Antibody-drug conjugate containing quaternary ammonium salt and medical use thereof

Abstract

The present application relates to an antibody-drug conjugate containing a quaternary ammonium salt, further relates to a pharmaceutical composition comprising the antibody-drug conjugate, further relates to an intermediate for preparing the antibody-drug conjugate, and further relates to a use of the antibody-drug conjugate for treatment and / or prevention of a disease or condition.

Description

Antibody-drug conjugate containing quaternary ammonium salt and its medical use

[0001] Cross-references

[0002] This application is based on the application with CN application number 202410145541.7 and application date February 1, 2024, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present application belongs to the field of pharmaceutical chemistry, and specifically relates to an antibody-drug conjugate containing a quaternary ammonium salt, a pharmaceutical composition comprising the antibody-drug conjugate, an intermediate for preparing the antibody-drug conjugate, and the use of the antibody-drug conjugate for treating and / or preventing diseases or conditions. Background Art

[0004] Immunotherapy has revolutionized the field of cancer treatment. Immunotherapy drugs are included in the guidelines for different types of cancer and have been tried in both adjuvant and neoadjuvant treatments. Current research focuses on identifying targets that may trigger or enhance anti-tumor immune responses. The stimulator of interferon genes (STING) receptor is an adaptor protein located in the endoplasmic reticulum. It exists as a dimer with its C-terminus located in the cytoplasm. It participates in the immune response through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Its activation can effectively induce the intracellular release of type I interferon, thereby exerting an immune effect to effectively inhibit tumor cells (Nature, 2008, 455, 674-678). STING agonists are one of the current hot spots in anti-tumor research, and many drugs have entered clinical research. In 2018, Professor Ramanjulu's team at the world-renowned pharmaceutical company GlaxoSmithKline discovered a novel immune agonist, diABZI, which exhibits improved stability, tissue penetrance, and efficacy compared to traditional cyclic dinucleotide stimulators (Nature. 2018 Dec;564(7736):439-443). Although STING agonists have been shown to have promising anti-tumor and antiviral effects, their use in human clinical trials is currently limited to intratumoral injection due to the potential for immunotoxicity associated with systemic administration (especially high-dose administration).

[0005] Traditional antibody-drug conjugates (ADCs) organically combine monoclonal antibodies and cytotoxins, combining the advantages of both antibodies and cytotoxic drugs, with the characteristics of strong targeting, high cytotoxicity, low toxic side effects, and long degradation half-life. ADCs structurally consist of three components: an antibody, a small molecule cytotoxin, and a linker. The role of the antibody is to achieve targeting, the role of the cytotoxin is to kill target cells, and the role of the linker is to achieve an organic combination of the antibody and cytotoxin structures, forming an organic whole. Studies have shown that compared with small molecule drugs, the targeting efficiency of ADCs is increased by more than 100 times (Clinical Cancer Research, 2015, 21(22): 5131-5138). Therefore, as a platform technology, ADCs have great advantages in targeted therapy. The FDA has approved 15 ADC drugs for marketing, including Mylotarg, Adcetris, Kadcyla, Besponsa, Lumoxiti, Polivy, Padcev, and Enhertu. There are more than 100 ADCs in clinical research, showing great application potential in multiple fields such as tumors, infections, and immunity.

[0006] With the rapid development of ADC research, a variety of new drug conjugate forms continue to emerge, and immunostimulating antibody conjugate (ISAC) is a new drug conjugate form (Nat Cancer. 2021 Jan; 2 (1): 18-33). The structure of ISAC consists of antibodies, linkers and small molecule immune agonists (innate immune agents or regulators). ISAC uses the targeting of antibodies to specifically deliver small molecule immune agonists to the tumor site, and uses antigen-mediated endocytosis to enter the cell, cleaving and releasing small molecule immune agonists under the action of tissue proteases to exert drug efficacy. Compared with small molecule immune agonists, ISAC can target tumor cells or immune cells around tumor cells to achieve local immune effects and kill tumor cells. At the same time, it avoids the systemic immune effects caused by small molecule immune agonists and reduces the risk of immune factor storms.

[0007] In 2022, Wu et al. published a new ISAC: αEGFR-172 (Proc Natl Acad Sci US A. 2022 Dec 6; 119(49): e2214278119), which uses a monoclonal antibody targeting epidermal growth factor receptor (EGFR) as the antibody end and a CDN-type STING agonist: IMSA172 as the payload, both of which are coupled with a cleavable linker. In vivo studies have shown that αEGFR-172 can effectively activate the cGAS-STING pathway in cells overexpressing EGFR and effectively inhibit tumor growth in a mouse B16F10 tumor model. Although ISAC perfectly combines the precision of antibody targeting tumors with the killing potential of immune small molecules, it has important clinical value and application prospects, but it still faces challenges in terms of safety. In March 2023, Mersana's ISAC candidate drug XMT-2056 experienced treatment-related grade 5 (fatal) serious adverse events in a Phase I clinical trial, and the clinical study was stopped. The reason for the failure of XMT-2056 is that the ester linker it used is not stable enough in the circulatory system. Therefore, it is urgent to develop new connection technologies to promote the development of ISAC. Summary of the Invention

[0008] The inventors discovered that coupling antibodies, linkers and small molecule immunostimulants through quaternary ammonium salts can effectively enhance the stability of ISACs in the circulatory system, improve the solubility of effector molecules, and accurately deliver immunostimulants.

[0009] Therefore, the first aspect of the present application relates to a compound represented by formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates,

[0010] in:

[0011] A represents a biological macromolecule or its fragment,

[0012] B 00 Represents a linker, used to connect A and -NR1R2- group,

[0013] X represents a negatively charged anion.

[0014] R1 and R2 are each independently C 1-8 Alkyl, C 1-8 Alkylcycloalkyl, C 4-8 Cycloalkyl, 4-8 membered heterocycloalkyl, C 1-8 Alkyl sugar, C 5-14 aryl, 5-14 membered heteroaryl, or

[0015] R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocyclyl or heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 alkoxy, fluorine, chlorine, bromine, iodine, hydroxy, cyano, amino or nitro,

[0016] M is -(CH2) o -、-(CH2) o -CH=CH-(CH2) p -、-O-(CH2) o -、-NH-(CH2) o -、-O-(CH2) o -CH=CH-(CH2) p -O-, -NH-(CH2) o -CH=CH-(CH2) p -NH- or -CH2-CHOH-CHOH-CH2-, wherein o and p are each independently an integer from 0 to 9,

[0017] f is 0 or 1,

[0018] R8 is -(CH2) a -、-(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-, wherein a is an integer of 1 to 8,

[0019] R9 is C 1-8 Alkyl, C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino,

[0020] R 10 With R 11 Each independently is C 1-4 Alkyl, -CONR a R b 、-CH2NR a R b 、-NR a R b 、-NR a C(=O)-R b 、-CH2NR a C(=O)-R b, -C(=O)OCH3 or -O-(CH2)e-OH, wherein R a and R b Each independently is H, hydroxyl or C 1-4 Alkyl, e is an integer from 1 to 9,

[0021] R 12 With R 13 Each independently represents -(CH2) g -CH3, -O-(CH2) g -OH, -O-(CH2) g -CH3, -CONH2, Where g is a number from 1 to 9.

[0022] d is a number between 1 and 20.

[0023] The second aspect of the present application relates to an intermediate compound for preparing the compound represented by the above formula II. Specifically, the intermediate compound is a compound represented by formula I, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates.

[0024] in:

[0025] B represents a linker, which is used to couple the -NR1R2- group to a biomacromolecule or a fragment thereof.

[0026] X represents a negatively charged anion.

[0027] R1 and R2 are each independently C 1-8 Alkyl, C 1-8 Alkylcycloalkyl, C 4-8 Cycloalkyl, 4-8 membered heterocycloalkyl, C 1-8 Alkyl sugar, C 5-14 aryl, 5-14 membered heteroaryl, or

[0028] R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocyclyl or heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 alkoxy, fluorine, chlorine, bromine, iodine, hydroxy, cyano, amino or nitro,

[0029] M is -(CH2) o -、-(CH2) o -CH=CH-(CH2) p-、-O-(CH2) o -、-NH-(CH2) o -、-O-(CH2) o -CH=CH-(CH2) p -O-, -NH-(CH2) o -CH=CH-(CH2) p -NH- or -CH2-CHOH-CHOH-CH2-, wherein o and p are each independently an integer from 0 to 9,

[0030] f is 0 or 1,

[0031] R8 is -(CH2) a -、-(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-, wherein a is an integer of 1 to 8,

[0032] R9 is C 1-8 Alkyl, C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino,

[0033] R 10 With R 11 Each independently is C 1-4 Alkyl, -CONR a R b 、-CH2NR a R b 、-NR a R b 、-NR a C(=O)-R b 、-CH2NR a C(=O)-R b , -C(=O)OCH3 or -O-(CH2)e-OH, wherein R a and R b Each independently is H, hydroxyl or C 1-4 Alkyl, e is an integer from 1 to 9,

[0034] R 12 With R 13 Each independently represents -(CH2) g -CH3, -O-(CH2) g -OH, -O-(CH2) g -CH3, -CONH2, Where g is a number from 1 to 9.

[0035] The third aspect of the present application relates to the use of the compound represented by the above formula I, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates in the preparation of antibody-drug conjugates (e.g., immunostimulatory antibody-drug conjugates).

[0036] The fourth aspect of the present application relates to a pharmaceutical composition or vaccine, which comprises the compound represented by the above-mentioned formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0037] The fifth aspect of the present application relates to the use of the compound represented by the above-mentioned formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates in the preparation of a medicament for treating and / or preventing a disease or condition or alleviating the severity of the disease or condition, wherein the disease or condition is selected from tumors, pathogen infections, diseases caused by pathogen infections, hematological diseases, metabolic diseases, inflammation, allergic diseases, autoimmune diseases, precancerous syndromes, tumor metastasis, and cardiovascular diseases.

[0038] The sixth aspect of the present application relates to a method for treating and / or preventing a disease or condition or alleviating the severity of the disease or condition, which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates, wherein the disease or condition is selected from tumors, pathogen infections, diseases caused by pathogen infections, hematological diseases, metabolic diseases, inflammation, allergic diseases, autoimmune diseases, precancerous syndromes, tumor metastasis, and cardiovascular diseases.

[0039] The seventh aspect of the present application relates to a compound represented by formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates, which are used to treat and / or prevent a disease or condition or alleviate the severity of the disease or condition, wherein the disease or condition is selected from tumors, pathogen infections, diseases caused by pathogen infections, hematological diseases, metabolic diseases, inflammation, allergic diseases, autoimmune diseases, precancerous syndromes, tumor metastasis, and cardiovascular diseases.

[0040] Detailed Description of the Invention

[0041] Immunostimulatory Antibody-Drug Conjugates

[0042] The first aspect of the present application relates to an immunostimulatory antibody-drug conjugate, which is a compound represented by Formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates.

[0043] in:

[0044] A represents a biological macromolecule or its fragment,

[0045] B 00 Represents a linker, used to connect A and -NR1R2- group,

[0046] X represents a negatively charged anion.

[0047] R1 and R2 are each independently C 1-8 Alkyl, C 1-8 Alkylcycloalkyl, C 4-8 Cycloalkyl, 4-8 membered heterocycloalkyl, C 1-8 Alkyl sugar, C 5-14 aryl, 5-14 membered heteroaryl, or

[0048] R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocyclyl or heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 alkoxy, fluorine, chlorine, bromine, iodine, hydroxy, cyano, amino or nitro,

[0049] M is -(CH2) o -、-(CH2) o -CH=CH-(CH2) p -、-O-(CH2) o -、-NH-(CH2) o -、-O-(CH2) o -CH=CH-(CH2) p -O-, -NH-(CH2) o -CH=CH-(CH2) p -NH- or -CH2-CHOH-CHOH-CH2-, wherein o and p are each independently an integer from 0 to 9,

[0050] f is 0 or 1,

[0051] R8 is -(CH2) a -、-(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-, wherein a is an integer of 1 to 8,

[0052] R9 is C1-8 Alkyl, C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino,

[0053] R 10 With R 11 Each independently is C 1-4 Alkyl, -CONR a R b 、-CH2NR a R b 、-NR a R b 、-NR a C(=O)-R b 、-CH2NR a C(=O)-R b , -C(=O)OCH3 or -O-(CH2)e-OH, wherein R a and R b Each independently is H, hydroxyl or C 1-4 Alkyl, e is an integer from 1 to 9,

[0054] R 12 With R 13 Each independently represents -(CH2) g -CH3, -O-(CH2) g -OH, -O-(CH2) g -CH3, -CONH2, Where g is a number from 1 to 9.

[0055] d is a number between 1 and 20.

[0056] In some embodiments, R 10 Located at the ortho, meta or para position of R8; and / or R 11 Located at the ortho, meta or para position of R9.

[0057] In some embodiments, R 10 Located adjacent to R8, and R 11 Located in the ortho position of R9.

[0058] In some embodiments, the compound of Formula II has a structure as shown in Formula II-1,

[0059] Among them, A, X, R1, R2, f, R8, R9, R 10 、R 11 、R 12 、R 13, d are as defined in any embodiment of this application,

[0060] -B1-VLW- represents a linker for connecting A and -NR1R2- groups, wherein

[0061] B1 is selected from as well as Where k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0062] wherein each i is independently an integer between 0 and 12, each j is independently an integer between 0 and 12, or V does not exist;

[0063] L is -(CH2CH2O) k -(CH2) l -, -CHZ-, -(CH2) m -,or Where Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, m is an integer between 0 and 30, or L is absent;

[0064] wherein R3, R4 and R6 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) c -NH2, -(CH2) c -NHC(NH)NH2, -CHCH(CH3)CH3 or -(CH2) c -NHC(=O)NH2, Where c is a number from 0 to 8; R5 is R 14 、R 15 Each is independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) h -NH2 or -(CH2) h , each h is independently a number from 0 to 8,

[0065] A is coupled to the site ** of the B1 group through an S atom or a N atom in the biomacromolecule or its fragment.

[0066] In some embodiments, in the compound of formula II, X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - , SO4 2- , PO 4, 3- ,HPO4 2- or H2PO4 - .

[0067] In some embodiments, in the compound of formula II, X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - .

[0068] In some embodiments, in the compound of formula II, X is Cl - , HCOO - , CH3COO - ,CF3COO - .

[0069] In some embodiments, in the compound of formula II, X is Cl - , Br - , I - .

[0070] In some embodiments, in the compound of formula II, X is Cl - .

[0071] In some embodiments, in the compound of formula II, R1 and R2 are each independently C 1-8 In some embodiments, in the compound represented by Formula II, R1 and R2 are each independently C 1-6 In some embodiments, in the compound represented by Formula II, R1 and R2 are each independently C 1-4 In some embodiments, in the compound of Formula II, R1 and R2 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, or n-hexyl.

[0072] In some embodiments, in the compound of formula II, R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms, wherein the other heteroatoms are selected from N, O or S atoms, and the heterocycloalkyl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 In some embodiments, in the compound of formula II, R1, R2 and the N atom to which they are attached form a pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl or thiomorpholinyl group. In some embodiments, in the compound of formula II, R1, R2 and the N atom to which they are attached form a pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl or thiomorpholinyl group.

[0073] In some embodiments, in the compound of formula II, R1, R2 and the N atom to which they are attached form a 5-7 membered heteroaryl group, wherein the heteroaryl group contains 1 N atom and 1, 2 or 3 other heteroatoms, wherein the other heteroatoms are selected from N, O or S atoms, and the heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 Alkoxy, fluorine, chlorine, bromine, iodine, hydroxyl, cyano, amino or nitro. In some embodiments, in the compound shown in Formula II, R1, R2 and the nitrogen atom to which they are attached form an imidazolyl, triazolyl, indolyl, tetrazolyl, pyridyl, pteridinyl, pyrimidinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl or thiazinyl.

[0074] In some embodiments, in the compound of formula II, R' is H, C 1-6 Alkyl, C 1-6 In some embodiments, in the compound of formula II, R' is H, C 1-4 Alkyl, C 1-4Alkoxy, fluorine, chlorine, bromine, iodine, hydroxyl, cyano, amino or nitro. In some embodiments, in the compound shown in formula II, R' is H. In some embodiments, in the compound shown in formula II, R' is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl or n-hexyl. In some embodiments, in the compound shown in formula II, R' is methyl, ethyl or n-propyl. In some embodiments, in the compound shown in formula II, R' is methoxy, ethoxy, propoxy or butoxy. In some embodiments, in the compound shown in formula II, R' is fluorine, chlorine, bromine or iodine. In some embodiments, in the compound shown in formula II, R' is fluorine, chlorine or bromine. In some embodiments, in the compound shown in formula II, R' is cyano, amino or nitro.

[0075] In some embodiments, in the compound of formula II, M is -(CH2) o -CH=CH-(CH2) p -, wherein o and p are each independently an integer from 0 to 9. In some embodiments, in the compound of Formula II, o and p are each independently an integer from 1 to 6, for example, 1, 2, 3, 4, 5 or 6.

[0076] In some embodiments, in the compound of Formula II, f is 0. In some embodiments, in the compound of Formula II, f is 1.

[0077] In some embodiments, in the compound of formula II, R8 is -(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-. In some embodiments, in the compound of formula II, R8 is -(CH2) a -O-.

[0078] In some embodiments, in the compound of Formula II, a is an integer from 1 to 6, for example, 1, 2, 3, 4, 5 or 6.

[0079] In some embodiments, in the compound of formula II, R9 is C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 In some embodiments, in the compound represented by formula II, R9 is C 1-8 In some embodiments, in the compound represented by formula II, R9 is C 1-6 In some embodiments, in the compound represented by formula II, R9 is C 1-4In some embodiments, in the compound shown in formula II, R9 is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy or 1,2-dimethylbutoxy. In some embodiments, in the compound shown in formula II, R9 is methoxy, ethoxy, n-propoxy or n-butoxy. In some embodiments, in the compound shown in formula II, R9 is C 1-6 In some embodiments, in the compound represented by formula II, R9 is C 1-4 In some embodiments, in the compound shown in formula II, R9 is methylthio, ethylthio, propylthio or butylthio. In some embodiments, in the compound shown in formula II, R9 is C 1-6 In some embodiments, in the compound represented by formula II, R9 is C 1-4 In some embodiments, in the compound represented by Formula II, R9 is methylamino, ethylamino, propylamino, or butylamino.

[0080] In some embodiments, in the compound of Formula II, R 10 With R 11 Each independently -CONR a R b 、-CH2NR a R b or -CH2NR a C(=O)-R b In some embodiments, in the compound of formula II, R 10 With R 11 Each independently -CONR a R b .

[0081] In some embodiments, in the compound of Formula II, R a and R b Each independently is H or C 1-4 In some embodiments, in the compound represented by Formula II, R a and R b Each is independently H. In some embodiments, in the compound of formula II, R a and R b Each is independently methyl, ethyl, n-propyl or n-butyl.

[0082] In some embodiments, in the compound of Formula II, R 12 With R 13 Each independently In some embodiments, in the compound of Formula II, R12 With R 13 Each independently In some embodiments, in the compound of Formula II, R 12 With R 13 Each independently

[0083] In some embodiments, in the compound of Formula II, d is a number between 1 and 8. In some embodiments, in the compound of Formula II, d is a number between 1 and 6, for example, about 1, about 2, about 3, about 4, about 5, or about 6.

[0084] In some embodiments, in the compound of Formula II, A is an antibody or an antigen-binding fragment thereof, a polypeptide, a protein, or an antigen.

[0085] In some embodiments, in the compound of Formula II, A is an antibody or an antigen-binding fragment thereof, a protein or an antigen.

[0086] In some embodiments, in the compound represented by Formula II, A is an antigen.

[0087] In some embodiments, in the compound represented by Formula II, A is an antibody or antigen-binding fragment thereof, polypeptide or protein that can specifically bind to the target.

[0088] In some embodiments, in the compound represented by Formula II, A is an antibody or antigen-binding fragment or protein that can specifically bind to the target.

[0089] In some embodiments, in the compound represented by Formula II, A is an antibody or an antigen-binding fragment thereof that can specifically bind to a target.

[0090] In some embodiments, in the compound represented by Formula II, A is a protein that can specifically bind to a target.

[0091] In some embodiments, the antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, IgG antibody, mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), bispecific antibody, multispecific antibody, and Probody.

[0092] In some embodiments, the antibody binding fragment is selected from the group consisting of: single-chain antibody, dAb, complementarity determining region fragment, Fv, single-chain Fv (scFv), Fd, Fab, Fab', F(ab')2, and VHH (Nanobody or Nb).

[0093] In some embodiments, the protein is albumin, preferably human serum albumin.

[0094] In some embodiments, in the compound of Formula II, A is a monoclonal antibody or an antigen-binding fragment thereof.

[0095] In some embodiments, in the compound of Formula II, A is a monoclonal antibody or antigen-binding fragment thereof with a thiol or amino group as a coupling site, or a monoclonal antibody or antigen-binding fragment thereof that is site-directed mutated or modified with a thiol or amino group as a coupling site.

[0096] In some embodiments, in the compound of Formula II, A is selected from: anti-HER2 humanized monoclonal antibody mil40, trastuzumab (HERCEPTIN), pertuzumab (PERJETA), cetuximab (ERBITUX), panitumumab (VECTIBIX), rituximab (RITUXAN), alemtuzumab (CAMPATH), ibritumomab tiuxetan (ZEVALIN), tositumomab (BEXXAR), ofatumumab (ARZERRA), bevacizumab (AVASTIN) , ipilimumab (YERVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi), sacituzumab, rovalvituzumab, antibacterial antibodies, their biosimilars or antigen-binding fragments, and site-directed mutagenesis or modification of the above-mentioned monoclonal antibodies or their antigen-binding fragments.

[0097] In some embodiments, in the compound of Formula II, A is trastuzumab or an antigen-binding fragment thereof.

[0098] In some embodiments, in the compound of formula II, B1 is In some embodiments, in the compound of formula II, B1 is In some embodiments, in the compound of formula II, B1 is

[0099] In some embodiments, in the compound of formula II, k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, in the compound of formula II, k1 is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, in the compound of formula II, k1 is 2, 3, 4, 5 or 6.

[0100] In some embodiments, in the compound of formula II, V is wherein i and j are as defined in any embodiment of the present application.

[0101] In some embodiments, in the compound of formula II, i and j are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, in the compound of formula II, i and j are each independently 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, in the compound of formula II, i and j are each independently 1, 2, 3, 4, 5 or 6.

[0102] In some embodiments, in the compound of formula II, L is -(CH2CH2O) k -(CH2) l -, -CHZ-, or -(CH2) m -, where Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, k, l, m, and n are as defined in any embodiment of the present application. In some embodiments, in the compound of Formula II, L is absent.

[0103] In some embodiments, in the compound of Formula II, each k is independently an integer between 0 and 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0104] In some embodiments, in the compound of formula II, each l is independently an integer between 0 and 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0105] In some embodiments, in the compound of Formula II, n is an integer between 0 and 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0106] In some embodiments, in the compound of formula II, m is an integer between 0 and 20. In some embodiments, in the compound of formula II, m is an integer between 0 and 16, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

[0107] In some embodiments, in the compound of Formula II, L is absent.

[0108] In some embodiments, in the compound of formula II, W is wherein R3, R4 and R5 are as defined in any embodiment of the present application.

[0109] In some embodiments, in the compound of formula II, R3 and R4 are each independently methyl, ethyl, n-propyl, isopropyl or -(CH2) c-NHC(=O)NH2, wherein c is as defined in any embodiment of the present application.

[0110] In some embodiments, in the compound of formula II, R5 is where R 14 、R 15 The definition of is as described in any embodiment of this application.

[0111] In some embodiments, in the compound of formula II, R3 and R4 are each independently methyl, isopropyl or -(CH2) c -NHC(=O)NH2.

[0112] In some embodiments, in the compound of Formula II, c is an integer between 0 and 6, for example, 0, 1, 2, 3, 4, 5, or 6.

[0113] In some embodiments, in the compound of Formula II, R 14 、R 15 Each is independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl. In some embodiments, in the compound of formula II, R 14 、R 15 Each is independently H, methyl, ethyl or n-propyl. In some embodiments, in the compound of formula II, R 14 、R 15 Each independently represents H or methyl. In some embodiments, in the compound represented by Formula II, R 14 、R 15 Each is independently H.

[0114] In some embodiments, in the compound of formula II, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined in any embodiment of the present application.

[0115] In some embodiments, in the compound of formula II, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined in any embodiment of the present application.

[0116] In some embodiments, the compound of formula II has the structure shown in formula II-2 or II-3,

[0117] wherein A, X, V, L, W, and k1 are defined as described in any embodiment of the present application, and A is coupled to site # via an S atom in the biomacromolecule or a fragment thereof, or is coupled to site ## via an N atom in the biomacromolecule or a fragment thereof.

[0118] In some embodiments, the compound of Formula II is selected from:

[0119] in, The definitions of A, A and d are as described in any embodiment of the present application.

[0120] In some embodiments, in the compound of Formula II, the S atom connected to A is derived from the sulfur atom in A (e.g., the sulfur atom of a cysteine ​​residue), and the -NH- group connected to A is derived from the amino group in A (e.g., the side chain amino group of a lysine, arginine, asparagine, or glutamine residue).

[0121] Intermediate compounds

[0122] The second aspect of the present application relates to an intermediate compound for preparing an immunostimulatory antibody-drug conjugate, which is a compound of formula I, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates.

[0123] in:

[0124] B represents a linker, which is used to couple the -NR1R2- group to a biomacromolecule or a fragment thereof.

[0125] X represents a negatively charged anion.

[0126] R1 and R2 are each independently C 1-8 Alkyl, C 1-8 Alkylcycloalkyl, C 4-8 Cycloalkyl, 4-8 membered heterocycloalkyl, C 1-8 Alkyl sugar, C 5-14 aryl, 5-14 membered heteroaryl, or

[0127] R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocyclyl or heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 alkoxy, fluorine, chlorine, bromine, iodine, hydroxy, cyano, amino or nitro,

[0128] M is -(CH2)o -、-(CH2) o -CH=CH-(CH2) p -、-O-(CH2) o -、-NH-(CH2) o -、-O-(CH2) o -CH=CH-(CH2) p -O-, -NH-(CH2) o -CH=CH-(CH2) p -NH- or -CH2-CHOH-CHOH-CH2-, wherein o and p are each independently an integer from 0 to 9,

[0129] f is 0 or 1,

[0130] R8 is -(CH2) a -、-(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-, wherein a is an integer of 1 to 8,

[0131] R9 is C 1-8 Alkyl, C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino,

[0132] R 10 With R 11 Each independently is C 1-4 Alkyl, -CONR a R b 、-CH2NR a R b 、-NR a R b 、-NR a C(=O)-R b 、-CH2NR a C(=O)-R b , -C(=O)OCH3 or -O-(CH2)e-OH, wherein R a and R b Each independently is H, hydroxyl or C 1-4 Alkyl, e is an integer from 1 to 9,

[0133] R 12 With R 13 Each independently represents -(CH2) g -CH3, -O-(CH2) g -OH, -O-(CH2) g -CH3, -CONH2, Where g is a number from 1 to 9.

[0134] In some embodiments, R 10 Located at the ortho, meta or para position of R8; and / or R 11 Located at the ortho, meta or para position of R9.

[0135] In some embodiments, R 10 Located adjacent to R8, and R 11 Located in the ortho position of R9.

[0136] In some embodiments, the compound of Formula I has a structure as shown in Formula I-1,

[0137] Among them, X, R1, R2, f, R8, R9, R 10 、R 11 、R 12 、R 13 , d are as defined in any embodiment of this application,

[0138] B0-VLW- represents a linker used to couple the -NR1R2- group to a biomacromolecule or a fragment thereof, wherein

[0139] B0 is selected from or H, wherein r is an integer between 1 and 4;

[0140] V is wherein each i is independently an integer between 0 and 12, each j is independently an integer between 0 and 12, or V does not exist;

[0141] L is -(CH2CH2O) k -(CH2) l -, -CHZ-, -(CH2) m -,or Where Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, m is an integer between 0 and 30, or L is absent;

[0142] W is wherein R3, R4 and R6 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) c -NH2, -(CH2) c -NHC(NH)NH2, -CHCH(CH3)CH3 or -(CH2) c -NHC(=O)NH2, Where c is a number from 0 to 8; R5 is R 14 、R 15 Each is independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) h -NH2 or -(CH2) h , each h is independently a number from 0 to 8.

[0143] In some embodiments, in the compound of formula I, X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - , SO4 2- , PO 4, 3- ,HPO4 2- or H2PO4 - In some embodiments, in the compound of formula I, X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - In some embodiments, in the compound of formula I, X is Cl - , HCOO - , CH3COO - ,CF3COO - In some embodiments, in the compound of formula I, X is Cl - , Br - , I - In some embodiments, in the compound of formula I, X is Cl - .

[0144] In some embodiments, in the compound of formula I, R1 and R2 are each independently C 1-8 In some embodiments, in the compound of formula I, R1 and R2 are each independently C 1-6 In some embodiments, in the compound of formula I, R1 and R2 are each independently C 1-4 In some embodiments, in the compound of Formula I, R1 and R2 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, or n-hexyl.

[0145] In some embodiments, in the compound of formula I, R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms, wherein the other heteroatoms are selected from N, O or S atoms, and the heterocycloalkyl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 In some embodiments, in the compound of formula I, R1, R2 and the N atom to which they are attached form a pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl or thiomorpholinyl group. In some embodiments, in the compound of formula I, R1, R2 and the N atom to which they are attached form a

[0146] In some embodiments, in the compound of formula I, R1, R2 and the N atom to which they are attached form a 5-7 membered heteroaryl group, wherein the heteroaryl group contains 1 N atom and 1, 2 or 3 other heteroatoms, wherein the other heteroatoms are selected from N, O or S atoms, and the heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 In some embodiments, in the compound of formula I, R1, R2 and the nitrogen atom to which they are attached form an imidazolyl, triazolyl, indolyl, tetrazolyl, pyridyl, pteridinyl, pyrimidinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl or thiazinyl.

[0147] In some embodiments, in the compound of formula I, R' is H, C 1-6 Alkyl, C 1-6 In some embodiments, in the compound of formula I, R' is H, C 1-4 Alkyl, C 1-4Alkoxy, fluorine, chlorine, bromine, iodine, hydroxyl, cyano, amino or nitro. In some embodiments, in the compound shown in Formula I, R' is H. In some embodiments, in the compound shown in Formula I, R' is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl or n-hexyl. In some embodiments, in the compound shown in Formula I, R' is methyl, ethyl or n-propyl. In some embodiments, in the compound shown in Formula I, R' is methoxy, ethoxy, propoxy or butoxy. In some embodiments, in the compound shown in Formula I, R' is fluorine, chlorine, bromine or iodine. In some embodiments, in the compound shown in Formula I, R' is fluorine, chlorine or bromine. In some embodiments, in the compound shown in Formula I, R' is cyano, amino or nitro.

[0148] In some embodiments, in the compound of formula I, M is -(CH2) o -CH=CH-(CH2) p -, wherein o and p are each independently an integer from 0 to 9.

[0149] In some embodiments, in the compound of Formula I, o and p are each independently an integer from 1 to 6, for example, 1, 2, 3, 4, 5 or 6.

[0150] In some embodiments, in the compound of Formula I, f is 0. In some embodiments, in the compound of Formula I, f is 1.

[0151] In some embodiments, in the compound of formula I, R8 is -(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-. In some embodiments, in the compound of formula I, R8 is -(CH2) a -O-.

[0152] In some embodiments, in the compound of Formula I, a is an integer from 1 to 6, for example, 1, 2, 3, 4, 5 or 6.

[0153] In some embodiments, in the compound of formula I, R9 is C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 In some embodiments, in the compound of formula I, R9 is C 1-8 In some embodiments, in the compound of formula I, R9 is C 1-6 In some embodiments, in the compound of formula I, R9 is C 1-4In some embodiments, in the compound of formula I, R9 is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy or 1,2-dimethylbutoxy. In some embodiments, in the compound of formula I, R9 is methoxy, ethoxy, n-propoxy or n-butoxy. In some embodiments, in the compound of formula I, R9 is C 1-6 In some embodiments, in the compound of formula I, R9 is C 1-4 In some embodiments, in the compound shown in formula I, R9 is methylthio, ethylthio, propylthio or butylthio. In some embodiments, in the compound shown in formula I, R9 is C 1-6 In some embodiments, in the compound of formula I, R9 is C 1-4 In some embodiments, in the compound of Formula I, R9 is methylamino, ethylamino, propylamino, or butylamino.

[0154] In some embodiments, in the compound of Formula I, R 10 With R 11 Each independently -CONR a R b 、-CH2NR a R b or -CH2NR a C(=O)-R b In some embodiments, in the compound of Formula I, R 10 With R 11 Each independently -CONR a R b .

[0155] In some embodiments, in the compound of Formula I, R a and R b Each independently is H or C 1-4 In some embodiments, in the compound of formula I, R a and R b are each independently H. In some embodiments, in the compound of formula I, R a and R b Each is independently methyl, ethyl, n-propyl or n-butyl.

[0156] In some embodiments, in the compound of Formula I, R 12 With R 13 Each independently In some embodiments, in the compound of Formula I, R 12 With R13 Each independently In some embodiments, in the compound of Formula I, R 12 With R 13 Each independently

[0157] In some embodiments, in the compound of formula I, B0 is In some embodiments, in the compound of formula I, B0 is In some embodiments, in the compound of formula I, B0 is In some embodiments, in the compound of formula I, B0 is In some embodiments, in the compound of formula I, B0 is

[0158] In some embodiments, in the compound of formula I, r is 1, 2, 3 or 4. In some embodiments, in the compound of formula I, r is 1 or 2;

[0159] In some embodiments, in the compound of formula I, V is wherein i and j are as defined in any embodiment of the present application.

[0160] In some embodiments, in the compound of formula I, i and j are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, in the compound of formula I, i and j are each independently 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, in the compound of formula I, i and j are each independently 1, 2, 3, 4, 5 or 6.

[0161] In some embodiments, in the compound of formula I, L is -(CH2CH2O) k -(CH2) l -, -CHZ-, or -(CH2) m -, where Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, k, l, m, and n are as defined in any embodiment of the present application.

[0162] In some embodiments, in the compound of Formula I, each k is independently an integer between 0 and 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0163] In some embodiments, in the compound of formula I, each l is independently an integer between 0 and 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0164] In some embodiments, in the compound of Formula I, n is an integer between 0 and 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0165] In some embodiments, in the compound of Formula I, m is an integer between 0 and 20. In some embodiments, in the compound of Formula I, m is an integer between 0 and 16, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

[0166] In some embodiments, in the compound of Formula I, L is absent.

[0167] In some embodiments, in the compound of formula I, W is wherein R3, R4 and R5 are as defined in any embodiment of the present application.

[0168] In some embodiments, in the compound of formula I, R3 and R4 are each independently methyl, ethyl, n-propyl, isopropyl or -(CH2) c -NHC(=O)NH2, wherein c is as defined in any embodiment of the present application.

[0169] In some embodiments, in the compound of formula I, R5 is where R 14 、R 15 The definition of is as described in any embodiment of this application.

[0170] In some embodiments, in the compound of formula I, R3 and R4 are each independently methyl, isopropyl or -(CH2) c -NHC(=O)NH2.

[0171] In some embodiments, in the compound of Formula I, c is an integer between 0 and 6, for example, 0, 1, 2, 3, 4, 5, or 6.

[0172] In some embodiments, in the compound of Formula I, R 14 、R 15 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl. In some embodiments, in the compound of formula I, R 14 、R 15 Each is independently H, methyl, ethyl or n-propyl. In some embodiments, in the compound of formula I, R14 、R 15 Each independently represents H or methyl. In some embodiments, in the compound of formula I, R 14 、R 15 Each is independently H.

[0173] In some embodiments, in the compound of formula I, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined in any embodiment of the present application.

[0174] In some embodiments, in the compound of formula I, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined in any embodiment of the present application.

[0175] In some embodiments, the compound of Formula I has the structure shown in Formula I-2,

[0176] wherein the definitions of B0, X, V, L, and W are as described in any embodiment of this application.

[0177] In some embodiments, the compound of Formula I is selected from:

[0178] Preparation method

[0179] The third aspect of the present application relates to the use of the intermediate compound in the preparation of an antibody-drug conjugate (eg, an immunostimulatory antibody-drug conjugate).

[0180] The immunostimulatory antibody-drug conjugate described in the present application can be coupled to the biomacromolecule or its fragment A by methods well known in the art.

[0181] In some embodiments, the immunostimulatory antibody-drug conjugate can be prepared illustratively by the following reaction scheme:

[0182] The reaction reagents and reaction conditions are as follows: (1) Using maleimide-polyethylene glycol-carboxylic acid and compound VA as raw materials and anhydrous DMF as solvent, compounds I-11a, I-12a, and I-13a are obtained under the action of EDCI, HOBt, and DIPEA; (2) compounds I-11b, I-12b, and I-13b are obtained under the action of thionyl chloride; (3) compounds I-11, I-12, and I-13 are obtained in anhydrous DMF under the action of DIPEA and TBAI; (4) the final ISAC products II-11, II-12, and II-13 are further coupled with antibodies.

[0183] In some embodiments, the immunostimulatory antibody-drug conjugate can also be prepared illustratively by the following reaction scheme:

[0184] The reaction reagents and reaction conditions are as follows: (1) Using maleimide-polyethylene glycol-carboxylic acid and compound VC as raw materials and anhydrous DMF as solvent, compounds I-21a, I-22a, and I-23a are obtained under the action of EDCI, HOBt, and DIPEA; (2) compounds I-21b, I-22b, and I-23b are obtained under the action of thionyl chloride; (3) compounds I-21, I-22, and I-23 are obtained in anhydrous DMF under the action of DIPEA and TBAI; (4) the final ISAC products II-21, II-22, and II-23 are further coupled with antibodies.

[0185] In some embodiments, the immunostimulatory antibody-drug conjugate can also be prepared illustratively by the following reaction scheme:

[0186] The reaction reagents and reaction conditions are as follows: (1) Using maleimide-polyethylene glycol-propionic acid and compound PAB as raw materials and anhydrous DMF as solvent, compound I-31a is obtained under the action of EDCI, HOBt and DIPEA; (2) compound I-31b is obtained under the action of thionyl chloride; (3) compound I-31 is obtained in anhydrous DMF under the action of DIPEA and TBAI; (4) further coupled with antibody to obtain the final ISAC product II-31.

[0187] In some embodiments, the immunostimulatory antibody-drug conjugate can also be prepared illustratively by the following reaction scheme:

[0188] The reaction reagents and reaction conditions are as follows: (1) using propargyl-octapolyethylene glycol-carboxylic acid and compound VA as raw materials, using anhydrous DMF as solvent, under the action of NHS, DIC and DIPEA to obtain compound I-41a; (2) under the action of thionyl chloride to obtain compound I-41b; (3) in anhydrous DMF, under the action of DIPEA and TBAI to obtain compound I-41; (4) further coupling the compound with the antibody through click chemistry reaction to obtain the final ISAC product II-41.

[0189] Pharmaceutical composition or vaccine

[0190] The fourth aspect of the present application relates to a pharmaceutical composition or vaccine comprising a compound of formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0191] In certain embodiments, in the pharmaceutical composition or vaccine, the compound of Formula II, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates are present in an effective amount, for example, in a therapeutically effective amount, or in a prophylactically effective amount.

[0192] The pharmaceutical composition or vaccine can be administered orally or parenterally, such as by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, nasal drops, and the like.

[0193] The pharmaceutical composition or vaccine can be prepared into various dosage forms according to conventional methods in the art, including but not limited to tablets, capsules, solutions, suspensions, granules or injections.

[0194] use

[0195] The immunostimulatory antibody-drug conjugates described herein can be used to treat or prevent STING-mediated diseases and conditions, specifically, can be used to treat and prevent diseases or conditions or to alleviate the severity of the diseases or conditions, wherein the diseases or conditions are selected from tumors, pathogen infections, diseases caused by pathogen infections, hematological diseases, metabolic diseases, inflammation, allergic diseases, autoimmune diseases, precancerous syndromes, tumor metastasis, and cardiovascular diseases.

[0196] In some embodiments, the tumor is a carcinoma, a lymphoma, a lymphoid tumor, a blastoma, a sarcoma, or a leukemia.

[0197] In some embodiments, the pathogen infection is a bacterial infection or a viral infection.

[0198] In some embodiments, the inflammation is inflammation of any tissue or organ in the body, including musculoskeletal inflammation, vascular inflammation, neurological inflammation, digestive system inflammation, ocular inflammation, reproductive system inflammation, and other inflammations.

[0199] In some embodiments, the viral infection is an infection caused by severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant strain thereof, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, dengue virus, Zika virus, yellow fever virus or hepatitis B virus.

[0200] In some embodiments, the bacterial infection is an infection caused by Gram-positive bacteria, penicillin-resistant bacteria, Staphylococcus aureus, Mycobacterium tuberculosis, drug-resistant Mycobacterium tuberculosis, Clostridium difficile, or Chlamydia trachomatis.

[0201] In some embodiments, the disease caused by the viral infection is COVID-19.

[0202] In some embodiments, the disease caused by the pathogen infection is a disease caused by bacterial infection or viral infection.

[0203] In some embodiments, the disease caused by the bacterial infection is selected from bacterial lung infection, sepsis caused by Staphylococcus aureus pneumonia, Pseudomonas aeruginosa, Clostridium difficile and the like, tuberculosis, bacterial eye infection, heart, brain or skin infection, gastrointestinal infection, bacterial meningitis, or abscess in any organ (such as muscle, liver, meninges, or lung), cellulitis, bacteremia, skin necrosis, eyelid infection, eye infection, neonatal conjunctivitis, osteomyelitis, impetigo, ecthyma, scalded skin syndrome, food poisoning, pneumonia, surgical infection, urinary tract infection, burn infection, meningitis, endocarditis, sepsis, toxic shock syndrome, and septic arthritis.

[0204] In some embodiments, the cancer is selected from the group consisting of breast cancer (e.g., HER2-positive breast cancer); squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; peritoneal cancer; liver cancer; stomach cancer; gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urethral cancer; hepatoma; intestinal cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; anal cancer; penile cancer; melanoma; multiple myeloma and B-cell lymphoma; brain cancer; gallbladder cancer; esophageal cancer; bile duct cancer; head and neck cancer and related metastases.

[0205] In some embodiments, the autoimmune diseases include but are not limited to STING-associated vasculitis of infancy (SAVI), Aicardi Goutieres syndrome (AGS), pernio lupus, ataxia telangiectasia (also known as LouisBar syndrome), retinal vasculopathy with cerebral leukodystrophy (RCVL), systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, psoriasis, diabetes (including insulin-dependent diabetes mellitus (IDDM)), dermatomyositis, human immunodeficiency virus (HIV), AIDS, polymyositis, systemic sclerosis (scleroderma) and Sjögren's syndrome (SS), rheumatoid arthritis, Rheumatoid arthritis, psoriatic arthritis, polyarthritis, osteoarthritis, myasthenia gravis, polyarteritis nodosa, vasculitis, cutaneous vasculitis, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, autoimmune hepatitis, primary sclerosing cholangitis, Wegener's granulomatosis, microscopic polyangiitis, Behçet's disease, spondylitis, giant cell arteritis, polymyalgia rheumatica, Raynaud's phenomenon, primary biliary cirrhosis, primary vasculitis of the central nervous system, neuromyelitis optica, and mixed connective tissue disease.

[0206] definition

[0207] As used herein, the term "linker" is a molecule with two reactive ends, one end of which can be coupled to a macromolecule (e.g., an antibody or antigen-binding fragment thereof, polypeptide, or protein that can specifically bind to a target), and the other end is used to couple to a small molecule active compound, such as a cytotoxin or an immunomodulator. The macromolecule coupling end of the linker is usually a site that can be coupled through the sulfhydryl group of the cysteine ​​or the lysine amine group on the antibody, and the small molecule active compound coupling end of the linker is usually an active site that can be coupled through the sulfhydryl, amino, carboxyl, or hydroxyl group on the toxin molecule. When the term linker is used to describe a coupled linker, since the linker has reacted with one or both of the antibody and the cytotoxin to form a covalent bond, it may no longer include one or two reactive terminal reaction sites (e.g., a leaving group of a sulfhydryl reactive group, a leaving group of an amine reactive group).

[0208] As used herein, the term "immunomodulator" refers to substances that can modulate the immune system and have an activating or inhibitory effect on the body's immune response, including immunostimulants and immunosuppressants. Immunomodulators of particular interest in this application include STING agonists, glucocorticoids, and immunostimulatory cytokines.

[0209] As used herein, the term "cytotoxin" refers to molecules that are toxic to cancer cells upon release thereof. Toxins of particular interest in this application include methyl auristatin E (MMAE), auristatin, maytansinoids or their derivatives (e.g., maytansinoids, DM1, DM3, DM4), calicheamicin, duocarmycin, doxorubicin, camptothecin, or PBD-type cytotoxins.

[0210] As used herein, the term "antibody-drug conjugate" or "ADC" is a product formed by coupling multiple molecules (usually 1-8) of cytotoxins to an antibody molecule, each of which is coupled via a linker. The antibody conjugated to one or more cytotoxins is typically a monoclonal antibody selective for a specific antigen.

[0211] As used herein, the term "immunostimulatory antibody-drug conjugate" or "ISAC" is a drug conjugate. The structure of an ISAC consists of an antibody, a linker, and a small molecule immune agonist (innate immunity agent or modulator); the antibody is usually a monoclonal antibody that is selective for a specific antigen.

[0212] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V LIt consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) form antibody binding sites respectively. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definition of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883 or. The term "antibody" is not limited by any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0213] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefrance et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0214] The CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure are preferably identified by the Kabat, Chothia or IMGT numbering systems.

[0215] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0216] As used herein, the term "Fd fragment" means an antibody fragment consisting of the VH and CH1 domains; the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means one of the two Fab' fragments formed by reducing the F(ab')2 fragment to break the disulfide bond at the F(ab')2 hinge region.

[0217] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0218] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody, which is a small antibody fragment with two antigen-binding sites, comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL) connected thereto in the same polypeptide chain (VH-VL or VL-VH), in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993), and Poljak RJ et al., Structure 2:1121 1123 (1994)).

[0219] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., the monoclonal antibodies ch13, Hu13H2L0, Hu13H2L3, Hu13H3L0, Hu13H3L3, and Hu13H3L5 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibodies can be screened for specificity in the same manner as for intact antibodies.

[0220] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0221] As used herein, the term "VHH," also known as a nanobody (Nb), is a heavy chain single-domain antibody containing only a fragment of the VHH domain. It is composed of the variable region of the heavy chain of camelids (camels, llamas, alpacas, and their relatives). Nanobody crystals are 2.5 nm in diameter and 4 nm long, with a molecular weight of approximately 12-15 kDa.

[0222] As used herein, the term "Probody" is a recombinant antibody drug that relies on protease cleavage for activation. It is mainly composed of three modules: a monoclonal antibody with anti-cancer activity or a fragment of its variable region, a masking peptide connected to the N-terminus of the light chain (masking peptide, which blocks the binding of the antibody Fab region to the antigen), and an enzyme-cleavable polypeptide linker (spacer) connected between the polypeptide and the antibody. Probody can use traditional recombinant antibody production technology. The mechanism of action of Probody mainly utilizes the difference in protease activity between normal tissues and tumors, so as to maximize the activity of the drug in the tumor microenvironment rather than normal tissues. In normal tissues, Probody maintains an intact structure. Due to the presence of the masking peptide, the antibody cannot bind to the antigen and exert its effect and has a longer half-life. When Probody enters the tumor microenvironment, the protease therein will cut the linker and release the masking peptide, exposing the Fab region of the antibody to normally bind to the antigen.

[0223] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, which usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, USP 4,816,567).

[0224] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)).

[0225] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues from the corresponding non-human antibody, or with amino acid residues from other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).

[0226] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity for at least two different epitopes on the same antigen or different antigens. A multispecific antibody can be a full-length antibody or a fragment of such an antibody. The term "bispecific antibody" refers to an antibody that has binding specificity for two different antigens or that can recognize two different epitopes on a single antigen.

[0227] As used herein, the term "human antibody" or "fully human antibody" includes antibodies with variable regions and constant regions (if present) derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies (i.e., humanized antibodies) in which CDR sequences derived from another mammalian species germline (e.g., mouse) are grafted onto human framework sequences. Fully human antibodies or human antibodies can be derived from transgenic mice carrying human antibody genes or from human cells.

[0228] As used herein, the terms "specific binding," "specificity," or "specific for" refer to a non-random binding reaction between two molecules, such as an antibody and its antigen. In some embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D ) binds to the antigen.

[0229] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies (e.g., monoclonal antibodies ch13, Hu13H2L0, Hu13H2L3, Hu13H3L0, Hu13H3L3 and Hu13H3L5 of the present disclosure) have a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 - 6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D) binds to an antigen (e.g., protein tyrosine kinase 7 (PTK7)), e.g., as determined using surface plasmon resonance (SPR) in a BIACORE instrument.

[0230] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0231] As used herein, the term "antigen" refers to any substance that can induce an immune response in the body, that is, a substance that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to corresponding products in vivo or in vitro.

[0232] As used herein, the term "polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a polypeptide may contain modifications, such as, but not limited to, optionally substituted polysarcosine residues, optionally substituted glycosylated polyethylene glycol, glycosylation, phosphorylation, or disulfide bond modifications. A "protein" may comprise one or more polypeptides.

[0233] As used herein, the term "tautomer" refers to functional group isomers produced by the rapid movement of an atom in a molecule between two positions, such as keto-enol tautomers, imine-enamine tautomers, etc. If tautomers exist in the compounds of the present application, they may exist in the form of a single tautomer or a mixture thereof, preferably in the form of a more stable tautomer as the main component.

[0234] The compound represented by formula I or formula II of the present application contains The group, therefore, exists as imine-enamine tautomers and can also exist in the form of formula I', formula I'", formula I'", formula II', formula II", and formula II'".

[0235] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable carriers and / or excipients include, but are not limited to, pH adjusters, surfactants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0236] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered therapies.

[0237] It should also be noted that the dosage and method of use of the compounds of this application depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, the potency of the compound, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.001 and 1000 mg / kg body weight / day.

[0238] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a tumor, a pathogen infection, a disease caused by a pathogen infection, a hematological disease, a metabolic disease, inflammation, an allergic disease, an autoimmune disease, a precancerous syndrome, a tumor metastasis, a cardiovascular disease) in a subject or to minimize its effects. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, a reduction in the rate of disease progression, an improvement or alleviation of the disease state, and a regression or improved prognosis, whether detectable or undetectable. The amount of therapeutic agent that is effective in alleviating any particular disease symptom can vary depending on factors such as the patient's disease state, age, and weight, and the ability of the drug to elicit a desired response in the subject. Whether the symptoms of the disease are alleviated can be assessed by any clinical measurement that is typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptoms.

[0239] As used herein, the term "patient" refers to any human or non-human animal, particularly a human, receiving prophylactic or therapeutic treatment. For example, the antibodies, methods, and compositions described herein can be used to treat a subject suffering from cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, horses, cats, chickens, mice, rats, amphibians, reptiles, and the like.

[0240] As used herein, the term "about" is understood to mean within + / -20%, + / -18%, + / -15%, + / -12%, + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1%, + / -0.5%, + / -0.4%, + / -0.3%, + / -0.2%, + / -0.1% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term "about."

[0241] As used herein, “C l-8 "Alkyl" refers to a saturated linear or branched monovalent hydrocarbon group having 1 to 8 carbon atoms, preferably 1 to 6, 1 to 4 or 1 to 3 carbon atoms. l-8Representative examples of "alkylalkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, heptyl, octyl, and the like.

[0242] As used herein, the term "C l-8 "Alkylamino" means a group C l-8 Alkyl-NH-, i.e., C l-8 Alkyl-substituted amino. l-8 Representative examples of "alkylamino" include, but are not limited to, methylamino, ethylamino, propylamino, butylamino, pentylamino, hexylamino, heptylamino, octylamino, and the like.

[0243] As used herein, the term "C l-8 "Alkylthio" means a group C l-8 Alkyl-S-. "C l-8 Representative examples of "alkylthio" include, but are not limited to, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, tert-butylthio, sec-butylthio, n-pentylthio, n-hexylthio, 1,2-dimethylbutylthio, n-heptylthio, n-octylthio, and the like.

[0244] As used herein, the term "C l-8 "Alkoxy" means a group C l-8 Alkyl-O-. "C l-8 Representative examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, n-heptyloxy, n-octyloxy, and the like.

[0245] As used herein, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon group having 4 to 8 carbon atoms and having a monocyclic or bicyclic or multiple fused rings (including fused and bridged ring systems), preferably having 5 to 7, or 5 to 6 carbon atoms. Typical examples of "cycloalkyl" include, but are not limited to, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc.; bicyclic structures such as bicyclo[2.2.1]heptyl, and polycyclic structures such as adamantyl, etc.

[0246] As used herein, the term "heterocycloalkyl" means a cycloalkyl group as defined herein containing one, two or more heteroatoms independently selected from N, O and S. Typical examples of "heterocycloalkyl" include, but are not limited to, pyrrolidinyl, piperazinyl, piperidinyl and morpholinyl.

[0247] As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclic group having 5 to 14 carbon atoms and a conjugated π-electron system, a single ring, or two or more fused rings. The aryl group preferably has 5 to 10, 5 to 8, 5 to 7, or 5 to 6 carbon atoms. Typical examples of "aryl" include, but are not limited to, phenyl, naphthyl, and anthracenyl.

[0248] As used herein, the term "heteroaryl" refers to a heteroaromatic ring group having 5-14 ring members, including monocyclic heteroaromatic rings and polycyclic aromatic rings in which the monocyclic aromatic ring is fused to one or more other aromatic rings. A heteroaryl group contains one nitrogen atom and one or two or more other heteroatoms selected from O, S, or N. Also included within the scope of the term "heteroaryl" herein are groups in which an aromatic ring is fused to one or more non-aromatic rings (carbocyclic or heterocyclic), where the radical or point of attachment is on the aromatic or non-aromatic ring. The heteroaryl group preferably has 5-10 ring members, more preferably 5-7 ring members, and even more preferably 5-6 ring members. Typical examples of "heteroaryl" include, but are not limited to, imidazolyl, triazolyl, indolyl, tetrazolyl, pyridinyl, pteridinyl, pyrimidinyl, triazolyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, thiazinyl, and the like.

[0249] In this paper, the structure of Compound 3 is or a tautomer thereof.

[0250] When the compound name used in this article is inconsistent with the chemical structural formula, the chemical structural formula shall prevail.

[0251] The compounds of the present invention can be used either per se or in the form of pharmaceutically acceptable hydrates or solvates thereof. References herein to the compounds of the present application include compounds of Formula II or Formula I and pharmaceutically acceptable hydrates or solvates thereof.

[0252] Abbreviations / acronyms ADC (antibody-drug conjugate): antibody-drug conjugate; ISAC (Immune-stimulating Antibody Conjugate): immunostimulating antibody-drug conjugate; CTSB (Cathepsin B): cathepsin B; DAR (Drug to antibody ratio): antibody-drug molar ratio; DIPEA (N,N-Diisopropylethylamine): diisopropylethylamine; DMAC (Dimethylacetamide): N,N-dimethylacetamide; DMF (N,N-Dimethylformamide): N,N-dimethylformamide; DMSO (Dimethyl Sulphoxide): dimethyl sulfoxide; Diox (1,4-Dioxane): 1,4-dioxane; DIC (Diisopropylcarbodiimide): diisopropylcarbodiimide; EDTA (Ethylenediamine tetraacetic acid): ethylenediaminetetraacetic acid; EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide) The following items were used as the reagents: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBT (1-Hydroxybenzotriazole); HCl (hydrogen chloride); HER2 (Human epidermal growth factor receptor 2); MAB (Monoclonal Antibody); NHS (N-Hydroxy succinimide); TCEP (Tris(2-carboxyethyl)phosphine); PAB (P-Aminobenzylalcohol); Tris (Tris(hydroxymethyl)aminomethane). Beneficial effects

[0253] The present application provides an immunostimulatory antibody-drug conjugate containing a quaternary ammonium salt having one or more of the following advantages:

[0254] 1) Good stability in the circulatory system,

[0255] 2) It can improve the solubility of effector molecules,

[0256] 3) It can induce the upregulation of gene expression of related immune factors,

[0257] 4) Can effectively activate the immune response of tumor tissue,

[0258] 5) Has the ability to precisely deliver immune stimulants,

[0259] 6) Effectively avoid the systemic side effects of immune agonists,

[0260] 7) It has significant in vitro and in vivo efficacy.

[0261] 8) It has ideal drug safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0262] Figure 1 shows the hydrophobic interaction chromatography test results of ISACs represented by formulas II-11, II-12, II-13, II-21, II-22, and II-23;

[0263] Figure 2 shows the polymerization degree test results of ISACs represented by formulas II-11, II-12, II-13, II-21, II-22, and II-23;

[0264] FIG3 shows the results of upregulation of IFN-β and CXCL10 expression induced by ISAC represented by Formula II-13;

[0265] FIG4 shows the plasma stability evaluation results of the ISAC represented by Formula II-13;

[0266] FIG5 shows the results of the efficacy experiment of ISAC represented by Formula II-41 in SKOV3 / THP-1 co-culture;

[0267] FIG6 shows the release results of the compound represented by Formula I-13 under the action of cathepsin;

[0268] FIG7 shows the release results of the compound represented by Formula I-41 under the action of cell lysate;

[0269] Figure 8 shows the efficacy evaluation results of ISAC represented by Formula II-13 in a BALB / c mouse xenograft model bearing NCI-N87 human gastric cancer cells. Figure A shows the tumor inhibition effect of ISAC at different doses. The results show that both dose groups can completely and continuously regress tumors, and the anti-tumor inhibitory activity shows a dose-dependent relationship, with significant differences in anti-tumor activity (P**** < 0.0001). Figure B shows the body weight change curves of animals after treatment with ISAC at different doses. The results show that no animals in any test group showed significant weight loss due to drug tolerance during treatment.

[0270] FIG9 shows the change curve of tumor volume in a C57BL / 6J mouse xenograft model of E0771-HER2 tumor cells after treatment with the ISAC of Formula II-41 at different dosages. *** indicates a significance level of P<0.001, **** indicates a significance level of P<0.0001, and ns indicates not significant.

[0271] FIG10 shows the changes in tumor weight (g) in a C57BL / 6J mouse xenograft model of E0771-HER2 tumor cells after treatment with the ISAC of Formula II-41 at different doses. *** indicates a significance level of P<0.001, **** indicates a significance level of P<0.0001, and ns indicates not significant.

[0272] FIG11 shows the body weight change curve of a C57BL / 6J mouse xenograft model of E0771-HER2 tumor cells after treatment with the ISAC of Formula II-41 at different doses. ns indicates not significant.

[0273] FIG12 is a curve showing changes in tumor volume in a C57BL / 6J mouse xenograft model of E0771-HER2 tumor cells after treatment with the ISAC of Formula II-41 at different doses. * indicates a significance level of P<0.05, and *** indicates a significance level of P<0.001.

[0274] FIG13 shows the body weight change curve of a C57BL / 6J mouse xenograft model of E0771-HER2 tumor cells after treatment with the ISAC of Formula II-41 at different doses. ns indicates not significant.

[0275] FIG14 is a curve showing the change in the number of green objects (cancer cells) over time. DETAILED DESCRIPTION

[0276] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions according to conventional conditions or manufacturer recommendations are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0277] Example 1: Preparation of Compounds I-11, I-12, and I-13 Conjugated with Valyl-Alanine-Containing Linkers and STING Agonists

[0278] 1) Preparation of Intermediates I-11a, I-12a, and I-13a

[0279] Maleimide-diethylene glycol-carboxylic acid (3-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-propionic acid) (500.00 mg, 1.52 mmol) was added to a 100 mL eggplant-shaped flask and dissolved in 20 mL of anhydrous DMF. Under stirring at room temperature, EDCI (437.08 mg, 2.28 mmol), HOBt (247.27 mg, 1.83 mmol) and DIPEA (294.44 mg, 2.28 mmol) were added successively. The reaction was carried out at room temperature for 2 h under argon protection. Compound VA (Val-Ala) (445.91 mg, 1.52 mmol) was added to the reaction solution and monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporator and purified by column chromatography to obtain intermediate I-13a as a colorless oil (773.98 mg, 84.35% yield). 1H-NMR (600 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.21 (dd, J = 11.9, 7.0 Hz, 1H), 8.03 (t, J = 5.5 Hz, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.59 (dd, J = 16.4, 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.00 (s, 2H), 4 .71(s,1H),4.41–4.36(m,1H),4.33(s,1H),4.23–4.19(m,1H),3.61–3.57(m,4H),3.48 –3.45(m,4H),3.34(t,J=5.9Hz,2H),3.24(s,1H),3.14(q,J=5.8Hz,2H),2.46(dd,J=14. 1,7.2Hz,1H),2.39(dd,J=13.6,7.2Hz,1H),2.33(t,J=7.3Hz,2H),1.96(dq,J=13.6,6. 8Hz,1H),1.31(d,J=7.1Hz,3H),0.87(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 29 H 42 N5O9 + [M+H] + 604.6730; found 604.3130.

[0280] Using maleimide-tetraethylene glycol-carboxylic acid (500.00 mg, 1.20 mmol) as raw material and referring to the synthesis method of compound I-11a, intermediate I-12a was prepared as a colorless oil (667.26 mg, 80.38% yield). 1H-NMR (600MHz, DMSO-d6) δ10.01(s,1H),8.21(dd,J=11.8,7.0Hz,1H),8.04(t,J=5.5Hz,1H),7.91(d,J=8.7Hz,1H),7.60(dd,J=16.7,8.5Hz ,2H),7.37(d,J=8.5Hz,1H),7.24(d,J=8.5Hz,1H),7.00(s,2H),4.72(s,1H),4.40(p,J=7.1Hz,1H),4.33(s,1H),4.21(dd,J=8.3,7.0Hz,1H), 3.59(dd,J=8.2,3.0Hz,8H),3.51–3.46(m,12H),3.36(t,J=5.9Hz,2H),3.25(s,1H),3.18–3.12(m,2H),2.47(dd,J=14.2,7.2Hz,1H),2.42–2. 35(m,1H),2.33(t,J=7.3Hz,2H),1.97(dq,J=13.5,6.7Hz,1H),1.31(d,J=7.1Hz,3H),0.88(d,J=6.8Hz,3H),0.84(d,J=6.8Hz,3H).Calcd.for C 33 H 50 N5O 11 + [M+H] + 692.7790; found 692.3657.

[0281] Using maleimide-octaethylene glycol-carboxylic acid (500.00 mg, 0.84 mmol) as raw material and referring to the synthesis method of compound I-11a, intermediate I-13a was prepared as a colorless oil (560.32 mg, 76.85% yield). 1H-NMR (600MHz, DMSO-d6) δ9.98 (s, 1H), 8.25–8.14 (m, 1H), 8.03 (t, J = 5.4Hz, 1H), 7.90 (d, J = 8.7Hz, 1H), 7.60 (dd, J = 16.5, 8.5Hz, 2H), 7.37 (d,J=8.5Hz,1H),7.24(d,J=8.4Hz,1H),7.00(s,2H),4.71(s,1H),4.39(t,J=7.1Hz,1H),4.33(s,1H),4.21(dd,J=8.2,7.2Hz,1H),3.59(t,J =6.0Hz,4H),3.49(dd,J=9.4,4.5Hz,28H),3.36(t,J=5.9Hz,2H),3.25(s,1H),3.15(q,J=5.8Hz,2H),2.47(dd,J=14.2,7.1Hz,1H),2.41–2.3 6(m,1H),2.33(t,J=7.3Hz,2H),1.97(dq,J=13.6,6.8Hz,1H),1.31(d,J=7.1Hz,3H),0.88(d,J=6.8Hz,3H),0.84(d,J=6.8Hz,3H).Calcd.for C 41 H 66 N5O 15 + [M+H] + 868.9910; found 868.3524.

[0282] 2) Preparation of intermediates I-11b, I-12b, and I-13b:

[0283] Intermediate I-11a (300.00 mg, 0.50 mmol) was added to a 100 mL eggplant-shaped flask as a raw material, dissolved in a mixed solvent of 5 mL of anhydrous DMF and 15 mL of anhydrous dichloromethane, cooled to -15 ° C and slowly added with stirring thionyl chloride (178.46 mg, 1.50 mmol) dissolved in 5 mL of anhydrous dichloromethane. The reaction was maintained at -15 ° C for 30 min and monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporator and purified by column chromatography to obtain intermediate I-11b as a light yellow oil (142.09 mg, 45.68% yield). 1H-NMR (600MHz, DMSO-d6) δ10.09(s,1H),8.28(d,J=6.9Hz,1H),8.08(t,J=5.6Hz,1H),7.96(d,J=8.7Hz,1H),7.66(d,J=8.5Hz,2H ),7.42(d,J=8.5Hz,2H),7.06(s,2H),4.77(s,2H),4.45(p,J=7.1Hz,1H),4.27(dd,J=8.5,7.0Hz,1H),3.68–3.62(m,5H),3.57–3.5 0(m,6H),3.40(dd,J=7.5,4.2Hz,3H),3.30(s,1H),3.20(dd,J=11.4,5.7Hz,3H),2.52(dd,J=14.1,7.2Hz,1H),2.48–2.42(m,1H), 2.39(t,J=7.3Hz,2H),2.02(dq,J=13.6,6.7Hz,1H),1.37(d,J=7.1Hz,3H),0.93(d,J=6.8Hz,3H),0.89(d,J=6.8Hz,3H).Calcd.for C 29 H 41 ClN5O8 + [M+H] + 622.2565.; found 622.2618.

[0284] Intermediate I-12b was prepared from intermediate I-12a (345.89 mg, 0.50 mmol) as a light yellow oil (147.80 mg, 41.62% yield) by referring to the synthesis method of intermediate I-11b. 1H-NMR (600MHz, DMSO-d6) δ9.99(s,1H),8.21(dd,J=11.9,7.0Hz,1H),8.03(t,J=5.5Hz,1H),7.90(d,J=8.7Hz,1H),7.59(dd,J=16.4,8.5Hz ,2H),7.36(d,J=8.5Hz,1H),7.24(d,J=8.5Hz,1H),7.00(s,2H),4.71( s,1H),4.42–4.36(m,1H),4.33(s,1H),4.24–4.17(m,1H),3.62–3.55( m,4H),3.50–3.43(m,4H),3.34(t,J=5.9Hz,2H),3.14(q,J=5.8Hz,2H),2.51–2.49(m,1H),2.46(dd,J=14.1,7.2Hz,1H),2.39(dd,J=13.6,7. 2Hz,1H),2.33(t,J=7.3Hz,2H),1.96(dq,J=13.6,6.8Hz,1H),1.31(d,J=7.1Hz,3H),0.87(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 33 H 49 ClN5O 10 + [M+H] + 710.3090.; found 710.3159.

[0285] Intermediate I-13b was prepared from intermediate I-13a (434.00 mg, 0.50 mmol) as a light yellow oil (192.58 mg, 43.45% yield) by referring to the synthesis method of intermediate I-11b. 1H-NMR (600MHz, DMSO-d6) δ9.98(s,1H),8.19(dd,J=11.6,7.0Hz,1H),8.02(t,J=5.5Hz,1H),7.89(d,J=8.7Hz,1H),7.59(dd,J=16.5,8.5H z,2H),7.37(d,J=8.5Hz,1H),7.24(d,J=8.4Hz,1H),7.00(s,2H),4.71(s,1H),4.39(p,J=7.1Hz,1H),4.21(dd,J=8.5,6.9Hz,1H),3.62–3.5 7(m,4H),3.49(dd,J=9.4,4.5Hz,28H),3.36(t,J=5.9Hz,2H),3.25(s,1H),3.14(q,J=5.8Hz,2H),2.46(dd,J=14.1,7.2Hz,1H),2.42–2.36 (m,1H),2.33(t,J=7.3Hz,2H),1.97(dq,J=13.5,6.8Hz,1H),1.31(d,J=7.1Hz,3H),0.87(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 41 H 65 ClN5O 14 + [M+H] + 886.4138.; found 886.4213.

[0286] 3) Preparation of compounds I-11, I-12, and I-13:

[0287] Compound 3 (20 mg, 23.53 mmol) and intermediate I-11b (16.10 mg, 25.88 mmol) were added to a 1.5 mL microcentrifuge tube and dissolved in 300 μL of anhydrous DMF. DIPEA (3.49 mg, 25.88 mmol) and TBAI (4.35 mg, 11.77 mmol) were added and the mixture was kept at 55°C for 7 days with TLC monitoring. After the reaction was complete, the solvent was removed by rotary evaporation and purified by column chromatography to obtain compound I-11 as a white solid (14.95 mg, 38.41% yield). Purity>95%, Calcd. for C 71 H98 N 18 O 15 + [M+H] + 1436.6906.; found 1435.6939.

[0288] Compound 3 (20 mg, 23.53 mmol) and intermediate I-12b (18.38 mg, 25.88 mmol) were used as raw materials and the synthesis method of compound I-11 was referred to to prepare compound I-12 as a white solid (13.61 mg, 34.96% yield). Purity>95%, Calcd.for C 75 H 100 N 18 O 17 + [M+H] + 1524.7430.; found 1523.7432.

[0289] Compound 3 (20 mg, 23.53 mmol) and intermediate I-13b (22.94 mg, 25.88 mmol) were used as raw materials and the synthesis method of compound I-11 was referred to to prepare compound I-13 as a white solid (12.54 mg, 31.33% yield). Purity>95%, Calcd.for C 83 H 115 N 13 O 21 + [M+H] + 1700.8479.; found 1700.8507. 1H NMR(500MHz,DMSO-d6)δppm 12.44-13.03(m,1H),10.22-10.28(m,1H),8.47-8.53(m,1H),8.26-8.35(m,1H),8.02-8.07(m,1H),7.94-8.01(m,2H),7.87-7.91(m,1H) ,7.69-7.76(m,2H),7.66-7.68(m,1H),7.61-7.65(m,1H),7.32-7.42(m,4H),7.29-7.32(m,1H),7.24-7.29(m,1H),6.97-7.03(m,2H),6. 51-6.56(m,1H),6.44-6.51(m,1H),5.69-5.82(m,2H),4.78-4.99(m,4H),4.55-4.65(m,2H),4.46-4.55(m,4H),4.33-4.40(m,1H),4.16- 4.23(m,1H),3.93-4.10(m,3H),3.78-3.92(m,2H),3.61-3.68(m,3H),3.55-3.61(m,4H),3.47-3.52(m,31H),3.12-3.18(m,4H),2.45(br d,J=2.3Hz,1H),2.38-2.41(m,1H),2.30(s,3H),2.11(s,3H),2.09(s,3H),1.91-1.95(m,1H),1.83-1.90(m, 1H), 1.53-1.59 (m, 1H), 1.21-1.35 (m, 13H), 0.92-0.96 (m, 2H), 0.86 (d, J = 6.7Hz, 3H), 0.81 (d, J = 6.9Hz, 3H).

[0290] Example 2: Preparation of Compounds I-21, I-22, and I-23 Conjugated with Valyl-Citrulline-Containing Linkers and STING Agonists

[0291] 1) Preparation of intermediates I-21a, I-22a, and I-23a:

[0292] Maleimide-diethylene glycol-carboxylic acid (500.00 mg, 1.52 mmol) was added to a 100 mL eggplant-shaped flask and dissolved in 20 mL of anhydrous DMF. EDCI (437.08 mg, 2.28 mmol), HOBt (247.27 mg, 1.83 mmol), and DIPEA (294.44 mg, 2.28 mmol) were added sequentially with stirring at room temperature. The reaction was allowed to proceed at room temperature for 2 h under argon protection. L-Val-L-Cit-PAB (577.92 mg, 1.52 mmol) was then added to the reaction solution and monitored by TLC. Upon completion of the reaction, the solvent was removed by rotary evaporation and purified by column chromatography to afford intermediate I-21a as a colorless oil (926.31 mg, 88.35% yield). 1H-NMR (600MHz, DMSO-d6) δ9.93 (s, 1H), 8.11 (d, J = 7.6Hz, 1H), 8.03 (t, J = 5.5Hz, 1H), 7.89(d,J=8.7Hz,1H),7.55(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),7.00(s,2H),6.02 (s,1H),5.42(s,2H),5.10(t,J=5.7Hz,1H),4.42(d,J=5.5Hz,2H),4.38(dd,J=13.6,8 .1Hz,1H),4.23(dd,J=8.4,7.0Hz,1H),3.60(dd,J=14.3,7.1Hz,4H),3.47(t,J=5.1Hz ,4H),3.35(t,J=5.9Hz,2H),3.14(q,J=5.8Hz,2H),3.01(dt,J=13.1,6.6Hz,1H),2.95 (dt,J=13.1,6.5Hz,1H),2.48–2.45(m,1H),2.41–2.36(m,1H),2.33(t,J=7.3Hz,2H), 1.96(dd,J=13.5,6.8Hz,1H),1.70(dt,J=14.7,7.5Hz,1H),1.62–1.55(m,1H),1.46–1 .40(m,1H),1.39–1.32(m,1H),0.86(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 32 H 48 N7O 10 + [M+H] + 690.7670; found 690.3456.

[0293] Using maleimide-tetraethylene glycol-carboxylic acid (500.00 mg, 1.20 mmol) as starting material, the intermediate I-22a was prepared as a colorless oil (788.20 mg, 84.44% yield) according to the synthesis method of intermediate I-21a. 1H-NMR (600MHz, DMSO-d6) δ9.94 (s, 1H), 8.11 (d, J = 7.6Hz, 1H), 8.03 (t, J = 5.4Hz, 1H),7.89(d,J=8.7Hz,1H),7.56(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),7.00(s,2 H),6.04(s,1H),5.42(s,2H),5.10(t,J=5.7Hz,1H),4.42(d,J=5.6Hz,2H),4.23( dd,J=8.5,6.8Hz,1H),4.03(q,J=7.1Hz,1H),3.59(t,J=7.2Hz,4H),3.51–3.46(m, 12H),3.36(t,J=5.9Hz,2H),3.17–3.11(m,2H),3.01(dt,J=13.1,6.6Hz,1H),2.9 4(td,J=12.9,6.3Hz,1H),2.47(t,J=6.9Hz,1H),2.41–2.36(m,1H),2.33(t,J=7.3 Hz,2H),1.99(s,1H),1.71(dt,J=13.7,6.0Hz,1H),1.63–1.55(m,1H),1.45–1.41( m,1H),1.40–1.34(m,1H),0.86(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 36 H 56 N7O 12 + [M+H] + 778.8730; found 778.3977.

[0294] Using maleimide-octaethylene glycol-carboxylic acid (500.00 mg, 0.84 mmol) as starting material, and referring to the synthesis method of intermediate I-21a, intermediate I-23a was prepared as a colorless oil (649.00 mg, 80.98% yield). NMR (600MHz, DMSO-d6) δ9.94 (s, 1H), 8.11 (s, 1H), 8.03 (t, J = 5.4Hz, 1H), 7.8 9(d,J=8.7Hz,1H),7.56(d,J=8.5Hz,2H),7.23(d,J=8.4Hz,2H),7.00(s,2H), 6.06(s,1H),5.44(s,2H),4.42(s,2H),4.37(s,1H),4.23(dd,J=8.5,6.8Hz, 1H),3.59(t,J=6.2Hz,4H),3.51–3.49(m,28H),3.36(s,2H),3.15(d,J=5.8Hz ,2H),3.03–2.99(m,1H),2.97–2.93(m,1H),2.47(d,J=6.9Hz,1H),2.39(dd, J=9.0,5.4Hz,1H),2.33(t,J=7.3Hz,2H),1.98(dt,J=13.5,6.7Hz,1H),1.71( dt,J=14.7,7.4Hz,1H),1.59(ddd,J=13.6,8.9,4.6Hz,1H),1.46–1.42(m,1H ),1.39–1.35(m,1H),0.86(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 44 H 72 N7O 16 + [M+H ]+ 954.4957.; found 954.5015.

[0295] 2) Preparation of intermediates I-21b, I-22b, and I-23b:

[0296] Intermediate I-21b was prepared from intermediate I-21a (344.89 mg, 0.50 mmol) as a light yellow oil (154.53 mg, 43.64% yield) by referring to the synthesis method of intermediate I-11b. 1H-NMR (600MHz, DMSO-d6) δ10.10 (s, 1H), 8.15 (d, J = 7.5Hz, 1H), 8.03 (t, J = 5.3H z,1H),7.90(d,J=8.6Hz,1H),7.62(d,J=8.4Hz,2H),7.36(d,J=8.5Hz,2H),7.00 (s,2H),6.09(s,1H),5.48(s,2H),4.72(s,2H),4.40–4.36(m,1H),4.22(d,J=7. 0Hz,1H),3.60–3.57(m,4H),3.47(d,J=4.1Hz,4H),3.35(t,J=5.9Hz,2H),3.15– 3.12(m,2H),3.01(dd,J=13.4,6.7Hz,1H),2.95(dd,J=13.3,6.5Hz,1H),2.47(t ,J=7.0Hz,1H),2.41–2.37(m,1H),2.32(d,J=7.2Hz,2H),1.97(dd,J=13.7,6.9H z,1H),1.70(dt,J=14.1,7.1Hz,1H),1.62–1.57(m,1H),1.45(dd,J=14.6,7.5Hz ,1H),1.39–1.34(m,1H),0.86(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 32 H 46 ClN7O9 + [M+H] + 708.3046.; found 708.3115.

[0297] Intermediate I-22b was prepared from intermediate I-22a (389.94 mg, 0.50 mmol) as a light yellow oil (158.55 mg, 39.82% yield) by referring to the synthesis method of intermediate I-11b. 1H-NMR (600MHz, DMSO-d6) δ10.11 (s, 1H), 8.15 (d, J = 7.5Hz, 1H), 8.04 (t, J = 5. 2Hz,1H),7.90(d,J=8.6Hz,1H),7.62(d,J=8.5Hz,2H),7.36(d,J=8.5Hz,2H), 7.00(s,2H),6.11(s,1H),5.42(s,2H),4.72(s,2H),4.38(d,J=5.4Hz,1H),4. 23(d,J=8.4Hz,1H),3.59(t,J=7.1Hz,4H),3.50–3.47(m,12H),3.36(t,J=5.9H z,2H),3.16–3.13(m,2H),3.03–2.99(m,1H),2.96(dd,J=13.2,6.5Hz,1H),2. 48–2.46(m,1H),2.40–2.36(m,1H),2.32(d,J=7.1Hz,2H),1.97(dd,J=12.7,6. 0Hz,1H),1.71(dd,J=14.4,8.5Hz,1H),1.62–1.58(m,1H),1.45(d,J=7.3Hz,1 H),1.39–1.35(m,1H),0.86(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).Calcd.for C 36 H 55 ClN7O 11 + [M+H] + 796.3570.; found 796.3639.

[0298] Intermediate I-23b was prepared from intermediate I-23a (477.05 mg, 0.50 mmol) as a light yellow oil (198.54 mg, 40.83% yield) by referring to the synthesis method of intermediate I-11b. 1H-NMR(600MHz,DMSO-d6)δ10.17(s,1H),8.17(d,J=7.6Hz,1H),8.05(t,J=5.3 Hz,1H),7.93(d,J=8.7Hz,1H),7.64(d,J=8.5Hz,2H),7.36(d,J=8.5Hz,2H),7. 00(s,2H),6.25(s,1H),5.33(s,2H),4.72(s,2H),4.42–4.38(m,1H),4.23(d,J =7.0Hz,1H),3.60(d,J=6.9Hz,4H),3.50(d,J=5.7Hz,28H),3.37(t,J=5.9Hz,2H ),3.15(d,J=5.8Hz,2H),3.04–3.00(m,1H),2.99–2.96(m,1H),2.50–2.46(m,1 H),2.40(dd,J=13.5,7.1Hz,1H),2.34(t,J=7.3Hz,2H),1.99(dd,J=13.4,6.7Hz ,1H),1.73(dd,J=14.5,7.8Hz,1H),1.62(dd,J=8.9,4.6Hz,1H),1.48–1.44(m, 1H),1.40–1.36(m,1H),0.87(d,J=6.7Hz,3H),0.84(d,J=6.7Hz,3H).Calcd.for C 44 H 71 ClN7O 15 + [M+H] + 972.4618.; found 972.4671.

[0299] 3) Preparation of compounds I-21, I-22, and I-23:

[0300] Compound 3 (20 mg, 23.53 mmol) and I-21b (18.33 mg, 25.88 mmol) were used as raw materials and the synthesis method of compound I-11 was referred to to prepare compound I-21 as a white solid (12.74 mg, 35.37% yield). Purity>95%, Calcd.for C 74 H 97 N 20 O 16+ [M+H] + 1522.7386.; found 1521.7432.

[0301] Compound 3 (20 mg, 23.53 mmol) and intermediate I-22b (20.61 mg, 25.88 mmol) were used as raw materials and the synthesis method of compound I-11 was referred to to prepare compound I-22 as a white solid (12.19 mg, 32.16% yield). Purity>95%, Calcd.for C 78 H 105 N 20 O 18 + [M+H] + 1610.7910.; found 1609.7896.

[0302] Compound 3 (20 mg, 23.53 mmol) and intermediate I-23b (25.17 mg, 25.88 mmol) were used as raw materials, and a similar synthetic method to compound I-11 was used to prepare compound I-23 as a white solid (12.04 mg, 28.64% yield). Purity>95%, Calcd. for C 86 H 121 N 20 O 22 2+ [M+2H] 2+ 893.4480.; found 893.9559.

[0303] Example 3: Preparation of Compound I-31 Containing a PEG8-PAB Linker Coupled with a STING Agonist

[0304] 1) Preparation of Intermediate I-31a

[0305] Maleimide-octaethylene glycol-carboxylic acid (500.00 mg, 0.84 mmol) was added to a 100 mL eggplant-shaped flask and dissolved in 20 mL of anhydrous DMF. EDCI (241.54 mg, 1.26 mmol), HOBt (136.47 mg, 1.01 mmol), and DIPEA (162.72 mg, 1.26 mmol) were added sequentially with stirring at room temperature. The reaction was allowed to proceed at room temperature for 2 h under argon protection. PAB (103.45 mg, 0.84 mmol) was then added to the reaction solution and monitored by TLC. Upon completion of the reaction, the solvent was removed on a rotary evaporator and purified by column chromatography to afford intermediate I-31a as a colorless oil (500.85 mg, 85.45% yield).1 H-NMR(600MHz,DMSO-d6)δ10.02(s,1H),8.02(t,J=5.5Hz,1H),7.62–7.53(m,2H),7.35 (d,J=8.5Hz,1H),7.22(dd,J=8.4,4.4Hz,1H),7.00(s,2H),4.71(s,1H),4.33(s,1H),3 .69(t,J=6.2Hz,2H),3.59(t,J=7.3Hz,3H),3.52–3.47(m,33H),3.36(t,J=5.9Hz,3H), 3.25(s,1H),3.14(q,J=5.8Hz,3H),2.58–2.53(m,2H),2.33(t,J=7.3Hz,2H).Calcd.for C 33 H 52 N3O 13 + [M+H] + 698.3422.; found 698.3390.

[0306] 2) Preparation of Intermediate I-31b

[0307] Intermediate I-31a (348.89 mg, 0.50 mmol) was added to a 100 mL eggplant-shaped flask as a raw material, dissolved in a mixed solvent of 5 mL of anhydrous DMF and 15 mL of anhydrous dichloromethane, cooled to -15 ° C and slowly added with stirring thionyl chloride (178.46 mg, 1.50 mmol) dissolved in 5 mL of anhydrous dichloromethane. The reaction was maintained at -15 ° C for 30 min and monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporator and purified by column chromatography to obtain intermediate I-31b as a light yellow oil (154.85 mg, 43.24% yield). 1 H-NMR (600MHz, DMSO-d6) δ10.04(s,1H),8.01(t,J=5.3Hz,1H),7.60(d,J=8.4Hz, 2H),7.35(d,J=8.4Hz,2H),7.00(s,2H),4.71(s,1H),3.69(t,J=6.2Hz,2H),3.60( d,J=7.2Hz,2H),3.49(dd,J=9.9,4.6Hz,28H),3.37(d,J=4.1Hz,2H),3.25(s,1H), 3.14(dd,J=11.4,5.7Hz,2H),2.57–2.54(m,2H),2.33(t,J=7.3Hz,2H).Calcd.for C 33 H50 ClN3O 12 + [M+H] + 716.3083.; found 716.3153.

[0308] 3) Preparation of Compound I-31

[0309] Compound 3 (20 mg, 23.53 mmol) and intermediate I-31b (18.54 mg, 25.88 mmol) were added to a 1.5 mL microcentrifuge tube and dissolved in 300 μL of anhydrous DMF. DIPEA (3.49 mg, 25.88 mmol) and TBAI (4.35 mg, 11.77 mmol) were added and the mixture was kept at 55°C for 7 days with TLC monitoring. After the reaction was complete, the solvent was removed by rotary evaporation and purified by column chromatography to obtain compound I-31 as a white solid (14.40 mg, 39.98% yield). Purity>95%, Calcd. for C 75 H 101 N 16 O 19 + [M+H] + 1530.7423.; found [M+NH4] + 1529.7416.

[0310] Example 4: Preparation of Compound I-41 Conjugated with a Linker and a STING Agonist

[0311] 1) Preparation of Intermediate I-41a

[0312] Propynyl-octaethylene glycol-carboxylic acid (500.00 mg) was added to a 100 mL eggplant-shaped flask and dissolved in 20 mL of anhydrous DMF. NHS (437.08 mg), DIC (247.27 mg), and DIPEA (294.44 mg) were added sequentially with stirring at room temperature. The reaction was allowed to proceed at room temperature for 30 h under argon protection. Compound VA (577.92 mg) was added to the reaction solution and monitored by TLC. Upon completion of the reaction, the solvent was removed by rotary evaporation and purified by column chromatography to obtain intermediate I-41a as a colorless oil. MS (ESI) m / z: Calcd. for [M+H] + 711.4, found.[M+NH4] + 729.4.

[0313] 1H NMR(600MHz,Chloroform-d)data are as follows: δ8.66(s,1H),7.67(d,J=8.5Hz,2H),7.32–7.23(m,3H),7.07(t,J=6.0Hz,2H),4.67(p,J=7.3Hz,1 H),4.63(s,2H),4.24(dd,J=7.0,5.6Hz,1H),4.19(d,J=2.4Hz,2H),3.88–3.78(m,1H),3.71–3.57(m,28H),2 .69(s,0H),2.64(ddd,J=14.8,9.0,4.1Hz,1H),2.48(ddd,J=14.7,5.8,3.4Hz,1H),2.44(s,0H),2.26(td,J =7.1,5.8Hz,1H),2.02(s,3H),1.45(d,J=7.2Hz,3H),1.26(s,1H),0.99(dd,J=15.6,6.9Hz,6H).Calculated for C 35 H 61 N4O 12 + [M+NH4] + 729.4; found 729.4.

[0314] 2) Preparation of Intermediate I-41b

[0315] Intermediate I-41a (300.00 mg) was added to a 100 mL eggplant-shaped flask and dissolved in a mixture of 5 mL of anhydrous DMF and 15 mL of anhydrous dichloromethane. The mixture was cooled to -8°C and slowly added with stirring to a mixture of 178.46 mg of thionyl chloride dissolved in 5 mL of anhydrous dichloromethane. The reaction was maintained at -8°C for 30 min and monitored by TLC. Upon completion of the reaction, the solvent was removed by rotary evaporation and purified by column chromatography to obtain intermediate I-41b as a light yellow oil. MS (ESI) m / z: [M+H] + 729.4, found [M+NH4] + 747.3

[0316] 1H NMR(600MHz,Chloroform-d)δ8.70(s,1H),7.73(d,J=8.2Hz,2H),7.33–7.30(m,2H),7.17–6.97(m, 2H),4.70–4.63(m,1H),4.56(s,2H),4.24–4.17(m,3H),3.84(dt,J=9.8,4.9Hz,1H),3.72–3.52(m,2 8H),3.35(s,0H),2.67(d,J=11.1Hz,1H),2.47(ddd,J=14.6,5.5,3.2Hz,1H),2.43(t,J=2.4Hz,1H), 2.28(h,J=6.6Hz,1H),1.45(d,J=7.2Hz,3H),1.25(s,4H),1.01(dd,J=15.9,6.8Hz,6H).Calculated for C 35 H 60 ClN4O 11 + [M+NH4] + 747.3.; found 747.3.

[0317] 3) Preparation of Compound I-41

[0318] I-41b (25 mg) and compound 3 (20 mg) were added to a 1.5 mL EP tube and dissolved in 400 μL of anhydrous DMF. DIPEA (19 mg, 0.15 mmol) was added dropwise to the reaction system, and the reaction was shaken at room temperature for 5 days. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the crude product. The target product, compound I-41, was purified by column chromatography as a black solid (10 mg, 20% yield). MS (ESI) m / z: [M+H] + 1235.61, found [M+NH4] + 1543.8.

[0319] 1H-NMR (600MHz, DMSO-d6): δ10.289-10.191(m,1H),8.235(s,1H),7.873-7.861(m,2H),7.761-7. 735(m,3H),7.506-7.303(m,4H),6.647(s,1H),6.513-6.435(s,1H),5.325(t,J=4.8Hz,1H),4.99 9-4.855(m,3H),4.593-4.374(m,5H),4.201-4.189(m,2H),4.132(s,3H),3.589-3.413(m,43H),2 .109-2.099(m,4H),2.021-1.943(m,6H),1.323-1.293(m,7H),0.884-0.817(m,12H).Calculated for C 83 H 115 N 13 O 21 + [M+NH4] + 1543.8; found 1543.778.

[0320] Example 5: Preparation of Antibody Conjugates II-11, II-12, and II-13 Containing Valyl-Alanine Linker-STING Agonist

[0321] In this example, compounds I-11, I-12, and I-13 prepared in Example 1 were coupled to an anti-HER2 humanized monoclonal antibody.

[0322] 1) Preparation of commonly used buffered saline solutions:

[0323] Buffer-1: Dissolve 3.11 g of L-histidine in 1 L of double-distilled water. Once completely dissolved, adjust the pH to approximately 5.50 (±0.05) with medical-grade glacial acetic acid. Sterilize the solution by filtration through a 0.22 μm filter membrane, bottle, and store at 4°C for a short period of time until ready for use.

[0324] Buffer-2: Dissolve 6.06 g of TRIS base and 0.93 g of EDTA-2Na in a solution, then adjust the volume to 100 mL. Dissolve 7.88 g of TRIS HCl and 0.93 g of EDTA-2Na in a solution, then adjust the volume to 100 mL. Add TRIS HCl to the TRIS base solution and adjust the pH to 8.50 (±0.05). Sterilize the solution by filtration through a 0.22 μm filter, then bottle and store at 4°C for short-term use.

[0325] Buffered saline solution-3 (buffer-3): Use a pipette to measure 1.715 mL of medical glacial acetic acid and dissolve it in 200 mL of double-distilled water. After thorough mixing, filter through a 0.22 μm filter membrane for sterilization, bottle and store at 4°C for short-term use.

[0326] 2) Antibody coupling reaction:

[0327] ① Replacement of pharmaceutical antibodies: The anti-HER2 humanized monoclonal antibody Herceptin (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd.) used for conjugation contained pharmaceutical excipients such as 0.616 mg / ml histidine hydrochloride (monohydrate), 0.364 mg / ml L-histidine, 22.727 mg / ml trehalose, and 100 mg / ml Tween-20. To remove excipient interference, the frozen-thawed antibody stock solution was first slowly thawed at room temperature and then replaced with buffer 1 via a G25 dextran gel column. After replacement, the solution was concentrated by ultrafiltration and centrifugation (final concentration > 5 mg / mL), and its concentration was measured by ultraviolet spectrophotometry.

[0328] ②Prepare the coupling reaction solution: Based on the desired amount of coupling antibody (1 eq), accurately pipette the antibody solution in Buffer-1 and add a sufficient amount of Buffer-1 to achieve an antibody concentration of approximately 10 mg / mL. Adjust the pH to approximately 6-8 with Buffer-2 and transfer the solution to a clean reaction vial using a pipette.

[0329] ③ Antibody reduction: Slowly stir the reaction solution in the vial (100 rpm) and add 2-5 eq of 2.87 mg / mL TCEP·HCl solution. After addition, slowly stir at room temperature and react for 60-180 min.

[0330] ④ Antibody Conjugation: Calculate the volume of organic solvent (DMAC or DMSO) needed to make up 5% to 15% of the total volume. Also calculate the mass of the ADC small molecule payload (linker-STING agonist) to be added. Typically, a slight excess of the small molecule payload (usually 8 eq) is required. This will then determine the desired concentration of the organic solvent for the payload. After accurately preparing the ADC small molecule payload solution, slowly add it dropwise to the reduced antibody reaction solution. Continue stirring gently at room temperature and allow the reaction to proceed for 0.5-5 hours, depending on the specific coupling conditions.

[0331] ⑤ Termination of the reaction: After the reaction solution reaches the predetermined coupling time, add an excess of a water-soluble small molecule N-acetylcysteine ​​solution (1.63 mg / mL) containing a reducing thiol group, and continue the reaction with slow stirring for 30 minutes.

[0332] ⑥ Preliminary purification of the product: After the termination reaction of the coupling is completed, buffer-3 is added to adjust the pH of the reaction solution to about 5.50; the resulting reaction solution is filtered and then preliminarily purified using a G25 dextran gel column. The effluent of the front section (about 80%) is collected, concentrated again by ultrafiltration, sterile filtered and sample packaging is performed; except for some samples reserved for product analysis and stored at 4°C for short-term storage, the rest of the products are stored at -80°C until use.

[0333] The prepared ISAC structure is shown in Table 1, where d (DAR value) can be determined by referring to the method described in the literature (J. Ouyang, in Antibody-Drug Conjugates, Vol. 1045 (Ed.: L. Ducry), 2013, pp. 275-283.).

[0334] Table 1. ISAC structures of synthesized II-11, II-12, and II-13

[0335] Example 6: Preparation of Antibody Conjugates II-21, II-22, and II-23 Containing Valyl-Citrulline Linker-STING Agonist

[0336] In this example, compounds I-21, I-22, and I-23 prepared in Example 2 were coupled to an anti-HER2 humanized monoclonal antibody.

[0337] The coupling method was the same as that in Example 5. The prepared ISAC structure is shown in Table 2, where d (DAR value) can be determined by referring to the method described in the literature (J. Ouyang, in Antibody-Drug Conjugates, Vol. 1045 (Ed.: L. Ducry), 2013, pp. 275-283.).

[0338] Table 2. ISAC structures of synthesized II-21, II-22, and II-23

[0339] Example 7: Preparation of Antibody Conjugate II-31 Containing PEG8-PAB Linker-STING Agonist

[0340] In this example, compound I-31 prepared in Example 3 was coupled to the anti-HER2 humanized monoclonal antibody Herceptin using the same coupling method as in Example 5. The structure of the prepared ISAC is shown in Formula II-31, where d represents the DAR value, which is approximately 2. The determination of the DAR value can be carried out according to the method described in the literature (J. Ouyang, in Antibody-Drug Conjugates, Vol. 1045 (Ed.: L. Ducry), 2013, pp. 275-283.).

[0341] Example 8: Preparation of Antibody Conjugate II-41 Containing a STING Agonist

[0342] 1) Preparation of azide-modified antibodies: Antibodies (26.25 mg, 1.75 × 10 -4 mmol) was added into a 5 mL EP tube, and the compound 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (0.54 mg, 1.4 × 10 -3 After stirring overnight at room temperature, the coupled product was replaced in PBS to prepare the azide-modified anti-HER2 humanized monoclonal antibody Herceptin. It is the anti-HER2 humanized monoclonal antibody Herceptin, where d represents the DAR value, which is approximately 2.

[0343] 2) Azide-modified anti-HER2 humanized monoclonal antibody (2.5 mg, 1.67 × 10 -5 mmol) was added to a 2 mL EP tube, and the DMA solution of compound I-41 (0.41 mg) prepared in Example 4 was added dropwise. Then, CuSO4 (0.13 mg, 5.01×10 -4 mmol), THPTA (0.87mg, 2.0×10 -3 mmol) and sodium ascorbate (1.49 mg, 7.51×10 -3 mmol) aqueous solution was added to the antibody, and the reaction was stirred at room temperature for 5 hours. The coupled product was replaced in PBS to prepare the target antibody-drug conjugate II-41, where d represents the DAR value, which is about 2.

[0344] Example 9: DAR value detection of ISACs represented by formula II-11, II-12, II-13, II-21, II-22, and II-23

[0345] This example evaluated the DAR values ​​(i.e., d in the structural formula) of the ISACs represented by Formulas II-11, II-12, II-13, II-21, II-22, and II-23. The DAR values ​​of each conjugate were determined using hydrophobic interaction chromatography ( FIG1 ). The main peaks shown in the chromatogram represent conjugation with 0, 2, 4, and 6 small molecule drugs, respectively. The DAR values ​​of the ISACs represented by Formulas II-11, II-12, II-13, II-21, II-22, and II-23 are shown in Table 3.

[0346] Table 3. DAR analysis results of antibody-drug conjugates

[0347] Example 10: Determination of the degree of polymerization of ISACs represented by formula II-11, II-12, II-13, II-21, II-22, and II-23

[0348] This example evaluated the degree of polymerization of the ISACs represented by Formulas II-11, II-12, II-13, II-21, II-22, and II-23 ( Figure 2 ). Size exclusion chromatography was used to examine the monomers and aggregates of each antibody conjugate. The results showed that the ISACs represented by Formulas II-11, II-12, II-13, II-21, II-22, and II-23 existed almost entirely in monomeric form (active monomer purity >95%).

[0349] Example 11: Stimulating Effects of ISAC Conjugates of Formula II-13 on Related Cytokines

[0350] In this example, the effectiveness of the ISAC shown in Formula II-13 in inducing immune factor gene expression regulation was evaluated by qPCR detection of changes in interferon beta (IFN-β) and chemokine ligand 10 (CXC motif chemokine ligand 10, CXCL10) mRNA in tumor cells after exposure to the test drug. The cell lines used in the test were HER2-positive SKOV3 and SKBR3 cell lines (purchased from ATCC). The test drugs were the control solvent (PBS), Compound 3, and the ISAC shown in Formula II-13. IFN-β is a marker that can be activated by cGAS-STING downstream activation signals, and CXCL10 is produced by type I interferon and serves as a marker that promotes the recruitment of immune cells to tumor tissue. The gene expression assessment of the above cytokines can be used to evaluate the degree of immune system activation of tumor tissue by the ADC.

[0351] The reagents, instruments and consumables used in the experiment are described in the following table:

[0352] RT-PCR primer sequences:

[0353] The test process is as follows:

[0354] ① Cell thawing

[0355] Thaw the vial containing target cells by gentle agitation in a 37°C water bath;

[0356] After the contents were thawed, the vials were removed from the water bath and decontaminated by immersion or spraying with 70% ethanol;

[0357] The contents of the vial were transferred to a centrifuge tube containing 9 mL of complete medium DMEM and centrifuged (200 g; 5 min);

[0358] Resuspend the cells in culture medium, pellet and distribute them onto a 75 cm 2 in culture flasks;

[0359] Incubate the culture at 37°C with 5% CO2 The cells were cultured in a CO2 incubator (48R, #CO48312044) with an oxygen concentration of 20% and a carbon dioxide concentration of 5%.

[0360] ② Expand cells

[0361] The cells were passaged three times a week at a ratio of 1:4 in culture medium containing 10% FBS (heat inactivated) and 1% penicillin / streptomycin solution;

[0362] For passaged cells, first rinse adherent cells with a trypsin / EDTA solution (3 mL). Then, add trypsin / EDTA (3 mL, T75 flask) and swirl to evenly coat the cells. Incubate the culture at 37°C until the cells detach. Verify detachment under a microscope, inactivate the trypsin by adding an equal volume of cell culture medium, collect the detached cells, centrifuge at 200 g for 5 minutes, and resuspend in fresh culture medium.

[0363] ③Cell inoculation

[0364] Collect cells and count the cell number;

[0365] 100 μL of cell suspension with adjusted density was added to the designated 6-well cell culture plate. The final cell density was approximately 5,000-10,000 cells / well.

[0366] The cells were covered with a lid and placed in a 37°C 5% CO2 incubator for incubation for 24 h with an oxygen concentration of 20%.

[0367] ④Prepare compounds

[0368] The compound solution was serially diluted at a ratio of 1:3, and the initial maximum concentration of the test drug ISAC represented by Formula II-13 and Compound 3 was about 400 nM.

[0369] ④ Co-incubation of drugs and cells

[0370] Remove the medium from the 6 wells seeded with cells and add 150 μL of fresh medium to each well. Then, add 50 μL of the prepared test drug to the 96-well plate. Incubate the drug and cells in a 37°C, 5% CO₂, 0.1% O₂ incubator for 24 hours. Wash away the drug with fresh medium, and incubate the cells in a 37°C, 5% CO₂, 20% O₂ incubator for an additional 72 or 120 hours.

[0371] ⑤ Cell RNA extraction

[0372] RNA extraction: Wash the cells with PBS (three times), add Trizol to dissolve the sample, and let it stand at room temperature for 5 minutes, then add chloroform, shake for 30 seconds, and let it stand at room temperature for 5 minutes; centrifuge in a 4℃ centrifuge (12000rpm) for 15 minutes, transfer the upper aqueous phase to an EP tube, add isopropanol to mix, and let it stand at room temperature for 10 minutes; centrifuge again (4℃, 12000rpm, 10 minutes), remove the supernatant, and add 75% alcohol to resuspend the precipitate, centrifuge again (4℃, 12000rpm, 5 minutes) and remove the supernatant, and dry at room temperature for 15 minutes; add DEPC water preheated at 65℃ to dissolve the precipitate, and store the dissolved RNA in a -20℃ refrigerator for later use.

[0373] ⑥gDNA removal and RNA reverse transcription:

[0374] Take 1 μL of total RNA and mix it with 1 μL of gDNA Remover, 1 μL of Oligo Primer, 10 μL of 2×TS Reaction Mix, and 1 μL of TransScript RT / RI Enzyme Mix to a total volume of 20 μL. Make up the remaining volume with RNase-free water. Incubate the PCR tube at 42°C for 15 minutes. At this point, the RNA has been reverse transcribed into cDNA.

[0375] The experimental results show (Figure 3) that under the same conditions, the ISAC represented by Formula II-13 caused the upregulation of gene expression of two cytokines, and the effect was better than or equal to that of the free payload Compound 3. These results show that the ISAC provided by this application can induce the upregulation of gene expression of related immune factors and can effectively activate the immune response of tumor tissue.

[0376] Example 12: In vitro plasma stability evaluation of ISAC represented by formula II-13

[0377] To determine the stability of the conjugate in human plasma and PBS, the released free payload Compound 3 was dissolved in 50% human plasma and PBS, respectively, to prepare standard samples. The conjugate test samples were prepared in the same manner as the standard. Both samples were incubated at 37°C for 10 days, and aliquots were taken at predetermined time points (0, 12h, 1, 2, 3, 4, 5, 6, 7, and 10d). The concentration of the total free payload Compound 3 released from the conjugate was determined at different time points based on the Compound 3 standard curve established using LC-MS.

[0378] Referring to Figure 4, the test results show that after 10 days, the ISAC represented by Formula II-13 released 0.23% and 0.01% of Compound 3 in PBS and 50% plasma, respectively, indicating that the ISAC represented by Formula II-13 can remain stable in PBS and 50% plasma at 37°C.

[0379] Example 13: Pharmacological efficacy of ISAC represented by Formula II-41 in SKOV3 / THP-1 co-cultured cell line

[0380] The induction of the STING pathway in immune cells by the HER2-targeting ISAC of Formula II-41 was evaluated using a SKOV3 / THP1-IRF3-luciferase reporter cell co-culture assay. SKOV3 human ovarian cancer cells were seeded in 96-well CellBIND surface tissue culture plates (approximately 15,000 cells / well) and allowed to attach for 6 hours in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. A series of ISAC dilutions (starting at 100 nM; three-fold serial dilutions in growth medium) was added to each well, and the plate was incubated at 37°C for 20 minutes. THP1 dual reporter cells (approximately 30,000 cells / well) were then added to each well and incubated for a further 20 hours at 37°C in a humidified atmosphere with 5% CO2. Cell culture supernatant (20 μL) from each incubation sample was added to the resuspended QUANTI-Luc (50 μL), and the luminescent signal was immediately measured using a SpectraMax M5 plate reader (Molecular Devices). EC was determined based on the dose-response curve. 50 value.

[0381] The experimental results are shown in Figure 5. The results show that the ISAC represented by formula II-41 can strongly activate the STING signal in THP-1 at very low doses. 50 =0.3nM.

[0382] Example 14: Study on the release of the compound represented by formula I-13 under the action of cathepsins

[0383] A 1 mM stock solution of the compound of Formula I-13 in DMA was mixed with a 1 mM stock solution of 2-mercaptoethanol in PBS at a volume ratio of 1:9 and stirred at room temperature for 1 hour to prepare a 100 μM stock solution of 2-mercaptoethanol-I-13. To 15 μL of 2-mercaptoethanol-I-13, 30 μL of CTSB (15 UN / mL) was added, followed by 105 μL of activity buffer (50 mM sodium acetate, 100 mM NaCl, 8 mM L-cysteine, 1 mM EDTA, pH 5.0). The mixture was mixed thoroughly and incubated in a 37°C incubator. Samples were collected at designated time points (t = 0, 1 h, 4 h, and 24 h), and 600 μL of methanol was added to the samples. The samples were centrifuged (12,000 rpm, 4°C, 20 min) to precipitate proteins. The supernatant was then analyzed by HPLC for the released STING agonist Compound 3 and the prototype compound of Formula I-13.

[0384] The experimental results are shown in Figure 6. Surprisingly, after 24 hours of incubation in 3UN CTSB, β-mercaptoethanol-I-13 remained almost entirely intact, with no significant release of Compound 3. This suggests that the dipeptide bond in the ISAC provided herein is not cleaved by cathepsins under standard conditions. Unlike conventional cleavable ISACs, the ISAC provided herein functions as a non-cleavable quaternary ammonium salt.

[0385] Example 15: Release study of the compound represented by formula I-41 in cell lysate

[0386] Collect SKBR3 (purchased from Wuhan Punosai Life Science Technology Co., Ltd.) about 1.5×10 6 Cells were collected in 1.5 mL EP tubes and centrifuged (1000 rpm, 4°C, 4 min). The culture medium was removed. 500 μL of RIPA lysis buffer (Product No. P0013B, Beyotime) was added to the collected cell sample, mixed thoroughly, and incubated on ice for 30 min before centrifugation (14000 rcf, 4°C, 15 min). 45 μL of the supernatant was mixed with 5 μL of a 100 μM DMSO stock solution of the compound represented by Formula I-41 and incubated in a 37°C incubator. Samples were taken at designated time points (0, 4 h, 24 h, and 48 h), and 150 μL of methanol was added to the sample. The sample was centrifuged (12000 rpm, 4°C, 20 min) to precipitate the protein. The supernatant was then analyzed by HPLC for the released STING agonist Compound 3 and the prototype compound represented by Formula I-41.

[0387] The experimental results are shown in Figure 7. Similar to those in Example 14, the results show that after incubation of the compound represented by Formula I-41 in cell lysate for 48 hours, the peak height and peak shape of the compound represented by Formula I-41 remained virtually unchanged, and no significant release of Compound 3 was observed. This indicates that the dipeptide bond in the ISAC provided herein is not cleaved by cathepsins under conventional conditions. Unlike traditional cleavable ISACs, it functions as a non-cleavable quaternary ammonium salt.

[0388] Example 16: In vivo efficacy study of ISAC represented by Formula II-13 in xenograft model

[0389] This example evaluated the in vivo efficacy of the ISAC represented by Formula II-13 in a xenograft animal model. The test drugs included II-13 (3 mg / mL), II-13 (6 mg / mL), a combination of Herceptin (6 mg / mL) and compound 3 (0.7 mg / mL), and PBS (control group).

[0390] Female BALB / c nude mice aged 6-8 weeks were purchased from SPF (Beijing) Biotechnology Co., Ltd. The mice were housed in an SPF animal breeding center with 3 mice per cage. Animal research followed the ARRIVE guidelines. NCI-N87 cells (approximately 1×10 7 , purchased from ATCC) were subcutaneously inoculated into mice when the tumor size was approximately 120 mm 3 or 180mm 3 At the time of the study, mice carrying NCI-N87 cell xenografts were randomly divided into groups of 6 per group. Mice with NCI-N87 cell xenografts were administered via the tail vein once every 4 days for a total of 4 doses. Starting from day 0, tumor size was measured with a digital caliper and twice a week. The general condition of the animals (mental state, activity, hair color) was observed. The equation (0.5×L×W 2 ) Tumor volume was calculated, where L and W represent the length and width of the tumor, respectively. At the end of the experiment, mice were sacrificed by cervical dislocation.

[0391] The experimental results are shown in Figure 8. The results show that both dose groups of the ISAC represented by Formula II-13 can completely and continuously regress tumors, and the anti-tumor inhibitory activity shows a dose-dependent relationship, with significant differences in anti-tumor activity (P**** < 0.0001). In addition, the ISAC represented by Formula II-13, when administered at 3 mg / kg, has a payload dose of 0.35 mg / kg, which significantly improves the anti-tumor activity compared to the reported effective dose of Compound 3 for tumor inhibition of 1.5 mg / kg. At the same time, both dose groups of the ISAC represented by Formula II-13 showed anti-tumor activity superior to the combined administration group (Herceptin 6 mg / kg + Compound 3 0.7 mg / kg), which also demonstrates its superior efficacy to small molecules. In this trial, none of the experimental animals in the treatment group showed significant weight loss, preliminarily indicating that the targeting of the ISAC represented by Formula II-13 avoids systemic side effects of the payload and has a good safety profile.

[0392] Example 17: In vivo efficacy study of ISAC represented by Formula II-41 in a xenograft model

[0393] This example evaluated the in vivo efficacy of the ISAC represented by Formula II-41 in a xenograft animal model. The test drugs included II-41 (6 mg / kg), II-41 (3 mg / kg), II-41 (1 mg / kg), a combination of Herceptin (6 mg / kg) and compound 3 (0.7 mg / kg), and PBS (control group).

[0394] 6-8 week old C57BL / 6J female mice were purchased from Nanjing Jieyao Technology Co., Ltd., Jiangsu, China. All mice were housed in a specific pathogen-free environment, and all procedures were performed in accordance with the guidelines approved by the Animal Ethics Committee of the Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences (approval ethics numbers IMB-20240912D801 and IMB-20241018D801). In the in vivo experiments, E0771-HER2 tumor cells (2 × 10 6 The cells were injected subcutaneously into the right abdomen of C57BL / 6J mice. The tumor volume reached 80 mm 3 Dosing began with weekly intravenous injections of PBS, TZ, and dSA3 or II-41 (1, 3, and 6 mg / kg) for a total of two doses. Tumor growth was monitored and recorded every two days, and mouse body weights were recorded every three days. Tumor volume was calculated as π / 6 × tumor length × (tumor width)². At the end of the experiment, mice were sacrificed by cervical dislocation.

[0395] The experimental results are shown in Figures 9-11. The results show that compared with the control group, the three dose groups of ISAC represented by Formula II-41 can significantly inhibit tumors (P*** < 0.001), and the anti-tumor inhibitory activity shows a dose-dependent relationship. It is worth noting that the administration of 6 mg / kg of ISAC represented by Formula II-41 almost completely achieved complete regression of the tumor. At the same time, the three dose groups of ISAC represented by Formula II-141 all showed anti-tumor activity that was better than the mixed administration group (Herceptin 6 mg / kg + Compound 3 0.7 mg / kg), which also shows that ISAC represented by Formula II-41 is more effective than small molecules. In this experiment, none of the experimental animals in the treatment group showed significant weight loss. It is preliminarily shown that ISAC represented by Formula II-41 has a good tumor inhibition effect and good safety.

[0396] Example 18: Immune Memory Effect of ISAC Represented by Formula II-41 in a Xenograft Model

[0397] This example evaluated the immune memory effect of ISAC represented by formula II-41 in a xenograft animal model. The test drugs were II-41 (3 mg / kg), II-41 (1 mg / kg), and PBS (control group).

[0398] In the immune memory experiment, E0771-HER2 tumors were implanted on the right side. When the tumor size reached 50-60 mm 3 ISAC of formula II-41 was used for treatment, and the drug was administered once every 3 days for a total of 3 times. On the 10th day, the tumors in all ISAC-treated groups completely disappeared. All mice with tumors in the control group underwent surgical resection of the right side of the tumor (primary tumor) and were immediately sutured. On the 14th day, all mice were injected intravenously with 2×10 6 E0771-HER2 cells were implanted into the left side of the mouse to detect memory effects.

[0399] The experimental results are shown in Figures 12-13. Compared to the control group, the ISAC-treated groups, including the II-41 (3 mg / kg) and II-41 (1 mg / kg) groups, all demonstrated a significant immune memory effect. After the second tumor inoculation, tumor volumes were significantly lower than those in the PBS control group. Notably, the II-41 (3 mg / kg) group experienced virtually no tumor recurrence after the second inoculation. None of the experimental animals in the treatment groups showed significant weight loss. This example demonstrates that treatment with the II-41 ISAC in mice establishes an immune memory effect that effectively prevents tumor recurrence.

[0400] Example 19: Pharmacological efficacy of ISAC represented by Formula II-41 in SKOV3 / PBMC co-culture cell line

[0401] This example evaluated the tumor cell cytotoxicity of the ISAC represented by Formula II-41 by activating the STING pathway in human peripheral blood cells (PBMCs) using a co-culture assay using SKOV3-GFP cancer cells. The test drugs included II-41 (16 nM), II-41 (1.6 nM), II-41 (Fab-blocked, 160 nM), II-41 (Fc-blocked, 160 nM), compound 3 (160 nM), compound 3 (16 nM), and PBS (control group).

[0402] The IncuCyte Cancer Cell Killing Assay assesses cancer cell death in co-culture with immune cells. SKOV3-GFP cells were plated and incubated overnight. The following day, each well was incubated with the test substance for 20 minutes at 37°C, followed by the addition of PBMCs (2x the concentration of SKOV3-GFP cells). The plate was then placed in an IncuCyte Live Cell Analyzer in an incubator (37°C, 5% CO2) and scanned every 2 hours for 24 hours. The number of green objects (cancer cells) was quantified over time using IncuCyte S3 (version 2022B) software.

[0403] The experimental results are shown in Figure 14. Compared to the control group, the ISAC-treated groups, including the II-41 (16 nM) and II-41 (1.6 nM) groups, strongly activated PBMC cells and demonstrated strong cytotoxicity against SKOV3-GFP tumor cells (disappearance of green fluorescence). Notably, II-41 (1.6 nM) exhibited significantly greater tumor cell cytotoxicity at a concentration 100-fold lower than that of compound 3 (160 nM), II-41 (Fab-blocked, 160 nM), and II-41 (Fc-blocked, 160 nM). This demonstrates that the combined immune activation of the Fab and Fc-termini of the ISAC represented by Formula II-41 significantly outperforms small molecules in immune activation.

[0404] To address the systemic side effects associated with the use of immune agonists, this application explored cleavable ISACs and, for the first time, designed an ISAC targeting the novel STING agonist diABZI-STING-agonist (Compound 3). Systematic evaluation tests of ISAC (II-13), including in vitro stability, activation of downstream cGAS-STING pathway signals, regulation of gene expression of related immune factors, induction of immunogenic cell death (ICD), plasma stability, and in vivo anti-tumor efficacy in xenograft models, have demonstrated the potential for this type of drug delivery model. The cleavable linker-based ISAC exhibits in vitro stability and immunoactivity that initially meet the drugability requirements for ADCs, while also exhibiting significant in vitro and in vivo efficacy and safety at therapeutic doses.

[0405] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present application can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present application. They should all be included in the scope of the technical solution for protection requested in this application.

Claims

1. A compound of formula II, its tautomers, geometric isomers, optical isomers, polymorphs, or pharmaceutically acceptable hydrates or solvates, in: A represents a biological macromolecule or its fragment, B 00 Represents a linker, used to connect A and -NR1R2- group, X represents a negatively charged anion. R1 and R2 are each independently C 1-8 Alkyl, C 1-8 Alkylcycloalkyl, C 4-8 Cycloalkyl, 4-8 membered heterocycloalkyl, C 1-8 Alkyl sugar, C 5-14 aryl, 5-14 membered heteroaryl, or R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocyclyl or heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 alkoxy, fluorine, chlorine, bromine, iodine, hydroxy, cyano, amino or nitro, M is -(CH2) o -、-(CH2) o -CH=CH-(CH2) p -、-O-(CH2) o -、-NH-(CH2) o -、-O-(CH2) o -CH=CH-(CH2) p -O-, -NH-(CH2) o -CH=CH-(CH2) p -NH- or -CH2-CHOH-CHOH-CH2-, wherein o and p are each independently an integer from 0 to 9, f is 0 or 1, R8 is -(CH2) a -、-(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-, wherein a is an integer from 1 to 8, R9 is C 1-8 Alkyl, C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino, R 10 With R 11 Each independently is C 1-4 Alkyl, -CONR a R b 、-CH2NR a R b 、-NR a R b 、-NR a C(=O)-R b 、-CH2NR a C(=O)-R b , -C(=O)OCH3 or -O-(CH2)e-OH, wherein R a and R b Each independently is H, hydroxyl or C 1-4 Alkyl, e is an integer from 1 to 9, R 12 With R 13 Each independently represents -(CH2) g -CH3, -O-(CH2) g -OH, -O-(CH2) g -CH3, -CONH2, Where g is a number from 1 to 9. d is a number between 1 and 20.

2. The compound of claim 1, its tautomer, geometric isomer, optical isomer, polymorph or pharmaceutically acceptable hydrate or solvate, wherein R 10 Located at the ortho, meta or para position of R8; and / or R 11 Located at the ortho, meta or para position of R9; Preferably, R 10 Located adjacent to R8, and R 11 Located in the ortho position of R9.

3. The compound of claim 1 or 2, its tautomer, geometric isomer, optical isomer, polymorph or pharmaceutically acceptable hydrate or solvate, wherein the compound has a structure as shown in Formula II-1, Among them, A, X, R1, R2, f, R8, R9, R 10 、R 11 、R 12 、R 13 d is defined as in claim 1 or 2, -B1-VLW- represents a linker for connecting A and -NR1R2- group, wherein B1 is selected from as well as Where k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; V is wherein each i is independently an integer between 0 and 12, each j is independently an integer between 0 and 12, or V is absent; L is -(CH2CH2O) k -(CH2) l -,-CHZ-,-(CH2) m -,or Where Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, m is an integer between 0 and 30, or L is absent; W is wherein R3, R4 and R6 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) c -NH2, -(CH2) c -NHC(NH)NH2, -CHCH(CH3)CH3 or -(CH2) c -NHC(=O)NH2, Where c is a number from 0 to 8; R5 is R 14 、R 15 Each is independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) h -NH2 or -(CH2) h , each h is independently a number from 0 to 8, A is coupled to the site ** of the B1 group through an S atom or a N atom in the biomacromolecule or its fragment.

4. The compound of any one of claims 1 to 3, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable salts, hydrates or solvates, characterized in that Any one or more of i) to x): i) X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - , SO4 2- , PO 4, 3- ,HPO4 2- or H2PO4 - ; Preferably, X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - ; Preferably, X is Cl - , HCOO - , CH3COO - ,CF3COO - ; Preferably, X is Cl - , Br - , I - ; Preferably, X is Cl - ; ii) R1 and R2 are each independently C 1-8 Alkyl, or R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocycloalkyl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 Alkoxy, fluorine, chlorine, bromine, iodine, hydroxyl, cyano, amino or nitro; preferably, R1, R2 and the N atom to which they are attached form a pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl or thiomorpholinyl; preferably, R1, R2 and the N atom to which they are attached form a or R1, R2 and the N atom to which they are attached form a 5-7 membered heteroaryl group, wherein the heteroaryl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 Alkoxy, fluorine, chlorine, bromine, iodine, hydroxyl, cyano, amino or nitro; preferably, R1, R2 and the nitrogen atom to which they are attached form an imidazolyl, triazolyl, indolyl, tetrazolyl, pyridyl, pteridinyl, pyrimidinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl or thiazinyl group; iii) M is -(CH2) o -CH=CH-(CH2) p -, wherein o and p are each independently an integer from 0 to 9; preferably, o and p are each independently an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6; iv) f is 0, or f is 1; v) R8 is -(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-; preferably, R8 is -(CH2) a -O-, preferably, wherein a is defined as in claim 1; further preferably, a is an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6; vi) R9 is C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino; preferably, R9 is C 1-8 Alkoxy; preferably, R9 is C 1-6 Alkoxy; preferably, R9 is C 1-4 Alkoxy; preferably, R9 is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy or 1,2-dimethylbutoxy; preferably, R9 is methoxy, ethoxy, n-propoxy or n-butoxy; preferably, R9 is C 1-6 Alkylthio; preferably, R9 is C 1-4 Alkylthio; preferably, R9 is methylthio, ethylthio, propylthio or butylthio; preferably, R9 is C 1-6 Alkylamino; preferably, R9 is C 1-4 Alkylamino; preferably, R9 is methylamino, ethylamino, propylamino or butylamino; vii) R 10 With R 11 Each independently is -CONR a R b 、-CH2NR a R b or -CH2NR a C(=O)-R b , preferably, R 10 With R 11 Each independently is -CONR a R b , where R a and R b The definition as claimed in claim 1; preferably, R a and R b Each independently is H or C 1-4 Alkyl; preferably, R a and R b are each independently H; preferably, R a and R b are each independently methyl, ethyl, n-propyl or n-butyl; viii)R 12 With R 13 Each independently Preferably, R 12 With R 13 Each independently Preferably, R 12 With R 13 Each independently ix) d is a number between 1 and 8; preferably, d is a number between 1 and 6, such as about 1, about 2, about 3, about 4, about 5 or about 6; x) A is an antibody or antigen-binding fragment thereof, a polypeptide, a protein, or an antigen; Preferably, A is an antibody or antigen-binding fragment thereof, a protein or an antigen; Preferably, A is an antibody or antigen-binding fragment thereof, polypeptide or protein that can specifically bind to the target; Preferably, A is an antibody or antigen-binding fragment or protein that can specifically bind to the target; Preferably, A is an antibody or antigen-binding fragment thereof that can specifically bind to the target; Preferably, A is an antigen; Preferably, A is a protein that can specifically bind to the target; Preferably, the antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, IgG antibody, mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), bispecific antibody, multispecific antibody, and probody; Preferably, the antibody binding fragment is selected from the group consisting of: single-chain antibody, dAb, complementarity determining region fragment, Fv, single-chain Fv (scFv), Fd, Fab, Fab', F(ab')2, and VHH (Nanobody or Nb). Preferably, the protein is albumin, preferably human serum albumin; Preferably, A is a monoclonal antibody or an antigen-binding fragment thereof; Preferably, A is a monoclonal antibody or antigen-binding fragment thereof with a thiol or amino group as a coupling site, or a monoclonal antibody or antigen-binding fragment thereof that has been site-directed mutated or modified with a thiol or amino group as a coupling site; Preferably, A is selected from the group consisting of: anti-HER2 humanized monoclonal antibody mil40, trastuzumab (HERCEPTIN), pertuzumab (PERJETA), cetuximab (ERBITUX), panitumumab (VECTIBIX), rituximab (RITUXAN), alemtuzumab (CAMPATH), ibritumomab tiuxetan (ZEVALIN), tositumomab (BEXXAR), ofatumumab (ARZERRA), bevacizumab (AVASTIN), ipilimumab (YE RVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi), sacituzumab, rovalvituzumab, antibacterial antibodies, their biosimilars or antigen-binding fragments, and site-directed mutagenesis or modification of the above monoclonal antibodies or antigen-binding fragments thereof; Preferably, A is trastuzumab or an antigen-binding fragment thereof.

5. The compound of claim 3 or 4, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates, characterized in that Any one or more of i) to iv): i) B1 is Preferably, B1 is Preferably, B1 is Wherein, k1 is defined as in claim 3; preferably, k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; further preferably, k1 is 1, 2, 3, 4, 5, 6, 7 or 8; further preferably, k1 is 2, 3, 4, 5 or 6; ii) V is wherein i and j are as defined in claim 3; preferably, i and j are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; further preferably, i and j are each independently 1, 2, 3, 4, 5, 6, 7 or 8; further preferably, i and j are each independently 1, 2, 3, 4, 5 or 6; iii) L is -(CH2CH2O) k -(CH2) l -, -CHZ-, or -(CH2) m -, or L is absent, wherein Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, k, l, m, n are defined as described in claim 3; preferably, each k is independently an integer between 0 and 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), each l is independently an integer between 0 and 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), n is an integer between 0 and 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), m is an integer between 0 and 20 (preferably m is an integer between 0 and 16 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)); preferably, L is absent; iv)W is wherein R3, R4 and R5 are defined as in claim 3; preferably, R3 and R4 are each independently methyl, ethyl, n-propyl, isopropyl or -(CH2) c -NHC(=O)NH2, wherein c is as defined in claim 3; preferably, R5 is where R 14 、R 15 The definition of as claimed in claim 3; Preferably, R3 and R4 are each independently methyl, isopropyl or -(CH2) c -NHC(=O)NH2; Preferably, c is an integer between 0 and 6, such as 0, 1, 2, 3, 4, 5, or 6; Preferably, R 14 、R 15 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; preferably, R 14 、R 15 Each independently is H, methyl, ethyl or n-propyl; preferably, R 14 、R 15 are each independently H or methyl; preferably, R 14 、R 15 Each independently is H; Preferably, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined above; Preferably, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined above.

6. The compound according to any one of claims 3 to 5, its tautomer, geometric isomer, optical isomer, polymorph or pharmaceutically acceptable hydrate or solvate, wherein the compound has the structure shown in formula II-2 or II-3, wherein A, X, V, L, W, and k1 are as defined in any one of claims 3 to 5, and A is coupled to site # via an S atom in a biomacromolecule or a fragment thereof, or is coupled to site ## via an N atom in a biomacromolecule or a fragment thereof.

7. The compound of any one of claims 1 to 6, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates, wherein the compound is selected from: in, The definitions of A, A and d are as described in any one of claims 1 to 6. Preferably, the S atom connected to A is derived from the sulfur atom in A (for example, the sulfur atom of a cysteine residue), and the -NH- connected to A is derived from the amino group in A (for example, the side chain amino group of a lysine, arginine, asparagine or glutamine residue).

8. A compound of formula I, its tautomers, geometric isomers, optical isomers, polymorphs, or pharmaceutically acceptable hydrates or solvates, in: B represents a linker, which is used to couple the -NR1R2- group to a biomacromolecule or a fragment thereof. X represents a negatively charged anion. R1 and R2 are each independently C 1-8 Alkyl, C 1-8 Alkylcycloalkyl, C 4-8 Cycloalkyl, 4-8 membered heterocycloalkyl, C 1-8 Alkyl sugar, C 5-14 aryl, 5-14 membered heteroaryl, or R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocyclyl or heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 alkoxy, fluorine, chlorine, bromine, iodine, hydroxy, cyano, amino or nitro, M is -(CH2) o -、-(CH2) o -CH=CH-(CH2) p -、-O-(CH2) o -、-NH-(CH2) o -、-O-(CH2) o -CH=CH-(CH2) p -O-, -NH-(CH2) o -CH=CH-(CH2) p -NH- or -CH2-CHOH-CHOH-CH2-, wherein o and p are each independently an integer from 0 to 9, f is 0 or 1, R8 is -(CH2) a -、-(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-, wherein a is an integer from 1 to 8, R9 is C 1-8 Alkyl, C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino, R 10 With R 11 Each independently is C 1-4 Alkyl, -CONR a R b 、-CH2NR a R b 、-NR a R b 、-NR a C(=O)-R b 、-CH2NR a C(=O)-R b , -C(=O)OCH3 or -O-(CH2)e-OH, wherein R a and R b Each independently is H, hydroxyl or C 1-4 Alkyl, e is an integer from 1 to 9, R 12 With R 13 Each independently represents -(CH2) g -CH3, -O-(CH2) g -OH, -O-(CH2) g -CH3, -CONH2, Where g is a number from 1 to 9.

9. The compound of claim 8, its tautomer, geometric isomer, optical isomer, polymorph or pharmaceutically acceptable hydrate or solvate, wherein R 10 Located at the ortho, meta or para position of R8; and / or R 11 Located at the ortho, meta or para position of R9; Preferably, R 10 Located adjacent to R8, and R 11 Located in the ortho position of R9.

10. The compound of claim 8 or 9, its tautomer, geometric isomer, optical isomer, polymorph, or pharmaceutically acceptable hydrate or solvate, wherein the compound has a structure as shown in Formula I-1, Among them, X, R1, R2, f, R8, R9, R 10 、R 11 、R 12 、R 13 d is defined as in claim 8 or 9, B0-VLW- represents a linker for coupling the -NR1R2- group to a biomacromolecule or a fragment thereof, wherein B0 is selected from or H, wherein r is an integer between 1 and 4; V is wherein each i is independently an integer between 0 and 12, each j is independently an integer between 0 and 12, or V is absent; L is -(CH2CH2O) k -(CH2) l -,-CHZ-,-(CH2) m -,or Where Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, m is an integer between 0 and 30, or L is absent; W is wherein R3, R4 and R6 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) c -NH2, -(CH2) c -NHC(NH)NH2, -CHCH(CH3)CH3 or -(CH2) c -NHC(=O)NH2, Where c is a number from 0 to 8; R5 is R 14 、R 15 Each is independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl, cyclobutane, cyclopentane, cyclohexane, -CH2(C6H5), -(CH2) h -NH2 or -(CH2) h , each h is independently a number from 0 to 8.

11. The compound of any one of claims 8 to 10, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable salts, hydrates or solvates, characterized in that Any one or more of i) to viii): i) X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - , SO4 2- , PO 4, 3- ,HPO4 2- or H2PO4 - ; Preferably, X is Cl - , Br - , I - , NO3 - , HCOO - , CH3COO - ,CF3COO - ; Preferably, X is Cl - , HCOO - , CH3COO - ,CF3COO - ; Preferably, X is Cl - , Br - , I - ; Preferably, X is Cl - ; ii) R1 and R2 are each independently C 1-8 Alkyl, or R1, R2 and the N atom to which they are attached form a 5-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heterocycloalkyl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 Alkoxy, fluorine, chlorine, bromine, iodine, hydroxyl, cyano, amino or nitro; preferably, R1, R2 and the N atom to which they are attached form a pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl or thiomorpholinyl; preferably, R1, R2 and the N atom to which they are attached form a or R1, R2 and the N atom to which they are attached form a 5-7 membered heteroaryl group, wherein the heteroaryl group contains 1 N atom and 1, 2 or 3 other heteroatoms selected from N, O or S atoms, and the heteroaryl group is optionally substituted by 1, 2, 3 or 4 R', each R' being independently H, C 1-8 Alkyl, C 1-8 Alkoxy, fluorine, chlorine, bromine, iodine, hydroxyl, cyano, amino or nitro; preferably, R1, R2 and the nitrogen atom to which they are attached form an imidazolyl, triazolyl, indolyl, tetrazolyl, pyridyl, pteridinyl, pyrimidinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl or thiazinyl group; iii) M is -(CH2) o -CH=CH-(CH2) p -, wherein o and p are each independently an integer from 0 to 9; preferably, o and p are each independently an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6; iv) f is 0, or f is 1; v) R8 is -(CH2) a -O-, -(CH2) a -S- or -(CH2) a -NH-; preferably, R8 is -(CH2) a -O-, preferably, wherein a is defined as in claim 1; further preferably, a is an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6; vi) R9 is C 1-8 Alkoxy, C 1-8 Alkylthio or C 1-8 Alkylamino; preferably, R9 is C 1-8 Alkoxy; preferably, R9 is C 1-6 Alkoxy; preferably, R9 is C 1-4 Alkoxy; preferably, R9 is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy or 1,2-dimethylbutoxy; preferably, R9 is methoxy, ethoxy, n-propoxy or n-butoxy; preferably, R9 is C 1-6 Alkylthio; preferably, R9 is C 1-4 Alkylthio; preferably, R9 is methylthio, ethylthio, propylthio or butylthio; preferably, R9 is C 1-6 Alkylamino; preferably, R9 is C 1-4 Alkylamino; preferably, R9 is methylamino, ethylamino, propylamino or butylamino; vii) R 10 With R 11 Each independently is -CONR a R b 、-CH2NR a R b or -CH2NR a C(=O)-R b , preferably, R 10 With R 11 Each independently is -CONR a R b , where R a and R b The definition as claimed in claim 1; preferably, R a and R b Each independently is H or C 1-4 Alkyl; preferably, R a and R b are each independently H; preferably, R a and R b are each independently methyl, ethyl, n-propyl or n-butyl; viii)R 12 With R 13 Each independently Preferably, R 12 With R 13 Each independently Preferably, R 12 With R 13 Each independently 12. The compound of claim 10 or 11, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates, characterized in that Any one or more of i) to iv): i) B0 is Preferably, B0 is Preferably, B0 is Preferably, B0 is Preferably, B0 is wherein r is as defined in claim 10; preferably, r is 1, 2, 3 or 4; preferably, r is 1 or 2; ii) V is wherein i and j are as defined in claim 3; preferably, i and j are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; further preferably, i and j are each independently 1, 2, 3, 4, 5, 6, 7 or 8; further preferably, i and j are each independently 1, 2, 3, 4, 5 or 6; iii) L is -(CH2CH2O) k -(CH2) l -, -CHZ-, or -(CH2) m -, or L is absent, wherein Z is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, k, l, m, n are defined as described in claim 3; preferably, each k is independently an integer between 0 and 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), each l is independently an integer between 0 and 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), n is an integer between 0 and 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), m is an integer between 0 and 20 (preferably m is an integer between 0 and 16 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)); preferably, L is absent; iv)W is wherein R3, R4 and R5 are defined as in claim 3; preferably, R3 and R4 are each independently methyl, ethyl, n-propyl, isopropyl or -(CH2) c -NHC(=O)NH2, wherein c is as defined in claim 3; preferably, R5 is where R 14 、R 15 The definition of as claimed in claim 3; Preferably, R3 and R4 are each independently methyl, isopropyl or -(CH2) c -NHC(=O)NH2; Preferably, c is an integer between 0 and 6, such as 0, 1, 2, 3, 4, 5, or 6; Preferably, R 14 、R 15 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; preferably, R 14 、R 15 Each independently is H, methyl, ethyl or n-propyl; preferably, R 14 、R 15 are each independently H or methyl; preferably, R 14 、R 15 Each independently is H; Preferably, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined above; Preferably, -VLW- is wherein i, j, k, l, m, R3, and R4 are as defined above.

13. The compound according to any one of claims 10 to 12, its tautomer, geometric isomer, optical isomer, polymorph or pharmaceutically acceptable hydrate or solvate, wherein the compound has the structure shown in Formula I-2, Wherein, the definitions of B0, X, V, L and W are as described in any one of claims 10-12.

14. The compound of any one of claims 1 to 6, its tautomers, geometric isomers, optical isomers, polymorphs, or pharmaceutically acceptable hydrates or solvates, wherein the compound is selected from:

15. Use of the compound according to any one of claims 8 to 14, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates in the preparation of an antibody-drug conjugate (e.g., an immunostimulatory antibody-drug conjugate).

16. A pharmaceutical composition or vaccine comprising a compound according to any one of claims 1 to 7, a tautomer, geometric isomer, optical isomer, polymorph or pharmaceutically acceptable hydrate or solvate thereof, and optionally one or more pharmaceutically acceptable carriers or excipients.

17. The use of a compound according to any one of claims 1 to 7, a tautomer, geometric isomer, optical isomer, polymorph or pharmaceutically acceptable hydrate or solvate thereof in the preparation of a medicament for treating and / or preventing a disease or condition or alleviating the severity of the disease or condition, wherein the disease or condition is selected from tumors, pathogen infections, diseases caused by pathogen infections, hematological diseases, metabolic diseases, inflammation, allergic diseases, autoimmune diseases, precancerous syndromes, tumor metastasis, cardiovascular diseases, Preferably, the tumor is a cancer, lymphoma, lymphoid tumor, blastoma, sarcoma or leukemia, Preferably, the pathogen infection is a bacterial infection or a viral infection; Preferably, the inflammation is inflammation of any tissue or organ in the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, eye inflammation, reproductive system inflammation and other inflammations; Preferably, the viral infection is an infection caused by severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, dengue virus, Zika virus, yellow fever virus or hepatitis B virus; Preferably, the bacterial infection is an infection caused by Gram-positive bacteria, penicillin-resistant bacteria, Staphylococcus aureus, Mycobacterium tuberculosis, drug-resistant Mycobacterium tuberculosis, Clostridium difficile or Chlamydia trachomatis; Preferably, the disease caused by the viral infection is COVID-19; Preferably, the disease caused by the pathogen infection is a disease caused by bacterial infection or viral infection; Preferably, the disease caused by the bacterial infection is bacterial lung infection, sepsis caused by Staphylococcus aureus pneumonia, Pseudomonas aeruginosa, Clostridium difficile and the like, tuberculosis, bacterial eye infection, heart, brain or skin infection, gastrointestinal infection, bacterial meningitis, or abscess in any organ (such as muscle, liver, meninges, or lung), cellulitis, bacteremia, skin necrosis, eyelid infection, eye infection, neonatal conjunctivitis, osteomyelitis, impetigo, ecthyma, scalded skin syndrome, food poisoning, pneumonia, surgical infection, urinary tract infection, burn infection, meningitis, endocarditis, sepsis, toxic shock syndrome, and septic arthritis; Preferably, the cancer is selected from the group consisting of breast cancer (e.g., HER2-positive breast cancer); squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; peritoneal cancer; liver cancer; stomach cancer; gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urethral cancer; hepatoma; intestinal cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; anal cancer; penile cancer; melanoma; multiple myeloma and B-cell lymphoma; brain cancer; gallbladder cancer; esophageal cancer; bile duct cancer; head and neck cancer and related metastases; Preferably, the autoimmune disease includes but is not limited to STING-associated vasculitis of infancy (SAVI), Aicardi Goutieres syndrome (AGS), pernio lupus, ataxia telangiectasia (also known as LouisBar syndrome), retinal vasculopathy with cerebral leukodystrophy (RCVL), systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, psoriasis, diabetes (including insulin-dependent diabetes mellitus (IDDM)), dermatomyositis, human immunodeficiency virus (HIV), AIDS, polymyositis, systemic sclerosis (scleroderma) and Sjögren's syndrome (SS), rheumatoid arthritis, Rheumatoid arthritis, psoriatic arthritis, polyarthritis, osteoarthritis, myasthenia gravis, polyarteritis nodosa, vasculitis, cutaneous vasculitis, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, autoimmune hepatitis, primary sclerosing cholangitis, Wegener's granulomatosis, microscopic polyangiitis, Behçet's disease, spondylitis, giant cell arteritis, polymyalgia rheumatica, Raynaud's phenomenon, primary biliary cirrhosis, primary vasculitis of the central nervous system, neuromyelitis optica, and mixed connective tissue disease.

18. Use of a compound according to any one of claims 1 to 7, its tautomers, geometric isomers, optical isomers, polymorphs or pharmaceutically acceptable hydrates or solvates in the preparation of a medicament for treating and / or preventing diseases and disorders mediated by STING.

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