Camptothecin compounds and their conjugates, their preparation methods and uses

Abstract

This relates to camptothecin compounds and their conjugates, or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, as well as pharmaceutical compositions thereof, methods of preparation thereof, and applications thereof. The compounds and conjugates may be used to treat proliferative diseases associated with abnormal cell activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to camptothecin compounds and their conjugates, their preparation methods, and their use in the prevention and / or treatment of proliferative diseases (including but not limited to tumors) associated with abnormal cell activity. Background Technology

[0002] Camptothecin (CPT) is a cytotoxic alkaloid isolated from the camptotheca tree (Campanula stenoptera), a plant in the Davidiaceae family. It forms a ternary complex with cellular DNA topoisomerase I, thereby inhibiting DNA unwinding, leading to DNA replication arrest and ultimately cell death (Cancer Res. 1989, 49, 6365), exhibiting broad-spectrum antiproliferative activity. However, its low solubility, instability, acquired tumor cell resistance, and significant toxicity make it unsuitable for clinical development. Camptothecin derivatives can increase water solubility and improve drugability by introducing water-soluble groups or preparing prodrugs. Several camptothecin derivatives with significantly improved solubility have been approved for marketing (Med. Res. Rev. 2015, 35, 753), such as topotecan, irinotecan, and beloteccan, for the treatment of various types of cancer.

[0003] Camptothecin derivatives are also used in antibody conjugation as small molecule toxins in antibody-drug conjugates (ADCs), also known as payloads. ADCs combine the high potency of cytotoxic small molecules with the high selectivity of antibodies for specific tumor cells. Compared to traditional chemotherapy drugs, ADCs can more precisely kill tumor cells and reduce the impact on normal cells. In recent years, ADCs using camptothecin derivatives as small molecule toxins have made significant progress. Two camptothecin-based ADCs have been approved for cancer treatment: DS-8101a, where the camptothecin analog dxd is conjugated to the anti-HER2 antibody trastuzumab via a cleavable tetrapeptide-based linker; and Immu-132, where the camptothecin analog SN-38 is conjugated to the anti-Trop-2 antibody cetuzumab via a hydrolyzable pH-sensitive linker.

[0004] However, ADCs using camptothecin-based drugs or derivatives as toxins generally have a high drug-to-antibody ratio (DAR), are difficult to manufacture, and are prone to instability. Furthermore, camptothecin compounds often exhibit bone marrow suppression leading to hematologic toxicity, such as neutropenia, leukopenia, thrombocytopenia, and anemia, as well as gastrointestinal side effects such as nausea, vomiting, and diarrhea.

[0005] Therefore, there is still a high clinical demand and application value for developing camptothecin compounds and their conjugates with novel structures that can improve efficacy and safety. Summary of the Invention

[0006] This application provides a camptothecin compound or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof, which may have one or more effects selected from the group consisting of: (1) having inhibitory activity against the in vitro proliferation of tumor cells; (2) having plasma stability; (3) having in vivo tumor-suppressive effects; (4) having a bystander effect; (5) having antitransporter transport capability; (6) having in vivo tumor-targeting capability; and (7) having good in vivo safety.

[0007] On the one hand, this application provides compounds of formula (Ia), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0008]

[0009] in,

[0010] R 1a Selected from hydrogen, halogen, amino, hydroxyl, -C 1-6 Alkyl and -C 1-6 Alkoxy;

[0011] R 2a and R 3a Each is independently selected from hydrogen, -(C(R) 2a-1 )2) p -H、-(C(R 2a-1 )2) p -OR 2a-2 、-(C(R 2a-1 )2) p -SR 2a-2 、-(C(R 2a-1 )2) p -S(O)2R 2a-2 、-(C(R 2a-1 )2) p -N(R 2a-2 )-S(O)2R 2a-2 and -C(=O)R 2a-2 Or R 2a and R 3a Together with the atoms connected thereto, they form a 3-10 membered heterocycle; said heterocycle is optionally bounded by one or more atoms selected from halogens, hydroxyl groups, amino groups, and -C atoms. 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl, -N(C) 1-6 alkyl)2 and -C1-6 Substitution of alkylene-OH groups;

[0012] p is selected from integers from 0 to 10;

[0013] R 2a-1 Each group is independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, and -C. 1-6 Alkyl and -halogenated C 1-6 alkyl;

[0014] Or, any two R 2a-1 Together with the atoms attached thereto, they form an oxo group, a 3-10 membered carbon ring, or a 3-10 membered heterocycle, wherein the 3-10 membered carbon ring or 3-10 membered heterocycle is optionally surrounded by a -C group. 1-6 Alkyl substitution;

[0015] R 2a-2 Selected from hydrogen, hydroxyl, amino, cyano, -C 1-6 Alkyl, -C 3-6 Cycloalkyl and 3-6 membered heterocyclic alkyl groups; wherein the alkyl, cycloalkyl, and heterocyclic alkyl groups are each optionally surrounded by one or more elements selected from hydrogen, halogen, hydroxyl, amino, and -C. 1-6 Alkyl substituents.

[0016] In some embodiments, compounds with the structure shown in formula (Ia) are provided, wherein R 1a Selected from hydrogen, halogens, -C 1-6 Alkyl; preferably, R 1a Selected from hydrogen and halogens; more preferably, R 1a Selected from hydrogen, F, and Cl.

[0017] In some embodiments, compounds with the structure shown in formula (Ia) are provided, wherein R 2a and R 3a Each is independently selected from hydrogen, -(C(R) 2a-1 )2) p -H、-(C(R 2a-1 )2) p -OR 2a-2 、-(C(R 2a-1 )2) p -S(O)2R 2a-2 、-(C(R 2a-1 )2) p -N(R 2a -2 )-S(O)2R 2a-2 and -C(=O)R 2a-2 ;

[0018] p is selected from an integer between 0 and 10; preferably, p is selected from an integer between 0 and 5.

[0019] R 2a-1 Each element is independently selected from hydrogen, halogen, hydroxyl, amino, or -C. 1-6 Alkyl; preferably, R 2a-1 Selected from hydrogen or -C 1-6 Alkyl; more preferably, R 2a-1 Selected from hydrogen or methyl;

[0020] Or, any two R 2a-1 Together with the atoms attached thereto, they form oxo groups, 3-6 membered carbon rings, or heterocycles; preferably, any two R groups... 2a-1 Together with the atoms attached to it, they form oxo groups and 3-6 membered carbon rings;

[0021] R 2a-2 Selected from hydrogen, -C 1-6 Alkyl, -C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl; preferably, R 2a-2 Selected from hydrogen or -C 1-6 Alkyl; more preferably, R 2a-2 Selected from hydrogen or methyl.

[0022] In some embodiments, compounds with the structure shown in formula (Ia) are provided, wherein R 2a and R 3a Together with the atoms bonded thereto, they form a 4-6 membered heterocycle; said heterocycle is optionally bonded by one or more elements selected from halogens, hydroxyl groups, amino groups, and -C atoms. 1-6 Alkyl, -C 1-6 Substituents of alkylene-OH groups.

[0023] In some embodiments, compounds with the structure shown in formula (Ia) are provided, wherein R 2a and R 3a Each is independently selected from hydrogen and -C 1-6 Alkyl, -C 1-6 alkylene-OH, -halogenated C 1-6 Alkyl and -C 1-6 Alkyl-OC 1-6 Alkyl; preferably, R 2a and R 3a Each is independently selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, -(CH2)2OH, -(CH2)3OH, -(CH2)2OCH3 and -(CH2)3OCH3

[0024] In some particularly preferred embodiments, compounds with the structure shown in formula (Ia) are provided, wherein R 1a Selected from hydrogen, F, and Cl; R 2a and R 3aEach is independently selected from hydrogen and -C 1-6 Alkyl, -C 1-6 alkylene-OH, -halogenated C 1-6 Alkyl and -C 1-6 Alkyl-OC 1-6 alkyl.

[0025] In some embodiments, the compounds with the structure shown in formula (Ia) are selected from:

[0026]

[0027]

[0028]

[0029] Preferably, the compound with the structure shown in formula (Ia) is selected from:

[0030]

[0031] On the other hand, this application provides compounds of formula (Ib), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0032]

[0033] in,

[0034] R 1b R 2b Each is independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl and -C 1-6 Alkoxy;

[0035] Or R 1b With R 2b Together with the atoms attached to it, they form 4-6 membered carbon rings or 4-6 membered heterocycles;

[0036] R 3b Selected from -(C(R) 3b-1 )2) t -H、-(C(R 3b-1 )2) t -OR 3b-2 、-(C(R 3b-1 )2) t -N(R 3b-2 )2、-(C(R 3b-1 )2) t -SR 3b-2 and -(C(R) 3b-1 )2) t -S(O)2R 3b-2;

[0037] t is selected from integers between 0 and 10;

[0038] R 3b-1 Each group is independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, and -C. 1-6 Alkyl and -halogenated C 1-6 alkyl;

[0039] Or, any two R 3b-1 Together with the atoms attached to it, they form oxo groups, 3-6 membered carbon rings, or 3-6 membered heterocycles;

[0040] R 3b-2 Selected from hydrogen, hydroxyl, -C 1-6 Alkyl, -C 3-6 Cycloalkyl, 3- to 6-membered heterocyclic alkyl; wherein the alkyl, cycloalkyl, and heterocyclic alkyl are each optionally surrounded by one or more elements selected from hydrogen, halogen, hydroxyl, amino, and -C. 1-6 Alkyl substituents;

[0041] R 4b Selected from hydrogen, hydroxyl, -C 1-6 Alkyl, -C 3-6 cycloalkyl, -halogenated C 1-6 Alkyl group. In some embodiments, compounds with the structure shown in formula (Ib) are provided, wherein R 1b Selected from hydrogen, halogens, -C 1-6 Alkyl, -C 1-6 Alkoxy; preferably, R 1b Selected from hydrogen, F, Cl, methyl and methoxy.

[0042] In some embodiments, compounds with the structure shown in formula (Ib) are provided, wherein R 2b Selected from hydrogen, halogens, -C 1-6 Alkyl, -C 1-6 Alkoxy; preferably, R 2b Selected from hydrogen and halogens; more preferably, R 2b Selected from F.

[0043] In some embodiments, compounds with the structure shown in formula (Ib) are provided, wherein R 1b With R 2b Together with the atoms bonded to it, they form a 5-membered heterocycle; preferably, the 5-membered heterocycle is

[0044] In some embodiments, compounds with the structure shown in formula (Ib) are provided, wherein R 3b Selected from hydrogen, -(C(R) 3b -1 )2)t -H、-(C(R 3b-1 )2) t -OR 3b-2 、-(C(R 3b-1 )2) t -NHR 3b-2 ;

[0045] t is selected from an integer between 0 and 10; preferably, t is selected from an integer between 3 and 5.

[0046] R 3b-1 Each element is independently selected from hydrogen, halogen, hydroxyl, amino, or -C. 1-6 Alkyl; preferably, R 3b-1 Selected from hydrogen or -C 1-6 Alkyl; more preferably, R 3b-1 Selected from hydrogen or methyl;

[0047] Or, any two R 3b-1 Together with the atoms attached thereto, they form oxo groups, 3-6 membered carbon rings, or 3-6 membered heterocycles; preferably, any two R groups... 3b-1 Together with the atoms attached to it, they form oxo groups and 3-6 membered carbon rings;

[0048] R 3b-2 Selected from hydrogen, -C 1-6 Alkyl, -C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl; preferably, R 3b-2 Selected from hydrogen or -C 1-6 Alkyl; more preferably, R 3b-2 Selected from hydrogen. In some embodiments, compounds with the structure shown in formula (Ib) are provided, wherein R 3b Selected from hydrogen, -C 1-6 Alkyl, -halogenated C 1-6 Alkyl and -C 1-6 Alkylene-OH.

[0049] In some embodiments, compounds with the structure shown in formula (Ib) are provided, wherein R 4b Selected from hydrogen, methyl, ethyl; preferably, R 4b Selected from hydrogen and methyl; preferably, R 4b Selected from hydrogen.

[0050] In some embodiments, compounds with the structure shown in formula (Ib) are provided, wherein R 3b and R 4b They are not both hydrogen.

[0051] In some embodiments, the compounds with the structure shown in formula (Ib) are selected from:

[0052]

[0053]

[0054] Preferably, the compound with the structure shown in formula (Ib) is selected from:

[0055]

[0056] On the other hand, this application provides compounds of formula (IIa), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0057]

[0058] in,

[0059] R 1a R 2a and R 3a As defined by compound (Ia);

[0060] L 1 For connection units;

[0061] L 2 -(C(RL) 21 )2) n -,

[0062] Where n is a natural number from 0 to 50,

[0063] L 2 Any C(R) in L21 The )2 unit can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -, -O-, -S-, -SO-, -SO2-, -P(R L22 )-、-P(=O)(R L22 -, -C(=S)-, -C(=NR) L22 )-, -N=N-, -C=N-, -N=C-,

[0064] -Cy- is selected from phenylene, 5- to 8-membered heteroaryl, 3- to 10-membered heterocyclic, or 3- to 10-membered cycloalkylene, wherein -Cy- is unsubstituted or independently formed by one or more R- atoms. cx replace,

[0065] R L21 R L22 R cx Each is independently selected from hydrogen, deuterium, halogens, -NO2, -CN, and -OR. L2a -SRL2a 、-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 Or be R L2a Optional substitution of -C 1-6 Alkyl, -C 1-6 alkenyl, -C 1-6 Alkyne, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl,

[0066] m and y are natural numbers from 0 to 50.

[0067] 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 or 3-10 membered heteroaryl;

[0068] 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;

[0069] Tr does not exist or is Or any combination of the above groups;

[0070] 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 Or be RTra Optional substitution of -C 1-6 Alkyl, -C 1-6 alkenyl, -C 1-6 Alkyne, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl,

[0071] R Tra For 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 Alkyne, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl,

[0072] z is a natural number that is 0 or greater.

[0073] On the other hand, this application provides compounds of formula (IIb), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0074]

[0075] in,

[0076] X is selected from: -O-, -N(R) 3b-2 )-

[0077] L 1 L 2 L 3 Tr is as defined by compound of formula (IIa);

[0078] R 1b R 2b R 3b-1 R 3b-2 and R 4b As defined by compound (Ib), and t is an integer selected from 1 to 10.

[0079] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein L 1 Selected from:

[0080]

[0081] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein L 1 Selected from: Preferably, L 1 Selected from

[0082] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein L 2 -(CHR) L21 ) n -;

[0083] Where n is a natural integer from 0 to 50;

[0084] L 2 Any CHR in L21 The unit can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -、-O-、

[0085] -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 -Cy- is unsubstituted or independently surrounded by 1 to 3 R- groups. cx replace;

[0086] Each R L21 R L22 R cx Each is independently selected from hydrogen, halogen, and -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;

[0087] m is a natural integer from 0 to 8;

[0088] y is 0, 1, 2, 3 or 4;

[0089] Each R L2a R L2b Each is independently selected from hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2, -C 1-6 alkyl.

[0090] In some implementations, L 2 -(CH2) n -;

[0091] Where n is a natural integer from 0 to 50;

[0092] L 2 Any methylene unit in the structure can be independently replaced by the following structural units: 4- to 6-membered heterocyclic groups, 3- to 6-membered cycloalkyl groups, -C(O)-, -NR- L22 -、-O-、

[0093] 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 or by R L2a Optional substitution of -C 1-6 alkyl;

[0094] m is a natural integer from 0 to 8;

[0095] y is 0, 1, 2, 3 or 4;

[0096] Each R L2a Each is independently selected from hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2, -C 1-6 alkyl.

[0097] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein L 2 Selected from:

[0098]

[0099] in,

[0100] n1, n2, n3, n4, and m are each independently selected from natural numbers from 0 to 8;

[0101] n5 and n6 are each independently selected from 0 or 1;

[0102] -Cy- is a 4- to 6-membered heterocyclic group or a 3- to 6-membered cycloalkyl group; preferably, -Cy- is... More preferably, -Cy- is

[0103] More preferably,

[0104] for

[0105] More preferably,

[0106] for Among them, c and L 1 Connected, d and L 3 Connected.

[0107] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein, Selected from:

[0108]

[0109] n1, n2, n3, n4, and m are each independently selected from natural numbers from 0 to 8;

[0110] n5 and n6 are each independently selected from 0 or 1;

[0111] -Cy- is a 4- to 6-membered heterocyclic group or a 3- to 6-membered cycloalkyl group; preferably, -Cy- is selected from: More preferably, -Cy- is selected from

[0112] Preferably, Selected from: More preferably, Selected from:

[0113] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein L 3Selected 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-L ys, D-Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Val-Glu, Val-Asp, Asn-Asn, Asp-Glu, Gly-Gly-Glu, Gly-Gly-Asp, Gly-Gly-Asn, Gly-Ala-Ala, Gly-Val-Ala, Gly-Val-Cit, Glu-Val-Cit , 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-Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Ala-Ala-Pro-Val, Ala-Ala-Pro-Nva, Or any combination of the above segments.

[0114] In some implementations, L 3 Selected from Lys, Gly, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asn-Asn, Asp-Glu, Gly-Glu-Gly, Gly-Gly-Phe-Gly, Or any combination of the above segments; preferably, L 3 Selected from Lys, Gly, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asn-Asn, Asp-Glu, Gly-Gly-Phe-Gly, Or any combination of the above segments; more preferably, L 3Selected from Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asp-Asp, Asp-Glu, Gly-Gly-Phe-Gly or

[0115] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein, Selected from:

[0116]

[0117]

[0118]

[0119] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein Tr is absent or is in

[0120] R Tr R Tr1 and R Tr2 Independently selected from hydrogen, halogens, -NO2, -CN, -OH, -NH2, -CO2H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)NH2, -CH2CO-(N(Me)CH2C(O)) z -NHMe, -(CH2CH2O) z -H, -CONH-(CH2CH2O) z -H;

[0121] z is a natural number from 0 to 8.

[0122] In some embodiments, compounds with the structures shown in formulas (IIa) and (IIb) are provided, wherein Tr is absent or is

[0123] In some embodiments, the compounds with the structure shown in formula (IIa) are selected from:

[0124]

[0125]

[0126]

[0127] In some embodiments, the compounds with the structure shown in formula (IIb) are selected from:

[0128]

[0129] On another front, this application 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

[0130] The structure shown in equation (IVa):

[0131]

[0132] in,

[0133] R 1a R 2a and R 3a As defined for compounds of formula (Ia), the wavy line indicates direct or indirect linkage to the ligand via a nitrogen atom; or

[0134] The structure shown in equation (IVb):

[0135]

[0136] in,

[0137] X is selected from: -O-, -N(R) 3b-2 )-;

[0138] R 1b R 2b R 3b-1 R 3b-2 and R 4b As defined in compound (Ib), t is an integer selected from 1 to 10, and the wavy line indicates direct or indirect connection with the ligand via X.

[0139] On another front, this application 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

[0140] The structure shown in equation (Va):

[0141]

[0142] in,

[0143] The wavy line indicates that it passes through L. 1a The nitrogen or carbon atom on the group is attached to a ligand;

[0144] L 1a Selected from:

[0145] L 2 L3 Tr is as defined by compound of formula (IIa);

[0146] R 1a R 2a and R 3a As defined by compounds of formula (Ia); or

[0147] The structure shown in equation (Vb):

[0148]

[0149] in,

[0150] The wavy line indicates that it passes through L. 1a The nitrogen or carbon atom on the group is attached to a ligand;

[0151] L 1a As defined by the compound of formula (Va);

[0152] L 2 L 3 Tr is as defined by compound of formula (IIa);

[0153] X is selected from: -O-, -N(R) 3b-2 )-;

[0154] R 1b R 2b R 3b-1 R 3b-2 and R 4b As defined by compound (Ib), and t is an integer selected from 1 to 10.

[0155] In some embodiments, ligand-drug conjugates comprising the structures shown in formulas (Va) and (Vb) are provided, wherein L 1a Selected from: Preferably, L 1a Selected from

[0156] In some embodiments, ligand-drug conjugates comprising the structures shown in formulas (Va) and (Vb) are provided, wherein Selected from:

[0157]

[0158] n1, n2, n3, n4, and m are each independently selected from natural numbers from 0 to 8;

[0159] n5 and n6 are each independently selected from 0 or 1;

[0160] -Cy- is a 4- to 6-membered heterocyclic group or a 3- to 6-membered cycloalkyl group; preferably, -Cy- is selected from: More preferably, -Cy- is selected from

[0161] In some embodiments, ligand-drug conjugates comprising the structures shown in formulas (Va) and (Vb) are provided, wherein, Selected from: More preferably, Selected from:

[0162] In some embodiments, ligand-drug conjugates comprising the structures shown in formulas (Va) and (Vb) are provided, wherein, Selected from:

[0163]

[0164]

[0165]

[0166] On the other hand, this application provides ligand-drug conjugates of formula (IIIa), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0167]

[0168] in,

[0169] L 2 L 3 Tr is as defined by compound of formula (IIa);

[0170] R 1a R 2a R 3a As defined by compound (Ia).

[0171] Ab is a ligand that binds to the target;

[0172] q represents the drug loading, which can be an integer or decimal from 1 to 16;

[0173] L 1a As defined by compound of formula (Va).

[0174] On the other hand, this application provides ligand-drug conjugates of formula (IIIb), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0175]

[0176] in,

[0177] Ab、q、L 1a As defined by compounds of formula (IIIa);

[0178] L 2 L 3 Tr is as defined by compound of formula (IIa);

[0179] X is selected from: -O-, -N(R) 3b2 )-;

[0180] R 1b R 2b R 3b-1 R 3b-2 and R 4b As defined by compound (Ib), and t is an integer selected from 1 to 10.

[0181] In some embodiments, ligand-drug conjugates of formulas (IIIa) and (IIIb) are provided, wherein Ab is a target-binding polypeptide, antibody, or antigen-binding fragment thereof.

[0182] In some implementations, Ab is an antibody or its antigen-binding fragment.

[0183] In some embodiments, the antibody is selected from: fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, bispecific antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

[0184] In some embodiments, the antigen-binding fragment is selected from: Fab, Fab', F(ab')2, Fv, scFv, biantibody, Fd, dAb, VHH, large antibody, and complementarity-determining region (CDR) fragment.

[0185] In some implementations, Ab is a monoclonal antibody.

[0186] In some implementations, the Ab specifically binds to antigens selected from the following group: 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, 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, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PSMA, PTK7, P-Cadherin, RNF43, NaPi2b, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB-1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9Epidermal growth factor, short proteoglycans, mesothelin, sodium phosphate cotransporter 2B, Claudin 18.2, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α4β6, trophoblast cell glycoproteins, or tissue factor.

[0187] In some preferred embodiments, the Ab specifically binds to antigens selected from the group consisting of: HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70, or EGFR.

[0188] In some embodiments, Ab is an antibody or antigen-binding fragment that specifically binds to HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70, or EGFR. In some embodiments, the antibody or antigen-binding fragment that specifically binds to HER2 is an anti-HER2 antibody or antigen-binding fragment, such as trastuzumab or pertuzumab, or a variant thereof.

[0189] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, wherein the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, each having one, two, three, or four amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 of the following molecules: trastuzumab or pertuzumab.

[0190] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, wherein the heavy chain variable region and the light chain variable region have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, and about 99% sequence identity with the heavy chain variable regions and light chain variable regions of the following molecules, respectively: trastuzumab or pertuzumab.

[0191] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the heavy chain and light chain have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, and about 99% sequence identity with the heavy chain and light chain of the following molecules, respectively: trastuzumab or pertuzumab.

[0192] The sequences of exemplary anti-HER2 antibodies or their antigen-binding fragments are provided in Table 1.

[0193] Table 1: Trastuzumab amino acid sequence

[0194]

[0195]

[0196]

[0197] In some implementations, the antibody or antigen-binding fragment that specifically binds to TROP2 is an anti-Trop-2 antibody or antigen-binding fragment, such as sacituzumab or a variant thereof.

[0198] In some embodiments, the anti-Trop-2 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, wherein the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, each having one, two, three, or four amino acid variations (e.g., amino acid substitutions or deletions) with one or more CDRs of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 of the molecule sacituzumab.

[0199] In some embodiments, the anti-Trop-2 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, wherein the heavy chain variable region and the light chain variable region have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, and about 99% sequence identity with the heavy chain variable regions and light chain variable regions of the following molecule: Sacituzumab.

[0200] In some embodiments, the anti-Trop-2 antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the heavy chain and light chain have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, and about 99% sequence identity with the heavy chain and light chain of the following molecule: Sacituzumab.

[0201] The sequences of exemplary anti-Trop-2 antibodies or their antigen-binding fragments are provided in Table 2.

[0202] Table 2: Amino acid sequence of sertozhumab

[0203]

[0204]

[0205]

[0206] In some implementations, the ligand-drug conjugate represented by formula (IIIa) is selected from:

[0207]

[0208]

[0209]

[0210]

[0211] Where Ab and q are as defined in compound (IIIa).

[0212] In some preferred embodiments, the ligand-drug conjugate represented by formula (IIIa) is selected from:

[0213]

[0214]

[0215]

[0216]

[0217]

[0218] Where q is defined as in compound (IIIa).

[0219] In some implementations, the ligand-drug conjugate represented by formula (IIIb) is selected from:

[0220]

[0221]

[0222]

[0223] Where Ab and q are as defined in compound (IIIa).

[0224] In some preferred embodiments, the ligand-drug conjugate represented by formula (IIIb) is selected from:

[0225]

[0226]

[0227]

[0228]

[0229] Where q is defined as in compound (IIIa).

[0230] In some implementations, q is an integer or decimal selected from 2 to 8.

[0231] The present invention also provides a pharmaceutical composition comprising compounds of the structures shown in formula (Ia) and (IIa) above, ligand-drug conjugates of formula (IIIa) or containing the structures shown in formula (IVa) or (Va), compounds of the structures shown in formula (Ib) and (IIb), ligand-drug conjugates of formula (IIIb) or containing the structures shown in formula (IVb) or (Vb), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, and a pharmaceutically acceptable carrier.

[0232] The present invention also provides a pharmaceutical formulation comprising compounds with structures shown in formula (Ia) and (IIa) above, ligand-drug conjugates shown in formula (IIIa) or ligand-drug conjugates containing structures shown in formula (IVa) or (Va), compounds with structures shown in formula (Ib) and (IIb), ligand-drug conjugates shown in formula (IIIb) or ligand-drug conjugates containing structures shown in formula (IVb) or (Vb), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof.

[0233] The present invention also provides the use of substance S in the preparation of a medicament for the prevention or treatment of cancer; said substance S is a compound with the structure shown in formula (Ia) or (IIa) above, a ligand-drug conjugate shown in formula (IIIa) or containing a ligand-drug conjugate shown in formula (IVa) or (Va), a compound with the structure shown in formula (Ib) or (IIb), a ligand-drug conjugate shown in formula (IIIb) or containing a ligand-drug conjugate shown in formula (IVb) or (Vb), or in the form of tautomers, enantiomers, diastereomers, or mixtures thereof, or in the form of a pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition thereof, or a pharmaceutical preparation thereof. The cancer is a solid tumor or a non-solid tumor. Examples include esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumors, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, stomach 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.

[0234] This invention also provides the use of substance S in the preparation of a drug for the prevention or treatment of diseases associated with abnormal cellular activity; said substance S is a compound with the structure shown in formula (Ia) or (IIa) above, a ligand-drug conjugate shown in formula (IIIa) or containing a ligand-drug conjugate shown in formula (IVa) or (Va), a compound with the structure shown in formula (Ib) or (IIb), a ligand-drug conjugate shown in formula (IIIb) or containing a ligand-drug conjugate shown in formula (IVb) or (Vb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition thereof, or a pharmaceutical preparation thereof. The disease associated with abnormal cellular activity may be cancer. The definition of cancer is as described above.

[0235] Terminology definition:

[0236] 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.

[0237] In this application, the term "pharmaceutical excipients" refers to 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.

[0238] 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. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound disclosed herein or its pharmaceutically acceptable salts or conjugates, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5-350 mg, and particularly preferably 1-250 mg.

[0239] 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.

[0240] In this application, the terms "pharmaceutically acceptable salt" or "medicinal salt" generally refer to salts of compounds or ligand-drug conjugates of this application, or salts of compounds described in this application, which are safe and / or effective when used in mammals and have the desired biological activity. The antibody-antibody-drug conjugates of this application can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0241] In this application, the terms "solvent" or "solvent compound" generally refer to a pharmaceutically usable solvate formed by the compound or ligand-drug conjugate of this application with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0242] 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, the drug loading is expressed as q, which can be, for example, an integer or decimal of 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10. For example, the drug loading q is 7.8 or 7.9. 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.

[0243] 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 antibody fragment linked to a biologically active cytotoxic drug via a stable linker unit.

[0244] 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 the following targets: HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70, or 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, 0772P, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B 7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD1lc, CD123, C D138, 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, CD 74, CD79a, CD79b, CD80, CDCP1, CDH11, CDllb, CEA, CEACAM5, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CT SD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, EN PP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, GD2, GEDA, GPC-1, 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, LY 6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Necti n-4, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP 1. PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PSMA, PTK7, P-cadherin, RNF43, NaPi2b, 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 α4β6, trophoblast cell glycoproteins, or tissue factor.

[0245] 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 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, and so on. The term "antigen-binding fragment" can refer to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. For example, fragments of full-length antibodies 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-B7-H3 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 Antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-ADAM9 antibody or anti-Mesothelin antibody, for example, trastuzumab or pertuzumab.

[0246] 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.

[0247] 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.

[0248] In this application, the terms "fully human antibody," "fully human antibody," or "completely human antibody" are used interchangeably, and the variable and constant regions of the antibody may both be of human origin, with immunogenicity and toxicity removed.

[0249] The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. 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.

[0250] 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.

[0251] 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.

[0252] 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 (in the form of “about a to b”, or equivalently, “approximately a to b”, or equivalently, “about ab”) should be understood to represent each numerical value and range encompassed within a wider range;

[0253] 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 C6-9 C 6-8 C 6-7 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.

[0254] 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, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl. The alkyl group in this invention may optionally be substituted with one or more substituents described herein.

[0255] In this application, the term "alkylene" refers to a saturated straight-chain or branched divalent hydrocarbon group. As used herein, the term "C" refers to... 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. The alkylene in this invention may optionally be substituted with one or more substituents described herein.

[0256] 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 (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.) having 2-6 carbon atoms and one, two, or three (preferably one) carbon-carbon double bonds, which is optionally substituted by one or more (e.g., 1-3) substituents described herein.

[0257] In this application, the term "alkenyl" refers to a straight-chain or branched divalent aliphatic hydrocarbon group having one or more carbon-carbon double bonds, wherein the two groups (or segments) to which it is attached may be attached to the same carbon atom or different carbon atoms. For example, the term "C" as used herein... 2-6 "Alkenyl" refers to alkenyl groups having 2-6 carbon atoms (e.g., alkenyl groups). (etc.), which may be optionally substituted by one or more (e.g., 1-3) substituents described herein.

[0258] 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.), which is optionally substituted by one or more (e.g., 1-3) substituents described herein.

[0259] In this application, the term "acetylenic" refers to a straight-chain or branched divalent aliphatic hydrocarbon group having one or more carbon-carbon triple bonds, wherein the two groups (or segments) to which it is attached are respectively attached to different carbon atoms. For example, the term "C" as used herein... 2-6 "Imyynyl" refers to an ynyl group having 2-6 carbon atoms (e.g., (etc.), which may be optionally substituted by one or more (e.g., 1-3) substituents described herein.

[0260] In this application, the term "aryl" refers to an aromatic hydrocarbon group having a monocyclic or fused ring with a conjugated π-electron system. For example, the term "6-10 aryl" as used herein refers to an aryl group (such as phenyl, naphthyl, etc.) having 6-10 carbon atoms, which is optionally substituted by one or more substituents described herein (such as halogen substitution, etc.).

[0261] In this application, the term "arylene" refers to a monocyclic or fused-ring divalent aromatic hydrocarbon group having a conjugated π-electron system. For example, the term "C" as used herein... 6-10 "Arylene" refers to an arylene group having 6-10 carbon atoms, which is optionally substituted by one or more substituents described herein (such as halogen substitution).

[0262] 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. 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, indolyl, etc., optionally substituted by one or more substituents described herein.

[0263] In this application, the term "hybrid aryl" refers to a monocyclic or fused-ring divalent aromatic group having a conjugated π-electron system, having one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 9, or 10 carbon atoms) and one or more (e.g., 1, 2, 3, or 4) heteroatoms, each independently selected from N, O, P, and S, for example, having a total of 5-12 (preferably 5-10, more preferably 5, 6, 9, or 10) ring atoms. For example, the term "5-12-membered hybrid aryl" as used herein refers to a hybrid aryl having 5-12 ring atoms, optionally substituted by one or more substituents described herein (e.g., substituted by C...). 1-6 Alkyl-substituted Replaced by halogen wait).

[0264] In this application, the terms "cycloalkyl" or "carbocyclic" refer to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic hydrocarbon group. 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 bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[5.2.0]nonyl, decahydronaphthyl, etc., which are optionally substituted by one or more substituents described herein.

[0265] In this application, the term "cycloalkylene" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic dicyclic group. For example, "C 3-12 "Cycloalkylene" or "3-12-membered cycloalkylene" refers to a cycloalkylene group having 3-12 ring carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). Common cycloalkylene groups include (but are not limited to) monocyclic cycloalkylene groups, such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkylene groups, including fused rings, bridged rings, or spirocyclic groups, such as bicyclic[1.1.1]pentylene, bicyclic[2.2.1]heptylene, bicyclic[3.2.1]octylene, bicyclic[5.2.0]nonylene, decahydronaphthylene, etc., which are optionally substituted by one or more substituents described herein.

[0266] The term "heterocyclic alkyl" or "heterocycle" 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 one, two, three, or four. Examples include 3-8-membered and 3-6-membered heterocyclic alkyl groups. Specific examples include, but are not limited to, ethylene oxide, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc., optionally substituted with one or more oxo groups or substituents described herein.

[0267] 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 1, 2, 3, or 4 heteroatoms. Examples include 3-8-membered and 3-6-membered heterocyclic alkylenes. Specific examples include, but are not limited to, ethylene oxide, cyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazineyl, tetrahydropyranyl, homopiperazineyl, etc., optionally substituted with one or more oxo groups or substituents described herein.

[0268] The term "fused ring (fused ring system)" refers to a polycyclic structure formed by two or more (e.g., 3, 4, or 5) carbon rings or heterocycles sharing a common ring edge, wherein the carbon rings include cycloalkyl and aryl groups, and the heterocycles include heteroaromatic and heterocyclic alkyl groups. The 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.

[0269] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, for example, fluorine or chlorine.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] In this application, the term "replaced" for zero or more (e.g., zero or more than one, zero or one, zero) methylene units generally means that when the structure contains one or more methylene units, the one or more methylene units may not be replaced, or may be replaced by one or more groups described herein (e.g., -NHC(O)-, -C(O)NH-, -C(O)-, -OC(O)-, -C(O)O-, -NH-, -O-, -S-, -SO-, -SO2-, -PH-, -P(=O)H-, -NHSO2-, -SO2NH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C- or -C(=N2)- etc.).

[0274] 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.

[0275] When used alone or in combination with other groups in this document, the term "oxo" refers to =O.

[0276] 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.

[0277] 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.

[0278] In this application, the term "non-natural amino acid" has the following structure: Where r is selected from 0, 1, 2, 3, 4, 5; where R a R bEach is independently selected from -C 1-6 Alkyl-NH2, -C 1-6 Alkyl-NH-C 1-6 Alkyl, -C 1-6 Alkyl-N(C) 1-6 Alkyl)2, -C 1-6 Alkyl-NH-C 3-10 cycloalkyl, -C 1-6 Alkyl-N (3-10 membered cycloalkyl) (C 1-6 Alkyl), -C 1-6 Alkyl-C 3-10 cycloalkyl, -C 1-6 Alkyl-(3-10 membered heterocyclic alkyl), -C 1-6 Alkyl-NHCOC 1-6 Alkyl, -C 1-6 Alkyl-NHCOOC 1-6 Alkyl, -C 1-6 Alkyl-NHS(O)2C 1-6 Alkyl, -C 1-6 Alkyl-S(O)2-C 1-6 Alkyl, -C 1-6 Alkyl-S(O)2-C 3-10 cycloalkyl, -C 1-6 Alkyl-S(O)2-NH2, -C 1-6 Alkyl-COOH, -C 1-6 Alkyl-CONH2, -C 1-6 Alkyl-CONHC 1-6 Alkyl, -C 1-6 Alkyl-CO (3-10 membered heterocyclic alkyl), The alkyl, cycloalkyl, and heterocyclic cycloalkyl groups are each optionally 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, halogen, -OH, -NH2, -SH, -NO2, CN, -COOH, and oxo groups;

[0279] 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.

[0280] 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%).

[0281] 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).

[0282] 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.

[0283] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0284] 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.

[0285] 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.

[0286] As used herein, the term "effective amount" (e.g., "therapeutic effective amount" or "preventive effective amount") refers to the amount of active ingredient that, when administered, will achieve the desired effect to a certain extent, such as relieving one or more symptoms of the treated condition or preventing the occurrence of the condition or its symptoms.

[0287] 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.

[0288] 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.).

[0289] 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 peroxyacids such as peracetic acid and m-chloroperoxybenzoic acid (mCPBA), 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).

[0290] 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.

[0291] 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.

[0292] 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.

[0293] The reagents and raw materials used in this invention are all commercially available.

[0294] The beneficial technical effects of this invention lie in the compounds of this invention:

[0295] (1) It has inhibitory activity against the in vitro proliferation of tumor cells;

[0296] (2) It has plasma stability;

[0297] (3) It has an in vivo tumor-suppressing effect;

[0298] (4) It has a bystander effect;

[0299] (5) It has the ability to resist transport by transporters;

[0300] (6) It has the ability to target tumors in vivo;

[0301] (7) It has good in vivo safety.

[0302] 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 cytotoxic agents, linkers, antibodies, and ADCs of this invention have significant clinical value.

[0303] Preparation method:

[0304] To achieve the synthetic objective of this disclosure, the present application employs the following synthetic technique:

[0305] Option 1: A method for preparing the compounds shown in formula (Ia) and (IIa) and the ligand-drug conjugate shown in formula (IIIa) or their pharmaceutically acceptable salts or solvates, the method comprising:

[0306]

[0307] in,

[0308] Ab、q、L 1a As defined by equation (IIIa);

[0309] L 1 L 2 L 3 Tr is as defined in equation (IIa);

[0310] R 1a R 2a R 3a As defined by equation (Ia);

[0311] X' is a halogen; preferably, X' is selected from Cl and Br;

[0312] Lg Tr ,Lg 1 The leaving group is selected from halogens, hydroxyl groups, etc. -OAc, etc.;

[0313] Pg 2 The protecting group is selected from: Boc, Fmoc, Cbz, Sem, etc.

[0314] Step 1 Compound Ia-1 and compound E are cyclized in a solvent under acidic conditions and catalysis (e.g., 80–130 °C) to give compound Ia-2.

[0315] In some embodiments, the solvent is selected from methanol, tetrahydrofuran, DMF, NMP, DMSO, toluene, n-hexane, n-heptane, etc.; toluene is preferred.

[0316] In some embodiments, the reagent providing the acidic conditions is selected from PTSA, acetic acid, hydrochloric acid, sulfuric acid, InCl3, p-toluenesulfonic acid, formic acid, and trifluoroacetic acid; acetic acid, hydrochloric acid, and InCl3 are preferred.

[0317] Step 2 Compound Ia-2 reacts with an acetylation agent (e.g., acetyl chloride or acetic anhydride) in acetic acid to give compound Ia-3.

[0318] Step 3 Compound Ia-3 undergoes oxidation in a polar solvent (such as dichloromethane, chloroform, ethyl acetate, etc.) in the presence of an oxidizing agent to give compound Ia-4.

[0319] In some embodiments, the oxidant in step 3 is m-CPBA, oxone, urea peroxide, hydrogen peroxide, etc., preferably m-CPBA.

[0320] Step 4 Compound Ia-4 reacts with a suitable organic solvent (e.g., toluene, DMF, NMP, DMSO, chloroform, etc.) in the presence of a halogenating agent to give compound Ia-5.

[0321] In some embodiments, the halogenating agent in step 4 is selected from oxalyl chloride, oxalyl bromide, thionyl chloride, phosphorus oxychloride, phosphorus tribromide, etc., preferably from oxalyl chloride.

[0322] Step 5 Compound Ia-5 undergoes a substitution reaction in a suitable organic solvent (e.g., acetonitrile, DMF, tetrahydrofuran, dioxane, dichloromethane, NMP, DMSO) in the presence of an organic base (e.g., DIPEA, triethylamine, DBU, pyridine, N-methylmorpholine, etc.) or an inorganic base (e.g., potassium carbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, etc.) to give compound Ib-6.

[0323] Step 6 Compound Ia-6 is reduced by reducing the nitro group in a suitable solvent (e.g., methanol, ethanol, ethyl acetate, dichloromethane, tetrahydrofuran, dioxane, etc.) in the presence of a reducing agent (e.g., palladium on carbon, iron powder / acetic acid, zinc powder / ammonium chloride, ammonium sulfide, SnCl2, etc.) to give the compound of formula (Ia-7).

[0324] Step 7 Compound Ia-7 is deacetylated in a suitable solvent (e.g., methanol, ethanol, DMF, tetrahydrofuran, dioxane, etc.) in the presence of an inorganic base (e.g., sodium carbonate, potassium carbonate, cesium carbonate, etc.) or an organic base (e.g., DBU, N-methylmorpholine, etc.), and then acidified with an acid (e.g., hydrochloric acid, trifluoroacetic acid, etc.) to obtain the compound of formula (Ia).

[0325] Step 8 Compound of formula (Ia) and compound F are reacted by condensation in the presence of a condensing agent or by substitution to obtain compound of formula (IIa).

[0326] Step 8a The compound of formula (Ia) and compound Fa are reacted by condensation or substitution in the presence of a condensing agent; subsequently, the protecting group is removed to give compound Ia-8.

[0327] Step 8b Compound Ia-8 and compound L 1 -Lg 1 The compound of formula (IIa) is obtained by condensation reaction or substitution reaction in the presence of a condensing agent.

[0328] In some embodiments, the condensing agent is selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 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 or benzotriazole-1-yl-oxytripyrrolidine phosphorus hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0329] Step 9 The compound of formula (IIa) reacts with Ab after disulfide bond reduction to give the ligand-drug conjugate of formula (IIIa).

[0330] In some embodiments, the reducing agent for reducing the antibody includes, but is not limited to, tris(2-carboxyethyl)phosphine, thiothreitol, cysteine, reduced glutathione, etc.; in particular, reducing the disulfide bonds on the antibody is preferred.

[0331] Option 2: A method for preparing the compounds of formula (Ib) and (IIb), the ligand-drug conjugate of formula (IIIb), or their pharmaceutically acceptable salts or solvates, comprising:

[0332]

[0333] in,

[0334] Ab、q、L 1a As defined by equation (IIIa);

[0335] X' is a halogen; preferably, X' is selected from Cl and Br;

[0336] X is selected from: -O-, -N(R) 3b-2 )-;

[0337] L1 L 2 L 3 Tr is as defined in equation (IIa);

[0338] R 1b R 2b R 3b-1 R 3b-2 and R 4d As defined by equation (Ib), and t is an integer selected from 1 to 10.

[0339] The specific preparation method of formula (Ib) is as described in Scheme 1.

[0340] The specific preparation methods for formulas (IIb) and (IIIb) are respectively carried out according to the methods described in steps 8, 8a, 8b and 9 of Scheme 1.

[0341] Those skilled in the art should understand that, depending on the desired product structure, one or more steps in the above preparation method can be omitted, and the order of reaction steps and the addition or omission of protection / deprotection reaction steps can be appropriately adjusted as needed. Detailed Implementation

[0342] 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.

[0343] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0344] The preparative high performance liquid chromatography (HPLC) method was performed using a Shimadzu LC-8A preparative liquid chromatograph (YMC, ODS, 250×20mm column).

[0345] Thin-layer chromatography purification was performed using GF 254 (0.4–0.5 nm) silica gel plates produced in Yantai.

[0346] 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.

[0347] 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.

[0348] Unless otherwise specified in the examples, the reaction temperature is room temperature (20℃~30℃).

[0349] 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.

[0350] 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.

[0351] The meanings of the abbreviations in the conventional synthesis methods, preparation examples, and intermediate synthesis examples are shown in the table below.

[0352]

[0353] Example 1: Preparation of camptothecin-like compounds

[0354] Example 1.1: Preparation of compound A1.

[0355]

[0356] Step 1: Preparation of compound A1-2.

[0357] Compound A1-1 (6 g, 36.13 mmol) and compound E (6 g, 22.81 mmol) were placed in a 2 L three-necked flask under nitrogen protection. 600 mL of glacial acetic acid was added to dissolve the compounds, followed by 120 mL of concentrated hydrochloric acid. The mixture was refluxed at 125 °C for 16 h. The reaction solution was then evaporated to dryness and purified by column chromatography to obtain compound A1-2 (8.6 g, 95.9% yield).

[0358] Step 2: Preparation of compound A1-3.

[0359] Compound A1-2 (8.60 g, 21.87 mmol) was placed in a 500 mL three-necked flask and dissolved in 123 mL of glacial acetic acid. Acetyl chloride (20.60 g, 262.52 mmol) was added under nitrogen protection, and the mixture was heated at 75 °C for 1 h. The reaction solution was evaporated to dryness, and the residue was purified by column chromatography to give compound A1-3 (9.4 g, 98.7%).

[0360] Step 3: Preparation of compound A1-4.

[0361] Compound A1-3 (9.4 g, 21.6 mmol) was placed in a 250 mL single-necked flask and dissolved in 108 mL of DCM. After cooling to 0 °C, m-CPBA (13.15 g, 64.81 mmol) was added, and the mixture was allowed to return to room temperature for 1 h. Subsequently, saturated sodium bicarbonate aqueous solution was added dropwise to adjust the pH to approximately 8, resulting in separation of the layers. The organic phase was collected, and the aqueous phase was extracted with DCM (80 mL × 3). The organic phases were combined, washed once with saturated brine, and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound A1-4 (7.5 g, yield 78.9%).

[0362] Step 4: Preparation of compound A1-5.

[0363] Compound A1-4 (3 g, 6.65 mmol) was placed in a 100 mL single-necked flask, dissolved in 38 mL of DMF, and cooled to 0 °C. Then, 8.3 mL of oxalyl chloride (2 M DCM solution) was slowly added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and reacted for 2 h. The reaction solution was poured into 400 mL of water and extracted with DCM (100 mL × 4). The organic phase was washed once with 150 mL of DCM, evaporated to dryness, and the residue was purified by column chromatography to give compound A1-5 (2.4 g, yield 77.1%).

[0364] Step 5: Preparation of compound A1-6.

[0365] Compound A1-5 (1.2 g, 2.55 mmol), 3-methylbut-1-amine (267 mg, 3.06 mmol), and K2CO3 (1.06 mg, 7.67 mmol) were placed in a 100 mL single-necked flask, dissolved in 1,4-dioxane, and stirred at 80 °C for 16 h. The reaction solution was evaporated to dryness, and the residue was purified by column chromatography to give compound A1-6 (0.42 g, yield 34.7%).

[0366] Step 6: Preparation of compound A1-7.

[0367] Compound A1-6 (360 mg, 0.69 mmol) and 10% Pd / C (360 mg) were placed in a 25 mL single-necked flask and dissolved in a 4 mL LCM / MeOH (10:1) mixed solvent. The reaction was carried out at 25 °C for 16 h under a hydrogen atmosphere. The reaction solution was filtered to remove Pd / C, and the organic phase was concentrated under reduced pressure to obtain compound A1-7 (317 mg crude product).

[0368] Step 7: Preparation of compound A1.

[0369] Compound A1-7 (310 mg, 0.63 mmol) was placed in a 50 mL single-necked flask, dissolved in 15 mL of methanol, and then 3 mL of 0.63 M K2CO3 aqueous solution was added. The mixture was stirred at room temperature for 2 h, and the pH was adjusted to approximately 4 with 5 M dilute hydrochloric acid and stirred for 10 min. The reaction solution was evaporated to dryness, and the crude product was subjected to HPLC to obtain compound A1 (22 mg, yield 7.7%).

[0370] MS m / z (ESI): 449.2 [M+H] +

[0371] 1 H-NMR (400MHz, DMSO-d6): δ7.72(d,J=9.0Hz,1H),7.59(s,1H),7.39–7.27(m,2H),6.66(s,1H),5.51(s,2H),5.44(s,2H), 3.74(d,J=7.4Hz,2H),1.92–1.72(m,3H),1.65(dd,J=14.8,6.9Hz,2H),0.99(d,J=6.6Hz,6H),0.88(dd,J=9.7,5.1Hz,3H).

[0372] Example 1.2: Preparation of compound A13.

[0373]

[0374] Compound A13 was synthesized using a method similar to that described in steps five and six of Example 1.1.

[0375] MS m / z(ESI): 423.2 [M+H] +

[0376] Example 1.3: Preparation of compound A14

[0377]

[0378] Compound A14 was synthesized using a method similar to that described in steps five and six of Example 1.1.

[0379] MS m / z (ESI): 437.2 [M+H] +

[0380] 1 H-NMR (400MHz, DMSO-d6): δ14.38(s,1H),8.62(s,1H),7.72(d,J=9.2Hz,1H),7.61(brs,1H),7.38–7.35(m,1H),7.38–7.35(m,1H),7.29(d,J=1. 6Hz,1H),6.67(s,1H),5.56(s,2H),5.43(d,J=1.6Hz,2H),3.85(q,J=6.4 Hz, 2H), 3.58 (t, J = 8.0 Hz, 2H), 1.98–1.80 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H).

[0381] The following compounds were synthesized using suitable raw materials, following the method described in Example 1.1.

[0382]

[0383]

[0384] Example 1.2: Preparation of compound B1.

[0385]

[0386] Steps 1 to 5: Preparation of compound B1-6.

[0387] Compound B1-6 was synthesized using a method similar to that described in steps one through five of Example 1.1.

[0388] Step 6: Preparation of compound B1.

[0389] Compound B1-6 (305 mg, 0.5 mmol) was placed in a 50 mL single-necked flask, dissolved in 15 mL of methanol, and then 3 mL of 0.63 M K2CO3 aqueous solution was added. The mixture was stirred at room temperature for 2 h, and the pH was adjusted to approximately 4 with 5 M dilute hydrochloric acid and stirred for 20 min. The reaction solution was evaporated to dryness, and the crude product was purified by reversed-phase HPLC to obtain compound B1 (4 mg, yield 17.6%).

[0390] MS m / z (ESI): 454.2 [M+H] +

[0391] The following compounds were synthesized using suitable raw materials, following the method described in Example 1.4.

[0392]

[0393]

[0394] Example 2: Preparation of linker intermediates

[0395] Example 2.1: Preparation of intermediate Int1

[0396]

[0397] Compound Int9-1 (500 mg, 1.42 mmol) was dissolved in DMF (5 ml), and HATU (810 mg, 2.13 mmol) and DIEA (549 mg, 4.26 mmol) were added. The mixture was stirred at room temperature for 30 minutes, and then compound Int9-2 (369 mg, 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 Int9 (422 mg, 50% yield).

[0398] The following intermediates were synthesized according to the method of Example 2.1:

[0399]

[0400] Example 3: Preparation of linker-payload for ligand-drug conjugates

[0401] Example 3.1: Preparation of compound AA1

[0402]

[0403] Compounds Int2 (100 mg, 0.28 mmol), A1 (125 mg, 0.28 mmol), HATU (159 mg, 0.42 mmol), and DIPEA (108 mg, 0.84 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 12 h. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate. The liquid phase was separated, dried over anhydrous sodium sulfate, and concentrated to dryness. The crude product was purified by reversed-phase HPLC to give compound AA1 (110 mg, 50% yield).

[0404] MS: [M+H] + , 784.4.

[0405] Example 3.2: Preparation of compound AA15

[0406]

[0407] Step 1: Preparation of compound AA15-1

[0408] Compound A1 (125 mg, 0.28 mmol), compound Int9 (105 mg, 0.28 mmol), HATU (159 mg, 0.42 mmol), and DIPEA (108 mg, 0.84 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 12 h. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate. The liquid phase was separated, dried over anhydrous sodium sulfate, and concentrated to dryness. The crude product was purified by column chromatography to give compound AA15-1 (200 mg, yield 74%).

[0409] Step 2: Preparation of compound AA15-2

[0410] Compound AA15-1 (200 mg, 0.21 mmol) was dissolved in piperidine / dichloromethane (5 mL, 1 / 5) and stirred at room temperature for 1 h. After the reaction was complete, ethyl acetate was added and the mixture was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to give crude compound AA15-2 (168 mg).

[0411] Step 3: Preparation of compound AA15-3

[0412] Compound AA15-2 (168 mg, 0.21 mmol), 2-(methanesulfonyl)pyrimidine-5-carboxylic acid (42 mg, 0.21 mmol), and HATU (121 mg, 0.32 mmol) were dissolved in anhydrous DMF (5 mL) and stirred at room temperature for 12 h. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate. The liquid phase was separated, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was purified by reversed-phase HPLC to give compound AA15-3 (50 mg, yield 24%).

[0413] Step 4: Preparation of compound AA15

[0414] Compound AA15-3 (50 mg, 0.05 mmol) was dissolved in TFA / dichloromethane (3 mL, 1 / 5) and stirred at room temperature for 1 h. After the reaction was complete, the solution was concentrated to dryness, and the residue was purified by reversed-phase HPLC to give compound AA15 (5 mg, 10% yield). MS: [M+H] + , 932.3.

[0415] The following compounds were synthesized according to the methods of Examples 3.1 and 3.2.

[0416]

[0417]

[0418] Example 3.3: Preparation of compound BB1

[0419]

[0420] Step 1: Preparation of compound B1-1

[0421] Compound B1 (500 mg, 1.150 mmol), [[2-(FMOC-amino)acetamido]methyl acetate (2.12 g, 5.75 mmol), and p-toluenesulfonic acid (20 mg, 0.115 mmol) were dissolved in DMAc (10 mL), and the mixture was heated to 50 °C and reacted for 17 h. After the reaction was complete as detected by LCMS, the mixture was cooled to room temperature, and dichloromethane and water were added. After stirring, the mixture was separated into liquid and liquid phases. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by reversed-phase HPLC to obtain compound B1-1 (600 mg, 70% yield).

[0422] Step 2: Preparation of compound B1-2

[0423] Compound B1-1 (600 mg, 0.80 mmol) was dissolved in piperidine / dichloromethane (10 mL, 1 / 5) and stirred at room temperature for 1 h. After the reaction was complete, ethyl acetate and water were added, the mixture was separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to give crude compound B1-2 (420 mg).

[0424] Step 3: Preparation of compound BB1

[0425] Compound BB1 was synthesized according to the method in Example 3.1. MS: [M+H] + , 980.4.

[0426] Example 3.4: Preparation of compound BB17

[0427]

[0428] Step 1: Preparation of compound BB17-1

[0429] Compounds Int15 (200 mg, 0.41 mmol), B1-2 (215 mg, 0.41 mmol), HATU (233 mg, 0.62 mmol), and DIPEA (158 mg, 1.23 mmol) were dissolved in DMF (8 mL) and stirred at room temperature for 12 h. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate. The liquid phase was separated, dried over anhydrous sodium sulfate, and concentrated to dryness. The crude product was purified by column chromatography to give compound BB17-1 (300 mg, 73% yield).

[0430] Step 2: Preparation of compound BB17

[0431] Compound BB17 was synthesized according to the method described in steps 2 and 3 of Example 3.2. MS: [M+H] + , 1084.4.

[0432] The following compounds were synthesized according to the methods described in Examples 3.3 and 3.4.

[0433]

[0434] Example 4: Preparation of ligand-drug conjugates

[0435] 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. Trastuzumab and Sacituzumab are examples of ligand-drug conjugates prepared.

[0436] The amino acid sequence of trastuzumab:

[0437] Light chain (SEQ ID NO:10)

[0438]

[0439] Heavy chain (SEQ ID NO:9)

[0440]

[0441] The amino acid sequence of cetozumab:

[0442] Light chain (SEQ ID NO:20)

[0443]

[0444] Heavy chain (SEQ ID NO:19)

[0445]

[0446] Example 4.1: Preparation of ADC-1A

[0447]

[0448] At 37°C, prepared tris(2-carbonylethyl) phosphate hydrochloride (10 mM, 0.135 mL, 1.35 μmol) was added to the trastuzumab antibody buffer (14.0 mM succinate-sodium hydroxide + 108 mM NaCl pH 6.0; 20 mg, 10.0 mg / mL, 0.135 μmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours. After the reaction was stopped, excess TCEP was removed by ultrafiltration using 14.0 mM succinate-sodium hydroxide + 108 mM NaCl pH 6.0 buffer.

[0449] Compound AA1 (1.29 mg, 1.65 μmol) was dissolved in 0.2 mL of 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-1A (20 mM histidine-hydrochloric acid, pH 5.5; 18.4 mg, 4.96 mg / mL, yield: 92%), which was stored at 4 °C.

[0450] LC-MS analysis and calculation yielded a DAR value of q = 7.82.

[0451] The following compounds were synthesized using a suitable linker-payload, following the method described in Example 4.1.

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459] Example 5: In vitro test of the compound's inhibitory effect on tumor cell proliferation

[0460] Test objective

[0461] To detect the inhibitory activity of the drug compound on the in vitro proliferation of NCI-N87 cells, JIMT-1 cells, and MBA-MB-231 tumor cells, cells were treated with different concentrations of the compound 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 is used to evaluate the in vitro activity of the compound.

[0462] Experimental methods

[0463] The following example, using the in vitro proliferation inhibition assay method for NCI-N87 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.

[0464] 1. Cell culture: NCI-N87 cells were cultured in 10% FBS RPMI-1640 medium.

[0465] 2. Cell preparation: Take NCI-N87 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 prepare a single-cell suspension.

[0466] 3. Cell plating: Mix the NCI-N87 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 9-6 well cell culture plate. Incubate the plates in an incubator for 18 hours (37°C, 5% CO2).

[0467] 4. Compound preparation: Dissolve the compound in DMSO to prepare a stock solution with an initial concentration of 10 mM.

[0468] There are eight concentrations of small molecule compounds: 300, 100, 30, 10, 3, 1, 0.3, and 0.1 nM.

[0469] 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).

[0470] 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.

[0471] 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.

[0472] Data Analysis

[0473] Use Microsoft Excel and Graphpad Prism 5 to process and analyze the data.

[0474] Table 3. IC50 of the small molecule fragment in this application on the in vitro proliferation inhibition of NCI-N87 cells. 50 value.

[0475]

[0476] Conclusion: According to the results in Table 3, the small molecule fragment in this application has significant inhibitory activity against the proliferation of NCI-N87 cells, JIMT-1 cells, and MDA-MB-231 cells.

[0477] Example 6: In vitro cell proliferation inhibition activity test of antibody-drug conjugates

[0478] Implementation: 6.1: In vitro proliferation inhibition activity test of NCI-N87 / JIMT-1 cells

[0479] use Chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effect of anti-Her2 antibody trastuzumab conjugated with camptothecin on cell proliferation in Her2-positive human gastric cancer cells NCI-N87 and human breast cancer cells JIMT-1 after incubation for 6 days.

[0480] 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. Complete culture medium was used as a blank control, and 3 replicates were set up. The plates were incubated at 37°C with 5% CO2 for 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%.

[0481] Experimental conclusion: The antibody-drug conjugate of this application has significant inhibitory activity against the proliferation of Her2-positive human gastric cancer cells NCI-N87 and human breast cancer cells JIMT-1.

[0482] Example 7: Intra-tumor inhibition test of antibody-drug conjugates

[0483] To evaluate the inhibitory effect of the ADC drug of the present invention on tumor formation in vivo, the antitumor effect of the ADC drug of the present invention was evaluated after Her2-positive human breast cancer cells JIMT-1 were used to form xenografts in mice.

[0484] Evaluation of the efficacy of antibody-drug conjugates against human breast cancer cell line JIMT-1 in tumor-bearing mice

[0485] 1. Test drug and materials

[0486] Blank control group (control group): physiological saline

[0487] ADC (treatment group): 5 mg / kg, single dose

[0488] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0489] 3. Experimental animals: 8-week-old female BALB / c-nude mice, purchased from Jicui Pharmaceutical Biotechnology Co., Ltd.

[0490] 4. Test methods:

[0491] 1×10 7 One JIMT-1 cell was subcutaneously injected into the right anterior scapula of an 8-week-old female BALB / c-nude mouse. When the tumor grew to approximately 125 mm... 3 Tumor-bearing mice were randomly assigned to StudyDirector™ groups and administered the ADC drug of this invention intravenously (iv) starting on day 0, once every 7 days for a total of 2 injections, at a dose of 5 mg / kg. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0492] Five mice were used in each of the solvent control group and the treatment group. The tumor inhibition rate was calculated by measuring tumor volume. Tumor inhibition rate (TGI%) = 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).

[0493] Experimental results show that the antibody-drug conjugate of the present invention exhibits significant antitumor activity after a single dose.

[0494] Example 8: Plasma stability test of antibody-drug conjugates

[0495] To evaluate the plasma stability of the antibody-drug conjugate of the present invention, the antibody-drug conjugate of the present invention was incubated in human, rat, and monkey plasma for 21 days. Samples were taken at 0 hours, 8 hours, 1 day, 4 days, 7 days, 14 days, and 21 days to detect the drug ligand conjugation rate (DAR value) and free drug.

[0496]

[0497] Incubation of antibody-drug conjugates in plasma

[0498] The antibody-drug conjugate of this invention was diluted with plasma to a final concentration of 150 μg / mL and incubated at 37°C in the dark for 21 days. Samples were collected at T0 (the sample was diluted within 30 minutes and immediately frozen to -70°C), 2 hours, 8 hours, day 1 (24 hours), day 4, day 7, day 14, and day 21. All samples were stored at -70°C before analysis. Only the DAR value of samples collected at 2 hours and 8 hours was analyzed.

[0499] LC-MS / MS analysis of free drug

[0500] Protein precipitation

[0501] Thaw the plasma samples and take 291.9 μL for free drug analysis. Add 400 μL of precipitant (containing 0.1% formic acid, 200 ng / mL tolbutamide, and 200 ng / mL labetalol in acetonitrile solution) to each sample and mix thoroughly. Shake the mixture for at least 20 minutes to precipitate proteins in the plasma. Centrifuge the samples at 4°C and 4000 rpm for 20 min, and take 150 μL of the supernatant for LC-MS / MS analysis.

[0502] The equipment used for free drug analysis is shown in the table below:

[0503]

[0504] The LC and MS parameters are as follows:

[0505]

[0506]

[0507] LC-MS determination of drug ligand coupling rate

[0508] Immobilized magnetic beads capture antibody-drug conjugates

[0509] Add 25 μL of streptavidin magnetic beads to each well of a 96-well plate and discard the stock buffer. Elute with 200 μL of PBS buffer and treat with 80 μL of biotin-labeled antigen protein and another 100 μL of PBS buffer for 120 min at room temperature. After complete immobilization of the antigen protein, elute twice with 200 μL of PBS buffer. Add 20 μL of antibody-drug conjugate plasma sample and 180 μL of PBS buffer to each well, then shake for 120 min at room temperature to ensure complete capture of the antibody-drug conjugate in the plasma, and then remove the supernatant. Wash twice with PBS buffer, then add 50 μL of elution buffer (1% formic acid aqueous solution) and treat for 20 min at room temperature. Then add 5 μL of neutralization buffer (1M NH4HCO3, pH 8.5) and 5 μL of DTT (1M) to allow the captured antibody-drug conjugate to regenerate at room temperature for 60 min, and then analyze by LC-MS.

[0510] Drug ligand coupling rate analysis

[0511] The LC-MS parameters are as follows:

[0512]

[0513]

[0514] The results showed that after 21 days of incubation in plasma, the antibody-drug conjugate of the present invention showed no or only trace amounts of drug detachment, and the drug ligand conjugation rate did not change significantly. The antibody-drug conjugate of the present invention has extremely high plasma stability, suggesting that the antibody-drug conjugate of the present invention has better safety.

[0515] Example 9: Stability of Antibody-Drug Conjugate Stock Solution (Liquid Formulation)

[0516] To evaluate the stability of the antibody-drug conjugate stock solution of the present invention, the ADC stock solution of the present invention was concentrated to 20 mg / mL, aliquoted into 2 mL cryovials, and incubated in a 40°C incubator. Samples were taken at 0 hours, 1 week, 2 weeks, 1 month, and 2 months to detect concentration, drug-ligand conjugation ratio (DAR value), SEC purity and aggregation, CE-SDS (non-reducing) purity, CE-SDS (reducing) purity, charge heterogeneity, and free drug. At the same time, the appearance of the liquid was visually observed to see if it was clear and whether any precipitation had formed.

[0517] The UV method was used to detect the sample concentration.

[0518] The drug ligand coupling rate (DAR value) was determined using hydrophobic HPLC (HIC) or reversed-phase HPLC (RP-HPLC).

[0519] The drug ligand conjugation ratio (DAR value) was determined using LC-MS.

[0520] The free drug was detected by reversed-phase HPLC (RP-HPLC).

[0521] The purity and aggregation degree of the SEC in the samples were determined by SEC-HPLC (TOSOH G3000 SW SEC column).

[0522] The purity of CE-SDS (non-reduced) and CE-SDS (reduced) was determined using Maurice.

[0523] Charge heterogeneity was detected using the imaging capillary isoelectric focusing electrophoresis (iCIEF) method.

[0524] The results showed that after incubation at 40°C for 1 week to 2 months, the antibody-drug conjugate stock solution of the present invention exhibited no significant changes in concentration, drug-ligand conjugation ratio (DAR), SEC purity and aggregation degree, CE-SDS (non-reducing) purity, CE-SDS (reducing) purity, charge heterogeneity, and free drug. The antibody-drug conjugate stock solution of the present invention possesses extremely high stability.

[0525] Example 10: Pharmacokinetic and toxicological studies of monkeys after single or multiple administrations

[0526] After a single or multiple intravenous infusions of the antibody-drug conjugate of the present invention into monkeys, the pharmacokinetic properties of the drug in the monkeys were investigated, and the toxic effects on the animals were observed.

[0527] Test methods

[0528] Pharmacokinetics: After a single intravenous infusion of different doses of the antibody-drug conjugate of this invention into monkeys, blood samples were collected at multiple consecutive time points, and the concentration of the drug in the blood was detected by appropriate specific detection methods.

[0529] Toxicological studies: After monkeys received single or multiple intravenous infusions of different doses of the antibody-drug conjugate of the present invention, the animals' tolerance to the antibody-drug conjugate of the present invention and drug-related toxicity were investigated through multiple aspects, including clinical observation, body weight and food intake, hematology, blood biochemistry, urine, gross anatomy, and histopathology.

[0530] The results showed that after single or multiple intravenous infusions of the antibody-drug conjugate of the present invention in monkeys, the pharmacokinetic properties of the total antibody and the ADC were similar, and the concentration of free toxin was very low, indicating that the antibody-drug conjugate of the present invention has high stability and good pharmacokinetic characteristics in vivo. The animals tolerated it well and did not exhibit serious or intolerable drug-related toxicity, indicating that the safety of the antibody-drug conjugate of the present invention is controllable.

Claims

1. A compound, or a medicinally usable salt thereof, wherein, The compound is .

2. A compound, or a medicinally usable salt thereof, wherein, The compound is selected from 3. A ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from: Where Ab is an antibody or its antigen-binding fragment; q represents the drug loading, which can be an integer or decimal from 1 to 16.

4. The ligand-drug conjugate according to claim 3, or a pharmaceutically usable salt thereof, wherein the Ab is selected from humanized antibodies and multispecific antibodies.

5. The ligand-drug conjugate according to claim 3, or a pharmaceutically usable salt thereof, wherein Ab is selected from fully human antibodies, chimeric antibodies, bispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

6. The ligand-drug conjugate of claim 3, or a pharmaceutically usable salt thereof, wherein the antigen-binding fragment is selected from Fab, F(ab')2, Fv, scFv, Fd, dAb, VHH, and complementarity-determining region (CDR) fragments.

7. The ligand-drug conjugate according to claim 3, or a pharmaceutically usable salt thereof, wherein Ab is an antibody or antigen-binding fragment that specifically binds to HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70 or EGFR.

8. The ligand-drug conjugate of claim 7, or a pharmaceutically usable salt thereof, wherein Ab is selected from trastuzumab and pertuzumab or variants thereof.

9. The ligand-drug conjugate according to any one of claims 3-8, or a pharmaceutically acceptable salt thereof, wherein, q is an integer or decimal selected from 2 to 8.

10. The ligand-drug conjugate according to any one of claims 3-8, or a pharmaceutically acceptable salt thereof, wherein, q is an integer or decimal selected from 4 to 8.

11. The ligand-drug conjugate according to claim 3, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from: Where q is as defined in claim 3.

12. A pharmaceutical composition comprising a ligand-drug conjugate or compound as described in any one of claims 1-11, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

13. A pharmaceutical formulation comprising a ligand-drug conjugate or compound as described in any one of claims 1-11, or a pharmaceutically acceptable salt thereof.

14. Use of the ligand-drug conjugate or compound of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 12 or the pharmaceutical formulation of claim 13, in the preparation for the prevention or treatment of diseases associated with abnormal cellular activity.

15. The use according to claim 14, wherein the disease associated with abnormal cell activity is cancer.

16. The use according to claim 15, wherein the cancer is a solid tumor or a non-solid tumor.

17. The use according to claim 16, wherein the cancer is selected from esophageal cancer, brain tumor, 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, or prostate cancer.

18. The use according to claim 16, wherein the cancer is selected from esophageal adenocarcinoma, esophageal squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, or sarcoma.

19. The use according to claim 16, wherein the cancer is selected from thyroid cancer and glioblastoma multiforme.

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