Linker-drug molecule and antibody-drug conjugate, and preparation method and application of linker-drug molecule and antibody-drug conjugate

Abstract

The invention discloses a compound represented by a formula (1), or a tautomer, a mesomer, a raceme, an enantiomer, a diastereoisomer, or a mixture form thereof, or a pharmaceutically acceptable salt, a prodrug or a solvate thereof, the structure of the formula (1) is represented as follows: # imgabs0. Strong hydrophilic sulfonic acid inner salt and / or phosphoric acid inner salt side chains are introduced at the position of (AAA and the like)-PAB (self-destruction group), so that the druggability (including hydrophilicity and stability) of the antibody drug conjugate corresponding to the compound is greatly enhanced, the in-vivo drug removal effect is reduced, and the tumor treatment effect is improved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a linker-drug molecule and an antibody-drug conjugate, and a preparation method and application thereof.

[0002] Cross-references

[0003] This application claims the benefit of PCT International Application No. PCT / CN2023 / 106385, filed on July 7, 2023, the contents of which are incorporated herein in their entirety. Background Art

[0004] By coupling biologically active drug compounds to tumor-targeting antibodies, proteins or peptides, the cancer treatment window of drug compounds can be increased. This is mainly achieved in two aspects. First, the drug conjugate cannot produce toxic side effects before reaching the cancer target lesions because the active drug cannot be smoothly detached. Second, the active drug can increase the concentration of the drug compound at the cancer target lesion through the coupled targeting antibody, protein or peptide, and the specially designed linker can be stimulated by the special environment of the cancer target lesion to break and release the active drug compound.

[0005] The specially designed cathepsin-sensitive amino acid peptides in the linker can be efficiently cleaved by the cathepsins in the lysosomes of cancer cells. The drug compound is connected to the amino acid peptide of the linker through a chemical self-destructing group. After the cathepsin cuts the peptide, the chemical self-destructing group releases the free drug compound through the "self-destructing effect", thereby producing a tumor-killing effect. Among them, the antibody or peptide drug conjugates with linkers in the form of peptide (VC, VA, AAA, etc.)-PAB (self-destructing group) have the most clinical validation data and approved drugs. However, since drug compounds, especially those with "bystander effects" that are important for tumor killing, have strong lipophilic characteristics, antibody, protein or peptide drug conjugates, especially when the drug ratio (DAR) is high, have poor drugability, show low hydrophilicity, form aggregates, and accelerate clearance in the body. By adding hydrophilic groups to the linker, such as polyethylene glycol (PEG) (Mol Cancer Ther, 2017, 16 (1): 116-123; Nat. Biotech. 2015, 33: 733-735, Toxicol. App. Pharmacol., 2020, 392: 114932; WO2023280227) polysarcosine (PSAR) (Chem. Sci, 2019, 10 (14): 4048-4053; WO2022228493) sugar alcohol (Cancer Res (2018) 78 (13_Supplement): 755; US 9907854), etc. to shield the drug compound, the drugability of the antibody, protein or peptide drug conjugate can be improved to a certain extent. However, the use of PEG polymers to modify proteins has exposed many shortcomings in clinical practice, such as a high proportion of patients with anti-PEG immunogenicity and related toxic side effects caused by it, and excessive modification leading to reduced protein function. Summary of the invention

[0006] Based on this, the present application introduces a strongly hydrophilic sulfonic acid inner salt and / or phosphate inner salt side chain (hereinafter sometimes referred to as the linker or inner salt linker of the present application, i.e., the compound represented by formula (1) described below) at the polypeptide (VC, VA, AAA, etc.)-PAB (self-destructive group) position of the linker. Since the sulfonic acid group or the phosphate group has both a hydrogen bond donor and a hydrogen bond acceptor, it is a strongly hydrophilic group, and the sulfonic acid inner salt or the phosphate inner salt has both a positive charge and a negative charge, the hydrophilicity can be further increased. At the same time, the water-soluble group in the form of the inner salt also has the potential to enhance the tumor targeting of the antibody drug conjugate and reduce nonspecific binding, thereby greatly enhancing the drugability (including hydrophilicity and stability) of the test antibody drug conjugate, reducing the drug clearance effect in the body, and improving the tumor treatment effect.

[0007] The technical solution of this application is as follows:

[0008] 1. A compound represented by formula (1), or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the structure of formula (1) is as follows:

[0009]

[0010] M is a linking group; Sp is -L a -Q a -; A is selected from non-existent, hydrogen, alkyl; T represents a linear or branched (C1-C8) trivalent alkyl group; Y, Y', Y" are -L b -Q b -;L a , L b are respectively selected from absence, alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene, polyethylene glycol group is optionally substituted with a substituent;

[0011] Q a , Q b are respectively selected from absent, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -S(=O)2NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, -O-, -NR-, -S-, S(=O)2-, S(=O)-, or -Se-, wherein R is selected from hydrogen or optionally substituted alkyl;

[0012] Z is selected from absent, hydrogen, optionally substituted alkyl or sulfonate;

[0013] Z', Z" are selected from absent, hydrogen, optionally substituted alkyl, sulfonate or phosphate;

[0014] At least one of Z, Z', and Z" is a sulfonic acid inner salt or a phosphate inner salt;

[0015] X comprises a self-destruct unit or does not exist; D comprises a drug molecule; i is an integer greater than or equal to 1; h is an integer from 1 to 6; o, p, and q are integers from 0 to 5, respectively.

[0016] 2. The compound according to item 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (1) is formula (1-1):

[0017]

[0018] Wherein, M, Sp, A, T, Y, h, o, i, X, D, and Z are defined as in item 1;

[0019] At least one of the i Zs is a sulfonate.

[0020] 3. The compound according to item 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (1-1) is formula (1-1-1):

[0021]

[0022] Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

[0023] 4. The compound according to item 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (1) is formula (1-2):

[0024]

[0025] Wherein, M, Sp, A, T, Y, Z, o, p, i, h, X, Y', and D are defined as in item 1;

[0026] Z' is a sulfonic acid inner salt or a phosphoric acid inner salt.

[0027] 5. The compound according to item 4, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (1) is formula (1-2-1):

[0028]

[0029] Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

[0030] 6. The compound according to item 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (1) is formula (1-3):

[0031]

[0032] Wherein, M, Sp, A, Y, T, Z, o, i, h, q, X, Y”, and D are defined as in item 1;

[0033] Z" is a sulfonic acid inner salt or a phosphoric acid inner salt.

[0034] 7. The compound according to item 6, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (1) is formula (1-3-1):

[0035]

[0036] Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

[0037] 8. The compound according to any one of items 1 to 7, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein when A and Z are both absent, N, Y, and T form a ring, that is

[0038] Preferably

[0039] 9. A compound according to any one of items 3, 5 or 7, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the amino acid is selected from one or more of alanine, arginine, asparagine, aspartic acid, γ-carboxyglutamate, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0040] 10. A compound according to any one of items 3, 5 or 7, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the amino acid is selected from one or more of valine, glutamic acid, citrulline, lysine, alanine, phenylalanine, arginine, glutamine, asparagine and glycine.

[0041] 11. A compound according to any one of items 3, 5 or 7, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the amino acid is selected from one or more of valine, citrulline, alanine, phenylalanine, glutamine and glycine.

[0042] 12. A compound according to any one of items 1 to 11, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein:

[0043] M is selected from N3, SH, ONH2, NH2, CO2H, (alkyl)-CO-, HCO-, BrCH 2- 、ICH2-、

[0044] 13. A compound according to any one of items 1 to 12, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein:

[0045] M is

[0046] 14. A compound according to any one of items 1 to 13, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein:

[0047] M is

[0048] 15. A compound according to any one of items 1 to 14, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein A is hydrogen, methyl, ethyl or propyl.

[0049] 16. A compound according to any one of items 1 to 15, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein A is hydrogen.

[0050] 17. A compound according to any one of items 1 to 16, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein T is one of the following structures:

[0051]

[0052] 18. The compound according to any one of items 1 to 17, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein T is

[0053] 19. The compound according to any one of items 1 to 18, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a One, two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol group; the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0054] 20. The compound according to any one of items 1 to 19, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a Selected from C1-C 10 Straight or branched alkylene, C2-C 12 Straight or branched alkenylene, C2-C 12One, two or more of a straight or branched alkynylene group, a saturated or unsaturated alicyclic group having 3-6 ring carbon atoms, a saturated or unsaturated heteroalicyclic group having 3-6 ring carbon atoms and 1 to 4 heteroatoms, or an arylene group having 6 to 10 carbon atoms in one or more rings, a heteroarylene group having 6 to 10 carbon atoms in one or more rings and 1 to 4 heteroatoms, or a polyethylene glycol group, wherein the alkylene group, alkenylene group, alkynylene group, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0055] 21. A compound according to any one of items 1 to 20, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein L a One, two or more selected from C1-C6 straight chain or branched alkylene, C2-C6 straight chain or branched alkenylene, C2-C6 straight chain or branched alkynylene, or polyethylene glycol group, wherein the alkylene, alkenylene and alkynylene are optionally substituted by a substituent.

[0056] 22. A compound according to any one of items 1 to 21, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein L a is one, two or more than three of a C1-C6 straight or branched alkylene group or a polyethylene glycol group,

[0057] The polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-,

[0058] -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n CH2-, n is an integer greater than or equal to 1.

[0059] 23. A compound according to any one of items 1 to 22, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein L a is ethylene, straight-chain propylene, straight-chain butylene, straight-chain pentylene, or -(CH2CH2O) nOne, two or more of CH2CH2-, wherein n is 2, 3, 4, 5, 6, 7 or 8.

[0060] 24. A compound according to any one of items 1 to 23, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein L a It is ethylene and / or -(CH2CH2O)4CH2CH2- or pentylene.

[0061] 25. The compound according to any one of items 1 to 24, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b One or two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0062] 26. The compound according to any one of items 1 to 25, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b Selected from C1-C 10 Straight or branched alkylene, C2-C 12 Straight or branched alkenylene, C2-C 12 One or two or more of a straight or branched alkynylene group, a saturated or unsaturated alicyclic group having 3-6 ring carbon atoms, a saturated or unsaturated heteroalicyclic group having 3-6 ring carbon atoms and 1 to 4 heteroatoms, or an arylene group having 6 to 10 carbon atoms in one or more rings, a heteroarylene group having 6 to 10 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol, wherein the alkylene group, alkenylene group, alkynylene group, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0063] 27. The compound according to any one of items 1 to 26, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is selected from C1-C6 straight chain or branched chain alkylene, C2-C6 straight chain or branched chain alkenylene, or C2-C6 straight chain or branched chain alkynylene, or polyethylene glycol group,

[0064] The polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-,

[0065] -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n One or two or more than three of CH2-, n is an integer greater than or equal to 1, and the alkylene, alkenylene and alkynylene groups are optionally substituted by a substituent.

[0066] 28. The compound according to any one of items 1 to 27, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is a C1-C6 straight or branched chain alkylene group, or a C6-C 10 One or two or more than three of the arylene groups.

[0067] 29. The compound according to any one of items 1 to 28, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b It is one or two or more of methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene, or benzylene.

[0068] 30. A compound according to any one of items 1 to 29, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein Q aOne, two or more selected from -C(=O)NR-, -NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, wherein R is selected from hydrogen, optionally substituted C1-C6 straight chain or branched alkyl.

[0069] 31. The compound according to item 30, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q a It is one or two or more of -C(=O)-, -NR-, -C(=O)NR-, -NRC(=O)NR-, -OC(=O)O-, -OC(=O)NR-, -NRC(=O)- or -OC(=O)-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight chain or branched alkyl.

[0070] 32. The compound according to item 30, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q a It is one or two or more of -C(=O)-, -NR-, -NRC(=O)- and -(C=O)NR-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight or branched alkyl group.

[0071] 33. The compound according to item 30, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q a It is -C(=O)-, -N(CH3)-, -NHC(=O)-, -C(=O)-NH- or -C(=O)-N(CH3)-.

[0072] 34. A compound according to any one of items 1 to 33, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein Q bis selected from one, two or more of the group consisting of absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, and -OC(=O)O-, wherein R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl.

[0073] 35. The compound according to item 34, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q b is absent, one or two or more of -C(=O)-, -C(=O)NR-, -OC(=O)NR-, -NRC(=O)-, -NRC(=O)O-, or -NRC(=O)NR-, and R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl.

[0074] 36. The compound according to item 34, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q b is absent, -C(=O)-, -NRC(=O)-, -NRC(=O)NR- or -C(=O)NR-, or two or more thereof; and R is hydrogen or methyl.

[0075] 37. The compound according to any one of items 1 to 36, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein X is selected from:

[0076]

[0077] The right wavy line in X connects D; R4 is selected from hydrogen, C1-C6 straight chain or branched alkyl, -(CH2CH2O) m R2, -CH2CH2NR2R3, -CH2CH2SO2R2, R2, R3 are independently selected from hydrogen, C1-C6 straight chain or branched alkyl, and m is an integer greater than 1.

[0078] 38. The compound according to item 37, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0079] X is

[0080] R4 is hydrogen, methyl, -CH2CH2OCH2CH2OH, -CH2CH2NMe2, -CH2CH2SO2Me.

[0081] 39. The compound according to item 37, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0082] X is R4 is hydrogen or methyl.

[0083] 40. A compound according to any one of items 1 to 39, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the sulfonic acid inner salt is

[0084]

[0085] A wavy line connects Y, Y' or Y"; c, d, e, f, g are all integers of 1, 2, 3, 4, 5 or 6;

[0086] R6, R7, R 11 , R 12 , R 13 , R 14 are independently selected from absence, hydrogen, and C1-C6 straight or branched chain alkyl.

[0087] 41. The compound according to item 40, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the sulfonic acid inner salt is

[0088] in,

[0089] R6, R7, R 11 , R 12 , R 13 and R 14 are selected from absence, methyl or ethyl, c, d, e, f, g are 1, 2 or 3 respectively.

[0090] 42. The compound according to item 40, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the sulfonic acid inner salt is one of the following structures:

[0091]

[0092] 43. A compound according to any one of items 1 to 42, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the phosphate inner salt is

[0093] in,

[0094] a and b are both integers of 1, 2, 3, 4, 5 or 6; R8, R9 and R 10 are selected from hydrogen, C1-C6 straight chain or branched alkyl.

[0095] 44. The compound according to item 43, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0096] The phosphate inner salt is

[0097] Among them, R8, R9, R 10 They are methyl or ethyl respectively; a is 1 or 2 or 3; b is 1 or 2 or 3.

[0098] 45. The compound according to item 43, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0099] The phosphate inner salt is

[0100] Among them, R8, R9, R 10 All are methyl; a is 2; b is 1.

[0101] 46. ​​A compound according to any one of items 1 to 45, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein D comprises a cytotoxin or an immunoagonist.

[0102] 47. A compound according to item 46, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the cytotoxin is selected from one or two of a microtubule inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor or an RNA polymerase 2 inhibitor.

[0103] 48. The compound according to item 46, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0104] The cytotoxin is selected from auristatins, maytansine alkaloids, calicheamicin, duocarmycin, pyrrolobenzodiazepines, One or more of the group consisting of camptothecin derivatives, camptothecin derivatives and α-amanitin.

[0105] 49. The compound according to item 46, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0106] The immune agonist is selected from TLR7 / 8 agonist or STING agonist.

[0107] 50. A compound according to any one of items 1-49, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein i is 2, 3, 4, 5, 6, 7 or 8.

[0108] 51. A compound according to any one of items 1-50, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein h is 1, 2, 3 or 4.

[0109] 52. A compound according to any one of items 1 to 51, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein o, p, q are 0, 1, 2 or 3, respectively.

[0110] 53. The compound according to any one of items 1 to 52, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a It is one, two or more of C1-C6 straight or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-、-(CH2CH2O) nCH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n CH2-, n is an integer greater than or equal to 1; Q a It is one or two or more of -C(=O)-, -NR-, -NR(C=O)- and -(C=O)NR-, wherein R is selected from hydrogen, C1-C6 straight chain or branched alkyl.

[0111] 54. The compound according to item 53, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a is ethylene, straight-chain propylene, straight-chain butylene, straight-chain pentylene, or -(CH2CH2O) n One, two or more of CH2CH2-, wherein n is 2, 3, 4, 5, 6, 7 or 8; Q a It is one, two, or more than three of -C(=O)-, -NR-, -NR(C=O)-, and -(C=O)NR-, wherein R is methyl and / or hydrogen.

[0112] 55. The compound according to item 53, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0113] L a is ethylene and / or -(CH2CH2O)4CH2CH2- or pentylene; Q a It is -C(=O)-, -N(CH3)-, -NH(C=O)-, -C(=O)NH- or -C(=O)-N(CH3)-.

[0114] 56. The compound according to any one of items 1 to 55, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is selected from a C1-C6 straight chain or branched alkylene group, a C2-C6 straight chain or branched alkenylene group, or a C2-C6 straight chain or branched alkynylene group, an arylene group having 6 to 10 carbon atoms in one or more rings, or a polyethylene glycol group, wherein the polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-、-(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O)n -, or -CH2O(CH2CH2O) n One or two or more than three of CH2-, n is an integer greater than or equal to 1, and the alkylene, alkenylene and alkynylene groups are optionally substituted by substituents; Q b is selected from one, two or more of the group consisting of absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -C(=O)-, and -OC(=O)O-, and R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl group.

[0115] 57. The compound according to item 56, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is a C1-C6 straight or branched chain alkylene group, or a C6-C 10 One or two or more than three kinds of arylene groups; Q b It is absent, one or two or more of -C(=O)-, -C(=O)NR-, -OC(=O)NR-, -NRC(=O)-, -NRC(=O)O-, or -NRC(=O)NR-, and R is selected from hydrogen, a C1-C6 straight chain or branched alkyl group.

[0116] 58. The compound according to item 56, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b Q is one or two or more of methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene or benzylene; b is absent, -C(=O)-, -NRC(=O)-, -NRC(=O)NR- or -C(=O)NR-, or two or more thereof; and R is hydrogen or methyl.

[0117] 59. A compound according to any one of items 1 to 58, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the substituent is selected from deuterium, tritium, a halogen atom, an amino group, a hydroxyl group, a cyano group, a nitro group, an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an optionally substituted heterocyclic group, an acyl group, an optionally substituted amino group, an optionally substituted carbamoyl group, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group, an optionally substituted hydroxyl group, an optionally substituted sulfanyl group (SH) and an optionally substituted silyl group.

[0118] 60. A compound according to any one of items 1 to 59, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the compound is selected from the following structures:

[0119]

[0120]

[0121]

[0122]

[0123] 61. A compound represented by formula (2), or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the structure of formula (2) is represented as follows:

[0124]

[0125] Ab comprises an antibody or an antigen-binding fragment thereof; M' is a linking group; Sp is -L a -Q a -; A is selected from non-existent, hydrogen, alkyl; T represents a linear or branched (C1-C8) trivalent alkyl group; Y, Y', Y" are -L b -Q b -;L a , L b Q is respectively selected from absence, alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group is optionally substituted with a substituent; Q a , Q bare respectively selected from the group consisting of absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -S(=O)2NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, -O-, -NR-, -S-, S(=O)2-, S(=O)-, or -Se-, wherein R is selected from hydrogen or an optionally substituted alkyl group; Z is selected from non-existence, hydrogen, an optionally substituted alkyl group or a sulfonate; Z' and Z" are selected from non-existence, hydrogen, an optionally substituted alkyl group, a sulfonate or a phosphate; at least one of Z, Z' and Z" is a sulfonate or a phosphate; X comprises a self-destruct unit or does not exist; D comprises a drug molecule; i is an integer greater than or equal to 1; h is an integer from 1 to 6; o, p and q are integers from 0 to 5 respectively; and j is an integer greater than or equal to 1.

[0126] 62. The compound according to item 61, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (2) is formula (2-1):

[0127]

[0128] wherein Ab, M', Sp, A, T, Y, h, o, i, j, X, D, and Z are defined as in item 61; and at least one of the i Zs is a sulfonate.

[0129] 63. The compound according to item 62, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (2-1) is formula (2-1-1):

[0130]

[0131] Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

[0132] 64. The compound according to item 61, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (2) is formula (2-2):

[0133]

[0134] wherein Ab, M', Sp, A, T, Y, Z, o, p, i, h, j, X, Y', and D are as defined in item 61; and Z' is a sulfonic acid salt or a phosphate salt.

[0135] 65. The compound according to item 64, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (2-2) is formula (2-2-1):

[0136]

[0137] Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

[0138] 66. The compound according to item 61, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (2) is formula (2-3):

[0139]

[0140] wherein Ab, M', Sp, A, Y, T, Z, o, i, h, j, q, X, Y", and D are as defined in item 61; and Z" is a sulfonic acid salt or a phosphate salt.

[0141] 67. The compound according to item 66, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the formula (2-3) is formula (2-3-1):

[0142]

[0143] Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

[0144] 68. The compound according to any one of items 61 to 67, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein, when A and Z are both absent, N, Y, and T form a ring, namely

[0145] Preferably

[0146] 69. A compound according to any one of items 63, 65 or 67, or its tautomer, racemate, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the amino acid is selected from one or more of alanine, arginine, asparagine, aspartic acid, γ-carboxyglutamate, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0147] 70. A compound according to any one of items 63, 65 or 67, or its tautomer, racemate, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the amino acid is selected from one or more of valine, glutamic acid, citrulline, lysine, alanine, phenylalanine, arginine, glutamine, asparagine and glycine.

[0148] 71. A compound according to any one of items 63, 65 or 67, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the amino acid is selected from one or more of valine, citrulline, alanine, phenylalanine, glutamine and glycine.

[0149] 72. A compound according to any one of items 61 to 71, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein M' is selected from one of the following structures:

[0150]

[0151] * Connect Ab.

[0152] 73. A compound according to any one of items 61 to 72, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein M' is selected from one of the following structures:

[0153]

[0154] 74. A compound according to any one of items 61 to 73, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein:

[0155] M' is

[0156] 75. A compound according to any one of items 61 to 74, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein A is hydrogen, methyl, ethyl or propyl.

[0157] 76. A compound according to any one of items 61 to 75, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein A is hydrogen.

[0158] 77. A compound according to any one of items 61 to 76, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein T is one of the following structures:

[0159]

[0160] 78. A compound according to any one of items 61 to 77, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein:

[0161] The T is

[0162] 79. The compound according to any one of items 61 to 78, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a One, two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol group; the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0163] 80. The compound according to any one of items 61 to 79, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a Selected from C1-C 10 Straight or branched alkylene, C2-C 12 Straight or branched alkenylene, C2-C 12 One, two or more of a straight or branched alkynylene group, a saturated or unsaturated alicyclic group having 3-6 ring carbon atoms, a saturated or unsaturated heteroalicyclic group having 3-6 ring carbon atoms and 1 to 4 heteroatoms, or an arylene group having 6 to 10 carbon atoms in one or more rings, a heteroarylene group having 6 to 10 carbon atoms in one or more rings and 1 to 4 heteroatoms, or a polyethylene glycol group, wherein the alkylene group, alkenylene group, alkynylene group, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0164] 81. The compound according to any one of items 61 to 80, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a One, two or more selected from C1-C6 straight chain or branched alkylene, C2-C6 straight chain or branched alkenylene, C2-C6 straight chain or branched alkynylene, or polyethylene glycol group, wherein the alkylene, alkenylene and alkynylene are optionally substituted by a substituent.

[0165] 82. The compound according to any one of items 61 to 81, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a is one, two or more than three of a C1-C6 straight or branched alkylene group or a polyethylene glycol group,

[0166] The polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-,

[0167] -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n CH2-, n is an integer greater than or equal to 1.

[0168] 83. The compound according to any one of items 61 to 82, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a is ethylene, straight-chain propylene, straight-chain butylene, straight-chain pentylene, or -(CH2CH2O) n One, two or more of CH2CH2-, wherein n is 2, 3, 4, 5, 6, 7 or 8.

[0169] 84. The compound according to any one of items 61 to 83, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a It is ethylene and / or -(CH2CH2O)4CH2CH2- or pentylene.

[0170] 85. The compound according to any one of items 61 to 84, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b One or two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0171] 86. The compound according to any one of items 61 to 85, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b Selected from C1-C 10 Straight or branched alkylene, C2-C 12 Straight or branched alkenylene, C2-C 12One or two or more of a straight or branched alkynylene group, a saturated or unsaturated alicyclic group having 3-6 ring carbon atoms, a saturated or unsaturated heteroalicyclic group having 3-6 ring carbon atoms and 1 to 4 heteroatoms, or an arylene group having 6 to 10 carbon atoms in one or more rings, a heteroarylene group having 6 to 10 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol, wherein the alkylene group, alkenylene group, alkynylene group, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0172] 87. The compound according to any one of items 61 to 86, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is selected from C1-C6 straight chain or branched alkylene, C2-C6 straight chain or branched alkenylene, or C2-C6 straight chain or branched alkynylene, or polyethylene glycol group, wherein the polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-、-(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n One or two or more than three of CH2-, n is an integer greater than or equal to 1, and the alkylene, alkenylene and alkynylene groups are optionally substituted by a substituent.

[0173] 88. The compound according to any one of items 61 to 87, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is a C1-C6 straight or branched chain alkylene group, or a C6-C 10 One or two or more than three of the arylene groups.

[0174] 89. The compound according to any one of items 61 to 88, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b It is one or two or more of methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene, or benzylene.

[0175] 90. The compound according to any one of items 61 to 89, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q a One, two or more selected from -C(=O)NR-, -NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, wherein R is selected from C1-C6 straight chain or branched alkyl optionally substituted with hydrogen.

[0176] 91. The compound according to item 90, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0177] Q a is -C(=O)-, -NR-, -C(=O)NR-, -NRC(=O)NR-, -OC(=O)O-, -OC(=O)NR-, -NRC(=O)-

[0178] or -OC(=O)-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight or branched alkyl.

[0179] 92. The compound according to item 90, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q a It is one or two or more of -C(=O)-, -NR-, -NRC(=O)- and -(C=O)NR-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight or branched alkyl group.

[0180] 93. The compound according to item 90, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q a It is -C(=O)-, -N(CH3)-, -NHC(=O)-, -C(=O)-NH- or -C(=O)-N(CH3)-.

[0181] 94. A compound according to any one of items 61 to 93, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein Qb is selected from one, two or more of the group consisting of absent, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, and -OC(=O)O-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight or branched alkyl group.

[0182] 95. The compound according to item 94, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q b is absent, -C(=O)-, -C(=O)NR-, -OC(=O)NR-, -NRC(=O)-, -NRC(=O)O-, or -NRC(=O)NR-, and R is selected from hydrogen or an optionally substituted C1-C6 straight or branched alkyl group.

[0183] 96. The compound according to item 94, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein Q b is absent, -C(=O), -NRC(=O)-, -NRC(=O)NR- or -C(=O)NR-, or two or more thereof; and R is hydrogen or methyl.

[0184] 97. The compound according to any one of items 61 to 96, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein X is selected from:

[0185]

[0186] The right wavy line in X connects D; R4 is selected from hydrogen, C1-C6 straight chain or branched alkyl, -(CH2CH2O) m R2, -CH2CH2NR2R3, -CH2CH2SO2R2-, R2, R3 are independently selected from hydrogen, C1-C6 straight chain or branched alkyl, and m is an integer greater than 1.

[0187] 98. The compound according to item 97, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0188] X is

[0189] R4 is hydrogen, methyl, -CH2CH2OCH2CH2OH, -CH2CH2NMe2, -CH2CH2SO2Me.

[0190] 99. The compound according to item 97, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0191] X is R4 is hydrogen or methyl.

[0192] 100. The compound according to any one of items 61 to 99, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the sulfonic acid inner salt is

[0193]

[0194] A wavy line connects Y, Y' or Y";

[0195] Wherein c, d, e, f, g are all integers of 1, 2, 3, 4, 5 or 6;

[0196] R6, R7, R 11 , R 12 , R 13 , R 14 are independently selected from absence, hydrogen, and C1-C6 straight or branched chain alkyl.

[0197] 101. The compound according to item 100, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the sulfonic acid inner salt is

[0198] Among them, R6, R7, R 11 , R 12 , R 13 and R 14 are selected from absence, methyl or ethyl, c, d, e, f, g are 1, 2 or 3 respectively.

[0199] 102. The compound according to item 100, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the sulfonic acid inner salt is one of the following structures:

[0200]

[0201] 103. A compound according to any one of items 61 to 102, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the phosphate inner salt is

[0202] Wherein, a and b are integers of 1, 2, 3, 4, 5 or 6, R8, R9 and R 10 are selected from hydrogen, C1-C6 straight chain or branched alkyl.

[0203] 104. The compound according to item 103, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the phosphate inner salt is

[0204] Among them, R8, R9, R 10 They are methyl or ethyl respectively; a is 1 or 2 or 3; b is 1 or 2 or 3.

[0205] 105. The compound according to item 103, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the phosphate inner salt is

[0206] Among them, R8, R9, R 10 All are methyl; a is 2; b is 1.

[0207] 106. A compound according to any one of items 61-105, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein D comprises a cytotoxin or an immunoagonist.

[0208] 107. A compound according to item 106, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the cytotoxin is selected from one or two of a microtubule inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor or an RNA polymerase 2 inhibitor.

[0209] 108. The compound according to item 106, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the cytotoxin is selected from auristatins, maytansine alkaloids DMs, calicheamicin, duocarmycin, pyrrolobenzodiazepines One or more of the group consisting of camptothecin derivatives, camptothecin derivatives and α-amanitin.

[0210] 109. A compound according to item 106, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the immune agonist is selected from a TLR7 / 8 agonist or a STING agonist.

[0211] 110. A compound according to any one of items 61-109, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein i is 2, 3, 4, 5, 6, 7 or 8.

[0212] 111. A compound according to any one of items 61-110, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein h is 1, 2, 3 or 4.

[0213] 112. A compound according to any one of items 61-111, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein o, p, q are 0, 1, 2 or 3 respectively.

[0214] 113. The compound according to any one of items 61 to 112, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a It is one, two or more of C1-C6 straight or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-,

[0215] -(CH2CH2O) nCH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n CH2-, n is an integer greater than or equal to 1; Q a It is one or two or more of -C(=O)-, -NR(C=O)- and -(C=O)NR-, wherein R is selected from hydrogen, C1-C6 straight chain or branched alkyl.

[0216] 114. The compound according to any one of items 61 to 113, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L a is ethylene, straight-chain propylene, straight-chain butylene, straight-chain pentylene, or -(CH2CH2O) n One, two or more of CH2CH2-, wherein n is 2, 3, 4, 5, 6, 7 or 8; Q a It is one, two, or more than three of -C(=O)-, -NR-, -NR(C=O)-, and -(C=O)NR-, wherein R is methyl and / or hydrogen.

[0217] 115. The compound according to item 114, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein:

[0218] L a is ethylene and / or -(CH2CH2O)4CH2CH2- or pentylene; Q a It is -C(=O)-, -N(CH3)-, -NH(C=O)-, -C(=O)NH- or -C(=O)-N(CH3)-.

[0219] 116. The compound according to any one of items 61 to 115, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is selected from a C1-C6 straight chain or branched alkylene group, a C2-C6 straight chain or branched alkenylene group, or a C2-C6 straight chain or branched alkynylene group, an arylene group having 6 to 10 carbon atoms in one or more rings, or a polyethylene glycol group, wherein the polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-、-(CH2CH2O) nCH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n One or two or more than three of CH2-, n is an integer greater than or equal to 1, and the alkylene, alkenylene and alkynylene groups are optionally substituted by substituents; Q b is selected from one, two or more of the group consisting of absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -C(=O)-, and -OC(=O)O-, and R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl group.

[0220] 117. The compound according to item 116, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b is a C1-C6 straight or branched chain alkylene group, or a C6-C 10 One or two or more than three kinds of arylene groups; Q b is absent, one or two or more of -C(=O)-, -C(=O)NR-, -OC(=O)NR-, -NRC(=O)-, -NRC(=O)O-, or -NRC(=O)NR-, and R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl.

[0221] 118. The compound according to item 116, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein L b Q is one or two or more of methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene or benzylene; b is absent, -C(=O)-, -NRC(=O)-, -NRC(=O)NR- or -C(=O)NR-, or two or more thereof; and R is hydrogen or methyl.

[0222] 119. A compound according to any one of items 61 to 118, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the substituent is selected from deuterium, tritium, a halogen atom, an amino group, a hydroxyl group, a cyano group, a nitro group, an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an optionally substituted heterocyclic group, an acyl group, an optionally substituted amino group, an optionally substituted carbamoyl group, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group, an optionally substituted hydroxyl group, an optionally substituted sulfanyl group (SH) and an optionally substituted silyl group.

[0223] 120. A compound according to any one of items 61 to 119, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein j is an integer from 1 to 20.

[0224] 121. A compound according to any one of items 61 to 120, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the compound is selected from the following structures:

[0225]

[0226]

[0227]

[0228]

[0229] 122. A compound according to any one of items 61-121, or its tautomer, racemate, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the antibody is selected from the following groups: murine antibody, chimeric antibody, humanized antibody and fully human antibody.

[0230] 123. A compound according to any one of items 61-122, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the antibody comprises a monoclonal antibody.

[0231] 124. A compound according to any one of items 61 to 123, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the antibody comprises a bispecific antibody.

[0232] 125. A compound according to any one of items 61-124, or its tautomer, racemate, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the antigen-binding fragment is selected from the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and VHH.

[0233] 126. A compound according to any one of items 61 to 125, or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the Ab is selected from the following group: anti-claudin 18.2 antibody, anti-human folate receptor antibody, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPC20 antibody, anti-CDH6 antibody, anti-EGFR antibody, anti-EGFRvⅢ antibody, anti-AXL antibody, anti-Nectin-4 antibody, anti-Tissue factor antibody, anti-TIM-1 antibody, anti-PSMA antibody, anti-EpCAM antibody, anti-MUC1 antibody, anti-STEAP1 antibody, anti-GPNMB antibody, anti-FGF2 antibody, anti-FOLR1 antibody, anti-c-MET antibody, anti-GFR antibody, anti-AGS-16, anti-Guanylyl cyclase C antibody, anti-Mesothelin antibody, anti-SLC44A4 antibody, anti-EphA2 antibody, anti-AGS-5 antibody, anti-GPC-3 antibody, anti-c-KIT antibody, anti-ROR1 antibody, anti-PD-L1 antibody, anti-CD27L antibody, anti-5T4 antibody, anti-Mucin 16 antibody, anti-NaPi2b antibody, anti-STEAP antibody, anti-SLITRK6 antibody, anti-ETBR antibody, anti-BCMA antibody, anti-CEACAM5 antibody, anti-SC-16 antibody, anti-SLC39A6 antibody, anti-Delta-like protein3 antibody, anti-Claudin18.2 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD45 antibody, anti-CD56 antibody, anti-CD66e antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD138 antibody, anti-CD147 antibody, anti-CD166 antibody, anti-CD223 antibody, anti-MUC16 antibody, anti-MSLN antibody, anti-ENPP3 antibody, anti-SLTRK6 antibody, anti-FGFR antibody, anti-LIV-1 antibody, anti-Lewis Y antibody, anti-av-integrin antibody, anti-ASCT2 antibody, anti-C4.4a antibody, anti-CA-IX antibody, anti-CD324 antibody, anti-CD352 antibody, anti-CD44v6 antibody, anti-CD48a antibody, anti-CLL-1 antibody, anti-Cripto antibody, anti-CS1 antibody, anti-DPEP3 antibody, anti-Ephrin-A2 antibody, anti-Ephrin-A4 antibody, anti-ETBR antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FLT3 antibody, anti-GD3 antibody, anti-GloboH antibody, anti-GPC3 antibody, anti-LAMP-1 antibody, anti-LRRC15 antibody, anti-Ly6E antibody, anti-MFI2 antibody, anti-NOTCH3 antibody, anti-p-cadherin antibody, anti-PRLR antibody and anti-RNF43 antibody, and antigen-binding fragments thereof.

[0234] 127. A compound according to any one of items 61 to 126, or its tautomer, racemate, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3 of Ab respectively comprise the heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3 of the antibody.

[0235] 128. A compound according to any one of items 61 to 127, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the heavy chain variable region VH and light chain variable region VL of the Ab respectively comprise the heavy chain variable region VH and light chain variable region VL of the antibody.

[0236] 129. A compound according to any one of items 61 to 128, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the heavy chain and light chain of Ab respectively comprise the heavy chain and light chain of an antibody.

[0237] 130. A compound according to any one of items 61 to 129, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the Ab is an anti-claudin 18.2 antibody or an antigen-binding fragment thereof.

[0238] 131. A compound according to any one of items 61 to 130, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the Ab is an anti-human folate receptor antibody or an antigen-binding fragment thereof.

[0239] 132. A pharmaceutical composition comprising a compound as described in any one of items 1 to 131, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof.

[0240] 133. Use of a compound as described in any one of items 1 to 132, or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof in the preparation of a medicament for treating and / or preventing tumors.

[0241] 134. The use according to item 133, wherein the tumor is selected from tumors associated with the expression of the following group of targets: claudin 18.2, human folate receptor, HER2, HER3, TROP2, B7-H3, GPC20, CDH6, EGFR, EGFRvⅢ, AXL, Nectin-4, Tissue factor, TIM-1, PSMA, EpCAM, MUC1, STEAP1, GPNMB, FGF2, FOLR1, c-MET, GFR, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CD138, CD147, CD166, CD223, MUC16, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, Lewis Y, av-integrin, ASCT2, C4.4a, CA-IX, CD324, CD352, CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2, Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, MFI2, NOTCH3, p-cadherin, PRLR, and RNF43.

[0242] 135. The use according to item 133, wherein tumors associated with the expression of the target include tumors with high expression of the target and / or tumors that are target positive.

[0243] 136. The use according to item 133, wherein the tumor is selected from solid tumors, hematological tumors and metastatic, refractory or recurrent lesions of cancer.

[0244] 137. The use according to claim 133, wherein the tumor is selected from the following group: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma and leukemia.

[0245] The antibody drug conjugate provided by the present application, compared with the anionic and cationic hydrophilic groups of PEG, the fluorophore-antibody drug conjugate modified by the water-soluble group in the form of sulfonic acid inner salt greatly increases the specific accumulation at the tumor site. The anti-conjugated drug of the present application introduces a strongly hydrophilic sulfonic acid inner salt and / or a phosphate inner salt side chain at the polypeptide (VC, VA, AAA, etc.)-PAB (self-destructive group) position of the linker. Since the sulfonic acid group or the phosphate group has both a hydrogen bond donor and a hydrogen bond acceptor, it belongs to a strongly hydrophilic group, and the sulfonic acid inner salt or the phosphate inner salt has both a positive charge and a negative charge, which can further increase the hydrophilic characteristics. At the same time, the water-soluble group in the form of inner salt also has the potential to enhance the tumor targeting of the antibody drug conjugate and reduce nonspecific binding, thereby greatly enhancing the drugability (including hydrophilicity, stability) of the test antibody drug conjugate, reducing the drug clearance effect in the body, and improving the tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0246] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application.

[0247] Figure 1 This is a molecular sieve exclusion chromatography (SEC) analysis diagram of the antibody drug conjugates of Comparative Example 2 and Experimental Example 6 provided in the present application.

[0248] Figure 2 This is a molecular sieve exclusion chromatography (SEC) analysis diagram of the antibody drug conjugates of Comparative Example 3 and Experimental Example 9 provided in the present application.

[0249] Figure 3 This is a molecular sieve exclusion chromatography (SEC) analysis chart of the antibody drug conjugates of Comparative Example 1 and Experimental Example 1, Experimental Example 5a, Experimental Example 7, Experimental Example 9 and Experimental Example 11 provided in the present application.

[0250] Figure 4 This is a hydrophobic interaction chromatography (HIC) analysis diagram of the antibody drug conjugates of Comparative Example 2 and Test Example 6 provided in the present application.

[0251] Figure 5This is a hydrophobic interaction chromatography (HIC) analysis diagram of the antibody drug conjugates of Comparative Example 3 and Test Example 9 provided in the present application.

[0252] Figure 6 This is a hydrophobic interaction chromatography (HIC) analysis chart of the antibody drug conjugates of Comparative Example 1, Test Example 1, Test Example 5a, Test Example 7, Test Example 9 and Test Example 11 provided in the present application.

[0253] Figure 7 This is the deconvoluted mass spectrum of the light chain of Experimental Example 5a provided in this application.

[0254] Figure 8 This is the deconvoluted mass spectrum of the heavy chain of Experimental Example 5a provided in this application.

[0255] Fig. 9 This is the reverse chromatogram of Experimental Example 5a provided in this application.

[0256] Fig.10 This is the in vitro cell killing curve of the 9D1 antibody-drug conjugate provided in the present application and the comparative sample on MC38-18.2 cells.

[0257] Fig.11 This is the tumor therapeutic effect curve of the 9D1 antibody-drug conjugate provided in the present application on the MC38-hClaudin18.2 mouse subcutaneous tumor model.

[0258] Fig.12 This is the tumor therapeutic effect curve of the 9D1 antibody-drug conjugate provided in the present application on the NUGC4-hClaudin18.2 mouse subcutaneous tumor model.

[0259] Fig.13 This is the tumor treatment effect curve of the Farletuzumab-LD-38 antibody drug conjugate provided in this application on the OVCAR-3 mouse subcutaneous tumor model.

[0260] Fig.14 This is the tumor treatment effect curve of the Farletuzumab-LD-38 antibody drug conjugate provided in this application on the IGROV1 mouse subcutaneous tumor model.

[0261] Fig.15 The pharmacokinetic curves of 9D1-LD-38 and 9D1-GGFG-Exatecan in rats provided in this application.

[0262] Fig.16 The killing activity of 9D1-LD-38, 9D1-LD-82, 9D1-LD-88 and 9D1-LD-89 against MC38-hClaudin18.2 cells.

[0263] Fig.17 Results showed that 9D1-LD-38 and 9D1-GGFG-Dxd induced immunogenic cell death in B16F10-hClaudin18.2 cells.

[0264] Fig.18 This is the bystander effect of 9D1-LD-38 and 9D1-GGFG-Dxd on HEK293-hClaudin18.2 / HEK293 cells.

[0265] Fig.19 This is the Exatecan result of releasing free toxin after 9D1-LD-38 digestion.

[0266] Fig. 20 These are the in vitro serum incubation stability results of 9D1-LD-38, 9D1-LD-65, 9D1-LD-73, 9D1-LD-74 and 9D1-Deruxtecan (9D1-GGFG-Dxd).

[0267] Fig.21 The results of the in vivo DAR value changes of 9D1-LD-38, 9D1-LD-65, and 9D1-Deruxtecan (9D1-GGFG-Dxd) are shown.

[0268] Fig. 22 The contents of total toxin (bound toxin + free toxin) and free toxin in the plasma of tumor-bearing mice administered 9D1-LD-38, 9D1-LD-65, and 9D1-Deruxtecan (9D1-GGFG-Dxd).

[0269] Fig.23 The contents of total toxin (bound toxin + free toxin) and free toxin in the tissues of tumor-bearing mice administered 9D1-LD-38, 9D1-LD-65, and 9D1-Deruxtecan (9D1-GGFG-Dxd).

[0270] Fig.24 These are the pharmacodynamic results of 9D1-LD-38, 9D1-Deruxtecan (9D1-GGFG-Dxd) and 9D1-vc-PAB-MMAE in the p-gp-mediated drug-resistant tumor-bearing mouse model.

[0271] Fig.25 The pharmacodynamic results of 9D1-LD-38 combined with ICI in an immunocompetent tumor-bearing mouse model.

[0272] Fig.26 This is the result of the bystander effect of 9D1-LD-38 in tumor-bearing mice. DETAILED DESCRIPTION

[0273] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0274] Unless otherwise stated, some of the terms used in the specification and claims of this application are defined as follows:

[0275] "Alkyl" is branched or unbranched, and preferably has 1 to about 12 carbon atoms. A more preferred class of alkyl has 1 to about 6 carbon atoms. Even more preferred are alkyls having 1, 2, 3 or 4 carbon atoms. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl can be substituted or unsubstituted.

[0276] "Alkenyl" is branched or unbranched, has one or more double bonds and 2 to about 12 carbon atoms. A more preferred class of alkenyls has 2 to about 6 carbon atoms. Even more preferred are alkenyls having 2, 3 or 4 carbon atoms. Alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 1-butenyl, 2-butenyl and 3-butenyl, etc., and alkenyl groups can be substituted or unsubstituted.

[0277] "Alkynyl" is branched or unbranched, has one or more triple bonds and 2 to about 12 carbon atoms. A more preferred class of alkynyl groups has 2 to about 6 carbon atoms, and even more preferred are alkynyl groups having 2, 3 or 4 carbon atoms, alkynyl groups include but are not limited to acetylene, propyne, 1-butyne, and the like.

[0278] The "alicyclic group" refers to a cyclic substituent group which does not have aromaticity, and may be a saturated hydrocarbon substituent group or an unsaturated hydrocarbon substituent group, for example, a cycloalkyl group, a cycloalkenyl group, or the like.

[0279] "Heteroalicyclic group" refers to a substituent group formed by a heterocyclic compound containing at least one heteroatom and not having aromaticity on the ring skeleton. The heteroatom can be oxygen, nitrogen, sulfur atoms, etc., including monocyclic, polycyclic, condensed ring, bridged ring and spirocyclic. For example, it has a 5-7-membered monocyclic or 7-10-membered bicyclic or tricyclic ring, which can contain 1,2 or 3 atoms selected from nitrogen, oxygen and / or sulfur. Heteroalicyclic groups include but are not limited to morpholinyl, oxetane, thiomorpholinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl, piperazinyl, hexahydropyrimidine. The heterocyclic group can be substituted or unsubstituted.

[0280] "Aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be linked together in a fused manner. The term "aryl" includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, and tetrahydronaphthyl aromatic groups. Preferably, the aryl group is a C6-C10 aryl group, more preferably, the aryl group is phenyl and naphthyl, and most preferably, naphthyl. The aryl group may be substituted or unsubstituted.

[0281] "Heteroaryl" refers to an aromatic 5-6-membered monocyclic or 8-10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and / or sulfur. The embodiments of "heteroaryl" include, but are not limited to, furanyl, pyridyl, 2-oxo-1,2-dihydropyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, benzodioxolyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, 1,3-dioxo-isoindolyl, quinolyl, indazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl. Heteroaryl may be substituted or unsubstituted.

[0282] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.

[0283] "Substitution" or "substituted" as used in this specification, unless otherwise specified, means that the group may be substituted by one or more groups selected from the following: alkyl, alkoxy, alkylthio, alkylamino, halogen, thio, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl substituents;

[0284] Whenever a group is described as "optionally substituted with substituents", the group may be unsubstituted or substituted with one or more of the substituents shown. Similarly, when a group is described as "unsubstituted or substituted", if substituted, the substituents may be selected from one or more of the substituents shown. If no substituents are specified, it means that the group shown as "optionally substituted with substituents" or "substituted" may be substituted with one or more groups, which are individually or independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclic, aralkyl, heteroaralkyl, (heteroalicyclic) alkyl, hydroxy, protected hydroxy, alkoxy, aryloxy, acyl, sulfhydryl, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N- -carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxyl, protected C-carboxyl, O-carboxyl, isocyanate, thiocyanate, isothiocyanate, nitro, silyl, sulfo, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, monosubstituted amino and disubstituted amino and protected derivatives thereof.

[0285] Halogen substituents include F, Cl, Br and I. "Hydroxyl" refers to the -OH group. "Halogen" refers to fluorine, chlorine, bromine and iodine.

[0286] "Amino" refers to -NH2. "Cyano" refers to -CN. "Nitro" refers to -NO2. "Carboxyl" refers to -C(O)OH. "Acyl" refers to -C(O)R.

[0287] The term "hydrogen" or "H" in the chemical structures used herein, unless otherwise specified, should be understood to include not only 1 H, also includes deuterium ( 2 H, D), tritium ( 3 H, T) or a mixture thereof. "Deuterated isotope derivative" refers to a corresponding compound obtained when one or more H atoms in the compound are replaced by D atoms.

[0288] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0289] The present application proposes a compound represented by formula (1), or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the structure of formula (1) is as follows:

[0290]

[0291] M is a linking group; Sp is -L a -Q a -; A is selected from non-existent, hydrogen, alkyl; T represents a linear or branched (C1-C8) trivalent alkyl group; Y, Y', Y" are -L b -Q b -;L a , L b Q is respectively selected from absence, alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group is optionally substituted with a substituent; Q a , Q b are respectively selected from absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -S(=O)2NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, -O-, -NR-, -S-, S(=O)2-, S(=O)-, or -Se -, wherein R is selected from hydrogen or an optionally substituted alkyl; Z is selected from non-existence, hydrogen, an optionally substituted alkyl or a sulfonate; Z', Z" are selected from non-existence, hydrogen, an optionally substituted alkyl, a sulfonate or a phosphate; at least one of Z, Z', Z" is a sulfonate or a phosphate; X comprises a self-destruct unit or does not exist; D comprises a drug molecule; i is an integer greater than or equal to 1, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. h is an integer from 1 to 6, for example, it can be 1, 2, 3, 4, 5, 6, etc. o, p, q are integers from 0 to 5, for example, they can be 0, 1, 2, 3, 4, 5, etc.

[0292] When h is greater than 1, multiple Sp can have different structures or the same structure, that is, multiple L a It can be different structures or the same structure; multiple Q a It can be different structures or the same structure. a and Q a The number of can be the same or different.

[0293] When i is greater than 1, multiple A's may be different or the same, multiple T's may be different or the same, multiple Y's may be different or the same, and multiple Z's may be different or the same.

[0294] When p is greater than 1, the multiple Y's may have different structures or the same structure.

[0295] When q is greater than 1, the multiple Y"s may have different structures or the same structure.

[0296] In the present application, the formula (1) is formula (1-1):

[0297]

[0298] Wherein, M, Sp, A, T, Y, h, o, i, X, D, and Z are defined as above; and at least one of the i Zs is a sulfonate.

[0299] In the present application, the formula (1-1) is formula (1-1-1):

[0300]

[0301] Wherein, AA is an amino acid residue, and w is an integer greater than or equal to 1, for example, 1, 2, 3, 4, 5, 6, etc. When w is greater than 1, multiple AAs may be residues of the same amino acid or residues of different amino acids.

[0302] In the present application, the formula (1) is the formula (1-2):

[0303]

[0304] Wherein, M, Sp, A, T, Y, Z, o, p, i, h, X, Y', and D are as defined above; and Z' is a sulfonic acid inner salt or a phosphate inner salt.

[0305] In the present application, the formula (1) is formula (1-2-1):

[0306]

[0307] Wherein, AA is an amino acid residue, and w is an integer greater than or equal to 1, for example, 1, 2, 3, 4, 5, 6, etc. When w is greater than 1, multiple AAs may be residues of the same amino acid or residues of different amino acids.

[0308] In the present application, the formula (1) is the formula (1-3):

[0309]

[0310] Wherein, M, Sp, A, Y, T, Z, o, i, h, q, X, Y", and D are as defined above; Z" is a sulfonic acid inner salt or a phosphate inner salt.

[0311] In the present application, when Z is hydrogen or an optionally substituted alkyl group, It is the residue of amino acid, also called AA.

[0312] In the present application, when Z is a sulfonic acid salt, for

[0313] In the present application, when A and Z are absent, N, Y, and T form a ring. for

[0314] Preferably

[0315] In the present application, when Z is selected from hydrogen or an optionally substituted alkyl group, the formula (1) is formula (1-3-1):

[0316]

[0317] Wherein, AA is an amino acid residue, and w is an integer greater than or equal to 1, for example, 1, 2, 3, 4, 5, 6, etc. When w is greater than 1, multiple AAs may be residues of the same amino acid or residues of different amino acids.

[0318] In the present application, the amino acid is selected from one or more of alanine, arginine, asparagine, aspartic acid, γ-carboxyglutamate, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0319] In the present application, the amino acid is selected from one or more of valine, glutamic acid, citrulline, lysine, alanine, phenylalanine, arginine, glutamine, asparagine and glycine.

[0320] In the present application, the amino acid is selected from one or more of valine, citrulline, alanine, phenylalanine, glutamine and glycine.

[0321] When w is greater than 1, multiple AAs can be the same or different, as follows:

[0322] In some embodiments, when w is 2, the two AAs may be independently a valine residue and a citrulline residue, respectively.

[0323] In some embodiments, when w is greater than 2, the w AAs may be independently valine residues and citrulline residues, wherein the number of valine residues may be 1 or 2 or more, or the number of citrulline residues may be 1 or 2 or more, for example, when w is 3, the three AAs are two valine residues and one citrulline residue, respectively. For another example, when w is 3, the three AAs are one valine residue and two citrulline residues, respectively.

[0324] In some embodiments, when w is 2, the two AAs may be independently a valine residue and an alanine residue, respectively.

[0325] In some embodiments, when w is greater than 2, the w AAs may be independently valine residues and alanine residues, wherein the number of valine residues may be 1 or 2 or more, or the number of alanine residues may be 1 or 2 or more, for example, when w is 3, the three AAs are two valine residues and one alanine residue, respectively. For another example, when w is 3, the three AAs are one valine residue and two alanine residues, respectively.

[0326] In some embodiments, when w is 2, the two AAs can be independently a valine residue and a phenylalanine residue, respectively.

[0327] In some embodiments, when w is greater than 2, the w AAs may be independently valine residues and phenylalanine residues, wherein the number of valine residues may be 1 or 2 or more, or the number of phenylalanine residues may be 1 or 2 or more, for example, when w is 3, the three AAs are two valine residues and one phenylalanine residue, respectively. For another example, when w is 3, the three AAs are one valine residue and two phenylalanine residues, respectively.

[0328] In some embodiments, when w is 2, the two AAs may be independently a valine residue and a glutamine residue, respectively.

[0329] In some embodiments, when w is greater than 2, the w AAs may be independently valine residues and glutamine residues, wherein the number of valine residues may be 1 or 2 or more, or the number of glutamine residues may be 1 or 2 or more, for example, when w is 3, the three AAs are two valine residues and one glutamine residue, respectively. For another example, when w is 3, the three AAs are one valine residue and two glutamine residues, respectively.

[0330] In some embodiments, when w is 2, the two AAs may be independently a valine residue and a glycine residue, respectively.

[0331] In some embodiments, when w is greater than 2, the w AAs may be independently valine residues and glycine residues, wherein the number of valine residues may be 1 or 2 or more, or the number of glycine residues may be 1 or 2 or more, for example, when w is 3, the three AAs are two valine residues and one glycine residue, respectively. For another example, when w is 3, the three AAs are one valine residue and two glycine residues, respectively.

[0332] In some embodiments, when w is 2, the two AAs can be independently a citrulline residue and an alanine residue, respectively.

[0333] In some embodiments, when w is greater than 2, the w AAs may be independently citrulline residues and alanine residues, wherein the number of citrulline residues may be 1 or 2 or more, or the number of alanine residues may be 1 or 2 or more, for example, when w is 3, the three AAs are respectively two citrulline residues and one alanine residue. For another example, when w is 3, the three AAs are respectively one citrulline residue and two alanine residues.

[0334] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues and phenylalanine residues, respectively, wherein the number of citrulline residues may be 1 or 2 or more, or the number of phenylalanine residues may be 1 or 2 or more.

[0335] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues and glutamine residues, respectively, wherein the number of citrulline residues may be 1 or 2 or more, or the number of glutamine residues may be 1 or 2 or more.

[0336] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues and glycine residues, respectively, wherein the number of citrulline residues may be 1 or 2 or more, or the number of glycine residues may be 1 or 2 or more.

[0337] In some embodiments, when w is greater than 1, the w AAs may be independently alanine residues and phenylalanine residues, respectively, wherein the number of alanine residues may be 1 or more than 2 or the number of phenylalanine residues may be 1 or more than 2.

[0338] In some embodiments, when w is greater than 1, the w AAs may be independently alanine residues and glutamine residues, respectively, wherein the number of alanine residues may be 1 or more than 2 or the number of glutamine residues may be 1 or more than 2.

[0339] In some embodiments, when w is greater than 1, the w AAs may be independently alanine residues and glycine residues, respectively, wherein the number of alanine residues may be 1 or more than 2 or the number of glycine residues may be 1 or more than 2.

[0340] In some embodiments, when w is greater than 1, the w AAs may be independently phenylalanine residues and glutamine residues, respectively, wherein the number of phenylalanine residues may be 1 or more than 2 or the number of glutamine residues may be 1 or more than 2.

[0341] In some embodiments, when w is greater than 1, the w AAs may be independently phenylalanine residues and glycine residues, respectively, wherein the number of phenylalanine residues may be 1 or more than 2 or the number of glycine residues may be 1 or more than 2.

[0342] In some embodiments, when w is greater than 1, the w AAs may be independently glutamine residues and glycine residues, respectively, wherein the number of glutamine residues may be 1 or 2 or more, or the number of glycine residues may be 1 or 2 or more.

[0343] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues and alanine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of citrulline residues may be 1 or more than 2, or the number of alanine residues may be 1 or more than 2.

[0344] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues and phenylalanine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of citrulline residues may be 1 or more than 2, or the number of phenylalanine residues may be 1 or more than 2.

[0345] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues and glutamine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of citrulline residues may be 1 or more than 2, or the number of glutamine residues may be 1 or more than 2.

[0346] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues and glycine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of citrulline residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0347] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, alanine residues and phenylalanine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of alanine residues may be 1 or more than 2, or the number of phenylalanine residues may be 1 or more than 2.

[0348] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, alanine residues and glutamine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of alanine residues may be 1 or more than 2, or the number of glutamine residues may be 1 or more than 2.

[0349] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, alanine residues and glycine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of alanine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0350] In some embodiments, when w is greater than 1, the w AAs may be independently a valine residue, a phenylalanine residue, and a glutamine residue, respectively, wherein the number of valine residues may be 1 or more than 2, the number of phenylalanine residues may be 1 or more than 2, or the number of glutamine residues may be 1 or more than 2.

[0351] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, phenylalanine residues and glycine residues, respectively, wherein the number of valine residues may be 1 or more than 2, the number of phenylalanine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0352] In some embodiments, when w is greater than 1, the w AAs may be independently a valine residue, a glutamine residue, and a glycine residue, respectively, wherein the number of valine residues may be 1 or more than 2, the number of glutamine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0353] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues, alanine residues and phenylalanine residues, respectively, wherein the number of citrulline residues may be 1 or more than 2, the number of alanine residues may be 1 or more than 2, or the number of phenylalanine residues may be 1 or more than 2.

[0354] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues, alanine residues and glutamine residues, respectively, wherein the number of citrulline residues may be 1 or more than 2, the number of alanine residues may be 1 or more than 2, or the number of glutamine residues may be 1 or more than 2.

[0355] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues, alanine residues and glycine residues, respectively, wherein the number of citrulline residues may be 1 or more than 2, the number of alanine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0356] In some embodiments, when w is greater than 1, the w AAs may be independently an alanine residue, a phenylalanine residue, and a glutamine residue, respectively, wherein the number of alanine residues may be 1 or more than 2, the number of phenylalanine residues may be 1 or more than 2, or the number of glutamine residues may be 1 or more than 2.

[0357] In some embodiments, when w is greater than 1, the w AAs may be independently an alanine residue, a phenylalanine residue, and a glycine residue, respectively, wherein the number of alanine residues may be 1 or more than 2, the number of phenylalanine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0358] In some embodiments, when w is greater than 1, the w AAs may be independently a phenylalanine residue, a glutamine residue, and a glycine residue, respectively, wherein the number of phenylalanine residues may be 1 or more than 2, the number of glutamine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0359] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues, alanine residues and phenylalanine residues, respectively, wherein the number of valine residues may be 1 or more, the number of citrulline residues may be 1 or more, the number of alanine residues may be 1 or more, or the number of phenylalanine residues may be 1 or more.

[0360] In some embodiments, when w is greater than 1, the w AAs may be independently a valine residue, a citrulline residue, an alanine residue, and a glutamine residue, wherein the number of valine residues may be 1 or more, the number of citrulline residues may be 1 or more, the number of alanine residues may be 1 or more, or the number of glutamine residues may be 1 or more.

[0361] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues, alanine residues and glycine residues, respectively, wherein the number of valine residues may be 1 or more, the number of citrulline residues may be 1 or more, the number of alanine residues may be 1 or more, or the number of glycine residues may be 1 or more.

[0362] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues, alanine residues, phenylalanine residues and glutamine residues, wherein the number of citrulline residues may be 1 or more, the number of alanine residues may be 1 or more, the number of phenylalanine residues may be 1 or more, or the number of glutamine residues may be 1 or more.

[0363] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues, alanine residues, phenylalanine residues and glycine residues, respectively, wherein the number of citrulline residues may be 1 or more than 2, the number of alanine residues may be 1 or more than 2, the number of phenylalanine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0364] In some embodiments, when w is greater than 1, the w AAs may be independently an alanine residue, a phenylalanine residue, a glutamine residue, and a glycine residue, wherein the number of alanine residues may be 1 or more than 2, the number of phenylalanine residues may be 1 or more than 2, the number of glutamine residues may be 1 or more than 2, or the number of glycine residues may be 1 or more than 2.

[0365] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues, alanine residues, phenylalanine residues and glutamine residues, wherein the number of valine residues may be 1 or more, the number of citrulline residues may be 1 or more, the number of alanine residues may be 1 or more, the number of phenylalanine residues may be 1 or more, or the number of glutamine residues may be 1 or more.

[0366] In some embodiments, when w is greater than 1, the w AAs may be independently valine residues, citrulline residues, alanine residues, phenylalanine residues and glycine residues, wherein the number of valine residues may be 1 or more, the number of citrulline residues may be 1 or more, the number of alanine residues may be 1 or more, the number of phenylalanine residues may be 1 or more, or the number of glycine residues may be 1 or more.

[0367] In some embodiments, when w is greater than 1, the w AAs may be independently citrulline residues, alanine residues, phenylalanine residues, glutamine residues and glycine residues, respectively, wherein the number of citrulline residues may be 1 or more, the number of alanine residues may be 1 or more, the number of phenylalanine residues may be 1 or more, the number of glutamine residues may be 1 or more, or the number of glycine residues may be 1 or more.

[0368] In the present application, M is N3, SH, ONH2, NH2, CO2H, (alkyl)-CO-, HCO-, BrCH 2- 、ICH2-、

[0369] Furthermore, M is

[0370] In some embodiments, M is

[0371] In some embodiments, M is

[0372] In some embodiments, M is

[0373] In some embodiments, M is

[0374] In the present application, A is hydrogen, methyl, ethyl or propyl.

[0375] In some embodiments, A is hydrogen. In some embodiments, A is methyl. In some embodiments, A is ethyl. In some embodiments, A is propyl.

[0376] In this application, T is one of the following structures:

[0377]

[0378] In some embodiments, T is

[0379] In this application, L a One, two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol group; the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur.

[0380] Furthermore, L a Selected from C1-C 10 Straight or branched alkylene, C2-C 12 Straight or branched alkenylene, C2-C 12 One, two or more of a straight or branched alkynylene group, a saturated or unsaturated alicyclic group having 3-6 ring carbon atoms, a saturated or unsaturated heteroalicyclic group having 3-6 ring carbon atoms and 1 to 4 heteroatoms, or an arylene group having 6 to 10 carbon atoms in one or more rings, a heteroarylene group having 6 to 10 carbon atoms in one or more rings and 1 to 4 heteroatoms, or a polyethylene glycol group, wherein the alkylene group, alkenylene group, alkynylene group, alicyclic group, heteroalicyclic group, arylene group, heteroarylene group are optionally substituted by a substituent, and the heteroatoms are selected from nitrogen, oxygen or sulfur. Further, L a One, two or more selected from C1-C6 straight chain or branched alkylene, C2-C6 straight chain or branched alkenylene, C2-C6 straight chain or branched alkynylene, or polyethylene glycol group, wherein the alkylene, alkenylene and alkynylene are optionally substituted by a substituent.

[0381] Furthermore, L a It is one, two or more of C1-C6 straight or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n-、-(CH2CH2O) n CH2-,

[0382] -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n CH2-, n is an integer greater than or equal to 1.

[0383] Furthermore, L a is ethylene, straight-chain propylene, straight-chain butylene, straight-chain pentylene, or -(CH2CH2O) n One, two or more of CH2CH2-, wherein n is 2, 3, 4, 5, 6, 7 or 8.

[0384] Furthermore, L a It is ethylene and / or -(CH2CH2O)4CH2CH2- or pentylene.

[0385] In some embodiments, L a The number of them is 2, one of which is ethylene and the other is -(CH2CH2O)4CH2CH2-.

[0386] In some embodiments, L a When the number of is 1, it is ethylene. a When the number of is 1, it is pentylene. a When the number is 1, it is -(CH2CH2O)4CH2CH2-.

[0387] In this application, L b One or two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol,

[0388] The alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group, heteroarylene group may be substituted with a substituent, and the heteroatom may be selected from nitrogen, oxygen or sulfur.

[0389] Furthermore, L b Selected from C1-C 10 Straight or branched alkylene, C2-C 12 Straight or branched alkenylene, C2-C 12One or two or more of a linear or branched alkynylene group, a saturated or unsaturated alicyclic group having 3 to 6 ring carbon atoms, a saturated or unsaturated heteroalicyclic group having 3 to 6 ring carbon atoms and 1 to 4 heteroatoms, or an arylene group having 6 to 10 carbon atoms in one or more rings, a heteroarylene group having 6 to 10 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol,

[0390] The alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group, heteroarylene group may be substituted with a substituent, and the heteroatom may be selected from nitrogen, oxygen or sulfur.

[0391] Furthermore, L b is selected from C1-C6 straight chain or branched chain alkylene, C2-C6 straight chain or branched chain alkenylene, or C2-C6 straight chain or branched chain alkynylene, or polyethylene glycol group,

[0392] The polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-,

[0393] -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n One or two or more than three of CH2-, n is an integer greater than or equal to 1, and the alkylene, alkenylene and alkynylene groups are optionally substituted by a substituent.

[0394] Furthermore, L b is a C1-C6 straight or branched chain alkylene group, or a C6-C 10 One or two or more than three of the arylene groups.

[0395] Furthermore, L b It is one or two or more of methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene, or benzylene.

[0396] When L b When the number is multiple, multiple L b It can be the same structure or different structures, as shown below:

[0397] In some embodiments, L b When the number is 1 or more, L b In some embodiments, L bWhen the number is 1 or more, L b In some embodiments, L b When the number is 1 or more, L b In some embodiments, L b When the number is 1 or more, L b In some embodiments, L b When the number is 1 or more, L b In some embodiments, L b When the number is 1 or more, L b In some embodiments, L b When the number is 1 or more, L b In some embodiments, L b When the number is 1 or more, L b In some embodiments, L b When the number is 1 or more, L b All are benzylene.

[0398] In some embodiments, L b When the number is multiple, multiple L b The number of methylene groups can be 1 or more than 2, and the number of ethylene groups can be 1 or more than 2. For example, L b When the number is 3, 3 L b There are two methylene groups and one ethylene group respectively. b When the number is 3, 3 L b One methylene and two ethylene groups respectively.

[0399] In some embodiments, L b When the number is multiple, multiple L b The number of methylene groups can be 1 or more than 2, and the number of n-propylene groups can be 1 or more than 2. For example, L b When the number is 3, 3 L b There are two methylene groups and one propylene group. b When the number is 3, 3 L b There is one methylene group and two n-propylene groups respectively.

[0400] In some embodiments, L b When the number is greater than 1, multiple L bIndependently there are methylene and isopropylene, wherein the number of the methylene can be 1 or more than 2, and the number of the isopropylene can be 1 or more than 2.

[0401] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene and n-butylene, wherein the number of methylene can be 1 or more than 2, and the number of n-butylene can be 1 or more than 2.

[0402] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene and isobutylene, wherein the number of methylene can be 1 or more than 2, and the number of isobutylene can be 1 or more than 2.

[0403] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene and sec-butylene, wherein the number of methylene can be 1 or more than 2, and the number of sec-butylene can be 1 or more than 2.

[0404] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene and tert-butylene groups, wherein the number of methylene groups can be 1 or more than 2, and the number of tert-butylene groups can be 1 or more than 2.

[0405] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene and benzylene, wherein the number of the methylene group may be 1 or more than 2, and the number of the benzylene group may be 1 or more than 2.

[0406] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are ethylene and n-propylene, wherein the number of ethylene can be 1 or more than 2, and the number of n-propylene can be 1 or more than 2.

[0407] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are ethylene and isopropylene, wherein the number of ethylene can be 1 or more than 2, and the number of isopropylene can be 1 or more than 2.

[0408] In some embodiments, L b When the number is greater than 1, multiple L bIndependently there are ethylene and n-butylene, wherein the number of ethylene can be 1 or more than 2, and the number of n-butylene can be 1 or more than 2.

[0409] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are ethylene and isobutylene, wherein the number of ethylene can be 1 or more than 2, and the number of isobutylene can be 1 or more than 2.

[0410] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are ethylene and sec-butylene, wherein the number of ethylene can be 1 or more than 2, and the number of sec-butylene can be 1 or more than 2.

[0411] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are ethylene and tert-butylene, wherein the number of ethylene can be 1 or more than 2, and the number of tert-butylene can be 1 or more than 2.

[0412] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are ethylene and benzylene, wherein the number of ethylene can be 1 or more than 2, and the number of benzylene can be 1 or more than 2.

[0413] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are n-propylene and isopropylene, wherein the number of n-propylene can be 1 or more than 2, and the number of isopropylene can be 1 or more than 2.

[0414] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are n-propylene and n-butylene, wherein the number of n-propylene can be 1 or more than 2, and the number of n-butylene can be 1 or more than 2.

[0415] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are n-propylene and isobutylene, wherein the number of n-propylene can be 1 or more than 2, and the number of isobutylene can be 1 or more than 2.

[0416] In some embodiments, L b When the number is greater than 1, multiple L bIndependently there are n-propylene and sec-butylene, wherein the number of n-propylene can be 1 or more than 2, and the number of sec-butylene can be 1 or more than 2.

[0417] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are n-propylene and tert-butylene, wherein the number of n-propylene can be 1 or more than 2, and the number of tert-butylene can be 1 or more than 2.

[0418] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are n-propylene and benzylene, wherein the number of n-propylene can be 1 or more than 2, and the number of benzylene can be 1 or more than 2.

[0419] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are isopropylene and n-butylene, wherein the number of isopropylene can be 1 or more than 2, and the number of n-butylene can be 1 or more than 2.

[0420] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are isopropylene and isobutylene, wherein the number of isopropylene can be 1 or more than 2, and the number of isobutylene can be 1 or more than 2.

[0421] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are isopropylene and sec-butylene, wherein the number of isopropylene can be 1 or more than 2, and the number of sec-butylene can be 1 or more than 2.

[0422] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are isopropylene and tert-butylene, wherein the number of isopropylene can be 1 or more than 2, and the number of tert-butylene can be 1 or more than 2.

[0423] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are isopropylene and benzylene, wherein the number of isopropylene can be 1 or more than 2, and the number of benzylene can be 1 or more than 2.

[0424] In some embodiments, L b When the number is greater than 1, multiple Lb Independently there are methylene, ethylene and n-propylene, wherein the number of methylene groups can be 1 or more than 2, the number of ethylene groups can be 1 or more than 2, and the number of n-propylene groups can be 1 or more than 2.

[0425] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene, ethylene and isopropylene, wherein the number of methylene groups can be 1 or more than 2, the number of ethylene groups can be 1 or more than 2, and the number of isopropylene groups can be 1 or more than 2.

[0426] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene, ethylene, n-propylene and isopropylene, wherein the number of methylene groups can be 1 or more than two, the number of ethylene groups can be 1 or more than two, the number of n-propylene groups can be 1 or more than two, and the number of isopropylene groups can be 1 or more than two.

[0427] In some embodiments, L b When the number is greater than 1, multiple L b Independently there are methylene, ethylene, n-propylene, isopropylene and n-butylene, wherein the number of methylene groups can be 1 or more, the number of ethylene groups can be 1 or more, the number of n-propylene groups can be 1 or more, the number of isopropylene groups can be 1 or more, and the number of n-butylene groups can be 1 or more.

[0428] In this application, Q a One, two or more selected from -C(=O)NR-, -NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, wherein R is selected from hydrogen, optionally substituted C1-C6 straight chain or branched alkyl.

[0429] Furthermore, Q a It is one or two or more of -C(=O)-, -NR-, -C(=O)NR-, -NRC(=O)NR-, -OC(=O)O-, -OC(=O)NR-, -NRC(=O)- or -OC(=O)-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight chain or branched alkyl.

[0430] Furthermore, Q aIt is one or two or more of -C(=O)-, -NR-, -NRC(=O)- and -(C=O)NR-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight or branched alkyl group.

[0431] Furthermore, Q a It is -C(=O)-, -N(CH3)-, -NHC(=O)-, -C(=O)-NH-, -C(=O)-N(CH3)-.

[0432] In some embodiments, Q a When the number of is 1, it is -C(=O)-.

[0433] In some embodiments, Q a When the number of is 2, one is -C(=O)- and the other is -C(=O)NH-.

[0434] In some embodiments, Q a When the number of is 2, one is -C(=O)-, the other is -C(=O)-NR-, and R is a methyl group.

[0435] In some embodiments, Q a When the number of is 2, one is -C(=O)-, the other is -NR-, and R is a methyl group.

[0436] In this application, Q b is selected from one, two or more of the group consisting of absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, and -OC(=O)O-, wherein R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl.

[0437] Furthermore, Q b is absent, one or two or more of -C(=O)NR-, -OC(=O)NR-, -NRC(=O)-, -NRC(=O)O-, or -NRC(=O)NR-, and R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl.

[0438] Furthermore, Q b is absent, -C(=O)-, -NRC(=O)-, -NRC(=O)NR- or -C(=O)NR-, or two or more thereof; and R is hydrogen or methyl.

[0439] In some embodiments, Q b In some embodiments, Qb is -C(=O)-. In some embodiments, Q b is -NRC(=O)-, R is hydrogen. In some embodiments, Q b is -NRC(=O)-, R is methyl. b is -NRC(=O)NR-, R is hydrogen. In some embodiments, Q b is -NRC(=O)NR-, R is methyl. In some embodiments, Q b is -C(=O)NR-, R is hydrogen. In some embodiments, Q b is -C(=O)NR-, R is methyl. In the present application, X is selected from:

[0440]

[0441] The right wavy line in X connects D;

[0442] R4 is selected from hydrogen, C1-C6 straight or branched alkyl, -(CH2CH2O) m R2, -CH2CH2NR2R3, -CH2CH2SO2R2, R2, R3 are independently selected from hydrogen, C1-C6 straight chain or branched alkyl, and m is an integer greater than 1.

[0443] Furthermore, X is

[0444] R4 is hydrogen, methyl, -CH2CH2OCH2CH2OH, -CH2CH2NMe2, -CH2CH2SO2Me.

[0445] In some embodiments, X is R4 is hydrogen.

[0446] In some embodiments, X is R4 is methyl.

[0447] In some embodiments, X is R4 is -CH2CH2OCH2CH2OH.

[0448] In some embodiments, X is R4 is -CH2CH2NMe2.

[0449] In some embodiments, X is R4 is -CH2CH2SO2Me.

[0450] In some embodiments, X is R4 is hydrogen.

[0451] In some embodiments, X is R4 is methyl.

[0452] In some embodiments, X is R4 is -CH2CH2OCH2CH2OH.

[0453] In some embodiments, X is R4 is -CH2CH2NMe2.

[0454] In some embodiments, X is R4 is -CH2CH2SO2Me.

[0455] In some embodiments, X is R4 is hydrogen.

[0456] In some embodiments, X is R4 is methyl.

[0457] In some embodiments, X is R4 is -CH2CH2OCH2CH2OH.

[0458] In some embodiments, X is R4 is -CH2CH2NMe2.

[0459] In some embodiments, X is R4 is -CH2CH2SO2Me.

[0460] In the present application, the sulfonic acid inner salt is

[0461] A wavy line connects Y, Y' or Y";

[0462] wherein c, d, e, f, g are all integers of 1, 2, 3, 4, 5 or 6; R6, R7, R 11 , R 12 , R 13 , R 14 are independently selected from absence, hydrogen, and C1-C6 straight or branched chain alkyl.

[0463] Furthermore, the sulfonic acid inner salt is

[0464] Among them, R6, R7, R 11 , R 12 , R 13 and R 14 are selected from absence, methyl or ethyl, c, d, e, f, g are 1, 2 or 3 respectively.

[0465] Furthermore, the sulfonic acid inner salt is one of the following structures:

[0466]

[0467] In some embodiments, the sulfonate is

[0468] In some embodiments, the sulfonate is

[0469] In some embodiments, the sulfonate is

[0470] In some embodiments, the sulfonate is

[0471] In the present application, the phosphate inner salt is

[0472] Wherein, a and b are integers of 1, 2, 3, 4, 5 or 6; R8, R9 and R 10 are selected from hydrogen, C1-C6 straight chain or branched alkyl.

[0473] Furthermore, the phosphate inner salt is

[0474] Among them, R8, R9, R 10 They are methyl or ethyl respectively; a is 1 or 2 or 3; b is 1 or 2 or 3.

[0475] Furthermore, the phosphate inner salt is

[0476] Among them, R8, R9, R 10 All are methyl; a is 2; b is 1.

[0477] In the present application, the D includes cytotoxins or immune agonists.

[0478] In the present application, the cytotoxin is selected from one or two of tubulin inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors or RNA polymerase 2 inhibitors.

[0479] In the present application, the cytotoxin is selected from auristatins, maytansine alkaloids DMs, calicheamicin, duocarmycin, pyrrolobenzodiazepines One or more of the group consisting of camptothecin derivatives, camptothecin derivatives and α-amanitin.

[0480] In the present application, the immune agonist is selected from TLR7 / 8 agonist or STING agonist.

[0481] In the present application, i is an integer of 2-8, for example, it can be 2, 3, 4, 5, 6, 7 or 8.

[0482] In the present application, h is an integer of 1-4, for example, it can be 1, 2, 3 or 4.

[0483] In the present application, o, p, and q are integers of 0-3, for example, 0, 1, 2, or 3.

[0484] In this application, L a It is one, two or more of C1-C6 straight or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-,

[0485] -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n CH2-, n is an integer greater than or equal to 1; Q a It is one or two or more of -C(=O)-, -NR(C=O)- and -(C=O)NR-, wherein R is selected from hydrogen, C1-C6 straight chain or branched alkyl.

[0486] Furthermore, L a is ethylene, straight-chain propylene, straight-chain butylene, straight-chain pentylene, or -(CH2CH2O) n One, two or more of CH2CH2-, wherein n is 2, 3, 4, 5, 6, 7 or 8; Q a It is one, two, or three or more of -C(=O)-, -NR(C=O)-, and -(C=O)NR-, wherein R is methyl and / or hydrogen.

[0487] Furthermore, L a is ethylene and / or -(CH2CH2O)4CH2CH2- or pentylene; Q a is -C(=O)-, -NR-, -NH(C=O)- and / or -C(=O)NH-.

[0488] In some embodiments, L a The number of is 2, one of which is ethylene and the other is -(CH2CH2O)4CH2CH2-. a The number of them is 2, one of which is -C(=O)- and the other is -C(=O)NH-.

[0489] In some embodiments, L a The number is 1, which is pentylene, Q a The number of is 1, which is -C(=O)-.

[0490] In some embodiments, L a The number is 1, which is -(CH2CH2O)4CH2CH2-, Q a The number of them is 2, one is -C(=O)- and the other is -C(=O)NH-.

[0491] In some embodiments, L a The number is 1, which is -(CH2CH2O)4CH2CH2-, Q a The number of them is 2, one is -C(=O)- and the other is -C(=O)N(CH3)-.

[0492] In some embodiments, L a The number of is 2, which are ethylene and pentylene, Q a The number of them is 2, one is -C(=O)- and the other is -N(CH3)-.

[0493] In this application, L b is selected from a C1-C6 straight chain or branched alkylene group, a C2-C6 straight chain or branched alkenylene group, or a C2-C6 straight chain or branched alkynylene group, an arylene group having 6 to 10 carbon atoms in one or more rings, or a polyethylene glycol group,

[0494] The polyethylene glycol group is -(CH2CH2O) n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-,

[0495] -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n -, or -CH2O(CH2CH2O) n One or two or more than three of CH2-, n is an integer greater than or equal to 1, and the alkylene, alkenylene, and alkynylene groups are optionally substituted with a substituent;

[0496] Q bis selected from one, two or more of the group consisting of absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -C(=O)-, and -OC(=O)O-, and R is selected from hydrogen, an optionally substituted C1-C6 straight chain or branched alkyl group.

[0497] Furthermore, L b is a C1-C6 straight or branched chain alkylene group, or a C6-C 10 One or two or more than three kinds of arylene groups; Q b It is absent, one or two or more of -C(=O)NR-, -C(=O)-, -OC(=O)NR-, -NRC(=O)-, -NRC(=O)O-, or -NRC(=O)NR-, and R is selected from hydrogen, a C1-C6 straight chain or branched alkyl group.

[0498] Furthermore, L b It is one or two or more of the following: methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene or benzylene;

[0499] Q b is absent, -C(=O)-, -NRC(=O)-, -NRC(=O)NR- or -C(=O)NR-, or two or more thereof; and R is hydrogen or methyl.

[0500] In some embodiments, in Y, L b When the number of is two, one is a methylene group and the other is an isopropylene group; b Does not exist.

[0501] In some embodiments, in Y, L b When the number of is three, they are respectively methylene, isopropylene and ethylene; Q b The number of is 1, which is -C(=O)NR-, and R is hydrogen.

[0502] In some embodiments, in Y, L b When the number of is two, one is ethylene and the other is isopropylene; b The number of is 1, which is -C(=O)NR-, and R is hydrogen.

[0503] In some embodiments, in Y, L b When the number of is two, one is n-propylene and the other is isopropylene; Q bThe number of is 1, which is -NRC(=O)NR-, and R is hydrogen.

[0504] In some embodiments, in Y, L b When the number is 1, it is a benzyl group, Q b Does not exist.

[0505] In some embodiments, in Y, L b When the number of is 2, they are ethylene and isopropylene respectively; Q b The number of is 1, which is -C(=O)-.

[0506] In the present application, the substituent is selected from deuterium, tritium, a halogen atom, an amino group, a hydroxyl group, a cyano group, a nitro group, an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an optionally substituted heterocyclic group, an acyl group, an optionally substituted amino group, an optionally substituted carbamoyl group, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group, an optionally substituted hydroxyl group, an optionally substituted sulfanyl group (SH) and an optionally substituted silyl group.

[0507] In the present application, the compound is selected from the following structures:

[0508]

[0509]

[0510]

[0511]

[0512] The present application also provides a compound represented by formula (2), or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein the structure of formula (2) is represented as follows:

[0513]

[0514] Ab comprises an antibody or an antigen-binding fragment thereof; M' is a linking group; Sp is -L a -Q a -; A is selected from non-existent, hydrogen, alkyl; T represents a linear or branched (C1-C8) trivalent alkyl group; Y, Y', Y" are -L b -Q b -;L a , L bQ is respectively selected from absence, alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group is optionally substituted with a substituent; Q a , Q b are respectively selected from absence, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -S(=O)2NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, -O-, -NR-, -S-, S(=O)2-, S(=O)-, or -Se -, wherein R is selected from hydrogen or an optionally substituted alkyl; Z is selected from non-existence, hydrogen, an optionally substituted alkyl or a sulfonate; Z' and Z" are selected from non-existence, hydrogen, an optionally substituted alkyl, a sulfonate or a phosphate; at least one of Z, Z' and Z" is a sulfonate or a phosphate; X comprises a self-destruct unit or does not exist; D comprises a drug molecule; i is an integer greater than or equal to 1, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0515] h is an integer of 1-6, for example, 1, 2, 3, 4, 5, 6, etc. o, p, q are integers of 0-5, for example, 0, 1, 2, 3, 4, 5, etc. j is an integer greater than or equal to 1, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0516] When h is greater than 1, multiple Sp can have different structures or the same structure, that is, multiple L a It can be different structures or the same structure; multiple Q a It can be different structures or the same structure. a and Q a The number of can be the same or different.

[0517] When i is greater than 1, multiple A's may be different or the same, multiple T's may be different or the same, multiple Y's may be different or the same, multiple o's may be different or the same, and multiple Z's may be different or the same.

[0518] When p is greater than 1, the multiple Y's may have different structures or the same structure.

[0519] When j is greater than 1, the parts in brackets may have the same structure or different structures.

[0520] In the present application, the formula (2) is formula (2-1):

[0521]

[0522] wherein Ab, M', Sp, A, T, Y, h, o, i, j, X, D, and Z are as defined in the aforementioned formula (1).

[0523] At least one of the i Zs is a sulfonate.

[0524] Furthermore, the formula (2-1) is formula (2-1-1):

[0525]

[0526] Among them, AA is the residue of amino acid,

[0527] w is an integer greater than or equal to 1, for example, 1, 2, 3, 4, 5, 6, etc. When w is greater than 1, the plurality of AAs may be residues of the same amino acid or residues of different amino acids.

[0528] In the present application, the formula (2) is formula (2-2):

[0529]

[0530] wherein Ab, M', Sp, A, T, Y, Z, o, p, i, h, j, X, Y', and D are as defined in the aforementioned formula (1); and Z' is a sulfonic acid inner salt or a phosphate inner salt.

[0531] Furthermore, the formula (2-2) is the formula (2-2-1):

[0532]

[0533] Wherein, AA is an amino acid residue, and w is an integer greater than or equal to 1, for example, 1, 2, 3, 4, 5, 6, etc. When w is greater than 1, multiple AAs may be residues of the same amino acid or residues of different amino acids.

[0534] In the present application, the formula (2) is the formula (2-3):

[0535]

[0536] wherein Ab, M', Sp, A, Y, T, Z, o, i, h, j, q, X, Y", and D are as defined in the aforementioned formula (1); and Z" is a sulfonic acid inner salt or a phosphate inner salt.

[0537] Furthermore, the formula (2-3) is formula (2-3-1):

[0538]

[0539] Wherein, AA is an amino acid residue, and w is an integer greater than or equal to 1, for example, 1, 2, 3, 4, 5, 6, etc. When w is greater than 1, multiple AAs may be residues of the same amino acid or residues of different amino acids.

[0540] In this document, all definitions described in formula (1) are applicable to formula (2); all definitions described in formula (1-1) are applicable to formula (2-1); all definitions described in formula (1-1-1) are applicable to formula (2-1-1); all definitions described in formula (1-2) are applicable to formula (2-2); all definitions described in formula (1-2-1) are applicable to formula (2-2-1); all definitions described in formula (1-3) are applicable to formula (2-3); all definitions described in formula (1-3-1) are applicable to formula (2-3-1); therefore, for formula (2), formula (2-1), formula (2-1-1), formula (2-2), formula (2-2-1), formula (2-3), and formula (2-3-1), Sp, A, Y, T, Z, o, i, h, j, q, X, Y', Z', Y", Z", and D in formula (2) can refer to the description in formula (1) and are not further limited here.

[0541] In the present application, M' is selected from one of the following structures:

[0542]

[0543] * Connect Ab.

[0544] Further, M' is selected from one of the following structures:

[0545]

[0546] In some embodiments, M' is

[0547] In the present application, the compound is selected from the following structures:

[0548]

[0549]

[0550]

[0551]

[0552] In the present application, the antibody is selected from the following groups: mouse antibody, chimeric antibody, humanized antibody and fully human antibody. In the present application, the antibody comprises a monoclonal antibody. In the present application, the antibody comprises a bispecific antibody. In the present application, the antigen-binding fragment is selected from the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and VHH.

[0553] In the present application, the Ab is selected from the following group: anti-claudin 18.2 antibody, anti-human folate receptor antibody, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPC20 antibody, anti-CDH6 antibody, anti-EGFR antibody, anti-EGFRvⅢ antibody, anti-AXL antibody, anti-Nectin-4 antibody, anti-Tissue factor antibody, anti-TIM-1 antibody, anti-PSMA antibody, anti-EpCAM antibody, anti-MUC1 antibody, anti-STEAP1 antibody, anti-GPNMB antibody, anti-FGF2 antibody, anti-FOLR1 antibody, anti-c-MET antibody, anti-GFR antibody, anti-AGS-16, anti-Guanylyl cyclase C antibody, anti-Mesothelin antibody, anti-SLC44A4 antibody, anti-EphA2 antibody, anti-AGS-5 antibody, anti-GPC-3 antibody, anti-c-KIT antibody, anti-ROR1 antibody, anti-PD-L1 antibody, anti-CD27L antibody, anti-5T4 antibody, anti-Mucin 16 antibody, anti-NaPi2b antibody, anti-STEAP antibody, anti-SLITRK6 antibody, anti-ETBR antibody, anti-BCMA antibody, anti-CEACAM5 antibody, anti-SC-16 antibody, anti-SLC39A6 antibody, anti-Delta-like protein3 antibody, anti-Claudin18.2 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD45 antibody, anti-CD56 antibody, anti-CD66e antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD138 antibody, anti-CD147 antibody, anti-CD166 antibody, anti-CD223 antibody, anti-MUC16 antibody, anti-MSLN antibody, anti-ENPP3 antibody, anti-SLTRK6 antibody, anti-FGFR antibody, anti-LIV-1 antibody, anti-Lewis antibody Y antibody, anti-av-integrin antibody, anti-ASCT2 antibody, anti-C4.4a antibody, anti-CA-IX antibody, anti-CD324 antibody, anti-CD352 antibody, anti-CD44v6 antibody, anti-CD48a antibody, anti-CLL-1 antibody, anti-Cripto antibody, anti-CS1 antibody, anti-DPEP3 antibody, anti-Ephrin-A2 antibody, anti-Ephrin-A4 antibody, anti-ETBR antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FLT3 antibody, anti-GD3 antibody, anti-GloboH antibody, anti-GPC3 antibody, anti-LAMP-1 antibody, anti-LRRC15 antibody, anti-Ly6E antibody, anti-MFI2 antibody, anti-NOTCH3 antibody, anti-p-cadherin antibody, anti-PRLR antibody and anti-RNF43 antibody, and antigen-binding fragments of the above antibodies.

[0554] In the present application, the heavy chain HCDR1, HCDR2 and HCDR3 and the light chain LCDR1, LCDR2 and LCDR3 of Ab comprise the heavy chain HCDR1, HCDR2 and HCDR3 and the light chain LCDR1, LCDR2 and LCDR3 of the antibody, respectively.

[0555] In the present application, the heavy chain variable region VH and light chain variable region VL of the Ab respectively comprise the heavy chain variable region VH and light chain variable region VL of the antibody. In the present application, the heavy chain and light chain of the Ab respectively comprise the heavy chain and light chain of the antibody. In the present application, the Ab is an anti-claudin 18.2 antibody or an antigen-binding fragment thereof. In the present application, wherein the Ab is an anti-human folate receptor antibody or an antigen-binding fragment thereof.

[0556] The present application also provides a pharmaceutical composition, which includes the aforementioned compound, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate.

[0557] The present application also provides the use of the aforementioned compounds, or their tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically acceptable salts, prodrugs or solvates in the preparation of drugs for treating and / or preventing tumors.

[0558] In the present application, the tumor is selected from tumors associated with the expression of the following group of targets: claudin 18.2, human folate receptor, HER2, HER3, TROP2, B7-H3, GPC20, CDH6, EGFR, EGFRvⅢ, AXL, Nectin-4, Tissue factor, TIM-1, PSMA, EpCAM, MUC1, STEAP1, GPNMB, FGF2, FOLR1, c-MET, GFR, AGS-16, Guanylylcyclase C, Mesothelin, SLC44A4, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16. NaPi2b, STEAP, SLITRK6, ETBR, BCMA, CEACAM5, SC-16, SLC39A6, Delta-likeprotein3, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CD138, CD147, CD166, CD 223, MUC16, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, LewisY, av-integrin, ASCT2, C4.4a, CA-IX, CD324, CD352 , CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2, Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, MFI2, NOTCH3, p-cadherin, PRLR and RNF43.

[0559] In the present application, tumors associated with the target expression include tumors with high expression of the target and / or target-positive tumors.

[0560] In the present application, the tumor is selected from solid tumors, blood tumors and metastatic, refractory or recurrent lesions of cancer.

[0561] In the present application, the tumor is selected from the following group: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma and leukemia.

[0562] Example

[0563] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0564] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0565] In the following examples, Fmoc-L-glutamic acid-5-tert-butyl ester, compound 12-1, HATU (Chinese name is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate), DIEA (Chinese name is N,N-diisopropylethylamine), di(p-nitrobenzene) carbonate, exitecan mesylate, Mal-amido-PEG4-acid (Chinese name is maleimide-amide-PEG4-propionic acid), DCM (Chinese name is dichloromethane), TFA ( Chinese name is trifluoroacetic acid), 6-maleimidocaproic acid, Fmoc-L-valyl-L-alanine, di(p-nitrobenzene) carbonate, Fmoc-L-glutamic acid-5-tert-butyl ester, 4-aminobenzyl alcohol, Fmoc-L-valine, MMAE (Chinese name is monomethyl auristatin E), HOBT (Chinese name is 1-hydroxybenzotriazole), methyl tert-ether, compound 65-1, BH3-Me2S, DMAP (Chinese name is 4-dimethylaminopyridine), (BOC)2O, EA (Chinese name ethyl acrylate), TBAF (Chinese name is tetra-n-butylammonium fluoride), EEDQ (Chinese name is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline), compound 71-1, 6-(maleimido)hexanoic acid succinimide ester, compound 72-1, HOSu (Chinese name is N-hydroxysuccinimide), EDCI (Chinese name is carbodiimide), Fmoc-glycine, nitrophenyl chloroformate, compound 73-1, BCN-PEG4-acid (Chinese name is re l-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecan-19-oic acid), Fmoc-NMe-PEG4-C2-Acid, N,N-diisopropylethylamine, 4-methylsulfonylbenzoic acid, DIPEA (Chinese name is N,N-diisopropylethylamine), exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride, and N-Boc-N-methylethylenediamine are all commercially available.

[0566] Example 1 (Preparation of LD-12)

[0567] The synthetic route of LD-12 is as follows:

[0568]

[0569] Synthesis of 12-2

[0570] Dissolve Fmoc-L-glutamic acid-5-tert-butyl ester (600 mg, 1.41 mmoL) and compound 12-1 (414 mg, 1.41 mmoL) in 18 mL of anhydrous DMF, add HATU (644 mg, 1.69 mmoL) and DIEA (365 mg, 2.82 mmoL), continue stirring at room temperature for 18 hours, and LCMS shows that the reaction is complete. Add water, extract with ethyl acetate three times, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate to obtain 0.9 g of crude product.

[0571] Synthesis of 12-3

[0572] Compound 12-2 (300 mg, 0.43 mmoL), di(p-nitrobenzene) carbonate (261 mg, 0.86 mmoL) and DIEA (166 mg, 1.28 mmoL) were dissolved in 4 mL of anhydrous DMF and stirred at room temperature for 18 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a thin layer TLC plate (MeOH / DCM, 0% to 3%) to obtain 0.3 g. MS+Na(+)889.4.

[0573] Synthesis of 12-4

[0574] Compound 12-3 (350 mg, 0.425 mmoL), DIEA (207 mg, 1.61 mmoL) and isotecan mesylate (600 mg, 0.69 mmoL) were dissolved in 2 mL of anhydrous DMF, reacted at room temperature for 6 hours, and then water was added. After extraction with ethyl acetate three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 470 mg. MS (+) 1162.4.

[0575] Synthesis of 12-5

[0576] Compound 12-4 (100 mg, 0.086 mmol) was dissolved in 2 mL of anhydrous DMF, piperidine (0.05 mL) was added at room temperature and stirred for 2 hours. After concentration under reduced pressure, methyl tert-ether was slurried and filtered to obtain 80 mg of crude product, which was directly used in the next reaction. MS (+) 940.4.

[0577] Synthesis of 12-6

[0578] Compound 12-5 (46 mg, 0.11 mmoL) was dissolved in 2 mL of anhydrous DMF, HATU (48.5 mg, 0.13 mmoL) was added, and the mixture was stirred at room temperature for five minutes. Mal-amido-PEG4-acid (80 mg, 0.085 mmoL) and DIEA (33 mg, 0.26 mmoL) were added in sequence, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a thin layer TLC plate (MeOH / DCM, 0% to 3%) to obtain 30 mg. MS (+) 1339.2.

[0579] Synthesis of LD-12

[0580] Compound 12-6 (30 mg, 0.022 mmol) was dissolved in 2 mL DCM, 0.3 mL TFA was added at room temperature and stirred for 2 hours. LCMS showed that the reaction was complete. After concentration under reduced pressure, the mixture was prepared by reverse phase (mobile phase containing 0.1% formic acid) to obtain 7.7 mg.

[0581] LCMS: m / z(ES+)(M+H)+=1282.4, Rt=1.385min

[0582] 1H NMR (400MHz, DMSO-d6) δ10.00(s,1H),8.23(d,J=6.8Hz,1H),8.11-8.03(m,3H),7.79(d,J=10.8Hz,1H),7.72(d,J=8.4Hz,1H),7. 60(d,J=8.4Hz,2H),7.38(d,J=8.4Hz,2H),7.33(s,1H),7.00(s,2H),6.55(s,1H),5.46(s,2H),5.36-5.25(m,3H),5.09(s,2H),4 .41-4.32(m,2H),4.23-4.17(m,1H),3.60(t,J=7.2Hz,2H),3.50-3.49(m,16H),3.37-3.33(m,2H),3.16-3.14(m,2H),2.45-2.32 (m,7H),2.27-2.10(m,4H),2.01-1.96(m,1H),1.96-1.81(m,3H),1.74(d,J=6.8Hz,1H),1.31(d,J=7.2Hz,3H),0.91-0.83(m,9H).

[0583] Example 2 (Preparation of LD-27)

[0584] The process flow of LD-27 is as follows:

[0585]

[0586] Synthesis of 27-1

[0587] Compound 12-5 (127.34 mg, 0.306 mmoL, the synthesis of compound 12-5 can refer to Example 1) and 6-maleimidocaproic acid (80 mg, 0.085 mmoL) were dissolved in 4 mL of anhydrous DMF, HATU (49 mg, 0.128 mmoLl) and DIEA (22 mg, 0.170 mmoL) were added, and stirring was continued at room temperature for 3 hours. LCMS showed that the reaction was complete. Water was added, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 80 mg of crude product. MS (+) = 1133.4.

[0588] Synthesis of LD-27

[0589] Compound 27-1 (70 mg, 0.062 mmol) was dissolved in 5 mL DCM, 0.8 mL TFA was added at room temperature and stirred for 2 hours. LCMS showed that the reaction was complete. After concentration under reduced pressure, the mixture was prepared by reverse phase (mobile phase containing 0.1% formic acid) to obtain 22.8 mg.

[0590] LCMS: m / z(ES+)(M+H)+=1077.4, Rt=1.454min

[0591] 1H NMR (400MHz, DMSO-d6) δ12.08(s,1H),9.97(s,1H),8.21(d,J=6.8Hz,1H),8.06-7.98(m,2 H),7.77(d,J=10.8Hz,1H),7.65-7.57(m,3H),7.44-7.26(m,3H),6.99(s,2H),6.51(s,1H ),5.45(s,2H),5.28(d,J=4.4Hz,3H),5.08(s,2H),4.42-4.15(m,3H),3.26-3.08(m,2H), 2.37(s,3H),2.29-1.63(m,12H),1.51-1.42(m,4H),1.36-1.12(m,6H),0.89-0.81(m,9H).

[0592] Example 3 (Preparation of LD-31)

[0593] The synthetic route of LD-31 is as follows:

[0594]

[0595] Synthesis of 31-2

[0596] Fmoc-L-valyl-L-alanine (220 mg, 0.54 mmoL) was dissolved in 5 mL of anhydrous DMF and HATU (244.5 mg, 0.643 mmoL) and DIEA (138.5 mg, 1.07 mmoL) were added. After stirring at room temperature for 15 minutes, compound 31-1 (119.67 mg, 0.536 mmoL, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2022058548A1) was added to the reaction solution. After stirring at room temperature for 2 hours, LCMS monitored the reaction to be complete. The crude product was obtained after concentration. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%). 320 mg was obtained, MS+Na(+)=638.4.

[0597] Synthesis of 31-3

[0598] Compound 31-2 (320 mg, 0.520 mmoL) was dissolved in 5 mL of DCM, and di(p-nitrobenzene) carbonate (474.30 mg, 1.559 mmoL) and DIEA (0.344 mL, 2.079 mmoL) were added in sequence, and stirred at room temperature for 2 hours. LCMS monitored the reaction to be complete, saturated brine was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed on a normal phase silica gel column (MeOH / DCM, 0% to 10%). 340 mg was obtained, MS+Na(+)=803.2.

[0599] Synthesis of 31-4

[0600] Compound 31-3 (340 mg, 0.462 mmoL) was dissolved in 2 mL of anhydrous DMF, and then ixetidecan mesylate (245.61 mg, 0.666 mmoL), DIEA (119.43 mg, 0.924 mmoL) and DMAP (5.65 mg, 122.17 mmoL) were added in sequence. After stirring at room temperature for 1 hour, the reaction was complete as monitored by LCMS. Saturated saline was added to precipitate a solid crude product. The crude product was slurried with tert-methyl ether to obtain 157 mg of compound 31-4, MS (+) = 1078.2.

[0601] Synthesis of 31-5

[0602] Compound 31-4 (440 mg, 0.408 mmol) was dissolved in 4 mL of anhydrous DMF, piperidine (69.56 mg) was added to the reaction solution, and stirred at room temperature for one hour. The solution was concentrated to obtain 292 mg of crude product, which was directly used in the next step, MS (+) = 855.2.

[0603] Synthesis of 31-6

[0604] Compound 31-5 (142.23 mg, 0.342 mmoL) and Mal-amido-PEG4-acid (292 mg, 0.342 mmoL) were dissolved in 4 mL of anhydrous DMF, DIEA (88.28 mg, 0.683 mmoL) and HATU (155.84 mg, 0.410 mmoL) were added, and stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Saturated brine was added to precipitate a solid crude product. The crude product was slurried with tert-methyl ether to obtain 350 mg of crude product, MS (+) = 1254.4.

[0605] Synthesis of 31-7

[0606] Compound 31-6 (90 mg crude product) was dissolved in 2.5 mL anhydrous DCM, TFA (0.5 mL) was added to the reaction solution, and stirred at room temperature for 1 hour. The solution was concentrated to obtain 80 mg crude product, which was directly used in the next step, MS (+) = 1198.4.

[0607] Synthesis of LD-31

[0608] Compound 31-7 (76.62 mg, 0.064 mmoL) was dissolved in 2 mL of anhydrous DMF, DIEA (16.54 mg, 0.128 mmoL) and HATU (29.20 mg, 0.077 mmoL) were added, and the mixture was stirred at room temperature for 15 minutes. Sulfonic acid quaternary ammonium salt A (14.81 mg, 0.070 mmoL, CAS No.: 78276-19-4, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2011146595A2) was added, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Reverse phase preparation (formic acid) was used for purification to obtain a white solid LD-31 (P1: 12.5 mg, P2: 11.3 mg).

[0609] LCMS: m / z(ES+)(M+H)+=1390.6, Rt=1.242min(P1), 1.279min(P2).

[0610] Example 4 (Preparation of LD-37)

[0611] The synthetic route of LD-37 is as follows:

[0612]

[0613] Synthesis of LD-37

[0614] Compound LD-12 (49 mg, 0.038 mmoL, the synthesis of LD-12 can refer to Example 1) was dissolved in 2 mL of anhydrous DMF, DIEA (9.88 mg, 0.076 mmoL) and HATU (17.43 mg, 0.046 mmoL) were added, and the mixture was stirred at room temperature for 15 minutes. Sulfonic acid quaternary ammonium salt A (8.04 mg, 0.038 mmoL, CAS No.: 78276-19-4, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2011146595A2) was added, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Reverse phase preparation (mobile phase containing 0.1% formic acid) was used for purification to obtain a white solid LD-37 (7.35 mg 13.04% yield).

[0615] LCMS: m / z(ES+)(M+H)+=1475.6, Rt=1.344min

[0616] 1H NMR (400MHz, DMSO) δ10.03(s,1H),8.24(d,J=6.4Hz,1H),8.16-8.07(m,3H),8.03(t,J=6.0Hz,1H),7.80(d,J=10.8Hz,1H),7.71(d,J=9.2Hz,1H), 7.62(d,J=8.0Hz,2H),7.38(d,J=8.4Hz,2H),7.33(s,1H),7.02(s,2H),6 .53(s,1H),5.47(s,2H),5.31(s,3H),5.10(s,2H),4.41(d,J=6.8Hz,1H), 4.31(s,1H),4.24-4.20(m,1H),3.61(t,J=7.2Hz,5H),3.46-3.51(m,16H ),3.16(dd,J=11.2,5.6Hz,4H),3.05(s,6H),2.69(s,3H),2.40(s,4H),2. 35(s,6H),2.18(s,4H),2.01(s,2H),1.90(d,J=9.2Hz,2H),1.79-1.69(m ,1H),1.32(d,J=7.2Hz,3H),0.89(d,J=6.8Hz,6H),0.84(d,J=6.8Hz,3H).

[0617] Example 5: (Preparation of LD-38)

[0618] The synthetic route of LD-38 is as follows:

[0619]

[0620] Synthesis of 38-1

[0621] Dissolve Fmoc-L-glutamic acid-5-tert-butyl ester (144.7 mg, 1.175 mmoL) and 4-aminobenzyl alcohol (500 mg, 1.175 mmoL) in 3 mL of anhydrous DMF, add DIEA (303.7 mg, 2.350 mmoL) and HATU (536.19 mg, 1.410 mmoL), stir at room temperature for 16 hours, LCMS shows that the reaction is complete. Add water, extract with ethyl acetate three times, wash the organic phases with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate to obtain a crude product. Purify with a normal phase silica gel column (EA / PE, 0% to 45%) to obtain 530 mg. MS (+) = 475.2.

[0622] Synthesis of 38-2

[0623] Compound 38-1 (530 mg, 0.999 mmol) was dissolved in 3 mL of anhydrous DMF, piperidine (0.2 mL) was added to the reaction solution, and stirred at room temperature for one hour. The solution was concentrated to obtain 600 mg of crude product, which was directly used in the next step, MS (+) = 309.2.

[0624] Synthesis of 38-3

[0625] Compound 38-2 (600 mg crude product) and Fmoc-L-valine (500 mg, 1.175 mmol) were dissolved in 3 mL of anhydrous DMF, DIEA ((334.13 mg, 2.585 mmol) and HATU (589.82 mg, 1.551 mmol) were added, and stirred at room temperature for 16 hours. LCMS showed that the reaction was complete. Water was added, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (EA / PE, 40% to 60%) to obtain 450 mg, 6. MS (+) = 630.4.

[0626] Synthesis of 38-4

[0627] Compound 38-3 (450 mg, 0.715 mmol) was dissolved in 1 mL of anhydrous DCM and 4 mL of anhydrous DMF, and di(p-nitrobenzene) carbonate (869.5 mg, 2.858 mmol) and 0.354 mL of DIEA were added in turn. After reacting at room temperature for 12 hours, water was added to the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (EA / PE, 30% to 70%) to obtain 200 mg. MS(+) = 543.2.

[0628] Synthesis of 38-5

[0629] Compound 38-4 (100 mg, 0.126 mmoL) and isotecan mesylate (54.78 mg, 0.126 mmoL) were dissolved in 2 mL of anhydrous DMF, and DMAP (0.15 mg, 0.0001 mmoL) and DIEA (32.52 mg, 0.252 mmoL) were added in turn. After reacting at room temperature for 1 hour, water was added to the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (EA / PE, 30% to 70%) to obtain 100 mg. MS (+) = 1091.4.

[0630] Synthesis of 38-6

[0631] Compound 38-5 (100 mg, 0.073 mmol) was dissolved in 3 mL of anhydrous DMF, piperidine (0.3 mL) was added to the reaction solution, and stirred at room temperature for one hour. The solution was concentrated to obtain 110 mg of crude product, which was directly used in the next step. MS (+) = 870.8.

[0632] Synthesis of 38-7

[0633] Compound 38-6 (110 mg crude product) and Mal-amido-PEG4-acid (30.19 mg, 0.073 mmoL) were dissolved in 2 mL of anhydrous DMF, DIEA (18.74 mg, 0.145 mmoL) and HATU (33.08 mg, 0.087 mmoL) were added, and stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 5% to 20%) to obtain 90 mg. MS (+) = 1267.6.

[0634] Synthesis of 38-8

[0635] Compound 38-7 (90 mg, 0.071 mmol) was dissolved in 3 mL of anhydrous DCM, 0.3 mL of TFA was added, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated to obtain 80 mg of crude product, which was directly used in the next step. MS (+) = 2111.4.

[0636] Synthesis of LD-38

[0637] Compound 38-8 (80 mg, crude product) and quaternary ammonium sulfonate salt A (13.89 mg, 0.066 mmol, CAS No.: 78276-19-4, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2011146595A2) were dissolved in 2 mL of anhydrous DMF, DIEA (17.07 mg, 0.132 mmoL) and HATU (30.14 mg, 0.079 mmoL) were added, stirred at room temperature for 1 hour, and LCMS showed that the reaction was complete. Reverse phase preparation (mobile phase containing 0.1% formic acid) was used for purification to obtain 13.5 mg of a white solid.

[0638] LCMS: m / z(ES+)(M+H)+=1404.6, Rt=1.314min

[0639] 1H NMR (400MHz, DMSO) δ9.89(s,1H),8.18(d,J=7.2Hz,2H),8.10-7.93(m,3H),7.78(d,J=10.8Hz,1H),7.63(d,J= 8.4Hz,2H),7.37(d,J=8.4Hz,2H),7.32(s,1H),6.99(s,2H),6.53(s,1H),5.45(s,2H),5.29(d,J=4.Hz,3H),5 .08(s,2H),4.32(dd,J=13.2,7.6Hz,1H),4.20-4.14(m,1H),3.59(dd,J=14.8,7.6Hz,6H),3.40-3.51(m,18H) ,3.14-3.20(m,4H),3.03(s,7H),2.42-2.30(m,7H),2.20-2.31(m,4H),2.03-1.82(m,8H),0.87-0.91(m,9H).

[0640] Example 6 (Preparation of LD-57)

[0641] The synthetic route of LD-57 is as follows:

[0642]

[0643] Synthesis of 57-1

[0644] Compound 38-4 (288 mg, 0.363 mmol, its synthesis method can refer to Example 5) and MMAE (200 mg, 0.279 mmol) were dissolved in 5 mL of anhydrous DMF, and pyridine (1 mL) and HOBT (59 mg, 0.279 mmol) were added in sequence. After reacting at room temperature for 18 hours, the crude product was obtained after concentration. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 320 mg. MS (+) = 1374.2.

[0645] Synthesis of 57-2

[0646] Compound 57-1 (260 mg, 0.189 mmol) was dissolved in 3 mL of anhydrous DMF, piperidine (0.05 mL) was added to the reaction solution, and stirred at room temperature for one hour. The solution was concentrated and slurried with tert-methyl ether to obtain 215 mg of crude product, which was directly used in the next step. MS (+) = 1152.5.

[0647] Synthesis of 57-3

[0648] Compound 57-2 (215 mg crude product) and 6-maleimidocaproic acid (47.3 mg, 0.224 mmoL) were dissolved in 3 mL of anhydrous DMF, DIEA (0.092 mL, 0.559 mmoL) and HATU (142 mg, 0.374 mmoL) were added, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 170 mg. MS (+) = 1345.7.

[0649] Synthesis of 57-4

[0650] Compound 57-3 (150 mg, 0.112 mmol) was dissolved in 3 mL of anhydrous DCM, TFA (0.6 mL) was added to the reaction solution, and stirred at room temperature for 2 hours. Concentration gave 150 mg of crude product, which was used directly in the next step. MS (+) = 1289.5.

[0651] Synthesis of LD-57

[0652] Compound 57-4 (143 mg, 0.111 mmoL) and quaternary ammonium sulfonate A (70 mg, 0.333 mmoL, CAS No.: 78276-19-4, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2011146595A2) were dissolved in 2 mL of anhydrous DMF, DIEA (0.092 mL, 0.554 mmoL) and HATU (126.5 mg, 0.333 mmoL) were added, and stirred at room temperature for 3 hours. LCMS showed that the reaction was complete. Reverse phase preparation (mobile phase containing 0.1% formic acid) was used for purification to obtain 30 mg of a white solid.

[0653] LCMS: m / z(ES+)(M+H)+=1481.2, Rt=1.475min

[0654] 1H NMR (400MHz, DMSO) δ9.89(s,1H),8.40-8.03(m,3H),7.92(t,J=7.6Hz,1H),7.63(d,J=6.4Hz,2H),7.34-7.13(m,8H),7.02(s,2H),5.06-5.10(m,3 H),4.85-3.92(m,10H),3.63-3.43(m,9H),3.30-2.97(m,18H),2.89(d,J =14.4Hz,3H),2.37-1.68(m,17H),1.61-1.45(m,6H),1.40-0.70(m,36H).

[0655] Example 7 (Preparation of LD-65)

[0656] The synthetic route of LD-67 is as follows:

[0657]

[0658] Synthesis of 65-2

[0659] Dissolve compound 65-1 (500 mg, 2.791 mmol) in 5 mL methanol, add methylamine methanol solution (1.73 g, 55.825 mmol). After reacting at room temperature for 1 hour, filter to obtain a crude product. Wash twice with water and dry under reduced pressure to obtain 550 mg. MS (+) 211.2.

[0660] Synthesis of 65-3

[0661] Compound 65-2 (500 mg, 2.379 mmol) was dissolved in 50 mL of anhydrous THF, and BH3-Me2S (457 mg, 5.947 mmol) (10.0 M in THF) was slowly added dropwise under ice bath. After 5 hours of reflux, LCMS showed that the reaction was complete, and 4 M HCl (12 mL) methanol solution was added to the reaction solution under ice bath, and the reaction was continued for 8 hours at 65 degrees. Filter and dry under reduced pressure to obtain 350 mg. MS (+) 197.08.

[0662] Synthesis of 65-4

[0663] Compound 65-3 (350 mg crude product) and imidazole (485.77 mg, 7.135 mmol) were dissolved in 5 mL of anhydrous DCM, TBDPS-Cl (735.44 mg, 2.676 mmol) was added at 0 degrees, and stirred for 2 hours. LCMS showed that the reaction was complete. Water was added, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (EA / PE, 10% to 50%) to obtain 710 mg. MS (+) 435.20.

[0664] Synthesis of 65-5

[0665] Compound 64-4 (1.5 g, 3.451 mmoL) and DMAP (0.21 g, 1.726 mmoL) were dissolved in DCM (20 mL), (BOC)2O (1.51 g, 6.903 mmoL) was added and stirred for 2 hours. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (EA / PE, 10% to 50%) to obtain 1.87 g. MS+Na(+)557.3.

[0666] Synthesis of 65-6

[0667] Compound 65-5 (1.7 g, 3.179 mmol) and Pd / C 5% (0.15 g, 1.410 mmol) were dissolved in EA (30 mL), the reaction solution was stirred for 16 hours under hydrogen atmosphere, filtered and dried to obtain 1.7 g. MS+Na(+)527.6.

[0668] Synthesis of 65-7

[0669] Compound 65-6 (350 mg, 0.693 mmol) was dissolved in anhydrous THF (4 mL), TBAF (1.387 mL, 1.387 mmol) was added and stirred at room temperature for 4 hours. The reaction solution was spin-dried and purified by normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 170 mg. MS+Na(+)=289.2.

[0670] Synthesis of 65-8

[0671] Compound 65-7 (170 mg, 0.638 mmoL), Fmoc-L-valyl-L-alanine (392.99 mg, 0.957 mmoL) and EEDQ (236.48 mg, 0.957 mmoL) were dissolved in 2 mL DCM and 1 mL methanol and stirred at room temperature for 16 hours. The reaction solution was spin-dried and purified by normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 367 mg. MS (+) Na (+) = 681.4.

[0672] Synthesis of 65-9

[0673] Compound 65-9 (367 mg, 0.557 mmoL), di(p-nitrobenzene) carbonate (224.57 mg, 1.114 mmoL) and pyridine (0.090 mL, 1.114 mmoL) were dissolved in 5 mL THF and stirred at room temperature for 16 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 380 mg. MS (+) Na (+) = 846.4.

[0674] Synthesis of 65-10

[0675] Compound 65-9 (350 mg, 0.425 mmoL), DMAP (5.19 mg, 0.043 mmoL) and isotecan mesylate (248.50 mg, 0.468 mmoL) were dissolved in 2 mL of anhydrous DMF, reacted at room temperature for 2 hours, and then water was added. After extraction with ethyl acetate three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 350 mg. MS (+) 1120.6.

[0676] Synthesis of 65-11

[0677] Compound 65-10 (350 mg, 0.312 mmol) was dissolved in 2 mL of anhydrous DMF, pyridine (0.05 mL) was added at room temperature and stirred for 2 hours. After concentration under reduced pressure, 380 mg of the crude product was directly used for the next reaction. MS (+) = 898.41.

[0678] Synthesis of 65-12

[0679] Compound 65-11 (127.34 mg, 0.306 mmoL) was dissolved in 3 mL of anhydrous DMF, HATU (116.28 mg, 0.306 mmoL) was added, and the mixture was stirred at room temperature for five minutes. Mal-amido-PEG4-acid (250 mg, 0.278 mmoL) and DIEA (3 mL) were added in sequence, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 185 mg. MS (+) = 1296.6.

[0680] Synthesis of 65-13

[0681] Compound 65-12 (40 mg, 0.031 mmol) was dissolved in 3 mL DCM, 0.6 mL TFA was added at room temperature and stirred for 2 hours, LCMS showed that the reaction was complete, and 37 mg was obtained after concentration under reduced pressure. MS+Na(+)=1218.6.

[0682] Synthesis of LD-65

[0683] Sulfonic acid quaternary ammonium salt B (37 mg, 0.031 mmol, CAS No.: 1349808-80-5, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2011146595A2) was dissolved in 2 mL of anhydrous DMF, HATU (12.94 mg, 0.034 mmoL) was added, and stirred at room temperature for five minutes. Compound 65-13 (11.75 mg, 0.046 mmoL) and DIEA (0.008 mL, 0.046 mmoL) were added in sequence, and stirring was continued at room temperature for 2 hours. LCMS showed that the reaction was complete. 5.2 mg of white solid was obtained by reverse phase preparation (mobile phase containing 10 mM NH4OAc).

[0684] LCMS: m / z(ES+)(M+H)+=1432.6, Rt=1.285min

[0685] 1H NMR (400MHz, DMSO) δ10.2-9.9(m,1H),8.19(t,J=7.2Hz,1H),8.12-8.00(m,2H),7.90-7.85(m,1H),7. 78(d,J=10.8Hz,1H),7.39-7.30(m,3H),7.00(s,2H),6.52(s,1H),5.44(d,J=4.0Hz,2H),5.29(s,4H) ,5.12(d,J=10.8Hz,2H),4.64-4.70(m,2H),4.39-4.34(m,1H),4.22-4.17(m,1H),3.48-3.47(m,16H) ,3.04-2.86(m,14H),2.39-2.30(m,11H),2.03-1.85(m,10H),1.29-1.30(m,3H),0.897-0.817(m,9H).

[0686] Example 8 (Preparation of LD-66)

[0687] The synthetic route of LD-66 is as follows:

[0688]

[0689] Synthesis of LD-66

[0690] Quaternary ammonium phosphate (8.93 mg, 0.046 mmol, Cas number: 853798-56-8, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2005114193) was dissolved in 2 mL of anhydrous DMF, DIEA (0.008 mL, 0.046 mmoL) and HATU (12.94 mg, 0.034 mmoL) were added, and stirred at room temperature for 5 minutes. Compound 65-13 (37 mg, 0.031 mmoL) was added and stirred for 2 hours, and LCMS showed that the reaction was complete. Reverse phase preparation (formic acid) was used for purification to obtain 11.5 mg of a white solid.

[0691] LCMS: m / z(ES+)(M+H)+=1420.6, Rt=1.246min

[0692] 1H NMR (400MHz, DMSO) δ10.11(s,1H),8.23-8.17(m,1H),8.07-7.87(m,2H),7.79-7.69(m,2H),7.45-7.1 3(m,4H),7.00(s,2H),6.51(s,1H),5.47-5.06(m,8H),4.83(dd,J=45.6,17.8Hz,1H),4.59(d,J=7.6Hz ,1H),4.48(d,J=8.4Hz,1H),4.37(d,J=6.8Hz,1H),4.21-4.09(m,4H),3.49-3.48(m,16H),3.19-3.10 (m,12H),2.96(s,5H),2.39-2.32(m,8H),1.99-1.83(m,4H),1.30(d,J=6.8Hz,3H),0.90-0.82(m,9H).

[0693] Example 9 (Preparation of LD-71)

[0694] The synthetic route of LD-71 is as follows:

[0695]

[0696] Synthesis of 71-2

[0697] Compound 31-1 (240 mg, 1.075 mmoL, its synthesis can refer to Example 1) and compound 71-1 (267 mg, 0.538 mmoL, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., the synthesis method refers to WO2022058548A1) were dissolved in 3 mL of anhydrous DCM and 1.5 mL of methanol, and EEDQ (266 mg, 1.075 mmoL) was added. Stir at room temperature for 3 hours, solid precipitated, and LCMS monitored the reaction to be complete. Filter, wash the filter cake three times with DCM and dry under reduced pressure to obtain 260 mg. MS (+) = 702.4.

[0698] Synthesis of 71-3

[0699] Compound 71-2 (240 mg, 0.342 mmol), di(p-nitrobenzene) carbonate (312.09 mg, 1.026 mmol) and DIEA (0.170 mL, 1.026 mmol) were dissolved in 5 mL DMF and stirred at room temperature for 16 hours. The reaction was complete after LCMS monitoring. Water was added, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 5% to 10%) to obtain 220 mg. MS (+) = 867.4.

[0700] Synthesis of 71-5

[0701] Under nitrogen protection at room temperature, triphosgene (136.1 mg, 0.459 mmol) was dissolved in 4 mL of anhydrous DCM, and triethylamine (0.128 mL, 0.917 mol) and 71-4 (172.6 mg, 0.917 mol) were added dropwise under ice bath. After stirring at room temperature for 3 hours, pyridine (2 mL), SN38 (120 mg, 0.306 mmol) and DMAP (7.47 mg, 0.061 mol) were added. The reaction was carried out at 50 ° C for 16 hours, and the reaction was complete after LCMS monitoring. After concentration and spin drying, the mixture was slurried with tertiary methyl ether and filtered to obtain 127 mg. MS (+) = 607.4.

[0702] Synthesis of 71-6

[0703] Compound 71-5 (140 mg, 0.231 mmol) was dissolved in 3 mL of anhydrous DCM, 0.6 mL of TFA was added at room temperature and stirred for 2 hours, LCMS showed that the reaction was complete, and 116.9 mg was obtained after concentration under reduced pressure. MS (+) = 507.2.

[0704] Synthesis of 71-7

[0705] Compound 71-6 (116.9 mg, 0.231 mmoL) and compound 71-3 (200 mg, 0.231 mmoL) were dissolved in 2 mL DMSO and 1 mL DMF, and HOBT (31.17 mg, 0.231 mmoL) and DIEA (59.64 mg, 0.461 mmoL) were added in sequence. After heating at 40°C and stirring for 3 hours, saturated brine was added to the reaction solution to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH, 0% to 10%) to obtain 200 mg. MS (+) = 1234.6.

[0706] Synthesis of 71-8

[0707] 100 mg of crude compound 71-7 was dissolved in 2 mL of anhydrous DMF, pyridine (0.05 mL) was added at room temperature and stirred for 2 hours. After concentration under reduced pressure, methyl tert-ether was slurried and filtered to obtain 100 mg of crude product, which was directly used in the next step. MS (+) 1012.6.

[0708] Synthesis of 71-9

[0709] Dissolve 100 mg of crude compound 71-8 and 6-(maleimido)hexanoic acid succinimide ester (43.9 mg, 0.142 mmol) in 2 mL of anhydrous DMF, add DIEA and stir at room temperature for 2 hours. Add water to the reaction solution, extract three times with ethyl acetate, wash the organic phases with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate to obtain a crude product. Purify with a thin layer silica gel plate (MeOH / DCM, 0% to 10%) to obtain 60 mg. MS (+) = 1205.6.

[0710] Synthesis of 71-10

[0711] Compound 71-9 (60 mg, 0.05 mmol) was dissolved in 3 mL DCM, 0.3 mL TFA was added at room temperature and stirred for 2 hours, LCMS showed that the reaction was complete, and 57 mg was obtained after concentration under reduced pressure. MS (+) = 1149.6.

[0712] Synthesis of LD-71

[0713] Compound 71-10 (57 mg, 0.050 mmoL) was dissolved in 2 mL of anhydrous DMF, HATU (20.76 mg, 0.055 mmoL) was added, and the mixture was stirred at room temperature for five minutes. Sulfonic acid quaternary ammonium salt A (15.64 mg, 0.074 mmol, CAS No.: 78276-19-4, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2011146595A2) and DIEA (0.025 mL, 0.149 mmoL) were added in sequence, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. 24.0 mg of white solid was obtained by reverse phase preparation (mobile phase containing 0.1% formic acid).

[0714] LCMS: m / z(ES+)(M+H)+=1341.6, Rt=1.210min

[0715] 1H NMR(400MHz,DMSO)δ10.04-9.96(m,1H),8.45(s,1H),8.185-8.085(m,2H),7.968-7.7 82(m,3H),7.63-7.32(m,9H),6.99(s,2H),6.53(s,1H),5.99(s,1H),5.70(d,J=20.6Hz ,1H),5.43-5.41(m,5H),5.35(s,3H),4.35-4.25(m,1H),4.19-4.17(m,1H),3.07-2.9 1(m,20H),1.97-1.83(m,8H),1.52-1.42(m,6H),1.32-1.15(m,9H),0.90-0.82(m,9H).

[0716] Example 10 (Preparation of LD-72)

[0717] The synthetic route of LD-72 is as follows:

[0718]

[0719] Synthesis of 72-2

[0720] Compound 72-1 (600 mg, 1.076 mmol) was dissolved in 4 mL of anhydrous DMF, and 0.1 mL of piperidine was added. The mixture was stirred at room temperature for one hour, and then distilled under reduced pressure and slurried with petroleum ether to obtain 350 mg of crude product. MS+Na(+)=357.9.

[0721] Synthesis of 72-3

[0722] Compound 72-2 (360 mg, 1.073 mmoL) and 6-maleimidocaproic acid (340 mg, 1.610 mmoL) were dissolved in 10 mL of anhydrous DMF, DIEA (0.532 mL, 3.219 mmoL) and HATU (817 mg, 2.150 mmoL) were added, and stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 5%) to obtain 400 mg. MS (+) = 529.6.

[0723] Synthesis of 72-4

[0724] Compound 72-3 (90 mg, 0.071 mmol) was dissolved in 3 mL of anhydrous DCM, TFA (0.8 mL) was added to the reaction solution, and stirred at room temperature for 18 hours. Concentration gave 80 mg of crude product, which was used directly in the next step. MS (+) = 473.5.

[0725] Synthesis of 72-5

[0726] Compound 72-4 (150 mg, 0.317 mmol) was dissolved in 2 mL, HOSu (40.2 mg, 0.349 mmol) and EDCI (67.2 mg, 0.351 mmol) were added, stirred at room temperature for 2 hours, and distilled under reduced pressure to obtain 160 mg of crude product, which was directly used in the next step. MS (+) = 569.8.

[0727] Synthesis of 72-6

[0728] Compound 65-7 (700 mg, 2.628 mmoL) and Fmoc-glycine (1.56 g, 5.25 mmoL) were dissolved in 20 mL of anhydrous THF, EEDQ (780 mg, 3.158 mmoL) was added, and the mixture was stirred at room temperature for 18 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 5%) to obtain 1000 mg. MS+Na(+)=567.8.

[0729] Synthesis of 72-7

[0730] Compound 72-6 (290 mg, 0.531 mmoL) was dissolved in 6 mL of anhydrous THF, and p-nitrophenyl chloroformate (129 mg, 0.640 mmoL) and pyridine (63 mg, 0.797 mmoL) were added in sequence. After reacting at room temperature for 3 hours, water was added to the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 5%) to obtain 300 mg. MS+Na(+)=732.7.

[0731] Synthesis of 72-8

[0732] Compound 72-7 (340 mg, 0.478 mmoL) and isotecan mesylate (254 mg, 0.478 mmoL) were dissolved in 8 mL of anhydrous DMF, and DMAP (8 mg, 0.025 mmoL) and DIEA (0.237 mL, 1.434 mmol) were added in turn. After reacting at room temperature for 12 hours, water was added to the reaction solution, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 380 mg. MS (+) = 1006.7.

[0733] Synthesis of 72-9

[0734] Compound 72-8 (300 mg, 0.298 mmol) was dissolved in 3 mL of anhydrous DMF, and 0.06 mL of piperidine was added. The mixture was stirred at room temperature for one hour, and then distilled under reduced pressure and slurried with petroleum ether to obtain 230 mg of crude product. MS (+) = 785.8.

[0735] Synthesis of 72-10

[0736] Compound 72-9 (180 mg, 0.316 mmoL) and compound 72-5 (207 mg, 0.264 mmoL) were dissolved in 3 mL of anhydrous DMF, DIEA was added and stirred at room temperature for 2 hours. Water was added to the reaction solution, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a thin layer silica gel plate (MeOH / DCM, 0% to 10%) to obtain 100 mg. MS (+) = 1240.8.

[0737] Synthesis of 72-11

[0738] Compound 71-10 (90 mg, 0.071 mmol) was dissolved in 3 mL of anhydrous DCM, TFA (0.3 mL) was added to the reaction solution, and stirred at room temperature for 1 hour. Concentration gave 36 mg of crude product, which was used directly in the next step. MS (+) = 1138.6.

[0739] Synthesis of LD-72

[0740] Compound 71-11 (36 mg, crude product) and quaternary ammonium phosphate (11.4 mg, 0.047 mmol, Cas number: 853798-56-8, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2005114193) were dissolved in 3 mL of anhydrous DMF, DIEA (0.015 mL, 0.093 mmol) and HATU (24 mg, 0.032 mmol) were added, and stirred at room temperature for 3 hours. LCMS showed that the reaction was complete. Reverse phase preparation (mobile phase containing 10 mM NH4OAc) was used for purification to obtain 15 mg of a white solid.

[0741] LCMS: m / z(ES+)(M+H)+=1361.5, Rt=1.114min

[0742] 1H NMR (400MHz, DMSO) δ10.01(d,J=12Hz,1H),9.04(d,J=8.0Hz,1H),8.40(s,2H),8.27-8.07(m,3H),7.7 9-7.60(m,2H),7.48-7.11(m,9H),6.99(s,2H),6.52(s,1H),5.57-5.00(m,8H),4.94-4.37(m,5H),4. 25-4.09(m,2H),3.99-3.48(m,10H),3.08(d,J=13.6Hz,7H),2.94(d,J=13.2Hz,9H),2.40-2.33(m,3H ),2.19-2.07(m,4H),1.91-1.81(m,2H),1.52-1.41(m,4H),1.33-1.00(m,3H),0.88(t,J=7.2Hz,3H).

[0743] Example 11 (Preparation of LD-73)

[0744] The synthetic route of LD-73 is as follows:

[0745]

[0746] Synthesis of LD-73

[0747] Compound 73-1 (25.19 mg, 0.055 mmoL) and HATU (20.98 mg, 0.055 mmoL) were dissolved in 2 mL of anhydrous DMF. After stirring at room temperature for 5 minutes, compound 65-13 (60 mg, 0.050 mmoL) and DIEA (0.008 mL, 0.075 mmoL) were added in sequence. After two hours, the reaction was complete as monitored by LCMS, and the reaction system was directly subjected to reverse phase preparation (mobile phase containing 0.1% formic acid) to obtain 19 mg of white solid.

[0748] LCMS: m / z(ES+)(M+H)+=1634.8, Rt=1.392min

[0749] 1H NMR (400MHz, DMSO) δ9.96 (d, J = 40.0Hz, 1H), 8.17 (d, J = 6.8Hz, 1H), 8.00 (s, 1H), 7.78 (d, J = 10. 8Hz,2H),7.62(s,1H),7.38-7.26(m,4H),6.99(s,2H),6.52(s,1H),5.44(s,3H),5.29(s,5H),5 .13-5.10(m,3H),4.67(s,1H),4.58(s,1H),4.36(d,J=7.2Hz,1H),4.22-4.19(m,1H),3.66-3.5 5(m,10H),3.52-3.40(m,50H),3.23(s,6H),3.18-3.10(m,5H),1.24(s,3H),0.89-0.81(m,9H).

[0750] Example 12 (Preparation of LD-74)

[0751] The synthetic route of LD-74 is as follows:

[0752]

[0753] Synthesis of 74-1

[0754] 6-Maleimidocaproic acid (18.59 mg, 0.088 mmoL) was dissolved in 1 mL of anhydrous DMF, HATU ((33.46 mg, 0.088 mmoL) was added, and the mixture was stirred at room temperature for five minutes. Compound 65-11 (72.11 mg, 0.080 mmoL) and DIEA (0.020 mL) were added in sequence, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. Water was added, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification was performed using a normal phase silica gel column (MeOH / DCM, 0% to 10%) to obtain 80 mg. MS (+) 1091.6.

[0755] Synthesis of 74-2

[0756] Compound 74-1 (65 mg, 0.073 mmol) was dissolved in 3 mL DCM, 0.6 mL TFA was added at room temperature and stirred for 2 hours, LCMS showed that the reaction was complete, and 65 mg was obtained after concentration under reduced pressure. MS (+) = 991.4.

[0757] Synthesis of LD-74

[0758] Compound 74-3 (20.00 mg, 0.026 mmoL, purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd., synthesis method reference WO2022228493) was dissolved in 1 mL of anhydrous DMF, HATU (10.85 mg, 0.029 mmoL) was added, and stirred at room temperature for five minutes. Compound 74-2 (25.71 mg, 0.026 mmoL) and DIEA (0.008 mL, 0.046 mmoL) were added in sequence, and stirring was continued at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction solution was prepared by reverse phase (mobile phase containing 10 mM NH4OAc) to obtain 9.5 mg of white solid.

[0759] LCMS: m / z(ES+)(M+H)+=1743.9, Rt=1.325min

[0760] 1H NMR (400MHz, DMSO) δ7.79(d,J=10.9Hz,2H),7.31(s,1H),6.99(s,2H),6.52(s,1H),5.45(s,2H),5.28(s,3H),5.12(s ,2H),4.33-4.04(m,26H),2.98-2.66(m,45H),1.90(m,7H),1.49-1.47(m,5H),1.31-1.15(m,7H),0.89-0.81(m,9H).

[0761] Example 13 (Preparation of LD-88)

[0762] The synthetic route of LD-88 is as follows:

[0763]

[0764] Synthesis of LD-88

[0765] Compound 38-8 (50 mg, 0.041 mmol, its synthesis method can refer to Example 3) and quaternary ammonium sulfonate C (16.34 mg, 0.078 mmol, CN115521998, Shanghai Haoyuan Pharmaceutical Co., Ltd.) were dissolved in 2 mL of anhydrous DMF, DIEA (0.010 mL, 0.062 mmol) and HATU (17.27 mg, 0.045 mmol) were added, and stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction liquid was directly subjected to reverse phase preparation (trifluoroacetic acid) for purification to obtain a light yellow solid LD-88 (10.0 mg, 17.28% yield).

[0766] LCMS: m / z(ES+)(M+H)+=1402.6, Rt=1.303min

[0767] 1H NMR (400MHz, DMSO) δ10.06(s,1H),9.95(s,1H),8.19(d,J=7.6Hz,1H),8.10-8.00(m,2H),7.91(d,J=8Hz,1H),7.79(d ,J=10.8Hz,1H),7.61(d,J=8.4Hz,2H),7.38(d,J=8.4Hz,2H),7.32(s,1H),7.00(s,2H),6.53(s,1H),5.45(s,2H),5. 29(s,3H),5.09(s,2H),4.45-4.41(m,2H),4.20-3.99(m,2H),3.62-3.57(m,4H),3.51-3.47(m,14H),3.19-3.13(m,5 H), 2.67 (dt, J = 9.2, 4.4Hz, 4H), 2.39 (s, 5H), 2.36-2.31 (m, 5H), 2.19 (s, 3H), 2.06-1.82 (m, 10H), 0.90-0.84 (m, 9H).

[0768] Example 14 (Preparation of LD-89)

[0769] The synthetic route of LD-89 is as follows:

[0770]

[0771] Synthesis of LD-89

[0772] Compound 38-8 (50 mg, 0.041 mmol, its synthesis method can refer to Example 3) and quaternary ammonium sulfonate D (17.58 mg, 0.078 mmol, Bioconjugate Chem. 2022, 33, 718-725, Shanghai Haoyuan Pharmaceutical Co., Ltd.) were dissolved in 2 mL of anhydrous DMF, DIEA (0.010 mL, 0.062 mmol) and HATU (17.27 mg, 0.045 mmol) were added, and stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction solution was directly subjected to reverse phase preparation (formic acid) for purification to obtain a white solid LD-89 (5.4 mg, 10.06% yield).

[0773] LCMS: m / z(ES+)(M+H)+=1418.6, Rt=1.288min

[0774] 1H NMR (400MHz, DMSO) δ9.86 (s, 1H), 8.11 (d, J = 7.2Hz, 1H), 8.01-7.89 (m, 3H), 7.71 (d, J = 10.8Hz, 1H), 7.55 (d, J =8.4Hz,2H),7.30(d,J=8.4Hz,2H),7.24(s,1H),6.93(s,2H),6.45(s,1H),5.38(s,2H),5.22(d,J=4.0Hz,3H ),5.01(s,2H),4.35-4.30(m,1H),4.15-4.12(m,1H),3.52(s,8H),3.42(d,J=3.6Hz,17H),3.25(s,5H),3.09 -2.99(m,14H),2.31(s,4H),2.26(d,J=7.2Hz,3H),2.16-2.07(m,2H),1.88-1.82(m,8H),0.82-0.76(m,9H).

[0775] Example 15 (Preparation of LD-94)

[0776] The synthetic route of LD-94 is as follows:

[0777]

[0778] Synthesis of Compound 94-1

[0779] Compound 38-5 (450 mg, 0.412 mmol, the synthesis method can be described in Example 3) was added to dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added dropwise to the system. The mixed system was stirred at room temperature for 2 hours. The system was directly concentrated to dryness and the crude product was used in the next step. m / z (ES+) (M+1) = 1035.4

[0780] Synthesis of Compound 94-2

[0781] Compound 94-1 (400 mg, 0.386 mmol), quaternary ammonium sulfonate A (121.90 mg, 0.580 mmol) and DIEA (74.91 mg, 0.580 mmol) were dissolved in DMF (4 mL), and the mixture was stirred at room temperature for 5 minutes, and HATU (161.63 mg, 0.425 mmol) was added. The reaction system was stirred at room temperature for 4 hours and then purified by reverse phase column (water: acetonitrile from 0% to 70%) to obtain 170 mg, m / z (ES+) (M+1) = 1227.6.

[0782] Synthesis of Compound 94-3

[0783] Compound 94-2 (70 mg, 0.057 mmol) was dissolved in DMF (1 mL), and diethylamine (14.30 mg, 0.196 mmol) was added dropwise to the system. The mixed system was stirred at room temperature for 2 hours, then concentrated and dried, slurried with petroleum ether and filtered. The filter cake was dried and used directly in the next step, m / z (ES+) (M+1) = 1005.6.

[0784] Synthesis of LD-94

[0785] Compound 94 (55 mg, 0.055 mmol), BCN-PEG4-acid (cas number: 1881221-47-1, 24.16 mg, 0.055 mmol) and DIEA (0.011 mL, 0.067 mmol) were dissolved in DMF (2 mL). After stirring at room temperature for 5 minutes, HATU (18.73 mg, 0.049 mmol) was added. After the mixed system was reacted at room temperature for 2 hours, the product was analyzed by Prep-HPLC (NH4HCO3 system) to obtain LD-94 (6 mg, yield: 7.68%).

[0786] LCMS: m / z(ES+)(M+H) + =1427.6,Rt=1.721min.

[0787] 1 H NMR (400MHz, DMSO) δ9.90(s,1H),8.19(s,2H),8.09-8.05(m,1H),8.00-7.96(m,1H),7.79(d,J=11.2Hz,1H),7.63(d,J=7.6 Hz,2H),7.37(d,J=8.2Hz,2H),7.31(s,1H),6.53(s,1H),5.45(s,1H),5.29(s,3H),5.08(s,2H),4.19-4.15(m,1H),4.02(d, J=8.6Hz,2H),3.62-3.58(m,2H),3.50-3.47(m,16H),3.10(s,2H),3.04-3.02(m,7H),2.69-2.66(m,4H),2.40-2.36(m,5H) ,2.34-2.32(m,4H),2.22-2.15(m,9H),1.99(s,4H),1.92-1.86(m,3H),1.55-1.46(m,2H),1.23(s,2H),0.90-0.83(m,14H).

[0788] Example 16 (Preparation of LD-98)

[0789] The synthetic route of LD-98 is as follows:

[0790]

[0791] Synthesis of Compound 98-1

[0792] Compound 65-11 (120 mg, 0.134 mmol, its synthesis method can refer to Example 5) and Fmoc-NMe-PEG4-C2-Acid (cas number: 2170240-98-7, 67.0 mg, 0.134 mmol) were dissolved in DMF (2 mL), HATU (55.9 mg, 0.147 mmol, 1.1 eq) and N, N-diisopropylethylamine (26 mg, 0.200 mmol, 1.5 eq) were added in sequence, and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM: MeOH, MeOH was 0 to 10%) to obtain 100 mg, m / z (ES+) (M+1) = 1381.6.

[0793] Synthesis of Compound 98-2

[0794] Compound 98-1 (100 mg, 0.072 mmol) was dissolved in DMF (2 mL), diethylamine (15.88 mg, 0.217 mmol) was added, and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to obtain a crude product of 100 mg, m / z (ES+) (M+1) = 1159.6.

[0795] Synthesis of Compound 98-3

[0796] Compound 98-2 (80 mg, 0.069 mmol) and 4-methylsulfonylbenzoic acid (13.95 mg, 0.069 mmol) were dissolved in DMF (2 mL), HATU (28.86 mg, 0.076 mmol) and DIPEA (13.38 mg, 0.104 mmol) were added in sequence, and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure, and the residue was purified by reverse phase chromatography (C18, 20-60% acetonitrile-H2O solution) to obtain 26 mg of yellow solid, m / z (ES+) (M+1) = 1343.49.

[0797] Synthesis of Compound 98-4

[0798] Compound 98-3 (25 mg, 0.019 mmol) was dissolved in DCM (2 mL) and TFA (0.4 mL) was added, and the reaction solution was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to obtain crude compound 18 (23 mg), which was directly used in the next step, m / z (ES+) (M+1) = 1243.4.

[0799] Synthesis of LD-98

[0800] Compound 98-4 (23 mg, 0.018 mmol) and quaternary ammonium sulfonate B (7.03 mg, 0.028 mmol) were dissolved in DMF (1 mL), HATU (7.74 mg, 0.020 mmol) and DIPEA (3.59 mg, 0.028 mmol) were added in sequence, and the reaction solution was stirred at room temperature for 2 hours. The reaction system was purified by pre-HPLC (TFA) to obtain LD-98 (6 mg, yield: 22.2%).

[0801] LCMS: m / z(ES+)(M+H) + =1479.6,Rt=1.340min.

[0802] 1 H NMR(400MHz, DMSO)δ9.97(d,J=40.2Hz,1H),9.14(d,J=12.0Hz,2H),8.19(t ,J=7.4Hz,1H),8.06(d,J=9.4Hz,1H),7.85(s,1H),7.78(d,J=11.2Hz,1H),7 .64(dd,J=49.2,8.4Hz,1H),7.33(d,J=12.4Hz,2H),6.52(s,1H),5.44(d,J= 4.0Hz,2H),5.30(s,3H),5.12(d,J=10.8Hz,2H),4.64(d,J=26.4Hz,2H),4.3 6(dd,J=13.6,6.8Hz,1H),4.23-4.14(m,1H),3.65(s,1H),3.59-3.54(m,3H) ,3.51-3.46(m,15H),3.43(s,4H),3.03(d,J=9.2Hz,7H),2.98(s,3H),2.92( s,2H),2.86(s,1H),2.68-2.67(m,1H),2.38(s,5H),2.33(dd,J=3.6,1.8Hz, 2H),2.20(s,2H),1.98-1.83(m,8H),1.31-1.23(m,4H),0.89-0.81(m,10H).

[0803] Example 17 (Preparation of LD-99)

[0804] The synthetic route of LD-99 is as follows:

[0805]

[0806] Synthesis of Compound 99-1

[0807] Exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride (CAS No.: 6118-51-0, 2g, 12.04mmol, 1.0eq) and N-Boc-N-methylethylenediamine (CAS No.: 121492-06-6, 2.52g, 14.46mmol, 1.2eq) were dissolved in methanol (60mL), triethylamine (2.43g, 24.1mmol, 2.0eq) was added at room temperature, and the reaction solution was heated to 65°C for 48h. The reaction solution was concentrated and diluted with ethyl acetate (60mL), dried with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column (petroleum ether: ethyl acetate = 1:1) to obtain 3g of white solid, m / z (ES+) (M+H) + =323.3.

[0808] Synthesis of Compound 99-2

[0809] Compound 99-1 (2.3 g, 7.135 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (4 mL) was added in an ice-water bath, and the reaction solution was warmed to room temperature for 1 hour. The reaction solution was concentrated to obtain 1.5 g of crude product, m / z (ES+) (M+H) + =223.2.

[0810] Synthesis of Compound 99-3

[0811] Compound 99-2 (1.58 g, 7.11 mmol, 1.0 eq) and tert-butyl 6-bromohexanoate (2.14 g, 8.52 mmol, 1.2 eq) were dissolved in DMF (10 mL) and reacted at room temperature for 72 hours. The reaction solution was concentrated and column purified (petroleum ether: ethyl acetate = 3:1) to obtain 2.5 g of the target product, m / z (ES+) (M+Na) + =393.2.

[0812] Synthesis of Compound 99-4

[0813] Compound 99-3 (1.5 g, 3.822 mmol) was dissolved in toluene (15 mL) and heated to 110°C for 18 hours. The reaction solution was concentrated to obtain the target product as a yellow oil (1 g), m / z (ES+) (M+H) + =325.2.

[0814] Synthesis of Compound 99-5

[0815] Compound 99-4 (100 mg, 0.308 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.7 mL) was added, and stirred at room temperature for 1 hour. The reaction solution was concentrated to obtain 82 mg of crude product as a yellow oil, m / z (ES+) (M+H)+ =269.2.

[0816] Synthesis of Compound 99-6

[0817] Compound 65-11 (88 mg, 0.098 mmol, 1.0 eq) and compound 99-5 (78.8 mg, 0.294 mmol, 3.0 eq) were dissolved in DMF (3 mL), and HATU (112 mg, 0.294 mmol, 3.0 eq) and diisopropylethylamine (0.081 mL, 0.489 mmol, 5.0 eq) were added in sequence, and reacted at room temperature for 2 hours. The reaction solution was filtered and purified by reverse column to obtain 80 mg of yellow solid, m / z (ES+) (M+H) + =1147.7.

[0818] Synthesis of Compound 99-7

[0819] Compound 99-6 (80 mg, 0.070 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (0.3 mL) was added dropwise, and stirred at room temperature for 1 hour. The reaction solution was concentrated to obtain 73 mg of crude product as a yellow oil, m / z (ES+) (M+H) + =1047.6.

[0820] Synthesis of LD-99

[0821] Compound 99-7 (73 mg, 0.070 mmol, 1.0 eq) and quaternary ammonium sulfonate B (35 mg, 0.14 mmol, 2.0 eq) were dissolved in DMF (2 mL), HATU (53 mg, 0.14 mmol, 2.0 eq) and N,N-diisopropylethylamine (0.058 mL, 0.35 mmol, 5.0 eq) were added in sequence, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was filtered and purified to obtain 9.2 mg of a white solid.

[0822] LCMS: m / z(ES+)(M+H) + =1284.6,Rt=1.217min

[0823] 1H NMR (400MHz, DMSO) δ10.01(d,J=38.5Hz,1H),9.24(s,1H),8.26-8.16(m,1H),8.13-8.01(m,1H),7.80(t,J=9.2Hz,2H),7.64(dd,J=47.3,8.1 Hz,1H),7.41-7.31(m,3H),7.17(s,1H),7.10(d,J=4.1Hz,2H),6.54(s,1H),5.44(d,J=4.0Hz,2H),5.29(s,3H),5.13(d,J=8.2Hz,2H),4.64(d ,J=27.5Hz,2H),4.36(d,J=7.5Hz,1H),4.22-4.16(m,1H),3.77(s,2H) ,3.22(s,2H),3.05(s,5H),2.99(d,J=3.8Hz,4H),2.94(s,2H),2.87(s, 2H),2.81(s,4H),2.36(d,J=18.7Hz,5H),2.19(dd,J=13.7,7.2Hz,5H) ,2.06-1.84(m,9H),1.53(s,5H),1.34-1.21(m,7H),0.89-0.83(m,9H).

[0824] Test Example 18 (Preparation of the compound of formula (2))

[0825] Coupling method 1 (full reduction, drug-antibody ratio of 8)

[0826] The antibody solution was changed to pH 7.4 phosphate buffer using an ultrafiltration centrifuge tube, 10-15 times the number of moles of the antibody was added with TCEP, and the solution was reacted at 37°C for 2 hours to open the interchain disulfide bonds of the antibody. After the reduction was completed, the antibody solution was changed to pH 7.4 phosphate buffer using an ultrafiltration centrifuge tube to remove excess TCEP, and 12-20 times the number of moles of the antibody were added to the Linker-Drug (the compound of formula (1), compound MC-GGFG-Exatecan, compound MC-GGFG-Dxd and compound ELC122 prepared in Examples 1-5, 7-17), respectively, and the solution was reacted at room temperature for 1 hour. After the reaction was completed, cysteine ​​was added at a mole of 2 times the number of moles of the Linker-Drug to quench the reaction, and the solution was changed to pH 5.5 acetate buffer using an ultrafiltration centrifuge tube. The antibody-drug conjugate sample after the solution change was filtered using a 0.22 micron sterilizing filter for standby use.

[0827] Coupling method 2 (partial reduction, drug-antibody ratio of 4)

[0828] Use an ultrafiltration centrifuge tube to replace the antibody solution in a pH 7.4 phosphate buffer, add 2.2-2.3 times the number of moles of the antibody TCEP, and react at 37°C for 2 hours to open the interchain disulfide bonds of the antibody. After the reduction is completed, add 6-8 times the number of moles of the antibody Linker-Drug (the compound of formula (1) prepared in Example 6 and the compound MC-VC-PAB-MMAE), and react at room temperature for 1 hour. After the reaction is completed, add 2 times the number of moles of Linker-Drug cysteine ​​to quench the reaction, and use an ultrafiltration centrifuge tube to replace the solution in a pH 5.5 acetate buffer. The antibody drug conjugate sample after the solution is filtered using a 0.22 micron sterilizing filter for standby use.

[0829] Conjugated antibody sequence

[0830] The amino acid sequence of the heavy chain of the anti-Claudin 18.2 humanized monoclonal antibody 9D1 is as follows:

[0831] QIQLVQSGAEVKKPGASVKISCKASGYSFTDYHMNWVRQAPGKGLEWIGNIDPYYGSPTYNHKFKGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCANYGRGNSFPYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0832] The amino acid sequence of the light chain of the anti-Claudin 18.2 humanized monoclonal antibody 9D1 is as follows:

[0833] DIVMTQSPSSLAVSLGERATINCKSSQSLLNSGNQRNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSFPFTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0834] The heavy chain amino acid sequence of the anti-human folate receptor (FRa) humanized monoclonal antibody Farletuzumab is as follows:

[0835] EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQG TPVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0836] The amino acid sequence of the light chain of the anti-human folate receptor (FRa) humanized monoclonal antibody Farletuzumab is as follows:

[0837] DIQLTQSPSSSLSASVGDRVTITTCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWS

[0838] SYPYMYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0839] Chromatographic Analysis of Antibody Drug Conjugates

[0840] The molecular sieve exclusion chromatography (SEC) analysis column was Tskgel G3000SWXL (TOSOH, 0008541), and the mobile phase was 0.1M PB pH7.0, 0.1M NaCl. Isocratic elution, flow rate 1mL / min, column temperature 25°C. The detection wavelengths were 280nm and 366nm. The hydrophobic interaction chromatography (HIC) analysis column was Butyl-NPR 4.6×100mm (TOSOH, 0042168), mobile phase A was 50mM PB pH7.0, 1.5M (NH4)2SO4, and mobile phase B was 50mM PB pH7.0, 20% acetonitrile. The separation gradient changed from 0% B to 100% B in 2-20 minutes, the column temperature was 30°C, and the flow rate was 0.6mL / min. The detection wavelengths were 280nm and 366nm, and the results are shown in Table 1 and Figure 1-Figure 6 As shown ( Figure 1 include Figure 1 A and Figure 1 B, Figure 2 include Figure 2 A and Figure 2 B, Figure 3 include Figure 3 A. Figure 3 B. Figure 3 C. Figure 3 D. Figure 3 E. Figure 3 F. Figure 3 G and Figure 3 H, Figure 4 include Figure 4 A and Figure 4 B, Figure 5 include Figure 5 A and Figure 5 B, Figure 6 include Figure 6 A. Figure 6 B. Figure 6 C. Figure 6 D. Figure 6 E. Figure 6 F. Figure 6 G and Figure 6 H).

[0841] Table 1 shows the molecular sieve exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) of each test example and comparative example.

[0842]

[0843] Summary: From Table 1 and Figure 1-Figure 6 It can be seen that the anti-claudin 18.2 (Claudin18.2) humanized monoclonal antibody 9D1 (WO2021160155) was used as a model antibody to couple with the designed linker drug compound and the control linker drug compound to prepare an antibody drug conjugate. The linker drug compounds MC-GGFG-Exatecan (Shanghai Haoyuan Biology, catalog number HY-114233), MC-VC-PAB-MMAE (Shanghai Haoyuan Biology, catalog number 646502-53-6) and MC-GGFG-Dxd (Deruxtecan) (Shanghai Haoyuan Biology, catalog number HY-13631E) were purchased; LD-74 was synthesized using the synthesis method of WO2022228493; ELC122 was synthesized using the synthesis method of WO2021225892.

[0844] The results of size exclusion chromatography (SEC) analysis of the obtained 9D1 antibody drug conjugates showed that the SEC purity of the antibody drug conjugates was greater than 97%, regardless of whether the conjugated drug was Exatecan, SN38 or MMAE. The purity of the antibody drug conjugate of the control linker LD-27 was only 89.22% (Table 1).

[0845] The results of hydrophobic interaction chromatography (HIC) analysis of the 9D1 antibody-drug conjugate showed that the HIC retention time of the internal salt linker designed and invented for the toxin MMAE, SN38 and Exatecan was significantly shortened relative to the control linker (excluding LD-74) 9D1 antibody-drug conjugate. The retention time of the main peak (antibody-drug conjugate ratio of 4) of the internal salt linker-MMAE antibody-drug conjugate (herein, the antibody-drug conjugate of LD-57) was shortened by about 1 minute (16.07 vs 16.99) compared with the control linker-MMAE (MC-VC-PAB-MMAE) antibody-drug conjugate (herein, the antibody-drug conjugate of Comparative Example 2), and the retention time was shortened more significantly for the components with increased antibody-drug conjugate ratios (Table 1, Figure 4 ); The retention time of the inner salt linker-SN38 antibody drug conjugate (LD-71 antibody drug conjugate) was shortened by about 4 minutes (12.93 vs 16.74 Table 1, Figure 5The HIC retention time of the internal salt linker-Exatecan antibody drug conjugates (LD-31, LD-37, LD-38, LD-65, LD-66, LD-72, LD-88, LD-89, LD-98 and LD-99 antibody drug conjugates) of the present application is between 13.12 and 14.17 min, which is significantly shorter than the retention time of the control linker-Exatecan (LD-12, LD-27, MC-GGFG-Exatecan) and MC-GGFG-Dxd antibody drug conjugates without hydrophilic side chains, and is also significantly shorter than the retention time of the LD-73 antibody drug conjugate containing polyethylene glycol (PEG 9) hydrophilic side chains (16.00 min), and is comparable to the retention time of the LD-74 antibody drug conjugate containing polysarcosine hydrophilic side chains (13.58 min) (Table 1, Figure 6 ). An interesting discovery is that for the internal salt linker-Exatecan, the closer the internal salt of the same structure is to the Exatecan toxin on the linker, the shorter the HIC retention time of the corresponding antibody-drug conjugate. For example, for the sulfonic acid internal salt linker-Exatecan, LD-37 (sulfonic acid internal salt is at the Z position of formula (1-1), which is far away from Exatecan), the corresponding antibody-drug conjugate HIC retention time is 14.07min, and the antibody-drug conjugate retention times of LD-38 (sulfonic acid internal salt is at the Z position of formula (1-1-1)), LD-65 (sulfonic acid internal salt is at the Z' position of formula) and LD-31 (P2) (sulfonic acid internal salt is at the Z" position of formula) are 13.57, 13.46 and 13.40min respectively, which are significantly shorter than the 14.07min of the antibody-drug conjugate corresponding to LD-37 (Table 1, Figure 6 ).

[0846] Hydrophobic interaction chromatography shows the hydrophobic characteristics of antibody-drug conjugates. This hydrophobicity is mainly caused by the hydrophobicity of the conjugated drug compound. The strong hydrophobicity of the antibody drug prolongs the retention time on the hydrophobic interaction chromatography. The water solubility and stability of the antibody-drug conjugate are poor and it is also easy to be cleared in the body. The super strong water solubility of the internal salt linker can greatly mask the hydrophobic characteristics of the drug compound, enhance the water solubility and stability of the antibody-drug conjugate, and shorten the retention time on the hydrophobic interaction chromatography.

[0847] In addition, the conjugates of the internal salt linker-Exatecan (LD-38) with the anti-human folate receptor (FRa) humanized monoclonal antibody Farletuzumab and the IgG1 isotype control (Shanghai Bio-Tech, Cat. No. B117901) monoclonal antibody were prepared with SEC purity >96% and HIC retention time of about 13min (Table 1), which is comparable to the retention time of the corresponding 9D1 antibody-drug conjugate.

[0848] The 9D1 antibody-drug conjugates (i.e., the compounds represented by formula (2)) in the following test examples were all prepared in Test Example 18.

[0849] Test Example 19 (Drug-Antibody Ratio (DAR))

[0850] Sample processing:

[0851] The 9D1-LD-38 prepared in Experimental Example 5a was diluted to 1 mg / mL with 0.1 M Tris-HCl, and 5 μL 0.5 M DTT was added for every 100 μg 9D1-LD-38. The mixture was incubated in a 37°C water bath for 30 min, and then 1% (v / v) 10% TFA was added to terminate the reaction.

[0852] LC-MS parameters:

[0853] Mobile phase A was 0.10% TFA and 0.02% FA in water, and mobile phase B was 0.08% FA and 0.02% TFA in acetonitrile. The separation gradient was 20% to 32% mobile phase B in 2-4 minutes, and 32% to 45% in 4-12 minutes. The flow rate was 0.4 mL / min. The chromatographic column was BioResolveTM RP mAb Polyphenyl Column (2.7 μm, 2.1×150 mm), and the column temperature was 70°C.

[0854] The mass spectrometer was Themo Q Exactive Plus in positive ion mode Full Scan, with a spray voltage of 3.8 kV and a scan range of 500-3500 m / z.

[0855] RP-HPLC parameters:

[0856] Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution. The separation gradient was 5-25 minutes, and mobile phase B changed from 30% to 50%. The flow rate was 0.6 mL / min. The chromatographic column was Proteomix RP-1000 (5 μm, 4.6×100 mm), and the column temperature was 60°C.

[0857] The results are shown in Table 2 and Figure 7-9 shown.

[0858] Table 2 shows the theoretical molecular weight and detected molecular weight of the main components of 9D1-LD-38 LC / MS

[0859]

[0860] Summary: From Table 2 and Figure 7-9It can be seen that since 9D1-LD-38 was coupled with cysteine, it was pre-treated to retain glycosylation in a reduced state. The results showed that the light chain and heavy chain each had one main component. According to mass spectrometry molecular weight identification, the main coupling form of the light chain was 1 LD-38 molecule, and the main coupling form of the heavy chain was 3 LD-38 molecules. The molecular weight deconvolution results are shown in Figure 7 and Figure 8 The results are consistent with the theoretical molecular weight. In addition, the partial ring opening of LD-38 molecules and the existence of heterogeneous forms with different coupling numbers of heavy chains were also found. The area of ​​the liquid phase spectrum ( Fig. 9 ) The DAR value of 9D1-LD-38 calculated by the normalization method was 7.8, which is equivalent to the theoretical drug-antibody conjugate ratio of 8.

[0861] Test Example 20 (In vitro tumor cell killing activity)

[0862] 1. Human claudin 18.2 positive MC38-hClaudin18.2 cell line

[0863] The 9D1 antibody-drug conjugates prepared in the above-mentioned test examples and comparative examples were used to perform in vitro killing on the MC38 cell line stably expressing human claudin 18.2. MC38-hClaudin18.2 cells were inoculated into a 96-well cell culture plate at 3000 cells / well and cultured overnight. The next day, each 9D1 ADC with gradient dilution was added and cultured for 48 to 72 hours. The fluorescence value of each experimental well was detected using AlamarBlue reagent (ThermoFisher, catalog number DAL1100) and the cell viability was calculated.

[0864] MC38-hClaudin18.2 cells were cultured in RPMI 1640 medium containing 10% FBS and supplemented with 1 μg / mL puromycin in a 37°C, 5% CO2 incubator for adherent culture. When the cell confluence was greater than 80%, the cells were passaged at a ratio of 1:10 to 1:20 every 3 days.

[0865] MC38-hClaudin18.2 cells were digested with recombinant enzyme digestion solution (TrypLETM Express Enzyme), and after counting, an appropriate amount of cells were taken and resuspended in RPMI 1640 + 10% FBS to adjust the cell density to 3×10 4 / mL, inoculated into a 96-well cell culture plate at 100μL / well, and cultured overnight in an incubator at 37°C and 5% CO2. The next day, the test drug was diluted 10 times in series, with a total of 7 to 8 concentration points; at the same time, a blank control with a sample concentration of 0 was set. The diluted sample was added to a 96-well cell culture plate containing cells at 100μL / well and incubated in a 37°C, 5% CO2 incubator for 48 to 72 hours. 20μL AlamarBlue detection reagent was added to each well, incubated at 37°C for 4 to 6 hours, and the fluorescence signal intensity was detected on a multifunctional microplate reader with an excitation wavelength of 560nm and an emission wavelength of 590nm.

[0866] The cell viability of the blank control was set as 100%, and the cell viability of the experimental wells was calculated according to the following formula (%):

[0867] Cell viability (%) = (fluorescence value of experimental well / fluorescence value of blank control well) × 100%

[0868] The average value of the measured cell viability was used as the ordinate, and the Sigmoidal dose-response (Variable Slope) method (GraphPadPrism software, GraphPad Software, San Diego, California) was used for nonlinear regression to obtain the cell proliferation inhibition curve and calculate the corresponding IC50 (HalfMaximal InhibitoryConcentration, half effective concentration) value. The results are shown in Table 3 and Fig.10 ( Fig.10 A. Fig.10 B. Fig.10 C. Fig.10 D. Fig.10 E).

[0869] Table 3 shows the in vitro cell killing half effective concentration (IC50) of 9D1 antibody drug conjugate and control antibody drug conjugate on MC38-hClaudin18.2 cells

[0870]

[0871] Summary: From Table 3 and Fig.10It can be seen that for Exatecan toxin, compared with the drug conjugates of IgG1 isotype control antibodies (Isotype IgG1-LD-12, Isotype IgG1-LD-38), 9D1 antibody drug conjugates all showed specific killing of MC38-hClaudin18.2, with IC50 values ​​of 0.7 to 11.03 nM, among which 9D1-GGFG-Exatecan had the worst pharmacodynamic activity, with an IC50 of 11.03 nM. The IC50 values ​​of 9D1 antibody drug conjugates containing internal salt hydrophilic side chains were between 0.8 and 4.4 nM, and except for 9D1-LD-31-P1, the activity was significantly stronger than that of Exatecan small molecule toxin (IC50 value 4.10 nM).

[0872] Against MMAE toxin, the IC50 value of the IgG1 isotype control antibody drug conjugate (Isotype IgG1-VC-PAB-MMAE) was 41.3nM in MC38-hClaudin18.2, and the 9D1 antibody drug conjugate (9D1-vc-PAB-MMAE and 9D1-LD-57) showed specific killing with IC50 values ​​of 0.41nM and 0.53nM, respectively.

[0873] 2. Human claudin 18.2 positive NUGC4-hClaudin18.2 cell line

[0874] Human gastric cancer cell line NUGC4 (NUGC4-hClaudin18.2) cells stably expressing human claudin 18.2 were cultured in RPMI 1640 medium containing 10% FBS and supplemented with 1 μg / mL puromycin in a 37°C, 5% CO2 incubator for adherent culture. When the cell confluence was greater than 80%, the cells were passaged at a ratio of 1:3 to 1:10 and every 3 to 4 days.

[0875] NUGC4-hClaudin18.2 cells were digested with recombinant enzyme digestion solution (TrypLETM Express Enzyme), and after counting, an appropriate amount of cells were taken and resuspended in RPMI 1640 + 10% FBS to adjust the cell density to 3×10 4 / mL, inoculated into 96-well cell culture plates at 100μL / well, and placed in an incubator at 37℃, 5% CO2 for overnight culture. The next day, 9D1 ADC and small molecule toxins were diluted 5 times from 1000nM (final concentration of 500nM), with a total of 7 to 8 concentration points; a negative control with a sample concentration of 0 and a blank control with no cells and only culture medium were set at the same time. The diluted samples were added to a 96-well cell culture plate containing cells at 100μL / well (double wells) and placed in a 37℃, 5% CO2 incubator for 120 hours. 20μL CCK-8 detection reagent (Dongren Chemical, Product No. CK04) was added to each well, incubated at 37℃ for 3 to 4 hours, and the OD value of each well in the ELISA plate was measured at a wavelength of 450nm and a reference wavelength of 630nm on a multifunctional microplate reader. The OD value of each well = OD450 nm-OD630 nm. The cell viability of the negative control was set as 100%, and the cell viability (%) of the experimental wells was calculated according to the following formula.

[0876] Cell viability (%) = (OD value of experimental well - OD value of blank well) / (OD value of negative control well - OD value of blank control well) × 100%

[0877] Sigmoidal dose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, San Diego, California) was used for nonlinear regression to calculate the corresponding IC50 (Half Maximal Inhibitory Concentration). The results are shown in Table 4.

[0878] Table 4 shows the in vitro cell killing half effective concentration (IC50) of 9D1 antibody drug conjugate and control antibody drug conjugate on NUGC4-hClaudin18.2 cells

[0879]

[0880] Summary: As shown in Table 4, for Exatecan toxin, compared with the drug conjugate of IgG1 isotype control antibody (Isotype IgG1-LD-38, IC50141.3 nM), 9D1 antibody drug conjugates showed specific killing of NUGC4-hClaudin18.2, with IC50 values ​​of 0.18-1.27 nM. The IC50 values ​​of 9D1 antibody drug conjugates containing internal salt hydrophilic side chains (0.55-1.00 nM) are better than those containing polyethylene glycol (PEG, 9D1-LD-73, IC501.27 nM) and polysarcosine (PSAR, 9D1-LD-74, IC501.16 nM) hydrophilic side chains.

[0881] Against MMAE toxin, the IC50 of 9D1-LD-57 antibody drug conjugate containing an internal salt hydrophilic side chain and the control antibody drug conjugate 9D1-vc-PAB-MMAE were comparable, 0.05 nM and 0.06 nM, respectively.

[0882] 3. Human claudin 18.2 positive HEK293-hClaudin18.2 cell line

[0883] HEK293-hClaudin18.2 cells were cultured in DMEM medium containing 10% FBS and supplemented with 250 μg / mL G418 in a 37°C, 5% CO2 incubator for adherent culture. When the cell confluence was greater than 80%, the cells were passaged at a ratio of 1:20 to 1:40 every 3 days.

[0884] HEK293-hClaudin18.2 cells were digested with recombinant enzyme digestion solution (TrypLETM Express Enzyme), and after counting, an appropriate amount of cells were taken and resuspended in DMEM + 10% FBS to adjust the cell density to 2×10 4 / mL, inoculated into 96-well cell culture plates at 100μL / well, and cultured overnight in an incubator at 37°C and 5% CO2. The next day, 9D1 ADC and small molecule toxins were diluted 10 times from 1000nM (final concentration of 500nM), with a total of 7 to 8 concentration points; at the same time, a negative control with a sample concentration of 0 and a blank control with only the same volume (200μL / well) of culture medium without cells were set. The diluted samples were added to a 96-well cell culture plate containing cells at 100μL / well (double wells) and incubated in a 37°C, 5% CO2 incubator for 72 hours. The aforementioned AlamarBlue detection method was used for cell activity detection and data processing, and the IC50 value was calculated. The results are shown in Table 5.

[0885] Table 5 shows the in vitro cell killing half effective concentration (IC50) of 9D1 antibody drug conjugate and control antibody drug conjugate on HEK293-hClaudin18.2 cells

[0886]

[0887] Note: NA means not applicable

[0888] Summary: As shown in Table 5, for Exatecan toxin, compared with the drug conjugate of IgG1 isotype control antibody (Isotype IgG1-LD-38, IC50688.2 nM), the 9D1 antibody-drug conjugate containing an internal salt hydrophilic side chain showed specific killing of HEK293-hClaudin18.2, with an IC50 value of 0.09-1.00 nM, and the IC50 of the small molecule Exatecan (0.51 nM).

[0889] For SN38 toxin, the 9D1 antibody-drug conjugate showed specific killing of HEK293-hClaudin18.2 compared with the drug conjugate of the IgG1 isotype control antibody (Isotype IgG1-ELC122, no killing). The killing of the 9D1 antibody-drug conjugate containing an internal salt hydrophilic side chain (9D1-LD-57) was slightly stronger than the control (9D1-ELC122), with IC50 values ​​of 0.40 and 0.63nM, respectively.

[0890] Test Example 21 (In vivo tumor therapeutic effect)

[0891] 1. Human claudin 18.2 positive MC38-hClaudin18.2 cell line mouse transplant tumor model

[0892] C57BL / 6 mice, female, 5-6 weeks old, were purchased from Shanghai Lingchang. The animals were housed in a laminar flow rack in an SPF animal room, 5 mice per cage, with 12 hours of alternating light, and free access to water and food. The mouse colorectal cancer cell line MC38-Claudin18.2 that stably expresses human claudin 18.2 was cultured in T75 square flasks in RPMI 1640 medium containing 10% FBS (Fetal Bovine Serum), placed in a 37°C, 5% CO2 incubator, and passaged every three days or so. MC38 h Claudin18.2 cells in the logarithmic growth phase were collected, centrifuged at 1000 rpm for 5 minutes, and resuspended in PBS. At 2×10 6 The cells were mixed with matrix gel at a volume ratio of 1:1 and then inoculated subcutaneously in C57BL / 6 mice. 3The tumor size was measured twice a week and the tumor volume (TV, mm 3 ) and draw the tumor growth curve.

[0893] TV(mm 3 )=(L×W 2 ) / 2, L represents the tumor length, W represents the tumor width

[0894] Tumor inhibition rate (%) = (TV control group - TV administration group) × 100 / TV control group

[0895] The number of days after the first administration was used as the horizontal axis and the tumor volume was used as the vertical axis. The mouse tumor curve was drawn using GraphPad Prism software (GraphPad Software, San Diego, California) in XY mode. Twoway ANOVA was used to analyze whether there were statistical differences between each group and the solvent control PBS group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0896] The treatment regimen of the 9D1 antibody-drug conjugate coupled to Exatecan on the MC38-hClaudin18.2 mouse subcutaneous tumor model is shown in Table 6. The tumor treatment effect and the corresponding tumor growth curve are shown in Table 6 and Fig.11 ( Fig.11 include Fig.11 A. Fig.11 B and Fig.11 C).

[0897] Table 6 shows the grouping, dosing regimen and tumor inhibition rate after drug treatment of MC38-hClaudin18.2 mouse subcutaneous tumor model

[0898] Grouping Route of administration Dosage (mg / kg) Dosing frequency Tumor inhibition rate (%) PBS control ip / 1 NA 9D1-ELC122 ip 10 1 39.82 9D1-GGFG-Exatecan ip 10 1 48.34 9D1-LD12 ip 10 1 48.63 PBS control ip / 1 NA 9D1-LD-31-P1 ip 10 1 27.8％ 9D1-LD-31-P2 ip 10 1 80.0％ 9D1-LD-37 ip 10 1 37.4％ 9D1-LD-38 ip 10 1 80.6％ PBS control ip 10 QUR NA 9D1-GGFG-Dxd ip 10 QUR 50.0％ 9D1-LD-38 ip 10 QUR 98.0％ IgG1Isotype-LD-38 ip 10 QUR 27.2％ PBS control ip / 1 NA 9D1-LD-65 ip 5 1 71.7％ 9D1-LD-66 ip 5 1 48.0％ 9D1-LD-72 ip 5 1 42.3％ 9D1-LD-73 ip 5 1 47.0％ 9D1-LD-74 ip 5 1 56.5％ 9D1-LD-38 ip 5 1 39.2％ IgG1Isotype-LD-38 ip 5 1 5.5％

[0899] Remarks: ip: Intraperitoneal injection; NA: not applicable.

[0900] Summary: From Table 6 and Fig.11 It can be seen that under the condition of a single dose of 10 mg / kg, the control antibody drug conjugates (9D1-ELC122, 9D1-GGFG-Exatecan, 9D1-LD-12) slowed down tumor growth ( Fig.11 A), the tumor inhibition rates (TGI) at the end of the experiment were 39.82%, 48.34% and 48.63% respectively. The antibody drug conjugates with internal salt groups in the linker (9D1-LD-31-P2 and 9D1-LD-38) showed better tumor therapeutic effects ( Fig.11 B), TGI were 80.0% and 80.6%, respectively.

[0901] At 10 mg / kg, once a week, for a total of 2 doses, the tumor inhibition rate of the control antibody drug conjugate 9D1-GGFG-Dxd was 50.0% at the end of the experiment, while the tumor of 9D1-LD-38 was almost completely eliminated (tumor inhibition rate 98.0%). The isotype control antibody drug conjugate (IgG1 Isotype-LD-38) only showed a slight tumor inhibition effect (Table 6).

[0902] In order to further compare and distinguish the tumor therapeutic effects of other 9D1 antibody drug conjugates containing internal salt groups, a low dose (5 mg / kg) was selected for drug efficacy experiments, and 9D1-LD-38 was set as an internal reference. Under low-dose (5 mg / kg) administration conditions, the isotype control antibody drug conjugate (IgG1 Isotype-LD-38) did not show obvious tumor therapeutic effects. 9D1-LD-38 has a significant tumor therapeutic effect, with a TGI of 39.2%. Other 9D1 antibody drug conjugates with internal salt groups in the linker (9D1-LD-65, 9D1-LD-66 and 9D1-LD-72) have better tumor therapeutic effects ( Fig.11 C), TGI reached 71.7%, 48.0% and 42.3% respectively. Especially for 9D1-LD-65, the tumor inhibition effect was the best, and the tumor inhibition growth curve ( Fig.11 C) and TGI are even significantly better than those of the reported 9D1 antibody drug conjugates containing other water-soluble groups on the linker, such as the 9D1-LD-73 (TGI 47.0%) control containing a polyethylene glycol (PEG) water-soluble group and the 9D1-LD-74 (TGI 56.5%) control containing a polysarcosine (PSAR) water-soluble group.

[0903] In summary, the 9D1 antibody drug conjugates (9D1-LD-31-P2, 9D1-LD-38, 9D1-LD-65, 9D1-LD-66 and 9D1-LD-72) with water-soluble groups on the linker are significantly more effective than the classic linker antibody drug conjugates without water-soluble groups, such as 9D1-ELC122, 9D1-GGFG-Exatecan and 9D1-LD12. In particular, for 9D1-LD-65, 9D1-LD-66 and 9D1-LD-72, the tumor treatment effect is comparable to or better than that of 9D1 antibody drug conjugates with other types of water-soluble groups on the linker (9D1-LD-73 containing PEG, 9D1-LD-74 containing PSAR), such as 9D1-LD-65.

[0904] 2. Human claudin 18.2-positive NUGC4-hClaudin18.2 cell line mouse transplant tumor model

[0905] The gastric cancer cell line NUGC4-hClaudin18.2 stably expressing human claudin 18.2 was cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2 in an incubator. The cells were subcultured every 4 to 5 days after they were fully grown, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

[0906] NUGC4-hClaudin18.2 tumor cells were adjusted to a concentration of 5×10 7 / mL, inoculated subcutaneously in the right flank of BALB / c nu / nu mice (female, 6-7 weeks old), 100 μL / mouse, and the tumor cell inoculation volume was 5×10 6 / mouse. When the tumor volume grows to about 150mm 3 The groups and dosing schedules are shown in Table 7. Tumor size was measured twice a week and tumor volume (TV, mm 3 ) and draw the tumor growth curve.

[0907] TV(mm 3 )=(L×W 2 ) / 2, L represents the tumor length, W represents the tumor width

[0908] The number of days after the first administration was used as the horizontal axis and the tumor volume was used as the vertical axis. The mouse tumor curve was drawn using GraphPad Prism software (GraphPad Software, San Diego, California) in XY mode. The results are shown in Fig.12 shown.

[0909] Table 7 shows the grouping and dosing regimen of the NUGC4-hClaudin18.2 mouse subcutaneous tumor model

[0910]

[0911] Note: ip: intraperitoneal injection; QW X 3: administration once a week, 3 times.

[0912] Summary: From Table 7 and Fig.12It can be seen that under the conditions of 5 mg / kg, once a week, and 3 times of administration, 9D1-LD-38, whose linker contains an internal salt-promoting water-soluble group, showed the best therapeutic effect, significantly better than 9D1-ECL122 and 9D1-GGFG-Exatecan. The tumor of 9D1-LD-38 basically disappeared after 24 days of treatment, while the tumor growth of the 9D1-GGFG-Exatecan treatment group was inhibited, but the tumor was not reduced. 9D1-ECL122 only showed a weak tumor inhibition effect, which may be because the toxin of 9D1-ECL122 is SN38, which is less toxic.

[0913] 3. Folate receptor positive OVCAR-3 cell mouse transplant tumor model

[0914] OVCAR-3 (OVCAR3) human ovarian cancer tumor cells expressing folate receptor (FRa) were cultured in RPMI-1640 medium containing inactivated 20% fetal bovine serum, 100 U / ml penicillin, and 100 μg / mL streptomycin in an incubator at 37°C and 5% CO2. The cells were subcultured every 4 to 5 days after they were fully grown, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

[0915] OVCAR3 tumor cells were mixed with PBS:Matrigel (volume ratio 1:1) to a concentration of 1×10 8 / mL, inoculated subcutaneously in the right flank of M-NSG experimental mice, 100uL / mouse, and the tumor cell inoculation volume was 1×10 7 / mice.

[0916] When the tumor grows to about 150 mm 3 The mice were divided into groups and given drugs (5 mice / group). The control group (Control) was given PBS, and the treatment group was given anti-human folate receptor (FRa) humanized monoclonal antibody Farletuzumab-LD-38 antibody drug conjugate. The dosage was 5 mg / kg, and the single dose was given. The tumor size was measured twice a week, and the tumor volume (TV, mm 3 ) and draw the tumor growth curve.

[0917] TV(mm 3 )=(L×W 2 ) / 2, L represents the tumor length, W represents the tumor width

[0918] Tumor inhibition rate (%) = (TV control group - TV administration group) × 100 / TV control group

[0919] The number of days after the first administration was used as the horizontal axis and the tumor volume was used as the vertical axis. The mouse tumor curve was drawn using GraphPad Prism software (GraphPad Software, San Diego, California) in XY mode. Twoway ANOVA was used to analyze whether there were statistical differences between each group and the solvent control PBS group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The results are shown in Fig.13 shown.

[0920] Summary: Fig.13 It can be seen that after a single administration of 5 mg / kg for 1 week, the Farletuzumab-LD-38 antibody drug conjugate showed a tumor growth inhibitory effect compared with the control group, and tumor shrinkage occurred thereafter. On the 24th day, the tumor inhibition rate (TGI) reached 93.5%, which was significantly different from the control group.

[0921] 4. Folate receptor-positive IGROV1 cell mouse transplant tumor model

[0922] IGROV1 human ovarian cancer tumor cells expressing folate receptor (FRa) were cultured in DMEM medium containing inactivated 10% fetal bovine serum, 100U / ml penicillin and 100μg / ml streptomycin in an incubator at 37°C and 5% CO2. The cells were sub-cultured every 3 to 4 days after they were fully grown, and tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

[0923] IGROV1 tumor cells were adjusted to a concentration of 1×10 8 / ml, inoculated subcutaneously in the right flank of BALB / c nude experimental mice, 100 μL / mouse, and the tumor cell inoculation volume was 1×10 7 / mice.

[0924] When the average tumor volume reaches 150 mm 3 The animals were divided into groups and administered at about 1:00 p.m. (the day of grouping was recorded as D0). Each model was divided into 2 groups, with 5 animals in each group. The control group (Control) was given PBS, and the treatment group was given the anti-human folate receptor (FRa) humanized monoclonal antibody Farletuzumab-LD-38 antibody drug conjugate. The dosage was 5 mg / kg, once a week, and the administration was 2 times. The tumor size was measured twice a week, and the tumor volume (TV, mm 3 ) and draw the tumor growth curve.

[0925] TV(mm 3 )=(L×W 2 ) / 2, L represents the tumor length, W represents the tumor width

[0926] Tumor inhibition rate (%) = (TV control group - TV administration group) × 100 / TV control group

[0927] The number of days after the first administration was used as the horizontal axis and the tumor volume was used as the vertical axis. The mouse tumor curve was drawn using GraphPad Prism software (GraphPad Software, San Diego, California) in XY mode. Twoway ANOVA was used to analyze whether there were statistical differences between each group and the solvent control PBS group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0928] The results are as follows Fig.14 As shown, after 2 doses of 5 mg / kg, once a week, the Farletuzumab-LD-38 antibody drug conjugate showed rapid tumor shrinkage relative to the control group, with a tumor inhibition rate (TGI) of 100% on the 30th day, and the efficacy was still maintained 50 days after administration.

[0929] Test Example 22 Pharmacokinetics in rats

[0930] The experiment selected 3 male SD rats and randomly divided them into 3 groups. They were given a single intravenous injection of 9D1-LD-38 prepared in the test sample test example and 9D1-GGFG-Exatecan prepared in the comparative example, and the dose was 10 mg / kg. Whole blood was collected from animals in each test sample group before administration, 5min, 1h, 5h, 24h (D2), 48h (D3), 120h (D6), 168h (D8), 240h (D11), 336h (D15), and 504h (D22) after the start of administration, and serum and plasma were separated. The concentrations of Total Ab (total antibody) and ADC (bound antibody) of each test sample in the serum sample were analyzed by ELISA. The pharmacokinetic parameters of each animal were calculated using the PK Solver non-compartmental model (NCA).

[0931] Determination of total antibody and bound antibody: ELISA method was used to determine the blood concentration of total antibody and bound antibody. The specific method is as follows:

[0932] a) Dilute anti-human IgG (Fab) 2 to 1 μg / mL with PBS, coat 96-well ELISA plate with 100 μL / well, and incubate at 4°C overnight.

[0933] b) Wash the plate three times and block with 3% BSA-PBST, 300 μL / well, at room temperature for 2 h.

[0934] c) Wash the plate 3 times, dilute 9D1-LD-38 / 9D1-GGFG-Exatecan with rat blank serum at a 2-fold ratio, with a starting concentration of 1 μg / mL, for a total of 7 concentrations, and set the "0" point as rat blank serum. At the same time, dilute the samples at different time points after drug administration with rat blank serum at appropriate multiples. Dilute all the above samples 50 times (9D1-GGFG-Dxd) and 100 times (9D1-LD-38) with 1% BSA-PBST, add 100 μL / well to the ELISA plate, and incubate at room temperature for 2 hours.

[0935] d) Wash the plate 6 times.

[0936] Detect total antibody: dilute HRP-goat anti-human IgGFC at 1:20000 with 1% BSA-PBST, add 100 μL / well to the ELISA plate, and incubate at room temperature for 1 hour.

[0937] ADC measurement: dilute anti-exatecan antibody to 0.5 μg / mL with 1% BSA-PBST, add 100 μL / well to the ELISA plate, and incubate at room temperature for 1 hour. Wash the plate 6 times, dilute HRP-goat anti-mouse IgG (H+L) with 1% BSA-PBST at 1:20000, add 100 μL / well to the ELISA plate, and incubate at room temperature for 1 hour.

[0938] Wash the plate 6 times, add TMB to the ELISA plate, 100 μL / well, and color develop at room temperature in the dark for about 10 minutes.

[0939] Add 50 μL / well of stop solution. Detect on an ELISA reader (wavelength 450 / 630 nm), the results are shown in Table 8 and Fig.15 .

[0940] Table 8 shows the pharmacokinetic parameters of 9D1-LD-38 and 9D1-GGFG-Exatecan in rats

[0941]

[0942] Summary: From Table 8 and Fig.15 It can be seen that after a single intravenous injection of 10 mg / kg to rats, the serum drug concentrations of total antibody and bound antibody (ADC) of 9D1-GGFG-Exatecan decreased rapidly ( Fig.15 ), the half-life is shorter, 65.1h and 57.8h respectively. The ratio of AUC of binding antibody to total antibody is 0.67,

[0943] The serum drug concentrations of 9D1-LD-38 total antibody and bound antibody decreased relatively slowly ( Fig.15 ), the half-lives were 145.4h and 119.8h respectively, and the ratio of bound antibody to total antibody AUC was 0.78.

[0944] The half-life of 9D1-LD-38 binding antibody is twice that of 9D1-GGFG-Exatecan; the AUC of 9D1-LD-38 binding antibody is 1.3 times that of 9D1-GGFG-Exatecan; the ratio of AUC of 9D1-LD-38 binding antibody to total antibody is greater than that of 9D1-GGFG-Exatecan (0.78 vs 0.67), indicating that 9D1-LD-38 is more stable and the small molecule is not easy to fall off.

[0945] In summary, the drug clearance of 9D1-LD-38 in rats, in which the linker contains an internal salt water-solubilizing group, is significantly weaker than that of 9D1-GGFG-Exatecan, in which the linker does not contain a water-solubilizing group, and its in vivo stability is better, as manifested by a significantly prolonged half-life of 9D1-LD-38, a significantly increased drug exposure, and a higher AUC ratio of bound antibody to total antibody.

[0946] Test Example 23 (In vitro cell killing effect of 9D1-LD-38, 9D1-LD-82, 9D1-LD-88 and 9D1-LD-89)

[0947] MC38-hClaudin18.2 cells were cultured in RPMI1640 (Thermo, Cat: 11875-085) medium containing 10% FBS (Thermo, Cat: 10099-141C) and 1 μg / mL puromycin (Thermo, Cat: A11138-03) in a 37°C, 5% CO2 incubator. When the cell confluence was greater than 80%, the cells were passaged at 1:10-1:20 and every 3 days. MC38-hClaudin18.2 cells were digested with recombinant enzyme digestion solution (TrypLETM Express Enzyme (Chinese name is trypsin), Themo, Cat: 12605028), and after counting, an appropriate amount of cells were taken and resuspended in RPMI 1640 + 10% FBS to adjust the cell density to 1.5×10 4 / mL, inoculated into 96-well cell culture plates at 100μL / well, and cultured overnight in an incubator at 37°C and 5% CO2. The next day, 9D1-LD-38, 9D1-LD-82, 9D1-LD-88 and 9D1-LD-89 were diluted 10 times from 500nM molar concentration, with a total of 8 concentration points; at the same time, a negative control with a sample concentration of 0 and a blank control with only the same volume (200μL / well) of culture medium without cells were set. The diluted samples were added to the 96-well cell culture plates containing cells at 100μL / well (double wells) and incubated in an incubator at 37°C and 5% CO2 for 72 hours. Add 20 μL of LamarBlue (Thermo, Cat: DAL1100) detection reagent to each well, incubate at 37°C for 6 hours, and detect the fluorescence signal intensity on a multifunctional microplate reader (Molecular Devices, M5) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Set the cell viability of the negative control to 100%, and calculate the cell viability (%) of the experimental wells according to the following formula;

[0948] Cell viability (%) = (OD value of experimental well - OD value of blank well) / (OD value of negative control well - OD value of blank control well) × 100%

[0949] The logarithm of ADC concentration was used as the abscissa, and the cell viability was used as the ordinate. Sigmoidal dose-response (Variable Slope) method (GraphPadPrism software, GraphPad Software, San Diego, California) was used for nonlinear regression to obtain a curve of cell proliferation inhibition, as shown in FIG. Fig.16 .

[0950] Summary: Fig.16 It can be seen that 9D1-LD-38, 9D1-LD-82, 9D1-LD-88 and 9D1-LD-89 can inhibit the proliferation of MC38-hClaudin18.2 cells in a dose-dependent manner. The inhibitory effects of 9D1-LD-38, 9D1-LD-88 and 9D1-LD-89 are comparable (IC50 values ​​are 0.632, 1.458 and 0.757 nM, respectively), while 9D1-LD-82 is slightly weaker (IC50 value is 8.013 nM).

[0951] Test Example 24 (Inducing immunogenic cell death in B16F10-hClaudin18.2 cells)

[0952] B16F10-hClaudin18.2 cells were revived and cultured in a medium containing DMEM (Thermo, cat: 10569044) + 10% FBS (Thermo, Cat: 10099-141C) + 0.5 μg / mL Puromycin in a 37°C, 5% CO2 incubator. When the cell confluence was greater than 80%, the cells were passaged at a ratio of 1:10 to 1:20 and every 3 to 4 days.

[0953] When the cells grew to a confluence of about 80%, they were digested with recombinant enzyme digestion solution (TrypLETM Express Enzyme, Thermo, cat: 12605028), resuspended in DMEM + 10% FBS, and an appropriate volume was taken for counting. The cell density was adjusted to 1×10 4 / mL, inoculated into 96-well cell plates at 100 μL / well, and cultured overnight at 37°C, 5% CO2 incubator. The next day, the ADC drug was diluted to a final concentration of 750nM with DMEM+10% FBS medium, the small molecule toxin Exatecan was diluted to a final concentration of 10nM, and a blank control with a sample concentration of 0 was set. The diluted sample was added to a 96-well cell culture plate containing cells at 100 μL / well and incubated in a 37°C, 5% CO2 incubator for 48 hours. The 96-well cell culture plate was removed from the incubator, digested with Accutase (Thermo, cat: 00-4555-56), washed once with 1% FBS PBS, and Anti-Calreticulin-AlexaFluor 647 antibody (Abcam, cat: AB196159, diluted 1:100 with 1% FBS PBS) was added at 100 μl / well, and incubated at room temperature in the dark for 30 minutes. After washing twice with 1% FBS PBS, 2 μl of PI dye (Thermo, cat: 00-4555-56) was added to each sample, incubated at room temperature in the dark for 10 min, and then tested by FACS (Bechman, CytoFLEX). Fig.17 .

[0954] Summary: Fig.17It can be seen that when the ADC concentration is 750nM, compared with the control ADC, the 9D1-LD-38 / 9D1-GGFG-Dxd treatment group has a significantly increased ratio of Calreticulin(+) / PI(-) cells, and the 9D1-LD-38 group is higher than the 9D1-GGFG-Dxd group; when the small molecule drug is loaded with Dxd at a concentration of 10nM, the ratio of Calreticulin(+) / PI(-) cells is significantly increased, and Exatecan(MCE, cat:HY-13631A)>Dxd(MCE, cat:HY-13631D). The results show that compared with 9D1-GGFG-Dxd, 9D1-LD-38 can induce stronger immunogenic cell death, providing a theoretical basis for its combination with immune checkpoint inhibitors.

[0955] Test Example 25 (bystander effect on HEK293-hClaudin18.2 / HEK293 cells)

[0956] AlarmaBlue detection method: hClaudin18.2 positive cells HEK293-hClaudin18.2 and negative cells HEK293 were revived and cultured in DMEM+10% FBS+250μg / mL G418 and DMEM+10% FBS culture media, respectively, in an incubator at 37°C and 5% CO2. When the cell confluence was greater than 80%, the cell was passaged at a ratio of 1:10-1:20 and every 3-4 days.

[0957] When the cells grew to a confluence of about 80%, the negative and positive cells were digested with recombinant enzyme digestion solution (TrypLETM Express Enzyme), and after counting, an appropriate amount of cell suspension was centrifuged and resuspended in DMEM + 10% FBS to adjust the cell density to 5×10 3 / mL, positive cells and negative cell groups were inoculated into 96-well cell culture plates at 100μL / well, and positive cells and negative cells were mixed in equal volumes at 1:1, then inoculated into 96-well cell culture plates at 200μL / well, and cultured overnight at 37°C, 5% CO2 incubator. The next day, the ADC drug was diluted with DMEM+10% FBS medium to a final concentration of 0.5nM, and a blank control with a sample concentration of 0 was set. 100μL of medium was discarded from each well of the positive and negative cell mixed groups, and the diluted ADC drug was added to the 96-well cell culture plate containing cells at 100μL / well, and incubated in a 37°C, 5% CO2 incubator for 120 hours. The 96-well cell culture plate was taken out from the incubator, 20 μL of AlamarBlue (Thermo, cat: DAL1100) detection reagent was added to each well, and the plates were incubated at 37°C for 4 hours. The fluorescence signal intensity was detected on a multifunctional microplate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

[0958] The cell viability of the blank control was set as 100%, and the cell viability of the experimental wells was calculated according to the following formula (%):

[0959] Cell viability (%) = (fluorescence value of experimental well / fluorescence value of blank control well) × 100%

[0960] FACS detection method: HEK293 cells were labeled. After counting the HEK293 cells in the logarithmic growth phase, an appropriate amount of cell suspension was centrifuged and washed twice with PBS. The density was then adjusted to 5×10 6 / mL, add an appropriate volume of CFSE (eBioscience, 65-0850-84) to a final concentration of 0.5nM, mix immediately, incubate at room temperature in the dark for 10 minutes, add 4-5 times the volume of pre-cooled DMEM + 10% FBS, incubate on ice for 5 minutes to terminate the reaction. After washing twice with pre-cooled DMEM + 10% FBS, adjust the cell density to 2×10 with DMEM + 10% FBS medium. 4 Pieces / mL.

[0961] Co-incubation and detection: HEK293-hClaudin18.2 positive cells were digested with recombinant enzyme digestion solution (TrypLETM Express Enzyme), and after counting, an appropriate amount of cell suspension was centrifuged and resuspended in DMEM + 10% FBS to adjust the cell density to 2×10 4 / mL, CFSE-labeled HEK293 cells and HEK293-hClaudin18.2 cells were mixed in equal volumes, inoculated into 96-well cell culture plates at 100 μL / well, and cultured overnight at 37°C, 5% CO2 incubator. The next day, the ADC drug was diluted to a final concentration of 0.5 nM with DMEM + 10% FBS medium, and a blank control with a sample concentration of 0 was set. The diluted sample was added to the 96-well cell culture plate containing cells at 100 μL / well and incubated in a 37°C, 5% CO2 incubator for 72 hours. The supernatant of the 96-well cell culture plate was discarded, and the cells were digested with recombinase digestion solution and washed once with 1% FBS PBS. Each sample was resuspended with 250 μL 1% FBS PBS and counted to calculate the total number of cells in each sample. 100 μL of each sample was taken for FACS detection to determine the ratio of hClaudin18.2-positive and -negative cells. Based on the total number of cells, the number of hClaudin18.2-positive and -negative cells in each sample was calculated.

[0962] Summary: After 5 days of co-culture of ADC drugs with HEK293, HEK293-hClaudin18.2 and HEK293+HEK293-hClaudin18.2 mixed culture cells, the cell viability was detected by AlarmaBlue method. The results are as follows: Fig.18 , Fig.18 include Fig.18 A and Fig.18 B, where Fig.18 A is the AlarmaBlue method, Fig.18 B is the flow cytometry method. Fig.18 As shown in A, the four ADC drugs had no killing effect on negative cells HEK293; compared with the control (Isotype IgG1) ADC, 9D1-LD-38 and 9D1-GGFG-Dxd had killing effects on positive cells HEK293-hClaudin18.2, and the killing effects of the two were comparable; compared with the control (Isotype IgG1) ADC, 9D1-LD-38 and 9D1-GGFG-Dxd had killing effects on both positive and negative mixed cells, and the killing effect of 9D1-LD38 was stronger than that of 9D1-GGFG-Dxd. The above results show that 9D1-LD-38 produces a stronger bystander killing effect than 9D1-GGFG-Dxd.

[0963] The ADC drug was co-cultured with HEK293 (CFSE-labeled) + HEK293-hClaudin18.2 mixed culture cells for 3 days, and the total amount and ratio of positive and negative cells were measured by cell counter and flow cytometer, respectively. Fig.18As shown in B, the isotype control (Isotype IgG1) ADC had no killing effect on positive cells HEK293-hClaudin18.2 and negative cells HEK293, while 9D1-LD-38 and 9D1-GGFG-Dxd had killing effects on both HEK293-hClaudin18.2 and negative cells HEK293, and the killing effect of 9D1-LD38 on negative cells HEK293 was stronger than that of 9D1-GGFG-Dxd. The above results show that 9D1-LD38 produces a stronger bystander killing effect than 9D1-GGFG-Dxd.

[0964] Test Example 26 (In vitro enzymatic release of free toxin molecules)

[0965] Preparation of activation buffer solution (10 mM L-cysteine ​​(ACRO, cat: 173600250), 15 mM EDTA, 0.1 M His-HCl, pH 5.5): Weigh 3.63 g of solid cysteine, add 3 mL of 0.1 M His-HCl, pH 5.5 solution to dissolve, then add 90 μL of 0.5 M EDTA solution and mix well.

[0966] Sample preparation: 1) Take 30 μL of activation buffer solution, add 5 μL of Cathepsin B enzyme solution (0.15 mg / mL, SinoBiological, cat: 10483-H08H), mix well and place in a 37°C water bath for 15 minutes. Add 300 μL of 0.1M His-HCl, pH 5.5 solution and 36 μL of 9D1-LD-38 (375 μg), and place in a 37°C water bath overnight; for confirmation of enzyme cleavage of free small molecules. 2) Dilute LD-38, LD-65 and Deruxtecan (MC-GGFG-Dxd) to 14 mg / mL with DMSO; take 10 μL of the diluted linker-toxin, add 20 μL activation buffer to each, then add 250 μL (LD-38), 244.5 μL (LD-65), 344.8 μL (MC-GGFG-Dxd), activation buffer, mix well and place in a 37°C water bath for 15 min; take 0.4 mg / mL Cathepsin B enzyme (SinoBiological, cat: 10483-H08H) 2.3μL, add 950μL activation buffer, 37℃ water bath for 15min; take 230μL of activated linker-toxin solution, add 230μL of activated enzyme solution (the molar ratio of linker-toxin to Cathepsin B is about 1200:1), 37℃ water bath for 0.5h, 2h, 4h, 20h. Take 50μL of enzyme solution at each time point, add 2.5μL 10% TFA solution to terminate the reaction; for enzyme cleavage kinetics detection, the results are as follows Fig.19 .

[0967] Preparation of negative control: 30 μL of activation buffer was placed in a 37°C water bath for 15 minutes. 300 μL of 0.1M His-HCl, pH 5.5 solution and 36 μL of 9D1-LD-38 (375 μg) were added and placed in a 37°C water bath overnight. RP-HPLC (Proteomix RP-1000) parameters are shown in Table A:

[0968] Table A

[0969]

[0970]

[0971] Summary: Fig.19It can be seen that the toxin molecules released by 9D1-LD-38 after Cathepsin B cleavage are consistent with the peak time of the standard Exatecan, and no interference peak appears in the control group (Contral) without adding Cathepsin B enzyme. Therefore, it is determined that Cathepsin B enzyme can effectively cleave the linker of 9D1-LD-38, and the cleavage release product is free Exatecan.

[0972] The enzymatic cleavage kinetics of LD-38, LD-65 and Deruxtecan (MC-GGFG-Dxd) are shown in Table 9 below. LD-38 can effectively release toxin molecules after being cleaved by Cathepsin B, and the cleavage is almost complete (95%) after 4 hours. LD-65 and MC-GGFG-Dxd are insensitive to the Cathepsin B enzyme. After 20 hours of cleavage, only a small amount of toxin is released from LD-65 (4%), and no cleavage product is released from MC-GGFG-Dxd.

[0973] Table 9 Cathepsin B cleavage kinetics of LD-38, LD-65 and Deruxtecan (MC-GGFG-Dxd)

[0974]

[0975] Test Example 27 (In vitro serum stability)

[0976] Sample incubation: 9D1-LD-38, 9D1-LD-65, 9D1-LD-73, 9D1-LD-74 and 9D1-Deruxtecan (9D1-GGFG-Dxd) prepared in the above test examples, with DAR values ​​of 8, were prepared with human, cynomolgus monkey and rat serum (9D1-LD-38 and 9D1-LD-65 were added with mouse serum), with a final concentration of 300 μg / mL, 200 μL / tube, three replicates, and negative controls (0.5% BSA-PBS) and standards were set at the same time. All samples were placed in a 37°C incubator for 0, 1d, 3d, 7d, 14d and 21d, and then free toxin Exatecan was determined.

[0977] Sample processing: 10.0 μL of standard curve samples, quality control samples, samples to be tested, double blank samples (10.0 μL matrix samples) and zero concentration samples (10.0 μL matrix samples) were added to the tube strips, and 25.0 μL internal standard working solution (Exatecan-D5 concentration was 3.00 ng / mL, and 25.0 μL internal standard diluent was added to the double blank samples); 90.0 μL methanol:acetonitrile (1:1, v / v) was added to each well, and oscillated at 2500 rpm for 5 min; centrifuged at 4°C, 3800 g for 5 min; 80.0 μL supernatant was taken and dried with nitrogen at 40°C; 100 μL 0.1% FA in 15% MeOH was added to each well, mixed evenly, and analyzed by LC-MS injection.

[0978] Sample analysis: Liquid chromatography (Nexera, Shimadzu) conditions: mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% acetonitrile solution; chromatographic column: ACE Excel 3μm 50 x 2.1 mm; flow rate: 0.6 mL / min; column temperature: 40°C; mass spectrometry analysis (Triple Quad 7500, AB / Sciex); data acquisition (Analyst, Version 1.6.2, Applied Biosystems / Sciex).

[0979] Data processing: Through formula C 毒素 = (toxin molecular weight * DAR) / ADC molecular weight * 300μg / mL to calculate the toxin content in ADC, serum toxin release rate = serum toxin content / C 毒素 A curve was drawn using Graphpad Prism 9 with the incubation time as the horizontal axis and the release rate as the vertical axis.

[0980] Summary: The exatecan toxin release rate-time curve in the in vitro serum stability study is shown in Fig. 20 , the toxin release of ADC increases with the extension of incubation time, Fig. 20 include Fig. 20 A. Fig. 20 B. Fig. 20 C. Fig. 20 D. Fig. 20 E and Fig. 20 F. 9D1-LD-38( Fig. 20 A) The toxin release rate in human, cynomolgus monkey and rat serum after incubation at 37°C for 21 days was only 2.8%, 0.9%, and 3.3%, respectively (the release peak in rat serum was about 7 days after incubation). Fig. 20B) Under the same conditions, the Dxd release rates after 21 days of incubation were 8.5% (human serum), 4.6% (cynomolgus monkey serum) and 5.3% (rat serum), which were 3, 5 and 1.5 times the toxin release of 9D1-LD-38 in the serum of the corresponding species. Fig. 20 C) 9D1-LD-73( Fig. 20 D) and 9D1-LD74( Fig. 20 E) The serum stability of 9D1-LD-38 in humans, cynomolgus monkeys and rats is basically similar to that of 9D1-LD-38. The highest release rate in human serum is about 3%, and the highest toxin release rate in cynomolgus monkey and rat serum is less than 1.5%. The toxin release of 9D1-LD-38 in mouse serum is slightly higher than that of 9D1-LD-65 ( Fig. 20 F), but the difference is not significant, and the maximum release rate of both in 21 days does not exceed 2%.

[0981] Test Example 28 (Dynamic changes of ADC DAR value in rats)

[0982] Three male SD rats were selected and randomly divided into three groups. The test products, i.e., 9D1-LD-38, 9D1-LD-65 and 9D1-Deruxtecan (9D1-GGFG-Dxd) prepared in the above test examples (DAR values ​​were 8, respectively), were administered by single intravenous injection, and the dose was 10 mg / kg. Whole blood was collected from the animals in each test product group before administration, 5min, 1h, 5h, 24h (D2), 48h (D3), 120h (D6), 168h (D8), 240h (D11), 336h (D15), and 504h (D22) after the start of administration, and the blood was divided into two portions, and serum and plasma were collected respectively, and frozen at -60°C.

[0983] Serum total antibody determination method: take 10.0 μL of standard curve samples, quality control samples, samples to be tested, double blank samples (10.0 μL matrix samples) and zero concentration samples (10.0 μL matrix samples) and add them to the row tubes; add 100 μL of precipitant (1% trichloroacetic acid in isopropanol solution) to each well, oscillate at 2000 rpm for 5 min, centrifuge at 10°C, 500 g for 5 min, and remove the supernatant; add 100 μL of ammonium bicarbonate solution (100 mM, pH = 8.0) to each well, vortex mix, resuspend the precipitate, centrifuge at 10°C, 1000 g for 5 min, and remove the supernatant; add 50.0 μL of ammonium bicarbonate solution (100 mM, PH = 8.0) to each well, vortex mix; add 10.0 μL of Sigma trypsin solution (Sigma, cat: Y0002311) (5 mg / mL), centrifuged at 3500 rpm for 30 s, vortexed at 1000 rpm for 3 min, placed in a shaking incubator, 60°C, 550 rpm, incubated for 1.5 hours; after the incubation, 20.0 μL of 10% FA in each tube was added ACN (terminator); for zero concentration samples, standard curve samples, quality control samples, and samples to be tested, add 10.0 μL of internal standard working solution (TTPP-IS, universal peptide of human Fc, 5000 ng / ml); for double blank samples, add 10.0 μL of internal standard preparation solvent (ammonium bicarbonate solution (100 mM, pH = 8.0)), centrifuge the above samples at 3800 g for 10 seconds, vortex at 1200 rpm for 2 minutes; centrifuge at 3800 g for 5 minutes, take 50.0 μL of supernatant, add 50.0 μL of water to each well, mix well, and analyze by LC-MS.

[0984] Serum-bound toxin determination method: 10.0 μL of standard curve samples, quality control samples, samples to be tested, double blank samples (10.0 μL matrix samples) and zero concentration samples (10.0 μL matrix samples) were added to the tubes in a row; 90 μL of precipitant (1% trichloroacetic acid in isopropanol solution) was added to each well, oscillated at 2000 rpm for 5 min, centrifuged at 10°C and 500 g for 5 min, and the supernatant was removed; 60 μL of ammonium bicarbonate solution (100 mM, pH = 6.0) was added to each well, vortexed to mix, the precipitate was resuspended, centrifuged at 10°C and 1000 g for 5 min, and the supernatant was removed; 60.0 μL of ammonium bicarbonate solution (100 mM, pH = 8.0) was added to each well, vortexed to mix; 60.0 μL of papain (Adamas life, cat: 88915D, 5 mg / mL), centrifuged at 3500 rpm for 30 s, vortexed at 1000 rpm for 3 min, placed in a shaking incubator, 40 ° C, 550 rpm, incubated for 2 hours; 10 μL of 1% formic acid aqueous solution was added to each well, centrifuged at 3500 rpm for 30 s, 40 ° C, 550 rpm, and continued to incubate for 0.5 hours; for zero concentration samples, standard curve samples, quality control samples, and samples to be tested, 25.0 μL of internal standard working solution ((Exatecan-D5, 1000 ng / ml)) was added, and for double blank samples, 25.0 μL of internal standard preparation solvent was added, and the above samples were centrifuged at 3800 g for 10 seconds and vortexed at 1200 rmp for 2 min; 200 μL of pre-cooled acetonitrile was added to each well, shaken at 2500 rpm for 5 min, centrifuged at 3800 g for 5 min, 50.0 μL of supernatant was taken, and 150 μL was added to each well 0.5% formic acid in water, mixed evenly, and analyzed by LC-MS.

[0985] The Total Ab (total antibody) and bound toxin concentration of each test sample in the serum samples were analyzed by LC / MS, and the bound toxin DAR value of the animal at each time point was calculated using DAR=bound toxin concentration (mmol / L) / total antibody concentration (mmol / L).

[0986] Summary: The DAR value-time curve of ADC in the animal serum of each test group is as follows Fig.21As shown: After a single intravenous injection of 10 mg / kg to rats, the DAR values ​​of 9D1-LD-38, 9D1-LD-65 and 9D1-Deruxtecan (9D1-GGFG-Dxd) gradually decreased over time. Among them, the DAR value curves of 9D1-LD-65 and 9D1-LD-38 were almost consistent and could be maintained at a higher DAR value level. Their DAR values ​​were maintained at around 7 at 48 hours after administration, and the DAR was still around 3-4 at 21 days. The DAR value of 9D1-Deruxtecan (9D1-GGFG-Dxd) decreased rapidly, and the DAR value dropped from about 7.5 at the beginning of administration to 5.5 at 48 hours after administration, and dropped to about 1.6 at 21 days. The above results indicate that after in vivo administration, 9D1-LD-38 and 9D1-LD-65 are more stable than 9D1-Deruxtecan (9D1-GGFG-Dxd), as shown by higher DAR.

[0987] Test Example 29 (In vivo tissue distribution in tumor-bearing mice)

[0988] Tumor-bearing mouse modeling and administration: Male C57BL / 6 mice (6 weeks old) were subcutaneously inoculated with MC38-hClaudin18.2 cells (1x 10 6 cell in 100 μL PBS), and when the mouse tumor volume grew to ~300 mm3, the mice were divided into groups and dosed progressively, and 9D1-LD-38, 9D1-LD-65, 9D1-Deruxtecan (9D1-GGFG-Dxd) and PBS prepared in the above test example were injected into the tail vein, with DAR values ​​of 8 and a dose of 10 mg / kg.

[0989] Sample collection: Blood and tumor tissue were collected at 24h, 72h, and 168h after drug administration, and 3 mice were sampled at each time point. Whole blood was collected from the eye sockets of tumor-bearing mice, placed in anticoagulant tubes containing EDTA-K2 anticoagulant, and centrifuged within 30 minutes to obtain plasma. Mice were killed by CO2 asphyxiation, and then tumors were collected. Plasma and tumor tissue were stored at -80℃.

[0990] Determination of free toxin in plasma: 10.0 μL of standard curve samples, quality control samples, samples to be tested, double blank samples (10.0 μL matrix samples) and zero concentration samples (10.0 μL matrix samples) were added to row tubes, and 25.0 μL internal standard working solution (Exatecan-D5 concentration was 3.00 ng / mL, and 25.0 μL internal standard diluent was added to the double blank samples); 90.0 μL methanol:acetonitrile (1:1, v / v) was added to each well, and oscillated at 2500 rpm for 5 min; centrifuged at 4°C, 3800 g for 5 min; 80.0 μL supernatant was taken and dried with nitrogen at 40°C; 100 μL 0.1% FA in 15% MeOH was added to each well, mixed evenly, and analyzed by LC-MS injection.

[0991] Determination of total toxins (bound toxins + free toxins) in blood: 10.0 μL of standard curve samples, quality control samples, samples to be tested, double blank samples (10.0 μL matrix samples) and zero concentration samples (10.0 μL matrix samples) were added to the tubes, 10.0 μL papain (Adamas life, cat: 88915D, 5 mg / ml) was added to each well, and centrifuged at 3500 rpm for 1 min; the samples were placed in a shaking incubator at 40°C, 550 rpm, and incubated for 6 to 8 hours; 1.00 μL 1% FA in was added to each well H2O, centrifuge at 3500rpm for 1min, 40℃, 550rpm, and continue incubation for 30min; for zero concentration samples, standard curve samples, quality control samples, and samples to be tested, add 10.0μL internal standard working solution (Exatecan-D5, 1000ng / ml), and for double blank samples, add 10.0μL internal standard preparation solvent; centrifuge the above samples at 3800g for 10 seconds, vortex at 1200rmp for 2min; add 100μL pre-cooled ACN to each well, oscillate at 2500rpm for 5min, and centrifuge at 3500g for 5min; take 30.0μL of supernatant, add 90.0μL 0.5% FA in H2O to each well, vortex for 3min, and analyze by LCMS.

[0992] Determination of free toxins in tumor tissues: Homogenization of tumor tissues (drug administration and blank PBS groups): Weigh the tumor tissues, add 10 times the volume of 50% methanol water, and homogenize. The homogenate of the blank PBS group is used as the matrix for standard curve and quality control sample configuration. Take 10.0 μL of standard curve samples, quality control samples, samples to be tested, double blank samples (10.0 μL matrix samples) and zero concentration samples (10.0 μL matrix samples) and add them to the row tubes, add 25.0 μL internal standard working solution (Exatecan-D5 concentration is 3.00 ng / mL, double blank samples add 25.0 μL internal standard diluent); add 90.0 μL methanol:acetonitrile (1:1, v / v) to each well, oscillate at 2500 rpm for 5 min; centrifuge at 4°C, 3800g for 5 min; take 80.0 μL supernatant and blow dry with nitrogen at 40°C; add 100 μL 0.1% FA in 15% MeOH to each well, vortex for 3 min, and analyze by LCMS.

[0993] Determination of total toxin (bound toxin + free toxin) in tumor tissue: 10.0 μL of standard curve samples, quality control samples, samples to be tested, double blank samples (10.0 μL matrix samples) and zero concentration samples (10.0 μL matrix samples) were added to the tubes in a row; 10.0 μL papain (Adamas life, cat: 88915D 5 mg / ml) was added to each well, and centrifuged at 3500 rpm for 1 min; placed in a shaking incubator, 40°C, 550 rpm, incubated for 6 to 8 hours; 1.00 μL 1% FA in each well was added H2O, centrifuge at 3500rpm for 1min, 40℃, 550rpm, and continue incubation for 30min; 5. For zero concentration samples, standard curve samples, quality control samples, and samples to be tested, add 10.0μL of internal standard working solution (Exatecan-D5, 1000ng / ml), and for double blank samples, add 10.0μL of internal standard preparation solvent; centrifuge the above samples at 3800g for 10 seconds, vortex at 1200rmp for 2min; add 100μL of pre-cooled ACN to each well, oscillate at 2500rpm for 5min; centrifuge at 3500g for 5min; take 30.0μL of supernatant, add 90.0μL of 0.5% FA in H2O to each well, vortex for 3min, and analyze by LCMS.

[0994] Sample analysis: Liquid chromatography (Nexera, Shimadzu) conditions: mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% acetonitrile solution; chromatographic column: ACE Excel 3μm 50x 2.1mm; flow rate: 0.6mL / min; column temperature: 40℃; mass spectrometry analysis (Triple Quad 7500, AB / Sciex); data acquisition (Analyst, Version 1.6.2, Applied Biosystems / Sciex). GraphPadPrism9 software was used to input the time point of sample collection and the corresponding toxin content to draw a toxin content curve in plasma and tumor tissue. The results are shown in Fig. 22 and Fig.23 , Fig. 22 include Fig. 22 A and Fig. 22 B, where Fig. 22 A is the total toxin (bound toxin + free toxin) in the plasma of tumor-bearing mice, Fig. 22 B is the content of free toxin in the plasma of tumor-bearing mice. Fig.23 include Fig.23 A and Fig.23 B, where Fig.23 A is the total toxin (bound toxin + free toxin) in the tissues of tumor-bearing mice, Fig.23 B is the content of free toxin in the tissues of tumor-bearing mice.

[0995] Summary: Fig. 22 It can be seen that the free and total toxins in the plasma of animals in each test group showed basically the same trend, reaching the highest level at 24 hours and then slowly decreasing. Fig. 22 A) The content was basically the same, at 24h the content was 3000-4000ng / mL, and after 7 days it dropped by about 70%-80%. The total toxin content in the plasma of 9D1-Deruxtecan (9D1-GGFG-Dxd) at each time point was only about half (1 / 2) of that of 9D1-LD-38 and 9D1-LD-65; the free toxin content and change trend in the plasma of animals in the 9D1-LD-38, 9D1-LD-65 and 9D1-Deruxtecan (9D1-GGFG-Dxd) groups were also basically the same ( Fig. 22 B), the highest at 24h, and then slowly decreased. Compared with the total toxin content in plasma, the free toxin content was extremely low, and the content at 24h was lower than 0.4ng / mL, accounting for 0.01%-0.02% of the total toxin content, and was also far lower than the IC50 value of in vitro target cell killing (3ng / mL). This shows that the toxins of the three ADC drugs are not easy to fall off in plasma and are relatively stable.

[0996] Depend on Fig.23 It can be seen that at 24h after drug administration, the total toxin level in the tumor tissue of each drug administration group was the highest ( Fig.23A), and then rapidly decreased to about 1 / 3 of the 24h content after 168h. The enrichment levels of 9D1-LD38 and 9D1-LD-65 in tumor tissues were significantly higher than those of 9D1-Deruxtecan (9D1-GGFG-Dxd). At 24h after drug administration, the free toxin level in tumor tissues of each drug administration group was the highest ( Fig.23 B), gradually decreased to 72h, and then basically maintained to 168h. The free toxin levels of 9D1-LD-38 and 9D1-LD-65 in tumor tissues were significantly higher than those of 9D1-Deruxtecan (9D1-GGFG-Dxd) (2 to 5 times) during the entire period, among which 9D1-LD-38 was the best. The above results of the content of total toxins (bound toxins + free toxins) and free toxins in tumor tissues of tumor-bearing mice indicate that 9D1-LD-38 and 9D1-LD-65 have better penetration and enrichment in tumor tissues, and the level of free toxins released is also higher, thus producing better tumor treatment effects compared with 9D1-Deruxtecan (9D1-GGFG-Dxd).

[0997] Test Example 30 (Efficacy in P-gp-mediated drug-resistant tumor-bearing mouse model)

[0998] Tumor-bearing mouse modeling and drug administration: BALB / c Nude mice (female, 5-6 weeks old) were subcutaneously inoculated with MC38hClaudin18.2 cells (1×10 6 When the tumor volume grew to about 120 mm3, the patients were randomly divided into groups and given medication according to Table 10. The tumor volume and body weight were measured twice a week and the tumor volume (TV, mm 3 ), and draw the tumor growth curve. TV (mm 3 )=(L×W 2 ) / 2, L represents tumor length, and W represents tumor width. GraphPad Prism software (GraphPad Software, San Diego, California) was used to plot the mouse tumor growth curve and body weight curve in XY mode. Tumor volume and body weight were expressed as mean ± standard error. Fig.24 , Fig.24 include Fig.24 A and Fig.24 B, where Fig.24 A is the mouse tumor growth curve. Fig.24 B is the mouse body weight curve.

[0999] Table 10 Grouping and dosing regimen of P-gp-mediated drug-resistant tumor-bearing mouse model

[1000]

[1001] Notes: ip, Intraperitoneal injection; po, Peros, oral administration; QW, once a week; QD, once a day; 5on+2off / cycle, a cycle of 7 days, 5 days of administration and 2 days of rest; 9D1-LD-38 and 9D1-Deruxtecan (9D1-GGFG-Dxd), DAR=8; 9D1-vd-PAB-MMAE, DAR=4; Tariquidar, p-gp inhibitor (Cat: HY-10550 / CS-0722 Lot: 242595).

[1002] Summary: P-glycoprotein (P-gp) transmembrane transporter causes chemotherapy resistance through active drug efflux, which is one of the main reasons for treatment failure. The mouse MC38 rectal cancer model has high expression of p-gp and is insensitive to many chemotherapy drugs. The tumor therapeutic effect of ADC drugs alone or in combination with p-gp inhibitor Tariquidar in the MC38-hClaudin18.2 tumor-bearing mouse model is shown in Figure 2. Fig.24 As shown in A, Tariquidar monotherapy had no tumor inhibitory effect compared with the PBS control group; the tumor in the 9D1-vc-PAB-MMAE group grew rapidly, and the tumor growth curve showed a weak inhibitory effect compared with the PBS control group (the tumor volume was approximately 2600mm on the 17th day after administration). 3 and 1900mm 3 ), 9D1-vc-PAB-MMAE+Tariquidar can greatly inhibit tumor growth, causing the tumor to shrink to almost disappear; 9D1-Deruxtecan (9D1-GGFG-Dxd) can significantly inhibit tumor growth, and the tumor inhibitory effect of 9D1-Deruxtecan (9D1-GGFG-Dxd) + Tariquidar is slightly enhanced compared with 9D1-Deruxtecan (9D1-GGFG-Dxd) alone, but neither can lead to tumor shrinkage (the tumor volume was approximately 1000mm on the 17th day after administration). 3 and 660mm 3 ); 9D1-LD-38 alone and 9D1-LD-38+Tariquidar can strongly inhibit tumor growth and cause tumors to shrink to near disappearance. The body weight of mice maintained normal fluctuation range after administration, and no weight loss caused by treatment was observed ( Fig.24 B). The above results show that compared with 9D1-vc-PAB-MMAE and 9D1-Deruxtecan (9D1-GGFG-Dxd), 9D1-LD-38 can resist tumor resistance caused by p-gp, and a single drug administration can produce a good tumor treatment effect.

[1003] Test Example 31 (Anti-tumor effect of combined administration of ICI on tumor-bearing mice)

[1004] Modeling and drug administration of tumor-bearing mice: 4T1-hClaudin18.2 cells were cultured in 1640 complete medium (supplemented with 10% FBS, 2 μg / mL puromycin, Gibco, Cat: A1113803) in square bottles and placed in a 37°C, 5% CO2 incubator. When the cell confluence was about 80%, they were digested with TRYPLE EXPRESS trypsin (Gibco, Cat: 12605028) and centrifuged to collect 2.5×10 7 Cells were collected and washed once with PBS. After discarding the supernatant, they were resuspended in PBS containing 50% Matrigel (Chinese name: matrix glue, Corning, Cat: 326234) to a total volume of 5 ml. 100 μL of cell suspension / mouse was inoculated into the right abdominal skin of BALB / c female mice (female, 5-6 weeks old). The tumor volume grew to about 150 mm3 and was randomly divided into groups and administered according to Table 11. The tumor size was measured twice a week with a vernier caliper. TV (mm 3 )=(L×W 2 ) / 2, L represents tumor length, and W represents tumor width. GraphPad Prism software (GraphPad Software, San Diego, California) was used to draw the mouse tumor growth curve in XY mode, and the tumor volume was expressed as mean ± standard error.

[1005] Table 11 Grouping and dosing regimen of ICI combination therapy in tumor-bearing mouse model

[1006]

[1007] Note: ip, intraperitoneal injection; QW, once a week; BIW, twice a week; αPD-1, anti-mouse PD-1 antibody (InVivoMAb (anti-mouse PD-1, Cat: BE0146).

[1008] Summary: Immune checkpoint inhibitors (ICI) have become one of the cornerstones of cancer treatment. The combined therapeutic effect of 9D1-LD-38 and anti-mouse PD1 drug αPD-1 in the 4T1-hClaudin18.2 tumor-bearing mouse model of immune-competent BALB / c mice is shown in Fig.25Compared with the PBS control group, 9D1-LD38 monotherapy and αPD-1 monotherapy were able to partially inhibit tumor growth (the tumor volume on the 14th day after administration was approximately 850 mm 3 、480mm 3 、426mm 3 ), the combined administration of 9D1-LD38+αPD-1 can greatly inhibit tumor growth and even produce significant tumor regression (the tumor volume was about 70mm on the 10th day after administration). 3 ).

[1009] Test Example 32 (Anti-tumor effect of bystander action on tumor-bearing mice)

[1010] Modeling and administration of tumor-bearing mice: MC38-hClaudin18.2 cells and MC38-luc (luciferase) were cultured in DMEM medium (Meilunbio, cat: MA0212-Nov-02H) containing inactivated 10% fetal bovine serum (Excell Bio, cat: 12A222) and 1x penicillin / streptomycin (Solarbio, cat: 20221231) at 37°C and 5% CO2 in an incubator. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo. MC38-hClaudin18.2 cells were mixed with MC38-luc cells (quantity ratio 1:1) and resuspended in PBS to adjust the cell concentration to 5×10 6 / ml, inoculated subcutaneously in the right abdomen of BALB / cnude mice, 100μL / mouse. 3 Around 14:00 p.m., the mice were divided into groups for drug administration (this day was recorded as PG-D0), with a total of 4 groups, 5 mice in each group. The specific drug administration schedule is shown in the table below:

[1011]

[1012] Table 12 Grouping and dosing regimen of bystander effect in tumor-bearing mouse model

[1013] Note: *, the administration volume is 10 μl / g body weight;

[1014] iv: intravenous injection; QW×2: administered once a week, for a total of 2 times.

[1015] Tumor volume was measured 2-3 times a week using a vernier caliper to measure the long and short diameters of the tumor, and the volume was calculated using the following formula: volume (TV) = 0.5 × long diameter × short diameter × short diameter. Luminescent signals were collected using a small animal in vivo imager (IVIS Lumina Series III, PerkinElmer) after intraperitoneal injection of luciferin substrate (15 mg / ml, 10 μl / g) into mice anesthetized with isoflurane.

[1016] Summary: Bystander killing is the ability of ADC drugs to release toxins after being internalized by target-positive tumor cells. The toxins can kill adjacent target-negative tumor cells through transmembrane action. This is considered to be one of the reasons why ADCs can produce good efficacy against tumors with heterogeneous target expression. In this experiment, target-positive (MC38-hClaudin18.2) cells and target-negative (MC38-luc) cells were mixed and inoculated on mice to produce a tumor-bearing mouse model with heterogeneous target expression. The bystander killing effect of the ADC drug was verified and tested. The results are as follows: Fig.26 , Fig.26 include Fig.26 A and Fig.26 B, Fig.26 A is the change in tumor volume of mice after administration. Fig.26 B is the bioluminescent signal of target-negative tumor cells in tumor-bearing mice after 3 and 7 days of drug administration. Fig.26 A shows that compared with the PBS control group (Contral), ISO (Isotype)-LD-38 grew faster and only produced a weak tumor inhibitory effect (16 days after tumor inoculation, the tumor volume was approximately 2700mm 3 and 1700mm 3 ); 9D1-Deruxtecan (9D1-GGFG-Dxd) was able to partially inhibit tumor growth (16 days after tumor inoculation, tumor volume was about 1000mm 3 ), but the tumor volume continued to increase; 9D1-LD-38 was able to greatly inhibit tumor growth, and the tumor was almost completely cleared on the 16th day, indicating that 9D1-LD-38 can kill not only target-positive MC38-hClaudin18.2 cells, but also target-negative MC38-luc cells. Bioluminescent signals of target-negative tumor cells in tumor-bearing mice collected on the 3rd day (PG-3, 3rd day after inoculation) and the 7th day (PG-7, 12th day after inoculation) after grouping ( Fig.26B) also clearly showed that compared with the control group and the ISO-LD38 and 9D1-Deruxtecan (9D1-GGFG-Dxd) administration groups, 9D1-LD38 could effectively kill target-negative tumor cells and cause the bioluminescence signal to be greatly weakened or disappeared.

[1017] Although the embodiments of the present application are described above in conjunction with the accompanying drawings, the present application is not limited to the above specific embodiments and application fields, and the above specific embodiments are merely illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present application, all of which belong to the protection of the present application.

Claims

1. A compound represented by formula (1), or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: The structure of formula (1) is shown below: M is a linking group; Sp is -L a -Q a -; A is selected from the group consisting of absent, hydrogen, and alkyl; T represents a linear or branched (C1-C8) trivalent alkyl group; Y, Y', and Y" are -L b -Q b -; L a , L b are respectively selected from absence, alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene, polyethylene glycol group is optionally substituted with a substituent; Q a , Q b are respectively selected from absent, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -S(=O)2NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, -O-, -NR-, -S-, S(=O)2-, S(=O)-, or -Se-, wherein R is selected from hydrogen or optionally substituted alkyl; Z is selected from absent, hydrogen, optionally substituted alkyl or sulfonate; Z', Z" are selected from absent, hydrogen, optionally substituted alkyl, sulfonate or phosphate; At least one of Z, Z', and Z" is a sulfonic acid inner salt or a phosphate inner salt; X contains a self-destruct unit or does not exist; D includes drug molecules; i is an integer greater than or equal to 1; h is an integer from 1 to 6; o, p, q are integers from 0 to 5 respectively, Preferably, the formula (1) is formula (1-1); wherein at least one of the i Zs is a sulfonic acid inner salt; or Preferably, the formula (1) is formula (1-2); wherein Z' is a sulfonic acid inner salt or a phosphate inner salt; or Preferably, the formula (1) is formula (1-3); Wherein Z" is a sulfonic acid inner salt or a phosphoric acid inner salt.

2. The compound according to claim 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: The formula (1-1) is formula (1-1-1): wherein AA is an amino acid residue, and W is an integer greater than or equal to 1; or The formula (1-2) is the formula (1-2-1): wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1; or The formula (1-3) is formula (1-3-1): Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

3. The compound according to claim 1 or 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: When A and Z do not exist, N, Y, and T form a ring, which is Preferably 4. The compound according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: The amino acids are selected from one or more of alanine, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; or M is selected from N3, SH, ONH2, NH2, CO2H, (alkyl)-CO-, HCO-, BrCH 2- 、ICH2-、 or A is hydrogen, methyl, ethyl or propyl; or The T is one of the following structures: or L a One, two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur; or L b One or two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur; or Q a One, two or more selected from -C(=O)NR-, -NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, wherein R is selected from hydrogen, an optionally substituted C1-C6 straight or branched alkyl group; or Q b is selected from one, two or more of absent, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, wherein R is selected from hydrogen, an optionally substituted C1-C6 straight or branched alkyl group; or X is selected from: The right wavy line in X connects D; R4 is selected from hydrogen, C1-C6 straight or branched alkyl, -(CH2CH2O) m R2, -CH2CH2NR2R3, -CH2CH2SO2R2, R2, R3 are independently selected from hydrogen, C1-C6 straight chain or branched alkyl, and m is an integer greater than 1; or The sulfonic acid salt is A wavy line connects Y, Y' or Y"; Wherein c, d, e, f, g are all integers of 1, 2, 3, 4, 5 or 6; R6, R7, R 11 , R 12 , R 13 , R 14 are independently selected from absence, hydrogen, C1-C6 straight or branched chain alkyl; or The phosphate inner salt is in, a and b are both integers of 1, 2, 3, 4, 5 or 6; R8, R9 and R 10 are selected from hydrogen, C1-C6 straight or branched alkyl; or The D comprises a cytotoxin or an immune agonist.

5. A compound according to any one of claims 1 to 4, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein: The substituent is selected from deuterium, tritium, a halogen atom, an amino group, a hydroxyl group, a cyano group, a nitro group, an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an optionally substituted heterocyclic group, an acyl group, an optionally substituted amino group, an optionally substituted carbamoyl group, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group, an optionally substituted hydroxyl group, an optionally substituted sulfanyl group (SH) and an optionally substituted silyl group.

6. A compound according to any one of claims 1 to 5, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein: The compound is selected from the following structures:

7. A compound represented by formula (2), or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: The structure of formula (2) is expressed as follows: Ab comprises an antibody or an antigen-binding fragment thereof; M' is a linking group; Sp is -L a -Q a -; A is selected from the group consisting of absent, hydrogen, and alkyl; T represents a linear or branched (C1-C8) trivalent alkyl group; Y, Y', and Y" are -L b -Q b -; L a , L b are respectively selected from absence, alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene, heteroarylene, polyethylene glycol group is optionally substituted with a substituent; Q a , Q b are respectively selected from absent, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -S(=O)2NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRS(=O)2-, -NRC(=O)NR-, -NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, -O-, -NR-, -S-, S(=O)2-, S(=O)-, or -Se-, wherein R is selected from hydrogen or optionally substituted alkyl; Z is selected from absent, hydrogen, optionally substituted alkyl or sulfonate; Z', Z" are selected from absent, hydrogen, optionally substituted alkyl, sulfonate or phosphate; At least one of Z, Z', and Z" is a sulfonic acid inner salt or a phosphate inner salt; X contains a self-destruct unit or does not exist; D includes drug molecules; i is an integer greater than or equal to 1; h is an integer from 1 to 6; o, p, q are integers from 0 to 5 respectively; j is an integer greater than or equal to 1; Preferably, the formula (2) is formula (2-1): wherein at least one of the i Zs is a sulfonate; or Preferably, the formula (2) is formula (2-2): wherein Z' is a sulfonic acid inner salt or a phosphate inner salt; or Preferably, the formula (2) is formula (2-3): Wherein Z" is a sulfonic acid inner salt or a phosphoric acid inner salt.

8. The compound according to claim 7, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: The formula (2-1) is formula (2-1-1): wherein AA is an amino acid residue, and W is an integer greater than or equal to 1; or The formula (2-2) is the formula (2-2-1): wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1; or The formula (2-3) is formula (2-3-1): Wherein, AA is an amino acid residue, and W is an integer greater than or equal to 1.

9. The compound according to claim 7 or 8, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: When A and Z do not exist, N, Y, and T form a ring, which is Preferably 10. The compound according to claim 8, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, wherein: The amino acids are selected from one or more of alanine, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; or M' is selected from one of the following structures: * Connect Ab; or A is hydrogen, methyl, ethyl or propyl; or The T is one of the following structures: or L a One, two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol group, wherein the alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group and heteroarylene group are optionally substituted by a substituent, and the heteroatom is selected from nitrogen, oxygen or sulfur; or L b One or two or more selected from alkylene, alkenylene, alkynylene, saturated or unsaturated alicyclic group having 3-10 ring carbon atoms, saturated or unsaturated heteroalicyclic group having 3-10 ring carbon atoms and 1 to 4 heteroatoms, arylene group having 6 to 18 carbon atoms in one or more rings, or heteroarylene group having 6 to 18 carbon atoms in one or more rings and 1 to 4 heteroatoms, or polyethylene glycol, The alkylene, alkenylene, alkynylene, alicyclic group, heteroalicyclic group, arylene group, heteroarylene group is optionally substituted by a substituent, wherein the heteroatom is selected from nitrogen, oxygen or sulfur; or Q a One, two or more selected from -C(=O)NR-, -NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, -OC(=O)O-, wherein R is selected from C1-C6 straight or branched alkyl optionally substituted with hydrogen; or Q b is selected from one, two or more of the group consisting of absent, -C(=O)NR-, -C(=O)O-, -OC(=O)NR-, -NRC(=O)-, -OC(=O)-, -NRC(=O)O-, -NRC(=O)NR-, NHC-(=NH)NH-, -C(=O)-, and -OC(=O)O-, wherein R is selected from hydrogen or an optionally substituted C1-C6 straight or branched alkyl group; or X is selected from: The right wavy line in X connects D; R4 is selected from hydrogen, C1-C6 straight or branched alkyl, -(CH2CH2O) m R2, -CH2CH2NR2R3, -CH2CH2SO2R2-, R2, R3 are independently selected from hydrogen, C1-C6 straight chain or branched alkyl, and m is an integer greater than 1; or The sulfonic acid salt is A wavy line connects Y, Y' or Y"; Wherein c, d, e, f, g are all integers of 1, 2, 3, 4, 5 or 6; R6, R7, R 11 , R 12 , R 13 , R 14 are independently selected from absence, hydrogen, C1-C6 straight or branched chain alkyl; or The phosphate inner salt is in, R8, R9, R 10 are methyl or ethyl respectively; a is 1 or 2 or 3; b is 1 or 2 or 3; or The D comprises a cytotoxin or an immune agonist.

11. A compound according to any one of claims 7 to 10, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein: The substituent is selected from deuterium, tritium, a halogen atom, an amino group, a hydroxyl group, a cyano group, a nitro group, an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an optionally substituted heterocyclic group, an acyl group, an optionally substituted amino group, an optionally substituted carbamoyl group, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group, an optionally substituted hydroxyl group, an optionally substituted sulfanyl group (SH) and an optionally substituted silyl group.

12. A compound according to any one of claims 7 to 11, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein: The compound is selected from the following structures:

13. A compound according to any one of claims 7 to 12, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein: The antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

14. Use of the compound according to any one of claims 7 to 12, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate in the preparation of a medicament for treating and / or preventing tumors.

15. A pharmaceutical composition, wherein It includes the compound according to any one of claims 1 to 14, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate.

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