Novel drug conjugate and use thereof

Abstract

Disclosed are a drug conjugate, such as an antibody-drug conjugate, and use thereof. The present invention pertains to the field of biopharmaceuticals. In some embodiments, the drug conjugate is a compound of formula I or a pharmaceutically acceptable salt or solvate thereof. The drug conjugate can be used for treating diseases such as cancer, autoimmune diseases, inflammatory diseases, or infectious diseases.

Description

Novel drug conjugates and uses thereof TECHNICAL FIELD

[0001] The present invention relates to drug conjugates such as antibody drug conjugates, linkers and intermediates for the preparation of the drug conjugates, and the preparation and use of the drug conjugates. BACKGROUND

[0002] Targeted therapy for cancer, immunodeficiency and infectious diseases is the core of current precision medicine. For many years, many literatures have reported that cell surface receptor binding molecules are used as drug delivery tools to form conjugates (conjugates) with cytotoxic molecules to attack various pathogenic cells (Allen, T. M. and Cullis, P. R., 2004 Science, 303(5665), 1818-22; Hu, Q. Y., et al. (2016), Chem Soc Rev 45(6): 1691-1719).

[0003] Antibody drug conjugates (ADC) are composed of three parts: antibody, cytotoxic molecule and linker connecting the two (Thomas, A., et al. (2016), Lancet Oncol 17(6):e254-e262). Each of the three has a unique function: the antibody needs to be specifically combined with tumor cells, the cytotoxic molecule needs to have enough activity and broad spectrum on tumor cells, and the linker needs to have unique functionality, stable in blood circulation, and effectively release cytotoxic molecules after reaching tumor cells (Chari, R. V. (2008), Acc Chem Res 41(1):98-107), and the three are reasonably constructed to achieve good clinical results (Singh, S. K., et al. (2015), Pharm Res 32(11):3541-3571; Hamilton, G. S. (2015), Biologicals 43(5):318-332).

[0004] It is clear that as a key component covalently linking the antibody and the cytotoxic molecule, the linker must ensure the chemical stability of ADC in the blood circulation and allow the rapid release performance at the target site after internalization. This seemingly contradictory requirement leads to the development of various types of linkers, which can be divided into two categories according to their cleavage mode: cleavable linkers and non-cleavable linkers. Therefore, how to optimize the selection of the appropriate linker, balance the effectiveness and toxicity of ADC drugs, cause minimal off-target effects, and improve the safety of ADC drugs, is particularly important for the design of ADC drugs (Sheyi, R., et al. (2022), Pharmaceutics 14(2):396; Su Z. et al. (2021), Acta Pharm Sin B 11(12):3889-3907). SUMMARY

[0005] The present application provides a drug conjugate having the structure shown in Formula I or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof:

[0006] wherein

[0007] Abu is an antigen binding unit, such as an antibody or an antigen binding fragment thereof;

[0008] D is a drug, such as an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases;

[0009] M is wherein * connects Abu, ** connects -NH-, R 1 is selected from: -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl, -O-(CH2) r -, arylene, -(CH2) r -arylene-, -arylene-(CH2) r -, -(CH2) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O)r -CH2-, -(CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2) r -; wherein each R m is independently H, C1-C6 alkyl, C3-C8 carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0010] R 4 is -NHCO- or -CONH-;

[0011] AA is an amino acid or a polypeptide;

[0012] each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, or 3;

[0013] X is O, NH, or S;

[0014] Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in a conjugated position with the C at the # position, i.e., when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is para or ortho to the C at the # position, and when Ar is a five-membered aryl or heteroaryl group, the nitro group on Ar is not ortho to the C at the # position;

[0015] R 5 is hydrogen or C1-C3 alkyl;

[0016] R 6 is hydrogen or C1-C3 alkyl;

[0017] each R A is independently C1-C3 alkyl, halogen, hydroxyl, or cyano, and b is 0, 1, 2, or 3;

[0018] w is 0, 1, 2, 3, 4, 5, or 6;

[0019] v is 0, 1, 2, 3, 4, 5, or 6;

[0020] n is an integer from 1-24, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0021] p is an integer or decimal number from 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0022] In one or more embodiments, w and v are not both 0.

[0023] In one or more embodiments, w is 0.

[0024] In one or more embodiments, v is 2.

[0025] In one or more embodiments, R 4 is -NHCO-.

[0026] In one or more embodiments, R 4 is -CONH-.

[0027] In one or more embodiments, w is 0, v is 2, and R 4 is -NHCO-.

[0028] In one or more embodiments, AA is where * is attached to -C(O)-, ** is attached to -NH-, and each R 8 is independently H, -CH3, -CH(CH3)CH3, -CH(CH3)CH2CH3, -CH2CH(CH3)CH3, -(CH2)3NH2, -(CH2)4NH2, -CH2COOH, -CH2CH2COOH, -(CH2)3NHC(O)NH2, -(CH2)3NHC(NH)NH2, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0029] In one or more embodiments, AA is selected from the following amino acids or polypeptides: Val-Cit, Val-Ala, Glu-Arg, Lys-Arg, Arg-Arg, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Trp-Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Glu-Gly-Cit, Gly-Gly-Phe-Gly, Gly-Phe-Arg-Gly, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.

[0030] In one or more embodiments, AA is Val-Cit, Val-Ala, Glu-Arg, Glu-Gly-Cit, or Gly-Gly-Phe-Gly.

[0031] In one or more embodiments, each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; X is O, NH, or S; Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in a conjugated position with the C at the # position, e.g., when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is para or ortho to the C at the # position, and when Ar is a five-membered aryl or heteroaryl group, the nitro group on Ar is not ortho to the C at the # position; R 5 is hydrogen or C1-C3 alkyl; R 6 is hydrogen or C1-C3 alkyl; each R A is independently C1-C3 alkyl, halogen, hydroxyl, or cyano.

[0032] In one or more embodiments, each R F is independently F, Cl, Br, or I.

[0033] In one or more embodiments, R F is F.

[0034] In one or more embodiments, each R F is independently -CH3, F, -NO2, or -OCH3.

[0035] In one or more embodiments, z is 0.

[0036] In one or more embodiments, z is 1 or 2.

[0037] In one or more embodiments, each R A is independently F, Cl, Br, or I.

[0038] In one or more embodiments, each R A is independently -CH3, F, Cl, or Br.

[0039] In one or more embodiments, R A is -CH3, and b is 1.

[0040] In one or more embodiments, b is 0.

[0041] In one or more embodiments, X is O.

[0042] In one or more embodiments, is

[0043] In one or more embodiments, is

[0044] In one or more embodiments, is

[0045] In one or more embodiments, the formula I is formula I-1:

[0046] wherein

[0047] Abu is an antigen binding unit, such as an antibody or antigen binding fragment thereof;

[0048] R 1 is selected from the group consisting of: -(CH2) r -, m -(CHR r )-, C3-C8carbocyclyl, r -arylene-, r -(CH2) r -arylene-(CH2) r -(C3-C8carbocyclyl)-, r C3-C8heterocyclyl, r -(CH2) r -(C3-C8heterocyclyl)-, r C(O)NR m (CH2) r -, r -(CH2CH2O)-CH2-, -(CH2CH20) r -CH2-, -(CH2CH20) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH20) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH20) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH20) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH20) r C(O)NR m (CH2) r -; wherein each R m is independently H, C1-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0049] D is a drug, such as an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease;

[0050] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0051] p is an integer or decimal number from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0052] In one or more embodiments, the formula I is formula I-2:

[0053] wherein

[0054] Abuis an antigen binding unit, such as an antibody or antigen binding fragment thereof;

[0055] R 1 is selected from the group consisting of -(CH2) r -, -(CHR m ) r -, C3-C8carbocyclyl, -O-(CH2) r -, arylene, -(CH2) r -arylene-, -arylene-(CH2)r -(CH2) r -(C3-C8carbocyclyl)-, -(C3-C8carbocyclyl)-(CH2) r -(CH2) r -(C3-C8heterocyclyl)-, -(C3-C8heterocyclyl)-(CH2) r -(CH2) r C(O)NR m (CH2) r -(CH2CH2O) r -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; wherein each R m is independently H, C1-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0056] D is a drug, such as an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease;

[0057] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0058] p is an integer or decimal number from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0059] In one or more embodiments, R 1 is -(CH2) r -

[0060] In one or more embodiments, R 1 is -(CH2) r r is 1 or 5.

[0061] In one or more embodiments, the formula I is formula I-3:

[0062] wherein

[0063] Abuis an antigen binding unit, such as an antibody or an antigen binding fragment thereof;

[0064] D is a drug, such as an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease;

[0065] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0066] p is an integer or a decimal number from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0067] In one or more embodiments, wherein the formula I is formula I-4:

[0068] wherein

[0069] Abuis an antigen binding unit, such as an antibody or an antigen binding fragment thereof;

[0070] D is a drug, such as an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease;

[0071] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0072] p is an integer or a decimal number from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0073] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0074] In one or more embodiments, the tubulin inhibitor is selected from the group consisting of dolastatins, auristatins, maytansines.

[0075] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F (AF).

[0076] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, a pyrrolobenzodiazepine (PBD) derivative.

[0077] In one or more embodiments, D is a DNA topoisomerase inhibitor or a salt thereof, such as camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, SN-38, a camptothecin derivative, 22-hydroxytriptolide, topotecan, lurtotecan, belotecan, irinotecan, exatecan, a derivative of exatecan (e.g., Dxd), homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-propenamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-propenamide, 12- -D-xylopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazol-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0078] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxytriptolide, exatecan, a derivative of exatecan (e.g., Dxd), or a salt thereof.

[0079] In one or more embodiments, D is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and analogs thereof, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, doxorubicin, methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, or chlorambucil derivative.

[0080] In one or more embodiments, D is wherein

[0081] X 1 and X 2 are each independently:

[0082] H,

[0083] hydroxy,

[0084] Ci-C6alkyl,

[0085] Ci-C6alkyl substituted by one or more hydroxy, halogen, nitro or cyano,

[0086] C2-C6alkenyl,

[0087] C2-C6alkynyl,

[0088] Ci-C6alkoxy,

[0089] Ci-C6aminoalkoxy,

[0090] halogen,

[0091] nitro,

[0092] cyano,

[0093] mercapto,

[0094] alkylthio,

[0095] amino, amino substituted by an amino protecting group, Ci-C6aminoalkyl optionally substituted in the amino moiety by an amino protecting group or Ci-C6alkyl,

[0096] Ci-C6aminoalkylamino optionally substituted in the amino moiety by an amino protecting group or Ci-C6alkyl,

[0097] Ci-C6alkyl attached to a heterocycle, which is optionally substituted by one or more Ci-C6alkyl, Ci-C6alkoxy, amino, halogen, nitro or cyano,

[0098] A C1-C6 alkylamino group attached to a heterocycle, wherein the heterocycle is optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, and the amino group is optionally substituted with an amino protecting group, a halogen group, a nitro group, a cyano group, or a protecting group.

[0099] An amino-substituted heterocyclic group, wherein the nitrogen atom or amino moiety of the heterocyclic portion is optionally replaced by a protecting group or one or more C1-C6 alkyl groups.

[0100] Heterocyclic amino groups, wherein the nitrogen atom or amino moiety of the heterocyclic portion is optionally substituted with a protecting group or a C1-C6 alkyl group.

[0101] The carbamoyl group may be optionally protected by a carbamoyl group or substituted with a C1-C6 alkyl group.

[0102] Morpholin-1-yl, or

[0103] Piperidin-1-yl;

[0104] Or, X 1 and X 2 Together with the attached atoms, they form unsubstituted or substituted dioxane heterocycles, such as... Where k is 1 or 2;

[0105] X 3 It is a C1-C6 alkyl group;

[0106] X 4 For H, -(CH2) q -CH3、-(CHR n ) q -CH3, C3-C8 carbon cyclic groups, -O-(CH2) q -CH3, aryl-CH3, -(CH2) q -arylene-CH3, -arylene-(CH2) q -CH3, -(CH2) q -(C3-C8 carbocyclic)-CH3, -(C3-C8 carbocyclic)-(CH2) q -CH3, C3-C8 heterocyclic groups, -(CH2) q -(C3-C8 heterocyclic group)-CH3, -(C3-C8 heterocyclic group)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O)q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3, or -(CH2CH2O) q C(O)NR n (CH2) q -CH3; wherein each R n is independently H, C1-C6 alkyl, C3-C8 carbocyclyl, phenyl, or benzyl; and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0107] ** is a point of attachment;

[0108] y is 0, 1, or 2;

[0109] Y is O, S, or CR 2 R 3 , wherein R 2 and R 3 are each independently H or C1-C6 alkyl;

[0110] s and t are each independently 0, 1, or 2, but not both 0.

[0111] In one or more embodiments, X 4 is H or C1-C6 alkyl.

[0112] In one or more embodiments, the heterocycle is azetidine, ethylenediamine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0113] In one or more embodiments, the amino protecting group is formyl, acetyl, trityl, t-butoxycarbonyl, benzyl, or p-methoxybenzyloxycarbonyl.

[0114] In one or more embodiments, D is wherein X 1 and X 2 are each independently C1-C6 alkyl, halogen, or -OH; or, X 1 and X 2 together with the atoms to which they are attached form an unsubstituted or substituted dioxane, such as wherein k is 1 or 2; ** is a point of attachment.

[0115] In one or more embodiments, D is wherein X 1 and X 2 each independently is C1-C6 alkyl, halogen, or -OH; or, X 1 and X 2 together with the atoms to which they are attached form an unsubstituted or substituted dioxacycle, such as wherein k is 1 or 2; ** is a point of attachment.

[0116] In one or more embodiments, X 1 and X 2 each is -CH3.

[0117] In one or more embodiments, X 1 and X 2 each independently is F, Cl, Br, or I.

[0118] In one or more embodiments, X 1 and X 2 each is F.

[0119] In one or more embodiments, X 1 and X 2 each independently is -CH3, F, or -OH.

[0120] In one or more embodiments, X 1 and X 2 each independently is F or -CH3.

[0121] In one or more embodiments, X 1 is -CH3and X 2 is F.

[0122] In one or more embodiments, X 1 , X 2 together with the atoms to which they are attached form wherein k is 1.

[0123] In one or more embodiments, X 1 , X 2 together with the atoms to which they are attached form wherein k is 2.

[0124] In one or more embodiments, the formula I is formula I-5:

[0125] wherein

[0126] Abu is an antigen binding unit, such as an antibody or antigen binding fragment thereof.

[0127] n is an integer from 1 to 24, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0128] p is an integer or decimal number from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0129] In one or more embodiments, n is 4, 8, 12, 16, or 20.

[0130] In one or more embodiments, Abu is an antigen binding unit containing a cysteine in the sequence and is linked to the other part of the drug conjugate (such as M of Formula I) through the sulfur atom of the cysteine.

[0131] In one or more embodiments, Abu is an antigen binding unit containing an Fc region. In one or more embodiments, Abu is linked to the other part of the drug conjugate (such as M of Formula I) through the Fc region.

[0132] In one or more embodiments, the target point to which Abu binds is selected from the group consisting of: HER2, TROP-2, Nectin-4, B7H3, B7H4, CLDN18, BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD20, CD21, CD22, CD30, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, FR alpha (FRα).

[0133] In one or more embodiments, the target point to which Abu binds is HER2, TROP-2, CLDN18.2, B7H3, or FRα.

[0134] In one or more embodiments, Abu is an antibody or an antigen binding fragment thereof, and the drug conjugate is an antibody drug conjugate.

[0135] In one or more embodiments, Abu is an antibody or antigen-binding fragment thereof comprising an Fc region. In one or more embodiments, the Fc region is a human IgGl, IgG2, IgG3, or IgG4 Fc region.

[0136] In one or more embodiments, Abu is linked to the other portion of the drug conjugate (e.g., M of Formula I) via the Fc region.

[0137] In one or more embodiments, Abu is a single domain antibody.

[0138] In one or more embodiments, Abu is a Fab fragment.

[0139] In one or more embodiments, Abu is a single chain antibody.

[0140] In one or more embodiments, Abu is a whole antibody.

[0141] In one or more embodiments, Abu is an anti-HER2 antibody, an anti-TROP-2 antibody, an anti-CLDN18.2 antibody, an anti-B7H3 antibody, or an anti-FRα antibody. In one or more embodiments, Abu is an anti-HER2 monoclonal antibody, an anti-TROP-2 monoclonal antibody, an anti-CLDN18.2 monoclonal antibody, an anti-B7H3 monoclonal antibody, or an anti-FRα monoclonal antibody.

[0142] In one or more embodiments, Abu is trastuzumab, pertuzumab, panitumumab, nimotuzumab, matuzumab, rituximab, or cetuximab.

[0143] In one or more embodiments, the antibody comprises a sequence that is at least 80% identical to any of the above-described antibodies, or an amino acid sequence that has one or more conservative amino acid substitutions compared to any of the above-described antibodies.

[0144] In one or more embodiments, p is an integer or decimal number from 2 to 8.

[0145] In one or more embodiments, p is an integer or decimal number from 4 to 8.

[0146] In one or more embodiments, p is an integer or decimal number from 6 to 8.

[0147] In one or more embodiments, p is an integer or decimal number from 7 to 8.

[0148] In one or more embodiments, p is an integer or decimal number from 4 to 5.

[0149] In one or more embodiments, p is an integer or decimal number from 6 to 7.

[0150] One or more embodiments provide a linker precursor for forming a drug conjugate, such as an antibody drug conjugate, which is a compound of Formula II, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0151] wherein

[0152] M' is wherein * is attached to -NH-, R 1 is selected from the group consisting of: -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl, -O-(CH2) r -, arylene, -(CH2) r -, arylene-, -arylene-(CH2) r -, -(CH2) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; wherein each R m is independently H, C1-C6 alkyl, C3-C8 carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0153] R 4 is -NHCO- or -CONH-;

[0154] AA is an amino acid or a polypeptide;

[0155] each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, or 3;

[0156] X is O, NH, or S;

[0157] Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in a conjugated position with the C at the # position of the aromatic system, e.g., when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is para or ortho to the C at the # position, and when Ar is a five-membered aryl or heteroaryl group, the nitro group on Ar is not ortho to the C at the # position;

[0158] R 5 is hydrogen or C1-C3 alkyl;

[0159] R 6 is hydrogen or C1-C3 alkyl;

[0160] each R A is independently C1-C3 alkyl, halogen, hydroxyl, or cyano, and b is 0, 1, 2, or 3;

[0161] R 7 is hydrogen or

[0162] w is 0, 1, 2, 3, 4, 5, or 6;

[0163] v is 0, 1, 2, 3, 4, 5, or 6;

[0164] n is 1-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0165] In one or more embodiments, w and v are not both 0.

[0166] In one or more embodiments, w is 0.

[0167] In one or more embodiments, v is 2.

[0168] In one or more embodiments, R 4 is -NHCO-.

[0169] In one or more embodiments, R 4 is -CONH-.

[0170] In one or more embodiments, w is 0, v is 2, R 4 is -NHCO-.

[0171] In one or more embodiments, AA is where * is attached to -C(O)-, ** is attached to -NH-, each R 8 is independently H, -CH3, -CH(CH3)CH3, -CH(CH3)CH2CH3, -CH2CH(CH3)CH3, -(CH2)3NH2, -(CH2)4NH2, -CH2COOH, -CH2CH2COOH, -(CH2)3NHC(O)NH2, -(CH2)3NHC(NH)NH2, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0172] In one or more embodiments, AA is selected from the following amino acids or polypeptides: Val-Cit, Val-Ala, Glu-Arg, Lys-Arg, Arg-Arg, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Trp-Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Glu-Gly-Cit, Gly-Gly-Phe-Gly, Gly-Phe-Arg-Gly, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.

[0173] In one or more embodiments, AA is Val-Cit, Val-Ala, Glu-Arg, Glu-Gly-Cit, or Gly-Gly-Phe-Gly.

[0174] In one or more embodiments, each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; X is O, NH, or S; Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in a conjugated position with the C of the # position of the aromatic system, e.g., when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is in the para or ortho position with the C of the # position, and when Ar is a five-membered aryl or heteroaryl group, the nitro group on Ar is not in the ortho position with the C of the # position; R 5is hydrogen or C1-C3alkyl; each R 6 is hydrogen or C1-C3alkyl; each R A is independently C1-C3alkyl, halo, hydroxyl, or cyano.

[0175] In one or more embodiments, each R F is independently F, Cl, Br, or I.

[0176] In one or more embodiments, R F is F.

[0177] In one or more embodiments, each R F is independently -CH3, F, -NO2, or -OCH3.

[0178] In one or more embodiments, z is 0.

[0179] In one or more embodiments, z is 1 or 2.

[0180] In one or more embodiments, each R A is independently F, Cl, Br, or I.

[0181] In one or more embodiments, each R A is independently -CH3, F, Cl, or Br.

[0182] In one or more embodiments, R A is -CH3and b is 1.

[0183] In one or more embodiments, b is 0.

[0184] In one or more embodiments, X is O.

[0185] In one or more embodiments, is

[0186] In one or more embodiments, is

[0187] In one or more embodiments, is

[0188] In one or more embodiments, the formula II is formula II-1A or formula II-1B:

[0189] wherein

[0190] R 1selected from the group consisting of: -(CH2) r - -(CHR m ) r - C3-C8carbocyclyl, -O-(CH2) r - arylene, -(CH2) r - arylene-, -arylene-(CH2) r - -(CH2) r - -(C3-C8carbocyclyl)-, -(C3-C8carbocyclyl)-(CH2) r - C3-C8heterocyclyl, -(CH2) r - -(C3-C8heterocyclyl)-, -(C3-C8heterocyclyl)-(CH2) r - -(CH2) r C(O)NR m (CH2) r - -(CH2CH2O) r - -(CH2CH2O) r - CH2-, -(CH2) r C(O)NR m (CH2CH2O) r - -(CH2) r C(O)NR m (CH2CH2O) r - CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r - -(CH2CH2O) r C(O)NR m (CH2CH2O) r - CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; wherein each R m is independently H, C1-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0191] n is an integer from 1 to 24, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0192] In one or more embodiments, the formula II is formula II-2A or formula II-2B:

[0193] wherein

[0194] R 1 Selected from: -(CH2) r -、-(CHR m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0195] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0196] In one or more embodiments, R 1 -(CH2) r-.

[0197] In one or more embodiments, R 1 is -(CH2) r -; r is 1 or 5.

[0198] In one or more embodiments, the formula II is formula II-3A or formula II-3B:

[0199] wherein n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0200] In one or more embodiments, the formula II is formula II-4A or formula II-4B:

[0201] wherein n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0202] In one or more embodiments, n is from 4 to 24.

[0203] In one or more embodiments, n is from 8 to 20.

[0204] In one or more embodiments, n is 4, 8, 12, 16, or 20.

[0205] One or more embodiments provide an intermediate for forming a drug conjugate (such as an antibody drug conjugate) which is a compound of formula III, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0206] D is a drug, such as an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease;

[0207] M’ is wherein * is attached to -NH-, R 1 is selected from the group consisting of -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl, -O-(CH2) r -, arylene, -(CH2) r -, arylene-, -arylene-(CH2) r -, -(CH2)r -(C3-C8carbocyclyl)-, -(C3-C8carbocyclyl)-(CH2) r -, C3-C8heterocyclyl, -(CH2) r -(C3-C8heterocyclyl)-, -(C3-C8heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; wherein each R m is independently H, C1-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0208] R 4 is -NHCO- or -CONH-;

[0209] AA is an amino acid or a polypeptide;

[0210] each R F is independently C1-C6alkyl, C1-C6alkoxy, -NO2, or halogen; z is 0, 1, 2, or 3;

[0211] X is O, NH, or S;

[0212] Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in a conjugated position with the C at the # position, i.e., when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is para or ortho to the C at the # position, and when Ar is a five-membered aryl or heteroaryl group, the nitro group on Ar is not ortho to the C at the # position;

[0213] R 5 is hydrogen or C1-C3alkyl;

[0214] R 6 is hydrogen or C1-C3alkyl;

[0215] each R A is independently C1-C3alkyl, halo, hydroxyl, or cyano, and b is 0, 1, 2, or 3;

[0216] w is 0, 1, 2, 3, 4, 5, or 6;

[0217] v is 0, 1, 2, 3, 4, 5, or 6;

[0218] n is 1-24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0219] In one or more embodiments, w and v are not both 0.

[0220] In one or more embodiments, w is 0.

[0221] In one or more embodiments, v is 2.

[0222] In one or more embodiments, R 4 is -NHCO-.

[0223] In one or more embodiments, R 4 is -CONH-.

[0224] In one or more embodiments, w is 0, v is 2, and R 4 is -NHCO-.

[0225] In one or more embodiments, AA is where * is attached to -C(O)-, ** is attached to -NH-, each R 8 is independently H, -CH3, -CH(CH3)CH3, -CH(CH3)CH2CH3, -CH2CH(CH3)CH3, -(CH2)3NH2, -(CH2)4NH2, -CH2COOH, -CH2CH2COOH, -(CH2)3NHC(O)NH2, -(CH2)3NHC(NH)NH2, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0226] In one or more embodiments, AA is selected from the following amino acids or polypeptides: Val-Cit, Val-Ala, Glu-Arg, Lys-Arg, Arg-Arg, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Trp-Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Glu-Gly-Cit, Gly-Gly-Phe-Gly, Gly-Phe-Arg-Gly, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.

[0227] In one or more embodiments, AA is Val-Cit, Val-Ala, Glu-Arg, Glu-Gly-Cit, or Gly-Gly-Phe-Gly.

[0228] In one or more embodiments, each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; X is O, NH, or S; Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in a conjugated position with the C of the # position of the aromatic system, e.g., when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is in the para or ortho position with the C of the # position, and when Ar is a five-membered aryl or heteroaryl group, the nitro group on Ar is not in the ortho position with the C of the # position; R 5 is hydrogen or C1-C3 alkyl; R 6 is hydrogen or C1-C3 alkyl; each R A is independently C1-C3 alkyl, halogen, hydroxyl, or cyano.

[0229] In one or more embodiments, each R F is independently F, Cl, Br, or I.

[0230] In one or more embodiments, R F is F.

[0231] In one or more embodiments, each R F is independently -CH3, F, -NO2, or -OCH3.

[0232] In one or more embodiments, z is 0.

[0233] In one or more embodiments, z is 1 or 2.

[0234] In one or more embodiments, each R A It can be F, Cl, Br or I.

[0235] In one or more embodiments, each R A It can be -CH3, F, Cl or Br.

[0236] In one or more embodiments, R A It is -CH3, and b is 1.

[0237] In one or more implementations, b is 0.

[0238] In one or more embodiments, X is 0.

[0239] In one or more embodiments, for

[0240] In one or more embodiments, for

[0241] In one or more embodiments, for

[0242] In one or more embodiments, Formula III is Formula III-1:

[0243] in

[0244] R 1 Selected from: -(CH2) r -、-(CHR m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) rC(O)NR m (CH2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0245] D represents drugs, such as anticancer drugs, cytotoxic drugs, cell differentiation factors, stem cell nutritional factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs, or drugs for treating infectious diseases.

[0246] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0247] In one or more embodiments, Formula III is Formula III-2:

[0248] in

[0249] R 1 Selected from: -(CH2) r -、-(CHR m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2)r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0250] D represents drugs, such as anticancer drugs, cytotoxic drugs, cell differentiation factors, stem cell nutritional factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs, or drugs for treating infectious diseases.

[0251] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0252] In one or more embodiments, R 1 -(CH2) r -

[0253] In one or more embodiments, R 1 -(CH2) r -, r is 1 or 5.

[0254] In one or more embodiments, Formula III is Formula III-3:

[0255] in

[0256] D is a drug, such as an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases;

[0257] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0258] In one or more embodiments, the formula III is formula III-4:

[0259] wherein

[0260] D is a drug, such as an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases;

[0261] n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0262] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0263] In one or more embodiments, the tubulin inhibitor is selected from the group consisting of dolastatins, auristatins, maytansines.

[0264] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F (AF).

[0265] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, a pyrrolobenzodiazepine (PBD) derivative.

[0266] In one or more embodiments, D is a DNA topoisomerase inhibitor or a salt thereof, such as camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxy camptothecin, 9-chloro-10-hydroxy camptothecin, camptothecin derivative SN-38, 22-hydroxyphellopterin, topotecan, lurtotecan, belotecan, irinotecan, exatecan, exatecan derivative (e.g. Dxd), silicon-based homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-propenamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-propenamide, 12- -D- glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H- indolocarbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0267] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxy camptothecin, topotecan, belotecan, irinotecan, 22-hydroxyphellopterin, exatecan, exatecan derivative (e.g. Dxd), or a salt thereof.

[0268] In one or more embodiments, D is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and analogs thereof, an Amatoxin, a spliceosome inhibitor, a phen(b)isobenzazepine (PBD) dimer, doxorubicin, methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, or chlorambucil derivative.

[0269] In one or more embodiments, D is wherein

[0270] X 1 and X 2 each independently is:

[0271] H,

[0272] hydroxy,

[0273] C1-C6 alkyl,

[0274] C1-C6 alkyl substituted by one or more hydroxy, halogen, nitro or cyano,

[0275] C2-C6alkenyl,

[0276] C2-C6alkynyl,

[0277] C1-C6alkoxy,

[0278] C1-C6aminoalkoxy,

[0279] halogen,

[0280] nitro,

[0281] cyano,

[0282] mercapto,

[0283] alkylthio,

[0284] amino, amino substituted with an amino protecting group, C1-C6aminoalkyl optionally substituted in the amino moiety with an amino protecting group or C1-C6alkyl,

[0285] C1-C6aminoalkylamino optionally substituted in the amino moiety with an amino protecting group or C1-C6alkyl,

[0286] C1-C6alkyl attached to a heterocycle optionally substituted with one or more C1-C6alkyl, C1-C6alkoxy, amino, halogen, nitro or cyano,

[0287] C1-C6alkylamino attached to a heterocycle optionally substituted with C1-C6alkyl, C1-C6alkoxy, the amino optionally substituted with an amino protecting group, halogen, nitro, cyano or protecting group,

[0288] heterocyclyl substituted with amino optionally substituted in the nitrogen atom of the heterocycle moiety or the amino moiety with a protecting group or one or more C1-C6alkyl,

[0289] heterocyclyl substituted with amino optionally substituted in the nitrogen atom of the heterocycle moiety or the amino moiety with a protecting group or C1-C6alkyl,

[0290] carbamoyl optionally substituted with a carbamoyl protecting group or C1-C6alkyl,

[0291] morpholin-1-yl, or

[0292] piperidin-1-yl;

[0293] or, X 1 and X 2 together with the atoms to which they are attached form an unsubstituted or substituted dioxolane, wherein k is 1 or 2;

[0294] X 3 is C1-C6alkyl;

[0295] X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -arylene-CH3, -arylene-(CH2) q -CH3, -(CH2) q -(C3-C8 carbocyclyl)-CH3, -(C3-C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl, -(CH2) q -(C3-C8 heterocyclyl)-CH3, -(C3-C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3, or -(CH2CH2O) q C(O)NR n (CH2) q -CH3; wherein each R n is independently H, C1-C6 alkyl, C3-C8 carbocyclyl, phenyl, or benzyl; and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0296] ** is a point of attachment;

[0297] y is 0, 1, or 2;

[0298] Y is O, S, or CR 2 R 3wherein R 2 and R 3 each independently is H or C1-C6 alkyl;

[0299] s and t each independently are 0, 1 or 2, but not both 0.

[0300] In one or more embodiments, X 4 is H or C1-C6 alkyl.

[0301] In one or more embodiments, the heterocycle is azetidine, ethylenediazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole or pyridine.

[0302] In one or more embodiments, the amino protecting group is formyl, acetyl, trityl, t-butoxycarbonyl, benzyl or p-methoxybenzyloxycarbonyl.

[0303] In one or more embodiments, D is wherein X 1 and X 2 each independently is C1-C6 alkyl, halogen or -OH; or, X 1 and X 2 together with the atoms to which they are attached form an unsubstituted or substituted dioxole, such as wherein k is 1 or 2; ** is the point of attachment.

[0304] In one or more embodiments, D is wherein X 1 and X 2 each independently is C1-C6 alkyl, halogen or -OH; or, X 1 and X 2 together with the atoms to which they are attached form an unsubstituted or substituted dioxole, such as wherein k is 1 or 2; ** is the point of attachment.

[0305] In one or more embodiments, X 1 and X 2 each is -CH3.

[0306] In one or more embodiments, X 1 and X 2 each independently is F, Cl, Br or I.

[0307] In one or more embodiments, X 1 and X 2 each is F.

[0308] In one or more embodiments, X 1 and X 2each independently -CH3, F, or -OH.

[0309] In one or more embodiments, X 1 and X 2 each independently F or -CH3.

[0310] In one or more embodiments, X 1 is -CH3and X 2 is F.

[0311] In one or more embodiments, X 1 , X 2 together with the atoms to which they are attached form wherein k is 1.

[0312] In one or more embodiments, X 1 , X 2 together with the atoms to which they are attached form wherein k is 2.

[0313] In one or more embodiments, Formula III is selected from the following structures:

[0314] wherein n is an integer from 1 to 24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0315] In one or more embodiments, n is from 4 to 24.

[0316] In one or more embodiments, n is from 8 to 20.

[0317] In one or more embodiments, n is 4, 8, 12, 16, or 20.

[0318] One or more embodiments provide a pharmaceutical composition comprising a drug conjugate (such as an antibody drug conjugate), or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier, excipient and / or adjuvant, and optionally other anti-cancer drugs. The pharmaceutical composition can be administered by any convenient route, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous membranes (e.g., linings of the oral cavity, rectum, and intestines) and can be administered together with other biologically active agents. Thus, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., as by powders, ointments, solutions or transdermal patches), bucally, or via oral or nasal sprays.

[0319] In some embodiments, the term "pharmaceutically acceptable carrier" refers generally to any type of nontoxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation auxiliary of like - agent that is nontoxic to recipients at the dosages and concentrations employed.

[0320] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active ingredient is administered to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of aminosalicylic acid, antioxidants, chelating agents, and agents for the adjustment of tonicity. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The compositions can be formulated as suppositories using a traditional binders and carriers such as glycerol, triglycerides, etc. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, by E. W. Martin, which is incorporated herein by reference. Such compositions will contain a pharmaceutically effective amount of the drug conjugate together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should be adapted to the mode of administration. The formulations can be packaged in ampoules, in pre- filled syringes or in multi-dose vials made of glass or plastic.

[0321] In some embodiments, the compositions are formulated in accordance with routine procedures as pharmaceutical compositions suitable for intravenous injection. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active ingredient. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by subcutaneous injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0322] One or more embodiments provide use of a drug conjugate in the manufacture of a medicament for treating a disease, such as a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0323] In one or more embodiments, the drug conjugate is used in combination with other drugs for treating a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0324] In one or more embodiments, the drug conjugate is used in combination with other anti-cancer drugs.

[0325] One or more embodiments provide use of a linker or intermediate, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a drug conjugate, such as an antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof.

[0326] Pharmaceutically acceptable salts include pharmaceutically acceptable salts of a drug conjugate, such as an antibody drug conjugate, with various organic and inorganic counterions well known in the art, illustrative salts include, when the molecule contains an acidic functionality, organic or inorganic salts such as sodium, potassium, calcium, magnesium, ammonium, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and tetraalkylammonium salts, and when the molecule contains a basic functionality, organic or inorganic acid salts such as hydrochlorides, hydrobromides, tartrates, mesylates, acetates, maleates, and oxalates. Other non-limiting examples of acids include sulfuric acid, nitric acid, phosphoric acid, propionic acid, glycolic acid, pyruvic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Solvates include hydrates. These salts can generally be prepared by conventional techniques by reacting, for example, the appropriate acid or base with an ADC of the application.

[0327] One or more embodiments provide a method of treating a disease, the method comprising administering to a patient an effective amount of a drug conjugate. An effective amount means an amount of an active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual, or human by a researcher, veterinarian, medical doctor, or other clinician, which includes the treatment of a disease.

[0328] Generally, a suitable dosage amount can range from about 0.1 to 100 mg / kg, and the frequency of administration can be, for example, once a month, once every two weeks, once every three weeks, twice every three weeks, three times every four weeks, once a week, twice a week, etc. For example, the mode of administration can be intravenous infusion, intravenous injection, subcutaneous injection, intramuscular injection, etc.

[0329] One or more embodiments provide a drug conjugate, such as an antibody drug conjugate, which can be used as a medicament.

[0330] One or more embodiments provide use of a drug conjugate, such as an antibody drug conjugate, or a pharmaceutical composition comprising a drug conjugate, such as an antibody drug conjugate, in the manufacture of a medicament for treating and / or preventing a disease.

[0331] In one or more embodiments, the disease is a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. In one or more embodiments, the cancer includes triple-negative breast cancer, glioblastoma, medulloblastoma, urothelial carcinoma, breast cancer, head and neck cancer, kidney cancer (clear cell renal cell carcinoma and papillary renal cell carcinoma), ovarian cancer (e.g., ovarian adenocarcinoma and ovarian teratocarcinoma), pancreatic cancer, gastric cancer, Kaposi sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), cervical cancer, colorectal cancer, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glioma, hepatobiliary cancer, T-cell lymphoma, uterine cancer, liver cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, neuroblastoma, sarcoma (e.g., synovial sarcoma and carcinosarcoma), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer (e.g., basal cell carcinoma and squamous cell carcinoma), pancreatic cancer, malignant melanoma, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, testicular cancer, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, or relapsed anaplastic large cell lymphoma).

[0332] One or more embodiments provide an article of manufacture comprising a drug conjugate or a pharmaceutical composition;

[0333] a container; and

[0334] a package insert, instructions, or a label indicating that the compound or composition is used for treating a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. DETAILED DESCRIPTION

[0335] "Alkyl" refers to saturated aliphatic hydrocarbon groups, including straight-chain and branched-chain hydrocarbon groups. For example, C1-C20 alkyl, such as C1-C6 alkyl. C1-C20 alkyl refers to alkyl groups having from 1 to 20 carbon atoms, for example, 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, or 20 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, neopentyl, n-hexyl, and the like. The alkyl group can be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxyl, carbonyl, carboxyl, aryl, heteroaryl, amine, halogen, sulfonyl, sulfinyl, phosphonyl, and the like.

[0336] "Carbocyclyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical composed only of carbon and hydrogen atoms, which can include fused or bridged ring systems, having from 3 to 15 carbon atoms, for example, having from 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) carbon atoms, and which is either saturated or unsaturated and connected to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, and the like. When specifically stated in the specification, a carbocyclyl group can be optionally substituted with one or more substituents independently selected from alkyl, halogen, haloalkyl, cyano, nitro, oxo, aryl, aralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl.

[0337] "Aryl" refers to an all-carbon monocyclic or fused-ring polycyclic ring system that is completely conjugated and has a delocalized pi electron system. Generally, aryl groups have from 5 to 14 carbon atoms, for example, 6, 10, 12, 14 carbon atoms. Aryl groups can be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, nitro, hydroxyl, carboxyl, aryl, aralkyl, amine, halogen, sulfonyl, sulfinyl, phosphonyl. Non-substituted aryl examples include, but are not limited to, phenyl, naphthyl, indenyl, anthryl, fluorenyl, phenanthryl, pyrenyl, biphenyl, and terphenyl.

[0338] "Alkyl" refers to a straight or branched hydrocarbon chain radical, which is saturated, non-saturated, or aromatic, which is non-substituted or substituted, and which contains from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Alkyl groups can be substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, nitro, hydroxyl, carbonyl, carboxyl, aryl, aralkyl, amine, halogen, sulfonyl, sulfinyl, phosphonyl. The definition of aryl is described above.

[0339] "Heteroaryl" refers to a monocyclic or fused ring radical having from 5 to 14 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ring atoms), containing from 1 to 4 (e.g., 1, 2, 3, or 4) heteroatoms selected from N, O, S, with the remaining ring atoms being C, and having a fully conjugated pi-electron system, including, but not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, indolyl, quinolinyl, isoquinolinyl, triazolyl, benzimidazolyl, benzotriazolyl, and the like. Heteroaryl groups can be non-substituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, nitro, hydroxyl, carbonyl, carboxyl, aryl, aralkyl, amine, halogen, sulfonyl, sulfinyl, phosphonyl.

[0340] "Heterocyclyl" refers to a stable 3- to 14-membered aromatic or nonaromatic ring radical, which is either saturated, unsaturated, or aromatic, which is composed of two to eight (e.g., two, three, four, five, six, seven, or eight) carbon atoms and from one to six (one, two, three, four, five, or six) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically noted in the specification, a heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or spiro ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, dioxinyl, thienyl[l,3]dithiane, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolinyl, octahydroisoindolinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, 1,2,4-thiadiazol-5(4H)-yl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiamorpholinyl, 1-oxo-thiamorpholinyl, and 1,1-dioxo-thiamorpholinyl. When specifically stated in the specification, a heterocyclyl group can be optionally substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, cyano, oxo, thioxo, nitro, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl.

[0341] "Alkoxy" means a group of the formula -O-(alkyl), wherein alkyl is alkyl as defined herein. Non- limiting examples of alkoxy groups are methoxy, ethoxy, n-propyloxy, 1-methylethoxy (isopropoxy), n-butyloxy, isobutyloxy, sec-butyloxy, t-butyloxy. Alkoxy groups can be substituted or unsubstituted.

[0342] "Halo" means fluoro (F), chloro (CI), bromo (Br), or iodo (I).

[0343] "Amino" means -NH2.

[0344] "Cyano" means -CN.

[0345] "Nitro" means -NO2.

[0346] "Hydroxy" means -OH.

[0347] "Carboxy" means -COOH.

[0348] "Mercapto" means -SH.

[0349] "Carbonyl" means -C=0.

[0350] "Polypeptide" means a molecule composed of two or more amino acid monomers linearly connected by amide bonds (also known as peptide bonds), and can include dipeptides, tripeptides, tetrapeptides, oligopeptides, etc.

[0351] "Amino acid" means an organic compound containing both an amino group and a carboxyl group, such as alpha-amino acids and beta-amino acids. Amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V), sarcosine (Sar), theanine (Thea), hydroxyproline (Hypro), hydroxylysine (Hylys), beta-aminoisobutyric acid (beta-AiBA), citrulline (Cit), and beta-alanine (beta-Ala), etc.

[0352] It is understood by one skilled in the art that when an amino acid or polypeptide is referred to as a component of a molecule (such as an antibody or ADC), the amino acid or polypeptide refers to the amino acid residue or polypeptide residue (whether or not written), i.e., the remainder of it after its part groups (such as one hydrogen atom of its amino group and / or the hydroxyl group of its carboxyl group) are lost as a result of its covalent bonding (such as amide bonding) to other parts of the molecule when it is combined with the other parts of the molecule.

[0353] Minor variations to the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that the identity of the amino acid sequence remains at least 75%, such as at least 80%, 90%, 95%, or 99%. In one or more embodiments, the variations are conservative amino acid substitutions. Conservative amino acid substitutions are substitutions occurring within a family of related amino acids in the side chains of which the functional properties are preserved in an identical or similar position. Genetically encoded amino acids are broadly grouped as follows: (1) acidic amino acids as aspartic acid and glutamic acid; (2) basic amino acids as lysine, arginine, and histidine; (3) non-polar amino acids as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids as glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine of the aliphatic-hydroxyl family; (ii) asparagine and glutamine of the amide family; (iii) alanine, valine, leucine, and isoleucine of the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine of the aromatic family. In one or more embodiments, the groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. For example, it is reasonable to predict that a substitution of leucine for isoleucine or valine, of glutamic acid for aspartic acid, of serine for threonine, or of one aliphatic amino acid for another, will not have a major effect on the properties of the resulting molecule, especially if the substitution occurs at a position remote from the active site of the molecule. Whether a change in an amino acid results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Fragments or analogs of an antibody or immunoglobulin molecule can be readily prepared by one of ordinary skill in the art.

[0354] In one or more embodiments, the amino acid substitutions have the effect of: (1) decreasing susceptibility to proteolysis, (2) decreasing susceptibility to oxidation, (3) altering binding affinity for forming protein complexes, (4) altering binding affinity, and (5) conferring or improving other physicochemical or functional properties of such analogs. The analogs can include various muteins having sequences that differ from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in naturally occurring sequences, preferably in portions of the polypeptide other than those forming intermolecular contacts. Conservative amino acid substitutions should not significantly alter the structural properties of the parent sequence (e.g., the substituted amino acid should not tend to disrupt a helical structure or other type of secondary structure present in the parent sequence). Examples of artificially recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0355] "Recombinant" with respect to a polypeptide or polynucleotide means a form of the polypeptide or polynucleotide that does not occur in nature, without limitation, examples of which can be produced by combining polynucleotides or polypeptides that normally do not occur together.

[0356] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology or identity can be determined by comparing positions in each sequence which can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "At least 80% identity" is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (inclusive of the endpoints) or any value therein. "At least 90% identity" is about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, or a range between any two of these values (inclusive of the endpoints) or any value therein.

[0357] "Antibody," "antigen binding unit," "antigen binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be an intact antibody and any antigen binding fragment thereof or a single chain thereof. Thus, the term "antibody" includes any protein or peptide that comprises at least a portion of an immunoglobulin molecule that has a biological activity of binding to an antigen. Antibodies, "antigen binding units," and antigen binding fragments include, but are not limited to, a complementarity determining region (CDR) of a heavy chain or light chain or a ligand binding portion thereof, a variable region of a heavy chain (VH), a variable region of a light chain (VL), a constant region of a heavy chain (CH), a constant region of a light chain (CL), a framework region (FR), or any portion thereof, or at least a portion of a binding protein. CDR regions include CDR regions of a variable region of a light chain (VL CDR1-3) and CDR regions of a variable region of a heavy chain (VH CDR1-3). An antibody or antigen binding unit thereof can be a polypeptide or polypeptide complex that specifically recognizes and binds one or more (e.g., two) antigens. An antibody or antigen binding unit thereof that specifically recognizes and binds multiple (e.g., two) antigens can be referred to as a multispecific (e.g., bispecific) antibody or antigen binding unit thereof.

[0358] "Antibody fragments" or "antigen binding fragments" refer to a portion of an antibody, and the antibody fragments of the present disclosure can be in a similar form as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc., in monospecific antibody fragments. Regardless of its structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. "Antibody fragments" include aptamers, mirror images, and diabodies. "Antigen binding fragments" also include any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a particular antigen.

[0359] Antibodies include a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the class of a heavy chain includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses therein (e.g., γ1-γ4). The "class" of an antibody is determined by the nature of the chain's constant region, which is either IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and the functional specificities conferred are known. All immunoglobulin classes are within the scope of the present disclosure. In one or more embodiments, the immunoglobulin molecule is of the IgG class. Two heavy chains and two light chains are linked by disulfide bonds in a "Y" configuration, with the light chain beginning at the "Y" mouth and continuing around the heavy chain through the variable region.

[0360] Antibodies, antigen binding fragments, or derivatives of the present disclosure include, but are not limited to, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies (scFv), epitope binding fragments (e.g., Fab, Fab', and F(ab')2).

[0361] Light chains can be classified as kappa (K) or lambda (l). Each heavy chain can be associated with either a K or l light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, its light and heavy chains are bound by covalent bonds, and the "tail" portions of the two heavy chains are bound by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The immunoglobulin K light chain variable region is Vκ; the immunoglobulin l light chain variable region is Vl.

[0362] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used according to their functional properties. The light chain variable region (VL) and the heavy chain variable region (VH) determine antigen recognition and specificity. The light chain constant region (CL) and the heavy chain constant region (CH) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant regions increases as they become more distal from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy terminus of the heavy and light chains, respectively.

[0363] The antibodies disclosed herein can be derived from any animal, including but not limited to fish, birds, and mammals. Preferably, the antibodies are human, murine, equine, rabbit, goat, camelid, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g., from a shark).

[0364] A "heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region can include a hinge region derived in part from an IgGl molecule and in part from an IgG3 molecule. In another embodiment, a portion of the heavy chain can include a chimeric hinge region derived in part from an IgGl molecule and in part from an IgG4 molecule.

[0365] A "light chain constant region" includes a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light-heavy pair" refers to a collection of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0366] The number of amino acids of conservative amino acid substitution of VL, VH is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values, inclusive of the endpoints, or any value therein. The number of amino acids of conservative amino acid substitution of heavy chain constant region, light chain constant region, heavy chain, or light chain is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values, inclusive of the endpoints, or any value therein.

[0367] An "antibody drug conjugate" or "ADC" refers to a binding polypeptide (such as an antibody or antigen binding unit thereof) linked to one or more chemical drugs, which can optionally be a therapeutic or cytotoxic agent. In preferred embodiments, an ADC comprises an antibody, a drug (e.g., a cytotoxic drug), and a linker capable of attaching or conjugating the drug to the antibody. Non-limiting examples of drugs that can be included in an ADC are mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, anti-hormones, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine / threonine kinase inhibitors), and radiosensitizers.

[0368] A "drug antibody conjugate ratio" or "DAR" refers to the number of drugs (e.g., exatecan) attached to an antibody of an ADC. The DAR of an ADC can range from 1 to 10, although higher loads (e.g., 20) are possible depending on the number of attachment sites on the antibody. The DAR can be used when referring to the number of drugs loaded onto a single antibody, or alternatively, to the average or mean DAR of a population of ADCs. In some embodiments, the value is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. When considering the average number of small molecule drugs bound per antibody, the DAR can also be referred to as the average drug antibody conjugate ratio, which value is selected from about 0 to about 10, or about 2 to about 8. In some embodiments, the drug antibody conjugate ratio is about 3 to about 6. In other embodiments, the drug antibody conjugate ratio is about 6 to about 8, or about 7 to about 8. The DAR value can be denoted herein by p.

[0369] The DAR value of ADCs can be determined using UV-Vis absorption spectroscopy (UV-Vis), high performance liquid chromatography-hydrophobic interaction chromatography (HPLC-HIC), high performance liquid chromatography-reverse phase chromatography (RP-HPLC), liquid chromatography mass spectrometry (LC-MS), and the like. These techniques are described in Ouyang, J. Methods Mol Biol, 2013, 1045: p. 275-83.

[0370] "Comprise", "comprising", and "comprises" and "comprised of" and "comprising" are intended to be open-ended terms i.e., the composition and methods encompass the elements listed, but not excluding additional elements. "Consisting essentially of" when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements of no essential significance to the claimed invention. "Consisting of" shall mean excluding more than trace amounts of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the disclosure.

[0371] "Patient" refers to a mammal, including humans and non-human mammals. In some embodiments, the patient is a human. In other embodiments, the patient is a non-human mammal, such as a wild, domesticated, and farm animals. In other embodiments, the patient is a dog, cat, mouse, rat, rabbit, guinea pig, or a primate such as a cynomolgus monkey.

[0372] "Treatment" of a disease in a patient refers to (1) preventing the disease from occurring in a patient that is predisposed or does not yet exhibit symptoms of the disease; (2) inhibiting the disease or its symptoms or arresting their development; or (3) relieving or causing the regression of the disease or its symptoms.

[0373] Other chemical terms used herein are used according to conventional usage in the art, such as The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0374] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0375] Antibody moieties

[0376] The antibodies described herein can be any antibody suitable for use in preparing an antibody drug conjugate, which can have the structure of a whole antibody or can include antibody fragments (polyclonal and monoclonal antibodies), such as Fab, Fab', F(ab)'2, and Fv, etc.

[0377] Antibodies are capable of specifically binding to antigens, such as tumor-specific antigens, etc. Tumor antigens can be used for the identification of tumor cells, can be potential indicators for tumor treatment, or can be targets for tumor treatment. Therefore, the selection of specific antibodies is mainly based on the type of disease and the cells and tissues as targets.

[0378] Examples of tumor antigens well known in the art include, but are not limited to, EGFR, HER2, CD20, CD30, CD33, CD47, CD52, CD133, CEA, VEGF, TROP-2, B7H3, FRalpha (FRa), Nectin-4, B7H4, CLDN18, BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD22, FcRH2, NCA, MDP, IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, etc.

[0379] In one or more embodiments, the antibody can be a humanized monoclonal antibody.

[0380] In one or more embodiments, the antibody is an anti-HER2 antibody, which can specifically act on human epidermal growth factor receptor 2, such as Trastuzumab, Pertuzumab, Margetuximab, ZW25, etc. The anti-HER2 antibody can be modified, such as one or more amino acid sequence changes, additions or reductions, to achieve the corresponding purposes, such as to enhance antibody-dependent cell-mediated cytotoxicity.

[0381] In one or more embodiments, the anti-HER2 antibody is the antibody disclosed in WO 2022 / 022526A1 (such as BAT0303F).

[0382] In one or more embodiments, the anti-HER2 antibody is Trastuzumab, and the heavy chain and light chain sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0383] The amino acid sequences corresponding to each sequence number are shown in Table 1.

[0384] Table 1 Amino acid sequences of trastuzumab

[0385] In one or more embodiments, the antibody is an anti-TROP-2 antibody that specifically acts on a TROP-2 protein, wherein the TROP-2 protein is trophoblast cell surface glycoprotein antigen 2. In one or more embodiments, the anti-TROP-2 antibody is a fully human monoclonal antibody, and in one or more embodiments, the anti-TROP-2 antibody is a humanized monoclonal antibody.

[0386] In one or more embodiments, the anti-TROP-2 antibody is an antibody disclosed in the following patent documents: WO 2019 / 029715 Al (e.g., BAT0806, BAT0807, BAT0808), WO 2021 / 147993 Al (e.g., PD3), CN 101264325B (e.g., RS7, hRS7), CN 105849126B (e.g., hTINA1-H1L1, hTINA1-H2L1, hTINA1-H2L2, hTINA1-H3L3), CN 110903395A (e.g., M1, M2, M3), CN 113896796A (e.g., 4D3, 7F11), US 2013 / 0089872 A (e.g., K5-70, K5-107, K5-116-2-1, T6-16, T5-86), US 5840854 A (e.g., BR110), US 2013 / 0122020 A (e.g., 3E9, 6G11, 7E6, 15E2, 18B1), US 2012 / 0237518 A (e.g., 77220, KM4097, KM4590).

[0387] In one or more embodiments, the anti-TROP-2 antibody is commercially available, including LS-C126418, LS-C178765, LS-C126416, LS-C126417 (LifeSpan BioSciences, Inc., Seattle, WA); 10428-MM01, 10428-MM02, 10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54 (eBioscience, San Diego, CA); sc-376181, sc-376746, Santa Cruz Biotechnology (Santa Cruz, CA); MM0588-49D6 (Novus Biologicals, Littleton, CO); ab79976 and ab89928 (Cambridge, MA).

[0388] In one or more embodiments, the anti-TROP-2 antibody is the anti-TROP-2 antibody 162-25.3 and 162-46.2 disclosed by Lipinski et al. (1981, Proc Natl. Acad Sci USA, 78:5147-50) or the Pr1E11 anti-TROP-2 antibody disclosed by Ikeda et al. (2015, Biochem Biophys Res Comm 458:877-82) that recognizes a unique epitope on TROP-2.

[0389] In one or more embodiments, the antibody is the hRS9 antibody, and the heavy and light chain sequences of the antibody are SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0390] The corresponding amino acid sequences of each sequence number are shown in Table 2.

[0391] Table 2. Amino acid sequences of hRS9 antibody

[0392] Antibodies can be produced by using conventional recombinant DNA technology. Techniques for selecting, constructing and cultivating vectors and cell lines that produce antibodies, etc. can be selected, constructed and cultivated using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, for example, Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D. L. Hacker, F. M. Wurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplements, are incorporated herein by reference.

[0393] In one or more embodiments, DNA encoding the antibody can be designed and synthesized according to the antibody amino acid sequences described herein in a conventional manner, placed into an expression vector, and then transfected into host cells, which are cultured in a medium to produce the monoclonal antibody. In some embodiments, the expression vector for the antibody includes at least one promoter element, the antibody coding sequence, a transcription termination signal and a poly A tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI and cytomegalovirus early promoters, and other promoters of some cells such as the actin promoter. Suitable expression vectors can include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian host cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, murine L cells and CHO cells, etc.

[0394] In one or more embodiments, the inserted gene fragment contains a selectable marker, such as a dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, or other selectable marker, to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into a host cell that lacks the gene, and the successfully transfected cells are allowed to grow in a selective medium, producing the desired protein. The antibody is purified by one or more purification steps. Purification can be performed using conventional methods, such as centrifugation and collection of the supernatant, followed by further centrifugation to remove impurities. Protein A affinity columns and ion exchange columns can be used to purify the antibody protein.

[0395] In one or more embodiments of the application, the number of small molecule drugs conjugated to an antibody in an antibody drug conjugate, i.e., the drug-to-antibody ratio (DAR) or p, is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the DAR or p is the average number of small molecule drugs conjugated to an antibody, i.e., the average drug-to-antibody ratio, which is selected from about 0 to 10, or 2 to 8. In some embodiments, the DAR or p is 3-6, and in other embodiments, the DAR or p is about 6 to about 8, or about 7 to about 8.

[0396] Synthetic methods

[0397] The present application also provides methods for preparing the drug conjugates (e.g., antibody drug conjugates) and intermediates. The drug conjugates (e.g., antibody drug conjugates) and intermediates of the present application can be prepared by known formulations and methods. In some embodiments, the methods for preparing are as described below.

[0398] A. Methods for preparing

[0399] 1. Methods for preparing compounds of general formula 1-9

[0400] Step 1: reacting a compound of general formula 1-1 and a compound of general formula 1-1’ or a compound of general formula 1-2 and a compound of general formula 1-2’ under basic conditions to obtain a compound of general formula 1-3;

[0401] Step 2: reacting a compound of general formula 1-3 and a compound of general formula 1-4 in the presence of a condensing agent under basic conditions to obtain a compound of general formula 1-5;

[0402] Step 3: removing the amino protecting group W of the compound of general formula 1-5 1 to obtain a compound of general formula 1-6;

[0403] Fourth step: the compound of formula 1-6 and the compound of formula 1-7 are reacted under basic or neutral conditions to obtain the compound of formula 1-8;

[0404] Fifth step: the compound of formula 1-8 and di(p-nitrophenyl)carbonate are reacted under basic conditions to obtain the compound of formula 1-9.

[0405] wherein W is an amino protecting group, for example 9-fluorenylmethyloxycarbonyl; W 1 , W 2 , W 3 each independently is hydrogen or an alcohol end group of a carboxylic acid active ester, for example succinimidyl pentafluorophenyl or benzotriazolyl

[0406] wherein n, AA, M', w, v, R 4 , R 5 , R 6 , R F , R A , Ar, X, z, b are as described herein.

[0407] 2. Preparation of the compound of formula 9

[0408] First step: the compound of formula 1 and the compound of formula 1' or the compound of formula 2 and the compound of formula 2' are reacted under basic conditions to obtain the compound of formula 3;

[0409] Second step: the compound of formula 3 and the compound of formula 4 are reacted in the presence of a condensing agent under basic conditions to obtain the compound of formula 5;

[0410] Third step: the amino protecting group W 1 of the compound of formula 5 is removed to obtain the compound of formula 6;

[0411] Fourth step: the compound of formula 6 and the compound of formula 7 are reacted under basic or neutral conditions to obtain the compound of formula 8;

[0412] Fifth step: the compound of formula 8 and di(p-nitrophenyl)carbonate are reacted under basic conditions to obtain the compound of formula 9.

[0413] wherein W is an amino protecting group, for example 9-fluorenylmethyloxycarbonyl; W 1 , W 2 , W 3 each independently is hydrogen or an alcohol end group of a carboxylic acid active ester, for example succinimidyl pentafluorophenyl or benzotriazolyl

[0414] wherein n, AA, M', w, v, R 4 , R 5 , R 6 , R F , R A , Ar, X, z, b are as described herein.

[0415] The above basic conditions can be provided with a reagent including an organic base and an inorganic base, the organic base including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, potassium acetate, sodium tert-butoxide, or potassium tert-butoxide, the inorganic base including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

[0416] The above condensing agent can be selected from N,N,N',N'-tetramethyl-0-(7-azabenzotriazol-1-yl)urea hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride salt, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 0-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, 0-benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1- yloxytris(dimethylamino)phosphonium hexafluorophosphate, or benzotriazol-1-yl-oxytrispyrrolidinophosphonium hexafluorophosphate.

[0417] B. Method for preparing

[0418] The compound of general formula 1-9 and D are reacted in the presence of a condensing agent under basic conditions to obtain the compound of general formula 1-10.

[0419] The compound of general formula 9 and D are reacted in the presence of a condensing agent under basic conditions to obtain the compound of general formula 10.

[0420] wherein n, AA, M', w, v, R 4 , R 5 , R 6 , R F , R A , Ar, X, z, b, D are as described herein.

[0421] The above basic conditions can be provided with reagents including organic and inorganic bases, the organic bases including but not limited to triethylamine, diethylamine, N- methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, potassium acetate, sodium or potassium tert-butoxide, and the inorganic bases including but not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.

[0422] The above condensing agent can be selected from N,N,N',N'-tetramethyl-O-(7- azabenzotriazol-1-yl)urea hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chloride salt, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride, N,N'-dicyclohexyl carbodiimide, N,N'-diisopropyl carbodiimide, O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, O-benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1- yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytrispyrrolidinophosphonium hexafluorophosphate.

[0423] C. Method for preparing

[0424] The compound of general formula 1-10 and Abu are coupled under weak acidic conditions to obtain the compound of general formula 1-11.

[0425] The compound of general formula 10 and Abu are coupled under weak acidic conditions to obtain the compound of general formula 11.

[0426] wherein n, AA, M', M, w, v, R 4 , R 5 , R 6 , R F , R A , Ar, X, z, b, D, Abu are as described herein.

[0427] The above weak acidic conditions can be provided with reagents including organic and inorganic acids, the organic acids including but not limited to acetic acid, benzoic acid, tartaric acid, oxalic acid, malic acid, citric acid, ascorbic acid, salicylic acid, caffeic acid, sorbic acid, quinic acid, oleanolic acid, succinic acid, chlorogenic acid, formic acid, propionic acid, and the inorganic acids including but not limited to carbonic acid, nitrous acid, hypochlorous acid, hydrofluoric acid, sulfurous acid, hydrosulfuric acid, silicic acid, metasilicic acid, phosphoric acid, metaphosphoric acid, sodium bicarbonate, sodium bisulfite.

[0428] The drug conjugate can be purified by conventional methods, such as by preparative high performance liquid chromatography (prep-HPLC) and the like.

[0429] Examples

[0430] Synthesis of compound C1 in Example 1

[0431] 1) Synthesis of compound C1-1

[0432] Compound C0-1 (5 g, 30 mmol) and tetrahydrofuran (THF, 50 mL) were added to a reaction bottle, after stirring and dissolving, di-tert-butyl dicarbonate (Boc2O, 20 g, 91 mmol) was added, nitrogen protection, stirring to reflux, reaction for 5 hours (h). After the reaction was complete, the reaction solution was concentrated to remove the solvent, ethyl acetate (20 mL), petroleum ether (180 mL) was added and stirred for 0.5 h, filtered, petroleum ether was rinsed, and the dried rinsed solid was obtained as a gray powder compound C1-1 (7.2 g). MS (ESI) m / z: 212 [M-55] + .

[0433] 2) Synthesis of compound C1-2

[0434] Compound C1-1 (7.2 g, 27 mmol) was added to a reaction bottle, N,N-dimethylformamide (DMF, 70 mL) was added and stirred and dissolved, then compound C0-2 (4.75 g, 27 mmol), potassium carbonate (5.5 g, 40.5 mmol), sodium iodide (2 g, 13.3 mmol) were added in turn, and stirred at room temperature for 3 h. After the reaction was complete, ethyl acetate (200 mL) was added, washed with saturated aqueous sodium chloride three times, washed with aqueous sodium sulfite once, and dried with sodium sulfate. Then filtered, the filtrate was concentrated to remove the solvent. Ethyl acetate (30 mL), petroleum ether (150 mL) was added and stirred for 0.5 h, filtered, petroleum ether was rinsed, and the dried rinsed solid was obtained as a gray powder C1-2 (10.2 g). MS (ESI) m / z: 407 [M+H] + .

[0435] 3) Synthesis of compound C1-3

[0436] Compound C1-2 (10.2 g, 25.1 mmol) and dichloromethane (DCM, 100 mL) were added into a reaction flask, which was protected by nitrogen. The solution was stirred and cooled to -50 °C, and diisobutylaluminum hydride (Dibal-H, 50 mL, 50 mmol) was added dropwise. After 2 h, methanol (20 mL) was added, followed by 20% aqueous citric acid solution (200 mL), and dichloromethane was extracted. The solvent was removed by concentration, and the product was purified by silica gel column chromatography to obtain compound C1-3 (3.5 g) as a white solid. MS (ESI) m / z: 379 [M+H] + .

[0437] 4) Synthesis of compound C1-4

[0438] Compound C1-3 (180 mg, 0.47 mmol) and N,N-dimethylformamide (DMF, 2 mL) were added into a reaction flask, which was stirred and cooled to 0-5 °C. Compound C0-3 (900 mg, 0.38 mmol), diisopropylethylamine (DIEA, 13 mg, 0.1 mmol) were added successively, and the solution was stirred at 0-5 °C for 1 h. After the reaction was completed, the product was purified by column chromatography to obtain compound C1-4 (210 mg) as a light yellow solid, which was directly used in the next reaction. MS (ESI) m / z: 544 [M+H] + .

[0439] 5) Synthesis of compound C1-5

[0440] Compound C1-4 (210 mg, 0.39 mmol), exatecan mesylate (207 mg, 0.39 mmol), 1-hydroxybenzotriazole (HOBt, 105 mg, 0.78 mmol), N,N-dimethylformamide (2 mL) were added into a reaction flask, which was stirred and cooled to 0-5 °C. Diisopropylethylamine (120 mg, 0.93 mmol) was added. The solution was stirred at room temperature for 2 h. After the reaction was completed, methyl tert-butyl ether was added, and the solution was stirred for 0.5 h. The solid was filtered, rinsed with methyl tert-butyl ether, and dried to obtain compound C1-5 (270 mg) as a gray powder. MS (ESI) m / z: 840 [M+H] + .

[0441] 6) Synthesis of compound C1

[0442] Compound C1-5 (100 mg, 0.12 mmol) and methanol (MeOH, 4 mL) were added into a reaction flask, which was stirred and dissolved. Hydrogen chloride / ethanol solution (2 mL, 20 mmol) was added, and the solution was stirred at room temperature for 1 h. The solvent was removed by concentration to obtain compound C1 (82 mg) as a brown solid. MS (ESI) m / z: 740 [M+H] +.

[0443] Synthesis of compound C2 of Example 2

[0444] 1) Synthesis of compound C2-1

[0445] Compound C1-3 (3.4 g, 9.0 mmol) and methanol (20 mL) were added to a reaction bottle, after stirring and dissolving, hydrogen chloride / isopropanol solution (20 mL, 100 mmol, 5M) was added, stirred at room temperature for 1 h, and the solvent was removed by concentration to obtain compound C2-1 crude (3.5 g), which was directly used in the next step. MS (ESI) m / z: 279 [M+H] + .

[0446] 2) Synthesis of compound C2-2

[0447] Compound C0-4 (3.2 g, 6.5 mmol), compound C2-1 crude (3.3 g) and N,N- dimethylformamide (30 mL) were added to a reaction bottle, and stirred to cool to 0-5 °C. Diisopropylethylamine (3.17 g, 24.6 mmol), 2-(7-azobenzotriazole)-N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU, 7.3 g, 19.2 mmol) were added, and stirred at 0-5 °C for 3 h. After the reaction was completed, ethyl acetate was added, and the mixture was washed with saturated aqueous sodium chloride solution three times, concentrated to dryness, and purified by column chromatography to obtain compound C2-2 (3.2 g) as a white solid. MS (ESI) m / z: 757 [M+H] + .

[0448] 3) Synthesis of compound C2-3

[0449] Compound C2-2 (3.2 g, 4.2 mmol) and tetrahydrofuran (50 mL) were added to a reaction, and after stirring uniformly, piperidine (10 mL) was added, and stirred at room temperature for 1 h. After the reaction was completed, methyl tert-butyl ether (500 mL) was added, stirred for 0.5 h, filtered, and the obtained solid was again slurried with methyl tert-butyl ether (100 mL), filtered, and dried to obtain compound C2-3 crude (2.5 g) as an oil, which was directly used in the next step. MS (ESI) m / z: 535 [M+H] + .

[0450] 4) Synthesis of compound C2-4

[0451] Compound C0-5 (8.14 g) was dissolved in N,N-dimethylformamide (DMF, 40 mL) under nitrogen protection at 0-5 °C, compound C0-6 (10 g) and DMF (10 mL) were added, 6.5 mL of diisopropylethylamine was added dropwise at 0-5 °C, and after 1 h of addition, the reaction was stirred at room temperature for 4 h. After the reaction was completed, DMF was removed by reduced pressure concentration, and silica gel column chromatography (dichloromethane and methanol at a volume ratio of 20:1 as eluent) gave 14.2 g of yellow oil liquid C2-4.

[0452] 5) Synthesis of compound C2-5

[0453] Compound C2-3 (2.5 g) and N,N-dimethylformamide (15 mL) were added to the reaction, stirred and cooled to 0-5 °C, compound C2-4 (3.7 g, 5.0 mmol), diisopropylethylamine (540 mg, 4.19 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.8 g, 12.6 mmol) were added in turn, and stirred at 0-5 °C for 1 h. After the reaction was completed, methyl tert-butyl ether (200 mL) was added, stirred for 0.5 h, filtered, methyl tert-butyl ether was rinsed, and the dried rinsed solid gave solid compound C2-5 crude (2.3 g), which was directly used in the next step reaction. MS (ESI) m / z: 626 [(M+2H) / 2] + .

[0454] 5) Synthesis of compound C2-6

[0455] Compound C2-5 crude (2.3 g) and tetrahydrofuran (40 mL) were added to the reaction, stirred uniformly, piperidine (10 mL) was added, and stirred at room temperature for 1 h. After the reaction was completed, methyl tert-butyl ether (250 mL) was added, stirred for 0.5 h, filtered, methyl tert-butyl ether was rinsed, and the dried rinsed solid gave solid compound C2-6 crude (1.8 g), which was directly used in the next step reaction. MS (ESI) m / z: 1029 [M+H] + .

[0456] 6) Synthesis of compound C2-7

[0457] Compound C2-6 crude (1.8 g) and N,N-dimethylformamide (40 mL) were added to the reaction, stirred uniformly, compound C0-7 (1.1 g, 4.4 mmol) was added, and stirred at room temperature for 1 h. After the reaction was completed, methyl tert-butyl ether (250 mL) was added, stirred for 0.5 h, filtered, methyl tert-butyl ether was rinsed, and the dried rinsed solid gave solid compound C2-7 crude (2.1 g), which was directly used in the next step reaction. MS (ESI) m / z: 1166 [M+H]+ .

[0458] 7) Synthesis of compound C2

[0459] Compound C2-7 crude (2.0 g) and N,N-dimethylformamide (15 mL) were added into the reaction, stirred and cooled to 0-5 °C, compound C0-3 (4.5 g, 5.0 mmol) and diisopropylethylamine (350 mg, 2.7 mmol) were added in turn, stirred at 0-5 °C for 1 h. After the reaction was completed, methyl tert-butyl ether was added, stirred for 0.5 h, filtered, methyl tert-butyl ether was rinsed, and the dried rinsed solid to obtain solid compound C2 crude (2.0 g). MS (ESI) m / z: 666 [(M+2H) / 2] + .

[0460] Example 3 Synthesis of compound C3

[0461] The preparation of compound C3 was according to the synthesis of compound C2 in Example 2, with compound C0-6 replaced by compound C0-8. White solid compound C3 was obtained finally. The structure of compound C0-8 was shown as follows.

[0462] Example 4 Synthesis of intermediate (C2-Exatecan)

[0463] Compound C2 crude (2.0 g), Exatecan mesylate (0.8 g, 1.5 mmol), 1-hydroxybenzotriazole (230 mg, 1.7 mmol) and N,N-dimethylformamide (15 mL) were added into the reaction, stirred and cooled to 0-5 °C, diisopropylethylamine (560 mg, 4.3 mmol) was added, stirred at room temperature for 2 h. After the reaction was completed, methyl tert-butyl ether was added, stirred for 0.5 h, filtered, methyl tert-butyl ether was rinsed, and the dried rinsed solid was purified by preparative HPLC to obtain white powder compound C2-Exatecan (280 mg). MS (ESI) m / z: 814 [(M+2H) / 2] + .

[0464] Example 5 Synthesis of intermediate (C3-Exatecan)

[0465] The preparation of compound C3-Exatecan was according to the synthesis of compound C2-Exatecan in Example 4, with compound C2 replaced by compound C3. White solid compound C3-Exatecan was obtained finally.

[0466] Example 6 Synthesis of ADC1

[0467] To Trastuzumab, 3 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) were added according to the mass of the substance of the antibody, 0.1 M disodium ethylenediaminetetraacetate (EDTA-2Na) was added to a final concentration of 2 mM, and then the pH of the system was adjusted to 8 with 1 M Tris base. Reduction was carried out at 25°C for 2 hours. TCEP and EDTA were removed by ultrafiltration with 10 mM succinic acid. The thiol antibody value was determined by measuring absorbance, and the content of free thiol was determined by the reaction of the thiol group with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and then measuring the absorbance at 412 nm.

[0468] During the coupling reaction, 7 molar equivalents of C2-Exatecan were added according to the mass of the substance of the antibody and the content of free thiol, and after stirring at 25°C for 1 hour, 0.1 M N-acetylcysteine was added to a final concentration of 2 mM, and stirring was continued for 15 minutes to terminate the reaction. The reaction mixture was filtered with a 0.22 micron filter, and then eluted and exchanged with 10 mM succinic acid through Sephadex G-25 resin.

[0469] The DAR value of ADC1, i.e., p, was 4.88, as determined by RP-HPLC.

[0470] Example 7 Synthesis of ADC2

[0471] To hRS9 antibody, 4 molar equivalents of TCEP were added according to the mass of the substance of the antibody, 0.1 M EDTA-2Na was added to a final concentration of 2 mM, and then the pH of the system was adjusted to 8 with 1 M Tris base. Reduction was carried out at 25°C for 2 hours. TCEP and EDTA were removed by ultrafiltration with 10 mM succinic acid. The thiol antibody value was determined by measuring absorbance, and the content of free thiol was determined by the reaction of the thiol group with DTNB, and then measuring the absorbance at 412 nm.

[0472] During the coupling reaction, 6.5 molar equivalents of C2-Exatecan were added according to the mass of the substance of the antibody and the content of free thiol, and after stirring at 25°C for 1 hour, 0.1 M N-acetylcysteine was added to a final concentration of 2 mM, and stirring was continued for 15 minutes to terminate the reaction. The reaction mixture was filtered with a 0.22 micron filter, and then eluted and exchanged with 10 mM succinic acid through Sephadex G-25 resin.

[0473] The DAR value of ADC2, i.e., p, was 6.11, as determined by RP-HPLC.

[0474] Example 8 In vitro biological activity of compound C1

[0475] The growth inhibition of tumor cells by compound C1 was evaluated using HER2-positive breast tumor cell line SK-BR-3 and HER2-negative cell line MDA-MB-468 (purchased from North Naibio). Briefly, SK-BR-3 and MDA-MB-468 cells were recovered in advance, and when the cell confluence reached about 60-80%, the cells were trypsinized to detach the cells, the viable cell density and viability were calculated, and the cell viability was greater than 90%. Then the cell density of SK-BR-3 and MDA-MB-468 was adjusted to 5x10 4 4 6 / mL and 5x10 4 6 / mL, 100 μL / well was plated in a 96-well plate, and incubated in a 37°C, 5% CO2 incubator for 3-5 h or overnight, then 100 μL of compound C1 diluted to different concentrations with detection diluent was added, and incubated in a 37°C, 5% CO2 incubator for 6 days. After removing the supernatant in the culture dish, cell proliferation analysis was performed with Cell Counting-Lite 2.0 Cell Viability Assay Kit (CCL2.0, Vazyme) reagent. The results showed that compound C1 had growth inhibition effect on SK-BR-3 cells and MDA-MB-468 cells.

[0476] Table 3 Growth inhibition of compound C1 on SK-BR-3 and MDA-MB-468 cells

[0477] Example 9 In vitro biological activity of ADC1

[0478] The growth inhibition of tumor cells by ADC1 was evaluated using HER2-positive breast tumor cell line SK-BR-3 and HER2-negative cell line MDA-MB-468 (purchased from North Naibio). Briefly, SK-BR-3 and MDA-MB-468 cells were recovered in advance, and when the cell confluence reached about 60-80%, the cells were trypsinized to detach the cells, the viable cell density and viability were calculated, and the cell viability was greater than 90%. Then the cell density of SK-BR-3 and MDA-MB-468 was adjusted to 1.5x10 5 5 6 / mL and 1x10 5 6 / mL, 100 μL / well was plated in a 96-well plate, and incubated in a 37°C, 5% CO2 incubator for 3-5 h or overnight, then 100 μL of compound C1 diluted to different concentrations with detection diluent was added, and incubated in a 37°C, 5% CO2 incubator for 6 days. After removing the supernatant in the culture dish, cell proliferation analysis was performed with Cell Counting-Lite 2.0 Cell Viability Assay Kit (CCL2.0, Vazyme) reagent. The results showed that compound C1 had growth inhibition effect on SK-BR-3 cells and MDA-MB-468 cells.

[0479] Table 4 Growth inhibition of ADC1 on SK-BR-3 and MDA-MB-468 cells Note: "-" represents no significant killing effect

[0480] Example 10 Bystander effect of ADC1

[0481] HER2-negative cells MDA-MB-468 were plated in advance at a density of 8000 cells per well, and after 3-5h of culture, the culture supernatant was spun dry, 150 μL of the supernatant after adding ADC1 for 4 days in Example 2 was transferred to MDA-MB-468 cells, and the incubator was continued to culture for 3 days. Then the supernatant in the culture plate was removed, and 10% CCK8-containing dilution medium was added, and after 1-2 hours of incubation in the incubator, the absorbance OD450 was read in the microplate reader. The results showed that for HER2-positive cell SK-BR-3 transfer supernatant, ADC1 showed bystander effect, and for HER2-negative cell MDA-MB-468 transfer supernatant, ADC1 showed no significant bystander killing effect.

[0482] Table 5 Bystander killing effect of ADC1 on SK-BR-3 and MDA-MB-468 cells Note: "-" represents no significant killing effect

[0483] Example 11 Pharmacodynamic evaluation of ADC1 in NCI-N87 model of subcutaneous transplantation in nude mice

[0484] This test evaluates the anti-tumor effect of ADC1 in a human gastric cancer cell NCI-N87 model of subcutaneous transplantation in nude mice.

[0485] Test animals: female BALB / c-nude mice, SPF level, 6 weeks old at the time of inoculation (Guangdong Yaoke Biotechnology Co., Ltd.). When the average tumor volume of the mice was 295.68 mm 3 , the grouping and dosing began, and the grouping day was defined as D1. There were 6 mice in each group, and the test design scheme was as follows:

[0486] Table 6 Dosing scheme in NCI-N87 model of subcutaneous transplantation in nude mice

[0487] Test results: After 25 days of dosing, the end-point survival mice in the solvent control group and the ADC1 drug group were both 6. The tumor volume changes are shown in Tables 7 and 8, and the results show that under the current test system, compared with the solvent control group, ADC1 showed a significant tumor inhibition effect.

[0488] Table 7 Tumor volume and tumor inhibition rate (TGI TV ) changes Note: a. TGI TV % = [1 - (T i - T0) / (C i - C0)] x 100, wherein T0and C0are the average tumor volumes of the dosing group and the vehicle control group on the day of grouping (day 1), Ti and Ci are the average tumor volumes of the dosing group and the vehicle control group on day i, respectively. i i

[0489] Table 8 Average tumor weight and tumor inhibition rate (TGI TW ) Note: a. TGI TW % = [1 - (T i - T0) / (C i - C0)] x 100, wherein T0and C0are the average tumor weights of the dosing group and the vehicle control group on the day of grouping (day 1), Ti and Ci are the average tumor weights of the dosing group and the vehicle control group on day 25, respectively. i i

[0490] Example 12 Toxicity test of ADC2 single intravenous infusion in Beagle dogs

[0491] The purpose of this test is to evaluate the possible toxic reactions after single intravenous infusion of ADC2 in Beagle dogs.

[0492] Grouping and test design

[0493] Test animals: Beagle dogs, 7-8 months old at the start of dosing, weighing between 6.8-8.6 kg before dosing (Jiangsu Mass Biotechnology Co., Ltd.).

[0494] According to the body weight of the animals determined before grouping, the animals were randomly grouped using the Provantis 10.3.0.0 computer system. The dosing regimens for the animals in each group are shown in Table 9.

[0495] Table 9 Animal grouping and dosing regimen

[0496] Among them, the vehicle control group was given normal saline (0.9% sodium chloride).

[0497] During the test, the animals were examined for body weight, food intake, pathology (blood cell count, blood cell count, blood biochemistry and coagulation function examination for animals at the point of death).

[0498] Test results

[0499] ​​​​During the test period, no test article related abnormal changes were observed in body weight and blood cell counts of animals in the vehicle control and ADC2 groups. No animal death or moribundity was observed in Beagle dogs given a single intravenous infusion of ADC2 at 30 and 45 mg / kg. In the 30 mg / kg dose group, food consumption was decreased by 18.6% to 32.3% in male animals on D2 to D5, and recovered at the end of the observation period. No abnormality was observed in food consumption of the rest of animals.

Claims

A drug conjugate having a structure as shown in Formula I or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Equation I is: in Abu is an antigen-binding unit; D represents a drug; M is Where * connects to Abu, ** connects to -NH-, and R 1 Selected from: -(CH2) r -、-(CHR m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R 4 It can be -NHCO- or -CONH-; AA can be an amino acid or a polypeptide; Each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, or 3; X is O, NH, or S; Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in the conjugate position of the C at the # position in the aromatic system. R 5 It is hydrogen or C1-C3 alkyl; R 6 It is hydrogen or C1-C3 alkyl; Each R A Independently, it can be a C1-C3 alkyl, halogen, hydroxyl, or cyano group, and b can be 0, 1, 2, or 3; w is 0, 1, 2, 3, 4, 5, or 6; v is 0, 1, 2, 3, 4, 5, or 6; n is an integer from 1 to 24; p is an integer or decimal between 1 and 10. A drug conjugate having a structure as shown in Formula I-1, Formula I-2, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Equation I-1 is: Equation I-2 is: in Abu is an antigen-binding unit; D represents a drug; R 1 Selected from: -(CH2) r -、-(CHR m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is an integer from 1 to 24; p is an integer or decimal between 1 and 10. A drug conjugate having a structure as shown in Formula I-3, Formula I-4, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Equation I-3 is: Equation I-4 is: in Abu is an antigen-binding unit; D represents a drug; n is an integer from 1 to 24; p is an integer or decimal between 1 and 10. A drug conjugate having a structure as shown in Formula I-5 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Equation I-5 is: in Abu is an antigen-binding unit; n is an integer from 1 to 24; p is an integer or decimal between 1 and 10. The drug conjugate according to any one of claims 1-4, wherein the amino acid sequence of Abu contains one or more cysteine ​​residues and is linked to other parts of the drug conjugate via the sulfur atom of the cysteine ​​residue. The drug conjugate according to any one of claims 1-5 is an antibody-drug conjugate, wherein Abu is an antibody or its antigen-binding fragment. The drug conjugate according to any one of claims 1-6, wherein the target of Abu binding is selected from: HER2, TROP-2, Nectin-4, B7H3, B7H4, CLDN18, BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD20, CD21, CD22, CD30, FcRH2, NCA, MDP, IL20Rα, and short proteoglycans (Brevican). ), EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, FRalpha. A compound having a structure as shown in Formula II or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Equation II is as follows: in M' is Where * connects -NH-, R 1 Selected from: -(CH2) r -、-(CH R m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH 2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R 4 It can be -NHCO- or -CONH-; AA can be an amino acid or a polypeptide; Each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, or 3; X is O, NH, or S; Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in the conjugate position of the C at the # position in the aromatic system. R 5 It is hydrogen or C1-C3 alkyl; R 6 It is hydrogen or C1-C3 alkyl; Each R A Independently, it can be a C1-C3 alkyl, halogen, hydroxyl, or cyano group, and b can be 0, 1, 2, or 3; R 7 For hydrogen or w is 0, 1, 2, 3, 4, 5, or 6; v is 0, 1, 2, 3, 4, 5, or 6; n is an integer from 1 to 24. A compound having a structure as shown in Formula II-1A, Formula II-1B, Formula II-2A or Formula II-2B, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Formula II-1A is: Formula II-1B is: Formula II-2A is: Formula II-2B is: in R 1 Selected from: -(CH2) r -、-(CHR m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is an integer from 1 to 24. A compound having a structure as shown in Formula II-3A, Formula II-3B, Formula II-4A or Formula II-4B, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Formula II-3A is: Formula II-3B is: Formula II-4A is: Formula II-4B is: in, n is an integer from 1 to 24. A compound having a structure as shown in Formula III or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Equation III is as follows: in D represents a drug; M' is Where * connects -NH-, R 1 Selected from: -(CH2) r -、-(CH R m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH 2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R 4 It can be -NHCO- or -CONH-; AA can be an amino acid or a polypeptide; Each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, or 3; X is O, NH, or S; Ar is a five- or six-membered aryl or heteroaryl group, and the nitro group on Ar is in the conjugate position of the C at the # position in the aromatic system. R 5 It is hydrogen or C1-C3 alkyl; R 6 It is hydrogen or C1-C3 alkyl; Each R A Independently, it can be a C1-C3 alkyl, halogen, hydroxyl, or cyano group, and b can be 0, 1, 2, or 3; w is 0, 1, 2, 3, 4, 5, or 6; v is 0, 1, 2, 3, 4, 5, or 6; n is an integer from 1 to 24. A compound having a structure as shown in Formula III-1, Formula III-2, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Equation III-1 is as follows: Formula III-2 is: in D represents a drug; R 1 Selected from: -(CH2) r -、-(CHR m ) r - C3-C8 carbon cycloyl group, -O-(CH2) r -, aryl, -(CH2) r -arylene-, -arylene-(CH2) r -、-(CH2) r -(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-(CH2) r - C3-C8 heterocyclic group, -(CH2) r -(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-(CH2) r -、-(CH2) r C(O)NR m (CH2) r -、-(CH2CH2O) r -、-(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -、-(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -、-(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; where each R m Each r is independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is an integer from 1 to 24. A compound having a structure as shown in Formula III-3, Formula III-4, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Formula III-3 is: Formula III-4 is: in D represents a drug; n is an integer from 1 to 24. A compound having a structure as shown in Formula III-5 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Formula III-5 is: in, n is an integer from 1 to 24. The drug conjugate of claim 1, the compound of claim 8 or 11, wherein, When Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is either para or ortho to the C group at the # position. The drug conjugate of claim 1, the compound of claim 8 or 11, wherein, When Ar is a five-membered aryl or heteroaryl group, the nitro group on Ar is not adjacent to the C at the # position. The drug conjugate according to any one of claims 1, 5-7, 15-16, and the compound according to any one of claims 8, 11, 15-16, wherein AA is Where * connects -C(O)-, ** connects -NH-, and each R 8 Independently, it can be H, -CH3, -CH(CH3)CH3, -CH(CH3)CH2CH3, -CH2CH(CH3)CH3, -(CH2)3NH2, -(CH2)4NH2, -CH2COOH, -CH2CH2COOH, -(CH2)3NHC(O)NH2, -(CH2)3NHC(NH)NH2. i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The drug conjugate according to any one of claims 1, 5-7, 15-16, and the compound according to any one of claims 8, 11, 15-16, wherein AA is selected from Val-Cit, Val-Ala, Glu-Arg, Lys-Arg, Arg-Arg, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Trp-Cit, Phe -Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Glu-Gly -Cit, Gly-Gly-Phe-Gly, Gly-Phe-Arg-Gly, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu and Gly-Phe-Leu-Gly. The drug conjugate according to any one of claims 1, 5-7, 15-16, and the compound according to any one of claims 8, 11, 15-16, wherein AA is Val-Cit, Val-Ala, Glu-Arg, Glu-Gly-Cit, or Gly-Gly-Phe-Gly. The drug conjugate according to any one of claims 1, 5-7, 15-16, and the compound according to any one of claims 11, 15-16, wherein... for The compound according to any one of claims 8, 15-16, wherein for The drug conjugate of any one of claims 1-3, 5-7, 15-20 or the compound of any one of claims 11-13, 15-20, wherein D is an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases. The drug conjugate of any one of claims 1-3, 5-7, 15-20 or the compound of any one of claims 11-13, 15-20, wherein D is a microtubule inhibitor, a DNA damaging agent or a DNA topoisomerase inhibitor. The drug conjugate of any one of claims 1-3, 5-7, 15-20 or the compound of any one of claims 11-13, 15-20, wherein D is selected from MMAE, MMAF, AF, calicheamicin derivatives, duocarmycin derivatives, pyrrolobenzodiazepine (PBD), camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivatives SN-38, 22-hydroxyeclipticine, topotecan, letopotecan, belotetan, irinotecan, ecilenotecan, ecilenotecan derivatives (e.g., Dxd), homosilatecan, 6 8-Dibromo-2-methyl-3-[2-(D-pyranoxysylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-pyranoglycosyl-12,1 3-Dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide, or pharmaceutically acceptable salts or solvates thereof. The drug conjugate of any one of claims 1-3, 5-7, 15-20 or the compound of any one of claims 11-13, 15-20, wherein D is in X 1 and X 2 Each independently is: H， hydroxyl group C1-C6 alkyl, C1-C6 alkyl groups substituted with one or more hydroxyl, halogen, nitro, or cyano groups. C2-C6 alkenyl, C2-C6 acetylene group, C1-C6 alkoxy groups, C1-C6 aminoalkoxy, halogen, Nitro, Cyano, Thiol, Alkylthio, Amino groups, amino groups substituted with amino protecting groups, or C1-C6 aminoalkyl groups optionally substituted with amino protecting groups or C1-C6 alkyl groups at the amino moiety. The amino moiety may be optionally replaced by an amino protecting group or a C1-C6 aminoalkyl amino group. A C1-C6 alkyl group attached to a heterocycle, wherein the heterocycle is optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogen groups, nitro groups, or cyano groups. A C1-C6 alkylamino group attached to a heterocycle, wherein the heterocycle is optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, and the amino group is optionally substituted with an amino protecting group, a halogen group, a nitro group, a cyano group, or a protecting group. An amino-substituted heterocyclic group, wherein the nitrogen atom or amino moiety of the heterocyclic portion is optionally replaced by a protecting group or one or more C1-C6 alkyl groups. Heterocyclic amino groups, wherein the nitrogen atom or amino moiety of the heterocyclic portion is optionally substituted with a protecting group or a C1-C6 alkyl group. The carbamoyl group may be optionally protected by a carbamoyl group or substituted with a C1-C6 alkyl group. Morpholin-1-yl, or Piperidin-1-yl; Or, X 1 and X 2 Together with the attached atoms, they form unsubstituted or substituted dioxane heterocycles, such as... Where k is 1 or 2; X 3 It is a C1-C6 alkyl group; X 4 For H, -(CH2) q -CH3、-(CHR n ) q -CH3, C3-C8 carbon cyclic groups, -O-(CH2) q -CH3, aryl-CH3, -(CH2) q -arylene-CH3, -arylene-(CH2) q -CH3, -(CH2) q -(C3-C8 carbocyclic)-CH3, -(C3-C8 carbocyclic)-(CH2) q -CH3, C3-C8 heterocyclic groups, -(CH2) q -(C3-C8 heterocyclic group)-CH3, -(C3-C8 heterocyclic group)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3, or -(CH2CH2O) q C(O)NR n (CH2) q -CH3; where each R n It can be independently H, C1-C6 alkyl, C3-C8 carbocyclic, phenyl, or benzyl; and each q can be independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** represents the connection point; y is 0, 1, or 2; Y represents O, S, or CR. 2 R 3 , where R 2 and R 3 Each is independently H or C1-C6 alkyl; s and t are each independently 0, 1 or 2, but not both simultaneously 0. The drug conjugate of any one of claims 1-3, 5-7, 15-20 or the compound of any one of claims 11-13, 15-20, wherein D is Where X 1 and X 2 Each can be independently a C1-C6 alkyl, halogen, or -OH; Or, X 1 X 2 Together with the attached atoms, they form unsubstituted or substituted dioxane heterocycles, such as... Where k is 1 or 2; ** is the connection point. The drug conjugate of any one of claims 1-7, 15-20, 22-26 or the compound of any one of claims 8-26, wherein p is 4, 8, 12, 16 or 20. A pharmaceutical composition comprising the pharmaceutical conjugate of any one of claims 1-7, 15-20, 22-27, and a pharmaceutically acceptable carrier, excipient, and / or excipient. Use of the drug conjugate of any one of claims 1-7, 15-20, 22-27 or the pharmaceutical composition of claim 28 in the preparation of a medicament for treating a disease. Use of the compound of any one of claims 8-27 in the preparation of drug conjugates; or use of the compound of any one of claims 8-27 in the preparation of antibody-drug conjugates of any one of claims 1-7, 15-20, and 22-27.

Images