Camptothecin compound, conjugate thereof, preparation method and application thereof

Abstract

The invention relates to a camptothecin compound and a conjugate thereof, or a tautomer, an enantiomer and a diastereoisomer thereof, or a mixture form thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof, and a preparation method and application thereof. The camptothecin compound can be used for treating proliferative diseases related to abnormal cell activity.

Description

Camptothecin compounds and conjugates thereof, preparation methods and uses thereof Technical Field

[0001] The present invention belongs to the field of medical technology and relates to camptothecin compounds and conjugates thereof, their preparation methods and uses in preventing and / or treating proliferative diseases associated with abnormal cell activity (including but not limited to tumor diseases). Background Art

[0002] Camptothecin (CPT) is a cytotoxic alkaloid isolated from the plant Camptotheca acuminata of the Davidiaceae family. It can form a ternary complex with cellular DNA topoisomerase I, thereby inhibiting DNA unwinding, leading to blocked DNA replication and, in turn, cell death (Cancer Res. 1989, 49, 6365). It has a broad spectrum of antiproliferative activity. However, due to its low solubility, instability, acquired tumor cell resistance and significant toxicity, it is not suitable for clinical development. Camptothecin derivatives can increase their water solubility by introducing water-soluble groups or preparing prodrugs, thereby improving their drugability. Several camptothecin derivatives with significantly improved solubility have been approved for marketing (Med. Res. Rev. 2015, 35, 753), such as topotecan, irinotecan and belotecan, for the treatment of various types of cancer.

[0003] Camptothecin derivatives are also used to conjugate with antibodies as small molecule toxins, also known as payloads, in antibody-drug conjugates (ADCs). 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 while minimizing the effects 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, in which the camptothecin analog dxd is conjugated to the anti-HER2 antibody trastuzumab via a cleavable tetrapeptide-based linker, and Immu-132, in which the camptothecin analog SN-38 is conjugated to the anti-Trop-2 antibody satuzumab via a hydrolyzable, pH-sensitive linker.

[0004] However, ADCs using camptothecins or their derivatives as toxins generally have a large drug-to-antibody ratio (DAR), are difficult to manufacture, and can easily lead to ADC instability. Furthermore, camptothecin compounds often exhibit hematotoxicity, such as neutropenia, leukopenia, thrombocytopenia, and anemia, due to myelosuppression, as well as gastrointestinal side effects such as nausea, vomiting, and diarrhea.

[0005] Therefore, there is still a high clinical demand and application value in developing camptothecin compounds and their conjugates with novel structures that can enhance effectiveness and improve safety issues.

[0006] Summary of the Invention

[0007] The present application provides a camptothecin compound or its tautomer, enantiomer, diastereoisomer, or mixture form, or its pharmaceutically acceptable salt or solvate, which may have one or more effects selected from the following groups: (1) having inhibitory activity on the proliferation of tumor cells in vitro; (2) having plasma stability; (3) having an in vivo tumor-suppressing effect; (4) having a bystander killing effect; (5) having anti-transporter transport ability; (6) having in vivo tumor targeting ability; and (7) having good in vivo safety.

[0008] In one aspect, the present application provides a compound represented by formula (I), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0009] in

[0010] R 1 、R 2 and R 3 are independently selected from hydrogen, halogen, hydroxy, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 alkoxy;

[0011] or R 1 With R 2 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring;

[0012] or R 1 With R 3 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring;

[0013] The condition is R 1 、R 2 and R 3 Not hydrogen at the same time.

[0014] In some embodiments, the present application provides a compound represented by formula (Ia), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0015] in,

[0016] R 1a and R 2a are independently selected from hydrogen, halogen, hydroxy, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 alkoxy;

[0017] or R 1a With R 2a and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring;

[0018] And R 1a and R 2a Not hydrogen at the same time.

[0019] In some embodiments, a compound of the structure shown in formula (Ia) is provided, wherein R 1a Selected from hydrogen, halogen, hydroxy, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy.

[0020] In some embodiments, a compound of the structure shown in formula (Ia) is provided, wherein R 1a is selected from hydrogen, halogen, methyl, methoxy, -OCF3 or -CF3; preferably, R 1a is selected from hydrogen, F, Cl, methyl- and -CF3; further preferably, R 1a is selected from hydrogen, F and methyl.

[0021] In some embodiments, a compound of the structure shown in formula (Ia) is provided, wherein R 2a Selected from hydrogen, halogen and -C 1-6 Alkyl; preferably, R 2a is selected from hydrogen, F or Cl; further preferably, R 2a is selected from hydrogen or F.

[0022] In some embodiments, a compound of the structure shown in formula (Ia) is provided, wherein R 2a is selected from hydrogen or halogen; preferably, R 2a is selected from hydrogen, F or Cl.

[0023] In some embodiments, a compound of the structure shown in formula (Ia) is provided, wherein R 1a With R 2a and the atoms to which it is attached together form a 5-membered heterocyclic ring containing one or two heteroatoms independently selected from O and N; preferably, the 5-membered heterocyclic ring is selected from Further preferably, the 5-membered heterocycle is

[0024] In some embodiments, the compound represented by formula (Ia) is selected from:

[0025] Preferably, the compound represented by formula (Ia) is selected from:

[0026] In other embodiments, the present application provides a compound represented by formula (Ib), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0027] in,

[0028] R 1b Selected from hydrogen, halogen, -C 1-6 Alkyl, -C 1-6 alkoxy;

[0029] R 2b Selected from halogen, -C 1-6 Alkyl, -halogenated C 1-6 alkyl.

[0030] In some embodiments, a compound of the structure shown in formula (Ib) is provided, wherein R 1b Selected from hydrogen and F.

[0031] In some embodiments, a compound of the structure shown in formula (Ib) is provided, wherein R 2b Selected from F, Cl, methyl and -CF3.

[0032] In some embodiments, the compound represented by formula (Ib) is selected from:

[0033] Preferably, the structural compound represented by formula (Ib) is selected from:

[0034] In another aspect, the present application provides a compound represented by formula (II), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0035] in,

[0036] L 1 is a connection unit;

[0037] L 2 -(C(RL 21 )2) n -,

[0038] Where n is a natural number from 0 to 50.

[0039] L 2 Any C(R L21 )2 units 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-,

[0040] -Cy- is selected from phenylene, 5 to 8 membered heteroarylene, 3 to 10 membered heterocyclylene, or 3 to 10 membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted by one or more R cx replace,

[0041] R L21 、R L22 、R cx Each independently selected from hydrogen, deuterium, halogen, -NO2, -CN, -OR L2a 、-SR L2a 、-N(R L2a )2、-N + (R L2a )3, -C(O)R L2a 、-CO2R L2a 、-C(O)C(O)R L2a 、-C(O)CH2C(O)R L2a 、-S(O)R L2a 、-S(O)2R L2a 、-C(O)N(R L2a )2、-SO2N(R L2a )2、-OC(O)R L2a 、-N(R L2a )SO2R L2b 、-N(R L2a )COR L2b 、-(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a 、-(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a 、-(CH2) y-CO-(N(Me)CH2C(O)) m -N + (R L2a )3, -(CH2) y -NHCOCH2(OCH2CH2)OR L2a 、-(CH2) y -NH(COCH2(N(Me)) m -R L2a 、-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-(CH2) y -NHCO-(CH2CH2O) m -R L2a 、-(CH2CH2O) m -R L2a 、-(COCH2N(Me)) m -R L2a 、-COCH2(OCH2CH2) m -OR L2a 、-CO-(CH2CH2O) m -R L2a 、-CO-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-CO-(CH2) y -NHCO-(CH2CH2O) m -R L2a Or by R L2a Optionally substituted -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,

[0042] m, y are natural numbers from 0 to 50,

[0043] R L2a 、R L2b Each independently selected from hydrogen, deuterium, halogen, -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;

[0044] L 3 Does not exist or is an amino acid residue, a short peptide consisting 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;

[0045] Tr does not exist or is or any combination of the above groups;

[0046] R Tr 、R Tr1 and R Tr2 Each independently selected from hydrogen, deuterium, halogen, -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 by R Tra Optionally substituted -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,

[0047] R Tra For hydrogen, deuterium, halogen, -NO2, -CN, -OH, -SH, -NH2, -N(Me)2, -S(O)2Me, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -C 1-6 Alkyl, -C 1-6 Alkenyl, -C 1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl,

[0048] z is a natural number 0 or greater;

[0049] where R 1 、R 2 and R 3 As defined for the compounds of formula (I).

[0050] In one embodiment, a compound of the structure shown in formula (IIa), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof is provided:

[0051] Among them L 1 , L 2 , L 3 Tr is as defined for the compound of formula (II), R 1a 、R 2a As defined for the compound of formula (Ia).

[0052] In one embodiment, a compound of the structure shown in formula (IIb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof is provided:

[0053] Among them, L 1 , L 2 , L 3 Tr is as defined for the compound of formula (II), R 1b 、R 2b As defined for the compound of formula (Ib).

[0054] In some embodiments, there are provided compounds of formula (II), formula (IIa) and formula (IIb), wherein L 1 Selected from:

[0055] In some embodiments, there are provided compounds of formula (II), formula (IIa) and formula (IIb), wherein L 1 Selected from: Preferably, L 1 Selected from

[0056] In some embodiments, compounds of formula (II), formula (IIa) and formula (IIb) are provided, wherein L 2 -(CHR L21 ) n -;

[0057] Wherein, n is a natural number from 0 to 50;

[0058] L 2 Any CHR in L21 The units can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -、-O-、

[0059] -Cy- is phenylene, 5- to 6-membered heteroarylene, 4- to 10-membered heterocyclylene, or 3- to 6-membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted by 1 to 3 R cx replace;

[0060] Each R L21 、R L22 、R cx are each independently selected from hydrogen, halogen, -OR L2a 、-N(R L2a )2、-C(O)R L2a 、-S(O)2R L2a 、-C(O)N(R L2a )2、-SO2N(R L2a )2、-N(R L2a )SO2R L2b 、-N(R L2a )COR L2b 、-(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a 、-(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a 、-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-(CH2) y -NHCO-(CH2CH2O) m -R L2a 、-(CH2) y -NHCOCH2(OCH2CH2)OR L2a 、-(CH2) y -NH(COCH2(N(Me)) m -R L2a 、-(CH2) y -NHCO-(CH2CH2O) m -R L2a 、-(CH2CH2O) m -R L2a 、-(COCH2N(Me)) m -R L2a 、-COCH2(OCH2CH2) m -OR L2a 、-CO-(CH2CH2O) m -R L2a , or by R L2a Optionally substituted -C 1-6Alkyl, -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;

[0061] m is an integer from 0 to 8;

[0062] y is 0, 1, 2, 3, or 4;

[0063] Each R L2a 、R L2b Each is independently selected from hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2 and -C 1-6 alkyl.

[0064] In some embodiments, L 2 -(CH2) n -;

[0065] Wherein, n is a natural integer from 0 to 50;

[0066] L 2 Any methylene unit in the group may be independently replaced by the following structural units: 4 to 6 membered heterocyclyl, 3 to 6 membered cycloalkyl, -C(O)-, -NR L22 -、-O-、

[0067] Each R L22 are each 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 Optionally substituted -C 1-6 alkyl;

[0068] m is a natural integer from 0 to 8;

[0069] y is 0, 1, 2, 3, or 4;

[0070] Each R L2a Each is independently selected from hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2 and -C 1-6 alkyl.

[0071] In some embodiments, compounds of the structures shown in formula (II), formula (IIa) and formula (IIb) are provided, wherein L 2 Selected from:

[0072] in,

[0073] n1, n2, n3, n4, and m are each independently selected from natural numbers from 0 to 8;

[0074] n5 and n6 are each independently selected from 0 or 1;

[0075] -Cy- is a 4- to 6-membered heterocyclylene or a 3- to 6-membered cycloalkylene; preferably, -Cy- is More preferably, -Cy- is

[0076] More preferably,

[0077] for

[0078] More preferably,

[0079] for Among them, c and L 1 Connected, d and L 3 connected.

[0080] In some embodiments, compounds of the structures shown in formula (II), formula (IIa) and formula (IIb) are provided, wherein Selected from:

[0081] n1, n2, n3, n4, and m are each independently selected from natural numbers from 0 to 8;

[0082] n5 and n6 are each independently selected from 0 or 1;

[0083] -Cy- is a 4- to 6-membered heterocyclylene or a 3- to 6-membered cycloalkylene; preferably, -Cy- is selected from: More preferably, -Cy- is selected from

[0084] In some embodiments, compounds of the structures shown in Formula (II), Formula (IIa) and Formula (IIb) are provided, wherein: Selected from: More preferably, Selected from:

[0085] In some embodiments, compounds of formula (II), formula (IIa) and formula (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-Lys, 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-A la-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-G ly, 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 fragments.

[0086] In some embodiments, 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 fragments; 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 fragments; More preferably, L 3Selected from Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asp-Asp, Asp-Glu, Gly-Gly-Phe-Gly or

[0087] In some embodiments, compounds of the structures shown in Formula (II), Formula (IIa) and Formula (IIb) are provided, wherein: Selected from:

[0088] In some embodiments, compounds of formula (II), formula (IIa) and formula (IIb) are provided, wherein Tr is absent or is in

[0089] R Tr 、R Tr1 and R Tr2 independently selected from hydrogen, halogen, -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;

[0090] z is a natural number from 0 to 8.

[0091] In some embodiments, compounds of formula (II), formula (IIa) and formula (IIb) are provided, wherein Tr is absent or is

[0092] In some embodiments, the compound represented by the structure of formula (IIa) is selected from:

[0093] Preferably, the compound represented by formula (IIa) is selected from:

[0094] In some embodiments, the compound represented by formula (IIb) is selected from:

[0095] In another aspect, the present 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 a structure represented by formula (IV):

[0096] Among them, R 1 、R 2 and R 3 As defined for compounds of formula (I), the wavy line indicates direct or indirect attachment to the ligand via a nitrogen atom.

[0097] In one embodiment, the present 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 a structure represented by formula (IVa):

[0098] Among them, R 1a 、R 2a As defined for compounds of formula (Ia), the wavy line indicates direct or indirect attachment to the ligand via a nitrogen atom.

[0099] In one embodiment, the present 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 a structure represented by formula (IVb):

[0100] Among them, R 1b 、R 2b As defined in formula (Ib), the wavy line indicates direct or indirect attachment to the ligand via a nitrogen atom.

[0101] In another aspect, the present 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 a structure represented by formula (V):

[0102] in,

[0103] The wavy line indicates the L 1a The nitrogen or carbon atom on the group is connected to the ligand;

[0104] L 1a Selected from:

[0105] L 2 , L3 , Tr is as defined for the compound of formula (II);

[0106] R 1 、R 2 and R 3 As defined for the compounds of formula (I).

[0107] In one embodiment, the present 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 a structure represented by formula (Va):

[0108] in,

[0109] The wavy line indicates the L 1a The nitrogen or carbon atom on the group is connected to the ligand;

[0110] L 1a As defined in the compound of formula (V);

[0111] L 2 , L 3 , Tr is as defined for the compound of formula (II);

[0112] R 1a 、R 2a As defined for the compound of formula (Ia).

[0113] In one embodiment, the present 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 a structure represented by formula (Vb):

[0114] in,

[0115] The wavy line indicates the L 1a The nitrogen or carbon atom on the group is connected to the ligand;

[0116] L 1a As defined in the compound of formula (V);

[0117] L 2 , L 3 , Tr is as defined for the compound of formula (II);

[0118] R 1b 、R 2b As defined for the compound of formula (Ib).

[0119] In some embodiments, ligand-drug conjugates comprising structures represented by formula (V), formula (Va), and formula (Vb) are provided, wherein L 1a Selected from: Preferably, L 1a Selected from

[0120] In some embodiments, ligand-drug conjugates comprising structures represented by Formula (V), Formula (Va), and Formula (Vb) are provided, wherein Selected from:

[0121] n1, n2, n3, n4, and m are each independently selected from natural numbers from 0 to 8;

[0122] n5 and n6 are each independently selected from 0 or 1;

[0123] -Cy- is a 4- to 6-membered heterocyclylene group or a 3- to 6-membered cycloalkylene group; preferably, -Cy- is selected from: More preferably, -Cy- is selected from

[0124] In some embodiments, ligand-drug conjugates comprising structures represented by Formula (V), Formula (Va), and Formula (Vb) are provided, wherein: Selected from: More preferably, Selected from:

[0125] In some embodiments, ligand-drug conjugates comprising structures represented by Formula (V), Formula (Va), and Formula (Vb) are provided, wherein: Selected from:

[0126] In another aspect, the present application provides a ligand-drug conjugate represented by formula (III), or its tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0127] in,

[0128] Ab is the ligand that binds to the target;

[0129] q is the drug loading capacity, which is an integer or decimal from 1 to 16;

[0130] L1a As defined in formula (V);

[0131] L 2 , L 3 and Tr are as defined for the compound of formula (II);

[0132] R 1 、R 2 and R 3 As defined for the compounds of formula (I).

[0133] In one embodiment, a ligand-drug conjugate of formula (IIIa) is provided, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0134] wherein Ab and q are as defined in formula (III), L 1a As defined in formula (V), L 2 , L 3 and Tr are as defined for the compound of formula (II), and R 1a 、R 2a As defined for the compound of formula (Ia).

[0135] In one embodiment, a ligand-drug conjugate of the structure represented by formula (IIIb) is provided, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0136] wherein Ab and q are as defined in formula (III), L 1a As defined in formula (V), L 2 , L 3 and Tr are as defined for the compound of formula (II), and R 1b 、R 2b As defined for the compound of formula (Ib).

[0137] In some embodiments, ligand-drug conjugates of structures represented by Formula (III), Formula (IIIa), and Formula (IIIb) are provided, wherein Ab is a target-binding polypeptide, antibody, or antigen-binding fragment thereof.

[0138] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof.

[0139] In some embodiments, the antibody is selected from the group consisting of: a fully human antibody, a humanized antibody, a chimeric antibody, a probody, a bispecific antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0140] In some embodiments, the antigen binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, scFv, diabody, Fd, dAb, VHH, big antibody, and complementarity determining region (CDR) fragment.

[0141] In some embodiments, Ab is a monoclonal antibody.

[0142] In some embodiments, the Ab specifically binds an antigen selected from the group consisting of 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, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, 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, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9Epidermal growth factor, brevican, mesothelin, sodium phosphate cotransporter 2B, claudin 18.2, endothrin receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α4β6, trophoblast glycoprotein, or tissue factor.

[0143] In some preferred embodiments, the Ab specifically binds to an antigen selected from the group consisting of HER2, HER3, B7H3, TROP2, Claudin 18.2, CD30, CD33, CD70, or EGFR.

[0144] 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, for example, trastuzumab or pertuzumab or its variant.

[0145] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each of which has one, two, three, or four amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following molecules, respectively: trastuzumab or pertuzumab.

[0146] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an 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%, or about 99% sequence identity with the heavy chain variable region and the light chain variable region of the following molecules, respectively: Trastuzumab or Pertuzumab.

[0147] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain of an antibody, wherein the heavy chain and the light chain have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to the heavy chain and light chain of the following molecules, respectively: Trastuzumab or Pertuzumab.

[0148] Sequences of exemplary anti-HER2 antibodies or antigen-binding fragments thereof are provided in Table 1.

[0149] Table 1: Trastuzumab amino acid sequence

[0150] In some embodiments, the antibody or antigen-binding fragment that specifically binds to TROP2 is an anti-Trop-2 antibody or antigen-binding fragment, for example, sacituzumab or a variant thereof.

[0151] In some embodiments, the anti-Trop-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, each of which has one, two, three, or four amino acid changes (e.g., amino acid substitutions or deletions) compared to one or more CDRs in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 of the following molecule: sacituzumab.

[0152] In some embodiments, the anti-Trop-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an 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%, or about 99% sequence identity to the heavy chain variable region and the light chain variable region of the following molecule, respectively: sacituzumab.

[0153] In some embodiments, the anti-Trop-2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain of an antibody, wherein the heavy chain and the light chain have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to the heavy chain and light chain of the following molecule, respectively: Sacituzumab.

[0154] Sequences of exemplary anti-Trop-2 antibodies or antigen-binding fragments thereof are provided in Table 2.

[0155] Table 2: Certolizumab amino acid sequence

[0156] In some embodiments, the ligand-drug conjugate of formula (IIIa) is selected from:

[0157] wherein Ab and q are as defined in formula (III).

[0158] In a preferred embodiment, the ligand-drug conjugate of the structure represented by formula (IIIa) is selected from:

[0159] wherein Ab and q are as defined in formula (III).

[0160] In a more preferred embodiment, the ligand-drug conjugate of the structure represented by formula (IIIa) is selected from:

[0161] wherein q is as defined in formula (III).

[0162] In some embodiments, the ligand-drug conjugate of formula (IIIb) is selected from:

[0163] wherein Ab and q are as defined in formula (III).

[0164] In some preferred embodiments, the ligand-drug conjugate of formula (IIIb) is selected from:

[0165] wherein q is as defined in formula (III).

[0166] In some embodiments, q is an integer or decimal selected from 2 to 8.

[0167] The present invention also provides a pharmaceutical composition comprising a compound having a structure represented by the above-mentioned formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa) or formula (IIb), a ligand-drug conjugate represented by formula (III), formula (IIIa) or formula (IIb), a ligand-drug conjugate having a structure represented by formula (IV), formula (IVa), formula (IVb), formula (V), formula (Va) or formula (IVb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[0168] The present invention also provides a pharmaceutical preparation comprising a compound having a structure represented by the above-mentioned formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa) or formula (IIb), a ligand-drug conjugate represented by formula (III), formula (IIIa) or formula (IIb), a ligand-drug conjugate comprising a structure represented by formula (IV), formula (IVa), formula (IVb), formula (V), formula (Va) or formula (IVb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0169] The present invention also provides a use of a substance S in the preparation of a drug for preventing or treating cancer; the substance S is a compound having a structure represented by the above-mentioned formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa) or formula (IIb), a ligand-drug conjugate represented by formula (III), formula (IIIa) or formula (IIb), a ligand-drug conjugate comprising a structure represented by formula (IV), formula (IVa), formula (IVb), formula (V), formula (Va) or formula (IVb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, the pharmaceutical composition, or the pharmaceutical preparation. The cancer disease is a solid tumor or a non-solid tumor. For example, selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumor (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0170] The present invention also provides a use of a substance S in the preparation of a drug for preventing or treating a disease associated with abnormal cell activity; the substance S is a compound represented by the structure of formula (I), formula (Ia), formula (Ib), formula (II), formula (IIa), or formula (IIb), a ligand-drug conjugate represented by formula (III), formula (IIIa), or formula (IIb), a ligand-drug conjugate comprising a structure represented by formula (IV), formula (IVa), formula (IVb), formula (V), formula (Va), or formula (IVb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, the pharmaceutical composition, or the pharmaceutical preparation. The disease associated with abnormal cell activity may be cancer. The definition of cancer is as described above.

[0171] Definition of terms:

[0172] Throughout this application, unless otherwise indicated, 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 conventional procedures widely used in the relevant fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0173] In this application, the term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These substances, in addition to the active ingredients, have been reasonably evaluated for safety and are included in pharmaceutical preparations. In addition to providing shape, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, dissolution assistance, and sustained-release control. They are important ingredients that may affect the quality, safety, and efficacy of drugs. Based on their source, they can be divided into natural, semi-synthetic, and fully synthetic. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0174] In this application, the term "pharmaceutical composition" refers to various suitable dosage forms that can be prepared according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of a compound of the present disclosure or a pharmaceutically acceptable salt or conjugate thereof, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5 to 350 mg, and particularly preferably 1-250 mg.

[0175] The pharmaceutical composition can be administered in the form of an injection, including an injection solution, sterile powder for injection, and concentrated solution for injection. Among them, the carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils such as monoglycerides or diglycerides can also be used as solvents or suspending media.

[0176] In this application, the term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" generally refers to a salt of the compound or ligand-drug conjugate of the present application, or a salt of the compound described in the present application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The antibody-antibody drug conjugate compound of the present application may form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 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.

[0177] In this application, the term "solvate" or "solvate compound" generally refers to a pharmaceutically acceptable solvate formed by a compound or ligand-drug conjugate compound of the present application and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0178] The term "drug loading" generally refers to the average amount of cytotoxic drug loaded per ligand and can also be expressed as the ratio of the amount of cytotoxic drug to the amount of antibody, for example, the drug / antibody ratio (DAR). The cytotoxic drug loading can range from 0 to 20 as an integer or decimal. In embodiments of the present 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 characterized by conventional methods such as UV / visible spectroscopy, mass spectrometry, HIC, ELISA assay, and HPLC.

[0179] In this application, the term "ligand-drug conjugate" generally refers to a ligand linked to a biologically active cytotoxic drug via a stable linker. In some embodiments of this application, the "ligand-drug conjugate" may be an antibody-drug conjugate (ADC), which may be a monoclonal antibody or antibody fragment linked to a biologically active cytotoxic drug via a stable linker.

[0180] In this application, the term "ligand" generally refers to small molecules, polypeptides, RNA, DNA, carbohydrates and macromolecular compounds that can recognize and bind to antigens or receptors associated with target cells. The function of the ligand can be to present the drug to the target cell population bound to 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, the ligand can be represented by Ab, and the ligand antigen forms a connection bond with the connecting unit (also called "linker" or "linker") through the heteroatom on the ligand. The ligand can be an antibody or an antigen-binding fragment thereof. The antibody can be selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; 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 can 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, brevican, mesothelin, sodium phosphate cotransporter 2B, Claudin18.2, endothrin receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α4β6, trophoblast glycoprotein, or tissue factor.

[0181] In this application, the term "antibody or its antigen-binding fragment" generally refers to an immunological binder, which extends to all antibodies from all species, including dimers, trimers and multimer antibodies; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and reconstructed antibodies and their fragments. The term "antibody or its fragment" can refer to any antibody-like molecule with an antigen-binding region, and the term includes small molecule fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), linear antibodies, diabodies, etc. The term "antigen-binding fragment" can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. For example, a fragment of a full-length antibody can be used to implement the antigen-binding function of an antibody. The 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, anti-Claudin antibody One or more antibodies selected from 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, may be trastuzumab or pertuzumab.

[0182] 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 reduce the immune response induced by the murine antibody. Methods for creating chimeric antibodies include, for example, establishing a hybridoma that secretes a murine-specific monoclonal antibody, cloning the variable region genes from the murine hybridoma cells, cloning the human antibody constant region genes as needed, and then ligating the murine variable region genes with the human constant region genes to form a chimeric gene, which is then inserted into an expression vector. Chimeric antibody molecules can then be expressed in eukaryotic or prokaryotic systems.

[0183] In this application, the term "humanized antibody", also referred to as CDR-grafted antibody, generally refers to an antibody produced by transplanting mouse CDR sequences into a human antibody variable region framework, i.e., into different types of human germline antibody framework sequences. Humanized antibodies can overcome the problem of chimeric antibodies inducing strong heterologous reactions due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the 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.

[0184] In this application, the terms "fully human antibody", "fully human antibody" or "completely human antibody" are used interchangeably, and both the variable and constant regions of the antibody may be of human origin, eliminating immunogenicity and toxic side effects.

[0185] 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 antibodies or ligands described in this application may be fully human monoclonal antibodies. Relevant technologies for preparing fully human antibodies may include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0186] In this application, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. The most commonly used 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 CDRs can be applied to CDR1, CDR2 and CDR3 of the light chain variable domain (CDRL1, CDRL2, CDRL3 or L1, L2, L3), and CDR1, CDR2 and CDR3 of the heavy chain variable domain (CDR H1, CDRH2, CDRH3 or H1, H2, H3).

[0187] The term "one or more" or the similar expression "at least one" may mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more;

[0188] When the lower limit and upper limit of a numerical range are disclosed, any value and any included range falling within the range are specifically disclosed. In particular, each range of values ​​disclosed herein (in the form "about a to b", or equivalently, "approximately a to b", or equivalently, "about a b") should be understood to mean each value and range encompassed within the broader range;

[0189] For example, the statement "C 1-6 " should be understood to include any sub-ranges therein and each point value, such as C 2-5 、C 3-4 、C 1-2 、C 1-3 、C 1-4 、C 1-5 etc., as well as C1, C2, C3, C4, C5, C6, etc. For example, the expression “C 3-10 ” should also be understood in a similar manner, for example, any sub-ranges and point values ​​contained therein may be included, for example, C 3-9 、C6-9 、C 6-8 、C 6-7 、C 7-10 、C 7-9 、C 7-8 、C 8-9 etc. and C3, C4, C5, C6, C7, C8, C9, C 10 etc. For another example, the expression "3-10 yuan" should be understood to include any sub-ranges and point values ​​therein, 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. For another example, the expression "5-10 yuan" should also be understood in a similar manner, such as it can include any sub-ranges and point values ​​contained therein, 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.

[0190] In this application, the term "alkyl" refers to a saturated straight or branched hydrocarbon group. 1-6 "Alkyl" refers to a saturated straight or branched chain hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5 or 6 carbon atoms). 1-6 "Alkyl" is, 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 the present invention is optionally substituted with one or more substituents described herein.

[0191] In this application, the term "alkylene" refers to a saturated straight-chain or branched divalent hydrocarbon group. 1-6 "Alkylene" refers to a saturated straight or branched divalent hydrocarbon group having 1 to 6 carbon atoms. 1-6 "Alkylene" includes, but is not limited to, methylene, ethylene, propylene or butylene. The alkylene in the present invention is optionally substituted with one or more substituents described in the present invention.

[0192] In this application, the term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more carbon-carbon double bonds. 2-6The term "alkenyl" refers to an alkenyl group having 2 to 6 carbon atoms and one, two or three (preferably one) carbon-carbon double bonds (such as ethenyl, 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.), which is optionally substituted by one or more (such as 1 to 3) substituents described herein.

[0193] In this application, the term "alkenylene" refers to a straight or branched divalent aliphatic hydrocarbon group having one or more carbon-carbon double bonds, and the two groups (or fragments) connected thereto may be connected to the same carbon atom or to different carbon atoms. For example, the term "C 2-6 "Alkenylene" refers to an alkenylene group having 2 to 6 carbon atoms (e.g. , etc.), which are optionally substituted with one or more (e.g., 1-3) substituents described herein.

[0194] In this application, the term "alkynyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more carbon-carbon triple bonds. 2-6 The term "alkynyl" refers to an alkynyl group having 2 to 6 carbon atoms and one, two or three (preferably one) carbon-carbon triple bonds (e.g., 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 with one or more (e.g., 1 to 3) substituents described herein.

[0195] In this application, the term "alkynylene" refers to a straight or branched divalent aliphatic hydrocarbon group having one or more carbon-carbon triple bonds, wherein the two groups (or fragments) connected thereto are respectively connected to different carbon atoms. For example, the term "C 2-6 "Alkyne" refers to an alkynyl group having 2 to 6 carbon atoms (e.g. , etc.), which are optionally substituted with one or more (e.g., 1-3) substituents described herein.

[0196] In this application, the term "aryl" refers to a monocyclic or condensed aromatic hydrocarbon group having a conjugated π electron system. For example, the term "6-10 membered aryl" as used herein refers to an aryl group having 6-10 carbon atoms (such as phenyl, naphthyl, etc.), which is optionally substituted with one or more substituents described herein (such as halogen substitution, etc.).

[0197] In this application, the term "arylene" refers to a monocyclic or condensed-ring divalent aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C 6-10 The term "arylene" refers to an arylene group having 6 to 10 carbon atoms, which is optionally substituted with one or more substituents described herein (eg, substituted with halogen, etc.).

[0198] As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to monocyclic and fused heterocyclic ring systems 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. A heteroaryl group or heteroaromatic ring can be characterized by the number of ring atoms. For example, a 5-12 membered heteroaryl group can 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 thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, indolyl, and the like; optionally substituted with one or more substituents described herein.

[0199] In the present application, the term "heteroarylene" refers to a monocyclic or fused-ring divalent aromatic group having a conjugated π electron system, wherein the ring has 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 independently selected from N, O, P and S, for example, a total of 5-12 (preferably 5-10, more preferably 5, 6, 9 or 10) ring atoms. For example, the term "5-12 membered heteroarylene" as used herein refers to a heteroarylene group having 5-12 ring atoms, which is optionally substituted with one or more substituents described herein (e.g., C 1-6 Alkyl substituted Halogen-substituted wait).

[0200] In this application, the term "cycloalkyl" or "carbocycle" refers 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, bridged 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, decalinyl, etc., which are optionally substituted with one or more substituents described herein.

[0201] In this application, the term "cycloalkylene" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic divalent cyclic group. For example, "C 3-12 "Cycloalkylene" 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, bridged or spiro rings, such as bicyclo[1.1.1]pentylene, bicyclo[2.2.1]heptylene, bicyclo[3.2.1]octylene, bicyclo[5.2.0]nonylene, decahydronaphthylene, etc., which are optionally substituted with one or more substituents described herein.

[0202] The term "heterocycloalkyl" or "heterocycle" refers to a saturated or partially saturated non-aromatic cyclic group containing at least one ring member selected from N, O, P, and S, preferably, the number of heteroatoms is 1, 2, 3, or 4. For example, a 3-8-membered or 3-6-membered heterocycloalkyl group. Specific examples include, but are not limited to, oxiranyl, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, and the like, which are optionally substituted with one or more oxo groups or substituents described herein.

[0203] The term "heterocycloalkylene" refers to a saturated or partially saturated, non-aromatic, divalent cyclic group containing at least one ring member selected from N, O, P, and S, preferably, the number of heteroatoms is 1, 2, 3, or 4. For example, a 3-8-membered or 3-6-membered heterocycloalkylene group. Specific examples include, but are not limited to, oxiranylene, oxocyclobutanylene, pyrrolidinylene, tetrahydrofuranylene, piperidinylene, piperazinylene, tetrahydropyranylene, homopiperazinylene, and the like, which are optionally substituted with one or more oxo groups or substituents described herein.

[0204] The term "fused ring (fused ring system)" refers to a polycyclic structure formed by two or more (e.g., 3, 4, or 5) carbocyclic or heterocyclic rings with a common ring edge, wherein the carbocyclic ring includes a cycloalkyl group and an aryl group, and the heterocyclic ring includes a heteroaromatic ring and a heterocycloalkyl group. The fused ring system includes, but is not limited to, a fused ring system formed by a cycloalkyl group and a cycloalkyl group, a fused ring system formed by a cycloalkyl group and a heterocycloalkyl group, a fused ring system formed by a cycloalkyl group and an aromatic ring, a fused ring system formed by a cycloalkyl group and a heteroaromatic ring, a fused ring system formed by a heterocycloalkyl group and a heteroaromatic ring, a fused ring system formed by a heterocycloalkyl group and an aromatic ring, a fused ring system formed by a heteroaromatic ring and a heteroaromatic ring, a fused ring system formed by a heteroaromatic ring and an aromatic ring, and the like.

[0205] In the present application, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, for example, it can be fluorine or chlorine.

[0206] In this application, the term "each independently" means that at least two groups (or fragments) present in a structure with the same or similar value ranges can have the same or different meanings in 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 either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl.

[0207] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance can but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group can but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0208] In this application, the term "substituted" and its other variant forms in this article refer to that one or more (such as 1,2,3 or 4) atoms or atomic groups (such as hydrogen atoms) on the specified atom are replaced by other equivalents, provided that the normal valence of the specified atom or atomic group in the current situation is not exceeded, and stable compounds can be formed. If a certain atom or atomic group is described as "optionally replaced by...", it can be substituted or unsubstituted. Unless otherwise indicated, The attachment site of the substituent herein can be from any suitable position of the substituent. When the link in the substituent is shown as a chemical bond between two atoms connected to each other through the ring system, it means that the substituent can be connected to any ring-forming atom in the ring system.

[0209] In this application, the term 0 or more (e.g., 0 or more, 0 or 1, 0) methylene units are "replaced" generally means that when the structure contains 1 or more methylene units, the one or more methylene units may be unsubstituted or 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-, -SONH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C- or -C(=N2)-, etc.).

[0210] This article uses wavy lines The bonds in the structural formulae represented are intended to indicate that the structure represents either a cis or trans isomer, or a mixture of cis and trans isomers in any ratio.

[0211] The term "oxo," as used herein alone or in combination with other groups, refers to =0.

[0212] In the present application, one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, are independently replaced 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) whether substitution is possible or not without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0213] In this application, the term "amino acid" includes natural amino acids and non-natural amino acids, and the writing of conventional amino acids follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this article, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala; arginine can be represented by R or Arg; glycine can be represented by G or Gly; glutamine can be represented by Q or Gln;

[0214] As used herein, the term "unnatural amino acid" has the following structure: wherein r is selected from 0, 1, 2, 3, 4, 5; wherein R a 、Rb Each 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 heterocycloalkyl), -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 heterocycloalkyl), The alkyl, cycloalkyl, and heterocycloalkyl groups are each optionally substituted with one or more substituents selected from H, halogen, -OH, -NH2, -SH, -NO2, CN, -COOH, and oxo; or any R a 、R b Together with the atoms to which it is attached, it forms a 3-10 membered heterocycloalkyl or a 3-10 membered cycloalkyl; each of the cycloalkyl and heterocycloalkyl groups is optionally substituted with one or more substituents selected from H, halogen, -OH, -NH2, -SH, -NO2, CN, -COOH, and oxo;

[0215] In this application, the term "compound" generally refers to a substance containing two or more different elements. For example, the compound of this application can be an organic compound, such as 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 connected by chemical bonds, such as a compound in which one or more molecules with a molecular weight of less than 1000 Daltons are connected to a biomacromolecule through chemical bonds. The biomacromolecule can be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of this application can include a protein connected to one or more molecules with a molecular weight of less than 1000 Daltons, a protein connected to one or more molecules with a molecular weight of less than 10,000 Daltons, or a protein connected to one or more molecules with a molecular weight of less than 100,000 Daltons.

[0216] In this application, the term "stereoisomer" refers to an isomer formed by at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can 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-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0217] In this article, solid lines can be used Solid wedge or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the indicated stereoisomers exist. When present in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, 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, atropisomers, and mixtures thereof. The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0218] In this application, the term "comprising" generally refers to including the features specified but not excluding other elements. The terms "above" and "below" generally refer to including the number.

[0219] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 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 the specified value.

[0220] Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds identical to the structures described herein except for the replacement of a hydrogen atom by deuterium or tritium, or the replacement of a carbon atom by carbon-13 or carbon-14, are within the scope of this application.

[0221] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating one or more symptoms of a target disorder or condition.

[0222] As used herein, the term "effective amount" (e.g., "therapeutically effective amount" or "prophylactically effective amount") refers to an amount of active ingredient that, after administration, will achieve the desired effect to some extent, such as alleviating one or more symptoms of the condition being treated or preventing the appearance of the condition or its symptoms.

[0223] As used herein, unless otherwise indicated, the terms "treat," ...

[0224] As used herein, "subject" includes humans and non-human animals. Exemplary human subjects include human subjects suffering from diseases (e.g., the diseases described herein) (referred to as patients) or normal individuals. "Non-human animals" herein include 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.).

[0225] Those skilled in the art will appreciate that, since nitrogen requires an available lone pair of electrons to be oxidized to oxides, not all nitrogen-containing heterocycles are capable of forming nitrogen oxides. Those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming nitrogen oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming nitrogen oxides. Synthetic methods for preparing nitrogen oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidizing heterocycles and tertiary amines with peroxyacids such as Peracetic Acid and Metachloroperbenzoic Acid (mCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxirane such as dimethyldioxirane. These methods for preparing nitrogen oxides have been extensively described and reviewed in the literature, see for example: TL Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750 (AR Katritzky and AJ Boulton, Eds., Academic Press); and GWH Cheeseman and ESGWerstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392 (AR Katritzky and AJ Boulton, Eds., Academic Press).

[0226] The present invention also encompasses compounds of the present invention that contain protecting groups. During any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved using conventional protecting groups, for example, those described in TW Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 2006, which references are incorporated herein by reference. Protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

[0227] The present invention also encompasses methods for preparing the compounds described herein. It should be understood that the compounds of the present invention can be synthesized using the methods described below, as well as synthetic methods known in the art of synthetic organic chemistry or variations thereof known to 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 that is appropriate for the reagents and materials used and suitable for the transformation to be achieved.

[0228] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0229] The reagents and raw materials used in the present invention are commercially available.

[0230] The beneficial technical effects of the present invention are that the compounds of the present invention:

[0231] (1) It has inhibitory activity on the proliferation of tumor cells in vitro;

[0232] (2) Having plasma stability;

[0233] (3) Has tumor-suppressing effect in vivo;

[0234] (4) Has a bystander effect;

[0235] (5) possessing anti-transporter transport capability;

[0236] (6) Possesses the ability to target tumors in vivo;

[0237] (7) Has good in vivo safety.

[0238] In addition, the coupling method disclosed herein has a wide range of applications and can be widely used for coupling with bioactive molecules such as antibodies or targeted small molecule ligands. In summary, the cytotoxic agents, linkers, antibodies and ADCs of the present invention have significant clinical value.

[0239] Preparation method:

[0240] In order to achieve the synthesis purpose of the present disclosure, the present application adopts the following synthesis technology scheme:

[0241] Scheme 1: A method for preparing the compound represented by formula (Ia), formula (IIa), and the ligand-drug conjugate represented by formula (IIIa), or a pharmaceutically acceptable salt or solvate thereof, comprising:

[0242] in,

[0243] Ab and q are as defined in formula (III);

[0244] L 1a As defined in formula (V);

[0245] L 1 , L 2 , L 3 , Tr is as defined in formula (II);

[0246] R 1a 、R 2a As defined in formula (Ia);

[0247] Lg Tr , Lg 1 is a leaving group selected from halogen, hydroxyl, -OAc, etc.;

[0248] Pg 2 is a protecting group selected from Boc, Fmoc, Cbz, Sem and the like.

[0249] Step 1: Compound Ia-1 and compound E are cyclized under acidic conditions and heated in a solvent (eg, 80-130° C.) to obtain a compound of formula (Ia).

[0250] In some embodiments, the solvent is selected from methanol, tetrahydrofuran, DMF, NMP, DMSO, toluene, n-hexane, n-heptane, etc.; preferably toluene.

[0251] In some embodiments, the reagent providing acidic conditions is selected from PTSA, acetic acid, hydrochloric acid, sulfuric acid, InCl3, p-toluenesulfonic acid, formic acid, trifluoroacetic acid; preferably acetic acid, hydrochloric acid, InCl3.

[0252] Step 2: The compound of formula (Ia) and compound F are reacted with a condensation reaction or a substitution reaction in the presence of a condensation reagent to obtain a compound of formula (IIa).

[0253] Step 2a: The compound of formula (Ia) reacts with compound Fa in the presence of a condensation reagent through a condensation reaction or a substitution reaction; then the protecting group is removed to obtain compound Ia-2.

[0254] Step 2b: Compound Ia-2 and Compound L 1 -Lg 1 The compound of formula (IIa) is reacted by a condensation reaction in the presence of a condensation agent or by substitution.

[0255] In some embodiments, the condensing agent is selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole- N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0256] Step 3: The compound of formula (IIa) reacts with Ab after disulfide bond reduction to obtain a ligand-drug conjugate of formula (IIIa).

[0257] In some embodiments, reducing agents for reducing Ab include but are not limited to tris(2-carboxyethyl)phosphine, mercaptoethanol, dithiothreitol, cysteine, reduced glutathione, etc.; in particular, it is preferred to reduce disulfide bonds on the antibody.

[0258] Scheme 2: A method for preparing the compound represented by formula (Ib), formula (IIb), and the ligand-drug conjugate represented by formula (IIIb), or a pharmaceutically acceptable salt or solvate thereof, comprising:

[0259] in,

[0260] Ab and q are as defined in formula (III);

[0261] L 1a As defined in formula (V);

[0262] L 1 , L 2 , L3 , Tr is as defined in formula (II);

[0263] R 1b 、R 2b As defined in formula (Ib);

[0264] Lg Tr , Lg 1 is a leaving group selected from halogen, hydroxyl, -OAc, etc.;

[0265] Pg 2 is a protecting group selected from: Boc, Fmoc, Cbz, Sem, etc.;

[0266] The specific preparation method refers to Scheme 1.

[0267] Those skilled in the art will appreciate that, depending on the desired product structure, one or more steps in the above-described preparation method may be omitted, and the order of the reaction steps may be appropriately adjusted, and protection / deprotection reaction steps may be added or omitted as needed. DETAILED DESCRIPTION

[0268] The present invention includes all combinations of the described specific embodiments. Further embodiments of the present invention and the full scope of applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. For all purposes, all publications, patents and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety. The present invention is further illustrated below by way of example, but the present invention is not limited to the scope of the described embodiments. The experimental methods for which specific conditions are not specified in the following examples are selected according to conventional methods and conditions, or according to the product specifications.

[0269] Mass spectrometry (MS) was measured using an Agilent (ESI) mass spectrometer, manufactured by Agilent, model: Agilent 6120B.

[0270] Preparative high performance liquid chromatography (HPLC) was performed using a Shimadzu LC-8A preparative liquid chromatograph (YMC, ODS, 250 × 20 mm column).

[0271] Thin layer chromatography purification was performed using GF 254 (0.4-0.5 nm) silica gel plates produced in Yantai.

[0272] The reaction is monitored by thin layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). The developing solvent systems used include, but are 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 solvents is adjusted according to the polarity of the compounds or by adding triethylamine.

[0273] Column chromatography generally uses Qingdao Ocean 200-300 mesh silica gel 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 solvents is adjusted according to the polarity of the compound, and a small amount of triethylamine can also be added for adjustment.

[0274] Unless otherwise specified in the examples, the reaction temperature is room temperature (20°C to 30°C).

[0275] 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.

[0276] The above embodiments do not limit the solutions of the present application in any way. In addition to those described herein, various modifications of the present 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. Each reference cited in this application (including all patents, patent applications, journal articles, books and any other disclosures) is incorporated by reference in its entirety.

[0277] In conventional synthesis methods, preparation examples, embodiments and intermediate synthesis examples, the meanings of the abbreviations are shown in the following table.

[0278] Example 1: Preparation of camptothecin compounds

[0279] Example 1.1: Preparation of Compound A1.

[0280] Compound A1-1 (1.1 g, 7.4 mmol), compound E (1.80 g, 14.8 mmol), and InCl3 (3.3 g, 29.6 mmol) were placed in a reaction flask. Toluene (30 mL) was added and the mixture was heated to 120°C and refluxed for 24 h. After completion of the reaction, as determined by LCMS, the reaction solution was concentrated under reduced pressure. The crude product was purified by HPLC to yield compound A1 (145 mg, 5%).

[0281] MS m / z(ESI):396.1[M+H] +

[0282] 1 H-NMR (400MHz, DMSO-d6): δ8.05(d,J=8.0Hz,1H),7.94(brs,2H),7.74(s,J=10.8Hz,1H),7.30(s,1H),6.5 7(s,1H),5.44(s,2H),5.06(s,2H),2.09(s,1H),2.61(s,3H),1.87(q,J=7.2Hz,2H),0.88(t,J=7.2Hz,3H).

[0283] Example 1.2: Preparation of Compound A2.

[0284] Compound A2 was synthesized by referring to the method of Example 1.

[0285] MS m / z(ESI):400.1[M+H] +

[0286] 1 H-NMR (400MHz, DMSO-d6): δ8.38 (dd, J=12.0Hz, 8.4Hz, 1H), 7.90 (dd,J=11.6Hz, 8.0Hz, 1H), 7.53 (brs, 2H),7.21(s,1H),6.48(s,1H),5.41(s,2H),5.03(s,2H),1.84(q,J=7.2Hz,2H),0.86(t,J=7.2Hz,3H).

[0287] Example 1.3: Preparation of Compound A13.

[0288] Compound A2 was synthesized by referring to the method of Example 1.

[0289] MS m / z(ESI):408.1[M+H] +

[0290] 1 H-NMR (400MHz, DMSO-d6): δ7.85(s,1H),7.46(s,1H),7.31(s,1H),6.63(s,1H),6. 32(s,2H),5.43(s,2H),5.05(s,2H),1.86(q,J=7.2Hz,2H),0.87(t,J=7.2Hz,3H).

[0291] Example 1.4: Preparation of Compound A18.

[0292] Compound A2 was synthesized by referring to the method of Example 1.

[0293] MS m / z(ESI):416.1[M+H] +

[0294] 1 H-NMR (400MHz, DMSO-d6): δ8.69(d,J=8.0Hz,1H),7.96(brs,2H),7.87(s,J=10.8Hz,1H),7.30(s,1H ),6.57(s,1H),5.44(s,2H),5.06(s,2H),2.09(s,1H),1.87(q,J=7.2Hz,2H),0.88(t,J=7.2Hz,3H).

[0295] Referring to the method of Example 1.1, the following compounds were synthesized using appropriate starting materials:

[0296] Example 1.2: Preparation of Compound B2.

[0297] Compound B2-1 (1.0 g, 6.5 mmol), compound E (1.58 g, 13.0 mmol), and InCl3 (2.89 g, 26.0 mmol) were placed in a reaction flask. Toluene (20 mL) was added and the mixture was heated to 120°C and refluxed overnight. After completion of the reaction by LCMS, the reaction solution was concentrated under reduced pressure. The crude product was purified by HPLC to yield compound B2 (100 mg, 3.9%).

[0298] MS m / z(ESI):399.2[M+H] +

[0299] 1 H-NMR (400 MHz, DMSO-d6): δ8.31–8.26 (m, 1H), 7.91–7.86 (m, 1H), 7.51 (brs, 2H), 7.19 (s, 1H), 6.47 (s, 1H), 5.39 (s, 2H), 5.01 (s, 2H), 1.82 (q, J = 7.2 Hz, 2H), 0.87 (t, J = 7.2 Hz, 3H). Compound B5 was synthesized using appropriate starting materials according to the method of Example 1.2.

[0300] Example 2: Preparation of linker intermediates

[0301] Example 2.1: Preparation of intermediate Int1

[0302] Step 1: Preparation of compound Int1-2

[0303] 1-BOC-azetidine-3-carboxylic acid (200 mg, 1 mmol) was dissolved in DMF (5 ml), and HATU (567 mg, 1.49 mmol), DIEA (513 mg, 3.98 mmol), and compound Int1-1 (222 mg, 1.09 mmol) were added, followed by stirring at room temperature for 30 minutes. After completion of the reaction, water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed twice with saturated brine, dried, concentrated, and purified by column chromatography to obtain compound Int1-2 (339 mg, 85%).

[0304] Step 2: Preparation of compound Int1

[0305] Compound Int1-2 (339 mg, 0.85 mmmol) was dissolved in methanol (3 mL) and then added to a 2N aqueous LiOH solution (10 mL). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the pH of the solution was adjusted to 8 with a 2N aqueous citric acid solution. The product was extracted with ethyl acetate, and the organic phase was dried and concentrated. This yielded a crude product of compound Int1 (327 mg).

[0306] The following intermediates were synthesized by referring to the method of Example 2.1:

[0307] Example 3: Preparation of linker-payload for ligand drug conjugates

[0308] Example 3.1: Preparation of Compound AA1

[0309] Compound Int2 (100 mg, 0.28 mmol), compound A1 (110 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 completion of the reaction, water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by reverse-phase HPLC to afford compound AA1 (60 mg, 29%).

[0310] MS: [M+H] + , 731.3.

[0311] Example 3.2: Preparation of Compound AA84

[0312] Step 1: Preparation of compound AA84-1

[0313] Compound A1 (110 mg, 0.28 mmol), compound Int11 (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 completion of the reaction, water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound AA84-1 (80 mg, 38%).

[0314] Step 2: Preparation of compound AA84-2

[0315] Compound AA84-1 (80 mg, 0.11 mmol) was dissolved in trifluoroacetic acid / dichloromethane (5 mL, 1 / 3) and stirred at room temperature for 1 hour. After completion of the reaction, ethyl acetate was added and the pH was adjusted to 8 with saturated sodium bicarbonate. The layers were separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to afford compound AA84-2 (71 mg, 99%).

[0316] Step 3: Preparation of compound AA84

[0317] Compound AA84-2 (71 mg, 0.10 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (20 mg, 0.10 mmol), and HATU (57 mg, 0.15 mmol) were dissolved in anhydrous DMF (5 mL) and stirred at room temperature for 12 h. After completion of the reaction, water was added, and the mixture was extracted with ethyl acetate. The layers were separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was purified by reverse-phase HPLC to afford compound AA84 (10 mg, 12%).

[0318] MS: [M+H] + , 835.2.

[0319] The following compounds were synthesized by referring to the methods of Examples 3.1 and 3.2 using appropriate intermediates, starting materials and payloads.

[0320] Example 4: Preparation of Ligand Drug Conjugates

[0321] The antibody used as the ligand is prepared according to conventional methods. For example, after constructing a vector, it can be transfected into eukaryotic cells such as HEK293 cells or CHO cells for purification and expression. Trastuzumab and certolizumab are used as examples to prepare ligand-drug conjugates.

[0322] Amino acid sequence of trastuzumab:

[0323] Amino acid sequence of certolizumab pegol:

[0324] Example 4.1: Preparation of ADC-1A

[0325] At 37°C, the prepared TCEP (10 mM, 0.135 mL, 1.35 μmol) was added to the buffer solution of the antibody trastuzumab (14.0 mM succinic acid-sodium hydroxide + 108 mM NaCl pH 6.0; 20 mg, 10.0 mg / mL, 0.135 μmol), and the mixture was placed in a water bath shaker and shaken at 37°C for 3 hours. After stopping the reaction, the excess TCEP was removed by ultrafiltration using a 14.0 mM succinic acid-sodium hydroxide + 108 mM NaCl pH 6.0 buffer solution.

[0326] Compound AA1 (1.278 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 shaker and shaken at 22° C. for 2 hours to stop the reaction. The reaction solution was desalted and purified 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; 17 mg, 4.59 mg / mL, yield: 85%), which was stored at 4° C.

[0327] The DAR value q was calculated by LC-MS analysis and detection = 7.8.

[0328] The following compounds were synthesized by referring to the method of Example 4.1 using appropriate linker-payload.

[0329] Example 5: In vitro tumor cell proliferation inhibition test of compounds

[0330] Purpose of the test

[0331] In order to detect the inhibitory activity of drug compounds on the proliferation of NCI-N87 cells, JIMT-1 and MBA-MB-231 tumor cells in vitro, cells were treated with different concentrations of compounds in vitro and cultured for 6 days. Luminescent Cell Viability Assay (Promega, Catalog No. G7558) was used to detect cell proliferation. 50The in vitro activity of the compound was evaluated.

[0332] Experimental methods

[0333] The following uses the in vitro proliferation inhibition test method of NCI-N87 cells as an example to illustrate the method for testing the in vitro proliferation inhibition activity of the compounds of the present application on tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

[0334] 1. Cell culture: NCI-N87 cells were cultured in RPMI-1640 medium supplemented with 10% FBS.

[0335] 2. Cell preparation: Take NCI-N87 cells in the logarithmic growth phase, wash once with PBS, add 2-3 ml of trypsin to digest for 2-3 minutes, wait until the cells are completely digested, add 10-15 ml of cell culture medium to elute the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 ml of cell culture medium to resuspend the cells to make a single-cell suspension.

[0336] 3. Cell plating: Mix the NCI-N87 single cell suspension and adjust the viable cell density to 6x10 4 After adjusting the density, the cell suspension was mixed and added to a 96-well cell culture plate at 50 μl / well. The culture plate was cultured in an incubator for 18 hours (37°C, 5% CO2).

[0337] 4. Compound preparation: Dissolve the compound in DMSO and prepare a storage solution with an initial concentration of 10 mM.

[0338] There are 8 concentrations of small molecule compounds, namely: 300, 100, 30, 10, 3, 1, 0.3, and 0.1 nM.

[0339] 5. Sample addition: Add different concentrations of the test sample to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37°C, 5% CO2).

[0340] 6. Color development: Take out the 96-well cell culture plate, add 50ul CTG reagent to each well, and incubate at room temperature for 10 minutes.

[0341] 7. Plate reading operation: Take out the 96-well cell culture plate, place it in a microplate reader, and measure the chemiluminescence using the microplate reader.

[0342] Data Analysis

[0343] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5.

[0344] Table 3. IC values ​​of the small molecule fragments in this application for inhibition of NCI-N87 cell proliferation in vitro 50 value.

[0345] Conclusion: According to the results in Table 3, the small molecule fragments in this application have obvious proliferation inhibitory activity on NCI-N87 cells, JIMT-1 cells, and MDA-MB-231 cells.

[0346] Example 6: In vitro cell proliferation inhibition activity test of antibody-drug conjugates

[0347] Example 6.1: In vitro proliferation inhibition activity test of NCI-N87 / JIMT-1 cells

[0348] use The chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effect of the anti-Her2 antibody trastuzumab (ADC) coupled with a camptothecin compound on cell proliferation in Her2-positive human gastric cancer cells NCI-N87 and human breast cancer cells JIMT-1 after incubation for 6 days.

[0349] Cells in the logarithmic growth phase were collected and plated in a 96-well cell culture plate at a density of 6000 cells / well. The cell plates were placed in a 37°C, 5% CO2 incubator for overnight culture. On the second day of the experiment, the ADC drug of the camptothecin compound was diluted 3-fold with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 300nM). After the drug was added, 100μL / well was added to the cell culture plate. The complete culture medium was used as a blank control, and 3 replicates were set up; the plate was continued to be incubated in a 37°C, 5% CO2 incubator for 6 days. After the incubation was completed, the cell culture plate was removed and equilibrated to room temperature. 50μL CTG detection reagent (Promega, Cat#: G7573) was added to each well. After shaking and mixing, the plate was placed in the dark for 10 minutes, and the signal value was read using an enzyme-linked microplate reader. GraphPad Prism software was used to draw a S-shaped dose-response curve using a nonlinear regression model and calculate the IC 50 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%.

[0350] Experimental conclusion: The antibody-drug conjugate of the present application has significant proliferation inhibitory activity on Her2-positive human gastric cancer cell NCI-N87 and human breast cancer JIMT-1 cells.

[0351] Example 7: In vivo tumor inhibition test of antibody drug conjugates

[0352] To evaluate the inhibitory effect of the ADC drug of the present invention on tumor formation in vivo, after forming transplanted tumors in mice using Her2-positive human breast cancer cells JIMT-1, the anti-tumor effect of the ADC drug of the present invention was evaluated.

[0353] Evaluation of the efficacy of antibody-drug conjugates in JIMT-1 human breast cancer cell-bearing mice

[0354] 1. Test drugs and materials

[0355] Blank control group (control group): normal saline

[0356] ADC (treatment group): 5 mg / kg, single dose

[0357] 2. Preparation method: All samples were diluted with physiological saline.

[0358] 3. Experimental animals: 8-week-old female BALB / c-nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0359] 4. Test method:

[0360] 1×10 7 JIMT-1 cells were inoculated subcutaneously at the right anterior scapula of 8-week-old female BALB / c-nude mice. 3 Tumor-bearing mice were randomly divided into groups using StudyDirect™. Starting on day 0, A1-1 was injected intravenously (iv) 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.

[0361] Each vehicle control group or treatment group consisted of 5 mice. Tumor inhibition rate (TGI%) was calculated by measuring tumor volume. Tumor inhibition rate (TGI%) = 100% - (tumor volume of treatment group on the day of measurement - tumor volume of treatment group on day 0) / (tumor volume of control group on the day of measurement - tumor volume of control group on day 0).

[0362] The experimental results showed that the antibody-drug conjugate AA1-1 exhibited significant tumor-suppressing activity after a single administration.

[0363] Example 8: Plasma stability test of antibody drug conjugates

[0364] To evaluate the plasma stability of the antibody drug conjugates of the present invention, the antibody drug conjugates of the present invention were 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 coupling rate (DAR value) and free drug.

[0365] Incubation of Antibody Drug Conjugates in Plasma

[0366] The antibody drug conjugate of the present 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 immediately frozen to -70°C after dilution within 30 minutes), 2 hours, 8 hours, day 1 (24 hours), day 4, day 7, day 14, and day 21. All samples were stored in a -70°C freezer before analysis. Only the DAR values ​​were analyzed for samples collected at 2 hours and 8 hours.

[0367] Analysis of free drugs by LC-MS / MS

[0368] Protein precipitation

[0369] Plasma samples were thawed, and 291.9 μL was collected for free drug analysis. A precipitant (400 μL of 0.1% formic acid, 200 ng / mL tolbutamide, and 200 ng / mL labetalol in acetonitrile) was added to each sample and mixed thoroughly. The mixture was shaken for at least 20 minutes to precipitate plasma proteins. The samples were centrifuged at 4°C, 4000 rpm, and 150 μL of the supernatant was collected for LC-MS / MS analysis.

[0370] The equipment used for free drug analysis is shown in the table below:

[0371] The LC and MS parameters are as follows:

[0372] LC-MS determination of drug-ligand coupling rate

[0373] Immobilized magnetic beads capture antibody drug conjugates

[0374] 25 μL of streptavidin magnetic beads were added to each well of a 96-well plate, and the stock buffer was discarded. The beads were eluted with 200 μL of PBS buffer and treated with 80 μL of biotinylated antigen protein and another 100 μL of PBS buffer at room temperature for 120 minutes. After complete immobilization of the antigen protein, the beads were eluted twice with 200 μL of PBS buffer. 20 μL of antibody-drug conjugate plasma sample and 180 μL of PBS buffer were added to each well, followed by shaking at room temperature for 120 minutes to ensure complete capture of the antibody-drug conjugate in the plasma, and the supernatant was removed. The beads were washed twice with PBS buffer, followed by the addition of 50 μL of elution buffer (1% formic acid in water) and treatment at room temperature for 20 minutes. Subsequently, 5 μL of neutralization buffer (1 M NH4HCO3, pH 8.5) and 5 μL of 1 M DTT were added, and the captured antibody-drug conjugate was reduced at room temperature for 60 minutes before analysis by LC-MS.

[0375] Drug-ligand coupling rate analysis

[0376] The LC-MS parameters are as follows:

[0377] The results showed that after 21 days of incubation in plasma, no drug or only a very small amount of drug was shed from the antibody-drug conjugate of the present invention, and there was no significant change in the drug-ligand coupling rate. The antibody-drug conjugate of the present invention has extremely high plasma stability, indicating that the antibody-drug conjugate of the present invention has better safety.

[0378] Example 9: Antibody Drug Conjugate Stock Solution (Liquid Preparation) Stability

[0379] To evaluate the stock solution stability of the antibody-drug conjugate of the present invention, the ADC stock solution of the present invention was concentrated to 20 mg / mL, dispensed into 2 mL cryovials, and incubated in a 40°C incubator. Samples were taken at 0 hour, 1 week, 2 weeks, 1 month, and 2 months for concentration, drug-ligand coupling ratio (DAR value), SEC purity and aggregation, CE-SDS (non-reducing) purity, CE-SDS (reducing) purity, charge heterogeneity, and free drug detection. The liquid was also visually inspected for clarity and precipitation.

[0380] The sample concentration was detected by UV method.

[0381] The drug-ligand coupling rate (DAR value) was detected by hydrophobic HPLC (HIC) or reverse phase HPLC (RP-HPLC).

[0382] The drug-ligand coupling rate (DAR value) was detected by LC-MS.

[0383] Reverse phase HPLC (RP-HPLC) was used to detect free drug.

[0384] The SEC purity and aggregation of the samples were determined by SEC-HPLC (TOSOH G3000 SW SEC column).

[0385] Maurice was used to detect the purity of CE-SDS (non-reduced) and CE-SDS (reduced).

[0386] Imaging capillary isoelectric focusing (iCIEF) was used to detect charge heterogeneity.

[0387] The results showed that after incubation at 40°C for one week to two months, the antibody-drug conjugate stock solution of the present invention showed no significant changes in concentration, drug-ligand coupling ratio (DAR), SEC purity and aggregation, CE-SDS (non-reducing) purity, CE-SDS (reducing) purity, charge heterogeneity, and free drug. The antibody-drug conjugate stock solution of the present invention has extremely high stability.

[0388] Example 10: Pharmacokinetic and toxicological studies of single or multiple doses in monkeys

[0389] After a single or multiple intravenous injections of the antibody drug conjugate of the present invention are given to monkeys, the pharmacokinetic properties of the drug in the monkeys are investigated, and the toxicity of the animals is observed.

[0390] Test methods

[0391] Pharmacokinetics: After a single intravenous infusion of different doses of the antibody-drug conjugate of the present invention into monkeys, blood samples were collected at multiple time points and the concentration of the drug in the blood was measured using an appropriate specific detection method.

[0392] Toxicology studies: After single or multiple intravenous infusions of different doses of the antibody-drug conjugate of the invention in monkeys, the animals' tolerance to the antibody-drug conjugate of the invention, as well as drug-related toxicity, were assessed through clinical observation, body weight and food intake, hematology, blood biochemistry, urine, gross anatomy, and histopathology.

[0393] Results showed that after single or multiple intravenous infusions of the antibody-drug conjugate of this invention in monkeys, the pharmacokinetic properties of the total antibody and ADC were similar, with very low free toxin concentrations, demonstrating that the antibody-drug conjugate of this invention has high stability and good pharmacokinetic properties in vivo. The animals were well tolerated, with no severe or intolerable drug-related toxicities, demonstrating that the safety of the antibody-drug conjugate of this invention is controllable.

Claims

1. 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 a structure represented by formula (IV): in, The wavy lines indicate direct or indirect attachment to the ligand through the nitrogen atom; R 1 , R 2 and R 3 are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1 With R 2 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; or R 1 With R 3 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; The condition is R 1 , R 2 and R 3 Not hydrogen at the same time.

2. The ligand-drug conjugate according to claim 1, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate comprises a ligand and a structure represented by formula (V): in: The wavy line indicates the L 1a The nitrogen or carbon atom on the group is connected to the ligand; L 1a Selected from: L 2 =-(C(RL 21 )2) n -, Where n is a natural number from 0 to 50. L 2 Any C(R L21 )2 units 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-, -Cy- is selected from phenylene, 5 to 8 membered heteroarylene, 3 to 10 membered heterocyclylene, or 3 to 10 membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted by one or more R cx replace, R L21 , R L22 , R cx Each independently selected from hydrogen, deuterium, halogen, -NO2, -CN, -OR L2a 、-SR L2a 、-N(R L2a )2. -N + (R L2a )3. -C(O)R L2a 、-CO2R L2a 、-C(O)C(O)R L2a 、-C(O)CH2C(O)R L2a 、-S(O)R L2a 、-S(O)2R L2a 、-C(O)N(R L2a )2、-SO2N(R L2a )2. -OC(O)R L2a 、-N(R L2a )SO2R L2b 、-N(R L2a )COR L2b 、-(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a 、-(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a 、-(CH2) y -CO-(N(Me)CH2C(O)) m -N + (R L2a )3, -(CH2) y -NHCOCH2(OCH2CH2)OR L2a 、-(CH2) y -NH(COCH2(N(Me)) m -R L2a 、-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-(CH2) y -NHCO-(CH2CH2O) m -R L2a 、-(CH2CH2O) m -R L2a 、-(COCH2N(Me)) m -R L2a 、-COCH2(OCH2CH2) m -OR L2a 、-CO-(CH2CH2O) m -R L2a 、-CO-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-CO-(CH2) y -NHCO-(CH2CH2O) m -R L2a or by R L2a Optionally substituted -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, m, y are natural numbers from 0 to 50, R L2a , R L2b Each is independently selected from hydrogen, deuterium, halogen, -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; L 3 Does not exist or is an amino acid residue, a short peptide consisting 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; Tr does not exist or is or any combination of the above groups; R Tr , R Tr1 and R Tr2 Each is independently selected from hydrogen, deuterium, halogen, -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 by R Tra Optionally substituted -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, R Tra Hydrogen, deuterium, halogen, -NO2, -CN, -OH, -SH, -NH2, -N(Me)2, -S(O)2Me, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -C 1-6 Alkyl, -C 1-6 Alkenyl, -C 1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl, z is a natural number 0 or greater; R 1 , R 2 and R 3 As defined in claim 1.

3. The ligand-drug conjugate according to claim 2, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate has a structure shown in formula (III): in, Ab is the ligand that binds to the target; q is the drug loading, which is an integer or decimal from 1 to 16; R 1 , R 2 and R 3 As defined in claim 1; and L 1a , L 2 , L 3 and Tr as defined in claim 2.

4. The ligand-drug conjugate according to claim 1, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate comprises a ligand and: -Structure represented by formula (IVa): in, The wavy lines indicate direct or indirect attachment to the ligand through the nitrogen atom; R 1a and R 2a are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1a With R 2a and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; And R 1a and R 2a are not simultaneously hydrogen; or -Structure represented by formula (IVb): Among them, the wavy line indicates direct or indirect connection to the ligand through the nitrogen atom; R 1b Selected from hydrogen, halogen, -C 1-6 Alkyl and -C 1-6 Alkoxy; R 2b Selected from halogen, -C 1-6 Alkyl and -halogenated C 1-6 alkyl.

5. The ligand-drug conjugate according to claim 2, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate comprises a ligand and: -Structure represented by formula (Va): in, The wavy line indicates the L 1a The nitrogen or carbon atom on the group is connected to the ligand; L 1a , L 2 , L 3 , Tr as defined in claim 2; R 1a and R 2a are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1a With R 2a and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; And R 1a and R 2a are not simultaneously hydrogen; or -Structure represented by formula (Vb): The wavy line indicates the L 1a The nitrogen or carbon atom on the group is connected to the ligand; L 1a , L 2 , L 3 , Tr as defined in claim 2; R 1b Selected from hydrogen, halogen, -C 1-6 Alkyl and -C 1-6 Alkoxy; R 2b Selected from halogen, -C 1-6 Alkyl and -halogenated C 1-6 alkyl.

6. The ligand-drug conjugate according to claim 3, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, which has: -Structure represented by formula (IIIa): wherein Ab and q are as defined in claim 3, L 1a , L 2 , L 3 , Tr as defined in claim 2; R 1a and R 2a are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1a With R 2a and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; And R 1a and R 2a are not simultaneously hydrogen; or -Structure represented by formula (IIIa): wherein Ab and q are as defined in claim 3, L 1a , L 2 , L 3 , Tr as defined in claim 2; R 1b Selected from hydrogen, halogen, -C 1-6 Alkyl and -C 1-6 Alkoxy; R 2b Selected from halogen, -C 1-6 Alkyl and -halogenated C 1-6 alkyl.

7. The ligand-drug conjugate according to claim 3 or 6, or its tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, wherein Ab is a target-binding polypeptide, an antibody or an antigen-binding fragment thereof, in particular an antibody or an antigen-binding fragment thereof, for example, selected from fully human antibodies, humanized antibodies, chimeric antibodies, probodies, bispecific antibodies, multispecific antibodies, monoclonal antibodies and polyclonal antibodies; the antibody or antigen-binding fragment thereof can be selected from Fab, Fab', F(ab')2, Fv, scFv, diabodies, Fd, dAb, VHH and antigen-binding fragments of a complementarity determining region (CDR) fragment.

8. The ligand-drug conjugate according to claim 7, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Ab is a monoclonal antibody.

9. The ligand-drug conjugate according to claim 7, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Ab is an antibody or antigen-binding fragment that specifically binds to HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70 and EGFR, for example, Trastuzumab or Pertuzumab or a variant thereof.

10. The ligand-drug conjugate according to any one of claims 3 and 6-9, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein: q is an integer or decimal selected from 2 to 8, in particular an integer or decimal selected from 4 to 8.

11. A compound represented by formula (I), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, in R 1 , R 2 and R 3 are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1 With R 2 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; or R 1 With R 3 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; The condition is R 1 , R 2 and R 3 Not hydrogen at the same time.

12. The compound according to claim 11, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the compound is a compound represented by formula (Ia), in, R 1a and R 2a are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1a With R 2a and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; And R 1a and R 2a are not simultaneously hydrogen; or The compound represented by formula (Ib): in, R 1b Selected from hydrogen, halogen, -C 1-6 Alkyl and -C 1-6 Alkoxy; R 2b Selected from halogen, -C 1-6 Alkyl and -halogenated C 1-6 alkyl.

13. A compound represented by formula (II), or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate: in, L 1 is a connection unit; L 2 =-(C(RL 21 )2) n -, Where n is a natural number from 0 to 50. L 2 Any C(R L21 )2 units 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-, -Cy- is selected from phenylene, 5 to 8 membered heteroarylene, 3 to 10 membered heterocyclylene, or 3 to 10 membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted by one or more R cx replace, R L21 , R L22 , R cx Each independently selected from hydrogen, deuterium, halogen, -NO2, -CN, -OR L2a 、-SR L2a 、-N(R L2a )2. -N + (R L2a )3. -C(O)R L2a 、-CO2R L2a 、-C(O)C(O)R L2a 、-C(O)CH2C(O)R L2a 、-S(O)R L2a 、-S(O)2R L2a 、-C(O)N(R L2a )2、-SO2N(R L2a )2. -OC(O)R L2a 、-N(R L2a )SO2R L2b 、-N(R L2a )COR L2b 、-(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a 、-(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a 、-(CH2) y -CO-(N(Me)CH2C(O)) m -N + (R L2a )3, -(CH2) y -NHCOCH2(OCH2CH2)OR L2a 、-(CH2) y -NH(COCH2(N(Me)) m -R L2a 、-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-(CH2) y -NHCO-(CH2CH2O) m -R L2a 、-(CH2CH2O) m -R L2a 、-(COCH2N(Me)) m -R L2a 、-COCH2(OCH2CH2) m -OR L2a 、-CO-(CH2CH2O) m -R L2a 、-CO-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-CO-(CH2) y -NHCO-(CH2CH2O) m -R L2a or by R L2a Optionally substituted -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, m, y are natural numbers from 0 to 50, R L2a , R L2b Each is independently selected from hydrogen, deuterium, halogen, -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; L 3 Does not exist or is an amino acid residue, a short peptide consisting 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; Tr does not exist or is or any combination of the above groups; R Tr , R Tr1 and R Tr2 Each is independently selected from hydrogen, deuterium, halogen, -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 by R Tra Optionally substituted -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, R Tra Hydrogen, deuterium, halogen, -NO2, -CN, -OH, -SH, -NH2, -N(Me)2, -S(O)2Me, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -C 1-6 Alkyl, -C 1-6 Alkenyl, -C 1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl, z is a natural number 0 or greater; R 1 , R 2 and R 3 are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1 With R 2 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; or R 1 With R 3 and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; The condition is R 1 , R 2 and R 3 Not hydrogen at the same time.

14. The compound according to claim 13, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the compound is a compound represented by formula (IIa): Where L 1 , L 2 , L 3 , Tr as defined in claim 13, R 1a and R 2a are each independently selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy; or R 1a With R 2a and the atoms to which they are attached together form a 4-6 membered carbocyclic ring or a 4-8 membered heterocyclic ring; And R 1a and R 2a are not simultaneously hydrogen; or The compound represented by formula (IIb): Where L 1 , L 2 , L 3 , Tr as defined in claim 13, R 1b Selected from hydrogen, halogen, -C 1-6 Alkyl and -C 1-6 Alkoxy; R 2b Selected from halogen, -C 1-6 Alkyl and -halogenated C 1-6 alkyl.

15. A compound according to claim 13 or 14, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: L 1 Selected from: Preferably, L 1 Selected from More preferably, L 1 Selected from 16. The ligand-drug conjugate according to any one of claims 4 to 10 or the compound according to any one of claims 12, 14 to 15, or its tautomers, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts or solvates, wherein: R 1a Selected from hydrogen, halogen, hydroxyl, nitro, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogenated C 1-6 Alkyl and -halogenated C 1-6 Alkoxy, preferably, R 1a is selected from hydrogen, halogen, methyl, methoxy, -OCF3 or -CF3; more preferably, R 1a is selected from hydrogen, F, Cl, methyl- and -CF3; further preferably, R 1a is selected from hydrogen, F and methyl.

17. The ligand-drug conjugate according to any one of claims 4 to 10 or the compound according to any one of claims 12, 14 to 15, or its tautomers, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts or solvates, wherein: R 2a Selected from hydrogen, halogen and -C 1-6 Alkyl; preferably, R 2a is selected from hydrogen, F or Cl; further preferably, R 2a Selected from hydrogen or F.

18. The ligand-drug conjugate according to any one of claims 4 to 10 or the compound according to any one of claims 12, 14 to 15, or its tautomers, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts or solvates, wherein: R 1a With R 2a and the atoms to which it is attached together form a 5-membered heterocyclic ring containing one or two heteroatoms independently selected from O and N; preferably, the 5-membered heterocyclic ring is selected from Further preferably, the 5-membered heterocyclic ring is 19. The ligand-drug conjugate according to any one of claims 4 to 10 or the compound according to any one of claims 12, 14 to 15, or its tautomers, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts or solvates, wherein: R 1b Selected from hydrogen, F.

20. The ligand-drug conjugate according to any one of claims 4 to 10 or the compound according to any one of claims 12, 14 to 15, or its tautomers, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts or solvates, wherein: R 2b Selected from F, Cl, methyl and -CF3.

21. The ligand-drug conjugate or compound according to any one of claims 2, 3, 5, 6-10 and 13-20, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein L 2 =CHR L21 ) n -; in, n is a natural number from 0 to 50; L 2 Any CHR in L21 The units can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -、-O-、 -Cy- is phenylene, 5- to 6-membered heteroarylene, 4- to 10-membered heterocyclylene, or 3- to 6-membered cycloalkylene, wherein the -Cy- is unsubstituted or independently substituted by 1 to 3 R cx replace; Each R L21 , R L22 , R cx are each independently selected from hydrogen, halogen, -OR L2a 、-N(R L2a )2. -C(O)R L2a 、-S(O)2R L2a 、-C(O)N(R L2a )2、-SO2N(R L2a )2、-N(R L2a )SO2R L2b 、-N(R L2a )COR L2b 、-(CH2) y -CO-(N(Me)CH2C(O)) m -OR L2a 、-(CH2) y -CO-(N(Me)CH2C(O)) m -NHR L2a 、-(CH2) y -CONH-(CH2CH2O) m -R L2a 、-(CH2) y -NHCO-(CH2CH2O) m -R L2a 、-(CH2) y -NHCOCH2(OCH2CH2)OR L2a 、-(CH2) y -NH(COCH2(N(Me)) m -R L2a 、-(CH2) y -NHCO-(CH2CH2O) m -R L2a 、-(CH2CH2O) m -R L2a 、-(COCH2N(Me)) m -R L2a 、-COCH2(OCH2CH2) m -OR L2a 、-CO-(CH2CH2O) m -R L2a , or by R L2a Optionally substituted -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; m is a natural number from 0 to 8; y is 0, 1, 2, 3, or 4; Each R L2a , R L2b are each independently selected from hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2, -C 1-6 alkyl.

22. The ligand-drug conjugate or compound according to any one of claims 2, 3, 5, 6-10 and 13-20, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: L 2 -(CH2) n -; Where n is a natural number from 0 to 50; L 2 Any methylene unit in the can be independently replaced by the following structural units: 4 to 6 membered heterocyclyl, 3 to 6 membered cycloalkyl, -C(O)-, -NR L22 -、-O-、 Each R L22 are each 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 Optionally substituted -C 1-6 alkyl; m is a natural number from 0 to 8; y is 0, 1, 2, 3, or 4; Each R L2a are each independently selected from hydrogen, halogen, -CN, -OH, -NH2, -N(Me)2, -CO2H, -C(O)NH2, -C 1-6 alkyl.

23. The ligand-drug conjugate or compound according to any one of claims 2, 3, 5, 6-10 and 13-20, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: L 2 Selected from: in, n1, n2, n3, n4, m are each independently selected from natural numbers from 0 to 8; n5 and n6 are each independently selected from 0 or 1; -Cy- is a 4- to 6-membered heterocyclylene group or a 3- to 6-membered cycloalkylene group; preferably, -Cy- is Further preferably, -Cy- is Preferably, for More preferably, for Among them, the c-terminus and L 1 or L 1a Connected, d end and L 3 connected.

24. A compound as defined in any one of claims 13 to 20, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Selected from: n1, n2, n3, n4, m are each independently selected from natural numbers from 0 to 8; n5 and n6 are each independently selected from 0 or 1; -Cy- is a 4- to 6-membered heterocyclylene or a 3- to 6-membered cycloalkylene; preferably, -Cy- is selected from: Further preferably, -Cy- is selected from Preferably, Selected from: More preferably, Selected from:

25. The ligand-drug conjugate or compound according to any one of claims 2-3, 5-10 and 13-24, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein L 3 Selected from Val, D-Val, Phe, Lys, Leu, Ile, Gly, Ala, D-Ala, Cit, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Leu-Lys, Leu-Lys(Ac), Ala-Ala, Ala-Lys, 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 fragments; Preferably, L 3 is 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 fragments; 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 fragments; More preferably, L 3 Selected from Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asp-Asp, Asp-Glu, Gly-Gly-Phe-Gly or 26. A compound as defined in claim 24, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Selected from: Preferably, Selected from:

27. The ligand-drug conjugate or compound according to any one of claims 2-3, 5-10, 16-23 and 25, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein Selected from: More preferably, Selected from:

28. The ligand-drug conjugate or compound according to any one of claims 2-3, 5-10, 16-23 and 25, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein Selected from: Preferably, Selected from 29. The ligand-drug conjugate or compound according to any one of claims 2-3, 5-10 and 13-28, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Tr does not exist or is in R Tr , R Tr1 and R Tr2 independently selected from hydrogen, halogen, -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; z is a natural number from 0 to 8; Preferably, Tr does not exist or is 30. The compound according to claim 11 or 12, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, selected from:

31. A compound according to claim 11 or 12, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: The compound is selected from:

32. A compound according to claim 13 or 14, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: The compound is selected from:

33. The compound according to claim 13, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein: The compound is selected from:

34. The ligand-drug conjugate according to claim 3 or 6, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein: The ligand-drug conjugate is selected from: wherein Ab and q are as defined in claim 3.

35. The ligand-drug conjugate according to claim 3 or 6, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein: The ligand-drug conjugate is selected from: wherein Ab and q are as defined in claim 3.

36. The ligand-drug conjugate according to claim 3 or 6, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein: The ligand-drug conjugate is selected from: wherein q is as defined in claim 3.

37. A pharmaceutical composition comprising the ligand-drug conjugate or compound as described in any one of claims 1 to 36, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, and a pharmaceutically acceptable carrier.

38. A pharmaceutical preparation comprising the ligand-drug conjugate or compound as claimed in any one of claims 1 to 36 or its tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

39. Use of the ligand-drug conjugate or compound as described in any one of claims 1 to 36, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, or the pharmaceutical composition as described in claim 37, or the pharmaceutical preparation as described in claim 38 in the preparation of a drug for preventing or treating a disease associated with abnormal cell activity.

40. The use according to claim 39, wherein the disease associated with abnormal cell activity is cancer.

41. The method of claim 40, wherein the cancer is a solid tumor or a non-solid tumor, for example, selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.