Triazine compound as well as conjugate, preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a triazine compound as well as a conjugate, a preparation method and application thereof. Particularly, the invention relates to an antibody drug conjugate as shown in a formula I, and also relates to a compound as shown in a formula IV or VI or pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorphic substance, solvate, N-oxide, isotope labeled compound, metabolite or prodrug thereof. The invention also relates to a preparation method of the antibody drug conjugate, the compound or the derivative thereof. The invention also relates to a pharmaceutical composition containing the drug conjugate. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a triazine compound, a conjugate thereof, a preparation method and use thereof. Background Art

[0002] Chemical modification of proteins plays a vital role in exploring the biological functions of proteins and improving their pharmacological effects. Due to the complexity and metastability of protein structures, selective modification of proteins is challenging and usually requires special modification reagents and mild reaction conditions. The modification sites mainly include lysine residues, cysteine ​​residues, nitrogen termini and carbon termini of proteins.

[0003] Antibody-drug conjugates (ADCs) couple antibodies and bioactive molecules through specific linkers. Antibodies act as carriers to transport bioactive molecules into target cells, thereby reducing systemic exposure and improving safety. They are a hot topic in the research and development of targeted therapy. Traditional ADCs use lysine residues of antibodies or cysteine ​​residues obtained by opening interchain disulfide bonds for conjugation. The resulting ADCs are heterogeneous mixtures with different drug loadings and different connection positions, which are likely to affect the pharmacokinetics, stability, tolerance and efficacy of the drug. The new generation of ADC conjugation technology has turned to site-specific conjugation technology with precise drug loading and controlled connection sites. The ADCs obtained by this technology have a controllable drug-antibody ratio (DAR), high uniformity, better stability and pharmacokinetic properties, and are also more suitable for large-scale production of ADCs.

[0004] In recent years, a variety of site-specific coupling methods have been developed to provide precise modification of specific amino acid residues or sugar residues, including engineered mutant cysteine ​​coupling technology, engineered mutant non-natural amino acid coupling technology, glycosyl coupling technology and peptide enzymatic coupling technology, etc. However, these methods require antibody engineering or change the glycosylation characteristics of antibodies, and there are problems such as difficulty in protein expression, easy immunogenicity and low coupling yield.

[0005] In comparison, the disulfide bridging technology based on natural antibodies has more advantages in terms of cost and convenience. IgG1 antibodies contain four interchain disulfide bonds. After treatment with a reducing agent, eight reactive cysteine ​​residues can be exposed. The disulfide bridge linker reacts with two thiol groups respectively to reconnect the polypeptide chains, thereby achieving site-specific coupling and obtaining an ADC with controllable DAR value and high uniformity. The disulfide bridge linkers that are currently being studied include bissulfone derivatives, a new generation of maleimides (NGMs), and pyridazinediones (PDs). In recent years, many new disulfide bridge linkers have also emerged, including aryl dipropionitrile (ADPN), divinyl pyrimidine (DVP), and dibromomethylquinoxaline (C-Lock TM) and dichloroacetone, etc.

[0006] Although significant progress has been made in disulfide bridging technology, new approaches are still urgently needed to facilitate the development of homogeneous ADCs. Summary of the invention

[0007] The present application provides a class of triazine compounds, which can be used as disulfide bridges for synthesizing antibody-drug conjugates, achieving site-specific conjugation, and obtaining ADCs with controllable DAR values ​​and high uniformity.

[0008] In the first aspect, the present application provides an antibody drug conjugate as shown in formula (I):

[0009]

[0010] in,

[0011] Ab is the targeting group;

[0012] S comes from the cysteine ​​residue on the targeting group;

[0013] A is an unsubstituted or substituted heterocyclic group;

[0014] L is a linker;

[0015] D is a fragment of a biologically active molecule, including but not limited to cytotoxic compounds and immunomodulators;

[0016] m is an integer or a decimal number selected from 1 to 5.

[0017] In certain embodiments, the antibody drug conjugate has a structure shown in formula (II),

[0018]

[0019] For the antibody drug conjugates represented by general formula (I) or (II), in certain embodiments, A is unsubstituted or substituted with one or more R 1 A substituted heterocyclic group, wherein the heterocyclic group contains at least one N atom and is linked to the triazine through the N atom.

[0020] For the antibody drug conjugate shown in (II), L 1 It can be a covalent bond or a in;

[0021] L 1a is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3 Alkylene), C 2-6 Alkenylene (e.g. C 2-3 Alkenylene), C 2-6 Alkyne (e.g. C2-3 Alkyne), -O-, -S-, -N(R 2 )-、-C 1-6 Alkylene-O- (e.g. -C 1-3 Alkylene-O-), -C(=O)-, -C(=S)-, -C(=O)-O-, -C(=O)-N(R 3 )-、-C(=S)-N(R 3 )-、-C(=O)-N(R 3 )-N(R 3 )-、-N(R 3 )-O-、-N(R 3 )-N(R 3 )-、-N(R 3 )-C(=O)-, -OC(=O)-, -ON(R 3 )-, -OC(=O)-O-, -OC(=O)-N(R 3 )-, -S(=O)-, -S(=O)2-, -S(=O)2-N(R 3 )-、 C 3-10 Cycloalkyl (e.g. C 3-6 cycloalkyl) and 3-10 membered heterocyclyl (e.g. 3-6 membered heterocyclyl);

[0022] L 1b is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3 Alkylene), C 1-6 Haloalkylene (e.g. C 1-3 Haloalkylene), C 2-6 Alkenylene (e.g. C 2-3 Alkenylene), C 2-6 Alkyne (e.g. C 2-3 Alkyne),

[0023] L 1c is a covalent bond or is selected from

[0024] In certain embodiments, R 1 are independently selected from halogen (e.g., fluorine, chlorine, bromine, iodine), hydroxyl, cyano, C 1-6 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.), C 1-6 Haloalkyl (eg, trifluoromethyl) and oxo.

[0025] In certain embodiments, R 2 Selected from H, hydroxyl and C 1-6 In certain embodiments, R 2 is H. In certain embodiments, R 2 It is methyl.

[0026] In certain embodiments, R 3 Each independently selected from H and C 1-6 In certain embodiments, R 3 is H. In certain embodiments, R 3 It is methyl.

[0027] In certain embodiments, R 4 Each independently selected from H, C 1-6 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.), C 1-6 Haloalkyl (e.g. trifluoromethyl) and In certain embodiments, R 4 is H. In certain embodiments, R 4 It is methyl.

[0028] In certain embodiments, each a is independently selected from 1, 2, 3, 4, 5, and 6. In certain embodiments, a is 1 or 2.

[0029] In certain embodiments, each b is independently selected from an integer of 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In certain embodiments, each b is independently 1, 2, or 3. In certain embodiments, each b is independently 7 or 8.

[0030] In certain embodiments, each c is independently selected from 0, 1, 2, 3, 4, 5, and 6. In certain embodiments, each c is independently 1 or 2.

[0031] For the antibody drug conjugates represented by general formula (I) or (II), in certain embodiments, L 2 is a covalent bond or is selected from an amino acid fragment and a short peptide fragment formed by two or more amino acids, wherein the amino acid is selected from Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn, and the amino acid is optionally bound by one or more selected from H, C 1-6Alkyl and polyethylene glycol groups are substituted with hydrophilic structural units.

[0032] For the antibody drug conjugates represented by general formula (I) or (II), in certain embodiments, L 2 is a covalent bond or is selected from the following structures:

[0033]

[0034] R 5 Each independently selected from H, C 1-6 Alkyl and polyethylene glycol hydrophilic structural unit; preferably, the polyethylene glycol hydrophilic structural unit is In certain embodiments, R 5 is H;

[0035] Each d is independently selected from an integer of 1-10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0036] For the antibody drug conjugates represented by general formula (I) or (II), in certain embodiments, L 3 It can be a covalent bond or selected from the following structures:

[0037]

[0038]

[0039] For the antibody-drug conjugates represented by general formula (I) or (II), in certain embodiments, D can be selected from cytotoxic compound fragments and immunomodulator fragments, wherein the cytotoxic compound includes but is not limited to tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, wherein the tubulin inhibitors include but are not limited to dolastatin and auristatin, maytansine, tubulysins and cryptomycins, wherein the DNA damaging agents include but are not limited to PBD, duocarmycin and calicheamicin, wherein the topoisomerase inhibitors include but are not limited to camptothecin and its derivatives, epipodophyllotoxin, anthracycline and anthraquinone; wherein the immunomodulator is selected from Toll-like receptors (TLRs) modulators, STING (stimulator of interferon genes) modulators, RIG-I-like receptors (retinoic acid-inducible gene-I (RIG-I)-like receptors, RLRs) modulators, Nod-like receptors (nucleotide oligomerization domain (NOD)-like receptors, NLRs) modulators and C-type lectin receptors (C-type lectin receptors, CLRs) modulators.

[0040] For the antibody drug conjugates represented by general formula (I) or (II), in certain embodiments, A is unsubstituted or substituted with one or more R 1 A substituted 3-20 membered heterocyclyl (preferably a 6-18 membered heterocyclyl, such as a 6-, 12- or 18-membered heterocyclyl) containing at least one N atom and connected to the triazine via the N atom. In certain embodiments, A is selected from piperazine, piperidine, 1,10-diaza-18-crown-6-ether and 4,10-diaza-12-crown-4-ether. In certain embodiments, A is piperazine. In certain embodiments, A is 1,10-diaza-18-crown-6-ether.

[0041] In certain embodiments, L 1 is a covalent bond or is in;

[0042] L 1a is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3Alkylene), -O-, -S-, -N(R 2 )-、-C 1-6 Alkylene-O- (e.g. -C 1-3 Alkylene-O-), -C(=O)-N(R 3 )-、-N(R 3 )-C(=O)-、-OC(=O)-N(R 3 )-、-S(=O)2-N(R 3 )-、 C 3-10 Cycloalkyl (e.g. 3-6 membered cycloalkyl) and 3-10 membered heterocyclyl (e.g. 3-6 membered heterocyclyl);

[0043] L 1b is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3 Alkylene),

[0044] L 1c is a covalent bond or is selected from

[0045] R 2 Selected from H, hydroxyl and C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.); in certain embodiments, R 2 is H; in certain embodiments, R 2 is methyl;

[0046] R 3 Each independently selected from H and C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.); in certain embodiments, R 3 is H; in certain embodiments, R 3 is methyl;

[0047] R 4 Each independently selected from H, C 1-6 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.), C 1-6 Haloalkyl (e.g. trifluoromethyl) and In certain embodiments, R 4 is H; in certain embodiments, R 4 is methyl;

[0048] a is each independently selected from 1, 2, 3, 4, 5 and 6; in certain embodiments, a is each independently 1 or 2;

[0049] b is each independently selected from an integer of 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); in certain embodiments, b is each independently 1, 2 or 3; in certain embodiments, b is each independently 7 or 8;

[0050] Each c is independently selected from 0, 1, 2, 3, 4, 5 and 6; in certain embodiments, each c is independently 1 or 2.

[0051] In certain embodiments, L 1 is a covalent bond or is selected from the following structures:

[0052]

[0053]

[0054] Among them, position 1 is connected to A, and position 2 is connected to L 2 connected;

[0055] R 2 Selected from H, hydroxyl and C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.); in certain embodiments, R 2 is H; in certain embodiments, R 2 is methyl;

[0056] R 4 Each independently selected from H, C 1-6 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.), C 1-6 Haloalkyl (e.g. trifluoromethyl) and In certain embodiments, R 4 is H; in certain embodiments, R 4 is methyl;

[0057] a is each independently selected from 1, 2, 3, 4, 5 and 6; in certain embodiments, a is each independently 1 or 2;

[0058] b is each independently selected from an integer of 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); in certain embodiments, b is each independently 1, 2 or 3; in certain embodiments, b is each independently 7 or 8;

[0059] Each c is independently selected from 0, 1, 2, 3, 4, 5 and 6; in certain embodiments, each c is independently 1 or 2.

[0060] In certain embodiments, L 1 is a covalent bond or is:

[0061]

[0062]

[0063] Among them, position 1 is connected to A, and position 2 is connected to L 2 connected;

[0064] R 2 or R 4 Each independently selected from H, hydroxyl and C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.); in certain embodiments, R 2 is H; in certain embodiments, R 2 is methyl; in certain embodiments, R 4 is H; in certain embodiments, R 4 is methyl;

[0065] a is each independently selected from 1, 2, 3, 4, 5 and 6; in certain embodiments, a is each independently 1 or 2;

[0066] b is each independently selected from an integer of 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); in certain embodiments, b is each independently 1, 2 or 3; in certain embodiments, b is each independently 7 or 8.

[0067] In certain embodiments, L 2 is a covalent bond or is selected from the following structures:

[0068]

[0069] In certain embodiments, L 3 is a covalent bond or is selected from the following structures:

[0070]

[0071] In certain embodiments, the formula is a covalent bond or is selected from the following structures:

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Among them, position 1 is connected to A, and position 4 is connected to D;

[0080] R 2 or R 4 Each independently selected from H, hydroxyl and C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.); in certain embodiments, R 2 is H; in certain embodiments, R 2 is methyl; in certain embodiments, R 4 is H; in certain embodiments, R 4 is methyl;

[0081] a is each independently selected from 1, 2, 3, 4, 5 and 6; in certain embodiments, a is each independently 1 or 2;

[0082] b is each independently selected from an integer of 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); in certain embodiments, b is each independently 1, 2 or 3; in certain embodiments, b is each independently 7 or 8.

[0083] In certain embodiments, the formula Select from the following structures:

[0084]

[0085] Among them, position 1 is connected to A, and position 4 is connected to D;

[0086] a is 1 or 2;

[0087] b are each independently 1, 2, 3, 7 or 8;

[0088] R 4 Select from H or C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.); preferably, R 4 Selected from H.

[0089] In certain embodiments, the formula Select from the following structures:

[0090]

[0091] In certain embodiments, the formula Select from the following structures:

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] wherein position 1 is connected to A and position 4 is connected to D. In certain embodiments, the formula Selected from:

[0103]

[0104] wherein position 1 is connected to A and position 4 is connected to D. In certain embodiments, the formula Selected from:

[0105]

[0106] Among them, position 1 is connected to A, and position 4 is connected to D.

[0107] In certain embodiments, the formula Selected from:

[0108]

[0109] Among them, position 1 is connected to A, and position 4 is connected to D.

[0110] For the antibody drug conjugates of the present invention, in certain embodiments, D is selected from a cytotoxic compound fragment, wherein the cytotoxic compound is selected from a tubulin inhibitor, a DNA damaging agent and a topoisomerase inhibitor, wherein the tubulin inhibitor includes dolastatin, auristatin, maytansine, tubulysins and cryptomycins, wherein the DNA damaging agent includes PBD, duocarmycin and calicheamicin, and the topoisomerase inhibitor includes camptothecin and its derivatives.

[0111] In certain embodiments, D is selected from a microtubule inhibitor fragment and a topoisomerase inhibitor fragment, wherein the microtubule inhibitor is selected from dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3 and DM4, and the topoisomerase inhibitor is selected from camptothecin, SN-38, exitecan, topotecan, belotecan, rubitecan, diflutecan, 10-hydroxycamptothecin, 9-aminocamptothecin, doxorubicin, epirubicin and PNU-159682.

[0112] In certain embodiments, D is selected from an immunomodulator fragment selected from a TLR modulator, a NOD1 modulator, a NOD2 modulator, a RIG-I modulator, an MDA5 modulator, a LGP2 modulator, a Dectin-1 modulator, a Dectin-2 modulator, an ALRs modulator, and a STING modulator.

[0113] In certain embodiments, the immunomodulator is selected from a TLR agonist; the TLR agonist is selected from a TLR2 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist, and a TLR9 agonist; in certain embodiments, the TLR agonist is selected from a TLR7 agonist, a TLR8 agonist, and a TLR7 / 8 agonist.

[0114] In certain embodiments, D is selected from the following structures:

[0115]

[0116]

[0117] In certain embodiments, D is selected from D 1 -1, D 3 -1, D 3 -4, D 4 -7,D 4 -8,D 4-9,D 4 -10 or D 4 -11.

[0118] For the antibody-drug conjugates of the present invention, in certain embodiments, Ab is a targeting group that targets a cell surface receptor or a tumor surface antigen.

[0119] In certain embodiments, Ab is an antibody or an antigen-binding fragment thereof; the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a murine antibody, a humanized antibody, a fully human antibody, and a fusion protein comprising the antigen-binding portion of an antibody; the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, a disulfide-linked Fv, and scFv.

[0120] In certain embodiments, the antibody or antigen-binding fragment thereof is an anti-Her-2 antibody.

[0121] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0122] (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0123] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 3 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 4 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof;

[0124] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0125] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 9 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 10 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof;

[0126] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0127] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 12 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 15 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; or

[0128] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0129] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof.

[0130] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: VH shown in SEQ ID NO: 18 or a variant thereof, and / or, VL shown in SEQ ID NO: 19 or a variant thereof.

[0131] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain of VH shown in SEQ ID NO: 18 or a variant thereof and a weight constant region (CH) shown in SEQ ID NO: 20 or a variant thereof, and / or a light chain of VL shown in SEQ ID NO: 19 or a variant thereof and a light chain constant region (CL) shown in SEQ ID NO: 21 or a variant thereof.

[0132] The antibody used in the present invention is preferably trastuzumab, the sequence of which has been disclosed in, for example, CN103319599A. The last Lys of the heavy chain of trastuzumab is easily deleted, but this deletion does not affect the biological activity, see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143. The above-mentioned trastuzumab and its sequence or fragment with the last Lys of the heavy chain deleted belong to the trastuzumab described in the present invention.

[0133] In certain embodiments, the heavy chain sequence of the trastuzumab is shown as SEQ ID NO:1, and the light chain sequence of the trastuzumab is shown as SEQ ID NO:2.

[0134] In certain embodiments, the antibody drug conjugate of the invention has the structure of formula (III-A) or formula (III-B) or formula (III-C) or formula (III-D):

[0135]

[0136] Among them: Ab, L 1 , L 2 , L 3 , D and m are as defined in any of the above.

[0137] In certain embodiments, the antibody drug conjugates of the present invention are selected from the following structures:

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] In a second aspect, the present application provides a compound of formula (IV) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0163]

[0164] A is a substituted or unsubstituted heterocyclic group;

[0165] L is a linker;

[0166] D is a fragment of a biologically active molecule, including but not limited to cytotoxic compounds and immunomodulators.

[0167] In certain embodiments, A, L, and D are as defined in any of the above.

[0168] In certain embodiments, the compound has the structure of Formula (VA) or Formula (VB) or Formula (VC) or Formula (VD):

[0169]

[0170] Among them, L 1 , L 2 and L 3 are independently selected linkers;

[0171] D is a fragment of a biologically active molecule, including but not limited to cytotoxic compounds and immunomodulators.

[0172] In certain embodiments, L 1 , L 2 , L 3 and D are as defined in any of the above.

[0173] In certain embodiments, the compound of the second aspect of the present application or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the compound is selected from the following structures:

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] In a third aspect, the present application provides a compound of formula (VI) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0197]

[0198] in,

[0199] A is unsubstituted or substituted with one or more R 6 A substituted heterocyclic group, wherein the heterocyclic group contains at least one N atom and is connected to the triazine through the N atom;

[0200] Q 1 is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3 Alkylene), C 2-6 Alkenylene (e.g. C 2-3 Alkenylene), C 2-6 Alkyne (e.g. C 2-3 Alkyne), -O-, -S-, -N(R 7 )-、-C 1-6 Alkylene-O- (e.g. -C 1-3 Alkylene-O-), -C(=O)-, -C(=S)-, -C(=O)-O-, -C(=O)-N(R 8 )-、-C(=S)-N(R 8 )-、-C(=O)-N(R 8 )-N(R 8 )-、-N(R 8 )-O-、-N(R 8 )-N(R 8 )-、-N(R 8 )-C(O)-、-OC(=O)-、-ON(R 8 )-, -OC(=O)-O-, -OC(=O)-N(R 8 )-, -S(=O)-, -S(=O)2-, -S(=O)2-N(R 8 )-、 C 3-10 Cycloalkyl (e.g. C 3-6cycloalkyl) and 3-10 membered heterocyclyl (e.g. 3-6 membered heterocyclyl);

[0201] Q 2 is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3 Alkylene), C 1-6 Haloalkylene (e.g. C 1-3 Haloalkylene), C 2-6 Alkenylene (e.g. C 2-3 Alkenylene), C 2-6 Alkyne (e.g. C 2-3 Alkyne),

[0202] Q 3 Selected from H, azide, C 2-6 Alkynyl, Hydroxyl, -OC 1-6 Alkyl (e.g. -OC 1-3 Alkyl), -OC 1-6 Haloalkyl (e.g. -OC 1-3 haloalkyl), -O-benzyl, -N(R 9 )-C 1-6 Alkylene-C 2-6 Alkynyl (e.g. -N(R 9 )-C 1-3 Alkylene-alkynyl), C 1-6 Alkyl (e.g. C 1-3 alkyl), benzyl, acetyl, trifluoroacetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, fluorenyloxycarbonyl, C 3-10 Cycloalkyl (e.g. C 3-6 cycloalkyl), 3-10 membered heterocyclic group (e.g. 3-6 membered heterocyclic group), C 6-10 Aryl and 5-12 membered heteroaryl (e.g. 5-6 membered heteroaryl);

[0203] R 6 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.), C 1-6 Alkyl halides (e.g. trifluoromethyl) and oxo;

[0204] R 7 Selected from H, hydroxyl and C 1-6 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.);

[0205] R 8 Each independently selected from H and C 1-6Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.);

[0206] R 9 Each independently selected from H, C 1-6 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.), C 1-6 Haloalkyl (e.g. trifluoromethyl), hydroxyl and

[0207] e is each independently selected from 1, 2, 3, 4, 5 and 6; in certain embodiments, e is 1 or 2;

[0208] f is each independently selected from an integer of 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); in certain embodiments, f is 1, 2 or 3; in certain embodiments, f is 7 or 8;

[0209] Each g is independently selected from 0, 1, 2, 3, 4, 5 and 6; in certain embodiments, g is 1 or 2.

[0210] In certain embodiments, the compound described in the third aspect of the present application or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, wherein:

[0211] A is unsubstituted or substituted with one or more R 6 a substituted 3-20 membered heterocyclyl (preferably a 6-18 membered heterocyclyl, such as a 6-, 12- or 18-membered heterocyclyl) containing at least one N atom and linked to the triazine via the N atom, such as piperazine, piperidine, 1,10-diaza-18-crown-6-ether and 4,10-diaza-12-crown-4-ether;

[0212] Q 1 is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3 Alkylene), -O-, -S-, -N(R 7 )-、-C 1-6 Alkylene-O- (e.g. -C 1-3 Alkylene-O-), -C(=O)-N(R 8 )-、-N(R 8 )-C(=O)-、-OC(=O)-N(R 8 )-、-S(=O)2-N(R 8 )-、 C3-10 Cycloalkyl (e.g. C 3-6 cycloalkyl) and 3-10 membered heterocyclyl (e.g. 3-6 membered heterocyclyl);

[0213] Q 2 is a covalent bond or is selected from C 1-6 Alkylene (e.g. C 1-3 Alkylene),

[0214] Q 3 Selected from H, azide, C 2-6 Alkynyl, Hydroxyl, -OC 1-6 Alkyl (e.g. -OC 1-3 Alkyl), -OC 1-6 Haloalkyl (e.g. -OC 1-3 haloalkyl), -O-benzyl, -N(R 9 )-C 1-6 Alkylene-C 2-6 Alkynyl (e.g. -N(R 9 )-C 1-3 Alkylene-alkynyl), C 1-6 Alkyl (e.g. C 1-3 alkyl), benzyl, acetyl, trifluoroacetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, fluorenyloxycarbonyl, C 3-10 Cycloalkyl (e.g. C 3-6 cycloalkyl), 3-10 membered heterocyclic group (e.g. 3-6 membered heterocyclic group), C 6-10 Aryl and 5-12 membered heteroaryl (e.g. 5-6 membered heteroaryl);

[0215] R 6 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl (e.g. C 1-3 Alkyl), C 1-6 Haloalkyl (e.g. C 1-3 haloalkyl) and oxo;

[0216] R 7 Selected from H, hydroxyl and C 1-6 Alkyl (e.g. C 1-3 alkyl);

[0217] R 8 Each independently selected from H and C 1-6 Alkyl (e.g. C 1-3 alkyl);

[0218] R 9 Each independently selected from H, C 1-6 Alkyl (e.g. C 1-3 Alkyl), C 1-6Haloalkyl (e.g. C 1-3 haloalkyl), hydroxyl and

[0219] e is each independently selected from 1, 2, 3, 4, 5 and 6; in certain embodiments, e is 1 or 2;

[0220] f is each independently selected from an integer of 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); in certain embodiments, f is 1, 2 or 3; in certain embodiments, f is 7 or 8;

[0221] Each g is independently selected from 0, 1, 2, 3, 4, 5 and 6; in certain embodiments, g is 1 or 2.

[0222] In certain embodiments, the compound has the structure of Formula (VII-A) or Formula (VII-B) or Formula (VII-C) or Formula (VII-D):

[0223]

[0224] Among them, Q 1 , Q 2 and Q 3 As defined in any of the above.

[0225] In certain embodiments, the compound described in the third aspect of the present application or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, the compound is selected from the following structures:

[0226]

[0227]

[0228]

[0229]

[0230] In a fourth aspect, the present application relates to a pharmaceutical composition comprising the antibody-drug conjugate described in the first aspect of the present application, and a pharmaceutically acceptable carrier; optionally, the pharmaceutical composition further comprises one or more other anticancer drugs such as chemotherapeutic agents and / or antibodies.

[0231] In a fifth aspect, the present application relates to use of the antibody-drug conjugate described in the first aspect of the present application in the preparation of a drug for preventing or treating diseases related to abnormal cell proliferation (eg, cancer).

[0232] In a sixth aspect, the present application relates to a method for preventing or treating a disease related to abnormal cell proliferation (eg, cancer), comprising administering an effective amount of the antibody-drug conjugate described in the first aspect of the present application to a subject in need thereof.

[0233] In certain embodiments, the cancer is selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer.

[0234] In the seventh aspect, the present application relates to the use of the compound described in the second aspect or the third aspect or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug in the preparation of an antibody drug conjugate.

[0235] In an eighth aspect, the present application relates to a method for preparing the antibody-drug conjugate described in the first aspect of the present application, the method comprising: conducting a coupling reaction between a reduced antibody or an antigen-binding fragment thereof and the compound described in the second aspect of the present application or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof to obtain the antibody-drug conjugate. The antibody or antigen-binding fragment thereof can be reduced by a thiol reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)).

[0236] definition

[0237] Unless otherwise defined below, the meanings of all technical terms and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the technology used herein is intended to refer to the technology generally understood in the art, including those changes in technology or replacement of equivalent technology that are obvious to those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0238] The terms "comprises," "comprising," "having," "containing," or "involving" and other variations thereof herein are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0239] The terms "conjugate" or "conjugate" are interchangeable and refer to a substance obtained by linking one or more heterologous molecules (heterologous molecule(s)) to an antibody, wherein the heterologous molecules include but are not limited to cytotoxic agents (cytotoxic agent), and the English name is ADC (antibody-drug conjugate).

[0240] The one or more heterologous molecules can be connected to the antibody via a linker. The linker can be connected to the antibody via various chemical bonds. For example, in some embodiments, the linker is connected by forming a thioether bond with the thiol group of the antibody. In some specific ADC molecular structures, -S- only represents the thioether bond formed by the linker and the thiol group of the antibody, and does not mean that -S- is part of the linker.

[0241] The structure of the antibody-drug conjugate of the present application can be represented by the general formula I, wherein m refers to the number of bioactive molecular fragments connected to each antibody molecule. During the preparation of the antibody-drug conjugate, each antibody molecule may be connected to a different number of bioactive molecular fragments. Therefore, in general, the antibody-drug conjugate is a mixture of antibody-drug conjugates with different drug-antibody coupling ratios. In practice, DAR is usually used to represent the average value of the number of drugs connected to the antibody.

[0242] As used herein, the term "antibody" is used in the broadest sense, including various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen binding activity. For example, an immunoglobulin molecule can be composed of two pairs of polypeptide chains (each pair having a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light chain and heavy chain, the variable region and the constant region 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 (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins 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. The VH and VL regions can also be subdivided into highly variable regions (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883, or related research by AbM, Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272).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.

[0243] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the antibody variable region 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, for example, 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), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or, Martin's related work (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272), which integrates part of the definitions of Kabat and Chothia, and was first used in the Oxford Molecular antibody modeling software (Martin AC R. Protein sequence and structure analysis of antibody variable domains [M] / / Antibody engineering. Springer, Berlin, Heidelberg, 2010: 33-51.). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to a person skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0244] The CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention 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 invention are preferably identified by the Chothia numbering system.

[0245] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody, which includes a portion of an intact antibody and binds to an antigen that the intact antibody binds to. For example, a polypeptide of a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds to, and / or competes with the full-length antibody for specific binding to the antigen, is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, 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 techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0246] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains". Wherein, "full-length heavy chain" or "heavy chain" refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N to C; and, when the full-length antibody is an IgE isotype, optionally also includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N to C. A "full-length light chain" or "light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N to C. Two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be from a single species, such as human; or may be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0247] Herein, the technology for obtaining antibodies can use conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) to obtain an antigen-binding fragment of the antibody (e.g., the above-mentioned antibody fragment) from a given antibody (e.g., an antibody provided by the present invention), and screen the antigen-binding fragment of the antibody for specificity in the same manner as for intact antibodies.

[0248] As used herein, the terms "monoclonal antibody", "single antibody", and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody 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, relative to monoclonal antibodies, generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

[0249] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0250] For example, monoclonal antibodies can be prepared as follows. First, mice or other suitable host animals are immunized with immunogens (adjuvants are added when necessary). The injection method of immunogens or adjuvants is usually subcutaneous multi-point injection or intraperitoneal injection. The immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). The hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium preferably contains one or more substances that can inhibit the growth of unfused, parent myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin and thymidine (HAT culture medium) to the culture medium will inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have characteristics such as high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among them, myeloma cells are preferably mouse myeloma, such as MOP-21 or MC-11 mouse tumor derivatives (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md. USA). In addition, there are also studies reporting the use of human myeloma and human-mouse heteromyeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium of growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).For example, the affinity of monoclonal antibodies can be determined by the Scatchard analysis method described by Munson et al. in Anal. Biochem. 107: 220 (1980). After determining the specificity, affinity and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned by the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Suitable culture fluids can be DMEM or RPMI-1640, etc. In addition, hybridoma cells can also grow in animals in the form of ascites tumors. Using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by subcloned cells can be separated from cell culture fluid, ascites or serum.

[0251] Monoclonal antibodies can also be obtained through genetic engineering recombination technology. Using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody for PCR amplification, DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells. The obtained DNA molecules are inserted into expression vectors, and then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins), and cultured under appropriate conditions to obtain recombinantly expressed target antibodies.

[0252] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (target molecule recognized by the antibody) or its antigenic epitope can be immobilized on a column and the immunospecific antibody can be purified by immunoaffinity chromatography. The purification of immunoglobulins can be referred to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0253] As used herein, the term "mouse antibody" refers to the fusion of B cells from immunized mice with myeloma cells, followed by screening of mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation and antibody purification. Or, after antigens invade the mouse body, B cells differentiate and proliferate to form plasma cells, which can produce secretory antibodies. Produced by specific antigen stimulation, the production of antibodies is due to the interaction of various immune cells caused by antigens invading the human body, causing B cells in lymphocytes to differentiate and proliferate to form plasma cells, which can produce secretory antibodies.

[0254] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0255] Humanized antibodies can retain the expected properties of non-human donor antibodies (e.g., mouse antibodies) and can effectively reduce the immunogenicity of non-human donor antibodies (e.g., mouse antibodies) in human subjects, and therefore are particularly advantageous. However, due to the matching problem between the CDR of the donor antibody and the FR of the acceptor antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response) are usually lower than those of non-human donor antibodies (e.g., mouse antibodies).

[0256] Therefore, although researchers in this field have conducted in-depth studies on antibody humanization and have made some progress (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)), the prior art does not provide detailed guidance on how to fully humanize a donor antibody so that the generated humanized antibody has the highest possible degree of humanization and can retain the expected properties of the donor antibody as much as possible. Technicians need to explore, investigate and modify specific donor antibodies and put in a lot of creative work to obtain humanized antibodies that have a high degree of humanization (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% humanization) and retain the expected properties of the specific donor antibodies.

[0257] In the present invention, in order to make the humanized antibody retain the properties of the donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, the ability to increase the activity of immune cells and / or the ability to enhance the immune response), the framework region (FR) in the humanized antibody of the present invention may contain both the amino acid residues of the human acceptor antibody and the amino acid residues of the corresponding non-human donor antibody.

[0258] The humanized antibodies of the present invention can be prepared according to the sequence of the mouse monoclonal antibody prepared above.DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques.

[0259] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of not producing endogenous immunoglobulins after immunization and are capable of producing a complete human antibody repertoire. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies when challenged with antigen (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of the above transgenic animals include, HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); or "KM mice TM" carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996, Nature Biotech 14: 309).

[0260] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50) of the interaction.

[0261] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, bioluminescence interferometry (e.g., ForteBio Octet method) can be used to measure the dissociation constant. In addition, surface plasmon resonance technology (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0262] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, 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.

[0263] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0264] As used herein, the term "prevention" refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, a tumor) in a subject.

[0265] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" may also refer to extending survival compared to the expected survival (if not receiving treatment).

[0266] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, or has a risk of suffering from the above-mentioned disease.

[0267] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has 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 to be 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 other treatments administered simultaneously, etc.

[0268] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes blood tumors, especially hematological malignancies.

[0269] As used herein, the term "alkyl" is defined as a straight or branched chain saturated aliphatic hydrocarbon group. For example, as used herein, the term "C 1-6 "Alkyl" refers to a straight or branched chain group having 1 to 6 carbon atoms (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl), which is optionally substituted with one or more (e.g. 1 to 3) suitable substituents such as halogen. The term "C 1-3 "Alkyl" refers to a straight or branched chain group having 1 to 3 carbon atoms (eg methyl, ethyl, n-propyl, isopropyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents such as halogen.

[0270] As used herein, the term "alkoxy" refers to "alkyl-O-", wherein "alkyl" is as defined above. For example, the term "C 1-6 "Alkoxy" refers to "C 1-6 Alkyl-O-", the "C 1-6 "C alkyl" is as defined above. 1-3 "Alkoxy" refers to "C 1-3 Alkyl-O-", the "C 1-3 "Alkyl" is as defined above. Exemplary C 1-6 Alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy or n-hexoxy.

[0271] The term "alkylene" refers to a divalent group derived from a straight or branched chain alkane losing two hydrogen atoms, including, for example, "C 1-20 Alkylene", "C 1-10 Alkylene", "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0272] The term "alkenylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond losing two hydrogen atoms, including, for example, "C 2-6 Alkenylene", "C 2-4 Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, and the like.

[0273] The term "alkynylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond losing two hydrogen atoms. 2-6 Alkynylidene", "C 4-6 Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, and the like.

[0274] As used herein, the term "aryl" or "aromatic ring" refers to an all-carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated π electron system. 6-10 Aryl" or "C 6-10 "Aromatic ring" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl (ring) or naphthyl (ring). The aryl group is optionally substituted by 1 or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO2, C 1-6 alkyl, etc.) substituted.

[0275] In this article, the term "heteroaryl" refers to a cyclic group with aromaticity, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the ring structure can be oxoed. Specific examples include but are not limited to 5-10 yuan heteroaryl, 5-10 yuan nitrogen-containing heteroaryl, 6-10 yuan oxygen-containing heteroaryl, 6-8 yuan nitrogen-containing heteroaryl, 5-8 yuan oxygen-containing heteroaryl, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl oxazolyl, pyridinyl, 2-pyridone, 4-pyridone, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0276] The term "heterocyclic group" refers to a group formed by a saturated or partially saturated non-aromatic cyclic structure containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 5-6 membered heterocyclic groups, 5-6 membered nitrogen-containing heterocyclic groups, 5-6 membered oxygen-containing heterocyclic groups, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.

[0277] As used herein, the term "halogen" group is defined to include fluorine, chlorine, bromine, or iodine.

[0278] As used herein, the term "halo" refers to substitution with one or more (such as 1 to 3) the same or different halogen atoms.

[0279] As used herein, the term "haloalkyl" refers to an alkyl group substituted by one or more (such as 1 to 3) the same or different halogen atoms. 1-6The term "haloalkyl" refers to a halogenated alkyl group having 1 to 6 carbon atoms, for example, -CF3, -C2F5, -CHF2, -CH2F, -CH2CF3, -CH2Cl or -CH2CH2CF3, etc.

[0280] Optionally, hydrogen in the groups referred to in the present invention may be replaced by deuterium.

[0281] The term "substituted" means that one or more (e.g., 1, 2, 3, or 4) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that the normal valence of the designated atom in the present context is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds.

[0282] If a substituent is described as being "optionally substituted with," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and / or together, with independently selected substituents or unsubstituted. If a nitrogen of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced, with an independently selected substituent or unsubstituted.

[0283] If a substituent is described as being "independently selected" from a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0284] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, where reasonable.

[0285] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0286] When a bond to a substituent is shown to pass through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

[0287] The present invention also includes all pharmaceutically acceptable isotope-labeled compounds, which are identical to the compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g. 2 H. 3 H, deuterium D, tritium T); isotopes of carbon (e.g.11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (e.g. 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); isotopes of oxygen (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (e.g. 32 P); and sulfur isotopes (e.g. 35 S). Certain isotopically-labeled compounds of the invention (e.g., those incorporating a radioactive isotope) are useful in drug and / or substrate tissue distribution studies (e.g., assays). 3 H) and carbon-14 (i.e. 14 C) are particularly useful for this purpose because of their ease of incorporation and ease of detection. 11 C. 18 F. 15 O and 13 N) can be used to examine substrate receptor occupancy in positron emission tomography (PET) studies. Isotope-labeled compounds of the present invention can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations by using appropriate isotope-labeled reagents instead of the non-labeled reagents previously employed. Pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent can be isotopically substituted, for example, D2O, acetone-d6 or DMSO-d6.

[0288] The term "stereoisomer" means an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., 1, 2, 3, or 4) asymmetric centers, it can produce racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of the present application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0289] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

[0290] It should also be understood that certain compounds of the present invention may exist in free form for treatment, or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites or prodrugs, which, after being administered to a patient in need thereof, can directly or indirectly provide a compound of the present invention or a metabolite or residue thereof. Therefore, when referring to "compounds of the present invention" herein, the above-mentioned various derivative forms of the compounds are also intended to be covered.

[0291] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. A review of suitable salts is provided in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002) by Stahl and Wermuth. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0292] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound. The amount of polar solvent, especially water, may be present in a stoichiometric or non-stoichiometric ratio.

[0293] Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires an available lone pair of electrons to oxidize to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, including oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and meta-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxirane such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see for example: TL Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750; AR Katritzky and AJ Boulton, Eds., Academic Press; and GWH Cheeseman and ES G Werstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, AR Katritzky and AJ Boulton, Eds., Academic Press.

[0294] Also included within the scope of the present invention are metabolites of the compounds of the present invention, i.e., substances formed in vivo upon administration of the compounds of the present invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the present invention, including compounds prepared by contacting the compounds of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0295] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may have less pharmacological activity or no pharmacological activity themselves, and when administered to the body or thereon, can be converted into compounds of the present invention having the desired activity by, for example, hydrolytic cleavage. Usually such prodrugs will be functional group derivatives of the compounds, which are easily converted into the desired therapeutically active compounds in vivo. For additional information on the use of prodrugs, see "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (EB Roche ed., American Pharmaceutical Association). The prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain parts known to those skilled in the art as "pro-moiety" (e.g., "Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).

[0296] The present invention also encompasses compounds of the present invention containing protecting groups. In any process of preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive groups or reactive groups on any related molecules, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & P. ​​GMWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Using methods known in the art, the protecting groups can be removed at an appropriate subsequent stage.

[0297] In this document (specification and claims), the singular forms "a", "an" and "the" include their plural forms unless the context clearly indicates otherwise, for example, "a host cell" includes a plurality of such host cells. In addition, there is no corresponding grammatical rule for singular and plural in Chinese. The singular and plural of a noun should be determined according to the context or actual situation. Therefore, in the English translation, adding "one or more than one" before a noun is likely to be correct. DETAILED DESCRIPTION

[0298] In order to make the purpose and technical scheme of the present invention clearer, the embodiments of the present invention are described in detail below in conjunction with examples. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Those who do not specify specific conditions in the examples are all carried out according to the conditions recommended by normal conditions or manufacturers. Those who do not specify manufacturers for reagents or instruments used are all conventional products that can be obtained commercially.

[0299] The structures of the compounds were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS). 1 The H NMR was measured using a JEOL Eclipse 400 nuclear magnetometer. The solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or hexadeuterated dimethyl sulfoxide (DMSO-d6). The internal standard was tetramethylsilane (TMS). The chemical shift (δ) was given in parts per million (ppm).

[0300] The MS measuring instrument is Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0301] Preparation method for high performance liquid chromatography:

[0302] Instrument model: Agilent 1260, chromatographic column: Waters SunFire Prep C18 OBD (19mm×150mm×5.0μm); chromatographic column temperature: 25°C; flow rate: 20.0mL / min; detection wavelength: 214nm; elution gradient: (0min: 10% A, 90% B; 16.0min: 90% A, 10% B); mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid aqueous solution.

[0303] Aluminum plates (20×20 cm) produced by Merck were used for thin layer chromatography silica gel plates (TLC), and the specifications used for thin layer chromatography separation and purification were GF 254 (1 mm) produced in Yantai.

[0304] The reaction is monitored by thin layer chromatography (TLC) or LC-MS; the developing solvent systems used include: dichloromethane and methanol system, n-hexane and ethyl acetate system, and petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound or adjusted by adding triethylamine.

[0305] Preparation method of reverse phase column chromatography:

[0306] Preparation method A:

[0307] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reverse column (Spherical; 20-35μm; 100A); chromatographic column temperature: 25°C; flow rate: 28.0mL / min; detection wavelength: 220nm; mobile phase A: acetonitrile; mobile phase B: water;

[0308] Preparation method B:

[0309] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reverse column (Spherical; 20-35 μm; 100A); chromatographic column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid aqueous solution;

[0310] Preparation Method C:

[0311] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reversed phase column (Spherical; 20-35 μm; 100A); chromatographic column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.05% hydrochloric acid aqueous solution;

[0312] The microwave reaction was carried out using a Biotage Initiator+ (400 W, RT-300° C.) microwave reactor.

[0313] Column chromatography generally uses 200-300 mesh silica gel as a carrier. The eluent system includes: dichloromethane and methanol system, and petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine can also be added for adjustment.

[0314] Unless otherwise specified in the examples, the reaction temperature is room temperature (20°C to 35°C).

[0315] The reagents used in the present invention were purchased from Acros Organics, Aldrich Chemical Company, Teber Chemical and other companies.

[0316] In the general synthesis methods, examples, and intermediate synthesis examples, the meanings of the abbreviations are as follows.

[0317]

[0318]

[0319] The information of the sequences involved in the present invention is described in the following table:

[0320]

[0321]

[0322] I. Compound Examples Intermediate Preparation Example 1: Preparation of Compound Int1

[0323] Step 1: Preparation of compound Int1-2

[0324] Compound Int1-1 (1.90 g, 3.51 mmol) and isotecan mesylate (2.05 g, 3.86 mmol) were added to N, N-dimethylformamide (20 mL) in sequence, cooled to 0-10°C, N, N-diisopropylethylamine (908 mg, 7.03 mmol) and 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (2.01 g, 5.27 mmol) were added, and reacted at room temperature for 2 hours. Water was added to the reaction solution, filtered, and the filter cake was dried to obtain the title compound of this step (2.44 g, yield: 72.7%).

[0325] MS m / z(ESI):958.4[M+H] + .

[0326] Step 2: Preparation of compound Int1

[0327] Compound Int1-2 (2.44 g, 2.54 mmol) and diethylamine (928 mg, 12.7 mmol) were added to N,N-dimethylformamide (20 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 40%-50%) to obtain the title compound of this step (3.93 g, yield: 80.8%).

[0328] MS m / z(ESI):736.4[M+H] + .

[0329] Intermediate Preparation Example 2: Preparation of Compound Int3

[0330]

[0331] Compound Int3-1 (0.68 g, 1.33 mmol) was dissolved in dichloromethane (15 mL), cooled to 0-10°C, and Dess-Martin periodinant (0.84 g, 1.99 mmol) was slowly added in batches, and reacted at 25°C for 12 hours. Saturated sodium thiosulfate solution and saturated sodium bicarbonate aqueous solution were added to the reaction solution, stirred until clear, extracted with dichloromethane three times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the residue obtained by concentrating the filtrate was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 92 / 8 (v / v)) to obtain the title compound of this step (0.6 g, yield: 88.2%).

[0332] MS m / z(ESI):512.4[M+H] + .

[0333] Intermediate Preparation Example 3: Preparation of Compound Int4

[0334]

[0335] Azidoacetic acid (13.5 mg, 133.64 μmol) and compound VC-PABC-MMAE (100.0 mg, 89.07 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (46.49 mg, 359.72 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (68.39 mg, 179.86 μmol) were added, and the mixture was reacted at 25° C. for 5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=50%-60%) to obtain the title compound of this step (66 mg, yield: 59.6%).

[0336] MS m / z(ESI):1206.7[M+H] + .

[0337] Intermediate Preparation Example 4: Preparation of Compound Int5

[0338]

[0339] Referring to the synthetic route of compound Int4, the starting material compound VC-PABC-MMAE was replaced with compound Int1 to obtain the title compound of this step (35 mg, yield: 50.8%).

[0340] MS m / z(ESI):819.3[M+H] + .

[0341] Example 1-1: Preparation of tert-butyl 4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-carboxylate (C-1)

[0342]

[0343] Step 1: Preparation of tert-butyl 4-(4,6-dichloro-1,3,5-triazine-2-yl)piperazine-1-carboxylate

[0344] Under ice bath conditions, piperazine-1-carboxylic acid tert-butyl ester (4.50 g, 24.1 mmol) in acetonitrile (50 mL) was slowly added dropwise to a solution of 2,4,6-trichloro-1,3,5-triazine (4.95 g, 26.84 mmol) and N,N-diisopropylethylamine (3.46 g, 26.8 mmol) in acetonitrile (50 mL), and reacted at 0°C for 30 minutes. The reaction solution was concentrated to obtain the crude product of the title compound of this step, which was directly used in the next step.

[0345] MS m / z(ESI):334.3[M+H] + .

[0346] Step 2: Preparation of tert-butyl 4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-carboxylate

[0347] tert-Butyl 4-(4,6-dichloro-1,3,5-triazine-2-yl)piperazine-1-carboxylate (8.07 g, 24.2 mmol), potassium vinyl trifluoroborate (11.3 g, 84.6 mmol), potassium carbonate (10.0 g, 72.5 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium(II) (1.77 g, 2.42 mmol) were added to 1,4-dioxane (100 mL) and water (10 mL), replaced with nitrogen three times, and reacted at 80 °C for 6 hours. After the reaction solution was cooled to room temperature, it was diluted with water (100 mL), extracted three times with ethyl acetate (100 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue which was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 88 / 12 (v / v)) to give the title compound of this step (4.75 g, yield: 61.9%).

[0348] MS m / z(ESI):318.3[M+H] + .

[0349] 1H NMR (400MHz, CDCl3) δ6.73 (dd, J=16.0, 1.6Hz, 2H), 6.61 (dd, J=16.0, 8.0Hz, 2H), 5.78(dd,J=12.0,2.0Hz,2H),3.99-3.81(m,4H),3.56-3.43(m,4H),1.49(s,9H).

[0350] Example 1-2: Preparation of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine (C-2)

[0351]

[0352] Dissolve tert-butyl 4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-carboxylate (475 mg, 1.50 mmol) in anhydrous dichloromethane (5 mL), add trifluoroacetic acid (1.54 g, 13.5 mmol), and react at 25° C. for 4 hours. The reaction solution is concentrated to obtain the crude product of the title compound of this step.

[0353] MS m / z(ESI):218.3[M+H] + .

[0354] Example 1-3: Preparation of tert-butyl 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetate (C-3)

[0355]

[0356] At 0°C, tert-butyl 2-hydroxyacetate (47.9 mg, 0.36 mmol) and chlorosulfonyl isocyanate (51.3 mg, 0.36 mmol) were added to anhydrous dichloromethane (2 mL), replaced with nitrogen three times, reacted at 0°C for 1 hour, and then a solution of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine (100 mg, 0.30 mmol) and N,N-diisopropylethylamine (214 mg, 6.03 mmol) in dichloromethane (1 mL) was added and reacted at 0°C for 1 hour. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50%-60%) to obtain the title compound of this step (85.4 mg, yield: 62.1%).

[0357] MS m / z(ESI):455.3[M+H] + .

[0358] 1H NMR (400MHz, CD3OD) δ6.73(d,J=2.0Hz,1H),6.69(d,J=2.0Hz,1H),6.60(d,J=10.4Hz,1H),6.56(d,J=10.4Hz,1H),5 .80(d,J=2.0Hz,1H),5.78(d,J=2.0Hz,1H),4.31(s,2H),4.01(d,J=5.2Hz,4H),3.28(d,J=5.2Hz,4H),1.37(s,9H).

[0359] Example 1-4: Preparation of 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (C-4)

[0360]

[0361] Dissolve tert-butyl 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetate (85.4 mg, 0.19 mmol) in anhydrous dichloromethane (1 mL), add trifluoroacetic acid (69.2 mg, 0.61 mmol), and react at 25°C for 4 hours. The reaction solution is concentrated to obtain a crude product of the title compound of this step.

[0362] MS m / z(ESI):399.2[M+H] + .

[0363] Example 1-5: Preparation of tert-butyl 2-(((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetate (C-7)

[0364]

[0365] Step 1: Preparation of tert-butyl 2-((((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetate

[0366] Using the synthetic route of compound C-3, the reaction raw material 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine was replaced by 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane to obtain the title compound of this step (98 mg, yield: 25.8%).

[0367] MS m / z(ESI):500.2[M+H] + .

[0368] Step 2: Preparation of tert-butyl 2-((((16-(4,6-dichloro-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetate

[0369] Tert-butyl 2-((((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetate (98 mg, 176.55 μmol), 2,4,6-trichloro-1,3,5-triazine (32.89 mg, 176.55 μmol) and N,N-diisopropylethylamine (34.16 mg, 264.83 μmol) were added to ethyl acetate (2 mL) in sequence and stirred at room temperature for 2 hours. The reaction solution was concentrated and the residue was purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 65%-70%) to obtain the title compound of this step (71 mg, yield: 62.1%).

[0370] MS m / z(ESI):647.2[M+H] + .

[0371] Step 3: Preparation of tert-butyl 2-(((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetate

[0372] Tert-butyl 2-((((16-(4,6-dichloro-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetate (60 mg, 91.73 μmol), potassium vinyl trifluoroborate (43.44 mg, 321.07 μmol), potassium phosphate (78.58 mg, 366.94 μmol) and bistriphenylphosphine palladium dichloride (6.50 mg, 9.17 μmol) were added to 1,4-dioxane (1 mL) in sequence, nitrogen was blown for about 20 seconds, and the reaction was carried out at 70° C. for 8 hours. After the reaction solution was cooled to room temperature, it was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=55%-60%) to obtain the title compound of this step (37 mg, yield: 63.3%).

[0373] MS m / z(ESI):631.3[M+H] + .

[0374] Example 1-6: Preparation of 2-((((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetic acid (C-8)

[0375]

[0376] Using the synthetic route of compound C-4, the reaction raw material 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)sulfonyl)carbamoyl)oxy)acetic acid tert-butyl ester was replaced with 2-(((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetic acid tert-butyl ester to obtain the title compound of this step (35 mg, yield: 86.4%).

[0377] MS m / z(ESI):575.2[M+H] + .

[0378] Example 1-7: Preparation of tert-butyl 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11-tetraoxatetradecane-14-carboxylate (C-16)

[0379]

[0380] At 0°C, tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate (133 mg, 0.49 mmol) and chlorosulfonyl isocyanate (69.0 mg, 0.49 mmol) were added to anhydrous dichloromethane (2 mL), replaced with nitrogen three times, reacted at 0°C for 1 hour, and then a solution of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine (161 mg, 0.49 mmol) and N,N-diisopropylethylamine (346 mg, 2.68 mmol) in dichloromethane (1 mL) was added and reacted at 0°C for 1 hour. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50%-60%) to obtain the title compound of this step (95.0 mg, yield: 32.4%).

[0381] MS m / z(ESI):601.3[M+H] + .

[0382] Example 1-8: Preparation of 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11-tetraoxatetradecane-14-oic acid (C-6)

[0383]

[0384] Dissolve tert-butyl 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11-tetraoxatetradecane-14-carboxylate (23.7 mg, 39.5 μmol) in anhydrous dichloromethane (0.5 mL), add trifluoroacetic acid (34.6 mg, 303 μmol), and react at 25° C. for 4 hours. The reaction solution is concentrated to obtain the crude product of the title compound of this step.

[0385] MS m / z(ESI):545.3[M+H] + .

[0386] Example 1-9: Preparation of tert-butyl 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11,14,17,20,23,26-nonaoxaconicosane-29-carboxylate (C-22)

[0387]

[0388] At 0°C, tert-butyl hydroxy-octaethylene glycol-propionate (95.8 mg, 0.19 mmol) and chlorosulfonyl isocyanate (27.2 mg, 0.19 mmol) were added to anhydrous dichloromethane (2 mL), replaced with nitrogen three times, reacted at 0°C for 1 hour, and then a dichloromethane (1 mL) solution of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine (63.7 mg, 0.19 mmol) and N,N-diisopropylethylamine (139 mg, 1.07 mmol) was added, and reacted at 0°C for 1 hour. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50%-60%) to obtain the title compound of this step (126 mg, yield: 79.9%).

[0389] MS m / z(ESI):821.5[M+H] + .

[0390] Example 1-10: Preparation of 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11,14,17,20,23,26-nonaoxaconicosane-29-oic acid (C-23)

[0391]

[0392] Dissolve tert-butyl 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11,14,17,20,23,26-nonaoxaconicosane-29-carboxylate (52.0 mg, 63.3 μmol) in anhydrous dichloromethane (0.5 mL), add trifluoroacetic acid (34.6 mg, 303 μmol), and react at 25° C. for 4 hours. The reaction solution is concentrated to obtain the crude product of the title compound of this step.

[0393] MS m / z(ESI):765.6[M+H] + .

[0394] Example 1-11: Preparation of 2-oxo-2-(prop-2-yn-1-ylamino)ethyl((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamate (C-13)

[0395]

[0396] 2-((((4-(4,6-divinyl-1,3,5-triazin-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (22.8 mg, 57.15 μmol) and propargylamine (3.2 mg, 57.15 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (11.09 mg, 85.73 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (70.86 mg, 142.89 μmol) were added, and the mixture was reacted at 25°C for 5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 25%-30%) to obtain the title compound of this step (20 mg, yield: 80.4%).

[0397] MS m / z(ESI):436.1[M+H] + .

[0398] Example 1-12: Preparation of tert-butyl 3-(2-(2-(3-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)urea)ethoxy)ethoxy)propanoate (C-31)

[0399]

[0400] Chlorosulfonic acid isocyanate (46.62 mg, 0.31 mmol) was added to dichloromethane (4 mL), and tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propionate (73 mg, 0.30 mmol) was added dropwise at -20°C. After reacting at -20°C for 1 hour, a dichloromethane solution (3 mL) of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine (126.70 mg, 0.34 mmol) and triethylamine (127.93 mg, 1.25 mmol) was added dropwise, and stirred at room temperature for 1 hour. The reaction was quenched with ice water, and the residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 60%-70%) to obtain the title compound of this step (65 mg, yield: 24.3%).

[0401] MS m / z(ESI):556.3[M+H] + .

[0402] Example 1-13: Preparation of 3-(2-(2-(3-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)urea)ethoxy)ethoxy)propanoic acid (C-32)

[0403]

[0404] 3-(2-(2-(3-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)ureido)ethoxy)ethoxy)propanoic acid tert-butyl ester (65 mg, 70.19 μmol) was added to a mixed solution of trifluoroacetic acid (0.4 mL) and dichloromethane (2 mL) and stirred at room temperature for 1 hour. The reaction solution was concentrated to obtain a crude product of the title compound of this step.

[0405] MS m / z(ESI):500.2[M+H] + .

[0406] Example 1-14: Preparation of 4-(4,6-divinyl-1,3,5-triazine-2-yl)-N-(prop-2-yn-1-ylcarbamoyl)piperazine-1-sulfonamide (C-28)

[0407]

[0408] Propargylamine (17.7 mg, 0.31 mmol) and chlorosulfonyl isocyanate (50 mg, 0.34 mmol) were added to anhydrous dichloromethane (2 mL), replaced with nitrogen three times, reacted at -15 ° C for 1.5 hours, and then a solution of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine (83.8 mg, 0.37 mmol) and triethylamine (97.5 mg, 0.92 mmol) in dichloromethane (2 mL) was added, and the temperature was naturally raised to room temperature for 2 hours. The residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-50%) to obtain the title compound of this step (80 mg, yield: 69.5%).

[0409] MS m / z(ESI):378.1[M+H] + .

[0410] Example 1-15: Preparation of 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl ((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamate (C-37)

[0411]

[0412] Referring to the synthetic route of compound C-3, tert-butyl 2-hydroxyacetate was replaced with 2-(2-(prop-2-yn-1-yloxy)ethoxy)-1-ethanol to obtain the title compound of this step (62 mg, yield: 56.4%).

[0413] MS m / z(ESI):467.2[M+H] + .

[0414] Example 1-16: Preparation of 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl ((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamate (C-38)

[0415]

[0416] Referring to the synthetic route of compound C-7, the starting material tert-butyl 2-hydroxyacetate was replaced with 2-(2-(prop-2-yn-1-yloxy)ethoxy)-1-ethanol to obtain the title compound of this step (64 mg, yield: 41.1%).

[0417] MS m / z(ESI):643.3[M+H] + .

[0418] Example 1-17: Preparation of 3-(2-(2-(2-(4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoic acid (C-44)

[0419]

[0420] Step 1: Preparation of 4-(14,14-dimethyl-12-oxo-3,6,9,13-tetraoxapentadecanyl)piperazine-1-carboxylic acid benzyl ester

[0421] Dissolve tert-butyl 3-(2-(2-(2-oxoethoxy)ethoxy)ethoxy)propanoate (800 mg, 2.17 mmol), benzyl piperazine-1-carboxylate (457.7 mg, 1.97 mmol) and glacial acetic acid (355.6 mg, 5.92 mmol) in dichloromethane (6 mL) and methanol (3 mL), add sodium cyanoborohydride (391.7 mg, 5.92 mmol), and react at room temperature for 3 hours. The reaction solution was filtered, and the residue obtained by concentration of the filtrate was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 92 / 8 (v / v)) to obtain the title compound of this step (525 mg, yield: 55.3%).

[0422] MS m / z(ESI):481.3[M+H] + .

[0423] Step 2: Preparation of tert-butyl 3-(2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethoxy)propionate

[0424] 4-(14,14-dimethyl-12-oxo-3,6,9,13-tetraoxapentadecanyl)piperazine-1-carboxylic acid benzyl ester (525 mg, 1.09 mmol) was dissolved in methanol (10 mL), 10% palladium carbon (40 mg) was added, and hydrogen was replaced, and the hydrogen atmosphere was maintained at 50°C for 4 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain a crude product, which was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 10%-15%) to obtain the title compound of this step (360 mg, yield: 95.1%).

[0425] MS m / z(ESI):347.2[M+H] + .

[0426] Step 3: Preparation of tert-butyl 3-(2-(2-(2-(4-(4,6-dichloro-1,3,5-triazine-2-yl)piperazine-1-yl)ethoxy)ethoxy)ethoxy)propionate

[0427] Dissolve 2,4,6-trichloro-1,3,5-triazine (189.7 mg, 1.03 mmol) in anhydrous tetrahydrofuran (5 mL), cool to -10°C, and slowly drop a solution of tert-butyl 3-(2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoate (297.9 mg, 0.86 mmol) and N,N-diisopropylethylamine (165.9 mg, 1.29 mmol) in anhydrous tetrahydrofuran (8 mL). After the dropwise addition, the reaction solution was concentrated, and the crude product was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-35%) to obtain the title compound of this step (315 mg, yield: 74.3%).

[0428] MS m / z(ESI):494.2[M+H] + .

[0429] Step 4: Preparation of 3-(2-(2-(2-(4-(4,6-dichloro-1,3,5-triazine-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propionic acid

[0430] Dissolve tert-butyl 3-(2-(2-(2-(4-(4,6-dichloro-1,3,5-triazine-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoate (145.4 mg, 0.294 mmol) in anhydrous dichloromethane (3 mL), cool to 0°C, add trifluoroacetic acid (1.5 mL), and react at 0°C for 2 hours. The reaction solution is concentrated to obtain a crude product of the title compound of this step (114 mg, yield: 88.5%).

[0431] MS m / z(ESI):438.1[M+H] + .

[0432] Step 5: Preparation of 3-(2-(2-(2-(4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)ethoxy)ethoxy)ethoxy)propionic acid

[0433] 3-(2-(2-(2-(4-(4,6-dichloro-1,3,5-triazin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoic acid (114 mg, 0.26 mmol), potassium vinyl trifluoroborate (139.4 mg, 1.04 mmol), potassium carbonate (161.5 mg, 1.17 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (19.1 mg, 0.026 mmol) were added to a mixed solvent of 1,4-dioxane (4.5 mL) and water (1.5 mL) in sequence, replaced with nitrogen three times, and reacted at 90°C for 4 hours. The reaction solution was filtered through celite pad, and the filtrate was concentrated to obtain a crude product which was purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (59.8 mg, yield: 54.6%).

[0434] MS m / z(ESI):422.2[M+H] + .

[0435] Example 1-18: Preparation of 3-(2-(2-(2-(10-(4,6-divinyl-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propanoic acid (C-48)

[0436]

[0437] Step 1: Preparation of tert-butyl 3-(2-(2-(2-(1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propanoate

[0438] Tert-butyl 3-(2-(2-(2-oxoethoxy)ethoxy)ethoxy)propanoate (412.5 mg, 1.49 mmol), 1,7-dioxa-4,10-diazacyclododecane (390.2 mg, 2.24 mmol) and glacial acetic acid (268.9 mg, 4.48 mmol) were dissolved in dichloromethane (6 mL) and methanol (3 mL), sodium cyanoborohydride (281.4 mg, 4.48 mmol) was added, and the mixture was reacted at 25°C for 3 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product which was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (390 mg, yield: 60.1%).

[0439] MS m / z(ESI):435.3[M+H] + .

[0440] Step 2: Preparation of tert-butyl 3-(2-(2-(2-(10-(4,6-dichloro-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propanoate

[0441] Dissolve 2,4,6-trichloro-1,3,5-triazine (207.1 mg, 1.12 mmol) in anhydrous tetrahydrofuran (5 mL), cool to -10°C, and slowly drop tert-butyl 3-(2-(2-(2-(1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propanoate (390 mg, 0.90 mmol) and N,N-diisopropylethylamine (181.1 mg, 1.40 mmol) in anhydrous tetrahydrofuran (8 mL). After the dropwise addition, concentrate the reaction solution, and purify the crude product by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-35%) to obtain the title compound of this step (250 mg, yield: 45.8%).

[0442] MS m / z(ESI):582.2[M+H] + .

[0443] Step 3: Preparation of 3-(2-(2-(2-(10-(4,6-dichloro-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propionic acid

[0444] Dissolve tert-butyl 3-(2-(2-(2-(10-(4,6-dichloro-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecan-4-yl)ethoxy)ethoxy)ethoxy)propanoate (250 mg, 0.429 mmol) in anhydrous dichloromethane (3 mL), cool to 0°C, add trifluoroacetic acid (1.5 mL), and react at 0°C for 2 hours. The reaction solution is concentrated to obtain a crude product of the title compound of this step (218.5 mg, yield: 96.7%).

[0445] MS m / z(ESI):526.2[M+H] + .

[0446] Step 4: Preparation of 3-(2-(2-(2-(10-(4,6-divinyl-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propionic acid

[0447] 3-(2-(2-(2-(10-(4,6-dichloro-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propanoic acid (218.5 mg, 0.415 mmol), potassium vinyl trifluoroborate (222.4 mg, 1.66 mmol), potassium carbonate (257.8 mg, 1.87 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (30.34 mg, 0.042 mmol) were added to a mixed solvent of 1,4-dioxane (4.5 mL) and water (1.5 mL) in sequence, the atmosphere was replaced with nitrogen three times, and the reaction was carried out at 90°C for 4 hours. The reaction solution was filtered through a pad of celite and the filtrate was concentrated to obtain a crude product which was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25% - 30%) to obtain the title compound of this step (109.3 mg, yield: 51.7%).

[0448] MS m / z(ESI):510.3[M+H] + .

[0449] Example 1-19: Preparation of 3-(2-(2-(2-(16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)ethoxy)ethoxy)ethoxy)propanoic acid (C-46)

[0450]

[0451] Using the synthetic route of compound C-48, the starting material 1,7-dioxa-4,10-diazacyclododecane was replaced with 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane to obtain the title compound (70 mg, yield: 70.8%).

[0452] MS m / z(ESI):598.3[M+H] + .

[0453] Example 2-1: Preparation of Compound DL-7

[0454]

[0455] 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (9.50 mg, 18.5 μmol), N,N-diisopropylethylamine (4.79 mg, 37.1 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (9.11 mg, 27.8 μmol) were added to dimethyl sulfoxide (0.2 mL) and tetrahydrofuran (0.2 mL), stirred at 25° C. for 0.5 hour, and compound Int1 (20.5 mg, 27.8 μmol) was added, and the reaction was carried out at 25° C. for 2 hours. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=25%-35%) to obtain the title compound of this step (14.2 mg, yield: 68.9%).

[0456] MS m / z(ESI):1116.4[M+H] + .

[0457] Example 2-2: Preparation of Compound DL-19

[0458]

[0459] 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11-tetraoxatetradecane-14-oic acid (18.2 mg, 27.6 μmol), N,N-diisopropylethylamine (5.36 mg, 41.4 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.0 mg, 55.3 μmol) were added to dimethyl sulfoxide (0.3 mL) and tetrahydrofuran (0.3 mL), stirred at 25° C. for 0.5 hour, and compound Int1 (30.5 mg, 41.4 μmol) was added, and the reaction was carried out at 25° C. for 2 hours. The residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25% - 35%) to obtain the title compound of this step (7.00 mg, yield: 20.1%).

[0460] MS m / z(ESI):1263.3[M+H] + .

[0461] Example 2-3: Preparation of Compound DL-28

[0462]

[0463] 1-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine)-1-sulfonamido)-1-oxo-2,5,8,11,14,17,20,23,26-nonaoxaconicosan-29-oic acid (42.7 mg, 48.6 μmol), N,N-diisopropylethylamine (12.6 mg, 92.8 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (27.7 mg, 73.0 μmol) were added to dimethyl sulfoxide (0.3 mL) and tetrahydrofuran (0.3 mL), stirred at 25° C. for 0.5 hour, and compound Int1 (53.7 mg, 73.0 μmol) was added, and the reaction was carried out at 25° C. for 2 hours. The residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25% -35%) to obtain the title compound of this step (6.87 mg, yield:

[0464] 9.5%).

[0465] MS m / z(ESI):1483.6[M+H] + .

[0466] Example 2-4: Preparation of Compound DL-1

[0467]

[0468] The synthetic route of compound DL-7 was adopted, and the reaction raw material compound Int1 was replaced by VC-PABC-MMAE to obtain the title compound of this step (17 mg, yield: 26.7%).

[0469] MS m / z(ESI):1503.8[M+H] + .

[0470] Example 2-5: Preparation of Compound DL-8

[0471]

[0472] Using the synthetic route of compound DL-7, the reaction raw material 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)sulfonyl)carbamoyl)oxy)acetic acid was replaced with 2-((((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetic acid to give the title compound of this step (5.5 mg, yield: 11.5%).

[0473] MS m / z(ESI):1292.5[M+H]+ .

[0474] Example 2-6: Preparation of Compound DL-10

[0475]

[0476] Step 1: Preparation of compound DL-10-2

[0477] Dissolve compound DL-10-1 (114 mg, 71.35 μmol) in dichloromethane (4 mL), cool to 0°C, slowly add trifluoroacetic acid (77.5 mg, 7.68 mmol), and react at 0°C for 1 hour. The reaction solution was concentrated and the crude product was purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 45%-50%) to obtain the title compound of this step (85.5 mg, yield: 94.9%).

[0478] MS m / z(ESI):1325.5[M+H] + .

[0479] Step 2: Preparation of compound DL-10

[0480] Compound DL-10-2 (19 mg, 14.33 μmol), 2-oxo-2-(prop-2-yn-1-ylamino)ethyl ((4-(4,6-divinyl-1,3,5-triazin-2-yl)piperazin-1-yl)sulfonyl)carbamate (6.3 mg, 14.33 μmol), sodium ascorbate (5.8 mg, 28.67 μmol) and anhydrous copper sulfate (7.3 mg, 28.7 μmol) were added to a mixed solvent of dimethyl sulfoxide (1.5 mL), tetrahydrofuran (1.5 mL) and water (0.5 mL) at 0°C and reacted for 2 hours at 0°C. The reaction solution was concentrated to obtain a crude product which was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-35%) to obtain the title compound of this step (0.79 mg, yield: 3.1%).

[0481] MS m / z(ESI):881.5[M / 2+H] + .

[0482] Example 2-7: Preparation of Compound DL-49

[0483]

[0484] 3-(2-(2-(3-((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)ureido)ethoxy)ethoxy)propionic acid (15 mg, 30.03 μmol), compound Int1 (29.46 mg, 36.03 μmol), N,N-diisopropylethylamine (11.74 mg, 90.08 μmol), 1-hydroxybenzotriazole (4.51 mg, 33.03 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.69 mg, 45.04 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (8.0 mg, yield: 20.0%).

[0485] MS m / z(ESI):1217.5[M+H] + .

[0486] Example 2-8: Preparation of Compound DL-113

[0487]

[0488] Step 1: Preparation of compound DL-113-2

[0489] Compound DL-113-1 (400 mg, 795.86 μmol), compound Int2 (343.75 mg, 795.86 μmol), N,N-diisopropylethylamine (311.11 mg, 2.39 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (458.22 mg, 2.39 mmol) were added to anhydrous N,N-dimethylformamide (8 mL) in sequence and reacted at 0°C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 30%-35%) to obtain the title compound of this step (650 mg, yield: 90.0%).

[0490] MS m / z(ESI):907.6[M+H] + .

[0491] Step 2: Preparation of compound DL-113-3

[0492] Compound DL-113-2 (585 mg, 644.87 mmol) was added to a 4M solution of dioxane hydrochloride (8 mL) and reacted at room temperature for 3 hours. The crude product obtained by concentration of the reaction solution was dissolved in N,N-dimethylformamide (3 mL) and purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 15%-25%) to obtain the title compound of this step (460 mg, yield: 88.4%).

[0493] MS m / z(ESI):807.5[M+H] + .

[0494] Step 3: Preparation of compound DL-113

[0495] Compound DL-113-3 (35.45 mg, 43.93 μmol), 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (17.5 mg, 43.93 μmol), N,N-diisopropylethylamine (11.35 mg, 87.85 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19.95 mg, 52.71 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) in sequence and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 15%-30%) to obtain the title compound of this step (12 mg, yield: 23.0%).

[0496] MS m / z(ESI):1188.7[M+H] + .

[0497] Example 2-9: Preparation of Compound DL-114

[0498]

[0499] Step 1: Preparation of compound DL-114-1

[0500] Compound Int3 (420 mg, 0.82 mmol), compound Int2 (234 mg, 0.55 mmol), glacial acetic acid (98.5 mg, 1.64 mmol) and sodium cyanoborohydride (172 mg, 2.74 mmol) were added to 1,2-dichloroethane (3 mL) and anhydrous methanol (3 mL) and reacted at room temperature for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted three times with dichloromethane (10 mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the residue obtained by concentrating the filtrate was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 20%-30%) to obtain the title compound of this step (142 mg, yield: 28.2%).

[0501] MS m / z(ESI):924.6[M+H] + .

[0502] Step 2: Preparation of compound DL-114-2

[0503] Dissolve compound DL-114-2 (135 mg, 0.15 mmol) in anhydrous dichloromethane (2 mL), add trifluoroacetic acid (596 mg, 5.22 mmol), and react at 25° C. for 4 hours. The reaction solution is concentrated to obtain the crude product of the title compound of this step, which is directly used in the next step.

[0504] MS m / z(ESI):823.6[M+H] + .

[0505] Step 3: Preparation of compound DL-114

[0506] 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (20.7 mg, 51.9 μmol), N,N-diisopropylethylamine (8.59 mg, 69.2 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (14.5 mg, 69.2 μmol) were added to dimethyl sulfoxide (0.1 mL) and tetrahydrofuran (0.3 mL), stirred at 25°C for 0.5 hour, and then compound DL-114-2 (28.5 mg, 34.6 μmol) was added and reacted at 25°C for 2 hours. The residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25% - 35%) to obtain the title compound of this step (13.3 mg, yield: 31.9%).

[0507] MS m / z(ESI):1204.5[M+H] + .

[0508] Example 2-10: Preparation of Compound DL-115

[0509]

[0510] Step 1: Preparation of compound DL-115-1

[0511] Compound Int4 (145.3 mg, 0.44 mmol) and compound Int3 (270 mg, 0.53 mmol) were dissolved in a mixed solvent of 1,2-dichloroethane (6 mL) and methanol (3 mL), cooled to 0°C, glacial acetic acid (54 mg, 0.88 mmol) and sodium cyanoborohydride (140 mg, 2.2 mmol) were added, and reacted at 25°C for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, and the crude product obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 20%-25%) to obtain the title compound of this step (273 mg, yield: 75.2%).

[0512] MS m / z(ESI):827.6[M+H] + .

[0513] Step 2: Preparation of compound DL-115-2

[0514] Dissolve compound DL-115-1 (246 mg, 297.45 μmol) in dichloromethane (4 mL), cool to 0°C, slowly add trifluoroacetic acid (2 mL), and react at 0°C for 1 hour. The reaction solution is concentrated and the crude product is purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 10%-15%) to obtain the title compound of this step (200 mg, yield: 92.6%).

[0515] MS m / z(ESI):363.8[M / 2+H] + .

[0516] Step 3: Preparation of compound DL-115

[0517] Compound DL-115-2 (51.9 mg, 71.54 μmol), 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (28.5 mg, 71.54 μmol), N,N-diisopropylethylamine (18.5 mg, 143.07 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (32.6 mg, 85.84 μmol) were added to a mixed solvent of tetrahydrofuran (2 mL) and dimethyl sulfoxide (1 mL) and reacted at 25°C for 2 hours. The reaction solution was concentrated and the crude product was purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 30% - 35%) to give the title compound of this step (16.6 mg, yield: 21.0%).

[0518] MS m / z(ESI):1107.5[M+H] + .

[0519] Example 2-11: Preparation of Compound DL-2

[0520]

[0521] 2-((((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetic acid (15 mg, 23.49 μmol), compound VC-PABC-MMAE (27.78 mg, 23.49 μmol), N,N-diisopropylethylamine (6.12 mg, 46.99 μmol), 1-hydroxybenzotriazole (3.52 mg, 25.84 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.80 mg, 35.24 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-50%) to obtain the title compound of this step (3.62 mg, yield: 8.5%)

[0522] MS m / z(ESI):1679.9[M+H] + .

[0523] Example 2-12: Preparation of Compound DL-5

[0524]

[0525] Using the synthetic route of compound DL-2, the starting material compound VC-PABC-MMAE was replaced by compound GGFG-DXD to obtain the title compound of this step (6.05 mg, yield: 17.0%).

[0526] MS m / z(ESI):1397.5[M+H] + .

[0527] Example 2-13: Preparation of Compound DL-11

[0528]

[0529] Step 1: Preparation of compound DL-11-1

[0530] Cool in an ice-water bath, add compound DL-10-1 (400 mg, 237.82 μmol), propargylamine (13.23 mg, 237.82 μmol), sodium ascorbate (95.18 mg, 475.65 μmol) and copper sulfate pentahydrate (119.96 mg, 475.65 μmol) to a mixed solution of dimethyl sulfoxide (8 mL) and water (1 mL) in turn, and react at room temperature for 3 hours. The reaction solution is directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-50%) to obtain the title compound of this step (225 mg, yield: 51.5%).

[0531] MS m / z(ESI):1652.8[M+H] + .

[0532] Step 2: Preparation of compound DL-11-2

[0533] 2-((((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamoyl)oxy)acetic acid (30 mg, 41.77 μmol), compound DL-11-1 (61.37 mg, 33.41 μmol), N,N-diisopropylethylamine (10.88 mg, 83.54 μmol), 1-hydroxybenzotriazole (6.27 mg, 45.94 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12.09 mg, 62.65 μmol) were added to N,N-dimethylformamide (2 mL) in sequence and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 48% - 55%) to obtain the title compound of this step (22 mg, yield: 21.5%).

[0534] MS m / z(ESI):1105.5[M / 2+H] + .

[0535] Step 3: Preparation of compound DL-11

[0536] Trifluoroacetic acid (10.32 mg, 89.61 μmol) was added to a solution of compound DL-11-2 (22 mg, 8.96 μmol) in dichloromethane (1.5 mL), cooled in an ice-water bath, and stirred at this temperature for about 1 hour. The crude product obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 45%-50%) to obtain the title compound of this step (8 mg, yield: 40.5%).

[0537] MS m / z(ESI):1936.8[M+H] + .

[0538] Example 2-14: Preparation of Compound DL-40

[0539]

[0540] Compound DL-10-2 (70.2 mg, 52.99 μmol), 4-(4,6-divinyl-1,3,5-triazine-2-yl)-N-(prop-2-yn-1-ylcarbamoyl)piperazine-1-sulfonamide (20 mg, 52.99 μmol), sodium ascorbate (21 mg, 105.98 μmol) and copper sulfate pentahydrate (26.5 mg, 105.98 μmol) were added to a mixed solvent of dimethyl sulfoxide (1.5 mL), tetrahydrofuran (1.5 mL) and water (0.5 mL) and reacted at 0°C for 2 hours. The crude product obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-35%) to obtain the title compound of this step (80 mg, yield: 77.9%).

[0541] MS m / z(ESI):1702.7[M+H] + .

[0542] Example 2-15: Preparation of Compound DL-61

[0543]

[0544] Compound Int4 (36 mg, 29.86 μmol), 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl ((4-(4,6-divinyl-1,3,5-triazin-2-yl)piperazin-1-yl)sulfonyl)carbamate (13.9 mg, 29.86 μmol), sodium ascorbate (11.8 mg, 59.72 μmol) and anhydrous copper sulfate (14.3 mg, 89.6 μmol) were added to a mixed solvent of dimethyl sulfoxide (1.5 mL), tetrahydrofuran (1.5 mL) and water (0.5 mL) at 0°C and reacted at 0°C for 2 hours. The reaction solution was concentrated and the crude product was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (21.0 mg, yield: 42.1%).

[0545] MS m / z(ESI):1672.9[M+H] + .

[0546] Example 2-16: Preparation of Compound DL-62

[0547]

[0548] Referring to the synthetic route of compound DL-61, the starting material was replaced with 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl ((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)sulfonyl)carbamate with 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl ((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamate to give the title compound of this step (7 mg, yield: 9.3%).

[0549] MS m / z(ESI):1849.0[M+H] + .

[0550] Example 2-17: Preparation of Compound DL-67

[0551]

[0552] Referring to the synthetic route of compound DL-61, the starting material compound Int4 was replaced by compound Int5 to obtain the title compound of this step (21 mg, yield: 36.2%).

[0553] MS m / z(ESI):1285.5[M+H] + .

[0554] Example 2-18: Preparation of Compound DL-68

[0555]

[0556] Compound Int5 (32 mg, 39.10 μmol), 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl ((16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)sulfonyl)carbamate (25.0 mg, 38.92 μmol), sodium ascorbate (11.8 mg, 59.72 μmol) and anhydrous copper sulfate (14.3 mg, 89.6 μmol) were added to a mixed solvent of dimethyl sulfoxide (1.5 mL), tetrahydrofuran (1.5 mL) and water (0.5 mL) at 0°C and reacted at 0°C for 2 hours. The reaction solution was concentrated and the crude product was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35% - 40%) to obtain the title compound of this step (7.0 mg, yield: 12.3%).

[0557] MS m / z(ESI):1461.6[M+H] + .

[0558] Example 2-19: Preparation of Compound DL-79

[0559]

[0560] 3-(2-(2-(2-(4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoic acid (23.7 mg, 56.35 μmol) and VC-PABC-MMAE (63.3 mg, 56.35 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (10.9 mg, 84.52 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23.6 mg, 61.98 μmol) were added, and the mixture was reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 35%-40%) to obtain the title compound of this step (3 mg, yield: 3.2%).

[0561] MS m / z(ESI):764.3[M / 2+H] + .

[0562] Example 2-20: Preparation of Compound DL-81

[0563]

[0564] Using the synthetic route of compound DL-79, the starting material 3-(2-(2-(2-(4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)ethoxy)ethoxy)ethoxy)propanoic acid was replaced with 3-(2-(2-(2-(10-(4,6-divinyl-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propanoic acid to give the title compound of this step (10 mg, yield: 9.2%).

[0565] MS m / z(ESI):1615.0[M+H] + .

[0566] Example 2-21: Preparation of Compound DL-85

[0567]

[0568] The synthetic route of compound DL-79 was adopted, and the reaction raw material compound VC-PABC-MMAE was replaced by compound Int1 to obtain the title compound of this step (2 mg, yield: 2.6%).

[0569] MS m / z(ESI):1139.5[M+H] + .

[0570] Example 2-22: Preparation of Compound DL-86

[0571]

[0572] 3-(2-(2-(2-(16-(4,6-divinyl-1,3,5-triazine-2-yl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)ethoxy)ethoxy)ethoxy)propanoic acid (33.3 mg, 55.63 μmol) and compound Int1 (40.9 mg, 55.63 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (10.9 mg, 84.52 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23.6 mg, 61.98 μmol) were added and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 35%-40%) to obtain the title compound of this step (4 mg, yield: 5.5%).

[0573] MS m / z(ESI):1315.6[M+H] + .

[0574] Example 2-23: Preparation of Compound DL-87

[0575]

[0576] 3-(2-(2-(2-(10-(4,6-divinyl-1,3,5-triazine-2-yl)-1,7-dioxa-4,10-diazacyclododecane-4-yl)ethoxy)ethoxy)ethoxy)propanoic acid (38 mg, 74.57 μmol) and compound Int1 (54.9 mg, 74.57 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (14.5 mg, 111.85 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (31.2 mg, 82.03 μmol) were added and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (4 mg, yield: 4.4%).

[0577] MS m / z(ESI):1227.6[M+H] + .

[0578] 2. Preparation of Antibody Drug Conjugates

[0579] The antibody-drug conjugate was prepared using Trastuzumab monoclonal antibody as the antibody. The heavy chain and light chain sequences of Trastuzumab monoclonal antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0580] Example 3-1 Trastuzumab-ADC-1

[0581]

[0582] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-1 with a molar equivalent of 6 times that of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.9 mg / mL, 1.5mL) of Trastuzumab-ADC-1, which was stored at 4°C.

[0583] Example 3-2 Trastuzumab-ADC-7

[0584]

[0585] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.59 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM, ) of compound DL-7 with an 8-fold molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.2 mg / mL, 1.5 mL) of Trastuzumab-ADC-7, which was stored at 4°C.

[0586] Example 3-3 Trastuzumab-ADC-8

[0587]

[0588] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-8 with a molar equivalent of 6 times that of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution of Trastuzumab-ADC-8 (3.1mg / mL, 1.5mL), which was stored at 4°C.

[0589] Example 3-4 Trastuzumab-ADC-10

[0590]

[0591] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-10 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (3.1mg / mL, 1.5mL) of Trastuzumab-ADC-10, which was stored at 4°C.

[0592] Example 3-5 Trastuzumab-ADC-19

[0593]

[0594] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.59 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-19 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (6.8mg / mL, 1.5mL) of Trastuzumab-ADC-19, which was stored at 4°C.

[0595] Example 3-6 Trastuzumab-ADC-28

[0596]

[0597] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.59 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-28 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (9.1mg / mL, 1.5mL) of Trastuzumab-ADC-28, which was stored at 4°C.

[0598] Example 3-7 Trastuzumab-ADC-49

[0599]

[0600] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.59 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-49 with a molar equivalent of 6 times that of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.9 mg / mL, 1.5mL) of Trastuzumab-ADC-49, which was stored at 4°C.

[0601] Example 3-8 Trastuzumab-ADC-113

[0602]

[0603] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-113 with a molar equivalent of 6 times that of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (3.3 mg / mL, 1.5mL) of Trastuzumab-ADC-113, which was stored at 4°C.

[0604] Example 3-9 Trastuzumab-ADC-114

[0605]

[0606] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-114 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (3.3mg / mL, 1.5mL) of Trastuzumab-ADC-114, which was stored at 4°C.

[0607] Example 3-10 Trastuzumab-ADC-115

[0608]

[0609] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-115 with a molar equivalent of 10 times that of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (3.3mg / mL, 1.5mL) of Trastuzumab-ADC-115, which was stored at 4°C.

[0610] Example 3-11 Trastuzumab-ADC-2

[0611]

[0612] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-2 with a molar equivalent of 10 times that of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.4mg / mL, 3.5mL) of Trastuzumab-ADC-2, which was stored at -20°C.

[0613] Example 3-12 Trastuzumab-ADC-5

[0614]

[0615] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-5 with 8 times the molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (1.3mg / mL, 3.5mL) of Trastuzumab-ADC-5, which was stored at -20°C.

[0616] Example 3-13 Trastuzumab-ADC-11

[0617]

[0618] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.59 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-11 with an 8-fold molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (1.6mg / mL, 3.5mL) of Trastuzumab-ADC-11, which was stored at -20°C.

[0619] Example 3-14 Trastuzumab-ADC-40

[0620]

[0621] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.59 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-40 with an 8-fold molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (1.7mg / mL, 3.5mL) of Trastuzumab-ADC-40, which was stored at -20°C.

[0622] Example 3-15 Trastuzumab-ADC-61

[0623]

[0624] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-61 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (3.1mg / mL, 3.5mL) of Trastuzumab-ADC-61, which was stored at -20°C.

[0625] Example 3-16 Trastuzumab-ADC-62

[0626]

[0627] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-62 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.6mg / mL, 3.5mL) of Trastuzumab-ADC-62, which was stored at -20°C.

[0628] Example 3-17 Trastuzumab-ADC-67

[0629]

[0630] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-67 with 8 times the molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.7mg / mL, 3.5mL) of Trastuzumab-ADC-67, which was stored at -20°C.

[0631] Example 3-18 Trastuzumab-ADC-68

[0632]

[0633] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-68 with 8 times the molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (1.7mg / mL, 3.5mL) of Trastuzumab-ADC-68, which was stored at -20°C.

[0634] Example 3-19 Trastuzumab-ADC-79

[0635]

[0636] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-79 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.5mg / mL, 3.5mL) of Trastuzumab-ADC-79, which was stored at -20°C.

[0637] Example 3-20 Trastuzumab-ADC-81

[0638]

[0639] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-81 with a molar equivalent of 10 times that of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.4mg / mL, 3.5mL) of Trastuzumab-ADC-81, which was stored at -20°C.

[0640] Example 3-21 Trastuzumab-ADC-85

[0641]

[0642] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-85 with 8 times the molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (4.6mg / mL, 3.5mL) of Trastuzumab-ADC-85, which was stored at -20°C.

[0643] Example 3-22 Trastuzumab-ADC-86

[0644]

[0645] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-86 with 8 times the molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.3mg / mL, 3.5mL) of Trastuzumab-ADC-86, which was stored at -20°C.

[0646] Example 3-23 Trastuzumab-ADC-87

[0647]

[0648] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.60 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 3 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (5.5eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound DL-87 with an 8-fold molar equivalent of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 2 hours. The mixture was purified with a NAP-25 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.5mg / mL, 3.5mL) of Trastuzumab-ADC-87, which was stored at -20°C.

[0649] 3. Analysis of Antibody Drug Conjugates (ADC):

[0650] 1. The product of the coupling reaction was analyzed by HIC-HPLC under the following conditions:

[0651] Chromatographic column: Tosoh TSK gel Butyl-NPR, 4.6×100mm

[0652] Mobile phase A: 1.5 M ammonium sulfate aqueous solution

[0653] Mobile phase B: 25 mM sodium phosphate aqueous solution, pH = 7.0, 25% (v / v) isopropanol aqueous solution

[0654] Flow rate: 0.5mL / min; Column temperature: 30℃; Sample chamber temperature: 8℃; Wavelength: UV 280nm; Injection volume: 40-80μL

[0655] Gradient: 0-3min: 5% mobile phase B + 95% mobile phase A

[0656] 3-40min: 5%-100% mobile phase B + 95%-0 mobile phase A

[0657] 40~40.1min: 100%~5% mobile phase B + 0~95% mobile phase A

[0658] 40.1~45min: 5% mobile phase B + 95% mobile phase A

[0659] The DAR4 ratio was calculated as: DAR4 peak area / sum of peak areas*100%, where the peak area was determined by the area under the curve of each peak in the HIC-HPLC spectrum.

[0660] Table 1 HIC-HPLC results of antibody drug conjugates

[0661]

[0662]

[0663] Table 1 shows that the antibody-drug conjugates synthesized using the novel disulfide bridge linker of the present invention can achieve site-specific conjugation to obtain ADCs with controllable DAR values ​​and high uniformity.

[0664] Biological tests

[0665] Experimental Example 1: Antibody-drug conjugates inhibit proliferation of HER2-positive tumor cells

[0666] Experimental steps:

[0667] 1. Collect HER2-positive tumor cells SK-OV-3 (Nanjing Kebai), NCI-N87 (ATCC) and SK-

[0668] BR-3 (Nanjing Kebai), and counting.

[0669] 2. The three tumor cells were inoculated into 96-well plates (Corning), with 1000 cells per well (SK-OV-3), 3000 cells per well (NCI-N87) or 8000 cells per well (SK-BR-3) in a volume of 80 μL, and cultured in a 37°C, 5% CO2 incubator overnight to allow the cells to adhere (18 to 24 h).

[0670] 3. Prepare 5× drug working solution with gradient concentration according to the experimental design. Take out the 96-well cell plate, transfer 20 μL working solution from the compound plate and add it to the cell plate. Place the cell plate in a 37°C, 5% CO2 incubator for 96 hours.

[0671] 4. Test plate: Take out the cell plate and observe the cell status under a microscope. 2.0 kit (Novozyme) instructions, add detection solution to the cell plate, and obtain luminescence value by PHERA Star FS.

[0672] 5. Calculate the inhibition rate (IR%) = (1-(mean luminescence value of experimental wells / mean luminescence value of DMSO control wells))

[0673] *100%. Graphpad Prism 8.0 software was used for data analysis and plotting, and IC was calculated by fitting the inhibition rate and the logarithm of drug concentration using a four-parameter curve. 50 value.

[0674] Table 2 Results of the inhibition of tumor cell proliferation by antibody-drug conjugates

[0675] ADC Name <h2 style=";text-align:left;direction:ltr"><![CDATA[IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> (NCI-N87,nM)]]><h2 style=";text-align:left;direction:ltr"> <![CDATA[IC 50 (SK-BR-3,nM)]]> Trastuzumab-ADC-7 4.91 0.23 Trastuzumab-ADC-5 1.3 0.04 Trastuzumab-ADC-19 1.41 0.20 Trastuzumab-ADC-49 2.21 0.29 Trastuzumab-ADC-67 0.27 0.13 Trastuzumab-ADC-68 0.05 0.07

[0676] Table 2 shows that the antibody-drug conjugate of the present invention has a significant proliferation inhibitory effect on HER2-positive tumor cells.

Claims

1. An antibody-drug conjugate as shown in formula (I): in, Ab is the targeting group; S comes from the cysteine ​​residue on the targeting group; A is an unsubstituted or substituted heterocyclic group; L is a linker; D is a fragment of a biologically active molecule, including but not limited to cytotoxic compounds and immunomodulators; m is an integer or decimal between 1 and 5; Preferably, the antibody-drug conjugate has a structure as shown in formula (II), in, A is unsubstituted or substituted with one or more R 1 A substituted heterocyclic group, wherein the heterocyclic group contains at least one N atom and is connected to the triazine through the N atom; L 1 is a covalent bond or is in; L 1a is a covalent bond or is selected from C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkyne, -O-, -S-, -N(R 2 )-、-C 1-6 Alkylene-O-, -C(=O)-, -C(=S)-, -C(=O)-O-, -C(=O)-N(R 3 )-、-C(=S)-N(R 3 )-、-C(=O)-N(R 3 )-N(R 3 )-、-N(R 3 )-O-、-N(R 3 )-N(R 3 )-、-N(R 3 )-C(=O)-, -OC(=O)-, -ON(R 3 )-, -OC(=O)-O-, -OC(=O)-N(R 3 )-, -S(=O)-, -S(=O)2-, -S(=O)2-N(R 3 )-、 C 3-10 Cycloalkyl and 3-10 membered heterocyclic group; L 1b is a covalent bond or is selected from C 1-6 Alkylene, C 1-6 Halogenated alkylene, C 2-6 Alkenylene, C 2-6 Alkyne group, L 1c is a covalent bond or is selected from R 1 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and oxo; R 2 Selected from H, hydroxyl and C 1-6 alkyl; R 3 Each independently selected from H and C 1-6 alkyl; R 4 Each independently selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl and a are each independently selected from 1, 2, 3, 4, 5 and 6; b are each independently selected from an integer of 0-10; c are each independently selected from 0, 1, 2, 3, 4, 5 and 6; L 2 is a covalent bond or is selected from an amino acid fragment and a short peptide fragment formed by two or more amino acids, wherein the amino acid is selected from Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn, and the amino acid is optionally bound by one or more selected from H, C 1-6 Alkyl and polyethylene glycol group substitution of hydrophilic structural units; Preferably, L 2 is a covalent bond or is selected from the following structures: R 5 Each independently selected from H, C 1-6 Alkyl and polyethylene glycol hydrophilic structural unit; preferably, the polyethylene glycol hydrophilic structural unit is Each d is independently selected from an integer from 1 to 10; L 3 is a covalent bond or is selected from the following structures: D is selected from the group consisting of cytotoxic compound fragments and immunomodulator fragments, wherein the cytotoxic compound includes but is not limited to tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, wherein the tubulin inhibitors include but are not limited to dolastatin and auristatin, maytansine, tubulysins and cryptomycins, wherein the DNA damaging agents include but are not limited to PBD, duocarmycin and calicheamicin, wherein the topoisomerase inhibitors include but are not limited to camptothecin and its derivatives, epipodophyllotoxin, anthracycline and anthraquinone; wherein the immunomodulator is selected from the group consisting of Toll-like receptors (TLRs) modulators, STING (stimulator of interferon genes) modulators, RIG-I-like receptors (retinoic acid-inducible gene-I (RIG-I)-like receptors, RLRs) modulators, Nod-like receptors (nucleotide oligomerization domain (NOD)-like receptors, NLRs) modulators and C-type lectin receptors (CLRs) modulators.

2. The antibody-drug conjugate of claim 1, wherein: A is unsubstituted or substituted with one or more R 1 A substituted 3-20 membered heterocyclic group, the heterocyclic group contains at least one N atom and is connected to the triazine through the N atom, preferably, A is selected from piperazine, piperidine, 1,10-diaza-18-crown-6-ether and 4,10-diaza-12-crown-4-ether; L 1 is a covalent bond or is in; L 1a is a covalent bond or is selected from C 1-6 Alkylene, -O-, -S-, -N(R 2 )-、-C 1-6 Alkylene-O-, -C(=O)-N(R 3 )-、-N(R 3 )-C(=O)-、-OC(=O)-N(R 3 )-、-S(=O)2-N(R 3 )-、 C 3-10 Cycloalkyl and 3-10 membered heterocyclic group; L 1b is a covalent bond or is selected from C 1-6 Alkylene, L 1c is a covalent bond or is selected from R 1 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and oxo; R 2 Selected from H, hydroxyl and C 1-6 alkyl; R 3 H and C independently 1-6 alkyl; R 4 Each independently selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl and a are each independently selected from 1, 2, 3, 4, 5 and 6; b are each independently selected from an integer of 0-10; c is each independently selected from 0, 1, 2, 3, 4, 5 and 6.

3. The antibody drug conjugate according to any one of claims 1 to 2, wherein L 1 is a covalent bond or is selected from the following structures: in, Position 1 is connected to A, position 2 is connected to L 2 connected; R 2 Selected from H, hydroxyl and C 1-6 alkyl; R 4 Each independently selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl and a are each independently selected from 1, 2, 3, 4, 5 and 6; b are each independently selected from an integer of 0-10; c are each independently selected from 0, 1, 2, 3, 4, 5 and 6; Preferably, L 1 is a covalent bond or is: Among them, position 1 is connected to A, and position 2 is connected to L 2 connected; R 2 or R 4 Each independently selected from H, hydroxyl and C 1-6 alkyl; a are each independently selected from 1, 2, 3, 4, 5 and 6; b are each independently selected from an integer of 0-10.

4. The antibody drug conjugate according to any one of claims 1 to 3, wherein L 2 is a covalent bond or is selected from the following structures:

5. The antibody drug conjugate according to any one of claims 1 to 4, wherein L 3 is a covalent bond or is selected from the following structures:

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the formula is a covalent bond or is selected from the following structures: in, Position 3 and L 1 Connected to, position 4 is connected to D.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein the formula Select from the following structures: in, Position 1 is connected to A, position 4 is connected to D; R 2 or R 4 Each independently selected from H, hydroxyl and C 1-6 Alkyl; a is each independently selected from 1, 2, 3, 4, 5 and 6; b are each independently selected from an integer from 0 to 10; Preferably, the formula Select from the following structures: Wherein, position 1 is connected to A, and position 4 is connected to D; preferably, the formula for: wherein position 1 is connected to A, and position 4 is connected to D; more preferably, the formula Selected from: Among them, position 1 is connected to A, and position 4 is connected to D.

8. The antibody-drug conjugate according to any one of claims 1 to 7, wherein: D is selected from the fragments of cytotoxic compounds, wherein the cytotoxic compounds are selected from tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, wherein the tubulin inhibitors include dolastatin, auristatin, maytansine, tubulysins and cryptomycins, wherein the DNA damaging agents include PBD, duocarmycin and calicheamicin, and wherein the topoisomerase inhibitors include camptothecin and its derivatives; Preferably, D is selected from a microtubule inhibitor fragment and a topoisomerase inhibitor fragment, the microtubule inhibitor is selected from dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3 and DM4, and the topoisomerase inhibitor is selected from camptothecin, SN-38, exitecan, topotecan, belotecan, rubitecan, diflutecan, 10-hydroxycamptothecin, 9-aminocamptothecin, doxorubicin, epirubicin and PNU-159682; More preferably, D is selected from an immunomodulator fragment, wherein the immunomodulator is selected from a TLR modulator, a NOD1 modulator, a NOD2 modulator, a RIG-I modulator, an MDA5 modulator, a LGP2 modulator, a Dectin-1 modulator, a Dectin-2 modulator, an ALRs modulator, and a STING modulator; Preferably, the immunomodulator is selected from TLR agonists; the TLR agonists are selected from TLR2 agonists, TLR4 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR7 / 8 agonists and TLR9 agonists; preferably, the TLR agonists are selected from TLR7 agonists, TLR8 agonists and TLR7 / 8 agonists; Further preferably, D is selected from the following structures:

9. The antibody-drug conjugate of any one of claims 1 to 8, wherein Ab is a targeting group that targets a cell surface receptor or a tumor surface antigen; Preferably, Ab is an antibody or an antigen-binding fragment thereof; the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a murine antibody, a humanized antibody, a fully human antibody, and a fusion protein comprising the antigen-binding portion of an antibody; the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, a disulfide-bonded Fv, and scFv. More preferably, the antibody or antigen-binding fragment thereof is an anti-Her-2 antibody; Further preferably, the antibody or antigen-binding fragment thereof comprises: (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 3 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 4 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 9 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 10 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 12 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 15 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; or (4) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; Further preferably, the anti-Her-2 antibody is trastuzumab; Further preferably, the heavy chain sequence of the trastuzumab is shown as SEQ ID NO: 1, and the light chain sequence of the trastuzumab is shown as SEQ ID NO:

2.

10. The antibody drug conjugate of any one of claims 1 to 9, which has a structure of formula (III-A) or formula (III-B) or formula (III-C) or formula (III-D), Ab, L 1 , L 2 , L 3 , D and m are as defined in any one of claims 1-9.

11. The antibody drug conjugate according to any one of claims 1 to 10, which is selected from the following structures:

12. A compound of formula (IV) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof: A is a substituted or unsubstituted heterocyclic group; L is a linker; D is a fragment of a biologically active molecule, including but not limited to cytotoxic compounds and immunomodulators; Preferably, A, L and D are as defined in any one of claims 1 to 8; Preferably, the compound has a structure of formula (VA) or formula (VB) or formula (VC) or formula (VD), wherein: L 1 , L 2 and L 3 are independently selected linkers; D is a fragment of a biologically active molecule, including but not limited to cytotoxic compounds and immunomodulators; Preferably, L 1 , L 2 , L 3 and D is as defined in any one of claims 2-8.

13. The compound of claim 12 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, wherein the compound is selected from the following structures:

14. A compound of formula (VI) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof: in, A is unsubstituted or substituted with one or more R 6 A substituted heterocyclic group, wherein the heterocyclic group contains at least one N atom and is connected to the triazine through the N atom; Q 1 is a covalent bond or is selected from C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkyne, -O-, -S-, -N(R 7 )-、-C 1-6 Alkylene-O-, -C(=O)-, -C(=S)-, -C(=O)-O-, -C(=O)-N(R 8 )-、-C(=S)-N(R 8 )-、-C(=O)-N(R 8 )-N(R 8 )-、-N(R 8 )-O-、-N(R 8 )-N(R 8 )-、-N(R 8 )-C(O)-、-OC(=O)-、-ON(R 8 )-, -OC(=O)-O-, -OC(=O)-N(R 8 )-, -S(=O)-, -S(=O)2-, -S(=O)2-N(R 8 )-、 C 3-10 Cycloalkyl and 3-10 membered heterocyclic group; Q 2 is a covalent bond or is selected from C 1-6 Alkylene, C 1-6 Halogenated alkylene, C 2-6 Alkenylene, C 2-6 Alkyne group, Q 3 Selected from H, azide, C 2-6 Alkynyl, Hydroxyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, -O-benzyl, -N(R 9 )-C 1-6 Alkylene-C 2-6 Alkynyl, C 1-6 Alkyl, benzyl, acetyl, trifluoroacetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, fluorenyloxycarbonyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl and 5-12 membered heteroaryl; R 6 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and oxo; R 7 Selected from H, hydroxyl and C 1-6 alkyl; R 8 Each independently selected from H and C 1-6 alkyl; R 9 Each independently selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, hydroxyl and e are each independently selected from 1, 2, 3, 4, 5 and 6; f are each independently selected from an integer of 0-10; g are each independently selected from 0, 1, 2, 3, 4, 5 and 6; Preferably, A is unsubstituted or substituted with one or more R 6 Substituted 3-20 membered heterocyclic groups containing at least one N atom and linked to the triazine via the N atom, for example piperazine, piperidine, 1,10-diaza-18-crown-6-ether and 4,10-diaza-12-crown-4-ether; Q 1 is a covalent bond or is selected from C 1-6 Alkylene, -O-, -S-, -N(R 7 )-、-C 1-6 Alkylene-O-, -C(=O)-N(R 8 )-、-N(R 8 )-C(=O)-、-OC(=O)-N(R 8 )-、-S(=O)2-N(R 8 )-、 C 3-10 Cycloalkyl and 3-10 membered heterocyclic group; Q 2 is a covalent bond or is selected from C 1-6 Alkylene, Q 3 Selected from H, azide, C 2-6 Alkynyl, Hydroxyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, -O-benzyl, -N(R 9 )-C 1-6 Alkylene-C 2-6 Alkynyl, C 1-6 Alkyl, benzyl, acetyl, trifluoroacetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, fluorenyloxycarbonyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl and 5-12 membered heteroaryl; R 6 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and oxo; R 7 Selected from H, hydroxyl and C 1-6 alkyl; R 8 Each independently selected from H and C 1-6 alkyl; R 9 Each independently selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl, hydroxyl and e are each independently selected from 1, 2, 3, 4, 5 and 6; f are each independently selected from an integer of 0-10; g are each independently selected from 0, 1, 2, 3, 4, 5 and 6; Further preferably, the compound has a structure of formula (VII-A) or formula (VII-B) or formula (VII-C) or formula (VII-D):

15. The compound of claim 14 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the compound is selected from the following structures:

16. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 11, and a pharmaceutically acceptable carrier; optionally, the pharmaceutical composition further comprises one or more other anticancer drugs such as chemotherapeutic agents and / or antibodies.

17. Use of the antibody-drug conjugate according to any one of claims 1 to 11 in the preparation of a drug for preventing or treating a disease associated with abnormal cell proliferation (eg, cancer); Preferably, the cancer is selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer.

18. Use of a compound according to any one of claims 13 to 15 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof in the preparation of an antibody drug conjugate according to any one of claims 1 to 11.

19. A method for preparing the antibody drug conjugate of any one of claims 1 to 11, the method comprising: The antibody-drug conjugate is obtained by coupling a reduced antibody or an antigen-binding fragment thereof with a compound according to any one of claims 12 to 13 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof.

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