A nitrogen-containing heterocyclic compound, conjugates thereof, and methods of making and using the same

Abstract

A kind of nitrogen-containing heterocyclic compound, its conjugate and preparation method and use thereof.A ligand-drug conjugate is provided, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate comprises a ligand and a structure shown in formula (I).The nitrogen-containing heterocyclic compound, its conjugate has one or more effects selected from the following group:(1) has in vitro proliferation inhibitory activity on tumor cells;(2) has plasma stability;(3) has in vivo antitumor effect;(4) has anti-transporter transport capacity;(5) has in vivo tumor targeting ability;And (6) has good in vivo safety.

Description

[0001] This application claims priority to Chinese patent application 2024101791649, filed on February 8, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to a nitrogen-containing heterocyclic compound, its couplings, its preparation method, and its uses. Background Technology

[0003] Immunomodulators (such as thalidomide, lenalidomide, pomalidomide, CC-885, CC-90009, etc.) act as "molecular glues," binding to cereblon (CRBN) through a glutarimide ring structure and promoting the formation of CRL4. CRBN E3 ubiquitin ligase-mediated recruitment and ubiquitination of substrate proteins (such as GSPT1, IKZF1 / 3, CK1α, MYC, etc.). Although these compounds are exciting as novel cancer therapies, they have so far been limited to hematologic malignancies such as multiple myeloma and myelodysplastic syndromes (MDS), and are associated with clinically serious adverse events (AEs).

[0004] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxic drugs via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of cytotoxic drugs, while avoiding the drawbacks of low efficacy of the former and excessive toxicity of the latter. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce their impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814), thus exhibiting greater efficacy and a wider therapeutic window.

[0005] By leveraging the differences in surface antigen expression between tumor cells and normal cells, antibody-drug conjugates can be constructed by combining protein degrading agents with antibodies that bind to tumor cell surface antigens. This not only improves the clinical efficacy and tolerability of protein degrading agents but also expands their potential therapeutic range, such as for the treatment of breast cancer, lung cancer, liver cancer, gastric cancer, and prostate cancer. This overcomes the limitations of small molecule protein degrading agents and provides patients with more options.

[0006] Therefore, it is necessary to design and synthesize a series of protein degradation agent conjugates with significant antitumor activity for the treatment of tumor-related diseases. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the deficiency of the limited variety of existing antibody-drug conjugates, thereby providing a nitrogen-containing heterocyclic compound, its conjugates, its preparation method, and its uses. The nitrogen-containing heterocyclic compound and its conjugates of this invention have one or more effects selected from the group consisting of: (1) inhibitory activity against the in vitro proliferation of tumor cells; (2) plasma stability; (3) in vivo tumor-suppressive effect; (4) anti-transporter transport capability; (5) in vivo tumor-targeting capability; and (6) good in vivo safety.

[0008] On one hand, the present invention provides a ligand-drug conjugate, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a ligand and the structure shown in formula (I):

[0009]

[0010] in,

[0011] R 1 Hydrogen, deuterium, halogens, C 1-6 Alkyl or halogenated C 1-6 alkyl;

[0012] Ring A is: In this configuration, 1 is connected to V, and 2 is connected to piperidinedione.

[0013] U is -CH2- or -C(O)-;

[0014] R a Each can be independently represented by hydrogen, halogen, -OH, -CN, -NH2, nitro, or C. 1-6 Alkyl, -OC 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-12 Cycloalkyl or 4- to 12-membered heterocyclic alkyl; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-12 The cycloalkyl or 4- to 12-membered heterocycloalkyl groups are each optionally substituted by one or more substituents selected from halogens, -OH, -CN, -NH2 and oxo groups;

[0015] k2 is 0, 1, 2 or 3;

[0016] V is -NH- or -O-;

[0017] L represents a chemical bond, -C 1-6 Alkylene or -halogenated C 1-6 alkylene-;

[0018] Ring B is C 6-10 Aryl, 5-10 membered heteroaryl or 4-12 membered heterocyclic alkyl;

[0019] R 2 Each can be independently represented by hydrogen, deuterium, halogen, nitro, -CN, -OH, -NH2, or C. 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 2-6 alkenyl, C 2-6 Alkyne group, -CONH2, -COOH, -QC 0-6 Alkylene-C 3-12 cycloalkyl, -QC 0-6 Alkylene (4-12 membered heterocyclic alkyl), -QC 0-6 Alkylene-C 6-10 Aryl or -QC 0-6 alkylene-(5-10-membered heteroaryl); the C 1-6 Alkyl, C 0-6 Alkylene, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-12 Cycloalkyl, 4-12 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl groups are optionally bonded to one or more halogens, -CN, -OH, -NH2, C 1-6 Alkyl, oxo, and halogenated C 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl and -N(C) 1-6 Alkyl)2-substituted;

[0020] Or, any two adjacent R 2 Together with the atoms attached to it, they form C 5-8 Cycloalkyl or 5- to 8-membered heterocyclic alkyl;

[0021] Q can be a chemical bond, -O-, -S-, or -NH-, each independently.

[0022] k1 can be 0, 1, 2, or 3;

[0023] W represents a chemical bond, -O-, -S-, or -NR. w -、-C 1-6 alkylene-or-(C 1-6 Alkylene) m1 -(X)m2 -(C 1-6 Alkylene) m3 -(Y) m4 -;

[0024] X and Y are each independently selected from -O-, -S-, and -NR-. w -、-C(O)-、-NR c C(O)-、-C(O)NR w -, -S(O)-, -S(O)2-, -S(O)2NR w -、-NR w S(O)2- or -OC(O)-; the C 1-6 The alkylene group may optionally be replaced by one or more halogens;

[0025] m1, m2, m3, and m4 are each independently 0 or 1; and m2 and m4 are not both 0 at the same time, and m1 and m3 are both 0 at the same time;

[0026] R w For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-12 Cycloalkyl, 5-10-membered heteroaryl, or 4-12-membered heterocycloalkyl; the C 1-6 Alkyl, C 6-10 Aryl, C 3-12 Cycloalkyl, 5-10-membered heteroaryl, or 4-12-membered heterocycloalkyl are each optionally separated by one or more elements selected from halogen, -CN, -OH, -NH2, C 1-6 Alkyl and Halogenated C 1-6 Alkyl substitution;

[0027] Or R w Can be used with R 2 Together with the N and C atoms attached thereto, they form a 5-12 membered heterocyclic alkyl group; said heterocyclic alkyl group is optionally bonded by one or more halogens, -CN, -OH, -NH2, C 1-6 Alkyl and Halogenated C 1-6 Alkyl substitution;

[0028] R 3 For chemical bonds, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-12 Cycloalkylene, 4- to 12-membered heterocycloalkylene, C 6-10 arylene or 5-10 quinone heteroarylene; the C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-12Cycloalkylene, 4- to 12-membered heterocycloalkylene, C 6-10 Each of the arylene or 5-10 heteroarylene groups is optionally surrounded by one or more groups selected from halogen, -OH, -CN, -NH2, -SH, nitro, -OC. 1-6 Alkyl, -O (halogenated C) 1-6 alkyl), -NHC 1-6 Alkyl, -N(C) 1-6 Alkyl)2, -SC 1-6 Alkyl, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, -C 1-6 Alkyl -OH, -C 1-6 Alkyl-OC 1-6 Alkyl, -C 1-6 Alkyl-SH, -C 1-6 Alkyl-SC 1-6 Alkyl, -C 1-6 Alkyl-NH2, -C 1-6 Alkyl-NH(C) 1-6 Alkyl), -C 1-6 Alkyl-N(C) 1-6 Alkyl group 2, -C(O)NH2, -COOH, -C(O)NHC 1-6 Alkyl, -C(O)N(C) 1-6 Alkyl)2、-NHC(O)C 1-6 Alkyl, -NHC(O)OC 1-6 Alkyl group, -S(O)2C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 Cycloalkyl and 4- to 6-membered heterocyclic alkyl substitutions;

[0029] Z represents a chemical bond, -C 0-6 Alkylene -NH-, -C 0-6 Alkylene-N(C) 1-6 alkyl)-, -C 0-6 alkylene-O- or -C 0-6 Alkylene-S-.

[0030] In some implementation schemes, R 1 It is hydrogen, deuterium, or -CH3; preferably, R 1 For H.

[0031] In some implementations, ring A is: Preferably, ring A is: More preferably, ring A is In this context, 1 is connected to V, and 2 is connected to piperidinedione; k2 is either 0 or 1.

[0032] In some implementation schemes, R a Each is independently hydrogen, deuterium, halogen, -OH, -CN, -NH2, nitro, -CH3, or -OCH3; preferably, R a It is hydrogen, F, -OH, -NH2 or -CH3; more preferably, R a It can be hydrogen or F.

[0033] In some implementations, V is -NH-.

[0034] In some implementations, V is -O-.

[0035] In some embodiments, L is a chemical bond or -CH2-; preferably, L is -CH2-.

[0036] In some implementations, ring B is C. 6-10 Aryl, 5-10 membered heteroaryl or 5-10 membered heterocyclic alkyl; preferably, ring B is phenyl or pyridyl; more preferably, ring B is phenyl.

[0037] In some implementations, k1 is 0, 1, or 2; preferably, k1 is 0 or 1.

[0038] In some implementation schemes, R 2 Each can be independently hydrogen, halogen, -CN, -OH, or C. 1-6 Alkyl, -OC 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-12 Cycloalkyl or 4-12 membered heterocyclic alkyl; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-12 Cycloalkyl or 4-12 membered heterocycloalkyl groups are each optionally converted by one or more halogens, C 1-6 Alkyl, -OC 1-6 Alkyl and Halogenated C 1-6 Alkyl substitution; preferably, R 2 Each of these groups is independently hydrogen, F, Cl, Br, -CN, -CH3, -iPr, -tBu, -CF3, -CHF2, -OCH3, -OCF3, -O-CH2-CH2-O-CH3, cyclopropyl, vinyl, or ethynyl; more preferably, R 2 Each can be independently hydrogen, F, Cl, -CH3, or -CF3.

[0039] In some implementation schemes, R 2 Each independently as -OC 6-10 Aryl, -O-CH2-C 6-10 Aryl, -CH2-C6-10 Aryl, -O-(5-6 heteroaryl), -O-CH2-(5-6 heteroaryl), -CH2-(5-6 heteroaryl), -SC 6-10 Aryl or -S- (5-6 membered heteroaryl); the -CH2-, C 6-10 Aryl or 5-6 heteroaryl groups are each optionally bonded by one or more F, Cl, C 1-6 Alkyl and Halogenated C 1-6 Alkyl substitution;

[0040] Preferably, R 2 Each of the following is independently -O-phenyl, -O-CH2-phenyl, -CH2-phenyl, -O-pyridyl, -O-CH2-pyridyl, -CH2-pyridyl, -S-phenyl or -S-pyridyl; wherein the phenyl and pyridyl groups are each optionally substituted with one or more F, Cl, -CH3 and -CF3;

[0041] In some implementations, W represents a chemical bond, -O-, -S-, -NH-, or -C. 1-3 Alkylene-, -N(C) 1-3 alkyl)-, -(C 1-3 Alkylene) m1 -(O) m2 -(C 1-3 Alkylene) m3 -(O) m4 -、-(C 1-3 Alkylene) m1 -(O) m2 -(C 1-3 Alkylene) m3 -(N(C 1-6 alkyl)) m4 -、-(C 1-3 Alkylene) m1 -(N(C 1-6 alkyl)) m2 -(C 1-3 Alkylene) m3 -(O) m4 -or-(C 1-3 Alkylene) m1 -(N(C 1-6 alkyl)) m2 -(C 1-3 Alkylene) m3 -(N(C 1-6 alkyl)) m4 -

[0042] In some implementations, W and R 2Together with its attached N and C atoms, it forms a 5-6 membered heterocyclic alkyl group; said heterocyclic alkyl group is optionally bonded by one or more halogens, -C 1-6 Alkyl and -halogenated C 1-6 Alkyl substitution.

[0043] In some implementation schemes, R 3 For chemical bonds, C 1-4 Alkylene, C 3-6 Cycloalkylene, 4- to 6-membered heterocycloalkylene, C 6-10 arylene or 5-6 quinone heteroarylene; the C 1-4 Alkylene, C 3-6 Cycloalkylene, 4- to 6-membered heterocycloalkylene, C 6-10 Each of the arylene or 5-6 heteroarylene groups is optionally surrounded by one or more elements selected from halogen, -OH, -CN, -OC. 1-6 Alkyl, -O (halogenated C) 1-6 alkyl), -NHC 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, -C 1-6 Alkyl-OC 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 Cycloalkyl and 4- to 6-membered heterocyclic alkyl substitutions.

[0044] In some implementation schemes, R 3 The chemical bond is phenylene, pyridylene, cyclobutylene, cyclopentylene, cyclohexylene, piperazine, piperidinyl, morpholinyl, aziridine, aziridine, oxadicyclobutyl, or oxadicyclopentyl; wherein the phenylene, pyridylene, cyclobutylene, cyclopentylene, cyclohexylene, piperazine, piperidinyl, morpholinyl, aziridine, aziridine, oxadicyclobutyl, or oxadicyclopentyl is optionally bonded by one or more elements selected from halogen, -OH, -CN, -OC. 1-6 Alkyl, -O (halogenated C) 1-6 Alkyl), C 1-6 Alkyl, Halogenated C 1-6 Alkyl or C 3-6 Cycloalkyl substitution.

[0045] In some implementation schemes, R 3The chemical bond is phenylene, pyridylene, cyclobutylene, cyclopentylene, cyclohexylene, piperazine, piperidinyl, morpholinyl, aziridine, aziridine, oxadicyclobutyl, or oxadicyclopentyl; wherein the phenylene, pyridylene, cyclobutylene, cyclopentylene, cyclohexylene, piperazine, piperidinyl, morpholinyl, aziridine, aziridine, oxadicyclobutyl, or oxadicyclopentyl is optionally bonded by one or more elements selected from F, Cl, -OH, -CN, -OC. 1-6 Alkyl, -O (halogenated C) 1-6 Alkyl), C 1-6 Alkyl, Halogenated C 1-6 Alkyl or C 3-6 Cycloalkyl substitution.

[0046] In some implementation schemes, R 3 The chemical bond is phenylene, pyridinylene, cyclobutylene, cyclopentylene, cyclohexylene, piperazine, piperidinylene, morpholinyl, aziridine, or aziridine; wherein the phenylene, pyridinylene, cyclobutylene, cyclopentylene, cyclohexylene, piperazine, piperidinylene, morpholinylene, aziridine, or aziridine is optionally substituted by one or more of the groups selected from F, Cl, -OH, -CN, -OCH3, -OCF3, -CH3, -iPr, -tBu, -CF3, or -cyclopropyl.

[0047] In some implementations, Z is a chemical bond, provided that W and R are... 3 They cannot both be chemical bonds.

[0048] In some implementations, Z is -NH- or -N(CH3)-; preferably, Z is -NH-.

[0049] In some implementations, each halogen is independently fluorine, chlorine, bromine, or iodine, preferably fluorine.

[0050] In some implementations, each C 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl, such as methyl.

[0051] In some implementations, each -OC 1-6 The alkyl group is independently -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-n-butyl, -O-tert-butyl, -O-isobutyl or -O-sec-butyl; for example -O-methyl.

[0052] In some implementations, each C 2-6 The alkenyl group can be vinyl, propenyl, allyl, butenyl, or pentenyl.

[0053] In some implementations, each C 2-6 The alkynyl group can be independently ethynyl, propynyl, propynyl, butynyl, or pentylyyl, for example...

[0054] In some implementations, each C 3-12 The cycloalkyl group is independently C 3-8 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0055] In some embodiments, the cycloalkyl group is a saturated cycloalkyl group.

[0056] In some embodiments, the cycloalkyl group is a partially saturated cycloalkyl group; the partially saturated cycloalkyl group has one or more unsaturated bonds.

[0057] In some implementations, each 4- to 12-membered heterocyclic alkyl group is independently a 5- to 8-membered heterocyclic alkyl group.

[0058] In some embodiments, each heteroatom of a 4- to 12-membered heterocyclic alkyl group is independently N, O, or S, and the number of heteroatoms is independently 1, 2, or 3; preferably, each heteroatom of a 4- to 12-membered heterocyclic alkyl group is independently N or O, and the number of heteroatoms may be independently 1 or 2.

[0059] In some embodiments, each 4- to 12-membered heterocyclic alkyl group is independently monocyclic or polycyclic, the polycyclic being a bridged ring, fused ring, or spirocyclic; the polycyclic being a bicyclic or tricyclic; preferably, each 4- to 12-membered heterocyclic alkyl group is independently a 5- to 6-membered monocyclic heterocyclic alkyl group, a 9- to 10-membered bicyclic heterocyclic alkyl group, or an 11- to 12-membered tricyclic heterocyclic alkyl group.

[0060] In some implementations, each C 1-6 The alkylene group is independently methylene, ethylene, n-propylene, isopropylene, n-butylene, tert-butylene, isobutylene, or sec-butylene, preferably methylene.

[0061] In some implementations, each C 6-10 The aryl group can be phenyl or naphthyl, for example, phenyl.

[0062] In some implementations, the heteroatom of each 5-10 aryl group is independently N, O, or S, and the number of heteroatoms is independently 1, 2, or 3.

[0063] In some implementations, each 5-10 nucleotide heteroaryl group is independently a monocyclic or bicyclic ring, wherein the bicyclic ring is a fused ring.

[0064] In some embodiments, each of the 4 to 14-membered heterocyclic alkyl groups is a 5 to 12-membered heterocyclic alkyl group.

[0065] In some embodiments, each of the 4 to 14-membered heterocyclic alkyl groups has a heteroatom of N, O, or S, and the number of heteroatoms is 1, 2, or 3; preferably, the heteroatom is N, and the number of heteroatoms is 1 or 2.

[0066] In some embodiments, each of the 4 to 14-membered heterocyclic alkyl groups is a monocyclic or polycyclic (e.g., bicyclic or tricyclic), and the polycyclic group may be a bridged ring, a fused ring, or a spirocyclic ring.

[0067] In some embodiments, each 4- to 14-membered heterocyclic alkyl group contains 0, 1, or 2 unsaturated rings, preferably 1 unsaturated ring and at least one saturated ring; the unsaturated ring is preferably an aromatic ring.

[0068] In some embodiments, when each 4 to 14-membered heterocyclic alkyl group contains one unsaturated ring and at least one saturated ring, the heteroatom is located on the saturated ring.

[0069] In some implementations, each 4- to 14-membered heterocyclic alkyl group contains 0, 1, or 2 unsaturated bonds.

[0070] In some embodiments, each 4- to 14-membered heterocyclic alkyl group is a monocyclic, bicyclic, or tricyclic heterocyclic alkyl group; preferably a 5- to 6-membered monocyclic heterocyclic alkyl group, a 9- to 10-membered bicyclic heterocyclic alkyl group, or an 11- to 14-membered tricyclic heterocyclic alkyl group; for example

[0071] In some embodiments, the heterocyclic alkyl group is a saturated heterocyclic alkyl group.

[0072] In some embodiments, the heterocyclic alkyl group is a partially saturated heterocyclic alkyl group; the number of unsaturated bonds in the partially saturated heterocyclic alkyl group is one or more.

[0073] In some implementations, each C 0-6 The alkylene group is independently a linker, methylene, ethylene, n-propylene, isopropylene, n-butylene, tert-butylene, isobutylene, or sec-butylene, preferably a linker, methylene, or ethylene.

[0074] In some implementations, the structure shown in equation (I) is the same as the structure shown in equation (Ia):

[0075]

[0076] in,

[0077] R 1 R a R 2 R 3 The definitions of V, L, W, Z, ring B, k1, and k2 are as described in any one of the formulas (I) of this invention.

[0078] In some implementations, the structures shown in equations (I) and (Ia) are the same as those shown in equation (Ib):

[0079]

[0080] in,

[0081] R a R 2 R 3 The definitions of W, Z, k1, and k2 are as described in any one of the present invention formula (I).

[0082] In some implementations, the structures shown in equations (I), (Ia), and (Ib) are the same as those shown in equation (Ib-1):

[0083]

[0084] in,

[0085] R a R 2 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I).

[0086] In some implementations, the structures shown in equations (I), (Ia), (Ib), and (Ib-1) are: wherein,

[0087] R 2 Each of the following groups is independently hydrogen, F, Cl, Br, -CN, -CH3, -iPr, -tBu, -CF3, -CHF2, -OCH3, -OCF3, -O-CH2-CH2-O-CH3, cyclopropyl, vinyl, or ethynyl; preferably, R 2 Each can be independently hydrogen, F, Cl, -tBu, or -O-CH2-CH2-O-CH3;

[0088] Or, any two adjacent R 2 Together with the atoms attached to it, they form C 5-8 Cycloalkyl, 5- to 8-membered heterocyclic alkyl; preferably, two adjacent R 2 It forms together with the atoms it is attached to. The C mentioned 5-8 Cycloalkyl, 5- to 8-membered heterocycloalkyl or Each is optionally influenced by one or more halogens, -CN, -OH, -NH2, C 1-6 Alkyl, oxo, and halogenated C 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl and -N(C) 1-6 Alkyl)2-substituted;

[0089] Or, R 2 Each of the following is independently -O-phenyl, -O-CH2-phenyl, -CH2-phenyl, -O-pyridyl, -O-CH2-pyridyl, -CH2-pyridyl, -S-phenyl, or -S-pyridyl; preferably, R 2 Each of the following is independently -O-phenyl, -O-CH2-phenyl, -CH2-phenyl, -O-pyridyl, -O-CH2-pyridyl, -S-phenyl; wherein the phenyl and pyridyl groups are each optionally substituted with one or more F, Cl, -CH3 and -CF3;

[0090] W represents -NH-, -N(C) 1-3 Alkyl group, -CH2CH2-O-CH2CH2-NH-, -CH2CH2-O-CH2CH2-N(CH3)-, -CH2-O-CH2CH2-N(CH3)-, -CH2-O-CH2CH2CH2-N(CH3)-, -O-CH2CH2-NH- or -O-CH2CH2-N(CH3)-; preferably, W is -NH-, -N(C 1-3 Alkyl)-, -CH2CH2-O-CH2CH2-N(CH3)-, -O-CH2CH2-N(CH3)- or -CH2-O-CH2CH2CH2-N(CH3)-;

[0091] Or, W and R 2 Together with the attached N and C atoms, they form Side a is connected to ring B in a parallel loop.

[0092] In some implementations, the structures shown in equations (I), (Ia), (Ib), and (Ib-1) are: wherein,

[0093] R 2 Each can be independently hydrogen, F, Cl, -CH3, -O-phenyl, or -S-phenyl.

[0094] W can be -NH-, -CH2CH2-O-CH2CH2-N(CH3)-, or -O-CH2CH2-N(CH3)-;

[0095] Or, W and R 2 Together with the attached N and C atoms, they form Side a is connected to ring B in a parallel loop.

[0096] In some embodiments, the structures shown in formulas (I), (Ia), (Ib), and (Ib-1) are compounds shown as (Ib-1a), (Ib-1b), (Ib-1c), (Ib-1d), or (Ib-1e):

[0097]

[0098] R a R 2 The definitions of W and W are as described in any one of the present invention formulas (I).

[0099] In some implementations, the structures shown in equations (I), (Ia), and (Ib) are the same as those shown in equation (Ib-2):

[0100]

[0101] in,

[0102] R a R 2 R 3 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I).

[0103] In some implementations, the structures shown in equations (I), (Ia), (Ib), and (Ib-2) are: wherein,

[0104] R 2 Each is independently hydrogen, F, Cl, Br, -CN, -CH3, -CF3, -CHF2, -OCH3, or -OCF3; preferably, R 2 Each can be independently hydrogen, F, or Cl;

[0105] W represents a chemical bond, -O-, -S-, or -NR. w -, -CH2-, -CH2CH2-O-, -O-CH2CH2-, -CH2-O-CH2-, -CH2-O- or -O-CH2-; preferably, W is a chemical bond, -O-, -S-, -CH2-, -CH2CH2-O-, -O-CH2CH2-, -CH2-O-CH2-, -CH2-O- or -O-CH2-;

[0106] R w It is hydrogen or -C 1-6 Alkyl; preferably hydrogen;

[0107] R 3 It is phenylene, pyridinyl, cyclobutylene, cyclopentylene, cyclohexylene, piperidinyl, aziridine, or aziridine; preferably, R 3It is phenylene, cyclobutylene, cyclohexylene, piperidinylene, or aziridine butylene; wherein each of the phenylene, pyridinylene, cyclobutylene, cyclopentylene, cyclohexylene, piperidinylene, aziridine butylene, or aziridine pentylene is optionally substituted by one or more of the following: F, Cl, -OH, -CN, -OCH3, -OCF3, -CH3, -iPr, -tBu, -CF3, or -cyclopropyl.

[0108] In some implementations, the structures shown in equations (I), (Ia), (Ib), and (Ib-2) are, wherein,

[0109] R 2 Each can be independently hydrogen, F, or Cl;

[0110] W can be -S-, -O-CH2-, or -CH2-O-;

[0111] R 3 It is a phenylene oxide.

[0112] In some embodiments, the compounds shown in formulas (I), (Ia), (Ib), and (Ib-2) are:

[0113] in,

[0114] for

[0115] In some embodiments, the structures shown in formulas (I), (Ia), (Ib), and (Ib-2) are compounds shown in (Ib-2a), (Ib-2b), or (Ib-2c):

[0116]

[0117] in,

[0118] R 2 R 3 The definitions of W and W are as described in any one of the present invention formulas (I).

[0119] In some implementations, the structures shown in equations (I), (Ia), and (Ib) are the same as those shown in equation (Ib-3):

[0120]

[0121] in,

[0122] R 3 for Wherein, the a-terminus is connected to W; ring D is a 4- to 12-membered heterocyclic alkyl group; each of the 4- to 12-membered heterocyclic alkyl groups is optionally surrounded by one or more groups selected from halogen, -OH, -CN, -NH2, nitro, -OC. 1-6 Alkyl, -O (halogenated C) 1-6 Alkyl), C 1-6 Alkyl, Halogenated C 1-6 Alkyl substitution;

[0123] R a R 2 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I).

[0124] In some embodiments, compounds with the structures shown in formulas (I), (Ia), (Ib), and (Ib-3) are:

[0125] in,

[0126] R 2 Each is independently hydrogen, F, Cl, Br, -CN, -CH3, -CF3, -CHF2, -OCH3, or -OCF3; preferably, R 2 Each can be independently hydrogen, F, Cl, or -CH3;

[0127] W is a chemical bond, -O-, -CH2CH2-O-CH2-, -CH2CH2-O-, -CH2-O-CH2CH2-, -CH2-O-CH2-, -CH2-O-, -O-CH2CH2-, or -OCH2-; preferably, W is a chemical bond, -O-, -CH2CH2-O-, -O-CH2CH2-, -CH2-O-CH2-, -CH2-O-, or -O-CH2-; more preferably, W is -CH2-O- or -O-CH2-;

[0128] R 3 for Preferably, R 3 for More preferably, R 3 for Among them, end a is connected to W.

[0129] In some embodiments, compounds with the structures shown in formulas (I), (Ia), (Ib), and (Ib-3) are:

[0130] in,

[0131] for Among them, end a is connected to ring A.

[0132] In some embodiments, the structures shown in formulas (I), (Ia), (Ib), and (Ib-3) are compounds shown in (Ib-3a), (Ib-3b), or (Ib-3c):

[0133]

[0134] in,

[0135] R 2 R 3 The definitions of W and W are as described in any one of the present invention formulas (I).

[0136] In some implementations, the structures shown in equations (I), (Ia), and (Ib) are any of the following structures:

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] In some embodiments, the present invention provides a ligand-drug conjugate, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a ligand and the structure shown in formula (II):

[0143]

[0144] in,

[0145] The linker is a connecting subunit that binds to the ligand; it can be cleavable or non-cleavable.

[0146] R 1 R 2 R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the formulas (I) of this invention.

[0147] In some implementations, the structure shown in equation (II) is the same as the structure shown in equation (II-1):

[0148]

[0149] in,

[0150] R 1 R 2R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the present invention formula (I);

[0151] The wavy line indicates that it passes through L. 1 The nitrogen or carbon atom on the group is attached to a ligand;

[0152] L 1 for : Where a end With L 2 Connected;

[0153] L 2 -(C(RL) 21 )2) n -,

[0154] Where n is a natural number from 0 to 50, for example 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0155] L 2 Any C(R) in L21 Each of the two units is independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -, -O-, -S-, -SO-, -SO2-, -P(R L22 )-、-P(=O)(R L22 )-、-(R L22 )P(=O)-, -C(=S)-, -C(=NR L22 )-, -N=N-, -C=N-, -N=C-,

[0156] -Cy- is a phenylene, a 5- to 8-membered heteroaryl, a 3- to 10-membered heterocyclic, or a 3- to 10-membered cycloalkylene, wherein each of the -Cy- is independently surrounded by one or more R- atoms. cx replace;

[0157] R L21 R L22 R cx Each is independently selected from hydrogen, deuterium, halogens, -NO2, -CN, and -OR. L2a -SR L2a -N(R) L2a )2、-N + (R L2a 3、-C(O)R L2a -CO2R L2a -C(O)C(O)R L2a -C(O)CH2C(O)R L2a-S(O)R L2a -S(O)2R L2a -C(O)N(R) L2a )2、-SO2N(R L2a )2、-OC(O)R L2a -N(R) L2a SO2R L2b -N(R) L2a )COR L2b -(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a -(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a -(CH2) y -CO-(N(Me)CH2C(O)) m -N + (R L2a 3、-(CH2) y -NHCOCH2(OCH2CH2)OR L2a -(CH2) y -NH(COCH2(N(Me)) m -R L2a -(CH2) y -CONH-(CH2CH2O) m -R L2a -(CH2) y -NHCO-(CH2CH2O) m -R L2a -(CH2CH2O) m -R L2a -(COCH2N(Me)) m -R L2a -COCH2(OCH2CH2) m -OR L2a -CO-(CH2CH2O) m -R L2a -CO-(CH2) y -CONH-(CH2CH2O) m -R L2a -CO-(CH2) y -NHCO-(CH2CH2O) m -R L2a and being R L2a Optional substitution of C 1-6 Alkyl, C 1-6 alkenyl, C1-6 Alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl;

[0158] m and y are each natural numbers from 0 to 50, for example, 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0159] R L2a R L2b Each is independently selected from hydrogen, deuterium, halogens, -NO2, -CN, -OH, -SH, -NH2, -N(Me)2, -CO2H, -S(O)2Me, -S(O)2OH, -C(O)NH2, -SO2NH2, C 1-6 Alkyl, C 1-6 alkenyl, C 1-6 Alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl and 3-10 membered heteroaryl;

[0160] L 3 It is absent or consists of amino acid residues, or short peptides composed of 2-10 amino acid residues. Or any combination of the above groups, wherein the amino acid residue is a natural amino acid residue or a non-natural amino acid residue; wherein the a-terminus is connected to the L-terminus. 2 Linked; preferably, the N-terminus of the amino acid residue and the short peptide composed of 2-10 amino acid residues is connected to L. 2 Connected;

[0161] Tr does not exist or is Or any combination of the above groups; wherein the a-terminus is connected to the L-terminus. 3 Connected;

[0162] R Tr R Tr1 and R Tr2 Each is independently selected from hydrogen, deuterium, halogens, -NO2, -CN, -OH, -SH, -NH2, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -OC(O)NH2, -CH2CO-(N(Me)CH2C(O))) z -OR Tra -CH2CO-(N(Me)CH2C(O)) z -NHR Tra -(CH2CH2O) z -R Tra -CONH-(CH2CH2O) z -R Tra and being R Tra Optional substitution of C 1-6 Alkyl, C 1-6alkenyl, C 1-6 Alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl;

[0163] R Tra Independently selected from hydrogen, deuterium, halogens, -NO2, -CN, -OH, -SH, -NH2, -N(Me)2, -S(O)2Me, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, C 1-6 Alkyl, C 1-6 alkenyl, C 1-6 Alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl and 3-10 membered heteroaryl;

[0164] z is an independent natural number from 0 to 50; for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0165] In some implementations, the structures shown in equations (II) and (II-1) are...

[0166] Among them, L 1 Selected from: Preferably, L 1 Selected from Where end a and L 2 Connected.

[0167] In some implementations, the structures shown in equations (II) and (II-1) are...

[0168] Among them, L 2 -(CHR) L21 ) n -;

[0169] n is a natural number from 0 to 50; for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0170] L 2 Each CH2 unit in the structure is independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -、-O-、

[0171] -Cy- is a phenylene, a 5- to 6-membered heteroaryl, a 4- to 10-membered heterocyclic group, or a 3- to 6-membered cycloalkylene, wherein the -Cy- is independently surrounded by 1 to 3 R- groups. cx replace;

[0172] Each R L21 R L22 R cx Each independently represents hydrogen, halogen, -OR L2a-N(R) L2a )2、-C(O)R L2a -S(O)2R L2a -C(O)N(R) L2a )2、-SO2N(R L2a )2、-N(R L2a SO2R L2b -N(R) L2a )COR L2b -(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a -(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a -(CH2) y -CONH-(CH2CH2O) m -R L2a -(CH2) y -NHCO-(CH2CH2O) m -R L2a -(CH2) y -NHCOCH2(OCH2CH2)OR L2a -(CH2) y -NH(COCH2(N(Me)) m -R L2a -(CH2) y -NHCO-(CH2CH2O) m -R L2a -(CH2CH2O) m -R L2a -(COCH2N(Me)) m -R L2a -COCH2(OCH2CH2) m -OR L2a -CO-(CH2CH2O) m -R L2a or by R L2a Optional substitution of C 1-6 Alkyl, C 1-6 alkenyl, C 1-6 Alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl;

[0173] m is a natural integer from 0 to 8;

[0174] y is 0, 1, 2, 3 or 4;

[0175] Each RL2a R L2b Each can be independently represented as hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2, C 1-6 alkyl.

[0176] In some implementations, the structures shown in equations (II) and (II-1) are...

[0177] Among them, L 2 -(CH2) n -;

[0178] n is a natural integer from 0 to 50; for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0179] L 2 Each methylene unit in the structure is independently replaced by the following structural units: 4- to 6-membered heterocyclic group, 3- to 6-membered cycloalkyl group, -C(O)-, -NR L22 -、-O-、

[0180] Each R L22 Each is independently selected from hydrogen, -OR L2a -C(O)R L2a -S(O)2R L2a -C(O)N(R) L2a )2、-SO2N(R L2a 2、-(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a -(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a -(CH2) y -CONH-(CH2CH2O) m -R L2a -(CH2) y -NHCOCH2(OCH2CH2)OR L2a -(CH2CH2O) m -R L2a -(COCH2N(Me)) m -R L2a -COCH2(OCH2CH2) m -OR L2a -CO-(CH2CH2O) m -R L2a and being R L2a Optional substitution of C 1-6alkyl;

[0181] m is a natural integer from 0 to 8;

[0182] y is 0, 1, 2, 3 or 4;

[0183] Each R L2a Each can be independently represented as hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2, C 1-6 alkyl.

[0184] In some implementations, the structures shown in equations (II) and (II-1) are...

[0185] Among them, L 2 for:

[0186]

[0187] Among them, the left side and L 1 Connected;

[0188] in,

[0189] n1, n2, n3, and n4 are each independent natural numbers from 0 to 8; for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0190] n5 and n6 are each independently 0 or 1;

[0191] -Cy- is a 4- to 6-membered heterocyclic group or a 3- to 6-membered cycloalkyl group; preferably, -Cy- is... More preferably, -Cy- is The a-terminus is connected to a carbonyl group;

[0192] Preferably,

[0193] for

[0194] Among them, the c-end and L 1 Connected, d end and L 3 Connected.

[0195] In some implementations, the structures shown in equations (II) and (II-1) are...

[0196] in, for:

[0197]

[0198] Among them, the right side and L 3 Connected;

[0199] n1, n2, n3, and n4 are each independent natural numbers from 0 to 8; for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0200] n5 and n6 are each independently 0 or 1;

[0201] Preferably, for: Among them, the f end and L 3 Connected.

[0202] In some implementations, the structures shown in equations (II) and (II-1) are...

[0203] Among them, L 3 Does not exist or is L 3a -L 3b ;

[0204] L 3a and L 3b Short peptides consisting of 2-10 amino acid residues or absent or absent amino acid residues. Among them, the a end and L 2 Connected.

[0205] In some embodiments, the structures shown in equations (II-1), (IIa), (IIb), (IIb-1), (IIb-2), and (IIb-3) are wherein, L 3a With L 2 Connected.

[0206] In some embodiments, the structures shown in equations (II-1), (IIa), (IIb), (IIb-1), (IIb-2), and (IIb-3) are wherein, L 3aIs absent or selected from Val, D-Val, Phe, Lys, Leu, Ile, Gly, Ala, D-Ala, Cit, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Leu-Lys, Leu-Lys(Ac), Ala-Ala, Ala-Ly s, D-Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Val-Glu, Val-Asp, Asn-Asn, Asp-Glu, Asp-Ser, Gly-Gly-Glu, Gly-Gly-Asp, Gly-Gly-Asn, Gly-Ala-Ala, Gly-Val-Ala, Gly-Val-Cit, Glu-Val-C it, Ala-Ala-Ala, Ala-(D-Ala)-Ala, Ala-Ala-Asn, Ala-(D-Ala)-Asn, Ala-Ala-Asp, Val-Lys-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Gly-Gly-Gly, Lys-Ala-Asn, Gly-Phe-Gly, Gly-Gly-Phe, Asn-Pro-Val, Ala-Lys-Gly, Gly-Lys-Gly, Gly-Glu-Gly, (Gly)4, (Gly)2-Phe-Gly, (Gly)2-Glu-Gly, Lys-(Ala)2-Asn, Lys-(Ala)2-Asp, (Ala)2-Pro-Val, and (Ala)2-Pro-Nva; wherein, the nitrogen terminus of the amino acid residue is related to L 2 Connected;

[0207] L 3b For non-existent or Among them, the a end and L 3a Connected.

[0208] In some embodiments, the structures shown in equations (II-1), (IIa), (IIb), (IIb-1), (IIb-2), and (IIb-3) are wherein, L 3a The amino acid residue is absent or selected from Lys, Gly, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asp-Glu, Gly-Glu-Gly, and (Gly)2-Phe-Gly; wherein the nitrogen terminus of the amino acid residue is connected to L 2 Connected;

[0209] L3b For non-existent or Among them, the a end and L 3a Connected;

[0210] More preferably, L 3a It is absent or selected from Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, and (aGly)2-Phe-Gly; wherein the nitrogen terminus of the amino acid residue is L 2 Connected;

[0211] L 3b For non-existent or Among them, the a end and L 3a Connected.

[0212] In some implementations, the structures shown in equations (II) and (II-1) are...

[0213] in, for:

[0214]

[0215]

[0216] Preferably, for

[0217] More preferably, for

[0218] In some implementations, the structures shown in equations (II) and (II-1) are...

[0219] Where Tr either does not exist or is Among them, the a end and L 3 Connected;

[0220] R Tr R Tr1 and R Tr2 Independently, it can be hydrogen, halogen, -NO2, -CN, -OH, -NH2, -CO2H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)NH2, or -CH2CO-(N(Me)CH2C(O)). z -NHMe, -(CH2CH2O) z -H, -CONH-(CH2CH2O) z -H;

[0221] z is a natural number from 0 to 8; for example, 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0222] In some implementations, the structures shown in equations (II) and (II-1) are...

[0223] Where Tr either does not exist or is Among them, the a end and L 3 Connected.

[0224] In some implementations, the structures shown in equations (II) and (II-1) are the same as those shown in equation (IIa):

[0225]

[0226] in,

[0227] R 1 R a R 2 R 3 The definitions of V, L, W, Z, ring B, k1, and k2 are as described in any one of the present invention formula (I);

[0228] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0229] In some implementations, the structures shown in equations (II), (II-1), and (IIa) are the same as those shown in equation (IIb):

[0230]

[0231] in,

[0232] R a R 2 R 3 The definitions of W, Z, k1, and k2 are as described in any one of the present invention formula (I);

[0233] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0234] In some implementations, the structures shown in equations (II), (II-1), (IIa), and (IIb) are the same as those shown in equation (IIb-1):

[0235]

[0236] in,

[0237] R a R 2 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0238] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0239] In some embodiments, the structures shown in formulas (II), (II-1), (IIa), (IIb), and (IIb-1) are the structures shown in formulas (IIb-1a), (IIb-1b), (IIb-1c), (IIb-1d), or (IIb-1e):

[0240]

[0241] in,

[0242] Ra、R 2 The definitions of W and W are as described in any one of the present invention formulas (I);

[0243] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0244] In some implementations, the structures shown in equations (II), (II-1), (IIa), and (IIb) are the same as those shown in equation (IIb-2):

[0245]

[0246] in,

[0247] R a R 2 R 3 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0248] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0249] In some embodiments, the structures shown in equations (II), (II-1), (IIa), (IIb), and (IIb-2) are the structures shown in equations (IIb-2a), (IIb-2b), or (IIb-2c):

[0250]

[0251] in,

[0252] R 2 R 3 The definition of W is as described in any one of the present invention formulas (I);

[0253] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0254] In some implementations, the structures shown in equations (II), (II-1), (IIa), and (IIb) are the same as those shown in equation (IIb-3):

[0255]

[0256] in,

[0257] R a R 2 R 3 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0258] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0259] In some embodiments, the structures shown in equations (II), (II-1), (IIa), (IIb), and (IIb-3) are the structures shown in equations (IIb-3a), (IIb-3b), or (IIb-3c):

[0260]

[0261]

[0262] R 2 R 3 The definitions of W and W are as described in any one of the present invention formulas (I);

[0263] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0264] In some implementations, the structure shown in equation (II), (II-1), or (IIa) is any of the following structures:

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271] In some embodiments, the present invention provides a ligand-drug conjugate of formula (III), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

[0272]

[0273] in,

[0274] Ab is the ligand that binds to the target.

[0275] q represents the drug loading (drug-antibody conjugate ratio);

[0276] R 1 R 2 R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the present invention formula (I);

[0277] The linker is as described in any of formula (II) of this invention.

[0278] Additionally, it should be noted that the linker is connected to the Ab via a sulfur atom. Those skilled in the art will understand that the linker is connected to the thiol group inherent in the Ab itself after the disulfide bond is broken; that is, the -S- group between the linker and the Ab is not an additional external sulfur atom. For example... In this context, -S- does not refer to an additional external sulfur atom, but rather to the thiol group inherent in the Ab itself after the disulfide bond is broken, and the linker, for example... -S- is formed by connecting the a-end.

[0279] In some implementations, the ligand-drug conjugate with the structure shown in formula (III) is wherein Ab is a target-binding polypeptide, antibody, or antigen-binding fragment thereof.

[0280] In some preferred embodiments, the ligand-drug conjugate with the structure shown in formula (III) is wherein Ab is an antibody or its antigen-binding fragment.

[0281] In some embodiments, in the ligand-drug conjugate Ab with the structure shown in formula (III), the antibody is selected from one or more of the following:

[0282] (1) Fully human antibodies, humanized antibodies, murine antibodies, and chimeric antibodies;

[0283] (2) Probody;

[0284] (3) Bispecific antibodies and multispecific antibodies;

[0285] (4) Monoclonal antibodies and polyclonal antibodies;

[0286] (5)IgG antibodies.

[0287] In some embodiments, in the ligand-drug conjugate Ab with the structure shown in formula (III), the antigen-binding fragment is selected from: Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, VHH, and complementarity-determining region (CDR) fragments.

[0288] In some implementations, the ligand-drug conjugate with the structure shown in formula (III) is wherein Ab is a monoclonal antibody.

[0289] In some preferred embodiments, the ligand-drug conjugate of formula (III) is wherein the Ab targets an antigen selected from the group consisting of HER2, HER3, B7H3, B7H4, DLL3, TROP2, Claudin18.2, CD30, CD33, CD70, GPC-3, ADAM9, and EGFR.

[0290] In some embodiments, the ligand-drug conjugate of formula (III) is wherein Ab is an antibody or antigen-binding fragment thereof targeting HER3, B7H3, Claudin18.2, CD30, CD33, CD70, GPC-3, ADAM9 and EGFR.

[0291] In some preferred embodiments, the ligand-drug conjugate of formula (III) is used, wherein Ab is an antibody targeting HER3 or an antigen-binding fragment thereof, such as Patritumab or a variant thereof.

[0292] In some embodiments, the ligand-drug conjugate of formula (III) is used, wherein Ab is an anti-GPC-3 antibody or its antigen-binding fragment, such as codrituzumab or a variant thereof.

[0293] In some embodiments, the ligand-drug conjugate of formula (III) comprises an anti-Her3 antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0294] In some embodiments, the ligand-drug conjugate of formula (III) comprises an anti-HER3 antibody or an antigen-binding fragment thereof containing a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8 or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with it;

[0295] In some embodiments, the ligand-drug conjugate of formula (III) is wherein the anti-HER3 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO:7 and the amino acid sequence of the light chain variable region being as shown in SEQ ID NO:8.

[0296] In some embodiments, the ligand-drug conjugate of formula (III) comprises an anti-HER3 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain is as shown in SEQ ID NO:10 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0297] In some embodiments, the ligand-drug conjugate of formula (III) is wherein the anti-HER3 antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9 and the amino acid sequence of the light chain is as shown in SEQ ID NO:10.

[0298] In some embodiments, the ligand-drug conjugate of the structure shown in formula (III) is preferred, wherein the Ab is preferably an anti-GPC-3 antibody or an antigen-binding fragment thereof, such as codrituzumab or a variant thereof.

[0299] In some embodiments, the ligand-drug conjugate of formula (III) comprises a heavy chain variable region and a light chain variable region, wherein the anti-GPC-3 antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively.

[0300] In some embodiments, the ligand-drug conjugate of formula (III) comprises an anti-GPC-3 antibody or an antigen-binding fragment thereof containing a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17 or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:18 or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with it;

[0301] In some embodiments, the ligand-drug conjugate of the structure shown in formula (III) is wherein the anti-GPC-3 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO:17 and the amino acid sequence of the light chain variable region being shown in SEQ ID NO:18.

[0302] In some embodiments, the ligand-drug conjugate of formula (III) comprises an anti-GPC-3 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:19 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain is as shown in SEQ ID NO:20 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0303] In some embodiments, the ligand-drug conjugate of formula (III) is wherein the anti-GPC-3 antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:19 and the amino acid sequence of the light chain is as shown in SEQ ID NO:20.

[0304] In some embodiments, the ligand-drug conjugate with the structure shown in formula (III) is wherein q is an integer or fractional of 1-32, preferably an integer or fractional of 1-16, more preferably an integer or fractional of 2-8, such as 2, 3, 4, 5, 6, 7, 8, 7.5, 7.6, 7.7, 7.8 or 7.9; and for example 7.53, 7.64, 7.67 or 7.89.

[0305] In some embodiments, the ligand-drug conjugate with the structure shown in formula (III) is the ligand-drug conjugate shown in formula (III-1):

[0306]

[0307] in,

[0308] R 1 R 2 R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the present invention formula (I);

[0309] L 1 L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0310] Ab and q are as described in any of formula (III) of this invention.

[0311] In some embodiments, the ligand-drug conjugates with structures shown in formulas (III) and (III-1) are ligand-drug conjugates with structures shown in formula (IIIa):

[0312]

[0313] in,

[0314] R 1 R a R 2 R 3 The definitions of V, L, W, Z, ring B, k1, and k2 are as described in any one of the present invention formula (I);

[0315] L 1 L 2 L 3And Tr as described in any one of the present invention formulas (II-1);

[0316] Ab and q are as described in any of formula (III) of this invention.

[0317] In some embodiments, the ligand-drug conjugates with structures shown in formulas (III), (III-1), and (IIIa) are ligand-drug conjugates with structures shown in formula (IIIb):

[0318]

[0319] in,

[0320] R a R 2 R 3 The definitions of W, Z, k1, and k2 are as described in any one of the present invention formula (I);

[0321] L 1 L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0322] Ab and q are as described in any of formula (III) of this invention.

[0323] In some embodiments, the ligand-drug conjugates with structures shown in formulas (III), (III-1), (IIIa), and (IIIb) are the ligand-drug conjugates shown in formula (IIIb-1):

[0324]

[0325] in,

[0326] R a R 2 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0327] L 1 L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0328] Ab and q are as described in any of formula (III) of this invention.

[0329] In some embodiments, the structures shown in equations (III), (III-1), (IIIa), (IIIb), and (IIIb-1) are the same as those shown in equations (IIIb-1a), (IIIb-1b), (IIIb-1c), (IIIb-1d), and (IIIb-1e):

[0330]

[0331]

[0332] in,

[0333] R a R 2 The definitions of W and W are as described in any one of the present invention formulas (I);

[0334] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0335] Ab and q are as described in any of formula (III) of this invention.

[0336] In some embodiments, the ligand-drug conjugates with structures shown in formulas (III), (III-1), (IIIa), and (IIIb) are ligand-drug conjugates with structures shown in formula (IIIb-2):

[0337]

[0338] in,

[0339] R a R 2 R 3 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0340] L 1 L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0341] Ab and q are as described in any of formula (III) of this invention.

[0342] In some implementations, the structures shown in equations (III), (III-1), (IIIa), (IIIb), and (IIIb-2) are the same as those shown in equations (IIIb-2a), (IIIb-2b), and (IIIb-2c):

[0343]

[0344]

[0345] in,

[0346] R 2 R 3The definition of W is as described in any one of the present invention formulas (I);

[0347] L 1 L 2 L 3 And Tr as described in any of the present invention formula (II-1).

[0348] Ab and q are as described in any of formula (III) of this invention.

[0349] In some implementations, the structures shown in equations (III), (III-1), (IIIa), and (IIIb) are the same as those shown in equation (IIIb-3):

[0350]

[0351] in,

[0352] R a R 2 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0353] R 3 As described in any of the present invention formulas (Ib-3);

[0354] L 1 L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0355] Ab and q are as described in any of formula (III) of this invention.

[0356] In some implementations, the structures shown in equations (III), (III-1), (IIIa), (IIIb), and (IIIb-3) are the same as those shown in equations (IIIb-3a), (IIIb-3b), and (IIIb-3c):

[0357]

[0358]

[0359] R 2 R 3 The definitions of W and W are as described in any one of the present invention formulas (I);

[0360] L 1 L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0361] Ab and q are as described in any of formula (III) of this invention.

[0362] In some embodiments, formulas (III), (III-1), (IIIa), (IIIb), (IIIb-1), (IIIb-2), and (IIIb-3) and the ligand-drug conjugate shown are any of the following compounds:

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369] Wherein, Ab and q are as described in any one of the formulas (III) of this invention.

[0370] In some preferred embodiments, formulas (III), (III-1), (IIIa), (IIIb), (IIIb-1), (IIIb-2), and (IIIb-3) and the ligand-drug conjugate shown are any of the following compounds:

[0371]

[0372]

[0373]

[0374]

[0375] in,

[0376] q As described in any one of the present invention;

[0377] Ab1 and Ab2 are each independently selected from Patritumab or its variants and Codrituzumab or its variants;

[0378] Preferably, Ab1 is pertrastuzumab; Ab1 is coutuzumab.

[0379] In another aspect, the present invention also provides a nitrogen-containing heterocyclic compound as shown in formula (IV), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

[0380]

[0381] in,

[0382] linker 1 For the linker subunit that binds to the ligand;

[0383] R 1 R 2 R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the formulas (I) of this invention.

[0384] In some embodiments, the compound with the structure shown in formula (IV) is the compound shown in formula (IV-1):

[0385]

[0386] in,

[0387] R 1 R 2 R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the present invention formula (I);

[0388] L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0389] L 1a for:

[0390] In some embodiments, compounds with the structures shown in formula (IV) and formula (IV-1),

[0391] Among them, L 1a for: Preferably, L 1a for

[0392] In some embodiments, compounds with the structures shown in formula (IV) and formula (IV-1),

[0393] in, for:

[0394]

[0395] n1, n2, n3, and n4 are each independent natural numbers from 0 to 8; for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0396] n5 and n6 are each independently 0 or 1;

[0397] Preferably, for: In some embodiments, compounds with the structures shown in formula (IV) and formula (IV-1),

[0398] in, for:

[0399]

[0400] Preferably, Selected from

[0401] More preferably, for

[0402] In some embodiments, the compounds with structures shown in formula (IV) and (IV-1) are the compounds shown in formula (IVa):

[0403]

[0404] in,

[0405] R 1 R a R 2 R 3 The definitions of V, L, W, Z, ring B, k1, and k2 are as described in any one of the present invention formula (I);

[0406] L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0407] L 1a As described in any of the present invention formula (IV-1).

[0408] In some embodiments, the compounds with structures shown in formula (IV), formula (IV-1), and formula (IVa) are compounds shown in formula (IVb):

[0409]

[0410] in,

[0411] R a R 2 R 3 The definitions of W, Z, k1, and k2 are as described in any one of the present invention formula (I);

[0412] L 2 L3 And Tr as described in any one of the present invention formulas (II-1);

[0413] L 1a As described in any of the present invention formula (IV-1).

[0414] In some embodiments, the compounds with structures shown in formula (IV), formula (IV-1), formula (IVa), and formula (IVb) are the compounds shown in formula (IVb-1):

[0415]

[0416] in,

[0417] R a R 2 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0418] L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0419] L 1a As described in any of the present invention formula (IV-1).

[0420] In some embodiments, the compounds with structures shown in formulas (IV), (IV-1), (IVa), (IVb), and (IVb-1) are the compounds shown in formulas (IVb-1a), (IVb-1b), (IVb-1c), (IVb-1d), and (IVb-1e):

[0421]

[0422]

[0423] in,

[0424] R 2 The definitions of W and W are as described in any one of the present invention formulas (I);

[0425] L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0426] L 1a As described in any of the present invention formula (IV-1).

[0427] In some embodiments, the compounds with structures shown in formulas (IV), (IV-1), (IVa), and (IVb) are compounds shown in formula (IVb-2):

[0428]

[0429] in,

[0430] R a R 2 R 3 The definitions of W, k1, and k2 are as described in any one of formulas (I) and (Ib-2) of this invention;

[0431] L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0432] L 1a As described in any of the present invention formula (IV-1).

[0433] In some embodiments, the compounds with structures shown in formulas (IV), (IV-1), (IVa), (IVb), and (IVb-2) are the compounds shown in formulas (IVb-2a), (IVb-2b), and (IVb-2c):

[0434]

[0435] in,

[0436] R 2 R 3 The definition of W is as described in any one of formulas (I) and (Ib-2) of this invention;

[0437] L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0438] L 1a As described in any of the present invention formula (IV-1).

[0439] In some embodiments, the compounds with structures shown in formulas (IV), (IV-1), (IVa), and (IVb) are compounds shown in formula (IVb-3):

[0440]

[0441] in,

[0442] R a R 2 The definitions of W, k1, and k2 are as described in any one of the present invention formula (I);

[0443] L 2 L 3And Tr as described in any one of the present invention formulas (II-1);

[0444] L 1a As described in any of the present invention formula (IV-1).

[0445] In some embodiments, the compounds with structures shown in formulas (IV), (IV-1), (IVa), (IVb), and (IVb-3) are the compounds shown in formulas (IVb-3a), (IVb-3b), and (IVb-3c):

[0446]

[0447] in,

[0448] R 2 The definition of W is as described in any one of the present invention formulas (I);

[0449] L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0450] L 1a As described in any of the present invention formula (IV-1).

[0451] In some embodiments, the nitrogen-containing heterocyclic compounds of formula (IV), formula (IV-1), formula (IVa), formula (IVb), formula (IVb-1), formula (IVb-2), and formula (IVb-3) shown are any of the following compounds:

[0452]

[0453]

[0454]

[0455]

[0456]

[0457] The present invention also provides a nitrogen-containing heterocyclic compound as shown in Formula V, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof:

[0458]

[0459] in,

[0460] R 1 R 2 R3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the formulas (I) of this invention.

[0461] In some embodiments, the compound with the structure shown in formula (V) is the compound shown in formula (Va):

[0462]

[0463] in,

[0464] R 1 R a R 2 R 3 The definitions of V, L, W, Z, ring B, k1, and k2 are as described in any one of the formulas (I) of this invention.

[0465] In some embodiments, the compounds with structures shown in formulas (V) and (Va) are the compounds shown in formula (Vb):

[0466]

[0467] in,

[0468] R a R 2 R 3 The definitions of W, Z, k1, and k2 are as described in any one of the present invention formula (I).

[0469] In some embodiments, the compounds with structures shown in formulas (V), (Va), and (Vb) are the compounds shown in formula (Vb-1):

[0470]

[0471] in,

[0472] R a R 2 The definitions of W, k1, and k2 are as described in any one of formulas (I) and (Vb) of this invention.

[0473] In some embodiments, compounds with structures shown in formulas (V), (Va), (Vb), and (Vb-1) are used, wherein...

[0474] R 2 Each can be independently H, F, Cl, -CH3, -O-phenyl, -S-phenyl or -O-CH2-phenyl;

[0475] W can be -NH-, -CH2CH2-O-CH2CH2-N(CH3)-, or -O-CH2CH2-N(CH3)-;

[0476] Or, W and R 2 Together with the attached N and C atoms, they form Side a is connected to ring B in a parallel loop.

[0477] In some embodiments, the compounds with structures represented by formulas (V), (Va), (Vb), and (Vb-1) are compounds represented by formulas (Vb-1a), (Vb-1b), or (Vb-1c):

[0478] R 2 The definitions of W and W are as described in any one of the present invention formulas (I).

[0479] In some embodiments, the compounds with structures shown in formulas (V), (Va), and (Vb) are the compounds shown in formula (Vb-2):

[0480]

[0481] in,

[0482] R a R 2 R 3 The definitions of W, k1, and k2 are as described in any one of formulas (I) and (Vb) of this invention.

[0483] In some embodiments, compounds with the structures shown in formulas (V), (Va), (Vb), and (Vb-2) are: wherein,

[0484] R 2 Each can be independently hydrogen, F, or Cl;

[0485] W can be -O-, -S-, or -CH2-O-;

[0486] R 3 It is a phenylene oxide.

[0487] In some embodiments, the compounds with structures shown in formulas (V), (Va), (Vb), and (Vb-2) are compounds shown in (Vb-2a), (Vb-2b), or (Vb-2c):

[0488]

[0489] in,

[0490] R 2 R 3 The definitions of W and W are as described in any one of the present invention formulas (I).

[0491] In some embodiments, the compounds with structures shown in formulas (V), (Va), and (Vb) are compounds shown in formula (Vb-3):

[0492]

[0493] in,

[0494] R a R 2 R 3 The definitions of W, k1, and k2 are as described in any one of formulas (I) and (Vb) of this invention;

[0495] In some embodiments, compounds with the structures shown in formulas (I), (Ia), (Ib), and (Ib-3) are:

[0496] in,

[0497] R 2 Each can be independently hydrogen, F, Cl, or -CH3;

[0498] W can be -CH2CH2-O-, -O-CH2CH2-, -CH2-O-, or -O-CH2-;

[0499] R 3 for Among them, end a is connected to W.

[0500] In some embodiments, the compounds with structures shown in formulas (V), (Va), (Vb), and (Vb-3) are the compounds shown in (Vb-3a), (Vb-3b), and (Vb-3c):

[0501]

[0502] in,

[0503] R 2 R 3 The definitions of W and W are as described in any one of the present invention formulas (I).

[0504] In some embodiments, the nitrogen-containing heterocyclic compound of formula (V), formula (Va), formula (Vb), formula (Vb-1), formula (Vb-2), and formula (Vb-3) shown is any of the following compounds:

[0505]

[0506]

[0507]

[0508]

[0509] The present invention also provides a method for preparing a ligand-drug conjugate as shown in formula (III-1), which includes the following steps: a nitrogen-containing heterocyclic compound as shown in formula (IV-1) is reacted with Ab-SH by substitution or addition reaction to obtain the ligand-drug conjugate as shown in formula (III-1);

[0510]

[0511] in,

[0512] R 1 R 2 R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the present invention formula (I);

[0513] L 1 L 2 L 3 And Tr as described in any one of the present invention formulas (II-1);

[0514] L 1a As described in any one of the present invention formulas (IV-1);

[0515] Ab and q are as described in any of the present invention formula (III);

[0516] The Ab-SH is a product in which the interchain disulfide bonds of Ab are reduced to thiol groups.

[0517] In one embodiment, the method for preparing the ligand-drug conjugate as shown in formula (III-1) further includes the following step: reacting a nitrogen-containing heterocyclic compound as shown in formula (V) with L... 1a -L 2 -L 3 -Tr-Lg 1 Through nucleophilic substitution or condensation reactions, nitrogen-containing heterocyclic compounds as shown in formula (IV-1) are obtained;

[0518]

[0519] in,

[0520] Lg 1 For leaving groups, such as halogens, hydroxyl groups, wait.

[0521] In one embodiment, in the method for obtaining the nitrogen-containing heterocyclic compound as shown in formula (IV-1), the condensation reaction further comprises a condensing agent, which may be a condensing agent conventional for such reactions in the art, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, or 1-hydroxybenzotriazole. One or more of the following: 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and benzotriazole-1-yl-oxytripyrrolidinephosphide hexafluorophosphate, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0522] In one embodiment, the method for obtaining a nitrogen-containing heterocyclic compound as shown in formula (IV-1) further comprises a base, which may be a base conventional to such reactions in the art, preferably triethylamine, NMM or DIPEA.

[0523] In one embodiment, the method for preparing the ligand-drug conjugate as shown in formula (III-1) further includes the following steps: 1. Formula (V)-1 and Formula (V)-2 undergo intermolecular condensation in the presence of a condensing agent to obtain Formula (V)-3; 2. Formula (V)-3 undergoes acid deprotection, base deprotection, or reduction reaction with the participation of a metal catalyst to obtain a nitrogen-containing heterocyclic compound as shown in formula (V);

[0524]

[0525] in,

[0526] Pg 1 It can be a nitrogen protecting group, such as Boc, Fmoc, or Cbz; or a nitro group;

[0527] Pg 2 It is a protecting group for hydrogen or nitrogen, such as Boc, Fmoc or Cbz.

[0528] In one embodiment, in the method for obtaining formula (V)-3, the condensing agent can be a condensing agent commonly used in such reactions in the art, preferably: CDI, phosgene or triphosgene.

[0529] In one embodiment, the base obtained as shown in formula (V)-3 may be a base conventional to such reactions in the art, preferably one or more of piperidine, ethanolamine, morpholine, ethylenediamine and piperazine.

[0530] In one embodiment, the acid in the obtained nitrogen-containing heterocyclic compound of formula (V) can be an acid conventional to such reactions in the art, preferably one or more of trifluoroacetic acid, hydrogen chloride, methanesulfonic acid and p-toluenesulfonic acid.

[0531] In one embodiment, the metal catalyst used to obtain the nitrogen-containing heterocyclic compound of formula (V) can be a conventional metal catalyst for such reactions in the art, preferably Pd / C, Pd(OH)2 or Ni.

[0532] The present invention also provides a method for preparing a nitrogen-containing heterocyclic compound as shown in formula (IV-1), comprising the following steps: 1. reacting a nitrogen-containing heterocyclic compound as shown in formula (V) with Pg 3 -L 2 -L 3 -Tr-Lg 1 Substitution reaction yields nitrogen-containing heterocyclic compounds as shown in formula (IV-1)-1; 2. Deprotection of the nitrogen-containing heterocyclic compound shown in formula (IV-1)-1 with acid or base yields nitrogen-containing heterocyclic compounds as shown in formula (IV-1)-2; 3. The nitrogen-containing heterocyclic compound shown in formula (IV-1)-2 reacts with L... a -Lg 2 The nitrogen-containing heterocyclic compound shown in formula (IV-1) is obtained through condensation or substitution reactions;

[0533]

[0534] in,

[0535] Lg 2 For leaving groups, such as halogens, hydroxyl groups,

[0536] Pg 3 Protecting groups for nitrogen, such as Boc, Fmoc, or Cbz;

[0537] R 1 R 2 R 3 The definitions of V, L, W, Z, ring A, ring B and k1 are as described in any one of the present invention formula (I);

[0538] L 2 L 3And Tr as described in any one of the present invention formulas (II-1);

[0539] L 1a As described in any of the present invention formula (IV-1).

[0540] In one embodiment, the substitution reaction in the method for obtaining formula (IV-1)-1 further comprises a base, which may be a base conventional for such reactions in the art, preferably triethylamine, DIEA or NMM.

[0541] In one embodiment, in the method for obtaining formula (IV-1)-2, the base may be a base conventional to such reactions in the art, preferably one or more of piperidine, ethanolamine, morpholine, ethylenediamine and piperazine.

[0542] In one embodiment, in the method for obtaining formula (IV-1)-2, the acid may be an acid conventional to such reactions in the art, preferably one or more of trifluoroacetic acid, hydrogen chloride, methanesulfonic acid and p-toluenesulfonic acid.

[0543] In one embodiment, the condensation reaction in obtaining the nitrogen-containing heterocyclic compound of formula (IV-1) further comprises a condensing agent, which can be a condensing agent conventional for such reactions in the art, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, One or more of 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and benzotriazole-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0544] The present invention also provides a method for preparing a ligand-drug conjugate as shown in formula (IV-1), comprising the following steps: reacting a nitrogen-containing heterocyclic compound as shown in formula (V) with L... 1a -L 2 -L 3 -Tr-Lg 1Through nucleophilic substitution or condensation reactions, nitrogen-containing heterocyclic compounds as shown in formula (IV-1) are obtained;

[0545]

[0546] In one embodiment, the conditions and steps for preparing the nitrogen-containing heterocyclic compound as shown in formula (IV-1) are the same as those described above.

[0547] The present invention also provides a method for preparing a ligand-drug conjugate as shown in formula (V), comprising the following steps: 1. Formula (V)-1 and Formula (V)-2 undergo intermolecular condensation in the presence of a condensing agent to obtain Formula (V)-3; 2. Formula (V)-3 undergoes a reduction reaction with acid deprotection, base deprotection or metal catalyst to obtain a nitrogen-containing heterocyclic compound as shown in formula (V);

[0548]

[0549] In one embodiment, the conditions and steps of the method for preparing the nitrogen-containing heterocyclic compound as shown in formula (V) are the same as those described above.

[0550] The present invention also provides a pharmaceutical composition comprising substance S and one or more pharmaceutical excipients;

[0551] The substance S is any of the ligand-drug conjugates described above, or in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof; or

[0552] Nitrogen-containing heterocyclic compounds as described above, or their tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically usable salts.

[0553] The present invention also provides the use of substance S in the preparation of a medicament for the prevention or treatment of cancer;

[0554] The substance S is any of the ligand-drug conjugates described above, or in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof; or

[0555] Nitrogen-containing heterocyclic compounds as described above, or their tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically usable salts.

[0556] Preferably, the cancer is a solid tumor or a non-solid tumor, such as 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, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, or thyroid cancer.

[0557] The present invention also provides the use of substance S in the preparation of a medicament for the prevention or treatment of diseases related to abnormal cell activity;

[0558] The substance S is any of the ligand-drug conjugates described above, or in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof; or

[0559] Nitrogen-containing heterocyclic compounds as described above, or their tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically usable salts.

[0560] Preferably, the disease associated with the abnormal cell activity may be cancer; more preferably, the definition of cancer is as described above.

[0561] The present invention also provides a method for preventing or treating cancer, the method comprising administering an effective amount of substance S to an individual in need;

[0562] The substance S is any of the ligand-drug conjugates described above, or in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof; or

[0563] Nitrogen-containing heterocyclic compounds as described above, or their tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically acceptable salts;

[0564] The definition of cancer is as described above.

[0565] The present invention also provides a method for preventing or treating diseases associated with abnormal cellular activity, the method comprising administering an effective amount of substance S to an individual in need;

[0566] The substance S is any of the ligand-drug conjugates described above, or in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof; or

[0567] Nitrogen-containing heterocyclic compounds as described above, or their tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically usable salts.

[0568] Preferably, the disease associated with the abnormal cell activity may be cancer. The definition of cancer is as described above.

[0569] In another aspect, the present invention provides a substance S for treating cancer; said substance S is any of the ligand-drug conjugates or nitrogen-containing heterocyclic compounds as described above, or in the form of tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically usable salt thereof, or a pharmaceutical composition thereof.

[0570] The disease associated with the abnormal cell activity may be cancer. The definition of cancer is as described above.

[0571] Terminology definition:

[0572] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0573] In this application, the term "pharmaceutical excipients" refers to the excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. These are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified as natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0574] In this application, the term "pharmaceutical composition" refers to a dosage form that can be formulated according to various suitable routes of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, sprays, etc.

[0575] The pharmaceutical composition can be administered in injectable form, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media.

[0576] In this application, the terms "pharmaceutically acceptable salt" or "medicinal salt" generally refer to salts of compounds or ligand-drug conjugates of this application that are safe and / or effective when used in mammals and have the desired biological activity.

[0577] The term "drug loading" typically refers to the average amount of cytotoxic drug loaded onto each ligand, and can also be expressed as the ratio of cytotoxic drug to antibody, such as the drug / antibody ratio (DAR). The range of cytotoxic drug loading can be an integer or decimal of 0-20. In embodiments of this application, drug loading is expressed as q, which can be, for example, an integer or decimal of 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. For example, drug loading q is 7.5, 7.6, 7.7, 7.8, or 7.9; further examples include q being 7.53, 7.64, 7.67, or 7.89. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, HIC, ELISA assays, and HPLC characterization.

[0578] In this application, the term "ligand-drug conjugate" generally refers to a ligand linked to a biologically active cytotoxic drug via a stable linker unit. In some embodiments of this application, "ligand-drug conjugate" can be an antibody-drug conjugate (ADC), whereby an ADC can refer to a monoclonal antibody or antigen-binding fragment linked to a biologically active cytotoxic drug via a stable linker unit.

[0579] In this application, the term "ligand" generally refers to small molecules, peptides, RNA, DNA, carbohydrates, and macromolecules that can recognize and bind to antigens or receptors associated with target cells. The role of a ligand can be to present a drug to a target cell population that has bound the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigens. In this application, a ligand can be represented as Ab. The ligand antigen forms a linker bond with a linker unit (also called a "linker" or "connector") through a heteroatom on the ligand. The ligand can be an antibody or its antigen-binding fragment. The antibody can be selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; the antibody can be a monoclonal antibody. For example, the antibody can be an antibody targeting a subset of the following targets: HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70, and EGFR. For example, the antibody may be an antibody targeting the following targets: 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, O772P, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD1lc, CD123, CD138, CD142, CD147, CD166, CD19, CD19.CD22, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDllb, CEA, CEACAM5, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4 , DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, GD2 , GEDA, GPC-1, GPC-3, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV-1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RN F43, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9, epidermal growth factor, short proteoglycans, mesothelin, sodium phosphate cotransporter 2B, Claudin18.2, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin αvβ6, trophoblast cell glycoproteins, and tissue factor.

[0580] In this application, the term "antibody or antigen-binding fragment thereof" generally refers to an immunological conjugate, extending to all antibodies from all species, including dimer, trimer, and multimer antibodies; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and modified antibodies, and fragments thereof. The term "antibody or antigen-binding fragment thereof" can refer to any antibody-like molecule having an antigen-binding region, including small molecule fragments such as Fab′, Fab, F(ab′)2, single-domain antibodies (DABs), Fv, scFv (single-chain Fv), linear antibodies, diabody antibodies, etc. The term "antigen-binding fragment" can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. For example, fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. The antibodies may include: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-TROP2 antibody, and anti-Claudin antibody. 18.2 Antibodies, one or more of the following: anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-ADAM9 antibody, anti-GPC-3 antibody, and anti-Mesothelin antibody, such as Patritumab and / or Codrituzumab.

[0581] In this application, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. Methods for establishing chimeric antibodies include, for example, constructing a hybridoma that secretes murine-specific monoclonal antibodies, then cloning the variable region gene from the murine hybridoma cells, cloning the constant region gene of the human antibody as needed, and then linking the murine variable region gene and the human constant region gene to form a chimeric gene, which is then inserted into an expression vector. The chimeric antibody molecule can then be expressed in eukaryotic or prokaryotic systems.

[0582] In this application, the term "humanized antibody," also known as a CDR-grafted antibody, generally refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., grafting it into a different type of human germline antibody framework sequence. Humanized antibodies can overcome the problem of chimeric antibodies inducing a strong heterologous response due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy chain variable region and light chain variable region genes can be found in the VBase human germline sequence database.

[0583] In this application, the terms "fully human antibody", "human antibody", "fully human antibody" or "completely human antibody" are used interchangeably, and the variable region and constant region of the antibody may both be of human origin, with immunogenicity and toxic side effects removed.

[0584] The antibody or ligand described in this application can be a fully human monoclonal antibody. Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology, etc.

[0585] In this application, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. The most common definitions of the six CDRs are provided, for example, by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242; Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196:901 (1987); and MacCallum et al., "Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography," J. Mol. Biol. 262:732 (1996). As used in this application, the Kabat definition of CDR can be applied to CDR1, CDR2, and CDR3 (CDRL1, CDRL2, CDRL3 or L1, L2, L3) of light chain variable structural domains, and CDR1, CDR2, and CDR3 (CDR H1, CDRH2, CDRH3 or H1, H2, H3) of heavy chain variable structural domains.

[0586] The term "one or more species" or similar expression "at least one species" can mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more species.

[0587] When the lower and upper limits of a numerical range are disclosed, any numerical value falling within that range and any included range are specifically disclosed. In particular, each range of values ​​disclosed herein should be understood to represent each numerical value and range encompassed within a wider range;

[0588] For example, the expression "C" 1-6 "This should be understood as encompassing any subrange and each point value, such as C." 2-5 C 3-4 C 1-2 C 1-3 C 1-4 C 1-5 And so on, as well as C1, C2, C3, C4, C5, C6, etc. For example, the expression "C 3-10 "It should also be understood in a similar way, for example, it can encompass any subrange and point value contained within it, such as C." 3-9 C 6-9 C 6-8 C6-7 C 7-10 C 7-9 C 7-8 C 8-9 And C3, C4, C5, C6, C7, C8, C9, C 10 For example, the expression "3-10 yuan" should be understood as encompassing any sub-range and each point value within it, such as 3-4 yuan, 3-5 yuan, 3-6 yuan, 3-7 yuan, 3-8 yuan, 3-9 yuan, 4-5 yuan, 4-6 yuan, 4-7 yuan, 4-8 yuan, 5-7 yuan, 5-8 yuan, 6-7 yuan, etc., as well as 3, 4, 5, 6, 7, 8, 9, 10 yuan, etc. Similarly, the expression "5-10 yuan" should also be understood in a similar way, for example, it can encompass any sub-range and point value included within it, such as 5-6 yuan, 5-7 yuan, 5-8 yuan, 5-9 yuan, 5-10 yuan, 6-7 yuan, 6-8 yuan, 6-9 yuan, 6-10 yuan, 7-8 yuan, etc., as well as 5, 6, 7, 8, 9, 10 yuan, etc.

[0589] In this application, the term "natural number" is used for example, 0-50, 0-40, 0-30, 0-20, 0-10, 0-8, 0-6, 0-6, 0-4, or 0-2; and for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0590] In this application, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group. As used herein, the term "C" refers to a saturated straight-chain or branched hydrocarbon group. 1-6 "Alkyl" refers to a saturated straight-chain or branched hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbon atoms). "C" 1-6 "alkyl" can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl, etc.

[0591] In this application, the term "C" 0-6 In the term "alkylene", when C0 is 0, the alkylene bond is a connecting bond.

[0592] In this application, the term "C" 0-6 In the alkyl group, when C0 is 0, alkyl is hydrogen.

[0593] In this application, the term "alkylene" refers to a saturated straight-chain or branched divalent hydrocarbon group. For example, the term "C 1-6 "Alkylene" refers to a saturated, straight-chain or branched divalent hydrocarbon group having 1-6 carbon atoms. 1-6 "Alkylene" includes, but is not limited to, methylene, ethylene, propylene, or butylene.

[0594] In this application, the term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more carbon-carbon double bonds. For example, the term "C" as used herein... 2-6 "Alkenyl" refers to an alkenyl group having 2-6 carbon atoms and one, two, or three (preferably one) carbon-carbon double bonds (such as vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl, etc.).

[0595] In this application, the term "alkynyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more carbon-carbon triple bonds. For example, the term "C" as used herein... 2-6 "Alynyl" refers to an alkynyl group having 2-6 carbon atoms and one, two, or three (preferably one) carbon-carbon triple bonds (such as ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, etc.).

[0596] In this application, the term "aryl" refers to a monocyclic or fused-ring aromatic hydrocarbon group having a conjugated π-electron system. For example, the term "C" as used herein... 6-10 "Aryl" refers to an aryl group having 6-10 carbon atoms (such as phenyl, naphthyl, etc.).

[0597] In this application, the term "heteroaryl" or "heteroary ring" refers to a monocyclic and fused heterocyclic system having one or more conjugated π-electron systems, wherein one or more (e.g., 1, 2, or 3) ring atoms are heteroatoms selected from N, O, P, and S, and the remaining ring atoms are C. Heteroaryl or heteroary rings can be characterized by the number of ring atoms (e.g., 5-10 members, 5-6 members). For example, a 5-12 membered heteroaryl may contain 5-12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) ring atoms, particularly 5, 6, 9, or 10 ring atoms. Examples of heteroaryl groups include thiophene, furanyl, pyrrole, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, indole, etc.

[0598] In this application, the term "cycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic hydrocarbon group. For example, C 3-12 cycloalkyl, C 3-6 Cycloalkyl. For example, "C 3-12"Cycloalkyl" or "3-12 membered cycloalkyl" refers to a cycloalkyl group having 3-12 ring carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). Common cycloalkyl groups include (but are not limited to) monocyclic cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkyl groups, including fused rings, bridged rings, or spiro rings, such as bicyclic [1.1.1]pentyl, bicyclic [2.2.1]heptyl, bicyclic [3.2.1]octyl, bicyclic [5.2.0]nonyl, decahydronaphthyl, etc.

[0599] In this application, the term "cycloalkylene" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic divalent cyclic group. For example, C 3-12 Cycloalkylene, C 3-6 Cycloalkylene. For example, "C 3-12 "Cycloalkylene" or "3-12-membered cycloalkylene" refers to cycloalkylene compounds having 3-12 ring carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). Common cycloalkylene compounds include (but are not limited to) monocyclic cycloalkylene compounds, such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkylene compounds, including fused rings, bridged rings, or spirocyclic compounds, such as bicyclic[1.1.1]pentylene, bicyclic[2.2.1]heptylene, bicyclic[3.2.1]octylene, bicyclic[5.2.0]nonylene, decahydronaphthylene, etc.

[0600] The term "heterocyclic alkyl" refers to a saturated or partially saturated non-aromatic cyclic group containing at least one heteroatom selected from N, O, P, and S as a ring member, preferably 1, 2, 3, or 4 heteroatoms. Examples include 4- to 12-membered, 4- to 6-membered, 5- to 12-membered, 5- to 8-membered, 5- to 6-membered, 9- to 10-membered, 11- to 12-membered, 3- to 8-membered, and 3- to 6-membered heterocyclic alkyl groups. Specific examples include, but are not limited to, ethylene oxide, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, and homopiperazinyl.

[0601] The term "heterocyclic alkylene" refers to a saturated or partially saturated, non-aromatic divalent cyclic group containing at least one heteroatom selected from N, O, P, and S as a ring member. Preferably, the number of heteroatoms is 1, 2, 3, or 4. Examples include 3-8 membered and 3-6 membered heterocyclic alkylenes. Specific examples include, but are not limited to, ethylene oxide, cyclobutane, pyrroleyl, tetrahydrofuranyl, piperidinyl, piperazineyl, tetrahydropyranyl, and homopiperazineyl.

[0602] The term "fused ring (fused ring system)" refers to a chemically feasible polycyclic structure formed by two or more (e.g., 3, 4, or 5) carbon rings or heterocycles sharing common ring edges or common atoms, wherein the carbon rings include cycloalkyl and aryl groups, and the heterocycles include heteroaromatic and heterocyclic alkyl groups. Fused ring systems include, but are not limited to: fused ring systems formed by cycloalkyl groups with cycloalkyl groups, fused ring systems formed by cycloalkyl groups with heterocyclic alkyl groups, fused ring systems formed by cycloalkyl groups with aromatic rings, fused ring systems formed by cycloalkyl groups with heteroaromatic rings, fused ring systems formed by heterocyclic alkyl groups with aromatic rings, fused ring systems formed by heteroaromatic rings with heteroaromatic rings, and fused ring systems formed by heteroaromatic rings with aromatic rings.

[0603] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, iodine, such as fluorine and chlorine.

[0604] In this application, the term "each independently" means that at least two groups (or segments) in the structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0605] In this application, the terms “optional” or “optionally” generally mean that the event or environment described below may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0606] In this application, the term "substitution" and its other variant forms herein refer to the replacement of one or more (e.g., 1, 2, 3, or 4) atoms or groups of atoms (e.g., hydrogen atoms) on a specified atom with other equivalents, provided that the replacement does not exceed the normal valence of the specified atom or group of atoms in the present case and is capable of forming a stable compound. If an atom or group of atoms is described as "optionally substituted," it may or may not be substituted. Unless otherwise stated, the linking site of a substituent herein may be derived from any suitable position of the substituent. When the linking bond in a substituent is shown as a chemical bond through two atoms connected to each other in a ring system, it indicates that the substituent may be linked to any one of the cyclic atoms in the ring system.

[0607] This article uses wavy lines. The bonds in the structural formula are intended to indicate that the structure represents a cis or trans isomer, or a mixture of cis and trans isomers in any proportion.

[0608] When used alone or in combination with other groups in this document, the term "oxo" refers to =O.

[0609] In this application, one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, are independently substituted by a corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (e.g., alkene) bond.

[0610] In this application, the term "amino acid" includes both natural and non-natural amino acids, and the common amino acid designation follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this application, amino acids are generally represented by single-letter and three-letter abbreviations well known in the art. For example, alanine may be represented by A or Ala; arginine by R or Arg; glycine by G or Gly; and glutamine by Q or Gln.

[0611] In this application, the term "non-natural amino acid" has the following structure: Where r is selected from 0, 1, 2, 3, 4, and 5; where R a R b Each is independently selected from -C 1-6 Alkylene -NH2, -C 1-6 Alkylene-NH-C 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 Alkyl)2, -C 1-6 Alkylene-NH-C 3-10 cycloalkyl, -C 1-6 alkylene-N(3-10 membered cycloalkyl)(C 1-6 Alkyl), -C 1-6 Alkylene-C 3-10 cycloalkyl, -C 1-6 Alkylene (3-10 membered heterocyclic alkyl), -C 1-6 Alkylene-NHCOC1-6 Alkyl, -C 1-6 Alkylene-NHCOOC 1-6 Alkyl, -C 1-6 Alkylene-NHS(O)2C 1-6 Alkyl, -C 1-6 Alkylene-S(O)2-C 1-6 Alkyl, -C 1-6 Alkylene-S(O)2-C 3-10 cycloalkyl, -C 1-6 Alkylene -S(O)2-NH2, -C 1-6 Alkylene -COOH, -C 1-6 Alkylene -CONH2, -C 1-6 Alkylene-CONHC 1-6 Alkyl, -C 1-6 Alkylene-CO (3-10 membered heterocyclic alkyl), The alkyl, alkylene, cycloalkyl, and heterocycloalkyl groups are each optionally and independently substituted by one or more substituents selected from H, halogens, -OH, -NH2, -SH, -NO2, CN, -COOH, and oxo groups; or any R a R b Together with the atoms attached thereto, they form 3-10 membered heterocyclic alkyl groups and 3-10 membered cycloalkyl groups; each of the cycloalkyl groups and heterocyclic alkyl groups is optionally substituted by one or more substituents selected from H, halogens, -OH, -NH2, -SH, -NO2, CN, -COOH and oxo groups;

[0612] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound in this application can be an organic compound, a compound with a molecular weight of less than 500 Daltons, a compound with a molecular weight of less than 1000 Daltons, a compound with a molecular weight of more than 1000 Daltons, or a compound with a molecular weight of more than 10,000 Daltons or more than 100,000 Daltons. In this application, a compound can also refer to a compound linked by chemical bonds. For example, it can be a compound in which one or more molecules with a molecular weight of less than 1000 Daltons are linked by chemical bonds to a biological macromolecule, which can be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound in this application can include a compound in which a protein is linked to one or more molecules with a molecular weight of less than 1000 Daltons, a compound in which a protein is linked to one or more molecules with a molecular weight of less than 100,000 Daltons, or a compound in which a protein is linked to one or more molecules with a molecular weight of less than 100,000 Daltons.

[0613] In this application, the term "stereoisomer" refers to an isomer formed by at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0614] Solid lines may be used in this article. solid wedge Or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. Solid lines are used to depict bonds to asymmetric carbon atoms to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or imaginary wedges are used to depict bonds to asymmetric carbon atoms to indicate the presence of the indicated stereoisomers. When present in racemic mixtures, solid and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0615] Those skilled in the art will understand that, according to conventions used in the art, the structural formulas of the groups described in this invention are... This refers to the connection of the corresponding group R to other segments or groups in the compound through this site. The "-" at the end of the group indicates that the group is connected to other segments in the molecule through this site. For example, CH3-C(=O)- means that the C(=O) in the acetyl group is connected to other segments in the molecule.

[0616] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements. The terms "above" and "below" generally refer to situations that include the stated number.

[0617] Unless otherwise specified, the structures described in this application may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds whose structures are identical to those of this application except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are within the scope of this application.

[0618] The terms “active ingredient,” “therapeutic agent,” “active substance,” or “active agent” refer to a chemical entity that can effectively treat one or more symptoms of a target condition or disease.

[0619] Unless otherwise stated, as used herein, the term “treatment” means to reverse, alleviate, or inhibit the progression of a disease or condition to which such term applies, or one or more symptoms of such a disease or condition, or to prevent such a disease or condition, or one or more symptoms of such a disease or condition.

[0620] As used herein, “individual” includes both human and non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described herein) (referred to as patients) or normal individuals. In this invention, “non-human animals” includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0621] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form nitrogen oxides because nitrogen requires available lone pairs of electrons to be oxidized. Those skilled in the art will identify nitrogen-containing heterocycles capable of forming nitrogen oxides. They will also recognize that tertiary amines can form nitrogen oxides. Synthetic methods for preparing nitrogen oxides of heterocycles and tertiary amines are well known to those skilled in the art, including the oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic acid and m-chloroperoxybenzoic acid (m-CPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing nitrogen oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750 (AR. Katritzky and A.J. Boulton, Eds., Academic Press); and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392 (AR. Katritzky and A.J. Boulton, Eds., Academic Press).

[0622] Those skilled in the art will understand that all compounds covered by this invention are chemically feasible compounds; and all chemical bonds are linked in a chemically feasible manner.

[0623] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in TW Greene & P. ​​GMWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 2006, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0624] This invention also covers methods for preparing the compounds described herein. It should be understood that the compounds of this invention can be synthesized using the methods described below, as well as synthetic methods known in the field of synthetic organic chemistry or variations thereof understood by those skilled in the art. Preferred methods include (but are not limited to) those described below. The reaction can be carried out in a solvent or solvent mixture suitable for the reagents and materials used and suitable for achieving the conversion.

[0625] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0626] The reagents and raw materials used in this invention are all commercially available.

[0627] The positive and progressive effects of this invention are that the compounds of this invention have one or more of the following advantages:

[0628] (1) It has inhibitory activity against the in vitro proliferation of tumor cells;

[0629] (2) It has plasma stability;

[0630] (3) It has an in vivo tumor-suppressing effect;

[0631] (4) It has the ability to resist transport by transporters;

[0632] (5) It has the ability to target tumors in vivo;

[0633] (6) It has good in vivo safety;

[0634] Furthermore, the conjugation methods described in this disclosure have a wide range of applications and can be widely used for conjugation with bioactive molecules such as antibodies or targeting small molecule ligands. In summary, the protein degrading agents, linkers, antibodies, and ADCs of this invention have significant clinical value. Detailed Implementation

[0635] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific embodiments indicate preferred embodiments of the invention, these descriptions and embodiments are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including in quotation marks, are incorporated herein by reference in their entirety. The invention is further illustrated below by way of examples, but this does not limit the invention to the scope of the examples described. Experimental methods in the following examples, unless specific conditions are specified, are performed according to conventional methods and conditions, or as selected according to the trade specification.

[0636] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0637] The preparative high performance liquid chromatography (HPLC) method was performed using a Shimadzu LC-8A preparative liquid chromatograph (YMC, ODS, 250×20mm column).

[0638] Thin-layer chromatography purification was performed using GF 254 (0.4–0.5 nm) silica gel plates produced in Yantai.

[0639] The reaction was monitored using thin-layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). The developing solvent systems used included, but were not limited to, dichloromethane and methanol systems, n-hexane and ethyl acetate systems, and petroleum ether and ethyl acetate systems. The volume ratio of the solvent was adjusted according to the polarity of the compound, or by adding triethylamine, etc.

[0640] Column chromatography typically uses 200-300 mesh silica gel from Qingdao Ocean as the stationary phase. Eluent systems include, but are not limited to, dichloromethane and methanol systems and n-hexane and ethyl acetate systems. 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.

[0641] Unless otherwise specified in the examples, the reaction temperature is room temperature (20℃~30℃).

[0642] Unless otherwise specified, the reagents used in the examples were purchased from Acros Organics, Aldrich Chemical Company, Nanjing Yaoshi Technology, or Shanghai Shuya Pharmaceutical Technology, etc.

[0643] The above embodiments do not limit the scope of this application in any way. In addition to those described herein, various modifications to the invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All references cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) are incorporated herein by reference in their entirety.

[0644] The meanings of the abbreviations in the conventional synthesis methods, preparation examples, and intermediate synthesis examples are shown in the table below.

[0645]

[0646] Synthesis of starting materials:

[0647] Preparation Example 1.1: Preparation of Compound S1

[0648]

[0649] Step 1: Synthesis of compound S1-2

[0650] Compound S1-1 (3.00 g, 12.73 mmol) and potassium carbonate (3.52 g, 25.46 mmol) were added to DMF (30 mL), stirred for 1 h, and then 2-chloro-N-methylacetamide (2.05 g, 19.10 mmol) was added. The mixture was then reacted overnight at room temperature. After the reaction was confirmed to be complete by TLC, an aqueous solution and ethyl acetate were added, stirred, and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give compound S1-2 (2.73 g, 70% yield).

[0651] Step 2: Synthesis of compound S1-3

[0652] Compound S1-2 (2.53 g, 8.24 mmol) was dissolved in anhydrous THF (25 mL) at room temperature, cooled to 0 °C, and a 2 M borane tetrahydrofuran solution (22 mL, 44.55 mmol) was added. The mixture was then heated to 70 °C and reacted overnight. The reaction was confirmed to be complete by LC-MS. The mixture was cooled to 0 °C, and the reaction was quenched by slow dropwise addition of methanol, followed by concentration under reduced pressure. The residue was added to a 2 M hydrochloric acid aqueous solution and stirred for 2 h. Methyl tert-butyl ether was then added, and the mixture was stirred and separated, retaining the aqueous phase. The pH of the aqueous phase was adjusted to 9 with saturated sodium bicarbonate, and the aqueous phase was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound S1-3 (2.22 g, 92% yield).

[0653] Step 3: Synthesis of compound S1-4

[0654] Compound S1-3 (2.61 g, 8.91 mmol) and triethylamine (2.70 g, 26.73 mmol) were dissolved in THF (25 mL) at room temperature, and (Boc)₂O (2.14 g, 9.80 mmol) was added. The mixture was stirred at room temperature for 2 h. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S1-4 (3.15 g, 90% yield).

[0655] Step 4: Synthesis of compound S1-5

[0656] Compound S1-4 (3.15 g, 8.02 mmol), zinc cyanide (1.04 g, 8.82 mmol), and PdCl2(PPh3)2 (0.56 g, 0.80 mmol) were dissolved in dioxane (25 mL) at room temperature and reacted at 80 °C for 6 h under nitrogen protection. LCMS showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S1-5 (2.18 g, 80% yield).

[0657] Step 5: Synthesis of compound S1

[0658] At room temperature, compound S1-5 (2.18 g, 6.42 mmol) was dissolved in THF (20 mL), and cobalt chloride (0.83 g, 6.43 mmol) in water (10 mL) was added and stirred until homogeneous. Sodium borohydride (0.49 g, 12.86 mmol) was then added, and the reaction mixture was heated to 35 °C for 12 h. LC-MS showed that the reaction was complete. Water and ethyl acetate were added to the reaction mixture, and after stirring, the mixture was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound S1 (0.88 g, 40% yield).

[0659] Preparation Example 1.2: Preparation of Compound S2

[0660]

[0661] Step 1: Synthesis of compound S2-2

[0662] Compound S2-1 (2.20 g, 10.00 mmol) and triethylamine (3.04 g, 30.00 mmol) were dissolved in THF (30 mL), and (Boc)₂O (2.62 g, 12 mmol) was added. The mixture was stirred at room temperature for 2 h. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S2-2 (3 g, 94% yield).

[0663] Step 2: Synthesis of compound S2-3.

[0664] Compound S2-2 (1.00 g, 3.12 mmol), p-nitrobenzenethiophenol (0.58 g, 3.74 mmol), potassium carbonate (0.86 g, 6.24 mmol), and copper powder (0.59 g, 9.36 mmol) were added to DMF (10 mL) and reacted at 140 °C for 5 h under nitrogen protection. After cooling to room temperature, the mixture was filtered, and ethyl acetate and water were added to the filtrate. After stirring, the mixture was separated, and the organic phase was dried over anhydrous sodium sulfate. The solution was then concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S2-3 (0.37 g, 30% yield).

[0665] Step 2: Synthesis of compound S2.

[0666] Compound S2-3 (0.37 g, 0.93 mmol) was dissolved in dichloromethane (5 mL), followed by the addition of trifluoroacetic acid (1 mL), and the reaction was carried out at room temperature for 30 min. The reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure, followed by the addition of dichloromethane. The pH was adjusted to 8 with saturated sodium bicarbonate. The mixture was separated, and the organic phase was concentrated to dryness to give compound S2 (0.21 g, 73% yield).

[0667] Preparation Example 1.3: Preparation of Compound S3

[0668]

[0669] Step 1: Synthesis of compound S3-2

[0670] Cs₂CO₃ (4.6 g, 14.32 mmol) was added to a solution of compound S3-1 (2 g, 13.02 mmol) in DMF (30 mL), followed by p-nitrofluorobenzene (1.9 g, 13.67 mmol). The mixture was stirred at 120 °C for 1 hour, then cooled to room temperature. Water and ethyl acetate were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S3-2 (2.5 g, 72% yield).

[0671] Step 2: Synthesis of compound S3-3.

[0672] Fe powder (3 g, 54.6 mmol) was added to a mixed solution of compound S3-2 (2 g, 7.28 mmol) in EtOH / H2O (15 mL / 2 mL), followed by NH4Cl (3 g, 54.6 mmol). The mixture was then stirred at 80 °C for 2 hours. After the reaction was complete, the mixture was cooled to room temperature and diluted with acetonitrile. The solution was filtered and then concentrated under reduced pressure to obtain crude compound S3-3 (1.75 g).

[0673] Step 3: Synthesis of compound S3-4.

[0674] Compound S3-3 (1.50 g, 6.13 mmol) and NaHCO3 (1.00 g, 12.26 mmol) were added to a CH3CNCN / H2O (15 mL / 10 mL) mixture, and Boc2O (2.61 g, 9.20 mmol) was added with stirring. The mixture was stirred at room temperature for 24 hours, then water and ethyl acetate were added, and the mixture was separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S3-4 (1.31 g, 72% yield).

[0675] Step 4: Synthesis of compound S3.

[0676] A solution of S3-4 (0.6 g, 1.74 mmol) in THF (6 mL) was added dropwise to a solution of LiAlH4 (132 mg, 3.48 mmol) in THF (6 mL) at 0 °C. The mixture was stirred at 0 °C for 0.5 hours, and then sodium sulfate decahydrate (500 mg) was added. The filtered solution was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S3 (0.3 g, 50% yield).

[0677] Preparation Example 1.4: Preparation of Compound S4

[0678]

[0679] Step 1: Synthesis of compound S4-3

[0680] Under nitrogen protection, sodium hydride (0.42 g, 17.33 mmol) was added to anhydrous DMF (10 mL) of S4-2 (2 g, 11.55 mmol) at 0 °C. After stirring for 0.5 hours, compound S4-2 (1.79 g, 11.55 mmol) was added to the reaction solution. After the addition was complete, the temperature was slowly raised to room temperature and the reaction was continued for 1 hour. Subsequently, ice water and ethyl acetate were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give compound S4-3 (1.78 g, 50% yield).

[0681] Step 2: Synthesis of compound S4-4

[0682] Compound S4-4 was synthesized according to step 4 of Preparation Example 1.3.

[0683] Example 1: Linker 1 Preparation of intermediates

[0684] Example 1.1: Preparation of intermediate INT1

[0685]

[0686] Step 1: Preparation of compound INT1-3

[0687] Compound INT1-1 (5.00 g, 29.38 mmol) was dissolved in DMF (50 mL) at room temperature. HATU (16.76 g, 44.07 mmol) and DIEA (11.39 g, 88.14 mmol) were added, and the mixture was stirred at room temperature for 1 h. Then, compound INT1-2 (3.85 g, 29.38 mmol) was added, and the reaction was continued for 2 h. The reaction was confirmed by LCMS. Ethyl acetate and water were added, and the pH was adjusted to approximately 6 with 2 M citric acid while stirring. The mixture was separated, and the organic phase was washed twice with saturated brine. The organic phase was dried, concentrated, and purified by column chromatography to obtain compound INT1-3 (4.58 g, 55% yield).

[0688] Step 2: Preparation of compound INT1-5

[0689] Compound INT1-3 (2 g, 7.06 mmol) was dissolved in DMF (20 mL) at room temperature. HATU (4.03 g, 10.59 mmol) and DIEA (2.73 g, 21.18 mmol) were added, and the mixture was stirred at room temperature for 40 min. Then, compound INT1-4 (1.84 g, 7.06 mmol) was added, and the reaction was continued overnight. LC-MS was used to detect the completion of the reaction. Ethyl acetate and water were added, and the pH was adjusted to approximately 6 with 2 M citric acid while stirring. The mixture was separated, and the organic phase was washed twice with saturated brine. The organic phase was dried, concentrated, and purified by column chromatography to obtain compound INT1-5 (2.23 g, 60% yield).

[0690] Step 3: Preparation of compound INT1

[0691] Compound INT1-5 (0.50 g, 0.95 mmol) was dissolved in anhydrous dichloromethane (10 mL) at room temperature, and m-CPBA (0.49 g, 2.85 mmol) was added. The mixture was then stirred at room temperature for 4 h. The reaction was confirmed by LCMS. The reaction mixture was filtered, and the filter cake was collected to give compound INT1 (0.16 g, 30% yield).

[0692] Example 1.2: Preparation of intermediate INT2

[0693]

[0694] Compounds INT2-1 (1.00 g, 3.24 mmol) and INT1-4 (0.84 g, 3.24 mmol) were dissolved in DMF (10 mL), followed by the addition of triethylamine (0.66 g, 6.48 mmol). The mixture was stirred overnight at room temperature. After the reaction was confirmed to be complete by TLC, ethyl acetate and water were added, and the pH was adjusted to approximately 6 with 2 M citric acid while stirring. The mixture was separated into liquid and liquid phases, and the organic phase was washed twice with saturated brine. The organic phase was dried, concentrated, and purified by column chromatography to obtain compound INT2 (1.18 g, 80% yield).

[0695] Example 1.3: Preparation of intermediate INT3

[0696]

[0697] Step 1: Synthesis of compound INT3-1

[0698] Compound INT2 (0.50 g, 1.10 mmol) and p-hydroxybenzylamine (0.27 g, 2.20 mmol) were dissolved in dichloromethane / methanol (15 mL, dichloromethane:methanol = 2:1). EEDQ (0.54 g, 2.20 mmol) was added under light-protected conditions, and the reaction was continued at room temperature under light-protected conditions for 30 h. After the reaction was confirmed to be complete by TLC, the solvent was removed by concentration under reduced pressure, and the residue was purified by column chromatography to give compound INT3-1 (0.49 g, yield 80%).

[0699] Step 2: Synthesis of compound INT3

[0700] Compound INT3-1 (0.49 g, 0.88 mmol) and di(p-nitrobenzene) carbonate (0.53 g, 1.75 mmol) were dissolved in anhydrous DMF (10 mL), followed by the addition of DIPEA (0.23 g, 1.75 mmol). The mixture was stirred at room temperature for 2 h. After the reaction was completed as detected by TLC, ethyl acetate and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give compound INT3 (0.45 g, 70% yield).

[0701] Example 1.4: Preparation of intermediate INT4

[0702]

[0703] Step 1: Synthesis of compound INT4-2

[0704] Compound INT4-1 (0.50 g, 1.42 mmol) was dissolved in DMF (5 mL), and HATU (0.81 g, 2.13 mmol) and DIEA (0.55 g, 4.26 mmol) were added. The mixture was stirred at room temperature for 30 minutes, and then compound INT1-4 (0.37 g, 1.42 mmol) was added. The reaction was continued for 2 hours. After the reaction was complete, ethyl acetate was added, and the pH was adjusted to approximately 6 with 2 M citric acid while stirring. The mixture was separated, and the organic phase was washed twice with saturated brine. The organic phase was dried, concentrated, and purified by reversed-phase HPLC to obtain compound INT4-2 (0.42 g, 50% yield).

[0705] Steps 2 to 3: Synthesis of compound INT4

[0706] Compound INT4 was synthesized using a method similar to steps 1 and 2 of Example 1.3.

[0707] Example 2: Preparation of protein degrading compounds

[0708] Example 2.1: Preparation of compound PA1

[0709]

[0710] Step 1: Synthesis of compound PA1-2

[0711] Triphosgene (0.30 g, 1.00 mmol) was dissolved in anhydrous DCM (3 mL), cooled to 0 °C, and triethylamine (0.20 g, 2.00 mmol) was added dropwise while maintaining this temperature. Then, a solution of 1-(4-methyl-3-nitrophenyl)methylamine (0.17 g, 1.00 mmol) in anhydrous DCM (3 mL) was added dropwise, and the reaction was maintained at this temperature for 1 h. The solvent was removed by vacuum concentration, and the residue was cooled to 0 °C. Anhydrous DCM (3 mL) was added again, and the mixture was stirred until homogeneous. Then, anhydrous DMF (3 mL) solution of compound PA1-1 (0.26 g, 1.00 mmol) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 1 h. The reaction solution was concentrated under reduced pressure, and DCM and water were added to the residue. After stirring, the mixture was separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give compound PA1-2 (0.14 g, 31% yield).

[0712] Step 2: Synthesis of compound PA1

[0713] Compound PA1-2 (0.14 g, 0.31 mmol) was dissolved in DMF (5 mL), and 10% Pd / C (0.02 g) was added. After three purgings with hydrogen, the mixture was reacted in hydrogen for 24 h. The reaction was confirmed to be complete by TLC. Palladium on carbon was removed by filtration, and the filtrate was concentrated under reduced pressure to remove DMF. The residue was purified by reverse HPLC to obtain compound PA1 (32 mg, yield 32%).

[0714] MS m / z(ESI): 422.2 [M+H] + .

[0715] 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),9.27(s,1H),7.78(d,J=1.8Hz,1H),7.58(d,J=7. 8Hz,1H),7.42(dd,J=7.8,1.8Hz,1H),7.26–7.17(m,2H),7.12(d,J=7.8Hz,1H),6.94(t, J=6.0Hz,1H),5.07(dd,J=13.2,5.0Hz,1H),4.38(d,J=17.2Hz,1H),4.31–4.22(m,3H),2 .95–2.86(m,1H),2.61–2.26(m,1H),2.44–2.29(m,1H),2.26(s,3H),2.00–1.96(m,1H).

[0716] Example 2.2: Preparation of compound PA23

[0717]

[0718] Step 1: Synthesis of compound PA23

[0719] Compound PA23-1 was synthesized using a method similar to step 1 of Example 2.1.

[0720] Chemical step 2: Synthesis of compound PA23

[0721] Compound PA23-1 (0.20 g, 0.32 mmol) was dissolved in dichloromethane (5 mL), followed by the addition of trifluoroacetic acid (1 mL), and the reaction was carried out at room temperature for 30 min. The reaction was confirmed to be complete by TLC. The solvent was removed by concentration under reduced pressure, followed by the addition of dichloromethane. The pH was adjusted to 8 with saturated sodium bicarbonate. The mixture was separated, and the organic phase was concentrated to dryness. The residue was purified by reverse HPLC to obtain compound PA23 (0.06 g, yield 36%).

[0722] MS m / z (ESI): 528.2 [M+H] + .

[0723] 1 H NMR (400MHz, DMSO-d6) δ9.72(s,1H),8.39(s,1H),7.81(s,1H),7.56(d,J=7.8Hz,2H),7.41–7.22(m,4H),5.06(dd,J=13.2,5.0 Hz,1H),4.46–4.18(m,4H),3.59(d,J=16.2Hz,4H),3.01–2.79(m,5H),2.70–2.55(m,2H),2.45–2.32(m,3H),2.06–1.90(m,1H).

[0724] Example 2.3: Preparation of compound PB11

[0725]

[0726] Compound PB11 was synthesized using a method similar to that in Example 2.1.

[0727] MS m / z (ESI): 550.1 [M+H] + .

[0728] 1H NMR (400MHz, DMSO-d6) δ10.97(s,1H),8.84(s,1H),7.68(d,J=8.2Hz,1H), 7.49(s,1H),7.44–7.40(m,2H),7.19–7.17(m,2H),6.84(s,1H),6.81–6.7 7(m,1H),6.64–6.77(m,2H),5.04(dd,J=13.2,5.2Hz,1H),4.39-4.18(m,4 H),2.92–2.84(m,1H),2.57(m,1H),2.38–2.27(m,1H),2.01–1.90(m,1H).

[0729] Example 2.4: Preparation of compound PB7

[0730]

[0731] Compound PB7 was synthesized using a method similar to that in Example 2.2.

[0732] MS m / z (ESI): 534.1 [M+H] + .

[0733] 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),9.20(s,1H),7.75(s,1H),7.57(d,J=7.6Hz,2H),7.38(dd,J=8.4,1.6Hz,1H),7.21-7.18(m,3H),7.06(s, 1H),7.01–6.89(m,3H),5.06(dd,J=13.2,5.2Hz,1H),4.40-4.21(m,4H) ,2.94–2.86(m,1H),2.59(m,1H),2.39–2.29(m,1H),2.03–1.92(m,1H).

[0734] Example 2.5: Preparation of compound PC6

[0735]

[0736] Compound PC6 was synthesized using a method similar to that in Example 2.2.

[0737] MS m / z (ESI): 498.1 [M+H] + .

[0738] 1H NMR (400MHz, DMSO-d6) δ10.97(s,1H),9.24(s,1H),7.76(d,J=1.8Hz,1H),7.56(d,J=7.8Hz,1H ),7.40(dd,J=7.8,1.8Hz,1H),7.26–7.17(m,2H),7.12(d,J=7.8Hz,1H),6.91(t,J=6.0Hz,1H), 5.07(dd,J=13.2,5.0Hz,1H),4.42(d,J=17.2Hz,1H),4.38(d,J=1.2Hz,1H),4.36–4.34(m,3H), 3.47–3.38(m,4H),3.15–3.05(m,1H),2.59–2.50(m,2H),2.10–2.01(m,1H),1.94–1.90(m,1H).

[0739] Example 3: Preparation of linker-payload for ligand-drug conjugates

[0740] Example 3.1: Preparation of compound LP15

[0741]

[0742] Step 1: Synthesis of compound LP15-1

[0743] Compounds INT1 (100.36 mg, 0.18 mmol), HATU (102.66 mg, 0.27 mmol), and DIPEA (34.89 mg, 0.27 mmol) were dissolved in anhydrous NMP (5 mL) and stirred at room temperature for 1 h. Then, compound PA1 (75.86 mg, 0.18 mmol) was added. After the reaction was complete, ice water and ethyl acetate were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by reverse HPLC to obtain compound LP15-1 (51.90 mg, 30% yield).

[0744] Step 2: Synthesis of compound II-1-2

[0745] Compound LP15-1 (19.22 mg, 0.02 mmol) was dissolved in dichloromethane / trifluoroacetic acid (3 mL) and stirred at room temperature for 0.5 h. After the reaction was completed, the solution was concentrated to dryness under reduced pressure, and the crude product was purified by reverse HPLC to obtain compound LP15 (3.62 mg, yield 20%).

[0746] MS m / z (ESI): 905.8 [M+H] + .

[0747] Example 3.2: Preparation of compound LP11

[0748]

[0749] Step 1: Synthesis of compound LP11-1

[0750] Compound PA23 (52.80 mg, 0.10 mmol), compound INT3 (72.37 mg, 0.10 mmol), and DIEA (25.85 mg, 0.20 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 2 h. After the reaction was complete, water and ethyl acetate were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to give compound LP11-1 (77.88 mg, 70% yield).

[0751] Step 2: Synthesis of compound LP11

[0752] Compound LP11 was synthesized using a method similar to step 2 in Example 3.1.

[0753] MS m / z (ESI): 1057.5 [M+H] + .

[0754] Example 3.3: Preparation of compound LP22

[0755]

[0756] Compound LP22 was synthesized according to the method in Example 3.1.

[0757] MS m / z (ESI): 929.3 [M+H] + .

[0758] Example 4: Preparation of ligand-drug conjugates

[0759] Antibodies used as ligands are prepared using conventional methods, such as vector construction followed by transfection into eukaryotic cells like HEK293 or CHO cells for purification and expression. Ligand-drug conjugates were prepared using anti-HER3 antibody Patritumab (prepared according to WO2007077028A2) and anti-GPC-3 antibody Codrituzumab (prepared according to WO2006006693) as examples.

[0760] Patritumab heavy chain amino acid sequence of anti-HER3 antibody

[0761]

[0762] The light chain amino acid sequence of the anti-HER3 antibody Patritumab

[0763]

[0764] Codrituzumab heavy chain amino acid sequence of anti-GPC-3 antibody

[0765]

[0766] Anti-GPC-3 antibody Codrituzumab light chain amino acid sequence

[0767]

[0768] Example 4.1: Preparation of ADC-6

[0769]

[0770] At 37°C, prepared TCEP (10mM, 0.135mL, 1.35μmol) was added to the buffer solution of Patritumab monoclonal antibody (14.0mM succinate-sodium hydroxide + 108mM NaCl pH 6.0; 20mg, 10.0mg / mL, 0.135μmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours, and then cooled to room temperature.

[0771] Compound LP15 (1.49 mg, 1.65 μmol) was dissolved in 0.1 mL DMSO and added to the above solution. The mixture was placed in a water bath and shaken at 22 °C for 2 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 20 mM histidine-hydrochloric acid, pH 5.5) to obtain a solution of the exemplary product ADC-5 (20 mM histidine-hydrochloric acid, pH 5.5; 18 mg, 3.6 mg / mL, yield: 90%), which was stored at 4 °C.

[0772] LC-MS analysis and calculation yielded a DAR value of q = 7.81.

[0773] Referring to the method in Example 4.1, the following compounds were synthesized using suitable linker-payloads, wherein Ab1 is Patritumab and Ab2 is Codrituzumab.

[0774]

[0775] Example 5: In vitro test of the compound's inhibitory effect on tumor cell proliferation

[0776] Test objective

[0777] To detect the inhibitory activity of drug compounds on the in vitro proliferation of BT474, HCC1569, and hepG2 tumor cells, cells were treated with different concentrations of the compounds in vitro and cultured for 6 days. CTG (Cellular Transmission Therapy) was then used to analyze the cell proliferation. The Luminescent Cell Viability Assay (Promega, catalog number: G7558) uses reagents to detect cell proliferation based on IC50. 50 The value was used to evaluate the in vitro activity of the compound.

[0778] Experimental methods

[0779] The following example, using the in vitro proliferation inhibition assay for BT474 cells, illustrates the method for testing the in vitro proliferation inhibition activity of the compounds in this application against tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

[0780] 1) Cell culture: BT474 cells were cultured in 10% FBS RPMI-1640 medium.

[0781] 2) Cell preparation: Take BT474 cells in the logarithmic growth phase, wash them once with PBS, add 2-3 ml of trypsin to digest for 2-3 min. After the cells are completely digested, add 10-15 ml of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and then add 10-20 ml of cell culture medium to resuspend the cells to make a single-cell suspension.

[0782] 3) Cell plating: Mix the BT474 single-cell suspension thoroughly, and adjust the viable cell density to 6 x 10⁻⁶ cells / mL using cell culture medium. 4 Cells / ml: After adjusting the cell density, mix the cell suspension thoroughly and add 50 μL / well to a 96-well cell culture plate. Incubate the plate in an incubator for 18 hours (37°C, 5% CO2).

[0783] 4) Compound preparation: Dissolve the compound in DMSO to prepare a stock solution with an initial concentration of 10 mM. There are 8 concentrations for the small molecule compound: 300, 100, 30, 10, 3, 1, 0.3, and 0.1 nM.

[0784] 5) Sample addition procedure: Add the prepared test samples at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% CO2).

[0785] 6) Color development procedure: Take out the 96-well cell culture plate, add 50 μL of CTG reagent to each well, and incubate at room temperature for 10 minutes.

[0786] 7) Plate reading procedure: Take out the 96-well cell culture plate, place it in the microplate reader, and use the microplate reader to measure the chemiluminescence.

[0787] Data Analysis

[0788] Use Microsoft Excel and Graphpad Prism 5 to process and analyze the data.

[0789] Table 1. IC50 values ​​of the small molecule fragments in this application inhibiting the in vitro proliferation of BT474, HCC1569, and HepG2 cells. 50 value.

[0790]

[0791] ++++:<1nM,+++:1~30nM,++:30~100nM,+:>100nM

[0792] Conclusion: The small molecule fragment in this application has significant inhibitory activity against the proliferation of BT474, HCC1569, and HepG2 cells.

[0793] Example 6: In vitro cell proliferation inhibition activity test of antibody-drug conjugates

[0794] Example 6.1: In vitro proliferation inhibition activity test of HCC1569 / HepG2 cells

[0795] use Chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effect of the anti-Her3 antibody Patritumab conjugate protein degrader compound ADC drug on cell proliferation after 6 days of incubation in Her3-positive human breast cancer cells HCC1569; and the inhibitory effect of the anti-GPC-3 antibody DB1002 conjugate protein degrader compound ADC drug on cell proliferation after 6 days of incubation in GPC-3-positive human liver cancer cells HepG2.

[0796] Logarithmic growth phase cells were collected and cultured at a density of 6000 cells / well in 96-well cell culture plates. The plates were incubated overnight at 37°C with 5% CO2. On the second day of the experiment, the ADC drug of camptothecin was diluted 3-fold with complete culture medium to obtain 9 concentration gradients (starting from the highest concentration of 300 nM). 100 μL of the drug was added to each well of the cell culture plate, with complete culture medium as a blank control. Three replicates were set up. The plates were incubated at 37°C with 5% CO2 for another 6 days. After incubation, the cell culture plates were removed and equilibrated to room temperature. 50 μL of CTG assay reagent (Promega, Cat#: G7573) was added to each well. After vortexing and incubation in the dark for 10 minutes, the signal value was read using a microplate reader. GraphPad Prism software was used to plot the sigmoid dose-response curve using a nonlinear regression model and the IC50 was calculated. 50 Value. Cell viability calculation formula = (Lum) 待测药 -Lum 空白对照 ) / (Lum 溶剂空白对照 -Lum 空白对照 )×100%.

[0797]

[0798]

[0799] ++++: <1nM, +++: 1~10nM, ++: 10~100nM

[0800] Experimental conclusions: The antibody-drug conjugate of this application has significant inhibitory activity against the proliferation of Her3-positive human breast cancer cells HCC1569; in addition, it has significant inhibitory activity against the proliferation of GPC-3-positive human liver cancer cells HepG2.

[0801] The sequence (Kabat numbering scheme) of this application:

[0802]

[0803]

Claims

1. A ligand-drug conjugate, wherein the ligand-drug conjugate has any of the following structures: in, q is an integer or decimal between 6 and 8; Ab1 is pertratuzumab; Ab2 is coutuzumab.

2. A compound: in, The compound is any one of the following compounds: 。 3. A nitrogen-containing heterocyclic compound, or a pharmaceutically acceptable salt thereof: in, The compound is any one of the following compounds: 。 4. A pharmaceutical composition comprising substance S and one or more pharmaceutical excipients; The substance S is the ligand-drug conjugate according to claim 1; Alternatively, the nitrogen-containing heterocyclic compound of claim 3, or a pharmaceutically acceptable salt thereof.

5. The use of a substance S in the preparation of a drug for treating cancer; The substance S is the ligand-drug conjugate according to claim 1; Or, the nitrogen-containing heterocyclic compound of claim 3, or a pharmaceutically acceptable salt thereof; The cancer in question is either breast cancer or liver cancer.

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