Immunomodulator antibody-drug conjugate and use thereof

Abstract

Disclosed in the present invention is an immunomodulator antibody-drug conjugate and use thereof. The present invention provides an antibody-drug conjugate represented by formula I. The antibody-drug conjugate has a good modulatory effect on TLR7 / 8 and can effectively treat and / or alleviate cancer. (D1-L1)m-Ab-(L2-ST1)n (I)

Description

Immunomodulatory antibody-drug conjugates and their applications

[0001] This application claims priority to Chinese Patent Application No. 202510198585.0, filed on February 23, 2025, and Chinese Patent Application No. 202610181734.7, filed on February 6, 2026. The full text of the above patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to an immunomodulatory antibody-drug conjugate and its applications. Background Technology

[0003] Toll-like receptors (TLRs) are a family of important proteins that recognize pathogen-associated molecular patterns, sensing and initiating innate immune responses and promoting the development of adaptive immune responses. TLRs are primarily expressed on immune cells, such as myeloid dendritic cells (mDCs), plasmacytic dendritic cells (pDCs), monocytes, and B cells (Kawai and Akira, 2010), as well as in the lungs. In humans, more than 10 TLRs are considered to have significant functions. TLRs 1 / 2 / 4 / 5 and 6 are located in the cell membrane and their main function is to recognize extracellular macromolecular ligands from bacteria and fungi. Conversely, TLRs 3 / 7 / 8 / 9 are located in the intracellular endosome membrane and their main function is to recognize exogenous nucleic acids from pathogen-derived cells. Although most TLRs function through specific signaling pathways (primarily the MyD88-dependent pathway), different TLRs can coordinate different downstream molecules. The addition of specific TLRs leads to the activation of different cell populations (Schreibelt, et al., 2010) and the production of different modalities of cytokines and other inflammatory mediators (Ghosh, et al., 2006), thereby evoking different immune responses. For example, upon ligand binding, TLR8 forms a dimer and undergoes a conformational change, leading to the involvement of the adaptor protein MyD88. MyD88 recruits interleukin-1 receptor-associated kinases, resulting in the activation of downstream signaling pathways, including mitogen-associated protein kinases and the transcription factor NF-κB.

[0004] TLRs located in endosomes, primarily TLR7 / 8 / 9, have been considered highly attractive new targets for anticancer immunotherapy (Kanzler et al., 2007; Kreig, 2008; Smits et al., 2008; Hennessy et al., 2010; Kaczanowska et al., 2013; Beesu et al., 2016). For example, TLR7 activates pDCs to respond to viral infections, induces high levels of interferon-α, and induces adaptive T cell responses in major cells to endogenous viral antigens (Liu et al., 2009). Compared to TLR7 / 9, TLR8 is more widely expressed in different subtypes of immune cells. Regulatory T cells (Tregs) possess potent immunosuppressive capabilities and are a major obstacle to effective cancer immunotherapy. The TLR8 signaling pathway has been shown to be a necessary and sufficient condition for reversing the suppressive function of Treg cells to lead to strong tumor suppression. TLR8 selective agonists effectively activate various immune cells, including mDCs and monocytes (Gorden et al., 2005), promoting adaptive immune responses against cancer cells (Krug et al., 2003; Schnurr et al., 2005). Activated mDCs engulf apoptotic and dead tumor cells, and then, compared to pDCs, more effectively cross-present tumor-associated antigens to CD8+ CTLs (Berard et al., 2000; Dalgaard et al., 2005). Furthermore, mDC activation leads to the release of TNFα and interleukin-12 (IL-12), which can stimulate the activation of T cells and NK cells. NK cell activation is a major mechanism of antibody-mediated cytotoxicity (ADCC). Therefore, enhancing tumor cell killing through ADCC may present an important therapeutic opportunity for TLR8 selective inhibitors (Lu et al., 2011). Some monoclonal antibody therapies, such as rituximab and trastuzumab, are widely used to treat cancer patients, exerting their therapeutic effects through ADCC (Ferris et al., 2010). In fact, adding a TLR8 agonist to mAb therapy can enhance ADCC, thereby increasing the efficacy of mAb treatment (Ferris et al., 2015). Furthermore, recent studies have shown that TLR8 agonists can exert their anti-tumor effects directly, independent of their immunomodulatory function (Ignatz-Hoover et al., 2015). Therefore, TLR8 agonists can not only function as monotherapy but also enhance the efficacy of various chemotherapy and targeted anticancer drugs by strengthening the host immune response.

[0005] Among the TLR family members that recognize the nucleic acids of pathogenic microorganisms, TLR7 and TLR8 share high homology and can recognize some synthetically produced small molecules with antiviral activity, such as imidazoquinolines (ligands of TLR7 and TLR8). Studies of imidazoquinolines in a guinea pig genital herpes model infected with HSV showed that this compound had a relatively low effect on viral replication in vitro, but a strong effect in vivo, indicating that this class of compounds promotes the production of pro-inflammatory factors and regulates cytokines by immune cells, leading to an antiviral response (Int Immunopharmacol 2002; 2:443-451). More importantly, TLR7 and TLR8 can recognize viral ssRNA. Studies have shown that ssRNA viruses are natural ligands of TLR7 and TLR8, such as human immunodeficiency virus (HIV), influenza virus, Sendai virus, dengue virus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), hepatitis B virus (HBV), and hepatitis C virus (HCV). TLR8 can recognize antiviral compounds, ssRNA viruses, and synthetic oligonucleotides, and induce Th1 and inhibit Th2 cytokine secretion and Treg proliferation through the MyD88-dependent signaling pathway, mediating antiviral immunity and exerting anti-infective and anti-allergic effects.

[0006] Antibodies (or antibody fragments) can be linked to a drug (effect molecule) payload to form antibody-drug conjugates (ADCs). The antibodies in ADCs bind to target cells and, through processes such as endocytosis, release the effector molecule, exerting a therapeutic effect while reducing systemic exposure and drug toxicity. Currently, the most common ADC effector molecules fall into two main categories: 1) tubulin inhibitors; such as auristatin 10 derivatives, maytansine derivatives, tubulolysin, or cryptomycin; 2) DNA-damaging drugs; such as pyrrole benzodiazepines and indolechlorobenzodiazepines, kazidromycin, ducamycin and their derivatives, or DNA topoisomerase I inhibitors (camptothecin and its derivatives). Currently, there are no marketed ADCs using TLR7 / 8 agonists, or combinations of TLR7 / 8 agonists and DNA topoisomerase I inhibitors / tubulin inhibitors, as effector molecules, indicating a significant unmet clinical need. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an immunomodulatory antibody-drug conjugate and its application. This antibody-drug conjugate may further contain a topoisomerase I inhibitor or a microtubule inhibitor, such as camptothecin and its derivatives or olretamine derivatives, as an effector molecule, which can effectively treat and / or alleviate cancer.

[0008] This invention provides an antibody-drug conjugate as shown in Formula I, (D1-L1). m -Ab-(L2-ST1) n

[0009] (I)

[0010] Where Ab represents antibody;

[0011] m can be any value from 0 to 8;

[0012] n is any value from 1 to 8;

[0013] D1 is a topoisomerase I inhibitor or a tubulin inhibitor;

[0014] L1 is a connector containing a pyrolytic linker;

[0015] L2 is

[0016] A is a benzene ring or a 5-6 membered heteroaromatic ring;

[0017] M is the connector that links to the antibody;

[0018] X is the connection key.

[0019] X1 is a linker bond, -NHC(O)-, -O-, or -NH-;

[0020] X2 is a linker, -C(O)-, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene;

[0021] B is the connection key, C 6-10 arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group; the C 6-10 The arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group is unsubstituted, or selectively replaced by 1-3 groups selected from halogens, oxo groups, C... 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 The alkoxy group can be substituted at any position;

[0022] ST1 is

[0023] U is -C(O)- or a connection key;

[0024] V is either NH or S;

[0025] Y is either -O- or -NH-;

[0026] R1 is a halogen, -OP(O)(OH)2, or a pyranose group;

[0027] R2 represents H and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or amino C 1-6 alkyl;

[0028] R3 is H or C 1-6 alkyl;

[0029] R4 is C 1-6 Alkyl, C 1-6 Alkoxy or C 1-3 Alkoxy C 1-4 alkyl;

[0030] R4' is C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or C 1-3 Alkoxy C 1-4 alkyl;

[0031] R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OR. a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -NR a C(O)OR b -NR a C(O)NR a R b or -C(O)OR b The substituents can be substituted at any position;

[0032] R6 represents H, halogen, carboxyl, or C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl;

[0033] A1, A2, and A3 are each independently CR7 or N;

[0034] R7 is H or halogen;

[0035] Each R a and R bIndependently, they are hydrogen and C respectively. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl, 5-10 heteroaryl, C 3-10 cycloalkyl C 1-6 Alkyl, 3-10 membered heterocyclic alkyl C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl;

[0036] x is 0, 1, or 2;

[0037] t can be 0, 1, 2, 3, 4, or 5;

[0038] Furthermore, the antibody-drug conjugate shown in Formula I satisfies one of the following conditions:

[0039] (1) m is any value from 1 to 8;

[0040] (2) m is 0; x is 1 or 2;

[0041] (3) m is 0; x is 0; X1 is a linking bond, -NHC(O)-, -O- or -NH-; X2 is a linking bond, -C(O)- or -C(R3)2(CH2) 0-2 -;

[0042] (4) m is 0, x is 0; A is a 5-6 member heteroaryl ring.

[0043] In some embodiments, the antibody-drug conjugate as shown in Formula I is defined as follows, and the remaining groups are defined as described in any other embodiment (hereinafter referred to as "in some embodiments"):

[0044] In some implementations, the antibody-drug conjugate shown in Formula I satisfies one of the following conditions:

[0045] (1) m is any value from 1 to 8;

[0046] (2) m is 0; x is 1 or 2;

[0047] (3) m is 0; x is 0; X1 is a linking bond, -NHC(O)-, -O- or -NH-; X2 is a linking bond, -C(O)- or -C(R3)2(CH2) 0-2 -

[0048] In some implementations, the antibody may include one or two antigen-binding domains that can bind to an antigen.

[0049] In some implementations, the antibody may include an antigen-binding domain that can bind to an antigen.

[0050] In some implementations, the antibody may contain only an antigen-binding domain that can bind to an antigen.

[0051] In some implementations, the antibody may include an Fc segment.

[0052] In some implementations, the antibody may contain only one Fc segment.

[0053] In some implementations, the antibody may contain only an antigen-binding domain that can bind to the antigen and an Fc segment.

[0054] In some implementations, the antibody may be a monoclonal antibody.

[0055] In some implementations, the antibody may be an anti-HER2 antibody or an anti-FRα antibody.

[0056] In some implementations, the anti-HER2 antibody is Trastuzumab or Pertuzumab.

[0057] In some embodiments, the anti-HER2 antibody is a Trastuzumab variant or a Pertuzumab variant, the Trastuzumab variant having at least equivalent binding ability to the Trastuzumab antibody, and the amino acid sequence of the Trastuzumab variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Trastuzumab antibody;

[0058] The Pertuzumab variant has at least the same binding capacity as the Pertuzumab antibody, and the amino acid sequence of the Pertuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Pertuzumab antibody.

[0059] In some embodiments, the Trastuzumab variant has the same HCDR and LCDR regions as the Trastuzumab antibody, and the amino acid sequence of the Trastuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Trastuzumab antibody.

[0060] In some embodiments, the Pertuzumab variant has the same HCDR and LCDR regions as the Pertuzumab antibody, and the amino acid sequence of the Pertuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Pertuzumab antibody.

[0061] In some embodiments, the anti-FRα antibody is huMov19.

[0062] In some embodiments, the anti-FRα antibody is a huMov19 variant, Farletuzumab, or a Farletuzumab variant; the huMov19 variant has at least equivalent binding ability to the huMov19 antibody, and the amino acid sequence of the huMov19 variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the huMov19 antibody;

[0063] The Farletuzumab variant has at least the same binding capacity as the Farletuzumab antibody, and the amino acid sequence of the Farletuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Farletuzumab antibody.

[0064] In some embodiments, the huMov19 variant has the same HCDR and LCDR regions as the huMov19 antibody, and the amino acid sequence of the huMov19 variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the huMov19 antibody; preferably, the huMov19 variant is huMov19-AAG, huMov19-K149.5C, huMov19-AAG-K149.5C, or huMov19-LALA; more preferably, the huMov19 variant is huMov19-AAG, huMov19-K149.5C, or huMov19-AAG-K149.5C.

[0065] In some embodiments, the Farletuzumab variant has the same HCDR and LCDR regions as the Farletuzumab antibody, and the amino acid sequence of the Farletuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Farletuzumab antibody; preferably, the Farletuzumab variant is Farletuzumab-AAG, Farletuzumab-AAG-K149.5C, Farletuzumab-K149.5C, or Farletuzumab-LALA; more preferably, the Farletuzumab variant is Farletuzumab-AAG, Farletuzumab-AAG-K149.5C, or Farletuzumab-LALA.

[0066] In some embodiments, the antibody is huMov19, Trastuzumab, or Pertuzumab, the amino acid sequence of the light chain of huMov19 is shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain of huMov19 is shown in SEQ ID NO:6.

[0067] In some embodiments, the antibody is Farletuzumab, huMov19-AAG, huMov19-K149.5C, huMov19-AAG-K149.5C, Farletuzumab-AAG, Farletuzumab-AAG-K149.5C, or Farletuzumab-LALA.

[0068] The amino acid sequence of the light chain of huMov19-AAG is shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain of huMov19-AAG is shown in SEQ ID NO:10.

[0069] The amino acid sequence of the light chain of Farletuzumab-AAG is shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain of Farletuzumab-AAG is shown in SEQ ID NO:12.

[0070] The amino acid sequence of the light chain of huMov19-K149.5C is shown in SEQ ID NO:13, and the amino acid sequence of the heavy chain of huMov19-K149.5C is shown in SEQ ID NO:14.

[0071] The amino acid sequence of the light chain of huMov19-AAG-K149.5C is shown in SEQ ID NO:15, and the amino acid sequence of the heavy chain of huMov19-AAG-K149.5C is shown in SEQ ID NO:16.

[0072] The amino acid sequence of the light chain of Farletuzumab-AAG-K149.5C is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain of Farletuzumab-AAG-K149.5C is shown in SEQ ID NO:18.

[0073] The amino acid sequence of the light chain of Farletuzumab-LALA is shown in SEQ ID NO:19, and the amino acid sequence of the heavy chain of Farletuzumab-LALA is shown in SEQ ID NO:20.

[0074] In the antibody-drug conjugate shown in Formula I, m or n can be an integer or a non-integer. When it is a non-integer, it means that the antibody-drug conjugate shown in Formula I is a mixture of antibody-drug conjugates with different conjugation ratios. When it is an integer, it can mean that the antibody-drug conjugate shown in Formula I is a single antibody-drug conjugate with a fixed conjugation ratio, or it can mean that the antibody-drug conjugate shown in Formula I is a mixture of antibody-drug conjugates with different conjugation ratios.

[0075] In some implementations, m is 0 and n is any value from 1 to 8; m is 0 and n is preferably any value from 1 to 4.

[0076] In some implementations, m is 0 and n is any value from 2 to 8.

[0077] In some implementations, m is any value from 1 to 8; n is any value from 1 to 4.

[0078] In some implementations, m is any value from 2 to 8, and n is any value from 1 to 3.

[0079] In some implementations, m+n≤10.

[0080] In some implementations, m+n≤8.

[0081] In some embodiments, D1 is camptothecin and its derivatives, preferably camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, irinotecan, SN-38, SN-38 derivatives, Dxd, Dxd derivatives, topotecan, letopotecan, belotecone, ixotecan or ixotecan derivatives.

[0082] In some implementations, D1 is (preferred) ), (preferred) ),

[0083] R 9-1 and R 9-2 H, D, and C are independent of each other. 3-5 cycloalkyl or cyclopropylmethyl; or, R 9-1 and R 9-2 Together with the C atoms they are attached to, they form 3-5 membered cycloalkyl groups;

[0084] R 9-3 C 1-6 Alkyl; R 9-4 and R 9-5 C, each independently 1-6 Alkyl, halogen or hydroxyl.

[0085] In some embodiments, D1 is a microtubule inhibitor, preferably an olistatin derivative, such as MMAE or MMAF.

[0086] In some implementations, D1 is (preferred) ); R 9-1 and R 9-2 Each is independently H, D, cyclopropyl, or cyclopropylmethyl; or, R 9-1 and R 9-2 Together with the C atom it is connected to, a 3-5 membered cycloalkyl group is formed; the 3-5 membered cycloalkyl group is preferably cyclopropyl.

[0087] In some implementation schemes, R 9-1 and R 9-2 C independently 3-5 Cycloalkyl.

[0088] In some implementations, D1 is

[0089] In some implementations, D1 is (preferred) ); R 9-4 and R 9- 5 are C independently 1-6 Alkyl, halogen, or hydroxyl; preferably methyl, chlorine, or hydroxyl.

[0090] In some implementations, D1 is

[0091] In some implementations, D1 is

[0092] In some implementations, D1 is R 9-3 It is H or ethyl.

[0093] In some implementation schemes, R 9-3 C 1-6 alkyl.

[0094] In some implementations, D1 is

[0095] In some implementations, L1 is -M'-L 1a -L 1b -L 1c -;-L 1a - is an extension group; -L 1b - is a cleavable polypeptide linker; -L 1c - represents a linking bond or a self-eliminating group; M' represents a linker.

[0096] In some implementations, L1 is -M'-L 1a -L 1c -;-L 1a - is an extension group; -L 1c - represents a linking bond or a self-eliminating group; M' represents a linker.

[0097] In some implementations, in L1, -L 1b - can be -Gly-Gly-Phe-Gly-, -Ala-Ala-Ala, -Val-Cit-, or -Val-Ala-; it can also be -Gly-Glu-Val-Ala-Gly-, -Gly-Glu-Val-Ala-, or -Glu-Val-Ala-, read from left to right, with the left side being the NH end, and the NH end being the L end. 1a connect.

[0098] In some implementations, in L1, -L 1a -Independently for-C 1-6 Alkylene -C(O)-, -C 1-6 Alkylene-C(O)-NH-C 1-6 Alkylene-(PEG) Z -C(O)-、-phenyl-C 1-6 alkylene-C(O)-, -phenyl-C 1-6 Alkylene-C(O)-NH-C 1-6 alkylene-C(O)- or -C 1-6 Alkylene-C(O)-NH-C 1-6alkylene-C(O)-; the -L 1a -Through the carbonyl end and -L 1b -or-L 1c - Interconnected.

[0099] In some implementations, in L1, -L 1a -Independently for-C 1-6 Alkylene -C(O)-; more preferably -C 3-6 alkylene-C(O)-; the -L 1a -Through the carbonyl end and -L 1b - Interconnected, for example

[0100] In some implementations, in L1, -L 1a -Independently for-C 1-6 Alkylene -C(O)-; more preferably -C 3-6 alkylene-C(O)-; the -L 1a - Interconnected via carbonyl ends and -Gly-Gly-Phe-Gly-.

[0101] In some implementations, in L1, -L 1a -Independently for-C 1-6 alkylene-C(O)- or -C 1-6 Alkylene-C(O)-NH-C 1-6 Alkylene-(PEG) Z -C(O)-; z is an integer from 1 to 20; the -L 1a -Through the carbonyl end and -L 1b - Interconnected.

[0102] In some implementations, in L1, -L 1a -Independently for-C 1-6 alkylene-C(O)- or -C 1-6 Alkylene-C(O)-NH-C 1-6 Alkylene-(PEG) Z -C(O)-; z is an integer from 1 to 20; the -L 1a - Interconnected via carbonyl ends and -Val-Ala- or -Val-Cit-.

[0103] In some implementations, in L1, -L 1a -Independently -phenyl-C 1-6 Alkylene-C(O)-NH-C 1-6 alkylene-C(O)-; the -L 1a -Through the carbonyl end and -L 1b -or-L 1c- Interconnected, for example

[0104] In some implementations, in L1, M' is The connector is connected to Ab via terminal c). In some embodiments, L1, -L 1c -Independent for connection keys, (preferred) ), L 1c Connect to D1 via terminal d), and to -L via terminal e). 1a -or-L 1b -connect.

[0105] In some implementations, in L1, -L 1c - Independently for The self-eliminating group is connected to D1 via the d) end and to -L via the e) end. 1b -connect.

[0106] In some implementations, in L1, -L 1c - Independently for The self-eliminating group is connected to D1 via the d) end and to -Gly-Gly-Phe-Gly- via the e) end.

[0107] In some embodiments, in L1, the self-eliminating group is The self-eliminating group is connected to D1 via the d) end and to -Val-Cit- or -Val-Ala- via the e) end.

[0108] In some implementations, L1 is -M'-L 1a -L 1b -L 1c -;

[0109] -L 1a -for-C 1-6 Alkylene-C(O)-;

[0110] -L 1b - can be -Gly-Gly-Phe-Gly-, -Ala-Ala-Ala, -Val-Cit-, -Val-Ala-, -Gly-Glu-Val-Ala-Gly-, -Gly-Glu-Val-Ala-, or -Glu-Val-Ala-, read from left to right, with the left side being the NH end, and the NH end being the L end. 1a connect;

[0111] -L 1c -For connection key, (preferred) M' is the connector.

[0112] In some implementations, L1 is -M'-L 1a -L 1c -;-L 1a - is -phenyl-C 1-6 Alkylene-C(O)-NH-C 1-6 alkylene-C(O)-; the -L 1a -Through the carbonyl end and -L 1c - Interconnected; -L 1c -for (preferred) M' is the connector.

[0113] In some implementations, L1 is The L1 is connected to the Ab via the c) end; furthermore, the L1 is connected to the thiol group in the Ab via the c) end.

[0114] In some implementations, L1 is The L1 is connected to the Ab via the c) end; furthermore, the L1 is connected to the thiol group in the Ab via the c) end.

[0115] In some implementations, L1 is

[0116] The L1 is connected to the Ab via the c) end; furthermore, the L1 is connected to the thiol group in the Ab via the c) end.

[0117] In some implementations, L1 is The L1 is connected to the Ab via the c) end; further, the L1 is connected to the thiol group in the Ab via the c) end.

[0118] In some implementations, -L1-D1 is:

[0119] In some embodiments, A is phenyl, pyridyl, pyrimidinyl, pyridazinyl, or oxazolyl, preferably A for A is connected to M via end b).

[0120] In some implementations, m is any value from 1 to 8; A for b) Connect the end to M.

[0121] In some implementation schemes, A for x is 1 or 2, and A is connected to M through end b).

[0122] In some implementation schemes, A for R1 is a halogen, -OP(O)(OH)2, or A is connected to M via end b).

[0123] In some embodiments, m is 0, x is 0, and A is pyridinyl, pyrimidinyl, pyridazinyl, or oxazolyl, preferably. for A is connected to M via end b).

[0124] In some implementation schemes, A is A is connected to M via end b).

[0125] In some embodiments, the thiol or amino groups in M ​​and Ab are linked; preferably, the thiol groups in M ​​and Ab are linked.

[0126] In some implementations, M is methylene, The M is connected to Ab via the c) end.

[0127] In some implementations, R2 is H or halogenated C. 1-4 Alkyl, hydroxyl C 1-4 Alkyl or amino C 1-4 Alkyl groups, such as H,

[0128] In some implementations, X is a connection key, X is connected to B via end a); t is 0, 1, 2 or 3.

[0129] In some implementations, X is a connection key, X is connected to B via end a).

[0130] In some implementations, X1 is -NHC(O)-; X2 is a linker bond or C 3-6 Cycloalkylene.

[0131] In some implementations, X1 is -NHC(O)-; X2 is a linker or

[0132] In some implementations, X1 is -NHC(O)-; X2 is

[0133] In some implementations, X1 is the connection key; X2 is the connection key.

[0134] In some implementations, X1 is a linker or -O-; X2 is -C(R3)2(CH2). 0-3 -; R3 is H or methyl.

[0135] In some implementations, -X1-X2- is a linker, -CH2-, -OC(CH3)2-CH2-, -NHC(O)-, or It can also be used for

[0136] In some embodiments, B is a linking bond, a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, or surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8-2 The substituted 5-6 nucleotide heteroaryl group may be replaced by one, two, or three R groups. 8-3 Substituted 8-12 fused cyclic groups;

[0137] In B, the 5-6 membered heteroaryl group and the group with 1, 2, or 3 R groups 8-2 In the substituted 5-6-membered heteroaryl group, the heteroatom is independently selected from one or more of N, O and S, and the number of heteroatoms is independently 1, 2 or 3; the heteroatom is preferably N, and the number of heteroatoms is preferably 1 or 2.

[0138] In B, the 8-12 fused cycloaliphatic group and the group surrounded by 1, 2, or 3 R groups 8-3 The 8-12 fused cyclic group in the substituted 8-12 cyclic group is independently a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring; in the 5-6 fused heteroaromatic ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3; in the 5-6 fused heteroene ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3.

[0139] R 8-1 R 8-2 and R 8-3 Independently halogen, oxo group, C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 Alkyl group.

[0140] In some embodiments, in B, the 8-12 fused cyclic group can be a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring, which is connected to X1 through the cyclic C1; in the 5-6 fused heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 5-6 fused heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2.

[0141] In some embodiments, B is an 8-12 membered cyclic group or surrounded by one, two, or three R groups. 8-3 The substituted 8-12 fused cyclic group; the 8-12 fused cyclic group is a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 6-membered heteroaromatic ring or a benzene ring, and the cyclic C2 is a 6-membered heteroene ring, which is connected to the X1 through the cyclic C1; in the 6-membered heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 6-membered heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2.

[0142] In some implementations, B is the connector key. The B is unsubstituted, or selectively substituted by 1 to 3 elements selected from halogens, C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 The alkoxy group is substituted at any position; B is connected to X1 via the d) end; preferably, B is unsubstituted.

[0143] In some implementations, -XBX 1 -X 2 -Choose from any of the following options:

[0144] Option 1:

[0145] X is the connection key.

[0146] B is a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8- 2-substituted 5-6-membered heteroaryl groups or substituted with 1, 2 or 3 R groups 8-3 Substituted 8-12 fused cyclic groups;

[0147] X1 is -NHC(O)-;

[0148] X2 is the linker, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene;

[0149] Preferably, X is a linking bond, B is an 8-12 membered cyclic group; X1 is -NHC(O)-; X2 is C 3-6 Cycloalkylene;

[0150] Alternatively, X is a linking bond, B is an 8-12 membered cyclic group; X1 is -NHC(O)-; X2 is a linking bond;

[0151] Option 2:

[0152] X is

[0153] B is a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8- 2-substituted 5-6-membered heteroaryl groups or substituted with 1, 2 or 3 R groups 8-3 Substituted 8-12 fused cyclic groups;

[0154] X1 is the connection key;

[0155] X2 is a linker or -C(R3)2(CH2) 0-3 -;

[0156] Option 3:

[0157] X is

[0158] B is the connection key;

[0159] X1 is either -O- or -NH-;

[0160] X2 is -C(R3)2(CH2) 0-3 -

[0161] In some implementations, R4 is C 1-6 Alkyl; R4 is preferably n-propyl, but may also be n-butyl.

[0162] In some implementations, R4 is C 1-3 Alkoxy C 1-4 Alkyl groups, preferably

[0163] In some implementations, R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH, -OC(O)NR. a R b -NR aC(O)OR b or -NR a C(O)NR a R b The substituents can be substituted at any position; R a For H; R b For H, C 1-6 Alkyl or C 3-6 cycloalkyl, such as C 1-6 Alkyl, C 1-6 Alkoxy, -C 1-6 alkylene -OH or -OC 1-6 Alkylene-NHC(O)NHR b For example, n-propyl,

[0164] In some implementations, R7 is H, F, or Cl, such as H.

[0165] In some implementations, ST1 is A1, A2, and A3 are each independently CR7 or N; R4 is C. 1-6 Alkyl, C 1-6 Alkoxy or C 1-3 Alkoxy C 1-4 Alkyl; R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OR. a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -NR a C(O)OR b -NR a C(O)NR a R b and -C(O)OR b The substituents can be substituted at any position; R7 is H or a halogen.

[0166] In some implementations, ST1 is A1, A2, and A3 are each independently CR7; R4 is C. 1-6 Alkyl; R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH or -NHC(O)NHR. b The substituents can be substituted at any position; R7 is H, F, or Cl; R b It is cyclopropyl or cyclobutyl.

[0167] In some implementations, ST1 is A1, A2, and A3 are each independently CH; R4 is n-propyl; R5 is C. 1- 6-alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH or -NHC(O)NHR. b The substituents can be substituted at any position; R b It is cyclobutyl.

[0168] In some implementations, R6 is H, carboxyl, or phenyl C. 1-6 Alkyl-, such as H, carboxyl or benzyl.

[0169] In some implementation schemes, for

[0170] In some implementations, Y is O.

[0171] In some implementations, V is NH.

[0172] In some implementations, R4' is C 1-6 Alkyl groups, such as ethyl or n-butyl.

[0173] In some implementations, ST1 is

[0174] In some implementations, -XB-X1-X2-ST1 has the structure shown in Table 1:

[0175] Table 1

[0176] In some implementations, -L2-ST1 is:

[0177] In some implementations, m is 0; the antibody-drug conjugate as shown in Formula I is:

[0178] Preferably, the antibody-drug conjugate as shown in Formula I is any of the compounds shown in Table A:

[0179] Table A

[0180] In some embodiments, the antibody-drug conjugate as shown in Formula I is an immunomodulatory antibody-drug conjugate as shown in Formula I-2:

[0181] Among them, Ab, R1, R 9-1 R 9-2 The definitions of X, X1, X2, B, ST1, M, m, and n are as described above.

[0182] In some embodiments, the antibody-drug conjugate as shown in Formula I is an antibody-drug conjugate as shown in Formulas I-3:

[0183] The definitions of Ab, R1, X, X1, X2, B, ST1, M, m, and n are as described above.

[0184] In some embodiments, the antibody-drug conjugate as shown in Formula I is an antibody-drug conjugate as shown in Formulas I-4:

[0185] The definitions of Ab, R1, X, X1, X2, B, ST1, M, m, and n are as described above.

[0186] In some embodiments, the antibody-drug conjugate as shown in Formula I is an antibody-drug conjugate as shown in Formulas I-5:

[0187] The definitions of Ab, R1, X, X1, X2, B, ST1, M, m, and n are as described above.

[0188] In some embodiments, the antibody-drug conjugate as shown in Formula I is an antibody-drug conjugate as shown in Formulas I-6:

[0189] The definitions of Ab, R1, X, X1, X2, B, ST1, M, m, and n are as described above.

[0190] In some embodiments, the antibody-drug conjugate as shown in Formula I is:

[0191] In some embodiments, the antibody-drug conjugate as shown in Formula I is any of the compounds shown in Table B.

[0192] Table B:

[0193] This invention provides a compound as shown in Formula II or a pharmaceutically acceptable salt thereof.

[0194] Wherein, A is a benzene ring or a 5-6 membered heteroaromatic ring;

[0195] MX is the connector front body;

[0196] X is the connection key.

[0197] X1 is a linker bond, -NHC(O)-, -O-, or -NH-;

[0198] X2 is a linker, -C(O)-, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene;

[0199] B is the connection key, C 6-10 arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group; the C 6-10 The arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group is unsubstituted, or selectively replaced by 1-3 groups selected from halogens, oxo groups, C... 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 The alkoxy group can be substituted at any position;

[0200] ST1 is

[0201] U is -C(O)- or a connection key;

[0202] V is either NH or S;

[0203] Y is either O or NH;

[0204] R1 is a halogen, -OP(O)(OH)2, or a pyranose group;

[0205] R2 represents H and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or amino C 1-6 alkyl;

[0206] R3 is H or C 1-6 alkyl;

[0207] R4 is C 1-6 Alkyl, C 1-6 Alkoxy or C 1-3 Alkoxy C 1-4 alkyl;

[0208] R4' is C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or C 1-3 Alkoxy C 1-4 alkyl;

[0209] R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OR. a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -NR a C(O)OR b -NR a C(O)NR a R b or -C(O)OR b The substituents can be substituted at any position;

[0210] R6 represents H, halogen, carboxyl, or C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl;

[0211] A1, A2, and A3 are each independently CR7 or N;

[0212] R7 is H or halogen;

[0213] Each R a and R b Independently, they are hydrogen and C respectively. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl, 5-10 heteroaryl, C 3-10 cycloalkyl C 1-6 Alkyl, 3-10 membered heterocyclic alkyl C 1-6 Alkyl, phenyl C1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl;

[0214] x is 0, 1, or 2;

[0215] t can be 0, 1, 2, 3, 4, or 5;

[0216] And the compound shown in Formula II satisfies one of the following conditions:

[0217] (1) X1 is -NHC(O)-; X2 is C 3-6 Cycloalkylene; A is a 5-6 membered heteroaryl ring;

[0218] (2) X1 is -NHC(O)-; X2 is C 3-6 Cycloalkylene; A is a benzene ring; x is 1 or 2;

[0219] (3) X1 is a linking bond, -NHC(O)-, -O-, or -NH-; X2 is a linking bond, -C(O)-, or -(CH2). 0-2 C(R3)2-.

[0220] In some embodiments, the 5-6 membered heteroaryl ring, 5-6 membered hypoaryl ring, 5-10 membered heteroaryl C 1-6 Alkyl, 3-10 membered heterocyclic alkyl and 3-10 membered heterocyclic alkyl C 1-6 The heteroatoms in the alkyl group are independently one, two, or three of N, O, and S, and the number of said heteroatoms is independently one, two, or three.

[0221] In some embodiments, the heteroatoms in the 5-10 aryl group are independently one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three.

[0222] In some embodiments, for compounds of Formula II or pharmaceutically acceptable salts thereof, certain functional groups are defined as follows, and the remaining functional groups are defined as in any other embodiment (hereinafter referred to as "in some embodiments"):

[0223] In some implementations, A is a benzene ring, a pyridine ring, a pyrimidine ring, a pyridazine ring, or an oxazole ring.

[0224] In some implementation schemes, for b) Connect to MX.

[0225] In some implementation schemes, A for A is connected to MX via end b); x is 1 or 2; X1 is a connecting bond, -NHC(O)-, -O- or -NH-;

[0226] X2 is a linker, -C(O)-, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene.

[0227] In some embodiments, A is a benzene ring, preferably, for x is 1 or 2, and end b) is connected to MX.

[0228] In some implementation schemes, A for R1 is a halogen, -OP(O)(OH)2, or A is connected to MX via terminal b); X1 is a connecting bond, -NHC(O)-, -O-, or -NH-; X2 is a connecting bond, -C(O)-, or -C(R3)2(CH2). 0-3 -or C 3-6 Cycloalkylene.

[0229] In some embodiments, A is pyridinyl, pyrimidinyl, pyridazinyl, or oxazolyl, preferably, x is 0, and the b) end is connected to MX.

[0230] In some implementation schemes, A is A is connected to MX via terminal b); X1 is a connecting bond, -NHC(O)-, -O-, or -NH-; X2 is a connecting bond, -C(O)-, or -C(R3)2(CH2). 0-3 -or C 3-6 Cycloalkylene.

[0231] In some embodiments, A is a benzene ring, preferably, for When x is 0, more preferably, A for A is connected to MX via terminal b); X1 is a connecting bond, -NHC(O)-, -O-, or -NH-; X2 is a connecting bond, -C(O)-, or -(CH2). 0-2 C(R3)2-.

[0232] In some implementations, R1 is F, -OP(O)(OH)2, or

[0233] In some implementations, MX is -CH2Br or R9 and R 9-1Independently, H, Br,

[0234] In some implementations, MX is -CH2Br or

[0235] In some implementations, R2 is H or halogenated C. 1-4 Alkyl, hydroxyl C 1-4 Alkyl or amino C 1-4 Alkyl groups, such as H,

[0236] In some implementations, X is a connection key, X is connected to B via end a); t is 0, 1, 2 or 3.

[0237] In some implementations, X is a connection key, X is connected to B via end a).

[0238] In some implementations, X1 is -NHC(O)-; X2 is a linker bond or C 3-6 Cycloalkylene.

[0239] In some implementations, X1 is -NHC(O)-; X2 is a linker or

[0240] In some implementations, X1 is -NHC(O)-; X2 is

[0241] In some implementations, X1 is the connection key; X2 is the connection key.

[0242] In some implementations, X1 is a linker or -O-; X2 is -C(R3)2(CH2). 0-3 -; R3 is H or methyl.

[0243] In some implementations, -X1-X2- is a linker, -CH2-, -OC(CH3)2-CH2-, -NHC(O)-, or It can also be used for

[0244] In some embodiments, B is a linking bond, a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, or surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8-2 The substituted 5-6 nucleotide heteroaryl group may be replaced by one, two, or three R groups. 8-3 Substituted 8-12 fused cyclic groups;

[0245] In B, the 5-6 membered heteroaryl group and the group with 1, 2, or 3 R groups 8-2 In the substituted 5-6-membered heteroaryl group, the heteroatom is independently selected from one or more of N, O and S, and the number of heteroatoms is independently 1, 2 or 3; the heteroatom is preferably N, and the number of heteroatoms is preferably 1 or 2.

[0246] In B, the 8-12 fused cycloaliphatic group and the group surrounded by 1, 2, or 3 R groups 8-3 The 8-12 fused cyclic group in the substituted 8-12 cyclic group is independently a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring; in the 5-6 fused heteroaromatic ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3; in the 5-6 fused heteroene ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3.

[0247] R 8-1 R 8-2 and R 8-3 Independently halogen, oxo group, C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 Alkyl group.

[0248] In some embodiments, in B, the 8-12 fused cyclic group can be a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring, which is connected to X1 through the cyclic C1; in the 5-6 fused heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 5-6 fused heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2.

[0249] In some embodiments, B is an 8-12 membered cyclic group or surrounded by one, two, or three R groups. 8-3 The substituted 8-12 fused cyclic group; the 8-12 fused cyclic group is a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 6-membered heteroaromatic ring or a benzene ring, and the cyclic C2 is a 6-membered heteroene ring, which is connected to the X1 through the cyclic C1; in the 6-membered heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 6-membered heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2.

[0250] In some implementations, B is the connector key. The B is unsubstituted, or selectively substituted by 1 to 3 elements selected from halogens, C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 The alkoxy group is substituted at any position; B is connected to X1 via the d) end; preferably, B is unsubstituted.

[0251] In some implementations, -XBX 1 -X 2 -Choose from any of the following options:

[0252] Option 1:

[0253] X is the connection key.

[0254] B is a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8- 2-substituted 5-6-membered heteroaryl groups or substituted with 1, 2 or 3 R groups 8-3 Substituted 8-12 fused cyclic groups;

[0255] X1 is -NHC(O)-;

[0256] X2 is the linker, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene;

[0257] Preferably, X is a linking bond, B is an 8-12 membered cyclic group; X1 is -NHC(O)-; X2 is C 3-6 Cycloalkylene;

[0258] Alternatively, X is a linking bond, B is an 8-12 membered cyclic group; X1 is -NHC(O)-; X2 is a linking bond;

[0259] Option 2:

[0260] X is

[0261] B is a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8- 2-substituted 5-6-membered heteroaryl groups or substituted with 1, 2 or 3 R groups 8-3 Substituted 8-12 fused cyclic groups;

[0262] X1 is the connection key;

[0263] X2 is a linker or -C(R3)2(CH2) 0-3 -;

[0264] Option 3:

[0265] X is

[0266] B is the connection key;

[0267] X1 is either -O- or -NH-;

[0268] X2 is -C(R3)2(CH2) 0-3 -

[0269] In some implementations, R4 is C 1-6 Alkyl; R4 is preferably n-propyl, but may also be n-butyl.

[0270] In some implementations, R4 is C 1-3 Alkoxy C 1-4 Alkyl groups, preferably

[0271] In some implementations, R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH, -OC(O)NR. a R b -NR a C(O)OR b or -NR a C(O)NR a R b The substituents can be substituted at any position; R a For H; R b For H, C 1-6 Alkyl or C 3-6 cycloalkyl, such as C 1-6 Alkyl, C 1-6 Alkoxy, -C 1-6 alkylene -OH or -OC 1-6 Alkylene-NHC(O)NHR b For example, n-propyl,

[0272] In some implementations, R7 is H, F, or Cl, such as H.

[0273] In some implementations, ST1 is A1, A2, and A3 are each independently CR7 or N; R4 is C. 1-6Alkyl, C 1-6 Alkoxy or C 1-3 Alkoxy C 1-4 Alkyl; R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OR. a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -NR a C(O)OR b -NR a C(O)NR a R b and -C(O)OR b The substituents can be substituted at any position; R7 is H or a halogen.

[0274] In some implementations, ST1 is A1, A2, and A3 are each independently CR7; R4 is C. 1-6 Alkyl; R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH or -NHC(O)NHR. b The substituents can be substituted at any position; R7 is H, F, or Cl; R b It is cyclopropyl or cyclobutyl.

[0275] In some implementations, ST1 is A1, A2, and A3 are each independently CH; R4 is n-propyl; R5 is C. 1- 6-alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH or -NHC(O)NHR. b The substituents can be substituted at any position; R b It is cyclobutyl.

[0276] In some implementations, R6 is H, carboxyl, or phenyl C. 1-6 Alkyl-, such as H, carboxyl or benzyl.

[0277] In some implementation schemes, for

[0278] In some implementations, Y is O.

[0279] In some implementations, V is NH.

[0280] In some implementations, R4' is C 1-6 Alkyl groups, such as ethyl or n-butyl.

[0281] In some implementations, -XB-X1-X2-ST1 has the structure shown in Table 1:

[0282] Table 1

[0283] In some embodiments, the compound shown in Formula II is:

[0284] Or its pharmaceutically acceptable salt.

[0285] The present invention provides a pharmaceutical composition comprising an antibody-drug conjugate as shown in Formula I as described in any embodiment of the present invention and pharmaceutically acceptable excipients.

[0286] In some implementations, the amount of the antibody-drug conjugate as shown in Formula I may be a therapeutically effective amount.

[0287] In the pharmaceutical composition, the pharmaceutically acceptable excipients may include pharmaceutically acceptable carriers, diluents, and / or excipients.

[0288] The pharmaceutical composition can be administered via conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, and local administration (e.g., intratumoral injection).

[0289] The present invention also provides the application of the antibody-drug conjugate shown in Formula I in the preparation of drugs for regulating T cells and other immune cells.

[0290] The present invention provides the use of an antibody-drug conjugate as shown in Formula I or the above-described pharmaceutical composition in the preparation of drugs for treating and / or alleviating tumors.

[0291] This invention provides the use of an antibody-drug conjugate as shown in Formula I or the above-described pharmaceutical composition in the preparation of a medicament for treating, alleviating, and / or preventing TLR7 / 8-mediated diseases.

[0292] In some implementations, the TLR7 / 8-mediated related diseases refer to tumors.

[0293] The tumor may be a malignant tumor, including metastatic and non-metastatic cancers, as well as familial and incidental cancers, and may also include solid and non-solid tumors.

[0294] In this invention, "tumor" and "cancer" have the same meaning; including but not limited to one or more of lung cancer, stomach cancer, pancreatic cancer, breast cancer, prostate cancer, brain cancer (including malignant glioma and medulloblastoma), ovarian cancer, colon cancer, small bowel cancer, rectal cancer, esophageal cancer, gallbladder cancer, and bile duct cancer; and may also be one or two of endometrial cancer and urothelial carcinoma.

[0295] In this invention, the tumor is preferably a HER2-expressing tumor (which can be a HER2-highly expressing tumor, a HER2-mediumly expressing tumor, or a HER2-lowly expressing tumor). More preferably, the tumor is a HER2-highly expressing tumor.

[0296] In this invention, the tumor is preferably a tumor expressing FRα (it can be a tumor with high FRα expression, a tumor with moderate FRα expression, or a tumor with low FRα expression). More preferably, the tumor is a tumor with high FRα expression.

[0297] In this invention, unless otherwise stated, the term "selectively substituted at any position by one or more groups" means that any one or more hydrogen atoms of one or more atoms specified on the group are substituted by the specified group, provided that the substitution does not exceed the normal valence of the specified atom, and the substitution at any position is a reasonable substitution commonly found in the art.

[0298] In this invention, when the bonding with a substituent intersects with the bonding of two atoms in the connecting ring, then such a substituent can be bonded to any bondable ring atom on the ring.

[0299] In this invention, any combination of variables is permitted only if such a combination produces a stable compound.

[0300] In this invention, when any variable appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, when R is substituted by one or more groups, each substituent is an independent substituent, which can be the same or different.

[0301] Unless otherwise stated, the following terms appearing in this specification and claims have the following meanings:

[0302] The term "antibody" refers to any form of antibody that exhibits desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting receptor signal transduction induced by a ligand). Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (including bispecific antibodies). Naturally occurring "antibodies" are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region and a light chain constant region. The light chain constant region contains a CL domain. The variable regions of both the heavy and light chains contain binding domains (antigen-binding domains) that interact with antigens. Antigen-binding domains may be provided by one or more variable regions on the antibody; specifically, the antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). In some cases, the antigen that the antibody can bind to is a tumor-associated antigen. In some cases, the antigens that the antibodies can bind to are tumor-specific antigens. The antibodies can be monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies. These antibodies can have any class (IgG, IgE, IgM, IgD, IgA, and IgY) or subtype (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). This invention includes not only complete antibodies but also fragments of immunologically active antibodies (including Fab, F(ab')2, scFv, or Fv fragments) or fusion proteins formed by antibodies and other sequences. Therefore, the term "antibody" in this invention also includes fragments, derivatives, and analogs of said antibodies. In some specific embodiments, the antibodies bind to antigens including, but not limited to, the following optional antigens: HER2 and FRα (FolRα).

[0303] In this invention, the "antibody" may further comprise engineered antibodies. The engineered antibody may 1) comprise one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both the heavy and light chains, wherein the one or more non-naturally encoded amino acids include, but are not limited to, one or more of the following non-natural amino acids: p-acetylphenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-acetyl-L-phenylalanine, p-propynyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, p-azidomethyl-phenylalanine, etc.; 2) insert cysteine ​​residues into different positions of the antibody heavy chain or light chain, or replace specific amino acid residues of the antibody heavy chain or light chain with cysteine ​​residues, thereby forming unpaired cysteine ​​residues for coupling.

[0304] The "antibody" or its antigen-binding fragment described in this invention may include an Fc region, which may be further modified. In some cases, one or more mutations in the Fc region lead to improvements in drugs containing such modified Fc regions, such as reduced effector function, altered regulation of drug metabolic half-life, and altered drug stability. In some cases, the modified Fc region may contain one or more mutations that reduce or eliminate the interaction between the antibody and the immune system. Key interactions may include the interaction between the antibody Fc and the Fcγ receptor, and the interaction with C1q of the complement system. When using IgG1 as an isotype of the antibody of this invention, effector function can be modulated by substituting a portion of the amino acid residues in the constant region. Variants of IgG1 that reduce or weaken effector function include, but are not limited to: IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), IgG1 AAG (IgG1-L234A, L235A, P329G), and other mutations that can reduce binding to FcγR and / or C1q receptors. L234A and L235A represent the substitution of alanine for leucine at positions 234 and 235, as determined by the EU index (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), p78-85); G237A represents the substitution of alanine for glycine at position 237, as determined by the EU index; and P329G represents the substitution of alanine for proline at position 329, as determined by the EU index. In some cases, Fc-terminal glycosylation modification can alter the pharmacodynamic and pharmacokinetic properties of drugs (Journal of Pharmaceutical Sciences. 2015, 104(6), 1866-1884). For example, naturally occurring antibodies produced by mammalian cells often contain branched, biantennary oligosaccharides, which are typically attached to Asn297 in the Fc-terminal CH2 domain via N-linked bonds. Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the stem of the biantennary oligosaccharide structure.Modifying oligosaccharides in antibodies can generate antibody variants with several improved properties. For example, defucylation can enhance antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP); reducing terminal sialylation or increasing terminal acetylglucosaminelation can enhance ADCC; treatment of CHO cells with mannosidase inhibitors can increase mannosylation and defucylation, thereby enhancing ADCC and slightly reducing CDC. Furthermore, glycosylation can be modified through mutation, such as the N297Q mutation, which mutates asparagine at position 297 to glutamine.

[0305] The term "monoclonal antibody" refers to a polypeptide having substantially the same amino acid sequence or originating from the same genetic source. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically include multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen. In some specific embodiments, the antigen that the monoclonal antibody can bind to is a tumor-associated antigen; in some specific embodiments, the antigen that the monoclonal antibody can bind to is a tumor-specific antigen; in some specific embodiments, the monoclonal antibody binds to antigens including, but not limited to, optional antigens such as HER2 or FRα.

[0306] The terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be a) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or b) polypeptides having substituent groups in one or more amino acid residues; or c) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or d) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag).

[0307] In this invention, the biosimilar of the monoclonal antibody refers to a biosimilar of the monoclonal antibody that is highly similar to the monoclonal antibody, despite minor differences in its inactive components in clinical practice, and has no clinically significant differences in safety and / or efficacy.

[0308] The term "HER2" (also known as ERBB2, NEU, NGL, TKR1, CD340, p185, MLN19, HER-2 / neu) refers to a transmembrane tyrosine kinase receptor of the epidermal growth factor (EGF) receptor family. HER2 comprises an extracellular binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. HER2 does not possess its own ligand-binding domain and therefore cannot bind growth factors; however, HER2 binds tightly to other ligand-binding EGF receptor family members (e.g., HER1 or HER3) to form a heterodimer, stabilizing ligand binding and enhancing activation of kinase-mediated downstream signaling pathways. In humans, species-specific HER2 isotypes exist: A, B, C, D, and E. The term "HER2" as used in this invention includes all HER2 isotypes.

[0309] The term "anti-HER2 antibody" refers to an antibody that targets the HER2 protein, and the anti-HER2 antibody can be derived from any species, such as humans, rats, mice, and rabbits. The anti-HER2 antibody is preferably a monoclonal anti-HER2 antibody, and more preferably a humanized anti-HER2 antibody. The anti-HER2 antibody includes, but is not limited to: pertuzumab, trastuzumab, trastuzumab biosimilars (e.g., inetetamab, coprelotamab, SIBP-01, HL02, TX05, ALT02, EG12014), and pertuzumab biosimilars (e.g., SYSA1901, TQB2440, HLX11, HS627, KM118), etc.

[0310] Trastuzumab (also known as Herceptin or Herclon) is a humanized monoclonal antibody that binds to the juxtamembrane region of the extracellular structure of the HER2 receptor (Hudis CA, N Engl J Med. 2007; 357(1):39-51). The amino acid sequences of the variable regions of the heavy and light chains of trastuzumab are disclosed in US Patent 5,821,337. Trastuzumab interacts with the tricyclic region formed by human HER2 residues 557-561, 570-573, and 593-603 (Cho et al., Nature 421:756-760, 2003). Trastuzumab can interfere with HER2 signaling by preventing HER2 receptor dimerization, promoting HER2 receptor endocytosis, and inhibiting the shedding of the extracellular domain. In addition, another important mechanism of anti-HER2 antibodies is to mediate antibody-dependent cytotoxicity (ADCC). In ADCC, anti-HER2 antibodies bind to tumor cells and then recruit immune cells, such as macrophages, through Fcγ receptor (FcγR) interaction. Trastuzumab possesses a conserved human IgG Fc region and is able to recruit immune effector cells responsible for antibody-dependent cytotoxicity (Hudis CA, N Engl J Med. 2007; 357(1):39-51). Trastuzumab was approved by the US FDA in September 1998 for the treatment of patients with tumors that overexpress HER2 and who have received one or more chemotherapy regimens for metastatic breast cancer; the sequence is as follows:

[0311] Trastuzumab light chain (SEQ ID NO:1):

[0312] Trastuzumab heavy chain (SEQ ID NO:2):

[0313] Pertuzumab (also known as Perjeta or Omnitarg) is a humanized monoclonal antibody that binds to the extracellular domain of the HER2 receptor and inhibits the dimerization of HER2 and the HER2 receptor. The amino acid sequences of the heavy and light chain variable regions of pertuzumab are disclosed in US Patent 7,560,111. Pertuzumab primarily interacts with residues in the 245-333 region of human HER2, particularly residues His245, Val286, Ser288, Leu295, His296, or Lys311 (Franklin et al., Cancer Cell 5:317-328, 2004). Studies have shown that pertuzumab is more effective than trastuzumab in disrupting the formation of HER1-HER2 and HER3-HER2 complexes in breast and prostate cancer cell lines (Agus et al., J Clin Oncol. 2005; 23(11):2534-43. Epub Feb 7, 2005). Pertuzumab was approved by the US FDA in June 2012 for use in combination with trastuzumab and docetaxel to treat patients with HER2-positive metastatic breast cancer who have not received anti-HER2 therapy or chemotherapy. The pertuzumab sequence is as follows:

[0314] Pertuzumab light chain (SEQ ID NO:3):

[0315] Pertuzumab heavy chain (SEQ ID NO:4):

[0316] In this invention, the anti-HER2 antibody is only required to specifically bind to HER2 (e.g., an anti-HER2 antibody that has the activity of internalization in HER2-expressing cells through HER2 binding), and is not limited to the antibodies listed above.

[0317] In this invention, the isotype of the "anti-HER2 antibody" includes IgG1, IgG2, IgG3, IgG4, etc., with IgG1, IgG2 or IgG4 being preferred.

[0318] The term "HER2 low expression" generally refers to a HER2 expression level of IHC 1+ or IHC 2+ / FISH negative (i.e., IHC 2+ and simultaneously negative for FISH) in clinical testing. The terms "HER2 high expression" and "HER2 positive" are used interchangeably, generally referring to a HER2 expression level of IHC 2+ / FISH positive (i.e., IHC 2+ and simultaneously positive for FISH) or IHC 3+ in clinical testing. When IHC staining intensity is reported as a range, the term "HER2 low expression" in this article includes not only IHC 1+ or IHC 2+ / FISH negative, but also IHC 0 to 1+ and IHC 1+ to 2+. The terms "HER2 high expression" and "HER2 positive" each include not only IHC 2+ / FISH positive or IHC 3+, but also IHC 2+ to 3+. In this invention, FISH negative means that the FISH test result shows no amplification of the HER2 gene, and FISH positive means that the FISH test result shows amplification of the HER2 gene.

[0319] The term "FRα," short for Folate receptor α, is a cell surface glycoprotein with a molecular weight of approximately 40 kDa encoded by the FOLR1 gene. Initially discovered as a folate-binding protein, it was later found to participate in regulating tumor cell proliferation and metastasis in addition to transporting folate. FRα is not expressed or expressed at very low levels in normal tissues, but it is widely and highly expressed in solid tumors, such as mesothelioma (72-100%), triple-negative breast cancer (35-68%), ovarian cancer (76-89%), and non-small cell lung cancer (14-74%). A certain proportion of FRα is expressed in non-malignant tissues such as the choroid plexus, as well as in bronchial epithelial cells, thyroid glands, salivary glands, breast tissue, colon, and bladder. Anti-FRα monoclonal antibodies include, but are not limited to, huMov19 and Farletuzumab.

[0320] huMov19 is the antibody used in the marketed FRα ADC drug Mirvetuximab soravtansine. Soravtansine is composed of huMov19 (or M9346A) antibody, a sulfonyl-SPDB linker, and DM4-type maytansine, with an average drug / antibody ratio of 3.5. The "huMov19" (or "M9346A") antibody contains an anti-FRα antibody (WO2022256507A1) with a full-length heavy chain (SEQ ID NO:6) and a full-length light chain (SEQ ID NO:5).

[0321] huMov19 light chain (SEQ ID NO:5)

[0322] huMov19 heavy chain (SEQ ID NO:6)

[0323] Farletuzumab (also known as MORAb-003) is a humanized monoclonal antibody targeting FRα. The "Farletuzumab" antibody comprises an anti-FRα antibody (WO2017151979A1) with a full-length heavy chain (SEQ ID NO:8) and a full-length light chain (SEQ ID NO:7).

[0324] Farletuzumab light chain (SEQ ID NO:7)

[0325] Farletuzumab heavy chain (SEQ ID NO:8)

[0326] In some implementations, a huMov19 variant containing the Fc silencing mutations L234A, L235A, and P329G, abbreviated as huMov19-AAG, can be obtained by the method described in PLoS ONE, 2021, 16(12):e0260954.:

[0327] huMov19-AAG light chain (SEQ ID NO:9)

[0328] huMov19-AAG heavy chain (SEQ ID NO:10)

[0329] In some implementations, a Farletuzumab variant containing the Fc silencing mutations L234A, L235A, and P329G, abbreviated as Farletuzumab-AAG, can be obtained by the method described in PLoS ONE, 2021, 16(12):e0260954.:

[0330] Farletuzumab-AAG (SEQ ID NO:11)

[0331] Farletuzumab-AAG (SEQ ID NO:12)

[0332] In some embodiments, a cysteine ​​residue can be inserted between positions 149 and 150 in the CL domain of the huMov19 monoclonal antibody as described in WO2022198335A1 to obtain a huMov19 variant, abbreviated as huMov19-K149.5C, with the following sequence:

[0333] huMov19-K149.5C light chain (SEQ ID NO:13)

[0334] huMov19-K149.5C heavy chain (SEQ ID NO:14)

[0335] In some implementations, the huMov19 monoclonal antibody can simultaneously contain the Fc silencing mutations L234A, L235A, and P329G, and an insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain to obtain the huMov19 variant, abbreviated as huMov19-AAG-K149.5C, with the following sequence:

[0336] huMov19-AAG-K149.5C light chain (SEQ ID NO:15)

[0337] huMov19-AAG-K149.5C heavy chain (SEQ ID NO:16)

[0338] In some implementations, the Farletuzumab monoclonal antibody can simultaneously contain the Fc silencing mutations L234A, L235A, and P329G, and an insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain to obtain the Farletuzumab variant, abbreviated as Farletuzumab-AAG-K149.5C, with the following sequence:

[0339] Farletuzumab-AAG-K149.5C light chain (SEQ ID NO:17)

[0340] Farletuzumab-AAG-K149.5C heavy chain (SEQ ID NO:18)

[0341] In some implementations, the Farletuzumab monoclonal antibody can be obtained as described in PLoS ONE, 2021, 16(12):e0260954, containing the Fc silencing mutations L234A and L235A, abbreviated as Farletuzumab-LALA, with the following sequence:

[0342] Farletuzumab-LALA light chain (SEQ ID NO:19)

[0343] Farletuzumab-LALA heavy chain (SEQ ID NO:20)

[0344] In some implementations, the huMov19 monoclonal antibody can be obtained as described in PLoS ONE, 2021, 16(12):e0260954, containing the Fc silencing mutations L234A and L235A, abbreviated as huMov19-LALA.

[0345] In some embodiments, a cysteine ​​residue may be inserted between position 149 and position 150 of the CL domain of the Farletuzumab monoclonal antibody as described in WO2022198335A1 to obtain a Farletuzumab variant, abbreviated as Farletuzumab-K149.5C.

[0346] The term "linker" refers to a degradable or non-degradable linker fragment used to link a small molecule drug D1 to an antibody, or ST1 to an antibody. An antibody molecule can be linked to multiple linkers containing small molecule drugs D1 and / or ST1. Typically, each linker can link one or more small molecule drugs. In this invention, it is preferred that each linker links to one small molecule drug D1 or ST1. Typically, each antibody can be linked to multiple linkers. In this invention, it is preferred that each antibody is linked to 1 to 10 linkers; more preferably, each antibody is linked to 1 to 8 linkers. In this invention, a "linker" is the basic unit constituting a "linker," and "linkers" include "non-degradable linkers" and "degradable linkers."

[0347] In this invention, the non-degradable linker refers to a linker that possesses enzymatic and / or chemical stability both in vivo and in vitro. The release of small molecule drugs D1 and / or ST1 may not depend on the differential properties of plasma and tumor tissue and intracellular enzyme levels. The release of small molecule drugs D1 and / or ST1 can be achieved through antigen-mediated phagocytosis, which internalizes the antibody-immunostimulation conjugate and degrades the antibody to the amino acid level, thereby releasing derivatives of small molecule drugs D1 and / or ST1. These derivatives consist of small molecule drugs D1 and / or ST1, the linker, and amino acid residues, or residues covalently linked to small molecule drugs D1 and / or ST1 and the linker. Antibody-immunostimulation conjugates constructed with such non-degradable linkers exhibit better stability. Non-degradable linkers include alkylene chains and their polymers (e.g., alkylene amide polymers, alkylene glycol polymers, or combinations including alkylene glycol and alkylene amide polymer fragments), ethylene glycol fragments and polyethylene glycol fragments, aromatic rings or non-aromatic rings, and combinations thereof.

[0348] In this invention, the degradable linker can be degraded in vivo or in vitro, and contains linkers that can be degraded by specific enzymes in vivo or in vitro, or linkers that are chemically unstable. The degradable linker can be degraded intracellularly to release the small molecule drug D1, for example, by reduction in the cytoplasm, degradation under lysosomal acidic conditions, or degradation by specific intracellular proteases or other enzymes. The degradable linker contains one or more enzymatically degradable linkers, chemically unstable linkers, or other degradable linkers, with other portions being non-enzymatically degradable or chemically stable linkers. The chemically unstable linkers include oxime, hydrazone, and / or disulfide groups. The enzyme-specifically degradable linkers are linkers based on 1) amino acid residues or peptides. Peptide bonds can have good serum stability because the activity of lysosomal proteolytic enzymes in the blood is much lower than in certain tumor tissues; therefore, the linker can be selectively degraded in certain tumor tissues or cells to release the small molecule drug D1. The lysosomal enzyme can be selected from cathepsin B, cathepsin S, plasmin, elastase, β-glucuronidase, or β-galactosidase, etc. Peptide linkers based on peptide formation (-(A)) v-) can be a tetrapeptide (including but not limited to: -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Gly-Phe-Gly-, -Leu-Ser-Gly-lys-, -Ala-Ala-Pro-Val-, -Glu-Val-Ala-Gly-, -Glu-Val-Cit-Gly-, -Leu-Ala-Glu-Gly-) or a tripeptide (including but not limited to: -Val-Leu-Lys-, -A la-Pro-Val-, -Ala-Ala-Asn-, -Glu-Val-Cit-, -Leu-Ala-Glu-, -Glu-Val-Ala-, -Val-Cit-Gly-, -Val-Ala-Gly-, -Leu-Ala-Glu-), dipeptides (including but not limited to: -Val-Cit-, -Cit-Val-, -Val-Ala-, -Ala-Val-, -Ala-Cit-, -Cit-Ala-, -Asn-) Cit-, -Cit-Asn-, -Cit-Cit-, -Val-Glu-, -Glu-Val-, -Ser-Cit-, -Cit-Ser-, -Lys-Cit-, -Cit-Lys-, -Asp-Cit -, -Cit-Asp-, -Ala-Val-, -Val-Ala-, -Phe-Lys-, -Lys-Phe-, -Val-Lys-, -Lys-Val-, -Ala-Lys-, -Lys-Ala-, - Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-, -Ile-Cit-, -Cit-Ile-, -Phe-Arg-, -Arg-Phe-, -Cit-Trp-, -Trp-Cit-, -Pro-Val-) or amino acid monomers (including but not limited to: -Lys-, -Gly-, -Cit-), wherein the polypeptide linker is preferably a dipeptide linker, a tripeptide linker, or a tetrapeptide linker, more preferably a dipeptide linker and a tetrapeptide linker. The cleavable linker may also include non-cleavable fragments, such as alkylene chains and their polymers, polyethylene glycol (PEG) and its related polymers, aromatic or non-aromatic rings, and combinations thereof.

[0349] In this invention, the "connector" may include a spacer. The connector head may be directly connected to the connector in the connector, or connected to the connector in the connector through the spacer. The spacer may be polyethylene glycol and related polymers, alkylene groups containing 1 to 10 carbon atoms, cyclohexyl groups, phenyl groups, 1,3-dioxane groups, amide groups, ester groups, oxo groups, substituted amino or triazole groups, and any combination of one or more of the above-mentioned spacers.

[0350] In this invention, the "linker" may include a self-immolative group, which spatially separates the small molecule drug D1 from the enzyme degradation site, for example, Self-eliminating groups can also be represented by aminomethylene groups or amino C groups. 3-5 The alkyl acyl group is linked to the hydroxyl group on D1.

[0351] In this invention, the "linker" further includes a linker head, which is connected to the antibody. The linker head can be formed by reacting the linker head precursor with groups such as thiol (e.g., cysteine), amino (e.g., lysine), carbonyl (e.g., p-acetylphenylalanine), aldehyde, azide (e.g., p-azidomethylphenylalanine), and phenolic (e.g., tyrosine) groups in the antibody.

[0352] In this invention, the "connector precursor" can react with the antibody to form a connector that links to the antibody.

[0353] In this invention, the linker precursor can react with thiol groups (e.g., cysteine), amino groups (e.g., lysine), carbonyl groups (e.g., p-acetylphenylalanine), aldehyde groups, azido groups (e.g., p-azidomethylphenylalanine), phenolic groups (e.g., tyrosine) in the antibody.

[0354] In this invention, the linker precursor can react with the thiol groups in the antibody to form a linker that connects to the antibody. Preferably, it couples to one, two, three, or four cysteine ​​side-chain thiol groups in the antibody, and more preferably, it couples to one or two cysteine ​​side-chain thiol groups in the antibody. The linker precursor can be: 1) a maleimide-based linker precursor, such as i) a marketed ADC drug. Maleimide was used in all of them. 1) Random conjugation of cysteine ​​in the antibody; 2) Substituted maleimide (including 3-substituted maleimide or 3-,4-disubstituted maleimide) linker precursors, such as i) a class of bromomaleimide linker precursors disclosed in CN103933575B and Mol. Pharmaceutics 2015, 12, 3986-3998. ii) A class of phenylthio-substituted maleimide linker precursors is disclosed in Org. Biomol. Chem., 2014, 12, 7261-7269. iii) A class of 2-pyridinethiolated maleimide linker precursors is disclosed in WO2014197871A2.

[0355] In this invention, the linker precursor can react with an amino group in the antibody to form a linker that is linked to the antibody. Preferably, it is coupled with an amino group on the lysine side chain of the antibody. This coupling is preferably achieved by reacting the lysine side chain amino group with an activated carboxyl group in the linker precursor to form an amide bond. The linker precursor can be: 1) an NHS ester-based linker precursor, such as a marketed ADC drug. All of them use NHS activated esters 1) Obtained by random conjugation of lysine in the antibody; 2) Phenolic ester linker precursors, such as a series of phenolic ester linker precursors disclosed in RSC Advances, 2012, 2, 908–914 and US20120201809A1.

[0356] The linker precursors that can react with the thiol groups on the side chains of antibody amino acid residues include, but are not limited to: R9 and R 9-1 The definition is as described above. The reaction between the linker precursor and the thiol group (e.g., cysteine) in the antibody is specific, for example, as shown in Formula 1 or 2:

[0357] When the connector is maleimide or succinimide, it can be further hydrolyzed to obtain hydrolysis products, such as those shown in reaction formula 1' or reaction formula 2':

[0358] The term “and / or” includes any and all combinations of one or more of the related listed items.

[0359] Term "C" A-B "" refers to the range from the starting point to the ending point, where A and B, and all points within the range, are integers representing the number of carbon atoms, such as C. 1-4 This indicates that the number of carbon atoms is 1, 2, 3, or 4; C 1-6 This indicates that the number of carbon atoms is 1, 2, 3, 4, 5, or 6; C 3-8 This indicates that the number of carbon atoms is 3, 4, 5, 6, 7, or 8; C A-B It can be used in conjunction with any group containing carbon atoms to specify the number of carbon atoms, such as C. 1-6Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl, C 1- 4-alkoxy group, C 3-8 cycloalkyl C 1-4 Alkyl groups, etc.

[0360] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group containing 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-8, 1-6, 1-4, or 1-3 carbon atoms. Representative examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl, n-hexyl, n-heptyl, octyl, nonyl, decyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, 1-ethyl-2-methylpropyl, and 1,1,2-trimethylpropyl. 1,1-Dimethylbutyl, 1,2-Dimethylbutyl, 2,2-Dimethylbutyl, 1,3-Dimethylbutyl, 2,3-Dimethylbutyl, 2-Ethylbutyl, 2-Methylpentyl, 3-Methylpentyl, 4-Methylpentyl, 4,4-Dimethylpentyl, 2-Methylhexyl, 3-Methylhexyl, 4-Methylhexyl, 5-Methylhexyl, 2,3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, 2,2,4-Trimethylpentyl, Undecyl, Dodecyl, and their various isomers, etc.

[0361] The term "alkylene" refers to a saturated straight-chain or branched non-bridged divalent alkyl group containing 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-8, 1-6 or 1-4 carbon atoms, examples including but not limited to -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2CH2-, -CH2C(CH3)2CH2CH2-, =CH2, =CHCH3, =C(CH3)2.

[0362] The term "cycloalkyl" refers to a monocyclic or polycyclic group containing 3-20 carbon atoms and either saturated or partially unsaturated (containing one or two double bonds). "Monocyclic cycloalkyl" is preferably a 3-10 member monocyclic cycloalkyl, more preferably a 3-8 member monocyclic cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and cyclohexenyl. "Polycyclic cycloalkyl" includes "bridged cycloalkyl," "fused cycloalkyl," and "spirocycloalkyl." Representative examples of "bridged cycloalkyl" include, but are not limited to, borneol, bicyclo[2.2.1]heptenyl, bicyclo[3.1.1]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.2]nonyl, bicyclo[3.3.1]nonyl, bicyclo[4.2.1]nonyl, and adamantyl, etc. "Fused cycloalkyl" refers to a cycloalkyl ring fused to a phenyl, cycloalkyl, or heteroaryl group. Fused cycloalkyl groups include, but are not limited to, benzocyclobutenyl, 2,3-dihydroindenyl, and decahydronaphthyl. Monocyclic or polycyclic cycloalkyl groups can be linked to the parent molecule via any carbon atom on the ring.

[0363] The term "cycloalkylene" refers to a divalent cycloalkyl group. Therefore, "cycloalkylene" encompasses the definition of cycloalkyl groups described above. Preferably, "cycloalkylene" is C10. 3-10 Cycloalkylene, more preferably C10, 3-8 Cycloalkyl or C 3-6 Cycloalkylene.

[0364] The term "heterocyclic alkyl" refers to a non-aromatic cyclic group consisting of a carbon atom and heteroatoms selected from nitrogen, oxygen, sulfur, boron, etc., and / or sulfur-containing heteroatom groups, which is saturated or partially unsaturated (containing one or two double bonds). This cyclic group can be monocyclic or polycyclic, wherein the sulfur-containing heteroatom groups are selected from, but not limited to, S(O), S(O)₂, and S(O)(NH). In this invention, the number of heteroatoms and / or heteroatom groups in the heterocyclic alkyl group is preferably 1, 2, 3, or 4, and the boron, nitrogen, or carbon atom in the heterocyclic alkyl group may optionally be oxidized. The nitrogen atom may optionally be further substituted by other groups to form a tertiary amine or quaternary ammonium salt. "Monocyclic heterocyclic alkyl" is preferably a 3-10 member monocyclic heterocyclic alkyl group, more preferably a 3-8 member monocyclic heterocyclic alkyl group. Examples include: pyrrolyl, dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, tetrahydrofuranyl, tetrahydropyrazinyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, tetrahydropyridyl, tetrahydropyrimidinyl, piperidinyl, aziridinyl, morpholinyl, thiomorpholinyl, thiomorpholin-S-oxide-4-yl, piperidinyl, piperazinyl, 1,4-dioxane, homopiperazine. The terms include alkyl groups, such as 1-imino-1-tetrahydro-2H-thiaranyl, 1,1-tetrahydrothiophenyl, 1,1-3,4-dihydro-2H-thiaranyl, 1-imino-1-oxy-3,4-dihydro-2H-thiaranyl, 1,1-2,3-dihydrothiaphenyl, and 1-imino-1-oxy-2,3-dihydrothiaphenyl. "Polycyclic heterocyclic alkyl groups" include "fused heterocyclic alkyl groups," "spirocyclic alkyl groups," and "bridged heterocyclic alkyl groups." "Fused heterocyclic alkyl" refers to monocyclic heterocyclic alkyl rings fused to phenyl, cycloalkyl, heterocyclic alkyl, or heteroaryl groups. Fused heterocyclic alkyl groups include, but are not limited to: 2,3-dihydrobenzofuranyl, 1,3-dihydroisobenzofuranyl, dihydroindolyl, 2,3-dihydrobenzo[b]thiophenyl, dihydrobenzopiperanyl, 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydronaphridinyl, 5,6,7,8-tetrahydronaphridinyl, 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborylpentaneyl, etc. Monocyclic and polycyclic heterocyclic alkyl groups can be linked to the parent molecule via any ring atom on the ring. The aforementioned ring atoms specifically refer to the carbon and / or nitrogen atoms that constitute the ring skeleton.

[0365] The term "heterocyclic alkylene" refers to a divalent heterocyclic alkylene. Thus, "heterocyclic alkylene" encompasses the definition of heterocyclic alkylene described above. "Hypercyclic alkylene" is preferably a 3-10 membered heterocyclic alkylene, more preferably a 3-8 membered heterocyclic alkylene or a 3-6 membered heterocyclic alkylene.

[0366] The term "cycloalkylalkyl" refers to a cycloalkyl group that is linked to the parent structure via an alkyl group. Therefore, "cycloalkylalkyl" encompasses the definitions of alkyl and cycloalkyl groups described above.

[0367] The term "heterocyclic alkyl alkyl" refers to a heterocyclic alkyl group that is linked to the parent core structure via an alkyl group. Therefore, "heterocyclic alkyl alkyl" encompasses the definitions of alkyl and heterocyclic alkyl groups described above.

[0368] The term "alkoxy" refers to an alkyloxy group having the stated number of carbon atoms connected by an oxygen bridge. Thus, "alkoxy" encompasses the definition of alkyl groups described above.

[0369] The term "hydroxyalkyl" refers to an alkyl group in which any one hydrogen atom is replaced by a hydroxyl group, including but not limited to: -CH2OH, -CH2CH2OH, and -CH2CH2C(CH3)2OH.

[0370] The term "heterocyclic alkenyl" or "heterocyclic alkenyl" refers to a cyclic, unsaturated monovalent hydrocarbon group having a specified number of ring atoms (e.g., 5 to 10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), having one or more (e.g., 1, 2, or 3) carbon-carbon sp. 2 A double bond, which is a monocyclic compound and lacks aromaticity. Heterocyclic alkenyl groups or heterocyclic rings are connected to the rest of the molecule via carbon atoms or heteroatoms. Heterocyclic rings include, but are not limited to: The "heterene ring" shares two atoms and one bond with the rest of the molecule.

[0371] The term "aryl" or "aromatic ring" refers to any stable 6-20 membered monocyclic or polycyclic aromatic group, such as phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindene, or biphenyl. The term "arylene" refers to a divalent aryl group. Thus, "aryl" includes the definition of "arylene." "Arylene" is preferably C10. 6-10 Arene, more preferably phenylene.

[0372] The term "heteroaryl" or "heteroary ring" refers to an aromatic ring group formed by replacing at least one carbon atom in a ring with a heteroatom selected from nitrogen, oxygen, or sulfur. It can be a 5-7 membered monocyclic structure or a 7-20 fused ring structure, preferably a 5-6 membered monocyclic structure. In this invention, the number of heteroatoms is preferably 1, 2, or 3. The "heteroaryl" includes: pyridinyl, pyridoneyl, pyrimidinyl, pyrimidin-2,4(1H,3H)-diketoyl, pyrimidoneyl, piperazinyl, pyridazinoneyl, furanyl, thiopheneyl, thiazolyl, pyrroleyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazole, 1,2,4-triazolyl, 1,2,3-triazolyl, and tetrazolyl. Indazole, isoindazole, indolyl, isoindolyl, benzofuranyl, benzothiophene, benzo[d][1,3]dioxolane, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, isoquinolinone, quinazolinyl, 4-hydroxythieno[3,2-c]pyridyl, 4,5-dihydro-4-oxofuran[3,2]pyridyl, 4-hydroxy-5-azaindolyl, furan[2,3-c]pyridin-7(6H)-keto, thien[2,3-c]pyridin-7(6H)-keto, etc. The term "heteroaryl" refers to a divalent heteroaryl. Therefore, "heteroaryl" includes the definition of "heteroaryl". "Heteroaryl" is preferably a 5-10 member heteroaryl, more preferably a 5-6 member heteroaryl.

[0373] The term "fused ring group" refers to a fused ring structure formed by two, three, or four ring structures sharing two adjacent atoms. The fused ring group may further include a spirocyclic or bridged ring group. The fused ring group referred to in this invention is a saturated, unsaturated, or partially saturated fused ring structure, preferably with at least one ring being an aromatic ring. More preferably, it is a bicyclic or tricyclic fused ring group, and at least one ring is an aromatic ring. In this invention, the fused ring group is preferably an 8-12 membered fused ring group, more preferably an 8-10 membered fused ring group. Specific examples of cyclic groups include, but are not limited to: 5,6-dihydro-4H-cyclopentyl[b]thiophene, 5,6-dihydro-4H-cyclopentyl[b]furanyl, 2,3-dihydrobenzofuranyl, 1,3-dihydroisobenzofuranyl, dihydroindolyl, 2,3-dihydrobenzo[b]thiophene, dihydrobenzopiperanyl, 1,2,3,4-tetrahydroquinolinyl, 2,3-dihydro-1,4-benzodioxane, 3,4-dihydro-2H-1,4-benzooxazinyl, naphthidyl, naphthyl, benzofuranyl, benzothiophene, benzopyrroleyl, benzothiazolyl, benzooxazolyl, indazole, benzopyridinyl, benzimidazolyl, indolyl, quinolinyl, isoquinolinyl, purine, pteridinyl, The cyclic group can be linked to the parent molecule via a cyclic carbon atom, preferably via an aromatic cyclic carbon atom.

[0374] The term "arylalkyl" refers to an aryl group that is linked to the parent structure via an alkyl group. Therefore, "arylalkyl" encompasses the definitions of alkyl and aryl groups mentioned above.

[0375] The term "heteroarylalkyl" refers to a heteroaryl group that is linked to the parent structure via an alkyl group. Therefore, "heteroarylalkyl" encompasses the definitions of alkyl and heteroaryl groups mentioned above.

[0376] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0377] The term "halogenated alkyl" refers to an alkyl group that has been arbitrarily substituted with a halogen. Thus, "halogenated alkyl" encompasses the definitions of halogen and alkyl group mentioned above.

[0378] The term "amino" refers to -NH2.

[0379] The term "aminoalkyl" refers to an alkyl group in which at least one hydrogen atom is replaced by an amino group, including but not limited to: -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -C(CH3)2NH2, and -CH2C(CH3)2NH2. Thus, "aminoalkyl" encompasses the definitions of alkyl and amino groups as described above.

[0380] The term "alkoxyalkyl" refers to an alkyl group in which at least one hydrogen atom is replaced by an alkoxy group, including but not limited to: Therefore, "alkoxyalkyl" includes the definitions of alkyl and alkoxy mentioned above.

[0381] The term "oxo group" refers to =O.

[0382] In this invention, the abbreviations for amino acids are conventional abbreviations (referencing the Chinese Chemical Society Organic Chemistry Nomenclature Principles 2017), such as: alanine (Ala), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), and valine (Val). In this invention, the peptide symbol (taking -Gly-(-NH-CH2-CO-) as an example) indicates that when the hyphen is to the right of Gly, it represents the removal of an OH group from the -COOH group of the amino acid; when the hyphen is to the left of Gly, it represents the removal of a H atom from the -NH2- group of the amino acid.

[0383] The "room temperature" mentioned in this invention refers to 15-30℃.

[0384] The substituents R1, R2, R3, R4, and R in the "immunomodulator antibody-drug conjugate", "compound", and "pharmaceutically acceptable salt" of this invention 4’ R5, R6, R7, R 8-1 R 8-2 R 8-3 R9, R 9-1 R a and R b If tautomers exist, they can exist as a single tautomer or a mixture thereof, preferably as the more stable tautomer.

[0385] The "pharmaceutically acceptable salts" described in this invention are discussed in Berge et al., "Pharmaceutically acceptable salts," J. Pharm. Sci., 66, 1-19 (1977), and are obvious to medicinal chemists. These salts are substantially non-toxic and provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion. The compounds described in this invention may have acidic, basic, or amphoteric groups, and typical pharmaceutically acceptable salts include those prepared by reacting the compounds of this invention with an acid.

[0386] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0387] The reagents and raw materials used in this invention are all commercially available. Detailed Implementation

[0388] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0389] The structures of all compounds in this invention can be determined by nuclear magnetic resonance (NMR). 1 Identification by 1H NMR and / or mass spectrometry (MS).

[0390] 1 10 H NMR chemical shifts (δ) were recorded in pPM (10 -6NMR was performed using a Bruker AVANCE-400 spectrometer. Suitable solvents included deuterated chloroform (CDCl3), deuterated methanol (CD3OD), deuterated dimethyl sulfoxide (DMSO-d6), and heavy water (D2O), with tetramethylsilane used as an internal standard (TMS).

[0391] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1200 HPLC / 6120 mass spectrometer with an Ultimate C18 column (3.0 × 50 mm, 3 μm) at 40 °C; or using a Thermo UltiMate 3000 HPLC / MSQ PLUS mass spectrometer with an Xbridge C18 column (3.0 × 50 mm, 3.5 μm) at 30 °C. Agilent gradient elution conditions: 95-5% solvent A1 and 5-95% solvent B1 (0-2.0 min), then 95% solvent B1 and 5% solvent A1 (hold for 1.1 min). Percentages represent the volume percentage of a specific solvent in the total solvent volume. Solvent A1: 0.01% aqueous solution of trifluoroacetic acid (TFA); Solvent B1: 0.01% acetonitrile solution of trifluoroacetic acid; percentages represent the volume percentage of the solute in the solution. Thermo gradient elution condition 2: 95-5% solvent A2 and 5-95% solvent B2 (0-2 min), then 95% solvent B2 and 5% solvent A2 (hold for 1.8 min), where the percentage is the volume percentage of a particular solvent in the total solvent volume. Solvent A2: 10 mM ammonium bicarbonate aqueous solution; Solvent B2: acetonitrile.

[0392] Preparative high-performance liquid chromatography (prep-HPLC) was performed using a Gilson GX-281 preparative HPLC system or an Agela FLEXA-HP preparative HPLC system. The chromatographic column was an Ultimate 21.2*250 mm, 10 μm column. Separation conditions 1: Mobile phase A: 0.05% hydrochloric acid aqueous solution, mobile phase B: acetonitrile; Separation conditions 2: Mobile phase A: 10 mmol / L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; Separation conditions 3: Mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile. Alternatively, a Gemini 5 μm, C18, 150*21.2 nm column was used. Mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile. Detection wavelengths were 214 nm & 254 nm; flow rate was 15.0–20.0 mL / min.

[0393] The ultra-high performance liquid chromatography (UPLC) system used was Waters ACQUITY Hclass; the column was a Waters ACQUITY UPLC BEH Shield RP18 2.1mm*100mm, 1.7μm, 5μm; mobile phase A: 5mm potassium dihydrogen phosphate buffer, adjusted to pH 2.5 with phosphoric acid; mobile phase B: acetonitrile. Gradient elution was performed: mobile phase B from 90% to 60% for 5 minutes; B from 60% to 10% for 2 minutes; B held at 10% for 6 minutes; B from 10% to 90% for 0.1 minutes; B held at 90% for 1.9 minutes. Detection wavelengths: 214 & 262nm; column temperature: 40℃; flow rate: 0.4mL / min.

[0394] The fast column chromatography (Flash system / Cheetah™) used an Agela Technologies MP200 system, with a matching normal-phase separation column of Flash column Silica-CS (25g, 40g, 80g, 120g or 330g), manufactured by Tianjin Bona Agela. The elution system was ethyl acetate / petroleum ether or dichloromethane / methanol. The reversed-phase separation column was a C18 reversed-phase column (Spherical C18, 40-75μm). Model: SW-040), the elution system is 10mM ammonium bicarbonate aqueous solution / acetonitrile or 0.1% trifluoroacetic acid aqueous solution / acetonitrile.

[0395] The antibody concentration in the antibody-drug conjugate of this invention can be determined by the following method:

[0396] The concentration of the conjugate drug in an antibody-drug conjugate can be calculated by measuring the UV absorbance of the aqueous solution of the antibody-drug conjugate at a wavelength of 280 nm and then performing the following calculations.

[0397] Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical substances present in the system (additive absorption), assuming that the molar absorptivity of the antibody and drug does not change before and after antibody-drug conjugation, the antibody concentration and drug concentration in the antibody-drug conjugate are as shown in the following formula.

[0398] Therefore, antibody-immunostimulation conjugate concentration

[0399] The drug-to-analyte ratio (DAR) of each antibody molecule in the antibody-drug conjugate of this invention can be determined by the following method:

[0400] Method 1: The average number of drug molecules linked to each antibody molecule in an antibody-drug conjugate can be determined by high-performance liquid chromatography (HPLC) using the following method. HPLC instrument: Waters / Waters e2695; Mobile phase A: 1.5M (NH4)2SO4 + 50mM potassium phosphate (pH 7.0); Mobile phase B: 50mM sodium phosphate (pH 7.0) / isopropanol (75:25V / V); Analytical column: Thermo MabPac TM HIC-Butyl 5μm 4.6×100mm, PN.088558; Injection volume: 5μL; Flow rate: 1mL / min; Column temperature: 30℃; Detector: PDA detector; Detection wavelength: 280nm; Elution gradient:

[0401] Hydrophobic interaction chromatography (HIPC) can be used to determine the drug-antibody ratio (DAR) in antibody-drug conjugates (ADCs). Unconjugated antibodies are the least hydrophobic and are eluted first; antibodies linked to eight drugs are the most hydrophobic and are eluted last. The peak area percentage represents the relative distribution of ADCs linked to a specific number of drugs. The weighted average DAR is calculated by combining the peak area percentage with the number of conjugated drugs: DAR = ∑(relative peak area * number of drug conjugates) / total peak area.

[0402] In this invention, the molecular size heterogeneity (SEC) of antibody-immunostimulation conjugates can be analyzed using the following methods:

[0403] HPLC instrument: Waters Acquity Arc; Mobile phase: 100mM PB + 200mM Arg·HCl + 5% IPA, pH 6.8; Analytical column: TOSOH TSKgel G3000 SWxl, 7.8*300mm, 5μM. PN0008541; Injection volume: 10 μL; Flow rate: 0.6 mL / min; Column temperature: 30℃; Detector: PDA detector; Detection wavelength: 280 nm; Gradient: isocratic elution.

[0404] Method 2: For antibody-drug conjugates formed by a single antibody and two different payloads, the DAR value can be determined using SEC-MS. The ultra-high performance liquid chromatograph used was a Waters H-Class Bio System; the mass spectrometer used was a Waters SYNAPT G2-Si. The chromatographic column was: ACQUITY UPLC Protein BEH SEC 200A 1.7μm, 2.1mm*150mm; mobile phase: 50mM ammonium acetate; injection volume: 2μg (adjusted according to signal intensity); flow rate: 0.065mL / min; column temperature: 30℃±2℃; detection wavelength: 280nm; gradient: isocratic elution.

[0405] The abbreviations used in this embodiment of the invention have the following meanings:

[0406] AIBN: Benzoyl peroxide; DMF: N,N-Dimethylformamide; DIPEA: N,N-Diisopropylethylamine; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-Tetramethylurea hexafluorophosphate; NBS: N-bromosuccinimide; T3P: 1-n-Propylphosphine; HOBT: 1-Hydroxybenzotriazole.

[0407] Synthesis of intermediates:

[0408] L-1: Synthesis of 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)phenyl)-1,3-dioxane-5-carboxylic acid

[0409] Step 1: To a solution of methyl 3-hydroxy-2-(hydroxymethyl)propionate (1.5 g, 11.2 mmol) and 4-nitrobenzaldehyde (1.69 g, 11.2 mmol) in toluene (50 mL), p-toluenesulfonic acid monohydrate (210 mg, 1.12 mmol) was added. The reaction mixture was stirred at 110 °C for 2 hours, cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give 1A (1.5 g) as a pale yellow solid. m / z: [M+H] + 268.2.

[0410] Step 2: To a mixed solution of 1A (1.4 g, 5.24 mmol) in tetrahydrofuran (20 mL) and water (10 mL), lithium hydroxide monohydrate (440 mg, 10.5 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and the residue was adjusted to pH 3 with hydrochloric acid (2 M). The mixture was filtered, and the filter cake was dried under vacuum to obtain 1B (1.42 g) as a pale yellow solid. m / z: [M+H] + 254.1.

[0411] Step 3: Add palladium on carbon (10% wt, 299 mg) to a 20 mL solution of ethyl acetate (300 mg, 1.18 mmol) of 1B. After purging the reaction system with hydrogen, stir at room temperature for 1 hour under a hydrogen atmosphere, filter, and concentrate the filtrate under reduced pressure to obtain 1C (300 mg) as a yellow solid. m / z: [M+H] + 224.2.

[0412] Step 4: Add 2,5-dihydrofuran-2,5-dione (171 mg, 1.74 mmol) to a 3 mL solution of DMF containing 1C (300 mg, 1.34 mmol). Stir the reaction mixture at room temperature for 1 hour, concentrate under reduced pressure, and purify the residue by Flash column chromatography (C18, eluent: 10%–60% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain 1D (150 mg) as an orange solid. m / z: [M+H] + 322.2.

[0413] Step 5: Add sodium acetate (38.6 mg, 0.47 mmol) to a solution of 1D (150 mg, 0.47 mmol) of acetic anhydride (480 mg, 4.7 mmol). Stir the reaction mixture at 50 °C for 0.5 hours. After cooling to room temperature, purify the solution directly by Flash column chromatography (C18, eluent: 10%–60% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain L-1 (30 mg) as a yellow solid. m / z: [M+H] + 304.2.

[0414] L-2: Synthesis of 2-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-fluorophenyl)-1,3-dioxane-5-carboxylic acid

[0415] Step 1: To a toluene (10 mL) solution of 2-fluoro-5-nitrobenzaldehyde (1 g, 5.91 mmol) and ethyl 3-hydroxy-2-(hydroxymethyl)propionate (1.31 g, 18.87 mmol), p-toluenesulfonic acid monohydrate (56 mg, 0.3 mmol) was added. The reaction mixture was stirred at 110 °C for 8 hours, cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give 2A (1.4 g) as a pale yellow solid. m / z: [M+H] + 300.1.

[0416] Step 2: Add palladium on carbon (10% wt, 180 mg) to a mixed solution of 2A (500 mg, 1.67 mmol) in methanol (10 mL) and ethyl acetate (10 mL). After purging the reaction system with hydrogen, stir at room temperature for 2 hours under a hydrogen atmosphere, filter, and concentrate the filtrate under reduced pressure to obtain 2B (450 mg) as a yellow oil. m / z: [M+H] + 270.2.

[0417] Step 3: Add 2,5-dihydrofuran-2,5-dione (210 mg, 2.17 mmol) to a solution of 2B (450 mg, 1.67 mmol) and triethylamine (250 mg, 2.5 mmol) in dichloromethane (10 mL). Stir the reaction mixture at room temperature for 1 hour, concentrate under reduced pressure, add water (10 mL) to the residue, and adjust the pH to 3 with dilute hydrochloric acid (2N). Filter and dry the filter cake to obtain 2C (500 mg) as a white solid. m / z: [M+H] + 368.2.

[0418] Step 4: Add lithium hydroxide monohydrate (120 mg, 2.93 mmol) to a mixed solution of 2C (600 mg, 1.63 mmol) in tetrahydrofuran (4 mL) and water (2 mL). Stir the reaction solution at room temperature for 1 hour, concentrate under reduced pressure, adjust the pH of the residue to 3 with hydrochloric acid (2 M), filter, and vacuum dry the filter cake to obtain 2D (450 mg) as a yellow solid. m / z: [M+H] + 340.2.

[0419] Step 5: Add sodium acetate (49 mg, 0.59 mmol) to a DMF (2 mL) solution of 2D (200 mg, 0.59 mmol) and acetic anhydride (109 mg, 1.06 mmol). Stir the reaction mixture at 90 °C for 0.5 hours. After cooling to room temperature, purify the solution directly by Flash column chromatography (C18, eluent: 10%–45% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain L-2 (70 mg) as a yellow solid. m / z: [M+H] + 322.0.

[0420] L-3: Synthesis of 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolidine-1-yl)-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1,3-dioxane-5-carboxylic acid

[0421] Step 1: Silver oxide (2.1 g, 8.97 mmol) was added to a solution of 3-hydroxy-4-nitrobenzaldehyde (1 g, 5.98 mmol) and (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-tris(triacetyl)acetate (2.46 g, 5.98 mmol) in acetonitrile (20 mL). The reaction mixture was stirred in the dark at room temperature for 3 hours. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 3 / 1) to give 3A (2.85 g) as a pale yellow solid. m / z: [M+Na] + 320.2.

[0422] Steps 2-6: Using the synthesis method of L-2, react with 3A to obtain L-3 as a white solid. m / z: [M + Na] + 504.1.

[0423] L-4: Synthesis of 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolidine-1-yl)-3-(phosphonooxy)phenyl)-1,3-dioxane-5-carboxylic acid

[0424] Steps 1-3: Using the 2C synthesis method, 3-hydroxy-4-nitrobenzaldehyde and ethyl 3-hydroxy-2-(hydroxymethyl)propionate are reacted to obtain 4C as a white solid.

[0425] Step 4: Under ice bath conditions, a solution of 4C (500 mg, 1.37 mmol) in dichloromethane (5 mL) was slowly added dropwise to a solution of phosphorus oxychloride (630 mg, 4.11 mmol) and DIPEA (1.06 g, 8.22 mmol) in 30 mL of dichloromethane. The reaction mixture was stirred at 0 °C for 30 minutes and concentrated under reduced pressure at low temperature. The residue was purified by Flash column chromatography (C18, eluent: 10%–45% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to give 4D (350 mg) as a yellow solid. m / z: [M+H] + 446.0.

[0426] Step 5: To a mixed solution of 4D (350 mg, 0.79 mmol) in tetrahydrofuran (4 mL) and water (2 mL), lithium hydroxide monohydrate (130 mg, 3.16 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, concentrated under reduced pressure at low temperature, and the residue was purified by Flash column chromatography (C18, eluent: 10%–45% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to obtain 4E (190 mg) as a yellow solid. m / z: [M+H] + 418.2.

[0427] Step 6: Add acetic anhydride (2 mL) to 4E (130 mg, 0.31 mmol) and sodium acetate (25 mg, 0.31 mmol). Stir the reaction mixture at 90 °C for 1 hour. After cooling to room temperature, purify L-4 (40 mg) directly by Flash column chromatography (C18, eluent: 10%–45% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain a yellow solid. m / z: [M+H] + 400.0.

[0428] L-5: Synthesis of 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)phenyl)-1,3-dioxane-5-carboxylic acid

[0429] Using the same synthetic method as L-2, L-5 was obtained by reacting 3-nitrobenzaldehyde with ethyl 3-hydroxy-2-(hydroxymethyl)propionate, resulting in a white solid. m / z: [M+H] + 304.2.

[0430] L-6: Synthesis of 2-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pyridin-2-yl)-1,3-dioxane-5-carboxylic acid

[0431] Using the same synthetic method as L-2, L-6 was obtained as a white solid by reacting 5-nitropyridine-2-carboxaldehyde with ethyl 3-hydroxy-2-(hydroxymethyl)propionate. m / z: [M+H] + 305.0.

[0432] L-7: Synthesis of 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolidine-1-yl)-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1,3-dioxane-5-carboxylic acid

[0433] Using the same synthetic method as L-3, L-7 was obtained as a white solid by reacting 4-hydroxy-3-nitrobenzaldehyde with ethyl 3-hydroxy-2-(hydroxymethyl)propionate. m / z: [M+Na] + 504.1.

[0434] L-8: Synthesis of 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolidine-1-yl)-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-1,3-dioxane-5-carboxylic acid

[0435] Using the synthetic method for L-3, L-8 was obtained by reacting 2-hydroxy-4-nitrobenzaldehyde with ethyl 3-hydroxy-2-(hydroxymethyl)propionate, resulting in a white solid. m / z: [M+Na] + 504.0.

[0436] L-9: Synthesis of 2-(3-(bromomethyl)isoxazol-5-yl)-1,3-dioxane-5-carboxylic acid

[0437] Step 1: Under ice bath conditions, tert-butyldiphenylchlorosilane (3.86 g, 14.0 mmol) was added dropwise to a solution of ethyl 5-hydroxymethylisoxazole-3-carboxylate (2.0 g, 11.7 mmol) and imidazole (2.47 g, 36.2 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then cooled to room temperature, quenched with ice water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 9A (4.79 g) as a yellow oil. m / z: [M+H] + 410.2.

[0438] Step 2: Under ice bath conditions, diisobutylaluminum hydride (20 mL, 30 mmol) was added dropwise to a tetrahydrofuran (50 mL) solution of 9A (4.79 g, 11.7 mmol). The reaction system was stirred at room temperature for 2 hours. Tetrahydrofuran (50 mL) was added, and the mixture was cooled to 0°C. Water (1.2 mL) was slowly added dropwise, followed by a 15% sodium hydroxide aqueous solution (1.2 mL) and water (3 mL). The mixture was heated to room temperature and stirred for 15 minutes. Anhydrous magnesium sulfate was added and stirred for another 15 minutes. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 4 / 1) to obtain 9B (2.59 g) as a yellow oil. m / z: [M+H] + 368.2.

[0439] Step 3: Under ice bath conditions, triphenylphosphine (2.03 g, 7.76 mmol) and carbon tetrabromide (2.57 g, 7.76 mmol) were added to a solution of 9B (2.59 g, 7.05 mmol) in dichloromethane (26 mL). The reaction mixture was stirred at 0 °C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give 9C (2.34 g) as a colorless oil. m / z: [M+H] + 430.0.

[0440] Step 4: Under ice bath conditions, triethylamine trihydrofluoride (3.93 g, 24.4 mmol) was added dropwise to a 20 mL solution of tetrahydrofuran containing 9C (2.1 g, 4.88 mmol). The reaction mixture was stirred at room temperature for 3.5 hours. Partial solvent was removed by low-temperature, reduced-pressure concentration. The residue was purified directly by Flash column chromatography (C18, eluent: 0%–30% acetonitrile in 0.02% trifluoroacetic acid aqueous solution) to obtain 9D (804 mg) as a colorless oil. m / z: [M+H] + 192.0.

[0441] Step 5: Under nitrogen protection, dimethyl sulfoxide (60.9 mg, 0.78 mmol) was slowly added dropwise to a solution of oxalyl chloride (66 mg, 0.52 mmol) in dichloromethane (2.5 mL) at -78 °C. After stirring at -78 °C for 15 minutes, a solution of 9D (50 mg, 0.26 mmol) in dichloromethane (2.5 mL) was slowly added dropwise. After stirring at -78 °C for another 15 minutes, triethylamine (132 mg, 1.30 mmol) was slowly added. The temperature was raised to -50 °C and stirred for 1 hour, then slowly raised to room temperature. The reaction solution was directly purified by prep-TLC (ethyl acetate) to give 9E (33.3 mg) as a yellow oil.

[0442] Step 6: To a solution of ethyl 3-hydroxy-2-(hydroxymethyl)propionate (40 mg, 0.27 mmol) and 9E (33.3 mg, 0.18 mmol) in toluene (3 mL), p-toluenesulfonic acid hydrate (1.6 mg, 0.01 mmol) was added. The reaction mixture was stirred at 110 °C for 4 hours. After cooling to room temperature, the reaction solution was concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0%–50% acetonitrile in 0.02% trifluoroacetic acid aqueous solution) to obtain 9F (17 mg) as a yellow oil. m / z: [M+H] + 320.0.

[0443] Step 7: Under ice bath conditions, lithium hydroxide monohydrate (4.5 mg, 0.11 mmol) was added to a mixed solution of 9F (17.0 mg, 0.053 mmol) in tetrahydrofuran (1 mL) and water (0.5 mL). The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0%–38% acetonitrile in 0.02% trifluoroacetic acid aqueous solution) to obtain L-9 (9.5 mg) as a white solid. m / z: [M+H] + 292.0.

[0444] L-10: Synthesis of 2-(2-(bromomethyl)pyridin-5-yl)-1,3-dioxane-5-carboxylic acid

[0445] Step 1: Under nitrogen protection, NBS (1.75 g, 9.86 mmol) and AIBN (240 mg, 0.99 mmol) were added to a solution of methyl 2-methylpyrimidin-5-carboxylate (1.5 g, 9.86 mmol) in carbon tetrachloride (15 mL). The reaction solution was stirred at 82 °C for 1 hour, cooled to room temperature, and purified directly by Flash column chromatography (eluent: 10%–45% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to obtain 10A (1.2 g) as a yellow solid. m / z: [M+H] + 231.0.

[0446] Step 2: Under nitrogen protection, at -50°C, diisobutylaluminum hydride (4.04 mL, 6.06 mmol, 1.5 M) was slowly added dropwise to a solution of 10A (700 mg, 3.03 mmol) in 20 mL of dichloromethane. The reaction mixture was slowly brought to room temperature and stirred for 1 hour. Sodium sulfate decahydrate (2 g) and ethyl acetate (30 mL) were added to the reaction mixture. The mixture was stirred at room temperature for another 1 hour. The solid was filtered off, and the filtrate was concentrated under reduced pressure to obtain 10B (350 mg) as an orange-yellow solid. m / z: [M+H] + 203.0.

[0447] Step 3: Under ice bath conditions, add Dysmart oxidant (730 mg, 1.72 mmol) to a solution of 10B (350 mg, 1.72 mmol) in 10 mL of dichloromethane. Stir the reaction mixture for 1 hour, dilute with ethyl acetate, wash the mixture with saturated brine and saturated sodium bicarbonate solution, separate the organic phase, and concentrate under reduced pressure. Purify the residue by Flash column chromatography (methanol / dichloromethane = 1 / 20) to give 10C (340 mg) as a yellow oil. m / z: [M+H] + 201.0.

[0448] Step 4: To a solution of 10C (340 mg, 1.69 mmol) and ethyl 3-hydroxy-2-(hydroxymethyl)propionate (380 mg, 2.54 mmol) in toluene (6 mL), p-toluenesulfonic acid hydrate (16 mg, 0.085 mmol) was added. The reaction mixture was stirred at 110 °C for 16 hours, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (eluent: 10%–60% acetonitrile in 0.02% trifluoroacetic acid aqueous solution) to give 10D (320 mg) as a brown solid. m / z: [M+H] + 331.0.

[0449] Step 5: Under ice bath conditions, lithium hydroxide monohydrate (28 mg, 0.66 mmol) was added to a mixed solution of 10D (110 mg, 0.33 mmol) in tetrahydrofuran (3 mL) and water (1.5 mL). The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 10%–45% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to obtain L-10 (42 mg) as a white solid. m / z: [M+H] + 303.0.

[0450] L-11: Synthesis of 2-(6-(bromomethyl)pyrazin-3-yl)-1,3-dioxane-5-carboxylic acid

[0451] Using the same synthetic method as L-10, L-11 was obtained as a white solid by reacting it with methyl 6-methylpyrazine-3-carboxylate. m / z: [M+H] + 303.0.

[0452] L-12: Synthesis of 2-(6-(bromomethyl)phenyl)-1,3-dioxane-5-carboxylic acid

[0453] Using the same synthetic method as L-10, L-12 was obtained by reacting 4-(bromomethyl)benzaldehyde as a white solid. m / z: [M+H] + 301.0.

[0454] Compound Synthesis

[0455] Example 1: Synthesis of 2-amino-N,N-dipropyl-8-(1-((5,6,7,8-tetrahydro-1,6-naphthid-3-yl)carbamoyl)cyclopropyl)-3H-benzo[b]azapyro-4-carboxamide (compound 1)

[0456] Step 1: A DMF solution of compound 2.6 (1.1 g, 2.8 mmol), HATU (1.6 g, 4.33 mmol), dipropylamine (580 mg, 22 mmol), and DIPEA (560 mg, 4.37 mmol) was stirred at room temperature for 3 hours. The reaction mixture was diluted with ethyl acetate (100 mL), washed with water and saturated brine, and the organic phase was separated. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (methanol / dichloromethane = 1 / 20) to give compound 2.7 (500 mg, yield: 39%) as a pale yellow solid.

[0457] Step 2: Compound 2.7 (2 g, 4.31 mmol), Xphos (0.2 g), and Pd2(dba)3 (0.2 g) were dissolved in freshly prepared tetrahydrofuran solution (30 mL) of (1-(methoxycarbonyl)cyclopropyl)zinc bromide (refer to WO2018 / 138356A1). The reaction system was purged with nitrogen three times, then stirred at 75 °C under nitrogen protection for 2 hours. After cooling to room temperature, the reaction was quenched with ice water. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 3 / 1) to give compound 2.8 (500 mg, yield: 24%) as a pale yellow solid. m / z: [M+H] + 484.2.

[0458] Step 3: Lithium hydroxide monohydrate (130 mg, 3.1 mmol) was added to a mixed solution of compound 2.8 (500 mg, 1.03 mmol) in tetrahydrofuran (5 mL), methanol (0.5 mL), and water (0.5 mL). The reaction system was stirred at room temperature for 16 hours. The reaction solution was then neutralized with hydrochloric acid (1 M), and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 2.9 (400 mg, yield: 82%) as an off-white solid. m / z: [M+H] + 484.2.

[0459] Step 4: To a DMF (1 mL) solution of compound 2.9 (100 mg, 0.21 mmol), tert-butyl 3-amino-7,8-dihydro-1,6-naphthyl-6(5H)-carboxylic acid (64 mg, 0.25 mmol), HATU (97 mg, 0.25 mmol), and DIPEA (55 mg, 0.42 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 4 hours. Then, compound 2.10 (28 mg, yield: 19%) was purified directly by Flash column chromatography (C18, eluent: 0%–85% acetonitrile in 10 mM ammonium bicarbonate aqueous solution) to obtain compound 2.10 (28 mg, yield: 19%) as a white solid. m / z: [M+H] + 701.3.

[0460] Step 5: Add trifluoroacetic acid (1 mL) to a dichloromethane (2 mL) solution of compound 2.10 (28 mg, 0.4 mmol). After stirring the reaction solution at room temperature for 2 hours, concentrate under reduced pressure. Neutralize the residue with ammonia-methanol solution (7 M) and purify by Flash column chromatography (C18, eluent: 0%–60% acetonitrile in 10 mM ammonium bicarbonate aqueous solution) to obtain compound 1 (17 mg, yield: 85%) as a white solid. m / z: [M+H] +501.3; 1 H NMR (400MHz, DMSO-d6): δ9.19(s,1H),8.42(s,1H),7.63(s,1H),7.25(d,J=7.6Hz,1H),7.00(s,1H),6.91( d,J=8.4Hz,1H),6.74(s,2H),6.69(s,1H),3.78(s,2H),2.97(br.s,2H),2.63(s,6H),1.55-0.74(m,17H).

[0461] Example 2: Synthesis of 2-amino-N-(2-hydroxyethyl)-N-propyl-8-(1-((5,6,7,8-tetrahydro-1,6-naphthid-3-yl)carbamoyl)cyclopropyl)-3H-benzo[b]azapyro-4-carboxamide (compound 2)

[0462] Using the same synthetic method as compound 1, replacing dipropylamine in step 1 with 2-(propylamino)ethanol yields compound 2, a white solid. m / z: [M+H] + 503.3; 1 H NMR (400MHz, CD3OD): δ8.40(s,1H),7.68(s,1H),7.38(d,J=8.0Hz,1H),7.26(s,1H),7.16(d,J=8.0Hz,1H),6.94(s,1H),3.94(s,2H),3.77(br.s, 2H),3.62(t,J=8.0Hz,2H),3.50(t,J=8.0Hz,2H),3.16(t,J=8.0Hz,2H), 2.93-2.80(m,3H),1.78-1.50(m,4H),1.44-1.15(m,3H),0.90(br.s,3H).

[0463] Example 3: Synthesis of 2-amino-8-(1-((6-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)phenyl)-1,3-dioxane-5-carbonyl)-5,6,7,8-tetrahydro-1,6-naphthidium-3-yl)carbamoyl)cyclopropyl)-N-(2-hydroxyethyl)-N-n-propyl-3H-benzo[b]azapyroxene-4-carboxamide (compound II-1)

[0464] Under ice bath conditions, HATU (24.2 mg, 0.064 mmol) and DIPEA (9.5 mg, 0.074 mmol) were added to anhydrous DMF (2 mL) solution of L-5 (15.0 mg, 0.049 mmol) and compound 2 (27.1 mg, 0.054 mmol), and the reaction mixture was stirred at 0 °C for 1 hour. The reaction solution was directly purified by prep-HPLC (eluent: 5%–50% acetonitrile in 0.1% trifluoroacetic acid aqueous solution, 20 min) to give compound II-1 (18.5 mg, yield: 40%) as a white solid. m / z: [M+H] + 788.2.

[0465] Example 4: Synthesis of 2-amino-8-(1-((6-(2-(3-(bromomethyl)isoxazol-5-yl)-1,3-dioxane-5-carbonyl)-5,6,7,8-tetrahydro-1,6-naphthidium-3-yl)carbamoyl)cyclopropyl)-N-(2-hydroxyethyl)-N-n-propyl-3H-benzo[b]azapyroxene-4-carboxamide (compound II-2)

[0466] Under ice bath conditions, a 50% ethyl acetate solution of T3P (63 mg, 0.1 mmol) was added to an anhydrous DMF solution (1.5 mL) of L-9 (9.5 mg, 0.033 mmol) and triethylamine (13.4 mg, 0.10 mmol), and the reaction mixture was stirred at 0 °C for 0.5 h. Compound II-2 (14.9 mg, 0.03 mmol) was added to the above reaction mixture, and the resulting mixture was stirred at 0 °C for 10 min. Under ice bath conditions, triethylamine (13.4 mg, 0.10 mmol) and a 50% ethyl acetate solution of T3P (63 mg, 0.1 mmol) were added, and the mixture was stirred at 0 °C for another 10 min. The reaction mixture was directly purified by prep-HPLC (eluent: 5%–50% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound II-2 (2.4 mg, yield: 8%) as a white solid. m / z: [M+H] + 776.2.

[0467] Example 5: Compounds as shown in Table 2 were prepared using the synthetic methods of Example 3 or 4:

[0468] Table 2

[0469] Example 6: Synthesis of Compound II-23

[0470] Synthesis of Intermediate A: Under ice bath conditions, DIPEA (0.74 g, 5.74 mmol) was added to a mixed solution of (S)-5-tert-butyl-1-(2,5-dioxopyrrolidone-1-yl)2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)glutarate (1.50 g, 2.87 mmol) and (S)-2-((S)-2-amino-3-methylbutyramamido)propionic acid (0.81 g, 4.30 mmol) in acetonitrile (10.0 mL) and water (13.0 mL). The reaction system was stirred at 0 °C for 1 hour, then slowly raised to room temperature and stirred for 3.5 hours. After removing some of the solvent by vacuum concentration, intermediate A (1.30 g, yield: 76%) was purified directly by Flash column chromatography (C18, eluent: 0%–60% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) as a white solid. m / z:[M+H] + 596.4.

[0471] Step 1: Under ice bath conditions, HATU (0.43 g, 1.13 mmol) and DIPEA (0.22 g, 1.74 mmol) were added to an 8 mL DMF solution of Fmoc-glycine (0.26 g, 0.87 mmol) and eczemab mesylate (0.50 g, 0.94 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was then purified directly by Flash column chromatography (C18, eluent: 0%–57% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to give compound 1.1 (0.60 g, yield: 96%) as a yellow solid. m / z: [M+H] + 715.2.

[0472] Step 2: Diethylamine (0.31 g, 4.20 mmol) was added to a DMF (6.0 mL) solution of compound 1.1 (0.60 g, 0.84 mmol), and the reaction mixture was stirred at room temperature for 1 hour. The reaction solution was then purified directly by Flash column chromatography (C18, eluent: 0%–37% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound 1.2 (178.0 mg, yield: 43%) as a yellow solid. m / z: [M+H] + 493.2.

[0473] Step 3: Under ice bath conditions, HATU (164.3 mg, 0.43 mmol) and DIPEA (93.1 mg, 0.72 mmol) were added to a DMF (5.0 mL) solution of compound 1.2 (178.0 mg, 0.36 mmol) and intermediate A (214.4 mg, 0.36 mmol). The reaction system was stirred at 0 °C for 1 hour. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0%–62% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound 1.3 (300.0 mg, yield: 78%) as a yellow solid. m / z: [M+H] + 1070.4.

[0474] Step 4: Diethylamine (102.4 mg, 1.40 mmol) was added to a DMF (3.0 mL) solution of compound 1.3 (300.0 mg, 0.28 mmol), and the reaction mixture was stirred at room temperature for 1 hour. The reaction solution was then purified directly by Flash column chromatography (C18, eluent: 0%–42% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound 1.4 (200.0 mg, yield: 84%) as a yellow solid. m / z: [M+H] + 848.4.

[0475] Step 5: Under ice bath conditions, DIPEA (62.0 mg, 0.48 mmol) was added to a DMF (3.0 mL) solution of compound 1.4 (200.0 mg, 0.24 mmol) and Boc-glycine-N-hydroxysuccinimide ester (65.3 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction solution was then purified directly by Flash column chromatography (C18, eluent: 0%–55% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound 1.5 (200.0 mg, yield: 84%) as a yellow solid. m / z: [1 / 2(M-100-56)+H] + 425.2.

[0476] Step 6: Under ice bath conditions, trifluoroacetic acid (0.5 mL) was added dropwise to a solution of compound 1.5 (200.0 mg, 0.20 mmol) in dichloromethane (2.0 mL). The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0%–39% acetonitrile in 0.05% aqueous trifluoroacetic acid) to give compound 1.6 (147.0 mg, yield: 95%) as a pale yellow solid. m / z: [M+H] + 849.4.

[0477] Step 7: Under ice bath conditions, DIPEA (28.4 mg, 0.22 mmol) was added to a DMF (3 mL) solution of compound 1.6 (95.0 mg, 0.11 mmol) and 3-maleimide propionic acid hydroxysuccinimide ester (32.2 mg, 0.12 mmol). The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly purified by prep-HPLC (eluent: 5%–40% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to give compound II-23 (49.4 mg, yield: 43%) as a pale yellow solid. m / z: [M+H] + 1000.3.

[0478] Example 7: Synthesis of Compound II-24

[0479] Step 1: Silver oxide (8.00 g, 34.5 mmol) was added to an acetonitrile (60.0 mL) solution of 2,3,4,6-tetraacetoxy-α-D-pyranose bromide (4.00 g, 9.73 mmol) and 4-hydroxy-3-nitrobenzaldehyde (1.79 g, 10.70 mmol). The reaction mixture was stirred overnight at 30 °C in the dark. The reaction mixture was then filtered through diatomaceous earth, the filter cake was washed with acetonitrile, the filtrate was concentrated under reduced pressure, the residue was dissolved again in dichloromethane, filtered as a solid, the filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (dichloromethane / methanol = 20 / 1) to give compound 3.2 (3.86 g, yield: 80%) as a white solid. m / z: [M+Na] + 520.2.

[0480] Step 2: Compound 3.2 (3.86 g, 7.76 mmol) was dissolved in ethyl acetate (193.0 mL), then 0.50 g of 10% palladium on carbon was added, followed by 6 drops of triethylamine. The reaction mixture was stirred overnight at 30°C under a hydrogen balloon. The reaction mixture was then filtered through diatomaceous earth and concentrated under reduced pressure to give compound 3.3 (3.423 g, yield: 94%) as a white solid. m / z: [M+H] + 470.2.

[0481] Step 3: Under nitrogen protection at room temperature, 1H-imidazolium (0.60 g, 8.75 mmol) and tert-butyldimethylchlorosilane (1.15 g, 7.65 mmol) were added to a dichloromethane (85.6 mL) solution of compound 3.3 (3.423 g, 7.29 mmol). The reaction system was stirred at room temperature for 2 hours. The reaction was then quenched with water (60 mL), and the organic phase was collected. The aqueous phase was extracted with dichloromethane (2 × 50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give compound 3.4 (3.332 g, yield: 78%) as a white solid. m / z: [M+H] + 584.2.

[0482] Step 4: Under ice bath conditions, HATU (0.43 g, 1.12 mmol) and DIPEA (0.22 g, 1.72 mmol) were added to a DMF (10 mL) solution of compound 3.4 (0.50 g, 0.86 mmol) and 4-[N-(tert-butoxycarbonyl)amino]butyric acid (0.18 g, 0.90 mmol). The reaction system was stirred at room temperature for 16 hours. The reaction solution was purified directly by Flash column chromatography (C18, eluent: 0%–67% acetonitrile in 0.05% trifluoroacetic acid aqueous solution). The collected fraction was stirred at 30 °C for 4 hours, and the solution was directly lyophilized to give compound 3.5 (495.0 mg, yield: 88%) as a white solid. m / z: [M-100+H] + 555.2.

[0483] Step 5: Compound 3.5 (495.0 mg, 0.76 mmol) was dissolved in methanol (39 mL). After cooling at 0°C for 5 minutes, a methanol solution of sodium methoxide (5.4 M, 0.84 mL, 4.56 mmol) was added. The reaction mixture was stirred at 0°C for 30 minutes, followed by stirring at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure at low temperature to remove some of the solvent, and then purified directly by Flash column chromatography (C18, eluent: 0%–33% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to obtain compound 3.6 (0.29 g, yield: 79%) as a white solid. m / z: [M+Na] + 509.2.

[0484] Step 6: Under ice bath conditions, di(p-nitrobenzene) carbonate (242 mg, 0.80 mmol) and DIPEA (206 mg, 1.59 mmol) were added sequentially to a DMF (5 mL) solution of compound 3.6 (260.0 mg, 0.53 mmol). The reaction mixture was stirred at room temperature for 3.5 hours. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0%–57% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound 3.7 (80.0 mg, yield: 23%) as a white solid. m / z: [M-100+H] + 552.2.

[0485] Step 7: Under ice bath conditions, DIPEA (31.0 mg, 0.24 mmol) was first added to eczemab mesylate (63.8 mg, 0.12 mmol in 2 mL of DMF). After the solution became clear, compound 3.7 (80.0 mg, 0.12 mmol) was added, and the reaction mixture was stirred at room temperature for 5 hours. The reaction solution was then purified directly by Flash column chromatography (C18, eluent: 0%–49% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound 3.8 (60.0 mg, yield: 52%) as a pale yellow solid. m / z: [M+H] + 948.3.

[0486] Step 8: Under ice bath conditions, trifluoroacetic acid (0.5 mL) was added dropwise to a solution of compound 3.8 (60.0 mg, 0.063 mmol) in dichloromethane (2.0 mL). The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0%–39% acetonitrile in 0.05% aqueous trifluoroacetic acid) to give compound 3.9 (43.0 mg, yield: 80%) as a pale yellow solid. m / z: [M+H] + 848.2.

[0487] Step 9: Under ice bath conditions, DIPEA (13.2 mg, 0.10 mmol) was added to a DMF (3 mL) solution of compound 3.9 (43.0 mg, 0.051 mmol) and 2-(4-maleiminophenyl)acetic acid (N-hydroxysuccinimide) ester (18.4 mg, 0.056 mmol). The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly purified by prep-HPLC (eluent: 5%–55% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound II-24 (6.07 mg, yield: 11%) as a white-yellow solid. m / z: [M+H] + 1061.2.

[0488] Example 8: Synthesis of Compound II-25

[0489] Step 1: Under ice bath conditions, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.98 g, 5.12 mmol) and HOBT (0.69 g, 5.12 mmol) were added to a DMF (15.0 mL) solution of compound 4.1 (1.45 g, 3.41 mmol) and compound 4.2 (1.0 g, 3.41 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was then purified directly by Flash column chromatography (C18, eluent: 0%–63% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to give compound 4.3 (1.6 g, yield: 67%) as a white solid. m / z: [M+Na] + 723.2.

[0490] Step 2: To a DMF (3.0 mL) solution of compound 4.3 (150 mg, 0.21 mmol), di(p-nitrobenzene) carbonate (128 mg, 0.42 mmol) and DIPEA (82 mg, 0.63 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 3.5 hours. The reaction solution was directly purified by prep-HPLC (eluting with 10%–75% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to give compound 4.4 (100 mg, yield: 54%) as a white solid.

[0491] Step 3: Under ice bath conditions, DIPEA (31.0 mg, 0.24 mmol) and HOBT (0.024 g, 0.18 mmol) were added sequentially to a DMF (3 mL) solution of compound 4.4 (100 mg, 0.12 mmol) and eczemab mesylate (70.0 mg, 0.13 mmol). The reaction system was stirred at room temperature for 3.5 hours. The reaction solution was directly purified by prep-HPLC (eluting with 10%–75% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to give compound 4.5 (95 mg, yield: 71%) as a yellow solid. m / z: [M+H] + 1162.2.

[0492] Step 4: At room temperature, diethylamine (0.05 mL) was added to a 3 mL solution of compound 4.5 (95 mg, 0.082 mmol) in DMF. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then purified directly by prep-HPLC (eluent: 10%–45% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain compound 4.6 (70 mg) as a white solid. m / z: [1 / 2 M + H] + 470.2.

[0493] Step 5: Diethylamine (0.05 mL) was added to a DMF (3 mL) solution of compound 4.6 (70 mg, 0.074 mmol) and Boc-glycine-N-hydroxysuccinimide ester (26 mg, 0.096 mmol). The reaction solution was stirred at 0 °C for 2 hours. The reaction solution was then purified directly by prep-HPLC (eluting with 10%–75% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain compound 4.7 (70 mg) as a white solid. m / z: [M+H] + 1097.2.

[0494] Step 6: Under ice bath conditions, add 1 mL of trifluoroacetic acid to a 4 mL solution of compound 4.7 (70 mg, 0.064 mmol) in dichloromethane. Slowly raise the reaction mixture to room temperature and continue stirring for 1 hour. The reaction mixture was then purified directly by prep-HPLC (eluent: 10%–45% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain compound 4.8 (55 mg, yield: 92%) as a pale yellow solid. m / z: [1 / 2 M + H] + 471.2.

[0495] Step 7: Under ice bath conditions, DIPEA (15 mg, 0.12 mmol) was added to a DMF (3 mL) solution of compound 4.8 (55 mg, 0.058 mmol) and 2,5-dioxopyrrolidone-1-yl 3-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionate (17 mg, 0.064 mmol). The reaction mixture was slowly heated to room temperature and stirred for 1 hour. The reaction mixture was then purified directly by prep-HPLC (eluting with 10%–55% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to give compound II-25 (46.8 mg, yield: 72%) as a white solid. m / z: [1 / 2 M + H] + 546.7.

[0496] Example 9: Antibody Preparation

[0497] The Farletuzumab antibody sequence comprising the full-length heavy chain SEQ ID NO:8 and the full-length light chain SEQ ID NO:7 was obtained from the WO2017151979A1 publication; the huMov19 antibody sequence comprising the full-length heavy chain SEQ ID NO:6 and the full-length light chain SEQ ID NO:5 was obtained from the WO2022256507A1 publication. Additionally, it can be obtained by pressing PLoS... ONE, 2021, 16(12):e0260954., describes the mutation positions to obtain huMov19 variants containing Fc silencing mutations L234A, L235A, and P329G, abbreviated as huMov19-AAG; obtain Farletuzumab variants containing Fc silencing mutations L234A, L235A, and P329G, abbreviated as Farletuzumab-AAG; also, according to the cysteine ​​insertion positions described in WO2022198335A1, cysteine ​​residues can be inserted between positions 149 and 150 in the CL domain of the huMov19 monoclonal antibody to obtain The following variants are available: huMov19 variant, abbreviated as huMov19-K149.5C; huMov19 variant containing the Fc silencing mutations L234A, L235A, P329G and the insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain, abbreviated as huMov19-AAG-K149.5C; and Farletuzumab variant containing the Fc silencing mutations L234A, L235A, P329G and the insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain, abbreviated as Farletuzumab-AAG-K149.5C.

[0498] Specific preparation method: Heavy and light chain gene fragments encoding the target antibody were obtained using gene amplification and site-directed mutagenesis PCR. These fragments were cloned into suitable mammalian expression vectors to construct recombinant expression plasmids. Positive clones were screened and verified by sequencing. The verified recombinant plasmids were then introduced into CHO host cells via electroporation. Transfected cells were cultured in selective media, and positive clones stably expressing the target antibody were selected using the selection marker pressure on the vector. The selected high-expression monoclonal cell lines were expanded and cultured for 4 to 6 days. The cell culture supernatant was collected, and after centrifugation or filtration to remove cells and debris, the target antibody was specifically captured using a Protein A affinity chromatography column. The purified antibody solution was concentrated by ultrafiltration and centrifugation. The antibody concentration was determined using NanoDrop or UV spectrophotometry. The molecular weight and integrity of the antibody were analyzed using reducing and non-reducing SDS-PAGE, and polymers and debris were quantitatively detected by SEC-HPLC to ensure product purity met standards (typically SEC monomer content >95%). The final product was tested for endotoxins, and the content was confirmed to be less than 1.0 EU / mg.

[0499] Synthesis of immunomodulator antibody-drug conjugates

[0500] Example 10: General Method 1

[0501] Add 2 mL of antibody solution (10.4 mg / mL) to a centrifuge tube and centrifuge at 3800 G for 5 minutes to concentrate the antibody solution. Equilibrate a Sephadex G-25 carrier NAP-25 column (5 mL / 12 mL) with phosphate buffer (50 mM, pH 6.5; PBS 6.5 / EDTA) containing sodium chloride (50 mM) and EDTA (2 mM). Pack 1.27 mL of antibody aqueous solution onto one NAP-25 column and separate the fraction eluted with PBS 6.5 / EDTA. Determine the antibody concentration as 13 mg / mL, then adjust the antibody concentration to 10 mg / mL using PBS 6.5 / EDTA. Transfer this solution to a 15 mL test tube, adjust the antibody concentration to 5 mg / mL using 3 mL of PBS 6.5 / EDTA solution, then add TCEP aqueous solution (2-10 equivalents per molecule of antibody) and incubate at 37°C for 2 hours. Add 300 μL of dimethyl sulfoxide (DMSO), then add a DMSO solution of the compounds (Linker-payload) shown in II-1 to II-22 (10 equivalents relative to one molecule of antibody), ultimately controlling the DMSO content to approximately 10%. Stir the solution at room temperature for 60 minutes. Equilibrate the NAP-25 column with phosphate buffer (PB7.4). Pack 6.6 mL of the antibody-drug conjugate aqueous solution onto one NAP-25 column, then separate the fraction eluted with PB7.4. Repeat this operation 2-3 times, collect the eluted fraction, concentrate, sterilize, and filter to obtain the immunomodulator antibody-drug conjugate.

[0502] The antibodies used in General Method 1 were Trastuzumab (Roche), Pertuzumab (Roche), huMov19 (Baiying Biotechnology), Farlezutumab (Baiying Biotechnology), and Farlezutumab-AAG (Baiying Biotechnology). The immunomodulatory antibody-drug conjugates shown in Table 3 were synthesized using General Method 1.

[0503] Table 3

[0504] Example 11: Preparation of I-28

[0505] I-25 was obtained by reacting Pertuzumab with MC-GGFG-Dxd using General Method 1, with a DAR value of 5.6 and a SEC of 98%.

[0506] Example 12: Preparation of I-40

[0507] I-40 was obtained by reacting huMov19 and MC-GGFG-Dxd using General Method 1, with a DAR value of 5.97 and an SEC value of 96.8%.

[0508] Example 13: Preparation of I-41

[0509] I-41 was obtained by reacting Farlezutumab-LALA and LNK4-S (synthesis reference WO2023170247A1) using general method 1, with a DAR value of 7.8 and an SEC value of 99.7%.

[0510] Example 14: General Method 2

[0511] Step 1: Add antibody solution to a centrifuge tube, then adjust the antibody concentration to 5 mg / mL using PBS pH=7.2. Take this solution (7.5 mg, 5 mg / mL) into a 2 mL sterile centrifuge tube, add TCEP aqueous solution (6 equivalents relative to one molecule of antibody) and ZnCl2 (2-4 equivalents relative to one molecule of antibody), incubate at 6°C for 15 hours, desalt column treatment once, add dehydroascorbic acid DHAA (1-9 equivalents relative to one molecule of antibody), stir at 12°C for 5 hours, finally add EDTA (5 mM), 150 μL of dimethyl sulfoxide (DMSO), and a dimethyl sulfoxide solution of MC-GGFG-Dxd (obtained using the method disclosed in WO2019238046A1) or II-24 (8 equivalents relative to one molecule of antibody), with the final DMSO ratio controlled at approximately 10%. The solution was stirred at 12°C for 60 minutes, treated with a 5 mL desalting column, and then concentrated using an Amicon ultrafiltration tube. This process was repeated 5 times. The fraction was then collected and sterilely filtered. The DAR value was then determined.

[0512] Step 2: Take the antibody-conjugate (3 mg, 5 mg / ml) obtained from ultrafiltration in Step 1, add TCEP aqueous solution (6 equivalents relative to one antibody molecule), and incubate at 25°C for 2 hours. Then add II-3 (5 equivalents relative to one antibody molecule) and DMSO (10% of the total volume). Continue stirring the system at 20°C for 1 hour. Finally, process with a 5 mL desalting column, then concentrate using an Amicon ultrafiltration tube. Repeat this operation 5 times, collect the fraction, and obtain the dual-loaded antibody-immunostimulation conjugate after sterile filtration. Detect the DAR value.

[0513] The antibodies used in General Method 2 were Pertuzumab (Roche), huMov19 (Baiying Biotechnology), Farlezutumab-AAG (Baiying Biotechnology), and Farlezutumab-LALA (Baiying Biotechnology). The immunomodulatory antibody-drug conjugates shown in Table 4 were synthesized using General Method 2.

[0514] Table 4

[0515] Example 15: General Method 3

[0516] Step 1: Add antibody solution to a centrifuge tube, then adjust the antibody concentration to 10 mg / mL using BES pH = 7.0. Transfer the above solution (5 mg, 1.8 mg / mL) to a 2 mL sterile centrifuge tube, add TCEP aqueous solution (6 equivalents relative to one antibody molecule) and ZnCl2 (4 equivalents relative to one antibody molecule), incubate at 6°C for 16 hours, desalt once, then add DHAA (9 equivalents relative to one antibody molecule), stir at 25°C for 2 hours, and finally add EDTA (5 mM), II-3 (8 equivalents relative to one antibody molecule) in N,N-dimethylacetamide (DMA) solution, controlling the final DMA ratio to approximately 10%. Continue stirring the solution at 25°C for 60 minutes, desalt with a 5 mL column, then concentrate using an Amicon ultrafiltration tube. Repeat this operation 5 times, collect the fraction, and filter sterilely. Detect the DAR value.

[0517] Step 2: Take the antibody-conjugate obtained in Step 1 (2 mg, 5 mg / mL), add TCEP aqueous solution (10 equivalents relative to one antibody molecule) and ZnCl2 (4 equivalents relative to one antibody molecule), incubate at 5°C for 20 minutes, then add the second linker payload (5 equivalents relative to one antibody molecule), and continue stirring at 5°C for 25 minutes to quench N-acetylcysteine. Finally, process with a 5 mL desalting column, then concentrate using an Amicon ultrafiltration tube. Repeat this operation 5 times, collect the fraction, sterilely filter to obtain the dual-loaded antibody-immunostimulation conjugate. Detect the DAR value.

[0518] The antibodies used in General Method 2 were huMov19 (Baiying Biotechnology) and Farlezutumab (Baiying Biotechnology). The immunomodulator antibody-drug conjugates shown in Table 5 were synthesized using General Method 3.

[0519] Table 5

[0520] Example 16: General Method 4

[0521] Step 1: Add antibody solution to a centrifuge tube and adjust pH to 7.0 using BES. Transfer the above solution (20 mg, 5 mg / mL) to a 15 mL sterile centrifuge tube, add TCEP aqueous solution (12 equivalents relative to one antibody molecule), incubate at 37°C for 2 hours, then add DHAA (30 equivalents relative to one antibody molecule), stir at 25°C for 2 hours, and finally add EDTA (5 mM) and II-3 (4 equivalents relative to one antibody molecule) in a DMA solution, maintaining the final DMA concentration at approximately 10%. Continue stirring the solution at 25°C for 60 minutes, process with a 5 mL desalting column, then concentrate using an Amicon ultrafiltration tube. Repeat this process 5 times, collect the fraction, and sterilely filter. Detect the DAR value.

[0522] Step 2: Take the antibody-conjugate obtained in Step 1 after ultrafiltration (2 mg, 5 mg / ml), add TCEP aqueous solution (10 equivalents relative to one antibody molecule), incubate at 35°C for 2 hours, then add the second linker payload (12 equivalents relative to one antibody molecule), and continue stirring at 25°C for 1 hour to quench N-acetylcysteine. Finally, process with a 5 mL desalting column, then concentrate using an Amicon ultrafiltration tube. Repeat this operation 5 times, collect the fraction, sterilely filter to obtain the dual-loaded antibody-immunostimulation conjugate. Detect the DAR value.

[0523] In General Method 2, the antibodies used were huMov19-AAG-K149.5C (Baiying Biotechnology) and Farlezutumab-AAG-K149.5C (Baiying Biotechnology). Immunomodulator antibody-drug conjugates as shown in Table 6 were synthesized using General Method 4.

[0524] Table 6

[0525] Example 17: General Method 5

[0526] Step 1: Add antibody solution to a centrifuge tube and adjust pH to 7.0 using BES. Transfer the above solution (20 mg, 5 mg / mL) to a 15 mL sterile centrifuge tube, add TCEP aqueous solution (12 equivalents relative to one antibody molecule), incubate at 37°C for 2 hours, then add DHAA (30 equivalents relative to one antibody molecule), stir at 25°C for 2 hours, and finally add EDTA (5 mM) and II-3 (4 equivalents relative to one antibody molecule) in a DMA solution, maintaining the final DMA concentration at approximately 10%. Continue stirring the solution at 25°C for 60 minutes, process with a 5 mL desalting column, then concentrate using an Amicon ultrafiltration tube. Repeat this process 5 times, collect the fraction, and filter sterilely. Detect the DAR value.

[0527] Step 2: Add the antibody-conjugate solution obtained in Step 1 to a centrifuge tube, and then adjust the antibody concentration to 10 mg / mL using BES pH = 7.0. Transfer this solution (5 mg, 1.8 mg / mL) to a 2 mL sterile centrifuge tube, add TCEP aqueous solution (6 equivalents relative to one antibody molecule) and ZnCl2 (4 equivalents relative to one antibody molecule), and incubate at 6°C for 16 hours. After one desalting column treatment, add DHAA (9 equivalents relative to one antibody molecule), and stir at 25°C for 2 hours. Finally, add EDTA (5 mM), a second linker payload (8 equivalents relative to one antibody molecule), and a DMA solution, maintaining the final DMA ratio at approximately 10%. Continue stirring the solution at 25°C for 60 minutes, treat with a 5 mL desalting column, and then concentrate using an Amicon ultrafiltration tube. Repeat this process 5 times, collect the fraction, and sterilely filter to obtain the dual-loaded antibody-immunostimulation conjugate. Detect the DAR value.

[0528] The antibody used in General Method 5 was huMov19-K149.5C, manufactured by Baiying Biotechnology. Immunomodulator antibody-drug conjugates as shown in Table 7 were synthesized using General Method 5.

[0529] Table 7

[0530] Example 1: Cell viability assay of TLR7 and TLR8 cells

[0531] In this experiment, the TLR7 and TLR8 bioactivity of the compounds shown in Formula I was determined using a cell assay. This method was performed in human embryonic kidney cells (HEK293) expressing TLR family members such as TLR4, TLR7, TLR8, or TLR9. TLR agonists activate TLRs, leading to downstream NF-κB activation, which in turn activates the secreted embryonic alkaline phosphatase (SEAP) reporter gene. SEAP activity was detected using Quanti-Blue (InvivoGen) reagent, thus reflecting the activity of the TLR7 and TLR8 agonists.

[0532] The detailed experimental method is as follows:

[0533] HEK-BLUE-hTLR7 and HEK-BLUE-hTLR8 cell lines were purchased from Invivogen and cultured in DMEM medium containing 4.5 g / L glucose (Sigma-Aldrich) and 10% fetal bovine serum under the following conditions: temperature 37°C, humidity 95%, and CO2 5%.

[0534] The compounds were tested at 10 concentration gradients ranging from 0.5 nM to 15 μM. A known active TLR7 or TLR8 agonist was added as a positive control, and 1 μL of DMSO was added as a negative control.

[0535] Cell processing was as follows: Cells were removed from the culture dish and centrifuged to remove the culture medium. They were resuspended in 10 mL of pre-warmed PBS in a T-150 flask, and 12 mL of pre-warmed culture medium was added. The cells were gently pipetted and counted under a microscope. Immediately, a single-cell suspension of 200,000 cells / mL was prepared using culture medium, and 200 μL / well (40,000 cells / well) was added to a 96-well plate. The final concentration of DMSO was 0.5%.

[0536] The compound was added and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0537] Pipette 20 μL of supernatant per well into 180 μL of preheated Quanti-Blue at 37 °C, incubate at 37 °C for 1.5 hours, and measure the absorbance (OD value) at 650 nm using a spectrophotometer. The agonist effect is calculated using the following formula:

[0538] Effect % = (mean OD of the drug group - mean OD of the DMSO group) / (mean OD of the positive control group - mean OD of the DMSO group) × 100

[0539] The concentration-response curve was fitted using Graphpad software, and the EC50 was calculated. 50 .

[0540] Example 2: Cytokine Detection

[0541] Frozen human peripheral blood mononuclear cells (Allcells) were rapidly thawed in a 37°C water bath and then added to 9 mL of RPMI 1640 medium (all from Gibco) containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 1 mM sodium pyruvate. The cells were centrifuged at 400 × g for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in the medium and the cell density was adjusted to 4 × 10⁶ cells / mL. 6 / mL. Add 50μL to each well of a 96-well Corning plate.

[0542] PBMC system: Add 50 μL of culture medium to the above culture system.

[0543] Coculture system: Human breast ductal carcinoma cells HCC1954 (Nanjing Kebai Biotechnology Co., Ltd.), human breast cancer cells SK-BR-3 (Nanjing Kebai Biotechnology Co., Ltd.), or human oral epidermal carcinoma cells KB (Nanjing Kebai Biotechnology Co., Ltd.) in normal growth state were collected and resuspended in RPMI 1640 complete medium, and the density was adjusted to 4×10⁻⁶. 5 / mL. Add 50μL to each well of a 96-well plate and mix well with hPBMC.

[0544] Prepare a 3-fold working concentration dilution of the test sample using culture medium, and add 50 μL to each well of the cell suspension in System 1 or System 2 above. The total system volume is 150 μL, and the final compound concentration is 500 nM. Add 50 μL of culture medium to the blank control and incubate at 37°C in a 5% CO2 incubator for 20 hours.

[0545] Human TNFα detection: The cell culture plate was centrifuged at 2000 rpm for 5 minutes at room temperature, and 100 μL of supernatant was transferred to a clean 96-well plate. The TNFα detection reagent (Cisbio, 62HTNFAPEG) was allowed to dissolve completely at room temperature. The two antibodies (TNFαEu Cryptate antibody and TNFαd2 antibody) were mixed with detection buffer at a ratio of 1:40. 4 μL of the antibody mixture and 16 μL of supernatant were added to the detection plate, and the plate was incubated at room temperature in the dark for 2 hours. The signal was read using HTRF mode on an Infinite M1000 PRO (TECAN). The test results are shown in Table 8-10.

[0546] Table 8:

[0547] Table 9:

[0548] Table 10:

[0549] Example 3: Cell Killing Assay

[0550] Prepare a single-cell suspension of human ovarian cancer Cov644 cells (ATCC) to be labeled: Wash cells twice with PBS to remove serum. Resuspend cells in PBS preheated to room temperature to a concentration of 5 × 10⁶ cells / mL. 6 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 7 Cells / mL. Add CFSE (Invitrogen, Cat. 65-0850-84; powder dissolved to 10 mM) to the desired final concentration (10 μM). Mix immediately and incubate in the dark at room temperature for 10 minutes. Stop labeling, add 4-5 volumes of cold complete culture medium (containing ≥10% serum), and incubate on ice for 5 minutes. Wash cells 3 times with complete culture medium.

[0551] Preparation of drug solution and effector cells: Add 50 μL of 1×10⁻⁶ cells to a 96-well analytical plate. 6 Target cell suspension per ml. Prepare test samples with assay buffer to a final concentration of 1 nM, adding 50 μl to each well of the assay plate. Thaw frozen PBMCs and wash once with experimental buffer. Resuspend to 1 × 10⁻⁶. 6 Add 100 μL per well to achieve a final concentration of 1 nM for the sample to be tested.

[0552] Flow cytometry analysis: Cells were collected after 48 h of culture. They were washed once with PBS. 100 μL / well of FVD780 (Invitrogen, Cat. 65-0865-14) diluted 1000-fold was added, and the cells were incubated at room temperature for 10 min. The cells were washed twice. Cells were resuspended in 200 μL of FACS buffer. Fluorescence density was detected using an LSR Fortessa (BD, 647794), and the data were analyzed. Results are shown in Table 11.

[0553] Table 11:

[0554] Example 4: In vivo efficacy experiment of MC38-HER2 mouse subcutaneous xenograft tumor model of colon cancer cells.

[0555] Cell culture: Mouse colon cancer MC38-HER2 cells were maintained as a monolayer in DMEM medium containing 10% fetal bovine serum and 4 μg / mL puromycin at 37°C in a constant temperature incubator containing 5% CO2. Tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for seeding.

[0556] Experimental animals: B6-hTLR8-HO mice, 6-8 weeks old, 18-22g.

[0557] Five experimental groups were set up for Vehicle, I-3, I-28, I-29, and I-3+I-28, as shown in Table 12 below:

[0558] Table 12

[0559] Experimental methods: MC38-HER2 cell line (5.0 × 10⁻⁶) was used. 6 (0.1 mL / mouse) was injected subcutaneously into the right back of each experimental mouse. The tumor growth was observed regularly until it grew to approximately 100 mm. 3 Mice were randomly grouped according to tumor size and body weight, and administered medication according to the dosing schedule shown in Table 12. Throughout the experiment, mouse body weight and tumor size were measured twice a week.

[0560] Formula for calculating tumor size: Tumor volume (mm) 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0561] RTV = Vt / V0 (V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment.)

[0562] T / C (%) = TRTV / CRTV * 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the solvent control group.)

[0563] TGI (%) = (1 - T / C) * 100%. Experimental results are shown in Table 13.

[0564] Table 13

[0565] Example 5: In vivo drug efficacy experiment in a mouse subcutaneous xenograft model of CT26-HER2 colon cancer cells.

[0566] Cell culture: Colon cancer CT26-HER2 cells were maintained in a monolayer culture in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and 20 μg / mL puromycin in a 37°C incubator with 5% CO2. Tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for seeding.

[0567] Experimental animals: BALB / c mice, 6-8 weeks old, 18-22g.

[0568] Three experimental groups were set up for Vehicle, I-17, and I-21, as shown in Table 14 below:

[0569] Table 14 Note: IV: intravenous injection; QW: once a week

[0570] Experimental methods: CT26-HER2 cell line (3.0×10⁻⁶) was used. 6 (0.1 mL / mouse) was injected subcutaneously into the right back of each experimental mouse. The tumor growth was observed regularly until it reached approximately 111 mm. 3 Mice were randomly grouped according to tumor size and body weight, and administered medication according to the dosing schedule shown in Table 14. Throughout the experiment, mouse body weight and tumor size were measured twice a week.

[0571] Formula for calculating tumor size: Tumor volume (mm) 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0572] RTV = Vt / V0 (V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment.)

[0573] T / C (%) = TRTV / CRTV * 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the solvent control group.)

[0574] TGI (%) = (1 - T / C) * 100%. Experimental results are shown in Table 15.

[0575] Table 15

[0576] Example 6: In vivo efficacy experiment of MC38-FRα mouse subcutaneous xenograft tumor model of colon cancer cells.

[0577] Cell culture: Mouse colon cancer MC38-FRα cells were maintained in a monolayer in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 1% hydroxyethylpiperazine ethanesulfonic acid in a 37°C incubator with 5% CO2. Tumor cells were passaged twice weekly. Cells in the exponential growth phase were harvested and counted for seeding.

[0578] Experimental animals: B6-hTLR8 transgenic mice, 7-8 weeks old, 16.3-23.1g.

[0579] Four experimental groups were set up for Vehicle, I-23, I-25, and I-34, as shown in Table 16 below:

[0580] Table 16

[0581] Three experimental groups were set up for Vehicle, I-23, and I-40, as shown in Table 17 below:

[0582] Table 17

[0583] Four experimental groups were set up for Vehicle, I-23, I-31, and I-33, as shown in Table 18 below:

[0584] Table 18

[0585] Five experimental groups were set up for Vehicle, I-30, I-40, I-23+I-40, and IMGN853, as shown in Table 19 below:

[0586] Table 19

[0587] Four experimental groups were set up for Vehicle, I-38, I-39, and I-41, as shown in Table 20 below:

[0588] Table 20

[0589] Two experimental groups were set up for Vehicle and I-37, as shown in Table 21 below:

[0590] Table 21

[0591] Five experimental groups were set up for Vehicle, I-39, I-43, I-44, and I-45, as shown in Table 22 below:

[0592] Table 22

[0593] Note: sc: subcutaneous administration; QW: once a week

[0594] Experimental methods: MC38-FRα cell line (2.0 × 10⁻⁶) was used. 6 One sample (0.1 mL, resuspended in PBS) was injected subcutaneously into the right back of each experimental mouse. Tumor growth was monitored periodically until the tumor reached approximately 176 mm. 3 Mice were randomly grouped according to tumor size and body weight, and administered medication according to the dosing schedule shown in Tables 16-22. Throughout the experiment, mouse body weight and tumor size were measured twice a week.

[0595] Formula for calculating tumor size: Tumor volume (mm) 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0596] RTV = Vt / V0 (V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment.)

[0597] T / C (%) = TRTV / CRTV * 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the solvent control group.)

[0598] TGI (%) = (1 - T / C) * 100%. Experimental results are shown in Tables 23-29.

[0599] Table 23

[0600] Table 24

[0601] Table 25

[0602] Table 26

[0603] Table 27

[0604] Table 28

[0605] Table 29

[0606] In Examples 2 and 6, the positive reference IMGN853 (MCE, HY-132258A) had a DAR value of 3.84.

Claims

1. An antibody-drug conjugate as shown in Formula I, (D1-L1) m -Ab-(L2-ST1) n (I)， in, Ab represents antibodies; m can be any value from 0 to 8; n is any value from 1 to 8; D1 is a topoisomerase I inhibitor or a tubulin inhibitor; L1 is a connector containing a pyrolytic linker; L2 is A is a benzene ring or a 5-6 membered heteroaromatic ring; M is the connector that links to the antibody; X is the connection key. X1 is a linker bond, -NHC(O)-, -O-, or -NH-; X2 is a linker, -C(O)-, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene; B is the connection key, C 6-10 arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group; the C 6-10 The arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group is unsubstituted, or selectively replaced by 1-3 groups selected from halogens, oxo groups, C... 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 The alkoxy group can be substituted at any position; ST1 is U is -C(O)- or a connection key; V is either NH or S; Y is either -O- or -NH-; R1 is a halogen, -OP(O)(OH)2, or a pyranose group; R2 represents H and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or amino C 1-6 alkyl; R3 is H or C 1-6 alkyl; R4 is C 1-6 Alkyl, C 1-6 Alkoxy or C 1-3 Alkoxy C 1-4 alkyl; R4' is C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or C 1-3 Alkoxy C 1-4 alkyl; R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OR. a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -NR a C(O)OR b -NR a C(O)NR a R b or -C(O)OR b The substituents can be substituted at any position; R6 represents H, halogen, carboxyl, or C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl; A1, A2, and A3 are each independently CR7 or N; R7 is H or halogen; Each R a and R b Independently, they are hydrogen and C respectively. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl, 5-10 heteroaryl, C 3-10 cycloalkyl C 1-6 Alkyl, 3-10 membered heterocyclic alkyl C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl; x is 0, 1, or 2; t can be 0, 1, 2, 3, 4, or 5; Furthermore, the antibody-drug conjugate shown in Formula I satisfies one of the following conditions: (1) m is any value from 1 to 8; (2) m is 0; x is 1 or 2; (3) m is 0; x is 0; X1 is a linking bond, -NHC(O)-, -O- or -NH-; X2 is a linking bond, -C(O)- or -C(R3)2(CH2) 0-2 -; (4) m is 0, x is 0; A is a 5-6 member heteroaryl ring.

2. The antibody-drug conjugate as shown in Formula I according to claim 1, characterized in that, It meets one or more of the following conditions: (1) The antibody contains one or two antigen-binding domains that can bind to an antigen; (2) The antibody contains an Fc segment; (3) The antibody described is a monoclonal antibody; (4) The antibody mentioned is an anti-HER2 antibody or an anti-FRα antibody; (5) m is 0 and n is any value from 1 to 8; preferably, m is 0 and n is any value from 1 to 4. (6) m is any value from 1 to 8; n is any value from 1 to 4; (7) D1 is camptothecin, MMAE, MMAF, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, irinotecan, topotecan, letopotecan, belotetane. (preferred) )、 (preferred) )、 R 9-1 and R 9-2 H, D, and C are independent of each other. 3-5 cycloalkyl or cyclopropylmethyl; or, R 9-1 and R 9-2 Together with the C atoms they are attached to, they form 3-5 membered cycloalkyl groups; R 9-3 For H or C 1-6 Alkyl; R 9-4 and R 9-5 C independently 1-6 Alkyl, halogen, or hydroxyl; (8) L1 is -M'-L 1a -L 1b -L 1c -,-L 1a - is an extension group; -L 1b - is a cleavable polypeptide linker; -L 1c - represents a linking bond or a self-eliminating group; M' represents a linker; or L1 represents -M'-L 1a -L 1c -;-L 1a - is an extension group; -L 1c - represents a linking bond or a self-eliminating group; M' represents a linker; (9) Option A is selected from any of the following: Option 1: A is phenyl, pyridyl, pyrimidinyl, pyridazinyl, or oxazolyl, preferably. for b) Connect the end to M; Option 2: m is 0, x is 0, and A is pyridinyl, pyrimidinyl, pyridazinyl, or oxazolyl, preferably. for b) Connect the end to M; (10) The mercapto or amino groups in M ​​and Ab are linked; preferably, the mercapto groups in M ​​and Ab are linked. (11) M is methylene, The M is connected to Ab via terminal c); (12) R2 is H, halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl or amino C 1-4 alkyl; (13) X is the connection key, X is connected to B via end a); t is 0, 1, 2 or 3; (14) X1 and X2 are any of the following schemes: Option 1: X1 is -NHC(O)-; X2 is a linker bond or C 3-6 Cycloalkylene; Option 2: X1 is the connection key; X2 is the connection key; Option 3: X1 is a connector or -O-; X2 is -C(R3)2(CH2) 0-3 -; R3 is H or methyl; (15) B is a linking bond, phenylene, 5-6 membered heteroaryl, 8-12 membered fused cyclic group, surrounded by 1, 2 or 3 Rs. 8-1 Substituted phenylene, with 1, 2 or 3 R 8-2 The substituted 5-6 nucleotide heteroaryl group may be replaced by one, two, or three R groups. 8-3 Substituted 8-12 fused cyclic groups; In B, the 5-6 membered heteroaryl group and the group with 1, 2, or 3 R groups 8-2 In the substituted 5-6-membered heteroaryl group, the heteroatom is independently selected from one or more of N, O and S, and the number of heteroatoms is independently 1, 2 or 3; the heteroatom is preferably N, and the number of heteroatoms is preferably 1 or 2; In B, the 8-12 fused cycloaliphatic group and the group surrounded by 1, 2, or 3 R groups 8-3 The 8-12 fused cyclic group in the substituted 8-12 cyclic group is independently a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring; in the 5-6 fused heteroaromatic ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3; in the 5-6 fused heteroene ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3. R 8-1 R 8-2 and R 8-3 Independently halogen, oxo group, C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 Alkoxy; (16) R4 is C 1-6 Alkyl; or R4 is C 1-3 Alkoxy C 1-4 alkyl; (17) R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH, -OC(O)NR. a R b -NR a C(O)OR b or -NR a C(O)NR a R b The substituents can be substituted at any position; R a For H; R b For H, C 1-6 Alkyl or C 3-6 cycloalkyl, such as C 1-6 Alkyl, C 1-6 Alkoxy, -C 1-6 alkylene -OH or -OC 1-6 Alkylene-NHC(O)NHR b ; (18)ST1 is (19) R6 is H, carboxyl, or phenyl C. 1-6 alkyl; (20) for (21) Y is O; (22) V is NH; (23) R4' is C 1-6 alkyl.

3. The antibody-drug conjugate as shown in Formula I according to claim 2, characterized in that, It meets one or more of the following conditions: (1) The anti-HER2 antibody is Trastuzumab, a Trastuzumab variant, Pertuzumab or a Pertuzumab variant, wherein the Trastuzumab variant has at least the same binding capacity as the Trastuzumab antibody, and the amino acid sequence of the Trastuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of the Trastuzumab antibody; The Pertuzumab variant has at least the same binding capacity as the Pertuzumab antibody, and the amino acid sequence of the Pertuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Pertuzumab antibody. Preferably, the Trastuzumab variant has the same HCDR and LCDR regions as the Trastuzumab antibody, and the amino acid sequence of the Trastuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Trastuzumab antibody. The Pertuzumab variant has the same HCDR and LCDR regions as the Pertuzumab antibody, and the amino acid sequence of the Pertuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Pertuzumab antibody. (2) The anti-FRα antibody is huMov19, a huMov19 variant, Farletuzumab, or a Farletuzumab variant; the huMov19 variant has at least the same binding ability as the huMov19 antibody, and the amino acid sequence of the huMov19 variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the huMov19 antibody; The Farletuzumab variant has at least the same binding capacity as the Farletuzumab antibody, and the amino acid sequence of the Farletuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Farletuzumab antibody. Preferably, the huMov19 variant has the same HCDR and LCDR regions as the huMov19 antibody, and the amino acid sequence of the huMov19 variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the huMov19 antibody. The Farletuzumab variant has the same HCDR and LCDR regions as the Farletuzumab antibody, and the amino acid sequence of the Farletuzumab variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the Farletuzumab antibody. (3) D1 is selected from any of the following schemes: Option 1: D1 is (preferred) ); R 9-1 and R 9-2 H, D, and C are independent of each other. 3-5 cycloalkyl or cyclopropylmethyl; or, R 9-1 and R 9-2 Together with the C atoms they are bonded to, they form C 3-5 cycloalkyl; the C 3-5 Cycloalkyl is preferably cyclopropyl. Option 2: D1 is (preferred) ); R 9-4 and R 9-5 C independently 1-6 Alkyl, halogen, or hydroxyl; preferably methyl, chlorine, or hydroxyl; for example, D1 is... Option 3: D1 is Option 4: D1 is R 9-3 For H or C 1-6 Alkyl group, preferably H or ethyl; Option 4: D1 is (3)-L 1a -Independently for-C 1-6 Alkylene -C(O)-, -C 1-6 Alkylene-C(O)-NH-C 1-6 Alkylene-(PEG) Z -C(O)-、-phenyl-C 1-6 alkylene-C(O)-, -phenyl-C 1-6 Alkylene-C(O)-NH-C 1-6 alkylene-C(O)- or -C 1-6 Alkylene-C(O)-NH-C 1-6 alkylene-C(O)-; the -L 1a -Through the carbonyl end and -L 1b -or-L 1c - Interconnected; Preferably, -L 1a -Independently for-C 1-6 Alkylene -C(O)-; or, -L 1a -Independently -phenyl-C 1-6 Alkylene-C(O)-NH-C 1-6 Alkylene-C(O)-; (4)-L 1b - can be -Gly-Gly-Phe-Gly-, -Ala-Ala-Ala, -Val-Cit-, -Val-Ala-, -Gly-Glu-Val-Ala-Gly-, -Gly-Glu-Val-Ala-, or -Glu-Val-Ala-. Following the reading order from left to right, the leftmost end is the NH terminator. The NH terminator is adjacent to the L terminator. 1a connect; (5)L 1c Independently for connection keys, (preferred) ), L 1c Connect to D1 via terminal d), and to -L via terminal e). 1a -or-L 1b -connect; (6) M' is The connector is connected to Ab via end c); (7) m is any value from 1 to 8; for b) Connect the end to M; or, for x is 1 or 2, and end b) is connected to M. Preferably, for b) Terminal is connected to M; R1 is a halogen, -OP(O)(OH)2, or... (8)B, the 8-12 fused cyclic group is a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring, which is connected to the X1 through the cyclic C1; in the 5-6 fused heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 5-6 fused heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; (9) B is an 8-12 membered cyclic group or surrounded by 1, 2 or 3 R groups. 8-3 The substituted 8-12 fused cyclic group; the 8-12 fused cyclic group is a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 6-membered heteroaromatic ring or a benzene ring, and the cyclic C2 is a 6-membered heteroene ring, which is connected to the X1 through the cyclic C1; in the 6-membered heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 6-membered heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; (10)ST1 is A1, A2, and A3 are each independently CR7; R4 is C. 1-6 Alkyl; R5 is C 1-6 Alkyl or C 1- 6-alkoxy group; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH or -NHC(O)NHR. b The substituents can be substituted at any position; R7 is H, F, or Cl; R b It is cyclopropyl or cyclobutyl; Preferably, ST1 is A1, A2, and A3 are each independently CH; R4 is n-propyl; R5 is C. 1-6 Alkyl or C 1- 6-alkoxy group; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH or -NHC(O)NHR. b The substituents can be substituted at any position; R b It is cyclobutyl.

4. The antibody-drug conjugate as shown in Formula I according to claim 1, characterized in that, It meets one or more of the following conditions: (1) L1 is any of the following schemes: Option 1: L1 is -M'-L 1a -L 1b -L 1c -; -L 1a -for-C 1-6 Alkylene-C(O)-; -L 1b - can be -Gly-Gly-Phe-Gly-, -Ala-Ala-Ala, -Val-Cit-, -Val-Ala-, -Gly-Glu-Val-Ala-Gly-, -Gly-Glu-Val-Ala-, or -Glu-Val-Ala-. Following the reading order from left to right, the leftmost end is the NH terminator. The NH terminator is adjacent to the L terminator. 1a connect; -L 1c -For connection key, (preferred) M' is the connector; Option 2: L1 is -M'-L 1a -L 1c -;-L 1a - is -phenyl-C 1-6 Alkylene-C(O)-NH-C 1-6 alkylene-C(O)-; the -L 1a -Through the carbonyl end and -L 1c - Interconnected; -L 1c -for (preferred) M' is the connector; (2)-XBX 1 -X 2 -Choose from any of the following options: Option 1: X is the connection key. B is a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8- 2-substituted 5-6-membered heteroaryl groups or substituted with 1, 2 or 3 R groups 8-3 Substituted 8-12 fused cyclic groups; X1 is -NHC(O)-; X2 is the linker, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene; Preferably, X is a linking bond, B is an 8-12 membered cyclic group; X1 is -NHC(O)-; X2 is C 3-6 Cycloalkylene; Alternatively, X is a linking bond, B is an 8-12 membered cyclic group; X1 is -NHC(O)-; X2 is a linking bond; Option 2: X is B is a phenylene group, a 5-6 membered heteroaryl group, an 8-12 membered cyclic group, surrounded by one, two, or three R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8- 2-substituted 5-6-membered heteroaryl groups or substituted with 1, 2 or 3 R groups 8-3 Substituted 8-12 fused cyclic groups; X1 is the connection key; X2 is a linker or -C(R3)2(CH2) 0-3 -; Option 3: X is B is the connection key; X1 is either -O- or -NH-; X2 is -C(R3)2(CH2) 0-3 - 5. The antibody-drug conjugate as shown in Formula I according to claim 1, characterized in that, It meets one or more of the following conditions: (1) The antibody mentioned is huMov19, Trastuzumab, Pertuzumab, Farletuzumab, huMov19-AAG, huMov19-K149.5C, huMov19-AAG-K149.5C, Farletuzumab-AAG, Farletuzumab-AAG-K149.5C, Farletuzumab-LALA, huMov19-LALA or Farletuzumab-K149.5C; The amino acid sequence of the light chain of huMov19-AAG is shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain of huMov19-AAG is shown in SEQ ID NO:

10. The amino acid sequence of the light chain of Farletuzumab-AAG is shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain of Farletuzumab-AAG is shown in SEQ ID NO:

12. The amino acid sequence of the light chain of huMov19-K149.5C is shown in SEQ ID NO:13, and the amino acid sequence of the heavy chain of huMov19-K149.5C is shown in SEQ ID NO:

14. The amino acid sequence of the light chain of huMov19-AAG-K149.5C is shown in SEQ ID NO:15, and the amino acid sequence of the heavy chain of huMov19-AAG-K149.5C is shown in SEQ ID NO:

16. The amino acid sequence of the light chain of Farletuzumab-AAG-K149.5C is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain of Farletuzumab-AAG-K149.5C is shown in SEQ ID NO:

18. The amino acid sequence of the light chain of Farletuzumab-LALA is shown in SEQ ID NO:19, and the amino acid sequence of the heavy chain of Farletuzumab-LALA is shown in SEQ ID NO:

20. (2) L1 is L1 is connected to Ab via terminal c); (3) for b) Connect the end to M; (4) R2 is H, Preferably, X is a connecting key. X is connected to B via end a); (5) -X1-X2- is a connecting bond, -CH2-, -OC(CH3)2-CH2-, -NHC(O)-, (6) B is the connector key. The B is unsubstituted, or selectively substituted by 1 to 3 elements selected from halogens, C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 The alkoxy group is substituted at any position; B is connected to X1 via the d) end; preferably, B is unsubstituted; (7) R4 is n-propyl, n-butyl, or (8) R5 is n-propyl, (9) R7 is H, F or Cl; (10) R6 is H, carboxyl, or benzyl; (9) R4' is ethyl or n-butyl; Preferably, ST1 is More preferably, -XB-X1-X2-ST1 is any of the structures in Table 1: Table 1:

6. The antibody-drug conjugate as shown in Formula I according to claim 1, characterized in that, The antibody-drug conjugate shown in Formula I is an antibody-drug conjugate shown in Formula I-2, I-3, I-4, I-5 or I-6: Among them, R 9-1 and R 9-2 The definitions of are as described in claim 3; the definitions of Ab, R1, X, X1, X2, B, ST1, M, m, and n are as described in any one of claims 1-5.

7. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate is any one of the following antibody-drug conjugates: Preferably, the antibody-drug conjugate is any of the compounds shown in Table A or Table B: Table A: Table B:

8. A compound as shown in Formula II or a pharmaceutically acceptable salt thereof, in, A is a benzene ring or a 5-6 membered heteroaromatic ring; MX is the connector front body; X is the connection key. X1 is a linker bond, -NHC(O)-, -O-, or -NH-; X2 is a linker, -C(O)-, -C(R3)2(CH2) 0-3 -or C 3-6 Cycloalkylene; B is the connection key, C 6-10 arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group; the C 6-10 The arylene, 5-6 membered heteroarylene, or 8-12 membered cyclic group is unsubstituted, or selectively replaced by 1-3 groups selected from halogens, oxo groups, C... 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 The alkoxy group can be substituted at any position; ST1 is U is -C(O)- or a connection key; V is either NH or S; Y is either O or NH; R1 is a halogen, -OP(O)(OH)2, or a pyranose group; R2 represents H and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or amino C 1-6 alkyl; R3 is H or C 1-6 alkyl; R4 is C 1-6 Alkyl, C 1-6 Alkoxy or C 1-3 Alkoxy C 1-4 alkyl; R4' is C 1-6 Alkyl, hydroxyl C 1-6 Alkyl or C 1-3 Alkoxy C 1-4 alkyl; R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OR. a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -NR a C(O)OR b -NR a C(O)NR a R b or -C(O)OR b The substituents can be substituted at any position; R6 represents H, halogen, carboxyl, or C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl; A1, A2, and A3 are each independently CR7 or N; R7 is H or halogen; Each R a and R b Independently, they are hydrogen and C respectively. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl, 5-10 heteroaryl, C 3-10 cycloalkyl C 1-6 Alkyl, 3-10 membered heterocyclic alkyl C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-10 heteroaryl C 1-6 alkyl; x is 0, 1, or 2; t can be 0, 1, 2, 3, 4, or 5; And the compound shown in Formula II satisfies one of the following conditions: (1) X1 is -NHC(O)-; X2 is C 3-6 Cycloalkylene; A is a 5-6 membered heteroaryl ring; (2) X1 is -NHC(O)-; X2 is C 3-6 Cycloalkylene; A is a benzene ring; x is 1 or 2; (3) X1 is a linking bond, -NHC(O)-, -O-, or -NH-; X2 is a linking bond, -C(O)-, or -(CH2). 0-2 C(R3)2-; The 5-6 membered heteroaryl ring, 5-6 membered hypoaryl, 5-10 membered heteroaryl, 5-10 membered heteroaryl C 1-6 Alkyl, 3-10 membered heterocyclic alkyl and 3-10 membered heterocyclic alkyl C 1-6 The heteroatoms in the alkyl group are independently one, two, or three of N, O, and S, and the number of said heteroatoms is independently one, two, or three.

9. The compound of formula II as claimed in claim 8, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) A is any of the following schemes: Option 1: A is a benzene ring, pyridine ring, pyrimidine ring, pyridazine ring, or oxazole ring, preferably, for b) Connect the end to MX; Option 2: A is a benzene ring, preferably, for x is 1 or 2, and end b) is connected to MX; more preferably, for R1 is a halogen, -OP(O)(OH)2, or b) Connect to MX; X1 is the connection key, -NHC(O)-, -O-, or -NH-; X2 is the connection key, -C(O)-, or -C(R3)2(CH2). 0-3 -or C 3-6 Cycloalkylene; Option 3: A is pyridinyl, pyrimidinyl, pyridazinyl, or oxazolyl, preferably. for When x is 0, terminal b) is connected to MX; more preferably, for b) Connect to MX; X1 is the connection key, -NHC(O)-, -O-, or -NH-; X2 is the connection key, -C(O)-, or -C(R3)2(CH2). 0-3 -or C 3- 6-cycloalkylene compounds; Option 4: A is a benzene ring, preferably, for When x is 0, more preferably... for A is connected to MX via terminal b); X1 is a connecting bond, -NHC(O)-, -O-, or -NH-; X2 is a connecting bond, -C(O)-, or -(CH2). 0-2 C(R3)2-; (2) MX is -CH2Br or R9 and R 9-1 Independently, H, Br, Preferably, MX is -CH2Br or (3) R2 is H or halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl or amino C 1-4 alkyl; (4) X is the connection key, X is connected to B via end a); t is 0, 1, 2 or 3; (5) X1 and X2 are any of the following schemes: Option 1: X1 is -NHC(O)-; X2 is a linker bond or C 3-6 Cycloalkylene; Option 2: X1 is the connection key; X2 is the connection key; Option 3: X1 is a connector or -O-; X2 is -C(R3)2(CH2) 0-3 -; R3 is H or methyl; (6) B is a linking bond, phenylene, 5-6 membered heteroaryl group, 8-12 membered fused cyclic group, surrounded by 1, 2 or 3 R groups. 8-1 Substituted phenylene, with 1, 2 or 3 R 8-2 The substituted 5-6 nucleotide heteroaryl group may be replaced by one, two, or three R groups. 8-3 Substituted 8-12 fused cyclic groups; In B, the 5-6 membered heteroaryl group and the group with 1, 2, or 3 R groups 8-2 In the substituted 5-6-membered heteroaryl group, the heteroatom is independently selected from one or more of N, O and S, and the number of heteroatoms is independently 1, 2 or 3; the heteroatom is preferably N, and the number of heteroatoms is preferably 1 or 2; In B, the 8-12 fused cycloaliphatic group and the group surrounded by 1, 2, or 3 R groups 8-3 The 8-12 fused cyclic group in the substituted 8-12 cyclic group is independently a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring; in the 5-6 fused heteroaromatic ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3; in the 5-6 fused heteroene ring, the heteroatom is selected from one or more of N, O, and S, and the number of the heteroatom is 1, 2, or 3. R 8-1 R 8-2 and R 8-3 Independently halogen, oxo group, C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1-3 Alkyl or halogenated C 1-3 Alkoxy; Preferably, in B, the 8-12 fused cyclic group is a cyclic C1 and cyclic C2, wherein the cyclic C1 is a 5-6 fused heteroaromatic ring or a benzene ring, and the cyclic C2 is a 5-6 fused heteroene ring or a 5-6 fused heteroaromatic ring, which is connected to X1 through the cyclic C1; in the 5-6 fused heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 5-6 fused heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2. More preferably, B is an 8-12 membered cyclic group or is surrounded by one, two or three R groups. 8-3 The substituted 8-12 fused cyclic group; the 8-12 fused cyclic group is a cyclic C1 and a cyclic C2, wherein the cyclic C1 is a 6-membered heteroaromatic ring or a benzene ring, and the cyclic C2 is a 6-membered heteroene ring, which is connected to the X1 through the cyclic C1; in the 6-membered heteroaromatic ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; in the 6-membered heteroene ring, the heteroatom is N, and the number of heteroatoms is 1 or 2; (7) R4 is C 1-6 Alkyl; or R4 is C 1-3 Alkoxy C 1-4 alkyl; (8) R5 is C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl or C 1-6 The alkoxy group is unsubstituted, or selectively replaced by one selected from -OH, -OC(O)NR. a R b -NR a C(O)OR b or -NR a C(O)NR a R b The substituents can be substituted at any position; R a For H; R b For H, C 1-6 Alkyl or C 3-6 cycloalkyl, such as C 1-6 Alkyl, C 1-6 Alkoxy, -C 1-6 alkylene -OH or -OC 1-6 Alkylene-NHC(O)NHR b ; (9)ST1 is (10) R6 is H, carboxyl, or phenyl C. 1-6 alkyl; (11) for (12) Y is O; (13) V is NH; (14) R4' is C 1-6 alkyl; Preferably, the A ring and R 1 The definitions of X, B, X1, X2 and ST1 may also be as described in claims 3, 4 or 5.

10. The compound of formula II as claimed in claim 8, or a pharmaceutically acceptable salt thereof, characterized in that, The compound shown in Formula II is any of the following compounds:

11. A pharmaceutical composition comprising an antibody-drug conjugate as described in any one of claims 1-7 and a pharmaceutically acceptable excipient.

12. The use of an antibody-drug conjugate as described in any one of claims 1-7 or a pharmaceutical composition as described in claim 11 in the preparation of a drug for treating and / or alleviating tumors; Preferably, the tumor meets one or two of the following conditions: (1) The tumor is a HER2 or FRα expressing tumor; (2) The tumor is one or more of the following: lung cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, brain cancer (including malignant glioma and medulloblastoma), ovarian cancer, endometrial cancer, colon cancer, small bowel cancer, rectal cancer, esophageal cancer, gallbladder cancer, bile duct cancer, and urothelial carcinoma.

13. The use of an antibody-drug conjugate as described in any one of claims 1-7 or a pharmaceutical composition as described in claim 11 in a medicament for treating, alleviating, and / or preventing TLR7 / 8-mediated diseases; Preferably, the TLR7 / 8-mediated related diseases refer to tumors. More preferably, the tumor satisfies one or two of the following conditions: (1) The tumor is a HER2 or FRα expressing tumor; (2) The tumor is one or more of the following: lung cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, brain cancer (including malignant glioma and medulloblastoma), ovarian cancer, endometrial cancer, colon cancer, small bowel cancer, rectal cancer, esophageal cancer, gallbladder cancer, bile duct cancer, and urothelial carcinoma.

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