionate (23 g, 65.2 mmol) in acetonitrile (200 mL), and the mixture was stirred at 80 °C for 16 h. TLC (in 20% ethyl acetate in petroleum ether, Rf = 0.8) showed that the reaction was complete. The mixture was partitioned between ethyl acetate (200 mL) and H2O (100 mL), the organic solution was concentrated and purified by column chromatography (solvent gradient: 0% to 3% ethyl acetate in petroleum ether) to give colorless oily tert-butyl 3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (15.4 g, yield 54%). 1H NMR (400 MHz, CDCl3): δ 7.44-7.32 (m, 5H), 6.75 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 2.8 Hz, 1H), 6.65-6.59 (m, 1H), 5 .03 (s, 2H), 2.91 (t, J = 8.0 Hz, 2H), 2.53 (t, J = 8.0 Hz, 2H), 1.43 (s, 9H), 0.98 (s, 9H), 0.17 (s, 6H).

[0271] Step 6, Preparation of intermediates 13 and 13g: Potassium bis(trimethylsilyl)amide (17.20 mL, 17.2 mmol) was added dropwise to a mixture of tert-butyl 3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (5.00 g, 11.3 mmol) in THF (50 mL) at -78°C under a nitrogen atmosphere. The mixture was stirred for 40 min. A solution of 3-phenyl-2-(benzenesulfonyl)-1,2-oxazolidinyl propane (4.10 g, 15.3 mmol) in THF (20 mL) was added dropwise to the above mixture at -78°C. The mixture was stirred at -65°C for 3 h. The reaction was then heated to 25°C. TLC (in 20% ethyl acetate in petroleum ether, Rf = 0.4) showed that the starting material had been consumed and new spots had formed. The mixture was partitioned between ethyl acetate (100 mL) and H₂O (50 mL), the organic solution was concentrated and purified by column chromatography (solvent gradient: 0% to 20% ethyl acetate in petroleum ether) to give a yellow, oily racemic mixture (2.10 g, yield 38.9%). The racemic mixture was separated by SFC (Daicel Chiralpak AS (250 mm 50 mm, 10 μm); mobile phase: 20% ethanol (0.1% ammonium hydroxide) in CO₂) to give (R)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)-2-hydroxypropionate tert-butyl ester (intermediate 13; 1.05 g, peak 2, desired isomer, RT = 2.769 min). 1H NMR (400 MHz, CDCl3): δ 7 .46 ‑ 7 .32 (m , 5H) , 6.79 (d , J = 8.8 Hz, 1H) , 6.74 (d , J = 2.8 Hz, 1 H) , 6.67 (dd , J = 8.8, 4.4 Hz, 1H) , 5.04 (s, 2H) , 4.40 ‑ 4.37 (m, 1H) , 3.15 (dd , J = 14.0, 4.8 Hz, 1H) , 2.89 ‑ 2.84 (m, 2H) , 1.41 (s, 9H) , 0.98 (s, 9H) , 0.17 (s, 6H).The stereoisomer tert-butyl-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)-2-hydroxypropionate was also obtained (intermediate 13 g; 0.94 g, peak 1, RT = 2.535 min). 1H NMR (400 MHz, CDCl3): δ 7 .46 ‑ 7 .32 (m , 5H) , 6.79 (d , J = 8.8 Hz, 1H) , 6.74 (d , J = 2.8 Hz, 1 H) , 6.67 (dd , J = 8.8, 4.4 Hz, 1H) , 5.04 (s, 2H) , 4.40 ‑ 4.37 (m, 1H) , 3.15 (dd , J = 14.0, 4.8 Hz, 1H) , 2.89 ‑ 2.84 (m, 2H) , 1.41 (s, 9H) , 0.98 (s, 9H) , 0.17 (s, 6H).

[0272] Example 4: Preparation of Intermediate 9 Step 1, Preparation of Intermediate 9c: (S)-(2,2-dimethyl-1,3-dioxolane-4-yl)methanol (18.70 mL, 151.3 mmol) was added to a mixture of sodium hydride (12.11 g, 302.7 mmol) and tetrahydrofuran (500 mL) at 0 °C under nitrogen atmosphere for 30 min. The reaction mixture was stirred at 0 °C for 0.5 h. Then, 3-bromoprop-1-ene (19.64 mL, 227 mmol) was added dropwise at 0 °C and stirred at 25 °C for 16 h. TLC (in 10% ethyl acetate in petroleum ether, Rf = 0.5) indicated that the reaction was complete. The reaction was quenched with water (500 mL), extracted with ethyl acetate (500 mL), and washed with brine (500 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain a yellow, oily crude (S)-4-((allyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (26 g), which was used directly in the next step.

[0273] Step 2, Preparation of intermediate 9d: 1 M hydrogen chloride (24.40 mL, 24.4 mmol) was added to a solution of (S)-4-((allyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (26.00 g, 151 mmol) in methanol (300 mL), and the mixture was stirred at 60 °C for 1 h. TLC (in 25% ethyl acetate in petroleum ether, Rf = 0.6) showed that the starting material had been consumed and new spots were found.The mixture was concentrated under vacuum to obtain a colorless, oily crude (R)-3-(allyloxy)propane-1,2-diol (19 g), which was used directly in the next step.

[0274] Step 3, Preparation of intermediate 9: Acetic anhydride (2.2 mL, 22.7 mmol) was added to a solution of (R)-3-(allyloxy)propane-1,2-diol (3.00 g, 22.7 mmol) and triethylamine (4.80 mL, 34.0 mmol) in dichloromethane (50 mL), and the reaction mixture was stirred at 25 °C for 3 h. TLC (in 50% ethyl acetate in petroleum ether, Rf = 0.5) showed that the starting material had been consumed and new spots were found. The mixture was concentrated to a residue, which was purified by column chromatography (solvent gradient: 0% to 25% ethyl acetate in petroleum ether) to give a yellow oil (S)-3-(allyloxy)-2-hydroxypropyl acetate (2.05 g, 51% yield). ¹H NMR (400 MHz, CDCl₃): δ 5.81–5.98 (m, 1 H), 5.16–5.35 (m, 2 H), 4.09–4.24 (m, 2 H), 4.05–4.02 (m, 3 H), 3.40–3.60 (m, 2 H), 2.09 (s, 3 H).

[0275] Example 5: Preparation of sqCit linker-compound 100 conjugates Compound 100 was used to prepare linker-drug conjugates, which were further used to prepare ADCs. The linker used in all conjugates was a square-cit (sq-Cit or sqCit) peptide mimic linker, which is described in more detail in PCT / US2014 / 070654. This linker was activated to form nitrobenzene carbonate, also as described in PCT / US2014 / 070654.To conjugate this activated linker to compound 100, racemic -(11R,20R)-14-[[2-[6-(2-aminoethylamino)-3-pyridyl]pyrimidin-4-yl]methoxy]-23,26-dichloro-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octadecane-1(24),2,5(28),6,8,13,15,17(27),22,25-decaen-11-carboxylic acid TFA salt (compound 100; 650 mg, 0.54 mg) was prepared at 25 °C. DIEA (0.28 mL, 1.61 mmol) and (4-nitrophenyl)[4-[[racemic-(2S)-2-[[1-[5-(2,5-dioxypyrrolo-1-yl)pentylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureo-pentanoyl]amino]phenyl]methyl carbonate (593 mg, 0.81 mmol) were added to a solution of DMF (4 mL), and the mixture was stirred for 2 hours. The reaction solution was purified by preparative HPLC (15% to 45% / water (TFA)-ACN) to give the title compound as a white solid (676.3 mg, 74% yield). LCMS (5-95 AB, 1.5 min): Rt = 0.858 min, m / z = 1576.54 [M+H]+.

[0276] Example 6: Synthesis specification of compound 101, pages 72 / 90, CN 122295346 A Preparation of [2-(4-aminophenyl)pyrimidin-4-yl]methanol: 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (1.01 g, 1.38 mmol) was added to a solution of (2-chloropyrimidin-4-yl)methanol (2.0 g, 13.84 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)phenylamine (3.64 g, 16.6 mmol) and sodium carbonate (4.4 g, 41.51 mmol) in dimethyl ether (80 mL) and water (8 mL) at 25 °C. The mixture was stirred at 80 °C for 12 h. The reaction solution was concentrated to obtain a crude product, which was then purified by silica gel rapid chromatography (eluting with 50% ethyl acetate in petroleum ether) to give a white solid [2-(4-aminophenyl)pyrimidin-4-yl]methanol (0.75 g, yield 27%).1H NMR (400MHz, DMSO‑d6) δ = 8.69 (d ,J= 5.2 Hz, 1H) , 8.08 (d ,J= 8.4 Hz, 2H) , 7.26 (d,J= 5.2 Hz, 1H) , 6.61 (d,J= 8.8 Hz, 2H) , 5.63 (s, 2H), 5.57 (t,J= 6.0 Hz, 1H), 4.55 (d,J= 5.6 Hz, 2H).

[0277] Preparation of N-[2-[4-[4-(hydroxymethyl)pyrimidin-2-yl]anilino]ethyl]tert-butyl carbamate: NaBH3CN (474 ​​mg, 2.24 mmol) was added to a solution of [2-(4-aminophenyl)pyrimidin-4-yl]methanol (300 mg, 1.49 mmol), N-(2-oxyethyl)carbamate (0.36 mL, 1.94 mmol), and acetic acid (44.76 mg, 0.75 mmol) in methanol (12 mL). The reaction was stirred at 25 °C for 3 h. The reaction solution was quenched with saturated NaHCO3 solution (10 mL) and extracted with dichloromethane (10 mL x 2). The organic layer was washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (eluting in dichloromethane with 0% to 6% methanol) to give a yellow oily N-[2-[4-[4-(hydroxymethyl)pyrimidin-2-yl]aniline]ethyl]carbamate tert-butyl ester (180 mg, 35% yield). 1H NMR (400MHz, DMSO‑d6)δ = 8.70 (d,J= 5.2 Hz, 1H) , 8.14 (d,J= 8.8 Hz, 2H) , 7.27 (d,J= 5.2 Hz, 1H) , 6.91 (s, 1H) , 6.64 (d,J= 8.8 Hz, 2H), 6.20 (s, 1H), 5.57 (t,J= 6.0 Hz, 1H), 4.55 (d,J= 6.0 Hz, 2H), 3.23-3.10 (m, 4H), 1.39 (s, 9H).

[0278] Preparation of methyl 4-methylbenzenesulfonic acid [2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methyl ester: TsCl (430 mg, 2.26 mmol) was added to a solution of N-[2-[4-[4-(hydroxymethyl)pyrimidin-2-yl]aniline]ethyl]carbamate tert-butyl ester (130 mg, 0.38 mmol) and N,N-diisopropylethylamine (0.52 mL, 3.01 mmol) in dichloromethane (5 mL) at 25 °C. The mixture was stirred for 16 h. The reaction solution was concentrated to obtain a crude product, which was purified by preparative TLC (in 50% ethyl acetate in petroleum ether, Rf = 0.5) to give a white solid methyl 4-methylbenzenesulfonic acid [2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methyl ester (15 mg, yield 8%). LCMS (5-95AB / 1.5 min): Rt = 1.000 min, m / z = 499.2 [M+H]+.

[0279] Preparation of (11R,20R)-14-[[2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methoxy]-23,26-dichloro-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octadecane-1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaen-11-carboxylic acid tert-butyl ester: according to Example 2 The macrocyclic intermediate 1 g was prepared using the following procedure. A mixture of 1 g of the intermediate (20 mg, 0.02 mmol), 4-methylbenzenesulfonic acid [2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methyl ester (12.93 mg, 0.03 mmol), and Cs₂CO₃ (24 mg, 0.07 mmol) in acetonitrile (2 mL) was stirred at 60 °C for 1 h.The reaction solution was concentrated to obtain a crude product, which was purified by preparative TLC (in 10% methanol in dichloromethane) to obtain a white solid (11R,20R)-14-[[2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methoxy]-23,26-dichloro-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octadec-1(25),2,5(28),6,8,13,15,17(27),22(26). 2,3-Decaene-11-carboxylic acid tert-butyl ester (15 mg, yield 54%). LCMS (5-95AB / 1.5 min): Rt = 1.066 min, m / z = 1135.5 [M+H]+.

[0280] Preparation of compound 101: 15 mg, 0.01 mmol of (11R,20R)-14-[[2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methoxy]-23,26-dichloro-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octadecano-1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaen-11-carboxylic acid tert-butyl ester was added to TFA (1 mL). The solution in dichloromethane (1 mL) was stirred at 25°C for 16 h. The reaction solution was concentrated to obtain a crude product, which was then purified by preparative HPLC (acetonitrile 18-48 / 0.025% TFA in water) to obtain a yellow solid (11R,20R)-14-[[2-[4-(2-aminoethylamino)phenyl]pyrimidin-4-yl]methoxy]-23,26-dichloro-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octadec-1(25),2,5(28),6,8,13,15,17(27),22(26) ,23-Decaene-11-carboxylic acid (10 mg, yield 69%; compound 101).1H NMR (400 MHz, DMSO‑d6) δ = 9.50‑9.36 (m, 1H), 8.77 (s, 1H), 8.76 (d,J= 4.8 Hz, 1H), 8.26‑8.19 (m, 2H), 7.90‑7.72 (m, 3H), 7.39-7.28 (m, 1H), 7.24-7.12 (m, 4H), 6.94 (s, 1H), 6.87-6.81 (m, 1H), 6.75-6.69 (m, 2H), 6.41 - 6.24 (m, 2H) , 5.83 ‑ 5.78 (m, 1H) , 5.25 ‑ 5.07 (m, 2H) , 4.98 (s, 1H) , 4.55 ‑ 4.49 (s, 2H) , 3.69 ‑ 3.57 (m, 2H) , 3.27 ‑ 3.19 (m, 3H), 3.15-2.93 (m, 8H), 2.93-2.77 (m, 6H), 2.01 (s, 3H), 1.97 (s, 3H). LCMS (5‑95AB / 1.5min): Rt= 0.844min, m / z=979.4 [M+H]+.

[0281] Example 7: Synthesis specification of compound 102, pages 74 / 90, CN 122295346 A. Preparation of tert-butyl 2-((5-bromopyrimidin-2-yl)amino)ethyl)carbamate: TEA (2 mL, 14.35 mmol) and tert-butyl (2-aminoethyl)carbamate (0.98 mL, 6.19 mmol) were added to a solution of 5-bromo-2-chloropyrimidin (1.0 g, 5.17 mmol) in EtOH (20 mL) at 25 °C. The mixture was stirred at 80 °C for 4 h. The reaction was concentrated and purified by column chromatography (silica gel, 100-200 mesh, 0% to 30% ethyl acetate in petroleum ether) to give a white solid of tert-butyl (2-((5-bromopyrimidin-2-yl)amino)ethyl)carbamate (1.4 g, yield 85%). 1H NMR (400M Hz, DMSO‑d6): δ = 8.35 (s, 2H) , 7.39 (t,J= 5.2 Hz, 1H) , 6.84 (t,J= 5.2 Hz, 1H) , 3.28‑3.21 (m, 2H) , 3.10‑3.04 (m, 2H), 1.36 (s, 9H).

[0282] Preparation of tert-butyl (2-((5-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)pyrimidin-2-yl)amino)ethyl)carbamate: A mixture of (2-((5-bromopyrimidin-2-yl)amino)ethyl)carbamate (1.4 g, 4.41 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxacyclopentaborane) (1.7 g, 6.69 mmol), PdCl2dppf (328 mg, 0.44 mmol) and AcOK (1.3 g, 13.25 mmol) in dimethyl ether (20 mL) was stirred at 85 °C for 16 h. The reaction was concentrated and purified by column chromatography (silica gel, 100-200 mesh, 10% to 80% ethyl acetate in petroleum ether) to give a white solid (2-((5-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)pyrimidin-2-yl)amino)ethyl)tert-butyl carbamate (1.4 g, 87% yield).

[0283] Preparation of tert-butyl (2-((4-(hydroxymethyl)-[2,5'-bipyrimidine]-2'-yl)amino)ethyl)carbamate: A mixture of tert-butyl (2-((5-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)pyrimidine-2-yl)amino)ethyl)carbamate (0.3 g, 0.82 mmol), (2-chloropyrimidine-4-yl)methanol (100 mg, 0.69 mmol), PdCl2dppf (52 mg, 0.07 mmol) and Na2CO3 (220 mg, 2.08 mmol) in dimethyl ether (5 mL) and water (0.5 mL) was stirred at 100 °C for 16 h. The reaction solution was concentrated and purified by rapid chromatography (0% to 2% MeOH in DCM) to give a white solid (0.2 g, 84% yield) of tert-butyl 2-((4-(hydroxymethyl)-[2,5'-bipyrimidine]-2'-yl)amino)ethyl)carbamate. 1H NMR (400 MHz, CDCl3): δ = 9.28 (s, 2H) , 8.68 (d,J= 5.2 Hz, 1H) , 7.14 (d,J= 5.2 Hz, 1H) , 5.94 (s, 1H) , 5.11 ‑ 4.95 (m, 1H) , 4.79 (s, 2H) , 3.65 (q,J= 6.0 Hz, 2H) , 3.47-3.36 (m, 2H) , 1.45 (s, 9H).Instructions for Use, pages 75 / 90, CN 122295346 A

[0284] Preparation of 4-methylbenzenesulfonic acid (2'-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-[2,5'-bipyrimidine]-4-yl)methyl ester: TsCl (220 mg, 1.15 mmol) was added to a solution of (2-((4-(hydroxymethyl)-[2,5'-bipyrimidine]-2'-yl)amino)ethyl)carbamate tert-butyl ester (200 mg, 0.58 mmol) and DIEA (0.4 mL, 2.31 mmol) in DCM (5 mL), and the mixture was stirred at 25 °C for 16 h. The mixture was concentrated and purified by a rapid chromatographic column (0% to 2% MeOH in DCM) to give a white solid methyl 4-methylbenzenesulfonic acid (2'-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-[2,5'-bipyrimidine]-4-yl) methyl ester (0.2 g, 69% yield). 1H NMR (400M Hz, CDCl3): δ = 9.21 (s, 2H), 8.77-8.72 (m, 1H), 7.87 (d,J= 8.4 Hz, 2H), 7.38 (d,J= 8.0 Hz, 2H), 7.29- 7.27 (m, 1H), 5.93 (s, 1H), 5.12 (s, 2H), 4.96 (s, 1H), 3.67-3.63 (m, 2H), 3.43-3.39 (m, 2H), 2.46 (s, 3H) , 1.45 (s, 9H). LCMS (5-95AB / 1.5min): Rt = 0.879 min, m / z = 501.1 [M+H]+.

[0285] Preparation of (4R,9R)-66-((2'-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-[2,5'-bipyrimidine]-4-yl)methoxy)-13,15-dichloro-26-(4-fluorophenyl)-12,16-dimethyl-9-((4-methylpiperazin-1-yl)methyl)-3,7,10-trioxa-2(5,4)-thieno[2,3-d]pyrimidine-1(1,4),6(1,3)-dibenzocyclodecane-4-carboxylic acid tert-butyl ester: 1 g of macrocyclic intermediate was prepared according to the procedure of Example 2.The mixture of 1 g (30 mg, 0.03 mmol) of intermediate, 4-methylbenzenesulfonic acid [2-[2-[2-(tert-butoxycarbonylamino)ethylamino]pyrimidin-5-yl]pyrimidin-4-yl]methyl ester (20.4 mg, 0.045 mmol) and Cs2CO3 (30 mg, 0.09 mmol) in MeCN (1 mL) was stirred at 60 °C for 1 h. The mixture was concentrated and purified by preparative TLC (in DCM at 10% MeOH) to give a white solid (4R, 9R)-66-((2'-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-[2,5'-bipyrimidin]-4-yl)methoxy)-13,15-dichloro-26-(4-fluorophenyl)-12,16-dimethyl-9-((4-methylpiperazin-1-yl)methyl)-3,7,10-trioxa-2(5,4)-thieno[2,3-d]pyrimidin-1(1,4),6(1,3)-dibenzocyclodecane-4-carboxylic acid tert-butyl ester (40 mg, 94% yield). LCMS (5-95AB / 1.5 min): Rt = 1.066 min, m / z = 1136.6 [M+H]+.

[0286] Preparation of compound 102: 20 mg, 0.02 mmol of (11R,20R)-14-[[2-[2-[2-(tert-butoxycarbonylamino)ethylamino]pyrimidin-5-yl]pyrimidin-4-yl]methoxy]-23,26-dichloro-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09, 28]octadecano-1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaen-11-carboxylic acid tert-butyl ester was added to DCM (1 TFA (0.3 mL) was added to the solution in mL, and the mixture was stirred at 25 °C for 16 h. The mixture was concentrated and purified by preparative HPLC (acetonitrile 18-48 / 0.025% TFA in water) to give a white solid (4R,9R)-66-((2'-((2-aminoethyl)amino)-[2,5'-bipyrimidin]-4-yl)methoxy)-13,15-dichloro-26-(4-fluorophenyl)-12,16-dimethyl-9-((4-methylpiperazin-1-yl)methyl)-3,7,10-trioxa-2(5,4)-thieno[2,3-d]pyrimidin-1(1,4),6(1,3)-dibenzocyclodecane-4-carboxylic acid (12 mg, 70% yield).1H NMR (400 MHz, DMSO‑d6): δ = 9.20 (s, 2H), 8.84 (d,J= 5.2 Hz, 1H), 8.77 (s, 1H), 7.92‑7.78 (m, 4H), 7.52‑7.45 (m, 1H), 7.23-7.11 (m, 4H), 6.94-6.89 (m, 1H), 6.86-6.81 (m, 1H), 6.27-6.21 (m, 1H), 5.82-5.78 (m, 1H), 5.25-5.10 (m, 2H), 5.00-4.92 (m, 1H), 4.52-4.15 (m, 3H), 3.47-3.34 (m, 4H), 3.30-3.16 (m, 2H), 3.13-2.93 (m, 7H), 2.87 - 2.80 (m, 5H), 1.97 (s, 6H). LCMS (5‑95AB / 1.5min): Rt= 0.797min, m / z=981.2 [M+H]+.

[0287] Example 8: Synthesis specification of compound 103, pages 76 / 90, CN 122295346 A. Preparation of 5-bromopicolinimidine hydrochloride: NaOMe (32.4 mg, 0.6 mmol) was added to a solution of 5-bromo-2-pyridinecarboxynitrile (1.8 g, 10 mmol) in MeOH (50 mL), and the mixture was stirred at 25 °C for 16 h. Then NH4Cl (630 mg, 11.7 mmol) was added. The reaction solution was stirred at 75 °C for 3 h. The mixture was concentrated and then EtOH (30 mL) was added. The suspension was stirred at 80 °C for 0.5 h, filtered and concentrated to obtain a residue, which was ground together with dichloromethane (20 mL) and filtered to obtain a white solid 5-bromopicolinimidine hydrochloride (2 g, yield 87%).

[0288] Preparation of 2-(5-bromopyridin-2-yl)-4-(dimethoxymethyl)pyrimidine: (E)-4-(dimethylamino)-1,1-dimethoxybut-3-en-2-one (878 mg, 5.07 mmol) and NaOMe (685 mg, 12.68 mmol) were added to a solution of 5-bromopyridin-2-formamidine hydrochloride (1 g, 4.23 mmol) in MeOH (30 mL), and the mixture was stirred at 75 °C for 16 h under N2 atmosphere.The reaction mixture was concentrated and diluted with ethyl acetate (50 mL). The solution was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography (silica gel, 100-200 mesh, 10% to 60% ethyl acetate in petroleum ether) to give a white solid 2-(5-bromopyridin-2-yl)-4-(dimethoxymethyl)pyrimidine (1 g, yield 76%).

[0289] Preparation of tert-butyl (2-((6-(4-(dimethoxymethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate: A mixture of 2-(5-bromopyridin-2-yl)-4-(dimethoxymethyl)pyrimidine (500 mg, 1.61 mmol), tert-butyl (2-aminoethyl)carbamate (0.38 mL, 2.42 mmol), Cs2CO3 (1.3 g, 4 mmol) and rac-Binap-Pd-G3 (160 mg, 0.16 mmol) in dimethyl ether (10 mL) was stirred at 100 °C for 16 h under a N2 atmosphere. The reaction solution was concentrated to obtain a crude product, which was then purified by column chromatography (silica gel, 100-200 mesh, see DCM manual page 77 / 90, 87 CN 122295346 A, 0% to 10% MeOH) to obtain a yellow solid crude (2-((6-(4-(dimethoxymethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate tert-butyl ester (300 mg, purity 60%). LCMS (5-95AB / 1.5 min): Rt = 0.802 min, m / z = 390.1 [M+H]+.

[0290] Preparation of 2-(5-((2-aminoethyl)amino)pyridin-2-yl)pyrimidine-4-carboxaldehyde: A mixture of tert-butyl (2-((6-(4-(dimethoxymethyl)pyrimidine-2-yl)pyridin-3-yl)amino)ethyl)carbamate (300 mg, 0.77 mmol) and concentrated HCl (1 mL) in THF (4 mL) and water (3 mL) was stirred at 50 °C for 1 h. The reaction solution was concentrated to obtain 2-(5-((2-aminoethyl)amino)pyridin-2-yl)pyrimidine-4-carboxaldehyde, which was used directly in the next step.

[0291] Preparation of [2-[5-(2-aminoethylamino)-2-pyridyl]pyrimidin-4-yl]methanol: NaBH4 (100 mg, 2.64 mmol) was added to a solution of 2-[5-(2-aminoethylamino)-2-pyridyl]pyrimidin-4-carboxaldehyde (300 mg, 1.23 mmol) in MeOH (3 mL) and THF (5 mL) at 0 °C, and the mixture was stirred at 25 °C for 1 h. The reaction solution was quenched with NH4Cl aqueous solution (2 mL) and concentrated to obtain crude [2-[5-(2-aminoethylamino)-2-pyridyl]pyrimidin-4-yl]methanol (300 mg), which was used directly in the next step.

[0292] Preparation of tert-butyl (2-((6-(4-(hydroxymethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate: (Boc)2O (267 mg, 1.22 mmol) and TEA (0.51 mL, 3.67 mmol) were added to a solution of [2-[5-(2-aminoethylamino)-2-pyridinyl]pyrimidin-4-yl]methanol (300 mg, 1.22 mmol) in DCM (10 mL), and the mixture was stirred at 25 °C for 16 h. The reaction solution was concentrated to obtain a crude product, which was purified by preparative TLC (in 10% methanol in dichloromethane, Rf = 0.5) to obtain a yellow oily tert-butyl (2-((6-(4-(hydroxymethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate (70 mg, yield 17%). LCMS (5-95AB / 1.5min RT = 0.7 min, m / z = 346.1 [M+H]+.

[0293] Preparation of tert-butyl (2-((6-(4-(chloromethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate: TsCl (77 mg, 0.41 mmol) was added to a solution of tert-butyl (2-((6-(4-(hydroxymethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate (70 mg, 0.2 mmol) and DIEA (0.14 mL, 0.81 mmol) in DCM (2 mL), and the mixture was stirred at 25 °C for 16 h. The reaction solution was concentrated under vacuum and passed through a rapid column (0% to 2% in DCM). (MeOH) purification to give a yellow oily tert-butyl (2-((6-(4-(chloromethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate (20 mg, yield 27%). LCMS (5-95AB / 1min): Rt = 0.475 min, m / z = 364.2 [M+H]+.

[0294] Preparation of (4R,9R)-66-((2-(5-((2-((tert-butoxycarbonyl)amino)ethyl)amino)pyridin-2-yl)pyrimidin-4-yl)methoxy)-13,15-dichloro-26-(4-fluorophenyl)-12,16-dimethyl-9-((4-methylpiperazin-1-yl)methyl)-3,7,10-trioxa-2(5,4)-thieno[2,3-d]pyrimidin-1(1,4),6(1,3)-dibenzocyclodecane-4-carboxylic acid tert-butyl ester: 1 g of macrocyclic intermediate was prepared according to the procedure of Example 2. The mixture of 1 g (20 mg, 0.02 mmol) of intermediate, (2-((6-(4-(chloromethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate tert-butyl ester (10 mg, 0.025 mmol) and Cs2CO3 (20 mg, 0.06 mmol) in MeCN (1 mL) was stirred at 60 °C for 1 h. The mixture was concentrated and purified by preparative TLC (in 10% MeOH in DCM) to give a white solid (4R,9R)-66-((2-(5-((2-((tert-butoxycarbonyl)amino)ethyl)amino)pyridin-2-yl)pyrimidin-4-yl)methoxy)-13,15-dichloro-26-(4-fluorophenyl)-12,16-dimethyl-9-((4-methylpiperazin-1-yl)methyl)-3,7,10-trioxa-2(5,4)-thieno[2,3-d]pyrimidin-1(1,4),6(1,3)-dibenzocyclodecane-4-carboxylic acid tert-butyl ester (10 mg, yield 36%).

[0295] Preparation of compound 103: A mixture of (4R,9R)-66-((2-(5-((2-((tert-butoxycarbonyl)amino)ethyl)amino)pyridin-2-yl)pyrimidin-4-yl)methoxy)-13,15-dichloro-26-(4-fluorophenyl)-12,16-dimethyl-9-((4-methylpiperazin-1-yl)methyl)-3,7,10-trioxa-2(5,4)-thieno[2,3-d]pyrimidin-1(1,4),6(1,3)-dibenzo[4,3]-cyclodecane-4-carboxylic acid tert-butyl ester (10 mg, 0.01 mmol) and TFA (0.3 mL) in DCM (1 mL) was stirred at 25 °C for 16 h. The mixture was concentrated and purified by preparative HPLC (acetonitrile 18-48 / 0.025% TFA in water) to give compound 103 (2.4 mg, 28% yield) as a white solid.1H NMR (400 MHz, DMSO‑ d6) δ = 8.92‑8.85 (m, 1H), 8.76 (s, 1H), 8.33‑8.28 (m, 1H), 8.19‑8.08 (m, 1H), 7.85‑7.80 (m, 2H), 7.57-7.49 (m, 1H), 7.27-7.11 (m, 6H), 6.93-6.83 (m, 2H), 6.29-6.20 (m, 1H), 5.83-5.68 (m, 1H) , 5.30-5 .17 (m, 2H), 5.01–4.87 (m, 1H), 4.56–4.38 (m, 3H), 3.12–2.95 (m, 11H), 2.86–2.74 (m, 7H), 2.06 (s, 3H), 1.95 (s, 3H).

[0296] Example 9: Preparation of Additional Loading Compounds Additional loading compounds were generally prepared according to the procedures outlined in Examples 1 to 8. For compound 104, the synthesis of intermediate 1g was generally performed, with 1-methylpiperazine replaced by 1-ethylpiperazine. For compounds 105 to 113, the synthesis of intermediate 1e was generally performed, with tert-butyl (2-aminoethyl)carbamate replaced by a boc-protected amino group.

[0297] Specification 79 / 90 pages 89 CN 122295346 A Specification 80 / 90 pages 90 CN 122295346 A Specification 81 / 90 pages 91 CN 122295346 A Example 10: General procedure for linker-drug intermediates The preparation of additional linker-drug intermediates using activated sq-Cit peptide mimic linkers and other payload drugs generally follows the procedure described in Example 5 for compound 100.

[0298] Example 11: General procedure for linker-drug intermediate conjugation with antibody The pH of a cysteine-engineered antibody (THIOMAB™, selected for use in ADCs) in 10 mM succinate (pH 5), 150 mM NaCl, and 2 mM EDTA is adjusted to pH 7.5–8.5 with 1 M Tris. Dissolve 10 mM linker-drug intermediates (with thiol-reactive maleimide groups) in DMF or DMSO in 10 to 18 equivalents and add them to the reduced, reoxidized and pH-adjusted antibody.The reaction was incubated at room temperature (37 °C) and monitored until completion (1 hour to approximately 24 hours), as determined by LC-MS analysis of the reaction mixture. Upon completion, the Ab-linker-loador conjugate was purified by one or more methods in any combination to remove any remaining unreacted linker-drug intermediates and aggregates (if present at significant levels). For example, the conjugate may have been diluted with 20 mM histidine-acetate (pH 5.5) to a final pH of approximately 5.5 and purified by S cation exchange chromatography using a HiTrap S column or S maxi centrifuge column (Pierce) connected to an Akta purification system (GE Healthcare). Alternatively, the conjugate may have been purified by gel filtration chromatography using an S200 column or Zeba centrifuge column connected to an Akta purification system. Alternatively, dialysis may have been used.

[0299] For the conjugates discussed and characterized in these examples, the Ab-linker-loador conjugates were formulated to 20 mM His / acetate (pH 5) and 240 mM sucrose using gel filtration or dialysis. The purified conjugates were concentrated by centrifugal ultrafiltration, filtered through a 0.2-μm filter under aseptic conditions, and stored frozen.

[0300] The resulting conjugates were characterized to determine protein concentration by BCA assay, aggregation level by SEC analysis, and DAR by LC-MS, as described below.

[0301] SEC: The Ab-linker-loador conjugates were subjected to particle size sieving chromatography using a Shodex KW802.5 column in 0.2 M potassium phosphate (pH 6.2) containing 0.25 mM potassium chloride and 15% IPA, at a flow rate of 0.65 ml / min. The aggregation state of the conjugate was determined by integrating the peak area absorbance eluted at 280 nm.

[0302] LC-MS: The conjugate was analyzed by LC-MS using an Agilent TOF 6530 ESI instrument. For example, the Ab-linker-loaded conjugate was treated with 25 mM DTT at 37 °C for 15 min, resulting in reduction between the heavy and light antibody chains. The resulting cleaved fragment was loaded onto a 1000 Å, 8 μm PLRP-S (highly cross-linked polystyrene) column heated to 80 °C and eluted with a gradient of 30% B to 40% B over 10 min.Mobile phase A was a 0.05% TFA H2O solution, and mobile phase B was a 0.05% TFA acetonitrile solution. The flow rate was 0.5 ml / min. Protein elution was monitored by UV absorbance detection at 280 nm prior to electrospray ionization and MS analysis. The obtained m / z spectra were deconvolved using Mass Hunter™ software (Agilent Technologies) to calculate the mass of the antibody fragments. Chromatographic separation of unconjugated LC, HC, and conjugated fragments was observed. In some cases, fragmentation within the linker drug source was observed. These fragments were considered for the final characterization and calculation of the drug-to-Ab ratio. The ratio was calculated by analyzing the peak intensity ratio of each observed fragment.

[0303] Specific example of an anti-Her2 7C2 ADC: The anti-Her2 7C2 antibody was prepared as described in PCT / US1997 / 018385. The sqCit linker-compound 100 conjugate was prepared according to Example 5 above. Four equivalents of the sqCit linker-compound 100 conjugate (concentration = 10 mM) dissolved in DMF or DMSO were added to an anti-Her2-7C2 antibody in 10 mM succinate (pH 5), 150 mM NaCl, and 2 mM EDTA. The pH of this mixture was adjusted to 7.5–8.5 with 1 M Tris and incubated at room temperature under monitoring until completion was confirmed by LC-MS. When the conjugation reaction was complete, the resulting anti-Her2-7C2-sqCit-compound 100 conjugate was purified to remove unreacted linker-drug intermediates and analyzed by particle size sieving chromatography (SEC) to determine the yield, drug-to-Ab ratio (DAR), and percentage of aggregation. The SEC analysis trace of the anti-Her2-7C2-sqCit compound 100 is shown in Figure 8A, with a yield of 64.83%. LC-MS analysis of the light and heavy chains of the conjugate is shown in Figures 8B and 8C, respectively. The drug-to-Ab ratio in the conjugate was calculated to be 1.9; the aggregation rate was determined to be 0.46%.

[0304] ADCs of compound 100 and additional payload compounds 101 to 107 were prepared and characterized in the biological examples provided herein. The general structures of the ADCs, exhibiting the payload and connection points of the sq-Cit linker, are provided below. Specific Abs and DARs used (if determined) are provided in specific examples.Specification 83 / 90 pages 93 CN 122295346 A

[0305] Specification 84 / 90 pages 94 CN 122295346 A Biological Example 1: Evaluation of the effects of comparative compounds on the in vitro viability of adenocarcinoma cells and breast cancer cells After incubation for 5 days, the cytotoxicity of comparative compounds A, B and C as free drugs in SK-BR-3 adenocarcinoma cells and their cytotoxicity alone in CAMA-1 breast cancer cells were evaluated. Figure 2 shows the structures of the compounds evaluated. Comparative compound A was prepared as described in PCT / US2018 / 000183. Comparative compound B was prepared as described in PCT / EP2014 / 078947. Comparative compound C was prepared as described in PCT / US2018 / 000180.

[0306] The human breast cancer cell line was obtained from the American Type Culture Collection (ATCC). Cells were seeded in 96-well black-walled plates in RPMI supplemented with 10% FBS and 2 mmol / L L-glutamine (4000 cells per well for SK-BR-3 and 8000 cells per well for CAMA1) and allowed to adhere overnight in a humid atmosphere at 37°C and 5% CO2. The medium was then removed and replaced with fresh RPMI medium containing different concentrations of free drug or conjugate (dose range = 0.0005 to 10 mM or mg / mL). After 5 days, 100 μL of Cell Titer-Glo reagent (Promega Corp.) was added to the wells at room temperature for 10 min, and the luminescence signal was measured using an EnVision Multilabel microplate reader (PerkinElmer). All cell assays were performed in quadruplicate. A DMSO control was included in all studies.

[0307] Figure 3A is a graph showing the viability of SK-BR-3 adenocarcinoma cells relative to compound concentration. Figure 3B is a graph showing the viability of CAMA-1 breast cancer cells relative to compound concentration. Table 1a provides a summary of the IC50 of each tested compound in each cell line. Compound A exhibited the highest cytotoxic potency in both cell lines (as measured by the lowest IC50). Acyclic compound B was the second most potent molecule in both cell lines. Compound C was the least potent molecule as assessed.

[0308] Table 1a. Summary of IC50 of comparative acyclic and cyclic MCL1 inhibitors in two different cell lines, where IC50 is reported in nanomolar units (CN 122295346 A, page 85 / 90 of the specification).

[0309] Following the same procedure, cyclic MCL1 inhibitors prepared according to the examples provided herein were evaluated in SK-BR-3 cells. The IC50 of those free compounds in SK-BR-3 cells is provided in Table 1b. Each molecule tested exhibited a higher IC50 than the compounds in Table 1a, with most molecules showing significantly poorer inhibitory activity. Compound 100 had an IC50 of 0.43 µM, which was less than 1 / 25th the activity of compound C (16.3 nM) and less than 1 / 2000th the activity of compound A (0.2 nM, estimated).

[0310] Table 1b. Summary of IC50 of selected cyclic MCL1 inhibitors described herein in SK-BR-3 cells, where IC50 is reported in micromoles.

[0311] Biological Example 2: General Procedures for In Vitro Cell Assays of Acute Myeloid Leukemia (AML) and Non-Hodgkin Lymphoma (NHL) The in vitro cell assays for evaluating the cytotoxic activity of the free compound and the compound as a conjugate were performed according to the same general procedures.

[0312] All acute myeloid leukemia and non-Hodgkin lymphoma cell lines (AML: MV-4-11, EOL-1, Molm-13, Nomo-1, HL-60, and OCL-AML-3; NHL: Su-DHL-5 and Su-DHL-10) were originally obtained from the ATCC or DSMZ cell banks. Cells were cultured in PRMI-1640 medium supplemented with 10% FBS and 2% glutamine. 2000 to 4000 cells were seeded at 100 µL / well in 96-well plates and incubated overnight at 37°C in a standard tissue incubator. Cells were then exposed to various concentrations of MCL1 inhibitors (free drug) or antibody-drug conjugates (ADCs) reagents, serially diluted 1:3 from stock solutions. After 5 days of incubation with the free drug and 7 days with the ADCs, Cell Titer-Glo reagent (Promega, Madison WI) was added to the wells at 100 µL / well, and the cells were incubated at room temperature for 10 min before recording the luminescence signal. Data represent one of three separate experiments (each experiment was repeated twice).

[0313] Biological Example 3: Evaluation of the effect of comparative compounds on the in vitro viability of lymphocytes The cytotoxicity of SU-DHL5 and SU-DHL10 cells as free drugs was evaluated according to the general procedures used for the above in vitro NHL cell assays.Figures 4A and 4B are graphs showing the activity of SU-DHL5 and SU-DHL10 relative to compound concentrations, respectively. The IC50 of each small molecule inhibitor in each cell line is summarized in Table 2. In this evaluation, in the SU-DHL5 cell line, compound A was the most cytotoxic, compound C was the second most cytotoxic, and compound B was the least cytotoxic; and in the SU-DHL10 cell line, compound A was the most cytotoxic, compound B was the second most cytotoxic, and compound C was the least cytotoxic.

[0314] Table 2. Summary of IC50 of comparative acyclic and cyclic MCL1 inhibitors in two different cell lines.

[0315] The results of biological examples 1 and 3 show that, across four cell lines and in two types of cancer models, compound A was the most cytotoxic molecule.

[0316] Biological Example 4: Evaluation and Comparison of the Effects of Compounds 100 on the In Viability of Acute Myeloid Leukemia (AML) Cell Lines Following the general procedures used for the above-described in vitro AML cell assays, the cytotoxicity of compounds 100, A, and B as free drugs in the AML cell lines MOLM-16, HL-60, OCI-AML3, and SU-DHL-10 was evaluated.

[0317] Figures 5A to 5C are graphs showing the cell viability of MOLM-16, HL-60, and OCI-AML3 relative to the compound concentrations, respectively. As a free drug, compound 100 was significantly less potent as a cytotoxic agent than either compound A or B in all four cell lines (less than 1 / 90 in MOLM-16; less than 1 / 250 in HL-60; less than 1 / 30 in OCI-AML3; and less than 1 / 70 in SU-DHL-10).

[0318] Table 3. Summary of IC50 comparison of compounds A and B and compound 100 in four different AML cell lines. Biological Example 5: Evaluation of anti-CD33 ADCs containing payloads of compound A, compound B or compound 100 in AML cell lines. Anti-CD33 antibody 15G15, sq-Cit linker and ADCs containing payloads of compound 100, compound A or compound B were prepared according to the procedures described in Examples 5 and 11. The 15G15 antibody is disclosed in more detail in PCT / US2014 / 069874.

[0319] Following the general procedures used for the above-described in vitro AML cell assays, the cytotoxicity of three different ADCs in the in vitro AML cell lines EOL-1, MV-4-11, and NOMO-1 was evaluated.

[0320] Figures 6A to 6C are graphs showing cell viability relative to compound concentrations in EOL-1, MV-4-11, and NOMO-1, respectively. The ADC containing compound 100 as the payload exhibited significantly improved cytotoxicity in the AML cell line EOL-1, as shown in Figure 6A. It maintained high cytotoxic potency in the cell line MV-4-11, as shown in Figure 6B. In the cell line NOMO-1, the compound 100 ADC showed reduced cytotoxic potency, but was still superior to the ADCs containing compound A or compound B as the payload. As described in the previous examples, the potency of the free drug compound 100 was significantly lower than that of the comparative compounds. However, when compound 100 was used as the payload in the ADC, the ADC showed comparable or better cytotoxic potency than the ADC using comparative compounds A or B. The significant increase in toxicity when the free drug was converted into the ADC was surprising and unexpected.

[0321] Biological Example 6: Evaluation of anti-HER2 7C2-sq-Cit-payload ADC using compound B, compound C or compound 100 as payload The ADC using anti-HER2 antibody 7C2, sq-Cit linker and payload compound B, compound C or compound 100 was prepared according to the procedures set forth in Examples 5 and 11. The 7C2 antibody is disclosed in more detail in PCT / US1997 / 018385. The in vitro cytotoxicity of the ADC in the breast cancer cell line SK-BR-3 was evaluated according to the procedures in Biological Example 1 for the above-described SK-BR-3 cell assay. Figure 7 is a graph of the activity of each ADC. As shown in the figure and the table below, the ADC using compound 100 as the payload has significantly better cytotoxic activity compared to ADCs using the same antibody and the same adapter but using either compound C or compound B as the payload. The DAR (drug loading rate) of the compound 100 and compound C ADCs is approximately 2; however, the DAR of the compound B ADC is approximately three times, i.e., approximately 6 drug molecules per antibody. Even with this higher drug loading, the cytotoxicity of the ADC containing compound B is still significantly lower than that of the ADC containing compound 100.This demonstrates a surprisingly improved cytotoxic efficacy of the ADC containing compound 100 as a payload in the breast cancer cell line SK-BR-3. The ADC using compound 100 exhibits better EC50 and maximum cell killing than either compound C or B. It is not wishful thinking to be bound by theory, but this is likely due to the improved cell retention of compound 100 compared to compounds B and C.

[0322] Table 4.7 Activity of C2-sq-Cit-payload ADCs on the SK-BR-3 breast cancer cell line.

[0323] Biological Example 7: Evaluation of the effect of anti-CD33 ADCs containing various exemplary payload compounds on the in vitro MV4-11 cell line. ADCs containing anti-CD33 antibody 15G15, sq-Cit linkers, and payload compounds 100 to 107 were prepared according to the procedures described in Examples 5 and 11. Following the procedure described in Biological Example 2, the cytotoxicity of these ADCs was assessed using an in vitro MV4-11 assay. Graphs of these results are shown in Figure 9. The ADC containing compound 100 as the payload had a DAR of approximately 6; all other ADCs had a DAR of approximately 4.

[0324] Biological Example 8: Comparison of the effect of anti-CD33 ADCs on tumor burden in an in vivo disseminated AML model. ADCs containing anti-CD33 antibody 15G15, a sq-Cit linker, and payload compound 100 (ADC-100) or compound B (ADC-B) were prepared following the procedures described in Examples 5 and 11, with a DAR of 6. For comparison, a non-target anti-gD ADC (gD is a viral protein not expressed on tumor cells) was also prepared. The cytotoxicity of these targeted ADCs at 30 mg / kg or 10 mg / kg, the non-targeted ADC at 30 mg / kg, and the mediator (control) was assessed in NSG mice using in vivo MV4-11 assays.

[0325] Mice were preconditioned by intraperitoneal injection of 20 mg / kg busulfan (Busilvex, Pierre Fabre, France) 24 h prior to inoculation with leukemia cells. Busulfan was pharmaceutical grade and diluted with sterile saline. Mice were then inoculated with MV-411 luciferase / GFP+ leukemia cells (2 million cells) under sterile conditions (cells suspended in HBSS and injected intravenously into recipient mice at 100 μL). Animals were monitored weekly by bioluminescence imaging to track tumor progression over time.When tumors were detected by imaging, animals were randomly assigned to groups and treatment was initiated (referred to as Day 0 of the study). All animals received an intraperitoneal injection of 30 mg / kg anti-ragweed to minimize nonspecific ADC uptake via Fc receptors expressed on tumor cells. Four hours later, either the ADC or the mediator (20 mM histidine acetate, 240 mM sucrose, 0.02% Tween 20, pH 5.5) was administered via a single intravenous injection into the tail vein of mice. At the end of the study, whole blood was collected from the animals to assess treatment efficacy.

[0326] These results are illustrated in Figure 10, which shows the percentage of tumor burden assessed 9 days after ADC administration. As shown in Figure 10, both anti-CD33 ADCs showed antitumor activity, with ADC-100 being more effective in reducing hematopoietic tumor burden. The corresponding non-target conjugate had the least effect compared to the mediator control.

[0327] Biological Example 9: Evaluation of the dose-response of compound 1 ADC to tumor burden in an in vivo disseminated AML model. The dose-response was evaluated in NSG mice using the DAR6 ADC with anti-CD33 antibody 15G15, sq-Cit linker, and payload compound 100 in an in vivo MV4-11 assay. The procedure was the same as in Biological Example 8 above, except that bone marrow from the femur was collected at the end of the study to assess the therapeutic effect. Bone marrow tumor burden on day 7 (in live tumor cells / 50kJ) is shown in Figure 11. The mean values ​​of the data are listed in Table 5.

[0328] Table 5. Mean values ​​of live tumor cell counts on day 7, plotted in Figure 11.

[0329] This data illustrates that the 15G15-compound 100 ADC exhibits dose-response inhibition in an in vivo disseminated AML model. Even at the lowest dose of 0.1 mg / kg, a reduction in tumor cells (relative to the catalyst) was observed.

[0330] Biological Example 10: Evaluation of the effect of anti-CD33 ADCs on tumor burden in an in vivo subcutaneous model. ADCs containing anti-CD33 antibody 15G15, a sq-Cit linker, and payload compound 100 (ADC-100) or compound B (ADC-B) were prepared according to the procedures described in Examples 5 and 11, wherein the DAR was 6. The cytotoxicity of these ADCs relative to the mediator or unmodified antibody (Ab) at 30 mg / kg was evaluated in SCID mice using an in vivo subcutaneous MV4-11 assay.Mice were subcutaneously inoculated with MV4-11 leukemia cells (10 million cells in 0.1 mL HBSS) in the flank region. When the tumor reached approximately 200 mm³, the animals were randomized and treatment began (referred to as Day 0 of the study). All animals received an intraperitoneal injection of 30 mg / kg anti-ragweed to minimize nonspecific ADC uptake via Fc receptors expressed on the tumor cells. Four hours later, the ADC or mediator (20 mM histidine acetate, 240 mM sucrose, 0.02% Tween 20, pH 5.5) was administered to the mice via a single intravenous injection in the tail vein. Two dimensions of the tumor (length and width, perpendicular to each other) were measured using calipers (Model 54-10-111; Fred V. Fowler Co.), and the tumor volume was calculated using the following formula: Tumor volume (mm³) = (length x width²) x 0.5.

[0331] A graph of tumor volume over time is shown in Figure 12. As shown in Figure 12, the ADC containing compound 100 was less effective in reducing tumor volume in the subcutaneous model compared to the ADC containing compound B. Without being bound by theory, this, combined with the results from Example 8, illustrates the unexpected advantage of compound 100 as an ADC in disseminated (or liquid) cancers compared to solid tumors. Without being bound by theory, this may be because compound 100 contains a net overall charge that inhibits the ability of passive diffusion across the membrane. This could lead to a reduced bystander effect (activity in non-targeting neighboring cells), which could result in reduced viability in solid tumors with lower tumor penetration or antigenic heterogeneity. However, this could also improve safety by not killing neighboring “healthy” cells and increase activity in liquid tumors (compared to free, diffusible, neutral payloads) by retaining the charged payload within the targeted cancer cells. The net overall charge is partly a result of the presence of amine functional groups.Instruction manual 90 / 90 pages 100 CN 122295346 A Figure 1 Instruction manual Figure 1 / 15 pages 101 CN 122295346 A Figure 2 Instruction manual Figure 2 / 15 pages 102 CN 122295346 A Figure 3A Figure 3B Instruction manual Figure 3 / 15 pages 103 CN 122295346 A Figure 4A Figure 4B Instruction manual Figure 4 / 15 pages 104 CN 122295346 A Figure 5A Figure 5B Instruction manual Figure 5 / 15 pages 105 CN 122295346 A Figure 5C Figure 6A Instruction manual Figure 6 / 15 pages 106 CN 122295346 A Figure 6B Figure 6C Instruction manual Figure 7 / 15 pages 107 CN 122295346 A Figure 7 Instruction manual Figure 8 / 15 pages 108 CN 122295346 A Figure 8A Figure 8B, Figure 8C, Figure 8B, Figure 8C, Figure 8C, Figure 8C, Figure 8B, Figure 8B, Figure 8B, Figure 8C ...

Claims

1. A compound of formula (X): (X)， Or its pharmaceutically acceptable salt, wherein: X is O or NH; Z is NC 1-6 Alkyl or NH; Ring A is phenyl or a 6-membered heteroaryl group; Ring B is phenyl or a 6-membered heteroaryl group; R 1 It is an alkyne or a phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R 2 It is an alkyl, cycloalkyl, heterocycloalkyl or -R 2a (OR 2a ) p -; wherein the alkyl, cycloalkyl, or heterocycloalkyl group is unsubstituted or is independently selected from one or more halogens, alkyl groups, -OH, -OR. 2b and -O(R) 2b O) q R 2c The substituents of the group are substituted, or two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R 2c It is independently hydrogen, alkyl, or haloalkyl; p and q are independent integers from 1 to 8; R 3 For –NH2, –NHR 3a –N(R) 3a )2 or –N + (R 3a )3, where each R 3a Independently alkyl, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy and haloalkoxy.

2. The compound according to claim 1, wherein the compound is a compound of formula (I): (I)， Or its pharmaceutically acceptable salt, wherein: X is O or NH; Z is NC 1-6 Alkyl or NH; Ring A is phenyl or a 6-membered heteroaryl group; Ring B is phenyl or a 6-membered heteroaryl group; R 1 It is an alkyne or a phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R 2 It is an alkyl, cycloalkyl, heterocycloalkyl or -R 2a (OR 2a ) p -; wherein the alkyl, cycloalkyl, or heterocycloalkyl group is unsubstituted or is independently selected from one or more halogens, alkyl groups, -OH, -OR. 2b and -O(R) 2b O) q R 2c The substituents of the group are substituted, or two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R 2c It is independently hydrogen, alkyl, or haloalkyl; p and q are independent integers from 1 to 8; and R 3 For –NH2, -NHR 3a -N(R) 3a )2 or –N + (R 3a )3, where each R 3a Each is independently an alkyl group, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogens and –OH.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein ring A and ring B are independently phenyl or heteroaryl containing one or two ring N atoms.

4. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein ring A is pyrimidine and ring B is phenyl, pyrimidine, or... ,in Indicates the connection point with ring A.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R 1 It is a phenyl group that has been substituted with one fluorine molecule.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R 2 It is a C1-C6 alkyl group, which is either unsubstituted or substituted by one or more substituents independently selected from the group consisting of –OH and halogen.

7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Z is N-CH3 or N-CH2CH3.

8. The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein R 3 It can be –NH2 or –NHCH3.

9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein X is O.

10. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein: Z is N-CH2CH3 or N-CH3; Ring A is a pyrimidine; Ring B is phenyl, pyrimidine, or... ,in Indicates the connection point with ring A; R 1 It is a phenyl group that has undergone one fluorine substitution; R 2 It is an unsubstituted or fluorinated C1-C6 alkyl group; R 3 For –NH2 or –NH(C) 1-6 Alkyl); and X is 0.

11. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein X is O, R 1 R is a phenyl group that has undergone one fluorine substitution. 2 It is an alkyl group, and R 3 It is –NH2.

12. The compound according to claim 2, wherein the compound is a compound of formula (II): (II), Or its pharmaceutically acceptable salt, wherein: X, R 1 R 2 and R 3 As defined for equation (X).

13. The compound of claim 12 or a pharmaceutically acceptable salt thereof, wherein R 2 It is an unsubstituted or fluorinated C1-C6 alkyl group; and R 3 For –NH2 or –NH(C) 1-6 alkyl).

14. The compound according to claim 1, wherein the compound is: , , , , , , , , , , , , or Or, for example, a pharmaceutically acceptable salt.

15. A conjugate of formula (A): Ab-(L-(DP) r ) m , Or its pharmaceutically acceptable salt, wherein: Ab represents antibodies; L stands for connector; DP represents the drug payload; r is an integer from 1 to 8; and m is an integer from 1 to 10; The drug payload is a compound of formula (X): (X)， Or its pharmaceutically acceptable salt, wherein: X is O or NH; Z is NC 1-6 Alkyl or NH; Ring A is phenyl or a 6-membered heteroaryl group; Ring B is phenyl or a 6-membered heteroaryl group; R 1 It is an alkyne or a phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R 2 It is an alkyl, cycloalkyl, heterocycloalkyl or -R 2a (OR 2a ) p -; wherein the alkyl, cycloalkyl, or heterocycloalkyl group is unsubstituted or is independently selected from one or more halogens, alkyl groups, -OH, -OR. 2b and -O(R) 2b O) q R 2c The substituents of the group are substituted, or two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R 2c It is independently hydrogen, alkyl, or haloalkyl; p and q are independent integers from 1 to 8; and R 3 For –NH2, –NHR 3a –N(R) 3a )2 or –N + (R 3a )3, where each R 3a Independently alkyl, wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy and haloalkoxy.

16. The conjugate according to claim 15, wherein the conjugate of formula (A) is the conjugate of formula (B): (B)， Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; m is an integer from 1 to 10; and X, R 1 R 2 R 3a Z, ring A and ring B are as defined with respect to equation (X).

17. The conjugate or a pharmaceutically acceptable salt thereof according to claim 15 or 16, wherein the linker is a peptide linker or a peptide mimic linker.

18. The conjugate according to any one of claims 15 to 17, wherein the conjugate is a conjugate of formula (B-1): R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; m is an integer from 1 to 10; X, R 1 R 2 R 3a Z, ring A, and ring B are as defined with respect to equation (X); Each R L1 Independently for C1-C 10 Alkyl, C1-C 10 alkenyl, C1-C 10 Alkyl NHC(NH)NH2 or C1-C 10 Alkyl NHC(O)NH2; and R L3 and R L2 Each independently constitutes H, C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl or heteroarylalkyl, or R L3 and R L2 Together they can form C3-C7 cycloalkyl groups.

19. The conjugate according to any one of claims 15 to 18, wherein: Z is N-CH2CH3 or N-CH3; Ring A is a pyrimidine; Ring B is phenyl, pyrimidine, or... ,in Indicates the connection point with ring A; R 1 It is a phenyl group that has undergone one fluorine substitution; R 2 It is an unsubstituted or fluorinated C1-C6 alkyl group; R 3 For –NH2 or –NH(C) 1-6 Alkyl); and X is 0.

20. The conjugate according to claim 16, wherein the conjugate of formula (A) is a conjugate of formula (C): (C)， Or its pharmaceutically acceptable salt, wherein: R 3 For –NH, –NR 3a or –N + (R 3a )2; m is an integer from 1 to 10; and And X, R 1 R 2 and R 3a As defined for equation (X).

21. The conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein R 2 It is an unsubstituted or fluorinated C1-C6 alkyl group; and R 3 For –NH2 or –NH(C) 1-6 alkyl).

22. The conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 15 to 18, wherein the drug payload is: , , , , , , or .

23. The conjugate according to claim 15, wherein the conjugate has the following structure: 。 24. The conjugate according to any one of claims 15 to 23, wherein the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from the group consisting of: CLL1, CD71, Trop2, MSLN, NaPi2b, Ly6E, EpCAM, STEAP1, STEAP2, TrpM4, CD21, CD79a, CD72, MUC16, HER2, CD33, CD22, CD79b, LIV1, CD123, CD74, BCMA, and FcRH5.

25. A pharmaceutical composition comprising: a conjugate according to any one of claims 15 to 24, and a pharmaceutically acceptable excipient.

26. A method of treating a condition in a subject who requires such treatment, the method comprising administering to the subject a therapeutically effective amount of the conjugate according to any one of claims 15 to 24.

27. The method of claim 26, wherein the condition is cancer, tumor or other malignant tumor.

28. The method of claim 27, wherein the disease is selected from the group consisting of: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), and myeloid cell leukemia (MCL).

29. A compound used in a method of treating a condition in a subject with such need, wherein said compound is a conjugate according to any one of claims 15 to 24.

30. The compound used according to claim 29, wherein the condition is cancer, tumor or other malignant tumor.

31. The compound used according to claim 30, wherein the disease is selected from the group consisting of: squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), and myeloid cell leukemia (MCL).

32. A drug payload-connector conjugate, wherein the drug payload-connector conjugate is of formula (B-L1): (B-L1), Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; where each R 3a Each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogens, –OH, alkoxy groups and haloalkoxy groups; Each R L1 Independently for C1-C 10 Alkyl, C1-C 10 alkenyl, C1-C 10 Alkyl NHC(NH)NH2 or C1-C 10 Alkyl NHC(O)NH2; R L3 and R L2 Each independently constitutes H, C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl or heteroarylalkyl, or R L3 and R L2 Together they can form C3-C7 cycloalkyl groups; X is O or NH; Z is NC 1-6 Alkyl or NH; Ring A is phenyl or a 6-membered heteroaryl group; Ring B is phenyl or a 6-membered heteroaryl group; R 1 It is an alkyne or a phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R 2 It is an alkyl, cycloalkyl, heterocycloalkyl or -R 2a (OR 2a ) p -; wherein the alkyl, cycloalkyl, or heterocycloalkyl group is unsubstituted or is independently selected from one or more halogens, alkyl groups, -OH, -OR. 2b and -O(R) 2b O) q R 2c The substituents of the group are substituted, or two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R 2c Independently hydrogen, alkyl, or haloalkyl; and p and q are independent integers from 1 to 8.

33. The drug payload-connector conjugate according to claim 32, wherein the drug payload-connector intermediate is of formula (C-L1): (C-L1), Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; wherein each alkyl group is independently unsubstituted or substituted by one or more substituents independently selected from the group consisting of halogen, –OH, alkoxy and haloalkoxy; Each R L1 Independently for C1-C 10 Alkyl, C1-C 10 alkenyl, C1-C 10 Alkyl NHC(NH)NH2 or C1-C 10 Alkyl NHC(O)NH2; R L3 and R L2 Each independently constitutes H, C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl or heteroarylalkyl, or R L3 and R L2 Together they can form C3-C7 cycloalkyl groups; X is O or NH; R 1 It is an alkyne or a phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R 2 It is an alkyl, cycloalkyl, heterocycloalkyl or -R 2a (OR 2a ) p -; wherein the alkyl, cycloalkyl, or heterocycloalkyl group is unsubstituted or is independently selected from one or more halogens, alkyl groups, -OH, -OR. 2b and -O(R) 2b O) q R 2c The substituents of the group are substituted, or two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl or haloheterocycloalkyl; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R 2c Independently hydrogen, alkyl, or haloalkyl; and p and q are independent integers from 1 to 8.

34. The drug payload-connector conjugate according to claim 32, wherein the drug payload-connector intermediate has the following structure: 。

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