Mcl-1 inhibitor compounds and uses in antibody drug conjugates

Abstract

This article describes compounds of formula (I) and their pharmaceutically acceptable salts. It further describes antibody-drug conjugates (ADCs) comprising compounds of formula (I) or their pharmaceutically acceptable salts as payloads. Methods for treating conditions are also described.

Description

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 Technology

[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-protein interactions with pro-apoptotic BH3 proteins (BIM, PUMA, NOXA) to regulate apoptosis. Inhibition of MCL1 by BH3 mimicry therapies 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, subsequently leading to apoptosis. MCL1 is amplified in approximately 10% of human cancers, widely expressed in both hematologic malignancies and solid tumors, and 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 (AML) is a type of blood cell cancer 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, eventually leading to death. Recent advances in targeted therapy have allowed a subset 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 in the successful application of antibody-drug conjugates (ADCs) for treating cancer patients has been the presence of toxicity at (or even below) effective doses. A narrow therapeutic index, or the absence of a therapeutic index, can result from a variety of factors. The therapeutic use of first-generation antibody-drug conjugates (such as Mylotarg) has been limited due to the unstable linker between the drug or payload and the antibody. Premature release of cytotoxic payloads reduces efficacy and increases off-target toxicity. However, even ADCs with stable linkers can 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 various types of cancer, including AML, significant unmet needs remain that directly impact patient lives. One approach to improving the safety profile of ADCs is to utilize novel payloads that exhibit improved selectivity between tumor cells and normal tissues. Summary of the Invention

[0006] In some respects, this paper provides 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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 halogens and –OH.

[0007] In other respects, this document provides sub-formulas of formula (I) and exemplary compounds of formula (I). Further, it provides conjugates comprising compounds of formula (I), and methods of using said compounds and conjugates. Thus, in another aspect, this document provides 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 effective payload of the drug is 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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 halogens and –OH.

[0008] In some aspects of the ADCs presented 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 an additional aspect, this document provides a pharmaceutical composition comprising any of the conjugates provided herein, and a pharmaceutically acceptable excipient. Further, a method is provided for treating a condition in a subject of need 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, a tumor, or other malignancy.

[0010] In addition, this paper provides 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 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; 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 They can together 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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.

[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. Attached Figure Description

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

[0013] Figure 2 The structures of compounds A, B, and C are provided for comparison and are evaluated in some of the biological examples presented herein.

[0014] Figure 3A and Figure 3B A graph illustrating 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) is shown as described in Biological Example 1.

[0015] Figure 4A and Figure 4B A graph illustrating the cytotoxic efficacy of different concentrations of compounds A, B, and C as free drugs against SU-DHL5 (Fig. 4A) and SU-DHL10 (Fig. 4B) acute myeloid leukemia (AML) cell lines is shown in Biological Example 3.

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

[0017] Figure 6A , Figure 6B and Figure 6C A graph illustrating 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 (Fig. 6A), MV-4-11 (Fig. 6B), and NOMO-1 (Fig. 6C) is shown as described in Biological Example 5.

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

[0019] Figure 8A 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 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 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 A graph illustrating the cytotoxicity of an exemplary anti-CD33-sqCit ADC containing the payload described herein against MV-4-11 cells in vitro is shown in Biological Example 7.

[0023] Figure 10 A graph illustrating 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 is provided, as described in Biological Example 8. This graph presents the percentage of tumor burden assessed 9 days after ADC administration.

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

[0025] Figure 12 A graph illustrating the cytotoxicity of anti-CD33-sqCit ADCs, including exemplary or comparative payloads, in SCID mice in an in vivo subcutaneous MV4-11 assay is provided, as described in Biological Example 10. This graph presents tumor volume over time. Detailed Implementation

[0026] This document discloses compounds of formulas (X) and (I), or pharmaceutically acceptable salts thereof, and antibody-drug conjugates (ADCs) comprising 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, exhibit poorer cell-killing activity compared to other previously known macrocyclic compounds, but as ADCs, exhibit comparable or better cell-killing activity. This reversal of cytotoxic activity between free drugs and ADCs is surprising and unexpected.

[0027] definition The compounds described herein (including conjugates) (e.g., compounds of formula (X), (I), or (A)) or their pharmaceutically acceptable salts 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. Similarly, 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.

[0028] The subject matter disclosed herein also includes isotopic notation forms of the compounds described herein, such as those in which one or more atoms are 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... 2 H, 3 H, 11 C 13 C 14 C 15 N、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 36 Cl、123 I and 125 I.

[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 by specific categories, subclasses, and species 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 “including,” “containing,” and “comprise” are used in their open, non-restrictive sense.

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

[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 a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or the immunoactive portion 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. The immunoglobulins disclosed herein can 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 can be derived from any species. However, in some aspects, immunoglobulins are immunoglobulins of human, mouse, or rabbit origin.

[0033] As used herein, the term “antibody fragment” encompasses 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 immunely 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 derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies contained in the group are identical, except for a small number of naturally occurring mutations that may be present. Monoclonal antibodies exhibit high specificity for a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which consist of 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 group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the subject matter described herein can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature, 256:495, or by a recombinant DNA method (see, for example: US 4816567, US5807715). 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] The monoclonal antibodies described in this article specifically include “chimeric” antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a specific species or belonging to a specific 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 they exhibit the desired biological activity (US 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Targeted chimeric antibodies described 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 originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0037] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided 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 called α, δ, ε, γ, and μ, respectively.

[0038] As used herein, the term "intact antibody" is an antibody that comprises VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may possess 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. This term includes both native sequence Fc regions and variant Fc regions.

[0040] A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from 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] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, the 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 the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, the humanized antibody may contain at least a portion of the antibody constant region derived from the human antibody. "Humanized form" antibodies, such as non-human antibodies, refer to antibodies that have already been humanized.

[0042] As used herein, the term "free cysteine ​​amino acid" refers to a cysteine ​​amino acid residue that has been modified into the parent antibody, has a thiol functional group (-SH), and is not paired as 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-C2). 12 Alkyl groups are alkyl groups with 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 carbon atoms, all geometric isomers having that number of carbon atoms 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, the alkoxy group comprises 1 to 12 carbon atoms (C1-C2). 12 Alkoxy groups are alkoxy groups with 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" refers to 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) and having a specified number of carbon atoms (i.e., C). 2-10This refers to having two to ten carbon atoms. Specific alkynyl groups include those 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 alkynyl groups include, but are not limited to, groups such as ethynyl (or acetylenyl), propynyl, propynyl (or propynyl), butynyl, butynyl, and butynyl. In particular, alkynylenes include propynyl-1-alkynyl.

[0047] As used herein, "cycloalkyl" refers to a monocyclic or polycyclic saturated or partially unsaturated non-aromatic hydrocarbon. In some embodiments, unless otherwise stated, cycloalkyl comprises 3 to 12 carbon atoms (C3-C4). 12 Cycloalkyl groups, having 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 nonaromatic 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 nonaromatic 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 includes, for example, C3-C6 membered cycloalkyl groups in which one or more hydrogen atoms are independently substituted with fluorine, chlorine, iodine, or bromine.

[0049] "Halogen" 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; therefore, 4-chloro-3-fluorophenyl falls within the dihaloaryl range.

[0050] "Halogenated alkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen, wherein each halogen is independently selected. Thus, halogenated alkyl includes, for example, C1-C6 alkyl groups in which one or more hydrogen atoms are independently replaced by fluorine, chlorine, iodine or bromine.

[0051] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are substituted with a halogen, wherein each halogen is independently chosen. 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-C6 alkoxy groups. 12 Halogenated alkoxy groups, C1-C8 halogenated alkoxy groups, C1-C6 halogenated alkoxy groups, or C1-C4 halogenated alkoxy groups. In some embodiments, the halogenated alkoxy 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 stated, 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). Heteroaryls may also include polycyclic groups having at least one aromatic ring comprising at least one cyclic heteroatom aromatic ring fused to a non-aromatic ring (e.g., 5,6,7,8-tetrahydroquinolinyl; 4,5,6,7-tetrahydroisobenzofuranyl). Heteroaryl groups may also include polycyclic groups having at least one aromatic ring containing at least one cyclic heteroatom (e.g., quinolinyl, quinoxalinyl, benzothiazolyl) fused to an aromatic ring. Heteroaryl groups may include polycyclic groups having two fused aromatic rings, wherein each ring contains at least one cyclic heteroatom (e.g., naphthidyl). Wherein the heteroaryl group is a polycyclic group, the connection point with another portion (e.g., with the remainder in the formula) may appear on any ring. Examples of heteroaryl groups include, but are not limited to, pyrroleyl, imidazolyl, triazolyl, furanyl, acezolyl, phenylthio, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, and indoleyl.

[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 groups containing 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 groups. 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. Thus, halocyclic heterocyclic alkyl includes, 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, 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.

[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 medications required to treat the condition; enhancing the efficacy of another medication; 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 product 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] "Pharmaceutical-acceptable salts" include salts that are generally safe and are neither biologically undesirable nor otherwise undesirable, and include those 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 acids, 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, where 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 the 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 (composed of two amino acids linked by a single peptide bond), followed by tripeptides, tetrapeptides, and so on. The peptide mimic chemical portion includes the non-amino acid chemical portion. The peptide mimic chemical portion may also contain one or more amino acids separated by one or more non-amino acid chemical units. The peptide mimic chemical portion may not contain two or more adjacent amino acids linked by peptide bonds at any point in its chemical structure.

[0060] The range of values ​​used in this article may include consecutive integers. For example, a range represented as "0 to 5" would include 0, 1, 2, 3, 4, and 5.

[0061] As used herein, the term “unsubstituted” may mean that the part 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, such as blocking its onset; alleviating a condition; causing a condition to subside; reducing symptoms caused by a condition; or terminating or reducing the 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, such as ±20% in some embodiments; ±10% in some embodiments; ±5% in some embodiments; ±1% in some embodiments; ±0.5% in some embodiments; and ±0.1% in some embodiments, because these variations are suitable for performing the disclosed methods or using 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 this invention. The upper and lower limits of these smaller ranges may be independently included within that smaller range and are also covered by this 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 this invention.

[0065] Chemical names can be generated based on the compound structures presented 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). For example, ChemDraw® software, such as ChemDraw® version 19.1, can also be used to generate chemical names. In case of any discrepancy between structure and name, the structure shall prevail.

[0066] The compounds (including conjugates) described herein, or their pharmaceutically acceptable salts, 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. Similarly, 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 subject matter disclosed herein includes combinations and subsets of the specific groups defined herein.

[0067] 1. Compound of formula (X) This article provides compounds 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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 halogens, –OH, alkoxy groups and haloalkoxy groups.

[0068] In some embodiments, the compound of formula (X) is a compound of formula (I) or a pharmaceutically acceptable salt thereof. Compounds of formula (I) are provided herein: (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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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 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 a phenyl group or a 6-membered heteroaryl group containing one to three ring N atoms; Ring B is a phenyl group or a 6-membered heteroaryl group containing one to three ring N atoms; R 1 It is an alkyne or a phenyl, wherein the phenyl is unsubstituted or substituted with one to three independently selected halogens; R 2It is a C1-C6 alkyl, C3-C8 cycloalkyl, a 3- to 8-membered heterocyclic alkyl containing one to three independently selected cyclic heteroatoms chosen from O, N, and S, or -R 2a (OR 2a ) p -; wherein the alkyl, cycloalkyl, or heterocycloalkyl group is unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Or, the 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 heterocycloalkyl containing one to three cyclic heteroatoms independently selected from O, N and S, or a 3- to 8-membered halocycloalkyl containing one to three cyclic heteroatoms independently selected from O, N and S; Each R 2a and each R 2b It is independently a C1-C6 alkyl or a C1-C6 haloalkyl; Each R 2c It is independently hydrogen, C1-C6 alkyl, or C1-C6 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 C1-C6 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.

[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... ,in Indicates the connection point with ring A. 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 pyridine. ,in The connection point with ring A is indicated. In other embodiments, ring A is pyrimidine and ring B is phenyl or... ,in The connection point with ring A is indicated. In some other embodiments, ring A is pyrimidine and ring B is... ,in Indicates the connection point with ring A. In some embodiments, Z is NH. 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 its pharmaceutically acceptable salt, wherein: X, R 1 R 2 and R 3 As defined for equation (I).

[0072] In some embodiments of formula (II) or its pharmaceutically acceptable salts: X is 0; Z is N-CH3, N-CH2CH3, 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 a C1-C6 alkyl, C3-C8 cycloalkyl, a 3- to 8-membered heterocyclic alkyl containing one to three independently selected cyclic heteroatoms chosen from O, N, and S, or -R 2a (OR 2a ) p -; wherein the alkyl, cycloalkyl, or heterocycloalkyl group is unsubstituted or substituted by one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2cOr, the 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 heterocycloalkyl containing one to three cyclic heteroatoms independently selected from O, N and S, or a 3- to 8-membered halocycloalkyl containing one to three cyclic heteroatoms independently selected from O, N and S; Each R 2a and each R 2b It is independently a C1-C6 alkyl or a C1-C6 haloalkyl; Each R 2c It is independently hydrogen, C1-C6 alkyl, or C1-C6 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 C1-C6 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.

[0073] In some embodiments of compounds of formula (X) or (I), or compounds of formula (II), or pharmaceutically acceptable salts thereof, R 1 It is a halogen-substituted phenyl group. In some embodiments, R 1 X is a phenyl group substituted with fluorine (such as one fluorine). 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, R 2 A C1-C6 alkyl group that is either unsubstituted or substituted with one or more substituents independently selected from the group consisting of –OH and halogens. In some embodiments, R 2 A C2-C4 alkyl group that is either unsubstituted or substituted with one or more substituents independently selected from the group consisting of –OH and halogens. In some embodiments, R 2 It is an unsubstituted or fluorinated C2-C4 alkyl group. In other embodiments, R 2 It is an unsubstituted C2-C4 alkyl group, such as –CH2CH2–, –CH2CH2CH2–, or –CH2CH2CH2CH2–. In some other embodiments, R 2Ethylene groups are either unsubstituted or substituted with one or more substituents independently selected from the group consisting of –OH and halogens (such as fluorine). In some embodiments, R 2 It is –CH2CH2–.

[0075] In some embodiments of the compound of formula (X) or a pharmaceutically acceptable salt thereof, 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 halogens, –OH, alkoxy groups, and haloalkoxy groups. In some embodiments, each R 3a Independently for C 1-6 Alkyl, wherein each C 1-6 Alkyl groups are either independently unsubstituted or substituted by one or more substituents selected independently from the group consisting of: halogens, -OH, C. 1-6 Alkoxy and C 1-6 Haloalkoxy. In some embodiments, each R 3a Independently for C 1-6 Alkyl groups, wherein each alkyl group is unsubstituted or substituted with a halogen, -OH, or –OCH3. In some embodiments of compounds of formula (X) or (I), or compounds of formula (II), or pharmaceutically acceptable salts thereof, 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 halogens and –OH. In some embodiments, R 3 For –NH2, –NHR 3a –N(R) 3a )2 or –N + (R 3a )3, where each R 3a Independently, it is an unsubstituted C1-C6 alkyl group. In some embodiments, R 3 It is –NH2 or –NH (C1-C6 alkyl), such as –NH2 or –NH (CH3).

[0076] In some examples of compounds of formula (I) or pharmaceutically acceptable salts thereof: Z is N-CH2CH3 or N-CH3; Ring A is a pyrimidine; Ring B is phenyl, pyrimidine, or pyridine; 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 –NH2 or –NH(C 1-6 Alkyl); and X is 0.

[0077] In some examples of compounds of formula (I) or pharmaceutically acceptable salts thereof: Z is NC 1-6 alkyl; 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 –NH2 or –NH(C 1-6 Alkyl); and X is 0.

[0078] In some examples of compounds of formula (I) or pharmaceutically acceptable salts thereof: 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 –NH2 or –NH(C 1-6 Alkyl); and X is 0.

[0079] In some examples of compounds of formula (I) or pharmaceutically acceptable salts thereof: Z is N-CH3; Ring A is a pyrimidine; Ring B is pyridine; R 1 It is a phenyl group that has undergone one fluorine substitution; R 2 It is an unsubstituted C1-C6 alkyl group; R 3 For –NH2; and X is 0.

[0080] In some examples of compounds of formula (II) or pharmaceutically acceptable salts thereof: R 1 It is a phenyl group that has undergone one fluorine substitution; R 2 It is an unsubstituted C1-C6 alkyl group; R 3 For –NH2; and X is 0.

[0081] In some embodiments of compounds of formula (X) or (I), or pharmaceutically acceptable salts thereof: X is O, Y is N, 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.

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

[0083] In some embodiments, the compound of formula (X) is selected from the group consisting of: , , , , , , , , , , , , and Or, a pharmaceutically acceptable salt.

[0084] In some embodiments, the compound of formula (I) is selected from the group consisting of: , , , , , , , , , , , and Or, a pharmaceutically acceptable salt.

[0085] In some embodiments, the compounds of formula (X), (I), or (II) are: , Or its pharmaceutically acceptable salt.

[0086] 2. Conjugates containing payloads This document further provides conjugates comprising a compound of formula (I) as a payload. The conjugates provided herein may comprise a single payload or multiple payloads linked to an antibody via a linker. 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 , 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; 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, a conjugate of formula (A) is provided herein: 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; 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 examples therein. For example, in some examples, 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 still other embodiments, ring A is pyrimidine and ring B is phenyl, pyrimidine, or pyridine. In some embodiments, Z is NH. In other embodiments, Z is NC. 1-6 Alkyl groups, such as, for example, N-CH3 or N-CH2CH3. In some other 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, R 2 A C1-C6 alkyl group that is either unsubstituted or substituted with one or more substituents independently selected from the group consisting of –OH and halogens. In some embodiments, R 2 A C2-C4 alkyl group that is either unsubstituted or substituted with one or more substituents independently selected from the group consisting of –OH and halogens. In some embodiments, R 2 It is an unsubstituted or fluorinated C2-C4 alkyl group. In other embodiments, R 2 It is an unsubstituted C2-C4 alkyl group, such as –CH2CH2–, –CH2CH2CH2–, or –CH2CH2CH2CH2–. In some other embodiments, R 2 Ethylene, either unsubstituted or substituted with one or more substituents independently selected from the group consisting of –OH and halogens. In some embodiments, R 2 For –CH2CH2–. In some embodiments, 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 halogens, –OH, alkoxy groups, and haloalkoxy groups. In some embodiments, 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 halogens and –OH. In some embodiments, R 3 For –NH2, –NHR 3a –N(R) 3a )2 or –N + (R 3a )3, where each R 3a Independently, it is an unsubstituted or substituted C1-C6 alkyl group selected independently from the following: halogen or C1-C6 alkoxy. In some embodiments, R 3 For –NH2, –NHR 3a –N(R) 3a )2 or –N + (R 3a )3, where each R 3a Independently, it is an unsubstituted C1-C6 alkyl group. In some embodiments, R 3 For –NH2, –NHR 3a –N(R) 3a )2 or –N + (R 3a )3, where each R 3a Independently, it is an unsubstituted or C1-C6 alkyl group substituted with one to four halogens. In some embodiments, R 3 For –NH2, –NHR 3a –N(R) 3a )2 or –N + (R 3a )3, where each R 3a Independently, it is an unsubstituted or C1-C4 alkyl group, substituted with one to four fluorine molecules. In some other embodiments, R 3 For –NHR 3a , where each R 3aIndependently, it is an unsubstituted or C1-C4 alkyl group substituted with one to four fluorine molecules. In some embodiments, R 3 It 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] In an ADC of formula (A), the one or more drug payloads can be linked to an 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 a compound of formula (X), a compound of formula (I), or a subform or compound thereof, the linker to the compound requires the formation of one or more covalent bonds with the compound, and the structure of the compound is modulated accordingly, such as by substitution of bonds by hydrogen atoms to the adapter, or by adding bonds to the adapter at chemically feasible sites and adding charges. In some embodiments, the adapter site is an N atom. In some embodiments, the adapter is linked via a single covalent bond through a variable Z, and the conjugate comprises wherein the variable Z is N (substitution of bonds by hydrogen atoms to the adapter) or N + C1-C6 alkyl compounds (where the bonds are formed by tertiary amines, which are then converted to quaternary amines). In other embodiments, this is achieved by bonding to variable R. 3 It is performed by a single covalent bond, and the variable R in the conjugate is... 3 For –NH–, –NR 3a – or –N + (R 3a )2–. Those skilled in the art will readily recognize how the structure of a compound is modulated when it is covalently linked to a linker at different sites.

[0091] Therefore, this paper provides the ADC of equation (A), where the ADC of equation (A) is the same as the ADC of equation (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 R2 R 3a Z, ring A and ring B are as defined with respect to equation (X).

[0092] In some embodiments, the compound of formula (X) is the compound of formula (I), and the ADC of formula (B) is: (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 (I).

[0093] This paper further provides the ADC of equation (A), where the ADC of equation (A) is the same as the ADC of equation (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 X, R 1 R 2 and R 3a As defined for equation (X).

[0094] In some embodiments, the compound of formula (X) is the compound of formula (I), and the ADC of formula (C) is: (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 X, R 1 R 2 and R3a As defined for equation (I).

[0095] In some other embodiments, this document provides an ADC of equation (A), wherein the ADC of equation (A) is the ADC of equation (D): (D), Or its pharmaceutically acceptable salt, wherein: m is an integer from 1 to 10; Z is N + -C 1-6 Alkyl or N; and X, R 1 R 2 and R 3 As defined for equation (X).

[0096] In some embodiments, the compound of formula (X) is the compound of formula (I), and the ADC of formula (D) is: (D), Or its pharmaceutically acceptable salt, wherein: m is an integer from 1 to 10; Z is N + -C 1-6 Alkyl or N; and X, R 1 R 2 and R 3 As defined for equation (I).

[0097] In other embodiments, this document provides an ADC of equation (A), wherein the ADC of equation (A) is the ADC of equation (E): (E), Or its pharmaceutically acceptable salt, wherein: m is an integer from 1 to 10; and X, R 1 R 2 and R 3 As defined for equation (X).

[0098] In some embodiments, the compound of formula (X) is the compound of formula (I), and the ADC of formula (E) is: (E), Or its pharmaceutically acceptable salt, wherein: m is an integer from 1 to 10; and X, R 1 R 2 and R 3 As defined for equation (I).

[0099] Each of the examples or combinations thereof 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 their adapter components are further described in detail below.

[0100] a. Antibody (Ab) As described herein, compounds of formula (I) (including compounds of formula (II)) can be used in combination with antibodies (Abs) and linkers to form antibody-drug conjugates (ADCs), such as ADCs of formulas (A), (B), (C), (D), or (E). In such embodiments, one or more compounds of formula (I) are covalently linked to an Ab via a linker to form an ADC. In the case of using multiple compounds of formula (X) or (I), each compound can be linked to an antibody via a separate linker. In ADCs, non-antibody, non-linker 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 can target cells expressing antigens, such as cell surface antigens.

[0102] In certain embodiments, antibodies 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 envisioned such that any antibody target can be combined with any suitable THIOMAB™ mutation (including LALAPG or NG2LH mutations) 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 an antibody derived from a library. 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. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0105] In some embodiments, the antibody is a chimeric antibody or a humanized antibody. Humanized antibodies and their manufacturing methods are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in 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) (describing "surface rework"); Dall'Acqua Methods 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach to 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 may bind to 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 prepare trispecific antibodies according to, for example, the description 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” containing antigen-binding sites that bind to both the target protein and another distinct 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 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 U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding antigen determinant 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; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 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 heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1). Antibody fragments can be produced using various techniques, including but not limited to the proteolytic digestion of intact antibodies and production through 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 possesses the desired characteristics, such as antigen binding.

[0109] In some embodiments, the antibodies used in the ADCs provided herein may be generated using recombinant methods and compositions, such as those 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 comprise 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 comprises 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. Trop2 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 the ADC comprising an anti-Trop2 antibody, 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 comprises an anti-Her2 antibody. Anti-Her2 antibodies are described in more detail in PCT / US1997 / 018385 and US7862817. In some embodiments, the ADC comprises the anti-Her2 antibody 7C2, which is described in more detail in PCT / US1997 / 018385, which is incorporated herein by reference in its entirety. In some embodiments of the ADC comprising an anti-Her2 antibody, the anti-Her2 antibody is humanized. In some embodiments, the antibody is a cysteine-engineered variant of the disclosed antibody.

[0113] In some embodiments, the ADC comprises an anti-CD33 antibody. CD33 is a sialylate-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 monocytes 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 some 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 comprising 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)) can 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 an antibody via a connector to form an ADC. When using multiple compounds of formula (X) or (I), each compound can be linked to an antibody via a separate connector.

[0116] A “connector” (L) is a bifunctional or multifunctional part that can be used to link one or more drug moieties (D) to an antibody (Ab) to form an ADC, wherein the drug moieties are compounds of formula (X) or (I) (and may also be referred to as a payload or drug payload, such as “DP”). In some embodiments, an ADC can be prepared using a connector having reactive functional groups for covalently linking to both a drug and an antibody. For example, in some embodiments, the cysteine ​​thiol of the Ab can 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 cuttable connectors (such as peptides, hydrazones, disulfides, peptide analogs, and glucuronides) and non-cuttable connectors (such as thioethers).

[0118] Methods of attaching a linker to an antibody are well known in the art and include the use of reactive functional groups on the linker, such as NHS esters, isothiocyanates, haloacetamides, mixed disulfides, and maleimides. Therefore, the ADCs provided herein include those in which the antibody is covalently attached 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. Non-limiting exemplary examples of such reactive functional groups 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 the conjugation method, and examples herein. Thus, in some embodiments, the Ab comprises a reactive cysteine ​​thiol, with which the linker forms a bond to attach the payload to the Ab.

[0120] In some embodiments, the linker has functional groups capable of reacting with electrophilic groups present on the antibody. Exemplary electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatoms of the reactive functional groups of the linker can react with the electrophilic groups 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, hydrazines, 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-carboxylate (“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 linker can be a “cleavable linker” that facilitates drug release. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photostable 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 on the linker, including at the junction of the linker with another component of the ADC, such as the linker to the antibody, the payload, or 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 enzymes of the thioredoxin family. Other types of enzymes can cleave linkers, including β-glucuronide linkers, which are those linkers having β-glucuronide glycosidic bonds that can be cleaved by the lysosomal enzyme β-glucuronidase.

[0123] In some embodiments, the adapter of the ADC provided herein is a cleavable adapter, wherein the cleavable adapter comprises a cleavable peptide bond, disulfide bond, or β-glucuronide bond. In some embodiments, the cleavable adapter is a peptide mimic adapter.

[0124] In some embodiments, the payload is linked to the antibody via a non-peptide mimic adapter, which can be cleaved by lysosomal enzymes. For example, the amide bond in the middle of the dipeptide (e.g., Val-Cit) can be replaced with an amide mimic; and / or an 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 inclevable linker. The inclevable linker includes a linker containing a peptide that cannot be cleaved by lysosomal proteases. The inclevable linker also includes a linker that does not contain a peptide and is not cleaved by lysosomal proteases.

[0126] In some other embodiments, the connector comprises a functional group covalently linked to the Ab; a spacer assembly; and a functional group covalently linked to the remainder of the ADC (e.g., to the payload, such as a compound of formula (I)). For example, the connector may be covalently linked to the Ab via a thio-succinimide, disulfide, ester, amide, or triazole functional group; comprise a spacer assembly; and then be linked to a 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 C1-C 10 Alkyl groups, or those comprising polyethylene glycol (PEG). In some embodiments, the spacer component comprises [-O-CH2CH2-]. 1-10 Connectors used in the methods and compositions provided herein include, for example, maleimide-PEG. n - Succinimide, where n is an integer from 1 to 20 (such as 1 to 10 or 3 to 6).

[0127] In some embodiments, the connector 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 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 connectors 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 "stretcher unit" that links an antibody to another adapter component or a portion of a drug. A non-limiting exemplary stretcher unit is shown below (where wavy lines indicate sites covalently linked to an antibody, drug, or additional adapter component; and in some embodiments, (Indicator of the binding point with the antibody) MC; MP; mPEG; and In some embodiments, the linker component comprises an “amino acid unit.” In some of these embodiments, the amino acid unit allows proteases to cleave the linker, thereby facilitating 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-gly (gly-gly-gly). The amino acid unit may comprise amino acid residues of naturally occurring and / or minor amino acids and / or non-naturally occurring amino acid analogs such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasminase.

[0129] In some embodiments, the adapter component includes "spacer" units that directly or indirectly link the antibody to the drug moiety via stretching units and / or amino acid units. The spacer units can be "self-eliminating" or "non-self-eliminating." A "non-self-eliminating" spacer unit is one in which a portion or all of the spacer units remain bound to the drug moiety after cleavage of the ADC. 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 the glycine-glycine spacer unit is cleaved by a tumor cell-associated protease, 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 unit from the drug moiety.

[0130] The “self-eliminating” spacer unit releases a portion of the drug. In some embodiments, the spacer unit of the connector comprises a p-aminobenzyl unit. 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 a p-aminobenzyloxycarbonyl (PAB). In some embodiments, the ADC comprising the self-consuming connector 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 may 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 the glycine residue is another example of a self-consuming spacer 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) in Str is a stretching element covalently connected to Ab; Sp is a spacer unit that is bonded or covalently attached to the drug moiety; PM is the non-peptide chemistry component, selected from the group consisting of the following: , and , W is -NH-heterocyclic alkyl- or heterocyclic alkyl; Y is a heteroaryl, aryl, -C(O)C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkyl or –C1-C6 alkyl-NH-; Each R 1 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 3 and R 2 Each independently constitutes H, C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl or heteroarylalkyl, or R 3 and R 2 They can together form C3-C7 cycloalkyl groups; and R 4 and R 5 Each independently is C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl, heteroarylalkyl, (C1-C 10 alkyl)OCH2-, or R 4 and R 5 They can be combined to form C3-C7 cycloalkyl rings.

[0134] In some such embodiments, PM is covalently linked to the Str unit via the left-hand side of the chemical moiety shown in the figure, i.e., via a bond to the carbon of the C(O) moiety of PM in the first two cases, or via a bond to the NHC(O)(CR) moiety of PM in the last case. 4 R 3 - Part of the nitrogen bonds.

[0135] In some embodiments of the ADC with a included (T) connector, PM is The Str unit is covalently bonded to PM via a nitrogen atom on the left-hand side of the portion shown in the figure. In some embodiments of the connector of formula (T), Sp is of formula (T). The interval basis, where each n is an independent integer from 1 to 6 and Indicates the connection point of the PM unit with the connector. In some embodiments of the connector of formula (T), Sp is of formula (T). The spacer basis, where n is 0 or 1 and Indicates the connection point of the PM unit with the connector.

[0136] In some embodiments of the ADCs (such as those of formula (A), (B), or (C)) provided herein, the connector has formula (T-1): (T-1), in: Each R 1 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 3 and R 2 Each independently constitutes H, C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl or heteroarylalkyl, or R 3 and R 2 They can together form C3-C7 cycloalkyl groups; and Indicates the connection point with Ab; and Sp is the spacer basis for the following equation. Where n is 0 or 1, and Indicates the connection point with the rest of the connector.

[0137] Therefore, in some embodiments, this document provides an ADC of formula (B-1): 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; 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; 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 They can together form C3-C7 cycloalkyl groups; and Ab represents antibodies.

[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: 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; X, R 1 R 2 R 3a Z, ring A, and ring B are as defined with respect to equation (I); Each R L1 Independently for C1-C10 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 They can together form C3-C7 cycloalkyl groups; and Ab represents antibodies.

[0139] This paper further provides an ADC of formula (C-1): 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; X, R 1 R 2 and R 3a As defined for 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; 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 They can together form C3-C7 cycloalkyl groups; and Ab represents antibodies.

[0140] In some embodiments, the compound of formula (X) is a compound of formula (I), and this document provides an ADC of formula (C-1): 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; X, R 1 R 2 and R 3a As defined for equation (I); 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 They can together form C3-C7 cycloalkyl groups; and Ab represents antibodies.

[0141] In some embodiments of the ADC of equations (B-1) and (C-1), R L1 For C1-C 10 Alkyl NHC(NH)NH2, and R L3 and R L2 Together they form C3-C7 cycloalkyl groups. In some embodiments, R L1 For C1-C 10 Alkyl NHC(NH)NH2, and R L3 and R L2 Together they form a C4 cycloalkyl group.

[0142] In some embodiments, the connector has the following structure: , in Indicates the site where the antibody is covalently linked.

[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 presented herein, the connector L can 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] A non-limiting exemplary connector is shown in the context of an ADC as provided herein in Equation (A): 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, where -S- is part 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 connector is replaced by a group that modifies solubility and / or reactivity. As a non-limiting example, charged substituents such as sulfonate (-SO3) groups are used. -Ammonium or other compounds can increase the water solubility of the adapter reagent and promote the coupling reaction of the adapter reagent with the antibody and / or drug moiety, or promote the coupling reaction of Ab-L (antibody-adaptor intermediate) with DP or DP-L (drug payload-adaptor intermediate) with Ab, depending on the synthetic route used to prepare the ADC. In some embodiments, a portion of the adapter is coupled with the antibody and a portion of the adapter is coupled with the drug, and then Ab- (adaptor moiety) is added. a Drug payload - (connector portion) b Coupling is performed to form an ADC of formula (A). In some such embodiments, the antibody contains more than one (connector portion). a Substituents allow more than one drug to couple 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 the antibody. In some embodiments, multiple DP-Ls are coupled with the antibody.

[0148] In some embodiments, a drug payload-connector conjugate of formula (B-L1) is provided: (B-L1), Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; 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 They can be combined to form C3-C7 cycloalkyl groups.

[0149] In some embodiments, a drug payload-connector conjugate of formula (B-L1) is also provided: (B-L1), Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; X, R 1 R 2 R 3a Z, ring A, and ring B are as defined with respect to equation (I); 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 They can be combined to form C3-C7 cycloalkyl groups.

[0150] This article further provides a drug payload-connector conjugate of formula (C-L1): (C-L1), Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; X, R 1 R 2 and R 3a As defined for 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 RL2 Each independently constitutes H, C1-C 10 Alkyl, C1-C 10 alkenyl, arylalkyl or heteroarylalkyl, or R L3 and R L2 They can be combined to form C3-C7 cycloalkyl groups.

[0151] This article also provides a drug payload-connector conjugate of formula (C-L1): (C-L1), Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; X, R 1 R 2 and R 3a As defined for equation (I); 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 They can be combined to form C3-C7 cycloalkyl groups.

[0152] In some embodiments of the drug payload-connector conjugates of formulas (B-L1) and (C-L1), X is 0. In still other embodiments of the drug payload-connector conjugates of formulas (B-L1) and (C-L1), X is 0, R L1 For C1-C 10 Alkyl NHC(NH)NH2, and R L3 and R L2 Together they form C3-C7 cycloalkyl groups. In some embodiments, R L1 For C1-C 10 Alkyl NHC(NH)NH2, and R L3 and R L2 Together they form a C4 cycloalkyl group.

[0153] This article provides drug payload-connector conjugates with the following structures: .

[0154] The compounds of this 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-carboxy-(6-acylaminohexanoic acid)succinimide (LC-SMCC), m-maleimide... Benzyl-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)butyric acid 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 succinimide-(4-vinyl sulfone)benzoate (SVSB), and including bis-maleimide reagents: dithiobismaleimide ethane (DTME), 1,4-bismaleimide butane (BMB), 1,4-bismaleimide-2,3-dihydroxybutane (BMDB), and bis... Maleiminohexane (BMH), bismaleiminoethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)3 (shown below); bifunctional derivatives of imino esters (such as dimethyl diimide adipate HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), 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 cysteine ​​in the antibody to be linked to a thiol-containing pharmaceutical moiety, linker, or linker-pharmaceutical intermediate.Other functional groups that react with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

[0155] Some 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, MA (1995) J. Org. Chem. 60:5352-5355; Frisch et al., (1996) Bioconjugate Chem. 7:180-186; US 6214345; WO 02 / 088172; US 2003130189; US2003096743; WO 03 / 026577; WO As described in 03 / 043583; and WO 04 / 032828.

[0156] c. Drug payload capacity of ADC Drug loading is the average amount of drug per antibody moiety. ADCs (such as those of formula (A), (B), (B-1), (C), (C-1), (D), or (E)) provided herein may include drug loadings ranging from 1 to 10 drug molecules (D) per antibody (Ab). Thus, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug moietyes may be covalently linked to an antibody, wherein the drug moiety is the drug payload of formula (I) (including compounds of formula (II)). Compositions of ADCs may include antibodies conjugated to a range of drugs (1 to 10) to form compositions having a range of drug loadings. In compositions of ADCs (including those drug compositions such as those described herein), the average drug loading per antibody is commonly referred to as the drug / antibody ratio or “DAR”. In the preparation of ADCs from conjugation reactions, the average amount of drug per antibody can be characterized by conventional methods such as mass spectrometry (including LCMS), ELISA assays, 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 means of 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, an antibody may have only one or a few cysteine ​​thiols, or only one or a few thiols with sufficiently high reactivity to link linkers. Higher drug loadings, such as m > 5, may lead to aggregation, insolubility, toxicity, or loss of cell permeability in some antibody-drug conjugates.

[0158] Typically, less than the theoretical maximum amount of drug moiety is conjugated to the antibody during the conjugation reaction. The antibody may contain, for example, numerous lysine residues that are unreactive with linker-drug intermediates or linker reagents. Additionally, in some embodiments, only the most reactive cysteine ​​thiol group reacts with thiol-reactive linker reagents or linker-drug intermediates. Generally, the antibody does not contain numerous (if any) free reactive cysteine ​​thiol groups that can be linked to the drug moiety. Most cysteine ​​thiol residues in the compound antibody are present as disulfide bonds and must be reduced with a reducing agent (such as dithiothreitol (DTT) or TCEP) under partial or complete reducing conditions.

[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) targeting the cysteine ​​thiol modification or limiting reduction conditions. When more than one nucleophilic or electrophilic group of an antibody reacts with a linker-drug intermediate or linker reagent, followed by a drug moiety reagent, the resulting product can be a mixture of ADC molecules containing drug moieties attached to the antibody, such as 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 attached to different sites on the antibody via linkers. Therefore, the antibody-drug conjugate compositions of the present invention comprise a mixture of antibody-drug conjugate compounds, wherein the antibody has one or more drug moieties and wherein the drug moieties are linked to the antibody at various amino acid residues.

[0160] 3. Methods for preparing compounds The compounds disclosed herein can be prepared by methods known in the field of organic synthesis, which are illustrated in part by the following synthetic schemes. In the schemes described herein, it should be understood that protecting groups are used where necessary for sensitive or reactive groups, based on general principles or chemical methods. The handling 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 procedures 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 of a stereocenter in the compounds disclosed herein. In some embodiments, the compounds of this disclosure may exist as either enantiomers or diastereomers. 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 cases where an advanced intermediate is purified to its individual enantiomers, each individual enantiomer may continue to participate in the reaction individually to provide the final enantiomerically pure compound of this disclosure. The determination 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" (EL Eliel, SH Wilen, and LN 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] ADC pharmaceutical compositions are typically prepared for parenteral administration, such as by bolus, intravenous, or intratumoral injection. Such pharmaceutical compositions may be prepared in aqueous solutions or 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 on the preparation of pharmaceutical compositions, including those for parenteral administration, aqueous solutions, or as lyophilized forms for reconstitution into an aqueous solution for administration, can be found, for example, in Remington's Pharmaceutical Sciences (2020), 23rd edition, edited by Adejare, A. 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. How to use This document provides methods for treating a condition in a subject in need, methods comprising administering to the subject a therapeutically effective amount of an ADC as described herein. Further methods for treating a condition in a subject in need are provided, methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an ADC as described herein and a pharmaceutically acceptable excipient. The ADC of the methods herein (including administering an ADC or administering a pharmaceutical composition comprising an ADC and a pharmaceutically acceptable excipient) may be an ADC of formula (A), formula (B), formula (B-1), formula (C), or formula (C-1). For example, in some embodiments of the methods 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] Also provided is a compound 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)). Further provided is the use of the compound as described herein in 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)). Also provided is the 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 being.

[0166] In some embodiments, the condition is cancer, tumor, or other malignant tumor. As used herein, cancer, tumor, and malignant tumor 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 lymphoma. In some embodiments, cancer, tumor, and malignant tumor 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 for treating a subject with a hyperproliferative disease, such as cancer, 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 herein 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 compounds are ADCs as described herein (e.g., ADCs 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, methods of using an ADC as described herein, comprising an anti-CD33 antibody, for treating hematologic malignancies such as non-Hodgkin's 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 disorders, are employed. In some embodiments, methods of using an ADC as described herein, comprising an anti-HER2 antibody, for treating HER2+ cancers such as HER2+ breast cancer or gastric cancer are employed.

[0169] For the prevention or treatment of disease, the appropriate dose of an ADC will depend on the type of disease to be treated, the severity and duration of the disease, the purpose of administering the molecule for prevention or treatment, 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 either once or in a series of treatments. Depending on the type and severity of the disease, an initial candidate dose of approximately 1 µg / kg to 15 mg / kg (e.g., 0.1–20 mg / kg) is given to the patient, for example, by single or multiple separate administrations or by continuous infusion. Based on the above factors, a typical daily dose can range from approximately 1 μg / kg to 100 mg / kg or more.

[0170] Enumerated Examples Example 1. A compound having 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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, -NHR3a -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 halogens 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 groups 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... ,in Indicates the connection point with ring A.

[0173] Example 4. The compound according to any one of Examples 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R 1 It is a phenyl group that has been substituted with one fluorine molecule.

[0174] Example 5. The compound according to any one of Examples 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R 2 C1-C6 alkyl groups that are unsubstituted or substituted by one or more substituents independently selected from the group consisting of –OH and halogens.

[0175] Example 6. The 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. The compound according to any one of Examples 1 to 6, or a pharmaceutically acceptable salt thereof, wherein R 3 It can be –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 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 –NH2 or –NH(C 1-6 Alkyl); and X is 0.

[0179] Example 10. The compound according to Example 1, 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.

[0180] Example 11. The compound according to Example 1, wherein the compound has formula (II): (II), Or its pharmaceutically acceptable salt, wherein: X, R 1 R 2 and R 3 As defined for equation (I).

[0181] Example 12. The compound according to Example 11, or a pharmaceutically acceptable salt thereof, wherein R 2 It is an unsubstituted or fluorinated C1-C6 alkyl group; and R 3 –NH2 or –NH(C 1-6 alkyl).

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

[0183] Example 13a. The compound according to Example 1, or a pharmaceutically acceptable salt thereof, wherein the compound is: , , , , , , , , , , , or Or, for example, a pharmaceutically acceptable salt.

[0184] Example 14. 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 effective payload of the drug is 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R2c 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 halogens 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 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 (I).

[0186] Example 16. The conjugate or a pharmaceutically acceptable salt thereof according to Example 14 or 15, 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): 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 (I); Each R L1 Independently for C1-C10 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 They can be combined to form C3-C7 cycloalkyl groups.

[0188] Example 18. The conjugate according to any one of Examples 14 to 17, 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 –NH2 or –NH(C 1-6 Alkyl); and X is 0.

[0189] Example 19. The conjugate according to Example 15, 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 (I).

[0190] Example 20. The conjugate or a pharmaceutically acceptable salt thereof according to Example 19, wherein R 2It is an unsubstituted or fluorinated C1-C6 alkyl group; and R 3 –NH2 or –NH(C 1-6 alkyl).

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

[0192] Example 21a. A conjugate or a pharmaceutically acceptable salt thereof according to any one of Examples 14 to 17, wherein the drug payload is: , , , , , , , , , , , or .

[0193] Example 22. The conjugate according to Example 14, wherein the conjugate has the following structure: .

[0194] Example 23. A 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 malignant tumor.

[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 for 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, 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): (B-L1), Or its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; 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: halogens and –OH; 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 They can together 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R 2cIndependently hydrogen, alkyl, or haloalkyl; and p and q are independent 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 its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; 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 They can together 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; Each R 2a and each R 2b It is independently an alkyl or haloalkyl group; Each R 2cIndependently hydrogen, alkyl, or haloalkyl; and p and q are independent 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 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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 3aIndependently alkyl, wherein 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.

[0206] Example 35. The compound according to claim 34, wherein the compound of formula (X) is the 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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 halogens 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... ,in Indicates the connection point with ring A.

[0209] Example 38. The compound according to any one of Examples 34 to 37, or a pharmaceutically acceptable salt thereof, wherein R 1 It is a phenyl group that has been substituted with one fluorine molecule.

[0210] Example 39. The compound according to any one of Examples 34 to 38, or a pharmaceutically acceptable salt thereof, wherein R 2 C1-C6 alkyl groups that are unsubstituted or substituted by one or more substituents independently selected from the group consisting of –OH and halogens.

[0211] Example 40. The 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. The compound according to any one of Examples 34 to 40, or a pharmaceutically acceptable salt thereof, wherein R 3 It can be –NH2 or –NHCH3.

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

[0214] Example 43. The compound according to Example 34 or 35, 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 –NH2 or –NH(C 1-6 Alkyl); and X is 0.

[0215] Example 44. The compound according to Example 34 or 35, 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.

[0216] Example 45. The compound according to Example 34 or 35, wherein the compound has formula (II): (II), Or its pharmaceutically acceptable salt, wherein: X, R 1 R 2 and R 3 As defined for equation (I).

[0217] Example 46. The compound according to Example 45, or a pharmaceutically acceptable salt thereof, wherein R 2 It is an unsubstituted or fluorinated C1-C6 alkyl group; and R 3 –NH2 or –NH(C 1-6 alkyl).

[0218] Example 47. The compound according to Example 34, or a pharmaceutically acceptable salt thereof, wherein the compound is: , , , , , , , , , , , , and Or, a pharmaceutically acceptable salt.

[0219] Example 48. 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 effective payload of the drug is compound of formula (X): (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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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 halogens, –OH, alkoxy groups and haloalkoxy groups.

[0220] Example 49. The conjugate according to Example 48, 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).

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

[0222] Example 51. The conjugate according to any one of Examples 48 to 50, wherein the conjugate has the 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 They can be combined to form C3-C7 cycloalkyl groups.

[0223] Example 52. The conjugate according to any one of Examples 48 to 51, 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 –NH2 or –NH(C 1-6 Alkyl); and X is 0.

[0224] Example 53. The conjugate according to Example 48, 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).

[0225] Example 54. The conjugate according to Example 53, or a pharmaceutically acceptable salt thereof, wherein R 2 It is an unsubstituted or fluorinated C1-C6 alkyl group; and R 3 –NH2 or –NH(C 1-6 alkyl).

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

[0227] Example 56. The conjugate according to Example 48, wherein the conjugate has the following structure: .

[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 the 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 the conjugate according to any one of Examples 48 to 57.

[0231] Example 60. The method according to Example 59, wherein the condition 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 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).

[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 malignant tumor.

[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 its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; 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; 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 RL2 They can together 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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.

[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 its pharmaceutically acceptable salt, wherein: R 3 For –NH–, –NR 3a – or –N + (R 3a )2–; 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 halogens, –OH, alkoxy groups and haloalkoxy groups.

[0238] Each R L1 Independently for C1-C 10 Alkyl, C1-C 10alkenyl, 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 They can together 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 substituted by one or more substituents independently selected from the group consisting of: halogen, alkyl, -OH, -OR 2b and -O(R) 2b O) q R 2c Alternatively, two substituents together with the atoms they are attached to form cycloalkyl, halocycloalkyl, heterocycloalkyl, or haloheterocycloalkyl groups; 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.

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

[0240] Example The abbreviations used in the examples below may include: Synthesis Example 1: Synthesis of Compound 100 Step 1: Preparation of intermediate 1b 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) at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was filtered, and the filtrate was used directly for 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 an aqueous solution of NH4Cl (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, 33% yield).

[0242] Step 3: Preparation of intermediate 1d 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-fluoropyridin-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 h. The mixture was filtered and concentrated to a residue, which was 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 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) at 25 °C. The mixture was then 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. 1 H 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 h. 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, yield 46%). LCMS (5-95AB, 1.5 min): R t = 0.823 min, m / z = 500.2[M+H] + .

[0245] Step 6: Preparation of intermediates over 1 hour 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 mmol), 4-methylbenzenesulfonic acid [2-[6-[2(tert-butoxycarbonylamino)ethylamino]-3-pyridyl]pyrimidin-4-yl]methyl ester (204 mg, 0.41 mmol) and Cs2CO3 (302 A solution of 0.93 mmol (mg, 0.93 mmol) in DMF (4 mL) was stirred at 25 °C for 1 hour. 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 hour. LCMS (5-95 AB, 1.5 min): R t = 0.930 min, m / z = 1136.5[M+H] + .

[0246] Step 7: Preparation of Compound 100 Add TFA (3 mL) to a solution of (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 mg, 0.28 mmol) in dichloromethane (2 mL), and stir the mixture at 25 °C for 16 minutes. The mixture was concentrated to obtain 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. 1 H 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): R t= 0.797 min, m / z = 980.4 [M+H] + .

[0247] Example 2: Synthesis of 1g of macrocyclic intermediate Figure 1 The example provides a synthetic scheme for producing 1 g of the macrocyclic intermediate. The intermediate compounds in this example are numbered according to... Figure 1 The corresponding numbers in the text.

[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 h. New spots were observed by TLC (in 50% ethyl acetate in petroleum ether, Rf = 0.4). The mixture was cooled to room temperature, and then MTBE (100 mL) was added and the mixture was further stirred at 10 °C for 30 min. 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). 1 H 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). 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 4-chloro-5,6-diiodo-thieno[2,3-d]pyrimidine (9.23 g, 88% yield) as a gray solid.1 H 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. 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%). 1 H 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, which 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, yield 29%). 1 H 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).

[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 min. Iodine (5.99 g, 23.6 mmol) was then added dropwise to a solution of tetrahydrofuran (20 mL), 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 NH4Cl aqueous solution and extracted with 30 mL x 3 ethyl acetate. The product 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, yield 52%). 1 H 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_1.5min): R T = 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) under a nitrogen atmosphere at 0 °C. 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): R T = 1.172 min, m / z = 642.8 [M+H] + .

[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(dibenzylideneacetone)dipalladium(0) (713 mg, 0.78 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (331 mg, 0.78 mmol) and K3PO4 (3.3 g, 15.6 mmol) were dissolved in tetrahydrofuran (40 mL) under N2. The mixture in water (10 mL) was stirred at 65 °C for 16 hours. 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, yield 84%). LCMS (AB_5-95_1.5min): R T = 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)thieno[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) were added to anhydrous tert-butanol (40 mL). (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 (3.88 g, 6.36 mmol) was added to the solution, and the mixture was 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. This 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)thiopheno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (2.4 g, yield 37%). LCMS (R₁₀₈₀₁₅₀₅ ... T =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. This 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, 83% yield). LCMS (10-80, R, 1.5 min): R T = 1.363 min, m / z = 989.4 [M+H] + .

[0259] Step 12, (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 (intermediate) Preparation of (16): 1,4-diazabicyclo[2.2.2]octane (DABCO; 3.10 mL, 27.8 mmol) and p-toluenesulfonyl chloride (4.24 g, 22.2 mmol) were added to a solution of (R)-tert-butyl-2-((5-(4-(((S)-1-(allyloxy)-3-hydroxypropane-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, 5.5 mmol) in anhydrous dichloromethane (50 mL) at 0 °C. 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, which 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)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (5.5 g, 86% yield). LCMS (85-100, AB, 2min): R T = 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)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-hydroxyphenyl)propionate (intermediate 17): at 0°C Tetrabutylammonium fluoride (5.30 mL, 5.3 mmol) was slowly 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)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)propionate (5.5 g, 4.8 mmol) 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)thieno[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), 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): R T = 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): R T = 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 (3.80 g, 2.8 mmol) from Step 15 and 1,4-diazabicyclo[2.2.2]octane (DABCO; 2.45 mL, 22.3 mmol) in dichloromethane (30 mL), 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 give 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): R T = 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): R T = 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 under H2 (15 psi). TLC (in 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): R T= 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 petroleum ether, 10% ethyl acetate, R) was performed. f = 0.3) indicates that the starting material has 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, which 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-hydroxybenzaldehyde (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 observed. 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, 1.5min): R T = 1.3min, 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. 1 H 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, 54% yield). 1 H 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, 1 H), 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 then subjected to SFC (Daicel Chiralpak AS (250 mm)). 50 mm, 10 μm); Mobile phase: 20% ethanol (0.1% ammonium hydroxide) in CO2 for separation to obtain (R)-3-(2-(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)phenyl)-2-hydroxypropionate tert-butyl ester (intermediate 13; 1.05 g, peak 2, desired isomer, R) T =2.769 min). 1 H 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, 4.4Hz, 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, R). T =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 observed. The mixture was concentrated under vacuum to give 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 observed. 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 oily (S)-3-(allyloxy)-2-hydroxypropyl acetate (2.05 g, 51% yield). 1 H 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 connector-compound 100 conjugate Compound 100 is used to prepare linker-drug conjugates, which are further used to prepare ADCs. The linker used in all conjugates is a square-cit (sq-Cit or sqCit) peptide mimic linker, which is described in more detail in PCT / US2014 / 070654. This linker is 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]octadec-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 mmol) and (4-nitrophenyl) were synthesized at 25 °C. [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) was added to a solution of DIEA (0.28 mL, 1.61 mmol) in 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 (676.3 mg, 74% yield) as a white solid. LCMS (5-95 AB, 1.5 min): R t = 0.858 min, m / z = 1576.54 [M+H] + .

[0276] Example 6: Synthesis of Compound 101 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%). 1 H 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 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 give a crude product, which was purified by preparative TLC (in 50% ethyl acetate in petroleum ether, Rf = 0.5) to give 4-methylbenzenesulfonic acid [2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methyl ester (15 mg, 8% yield) as a white solid. LCMS (5-95AB / 1.5min): Rt =1.000 min, m / z=499.2 [M+H] + 。

[0279] (11R,20R)-14-[[2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methoxy]-2 3 ,2 6 -Dichloro-3-(4-fluorophenyl)-2 4 ,2 5Preparation of tert-butyl 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: 1 g of macrocyclic intermediate was prepared according to the procedure in Example 2. A mixture of 1 g (20 mg, 0.02 mmol) of intermediate, 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 give a crude product, which was purified by preparative TLC (in 10% methanol in dichloromethane) to give a white solid (11R,20R)-14-[[2-[4-[2-(tert-butoxycarbonylamino)ethylamino]phenyl]pyrimidin-4-yl]methoxy]-2 3 ,2 6 -Dichloro-3-(4-fluorophenyl)-2 4 ,2 5 -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-decaen-11-carboxylic acid tert-butyl ester (15 mg, yield 54%). LCMS (5-95AB / 1.5min): Rt =1.066 min, m / z = 1135.5 [M+H] + .

[0280] Preparation of compound 101: A solution 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 (15 mg, 0.01 mmol) in TFA (1 mL) and 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 give a yellow solid (11R,20R)-14-[[2-[4-(2-aminoethylamino)phenyl]pyrimidin-4-yl]methoxy]-2 3 ,2 6 -Dichloro-3-(4-fluorophenyl)-2 4 ,2 5 -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-decaen-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 of Compound 102 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, and 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, 85% yield). 1 H NMR(400M Hz, DMSO-d6): δ = 8.35 (s, 2H), 7.39 (t,J= 5.2 Hz, 1H), 6.84 (t,J= 5.2Hz, 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 tert-butyl (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-dioxaneborane-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'-bipyrimidin]-2'-yl)amino)ethyl)carbamate: A mixture of tert-butyl (2-((5-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)pyrimidin-2-yl)amino)ethyl)carbamate (0.3 g, 0.82 mmol), (2-chloropyrimidin-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 (2-((4-(hydroxymethyl)-[2,5'-bipyrimidine]-2'-yl)amino)ethyl)carbamate tert-butyl ester (0.2 g, yield 84%). 1 H 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).

[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 tert-butyl (200 mg, 0.58 mmol) of (2-((4-hydroxymethyl)-[2,5'-bipyrimidine]-2'-yl)amino)ethyl)carbamate in DCM (5 mL), and the mixture was stirred at 25 °C for 16 h. The mixture was concentrated and purified by rapid chromatography (0% to 2% MeOH in DCM) to give 4-methylbenzenesulfonic acid (2'-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-[2,5'-bipyrimidine]-4-yl)methyl ester (0.2 g, yield 69%) as a white solid. 1 H NMR (400MHz, 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] (4R,9R)-6 6 -((2'-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-[2,5'-bipyrimidin]-4-yl)methoxy)-1 3 ,1 5 -dichloro-2 6 -(4-Fluorophenyl)-1 2 ,1 6Preparation of tert-butyl 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: 1 g of macrocyclic intermediate was prepared according to the procedure in Example 2. A mixture of 1 g of intermediate (30 mg, 0.03 mmol), 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: TFA (0.3 mmol) was added to a solution 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]octadec-1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaen-11-carboxylic acid tert-butyl ester (20 mg, 0.02 mmol) in DCM (1 mL). 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)-6. 6 -((2'-((2-aminoethyl)amino)-[2,5'-bipyrimidin]-4-yl)methoxy)-1 3,1 5 -dichloro-2 6 -(4-Fluorophenyl)-1 2 ,1 6 -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 (12 mg, 70% yield). 1 HNMR (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 of Compound 103 Preparation of 5-bromopicolinamidine hydrochloride: NaOMe (32.4 mg, 0.6 mmol) was added to a solution of 5-bromo-2-pyridinium carboxynitrile (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 give 5-bromopicolinamidine hydrochloride (2 g, 87% yield) as a white solid.

[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). The mixture was stirred at 75 °C for 16 h under a N2 atmosphere. The reaction 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 obtain 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 obtain 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, 0% to 10% MeOH in DCM) to give 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 give 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. 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 give a crude product, which was purified by preparative TLC (in 10% methanol in dichloromethane, Rf = 0.5) to give 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 purified by rapid chromatography (0% to 2% MeOH in DCM) to give a yellow oily tert-butyl (20 mg, 27% yield) of (2-((6-(4-(chloromethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate. LCMS (5-95AB / 1min): Rt =0.475 min, m / z=364.2 [M+H] + 。

[0294] (4R,9R)-6 6 -((2-(5-((2-((tert-butoxycarbonyl)amino)ethyl)amino)pyridin-2-yl)pyrimidin-4-yl)methoxy)-1 3 ,1 5 -dichloro-2 6 -(4-Fluorophenyl)-1 2 ,1 6Preparation of tert-butyl 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: 1 g of macrocyclic intermediate was prepared according to the procedure in Example 2. A mixture of 1 g of intermediate (20 mg, 0.02 mmol), (10 mg, 0.025 mmol) of tert-butyl (2-((6-(4-(chloromethyl)pyrimidin-2-yl)pyridin-3-yl)amino)ethyl)carbamate (1 mL) and Cs₂CO₃ (20 mg, 0.06 mmol) in MeCN 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)-dibenzocyclodecane-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 Compounds with Additional Payload The additional payload compounds are generally prepared according to the procedures outlined in Examples 1 to 8. For compound 104, the synthesis is generally carried out according to intermediate 1g, while 1-methylpiperazine is replaced with 1-ethylpiperazine. For compounds 105 to 113, the synthesis is generally carried out according to intermediate 1e, while tert-butyl (2-aminoethyl)carbamate is replaced with an substituted Boc-protected ammonia.

[0297] Example 10: General Procedures for Connectors-Drug Intermediates Additional linker-drug intermediates are prepared using activated sq-Cit peptide mimic linkers and other payload drugs, generally following the procedure described in Example 5 for compound 100.

[0298] Example 11: General Procedure for Connector-Drug Intermediate-Antibody Conjugation The pH of the cysteine-engineered antibody (THIOMAB™, selected for use in ADCs) in 10 mM succinate (pH 5), 150 mM NaCl, and 2 mM EDTA was adjusted to pH 7.5–8.5 with 1 M Tris. 10 to 18 equivalents of 10 mM linker-drug intermediate (with a thiol-reactive maleimide group) were dissolved in DMF or DMSO and added to the reduced, re-oxidized, 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. When the reaction was complete, the Ab-linker-loador conjugate was purified by one or more methods in any combination to remove any remaining unreacted linker-drug intermediate and aggregates (if present at significant levels). For example, the conjugate may have been diluted with 20 mM histidine-acetate (pH 5.5) until a final pH of approximately 5.5, and purified by 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-connector-loador conjugates were formulated into 20 mM His / acetate (pH 5) and 240 mM sucrose solutions using gel filtration or dialysis. The purified conjugates were concentrated by centrifugal ultrafiltration, filtered aseptically through a 0.2-μm filter, 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: Ab-connector-loaded conjugates were subjected to particle size sieving chromatography using a Shodex KW802.5 column in 0.2M potassium phosphate (pH 6.2) containing 0.25 mM potassium chloride and 15% IPA, with a flow rate of 0.65 ml / min. The aggregation state of the conjugates was determined by integrating the absorbance of the eluting peak area at 280 nm.

[0302] LC-MS: Conjugates were analyzed using an Agilent TOF 6530 ESI instrument. For example, the Ab-linker-loaded conjugate was treated at 37 °C with 25 mM DTT for 15 min, resulting in reduction between the heavy and light antibody chains. The resulting cleaved fragments were 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 solution in H₂O, and mobile phase B was a 0.05% TFA solution in acetonitrile. 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 quality 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 examples of anti-Her2 7C2 ADCs: 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 sqCit linker-compound 100 conjugate (concentration = 10 mM) dissolved in DMF or DMSO were added to the 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 and monitored until completion was confirmed by LC-MS. Upon completion of the conjugation reaction, the resulting anti-Her2-7C2-sqCit-compound 100 conjugate was purified to remove unreacted linker-drug intermediates and analyzed by particle size separation chromatography (SEC) to determine the yield, drug-to-Ab ratio (DAR), and percentage of aggregates. The SEC trace of anti-Her2-7C2-sqCit-compound 100 is shown in [image / image / description]. Figure 8A The yield was 64.83%. LC-MS analysis of the light and heavy chains of the conjugate showed [data missing]. Figure 8B and Figure 8CIn the conjugate, the drug-to-Ab ratio was calculated to be 1.9; the aggregation rate was determined to be 0.46%.

[0304] In the biological examples provided herein, ADCs of compound 100 and additional payload compounds 101 through 107 were prepared and characterized. The general structures of the ADCs, exhibiting the payload and connection points of the sq-Cit connector, are provided below. Specific Abs and DARs used (if identified) are provided in the specific examples.

[0305] Biological Example 1: Evaluating and comparing the effects of compounds on the in vitro viability of adenocarcinoma cells and breast cancer cells. After 5 days of incubation, the cytotoxicity of 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 and compared. Figure 2 The structures of the evaluated compounds are provided. 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] Human breast cancer cell lines were 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). Five days later, 100 μL of CellTiter-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 A graph showing the viability of SK-BR-3 adenocarcinoma cells relative to compound concentration. Figure 3BA graph showing the viability of CAMA-1 breast cancer cells relative to compound concentrations. Table 1a provides the IC50 values ​​for each tested compound in each cell line. 50 In summary, compound A exhibited the highest cytotoxic potency in both cell lines (e.g., by reaching the lowest IC50 threshold). 50 (Measured). 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. Comparative IC50 values ​​of acyclic and cyclic MCL1 inhibitors in two different cell lines 50 The summary, including IC 50 Reports are given in nanomoles.

[0309] Following the same procedure, cyclic MCL1 inhibitors prepared according to the examples provided herein were evaluated in SK-BR-3 cells. Table 1b provides the IC50 values ​​of the free compounds in SK-BR-3 cells. 50 Each molecule tested exhibited a higher IC value than the compounds in Table 1a. 50 Most of these molecules exhibited significantly poor inhibitory activity. Compound 100 had an IC50 of 0.43 µM. 50 Its activity is less than 1 / 25th that of compound C (16.3 nM) and less than 1 / 2000th that of compound A (0.2 nM, estimated value).

[0310] Table 1b. IC50 of the selected cyclic MCL1 inhibitors described in this paper in SK-BR-3 cells. 50 The summary, including IC 50 Reports should be submitted in micromoles.

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

[0312] All acute myeloid leukemia and non-Hodgkin's 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 of the free drug and 7 days of incubation of the antibody-drug conjugate (ADC), 100 µL of Cell Titer-Glo reagent (Promega, Madison WI) was added to each well, and the cells were incubated at room temperature for 10 min before the luminescence signal was recorded. The data represent one of three separate experiments (each experiment was repeated twice).

[0313] Biological Example 3: Evaluating and Comparing the Effects of Compounds on Lymphocyte Viability in Vitro Following the general procedures used for the above in vitro NHL cell assays, the cytotoxicity of compounds A, B, and C as free drugs in SU-DHL5 and SU-DHL10 cells was evaluated and compared. Figure 4A and Figure 4B The graphs show 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 exhibited the strongest cytotoxicity, followed by compound C, and compound B showed the weakest cytotoxicity; and in the SU-DHL10 cell line, compound A showed the strongest cytotoxicity, followed by compound B, and compound C showed the weakest cytotoxicity.

[0314] Table 2. Comparative IC50 values ​​of acyclic and cyclic MCL1 inhibitors in two different cell lines 50 Summary.

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

[0316] Biological Example 4: Evaluation and comparison of the effects of compound 100 and compound 100 on the in vitro viability of acute myeloid leukemia (AML) cell lines Following the general procedures used for the above 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 The graphs show cell viability relative to compound concentration in MOLM-16, HL-60, and OCI-AML3, respectively. As a free drug, compound 100 was significantly less potent as a cytotoxic agent in all four cell lines than compound A or compound B (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. Comparison of IC50 values ​​of compounds A and B, and compound 100, in four different AML cell lines. 50 Summary Biological Example 5: Evaluation of anti-CD33 ADCs containing payload compounds A, B, or 100 in AML cell lines The ADC was prepared using anti-CD33 antibody 15G15, a sq-Cit linker, and a payload compound 100, compound A, or compound B, following 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 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 The graphs show cell viability relative to compound concentration for EOL-1, MV-4-11, and NOMO-1, respectively. ADCs containing compound 100 as the payload showed significantly improved cytotoxicity in the AML cell line EOL-1, such as... Figure 6A As shown. It maintained high cytotoxicity in the cell line MV-4-11, such as Figure 6BAs shown. In the NOMO-1 cell line, the compound 100 ADC exhibited reduced cytotoxic potency, but was still superior to the ADCs using compounds A or B as payloads. 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 an ADC, the ADC showed comparable or better cytotoxic potency than the ADCs using comparative compounds A or B. This significant increase in cytotoxicity when the free drug was converted to an ADC was surprising and unexpected.

[0321] Biological Example 6: Evaluation of anti-Her2 7C2-sq-Cit-load ADCs using compound B, compound C, or compound 100 as payloads The ADC was prepared using the anti-HER2 antibody 7C2, the sq-Cit linker, and the payload compound B, compound C, or compound 100, according to the procedures described 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 A graph showing the activity of each ADC. As shown in the figure and table below, the ADC using compound 100 as the payload exhibits significantly better cytotoxic activity compared to ADCs using the same antibody and the same adapter but with either compound C or compound B as the payload. The DAR (drug loading) 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 significantly lower than that of the ADC containing compound 100. This demonstrates the surprisingly improved cytotoxic efficacy of the ADC containing compound 100 as the payload in the breast cancer cell line SK-BR-3. The ADC using compound 100 has a better EC50 and maximal cell killing than either compound C or B. Without being bound by theory, 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 ADC on SK-BR-3 breast cancer cell line.

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

[0324] Biological Example 8: Comparing the effects of anti-CD33 ADCs on tumor burden in an in vivo model of disseminated AML The ADCs, using 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, with DAR 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-target ADC at 30 mg / kg, and the mediator (control) was assessed in NSG mice using in vivo MV4-11 assays.

[0325] Twenty-four hours prior to inoculation with leukemia cells, mice were preconditioned by intraperitoneal injection of 20 mg / kg busulfan (Busilvex, Pierre Fabre, France). Busulfan was pharmaceutical grade and diluted with sterile saline. Mice were then aseptically inoculated with 2 million MV-411 luciferase / GFP+ leukemia cells (cells suspended in HBSS and administered intravenously to 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 randomized and treatment began (designated as Day 0 of the study). All animals received intraperitoneal injection of 30 mg / kg anti-ragweed to minimize nonspecific ADC uptake via Fc receptors expressed on tumor cells. Four hours later, ADC or 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. At the end of the study, whole blood was collected from the animals to assess the treatment effect.

[0326] The graphs of these results are shown in Figure 10 The figure shows the percentage of tumor burden assessed 9 days after ADC administration. Figure 10 As shown, both anti-CD33 ADCs exhibited antitumor activity, with ADC-100 being more effective in reducing hematologic tumor burden. The corresponding non-target conjugate showed minimal effect compared to the catalytic control.

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

[0328] Table 5. Mean number of surviving tumor cells on day 7, as shown in Table 5. Figure 11 The drawing.

[0329] These data illustrate the dose-responsive inhibitory effect of 15G15-compound 100 ADC in an in vivo model of disseminated AML. 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 using anti-CD33 antibody 15G15, a sq-Cit linker, and either payload compound 100 (ADC-100) or compound B (ADC-B) were prepared according to the procedures described in Examples 5 and 11, with a DAR of 6. The cytotoxicity of these ADCs relative to the mediator or unmodified antibody (Ab) at 30 mg / kg was assessed in SCID mice using in vivo subcutaneous MV4-11 assays. MV4-11 leukemia cells (10 million cells in 0.1 mL HBSS) were subcutaneously inoculated into the flank region of mice. When the tumor reached approximately 200 mm... 3 At the time of study, animals were randomly assigned to groups 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 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 via a single intravenous injection into the tail vein of mice. 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 (mm²) 3 = (length x width) 2 ) x0.5.

[0331] The graph showing tumor volume over time is shown in Figure 12 In the middle. For example Figure 12As shown, 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 advantages of compound 100 as an ADC in disseminated (or liquid) cancers compared to solid tumors. This may be because compound 100 contains a net overall charge that inhibits passive diffusion across the membrane. This could lead to a reduced bystander effect (activity in non-targeting neighboring cells), potentially resulting in reduced activity 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.

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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