Drug conjugate, and preparation method therefor and use thereof

Abstract

The present invention belongs to the field of biomedicine. Provided are a drug conjugate, such as an antibody-drug conjugate, and the use thereof. 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 cancers, autoimmune diseases, inflammatory diseases, or infectious diseases.

Description

Drug conjugates, methods of making and uses thereof TECHNICAL FIELD

[0001] The present application relates to drug conjugates such as antibody drug conjugates, linkers and intermediates for making the drug conjugates, and uses of the drug conjugates. BACKGROUND

[0002] An antibody drug conjugate is composed of three parts: an antibody, a cytotoxic molecule and a linker connecting the two. Each of the three has a unique function: the antibody needs to specifically bind to tumor cells, the cytotoxic molecule needs to have sufficient activity and broad spectrum to tumor cells, and the linker needs to have unique functionality, stable in blood circulation, and effectively release the cytotoxic molecule after reaching tumor cells (Chari, R.V. (2008), Acc Chem Res 41(1): 98-107). A reasonable construction of the three can 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).

[0003] There is still a need in the art to develop drug conjugates that can improve plasma stability while maintaining lysosomal cleavage of the ADC linker. SUMMARY

[0004] 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:

[0005] Abu-[M-W-AA-FF-D] p Formula I

[0006] wherein Abu is an antigen binding unit, D is a drug;

[0007] AA is a short peptide consisting of 2-5 amino acid residues;

[0008] FF is a spacer unit, or a self-immolative spacer unit;

[0009] M is an antigen binding unit covalent binding moiety;

[0010] W is an optional extension unit;

[0011] p is 1-10.

[0012] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, lie-Cit, Trp-Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, lie-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly.

[0013] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly.

[0014] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly.

[0015] In one or more embodiments, the AA is Glu-Arg.

[0016] In one or more embodiments, the AA is Gly-Gly-Phe-Gly.

[0017] In one or more embodiments, the AA is Gly-Gly-Glu-Gly.

[0018] In one or more embodiments, the FF is a self-immolative spacer unit.

[0019] In one or more embodiments, the FF is a non-self-immolative spacer unit.

[0020] In one or more embodiments, the FF is wherein each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4; wherein * connects AA, and ** connects D.

[0021] In one or more embodiments, W is (as ), wherein * is attached to M, ** is attached to AA, and 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.

[0022] In one or more embodiments, M is wherein * is attached to Abu, ** is attached to W, and R is selected from the group consisting of: -(CH2) r -, m ) r -, C3-C8carbocyclyl, -O-(CH2) r -, arylene, -(CH2) r -arylene-, r -(CH2) r -(C3-C8carbocyclyl)-, r -(C3-C8heterocyclyl)-, r -(C3-C8heterocyclyl)-, r -(CH2) r C(O)NR m (CH2) r -, r -(CH2CH2O) r -, r C(O)NR m (CH2CH2O) r -, r C(O)NR m (CH2CH2O) r -CH2-, r C(O)NR m (CH2CH2O) r -, r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -; wherein each R mindependently H, C1-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0023] In one or more embodiments, R is -(CH2) r - In one or more embodiments, R is -(CH2) r - and r is 1-5. In one or more embodiments, R is -(CH2)3-. In one or more embodiments, R is -CH2-.

[0024] In one or more embodiments, M is wherein * is attached to Abu and ** is attached to W.

[0025] In one or more embodiments, M is wherein * is attached to Abu and ** is attached to W.

[0026] One or more embodiments of the present application provide a drug conjugate having a structure represented by Formula I-1 or Formula I-1', or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0027] wherein

[0028] Abu is an antigen binding unit;

[0029] 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;

[0030] AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly;

[0031] FF is wherein each R F is independently C1-C6alkyl, C1-C6alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4; wherein * is attached to AA and ** is attached to D;

[0032] M is wherein * is attached to Abu and R is selected from the group consisting of: -(CH2) r -, -(CHR m ) r, C3-C8carbocyclyl, -0-(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;

[0033] 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;

[0034] p is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0035] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly.

[0036] In one or more embodiments, AA is Glu-Arg.

[0037] In one or more embodiments, AA is Gly-Gly-Phe-Gly.

[0038] In one or more embodiments, AA is Gly-Gly-Glu-Gly.

[0039] In one or more embodiments, FF is where * connects AA and ** connects D, where each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, 3, or 4.

[0040] In one or more embodiments, halogen is F.

[0041] In one or more embodiments, each R F is independently -CH3, F, -NO2, or -OCH3.

[0042] In one or more embodiments, z is 0.

[0043] In one or more embodiments, z is 1 or 2.

[0044] In one or more embodiments, FF is where * connects AA and ** connects D.

[0045] In one or more embodiments, FF is where * connects AA and ** connects D.

[0046] In one or more embodiments, FF is where * connects AA and ** connects D.

[0047] In one or more embodiments, FF is where * connects AA and ** connects D.

[0048] In one or more embodiments, FF is where * connects AA and ** connects D.

[0049] In one or more embodiments, AA-FF is where ** connects D.

[0050] In one or more embodiments, AA-FF is wherein ** is connected to D.

[0051] In one or more embodiments, AA-FF is wherein ** is connected to D.

[0052] In one or more embodiments, AA-FF is wherein ** is connected to D.

[0053] In one or more embodiments, AA-FF is wherein ** is connected to D.

[0054] In one or more embodiments, AA-FF is wherein ** is connected to D.

[0055] In one or more embodiments, R is -(CH2) r -. In one or more embodiments, R is -(CH2) r -, r is 1-5. In one or more embodiments, R is -(CH2)3-. In one or more embodiments, R is -CH2-.

[0056] In one or more embodiments, M is wherein * is connected to Abu.

[0057] In one or more embodiments, M is wherein * is connected to Abu.

[0058] In one or more embodiments, M is wherein * is connected to Abu. In one or more embodiments, R is -(CH2) r -.

[0059] In one or more embodiments, M is wherein * is connected to Abu.

[0060] In one or more embodiments, the drug conjugate has a structure according to Formula I-2, Formula I-2A, Formula I-7, or Formula I-7A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0061] wherein,

[0062] Abu is an antigen binding unit;

[0063] R 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 , 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;

[0064] 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 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 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0067] In one or more embodiments, the drug conjugate has a structure according to Formula I-3, Formula I-3A, Formula I-8, or Formula I-8A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0068] wherein,

[0069] Abuis an antigen binding unit;

[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 autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases;

[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 1-10, 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, a DNA topoisomerase inhibitor, or a DNA damage response (DDR) 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), beta-glucuronyl-monomethyl auristatin E (MMAU), monomethyl auristatin F (MMAF), auristatin F (AF). The structure of MMAU is as follows:

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

[0077] In one or more embodiments, D is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-amino camptothecin, 9-nitro camptothecin, 10-hydroxy camptothecin, 9-chloro-10-hydroxy camptothecin, the camptothecin derivative SN-38, 22-hydroxytriptolide, topotecan, lurtotecan, belotecan, exatecan, exatecan derivatives (such as 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-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- hydroxy camptothecin, topotecan, belotecan, irinotecan, 22-hydroxytriptolide, exatecan, exatecan derivatives (such as 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 phen(anti)benzodiazepine (PBD) dimer, doxorubicin, methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, or a phenylbutyric acid mustard derivative.

[0080] In one or more embodiments, D is a DNA damage response (DDR) inhibitor, for example, a PARP inhibitor, an ATR inhibitor, an ATM inhibitor, a DNA-PK inhibitor, a CHK1 inhibitor, a WEE1 inhibitor, a POLQ inhibitor, a CDK12 inhibitor, a USP1 inhibitor, a PKMYT1 inhibitor, or a RAD51 inhibitor. In one or more embodiments, the PARP inhibitor is Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, Senaparib, Veliparib, A-966492, Saruparib, Venadaparib, Stenoparib, Nesuparib, Mefuparib, or an analog thereof. In one or more embodiments, the ATR inhibitor is Berzosertib, Gartisertib, Ceralasertib, Elimusertib, Camonsertib, Tuvusertib, or an analog thereof. In one or more embodiments, the CHK1 inhibitor is Prexasertib, Rabusertib, AZD7762, MK-8776, CHIR-124, PF-477736, CCT245737 (SRA737), GDC-0575 (ARRY-575), LY2880070, or an analog thereof. In one or more embodiments, the WEE1 inhibitor is Azenosertib (ZN-c3), Adavosertib, Debio 0123, IMP7068, or an analog thereof.

[0081] In one or more embodiments, D is wherein

[0082] X 1 and X 2 each independently is:

[0083] H,

[0084] hydroxyl,

[0085] C1-C6 alkyl,

[0086] C1-C6 alkyl substituted by one or more hydroxyl, halogen, nitro, or cyano,

[0087] C2-C6 alkenyl,

[0088] C2-C6 alkynyl,

[0089] C1-C6 alkoxy,

[0090] C1-C6alkyl,

[0091] halogen,

[0092] nitro,

[0093] cyano,

[0094] mercapto,

[0095] alkylthio,

[0096] amino, amino substituted by an amino protecting group, C1-C6aminoalkyl optionally substituted in the amino moiety by an amino protecting group or C1-C6alkyl,

[0097] C1-C6aminoalkyl optionally substituted in the amino moiety by an amino protecting group or C1-C6alkyl,

[0098] C1-C6alkyl attached to a heterocycle optionally substituted by one or more C1-C6alkyl, C1-C6alkoxy, amino, halogen, nitro or cyano,

[0099] C1-C6alkylamino attached to a heterocycle optionally substituted by C1-C6alkyl, C1-C6alkoxy, said amino optionally substituted by an amino protecting group, halogen, nitro, cyano or a protecting group,

[0100] heterocyclyl substituted by amino optionally substituted in the nitrogen atom of the heterocycle moiety or in the amino moiety by a protecting group or one or more C1-C6alkyl,

[0101] heterocyclyl substituted by amino optionally substituted in the nitrogen atom of the heterocycle moiety or in the amino moiety by a protecting group or one or more C1-C6alkyl,

[0102] carbamoyl optionally substituted by a carbamoyl protecting group or C1-C6alkyl,

[0103] morpholin-1-yl, or

[0104] piperidin-1-yl;

[0105] 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;

[0106] X 3 is C1-C6alkyl;

[0107] X 4 is H, -(CH2) q -CH3, -(CHR n ) q-CH3, C3-C8carbocyclyl, -0-(CH2) q -CH3, arylene-CH3, -(CH2) q -arylene-CH3, -arylene-(CH2) q -CH3, -(CH2) q -(C3-C8carbocyclyl)-CH3, -(C3-C8carbocyclyl)-(CH2) q -CH3, C3-C8heterocyclyl, -(CH2) q -(C3-C8heterocyclyl)-CH3, -(C3-C8heterocyclyl)-(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-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0108] ** is the point of attachment;

[0109] y is 0, 1, or 2;

[0110] Y is O, S, or CR 1 R 2 , wherein R 1 and R 2 are each independently H or C1-C6alkyl;

[0111] s and t are each independently 0, 1 or 2, but not both 0.

[0112] In one or more embodiments, X 4 is H or C1-C6 alkyl.

[0113] In one or more embodiments, the heterocycle is azetidine, ethylenediamine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole or pyridine.

[0114] In one or more embodiments, the amino protecting group is formyl, acetyl, trityl, t-butoxycarbonyl, benzyl or p-methoxybenzyloxycarbonyl.

[0115] 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 dioxole, such as wherein k is 1 or 2; ** is the point of attachment.

[0116] 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 dioxole, such as wherein k is 1 or 2; ** is the point of attachment.

[0117] In one or more embodiments, X 1 and X 2 are each -CH3.

[0118] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0119] In one or more embodiments, X 1 and X 2 are each F.

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

[0121] In one or more embodiments, X 1 and X 2each independently F or -CH3.

[0122] In one or more embodiments, X 1 is -CH3and X 2 is F.

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

[0124] In one or more embodiments, X 1 , X 2 together with the atom to which they are attached form wherein k is 2.

[0125] In one or more embodiments, the drug conjugate has a structure according to Formula I-4, Formula I-4A, Formula I-9, or Formula I-9A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0126] wherein,

[0127] Abu is an antigen binding unit;

[0128] 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;

[0129] p is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0130] In one or more embodiments, n is 16.

[0131] In one or more embodiments, Abu is an antigen binding unit containing a cysteine in the sequence and is linked to the rest of the drug conjugate (e.g., M of Formula I and Formula I-1) through the sulfur atom of the cysteine.

[0132] 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 rest of the drug conjugate (e.g., M of Formula I and Formula I-1) through the Fc region.

[0133] In one or more embodiments, the target bound by the Abu 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, CD25, CD30, FcRH2, NCA, MDP, IL20R alpha, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ral, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, FR alpha (FRa).

[0134] In one or more embodiments, the target bound by the Abu is HER2, TROP-2, CLDN18.2, B7H3, or FRa.

[0135] In one or more embodiments, the Abu is an antibody or antigen-binding fragment thereof, and the drug conjugate is an antibody drug conjugate.

[0136] In one or more embodiments, the Abu is an antibody or antigen-binding fragment thereof containing an Fc region. In one or more embodiments, the Fc region is a human IgGl, IgG2, IgG3, or IgG4 Fc region.

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

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

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

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

[0141] In one or more embodiments, Abu is an anti-HER2 antibody (such as Trastuzumab, Pertuzumab, Margetuximab, ZW25, etc.), an anti-Trop2 antibody, an anti-CLDN18.2 antibody, an anti-B7H3 antibody, or an anti-FRa antibody. In one or more embodiments, Abu is an anti-HER2 monoclonal antibody, an anti-Trop2 monoclonal antibody, an anti-CLDN18.2 monoclonal antibody, an anti-B7H3 monoclonal antibody, or an anti-FRa monoclonal antibody.

[0142] In one or more embodiments, the anti-HER2 antibody is an antibody disclosed in WO 2022 / 022526A1 (such as BAT0303F).

[0143] In one or more embodiments, Abu is Trastuzumab, Pertuzumab, Panitumumab, Nintedanib, Matuzumab, Rituximab, or Cetuximab.

[0144] In one or more embodiments, Abu is Trastuzumab.

[0145] In one or more embodiments, the antibody is an anti-Trop2 antibody that specifically acts on a Trop2 protein, wherein the Trop2 protein is trophoblast cell surface glycoprotein antigen 2. In one or more embodiments, the anti-Trop2 antibody is a fully human monoclonal antibody. In one or more embodiments, the anti-Trop2 antibody is a humanized monoclonal antibody.

[0146] In one or more embodiments, the anti-Trop2 antibody is an antibody disclosed in 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), US2013 / 0089872A (e.g., K5-70, K5-107, K5-116-2-1, T6-16, T5-86), US 5840854A (e.g., BR110), US2013 / 0122020A (e.g., 3E9, 6G11, 7E6, 15E2, 18B1), US2012 / 0237518A (e.g., 77220, KM4097, KM4590), WO 2022 / 253284 Al (e.g., hRS9 antibody).

[0147] In one or more embodiments, the anti-Trop2 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).

[0148] In one or more embodiments, the anti-Trop2 antibody is the anti-Trop2 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-Trop2 antibody disclosed by Ikeda et al. (2015, Biochem Biophys Res Comm 458:877-82) which recognizes a unique epitope on Trop2.

[0149] In one or more embodiments, the drug conjugate has a structure as shown in Formula I-5, Formula I-5A, Formula I-6, Formula I-6A, Formula IV-2, or Formula IV-2A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0150] wherein p is 7-9; or, p is about 7; or, p is about 8.

[0151] In another aspect, the present application provides 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:

[0152] M’-W-AA-FF’ Formula II

[0153] wherein,

[0154] AA is a short peptide consisting of 2-5 amino acid residues;

[0155] FF’ is a spacer unit, or a self-immolative spacer unit;

[0156] M’ is a precursor moiety of a covalently binding moiety to an antigen binding unit;

[0157] W is an optional extension unit.

[0158] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly.

[0159] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly.

[0160] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly.

[0161] In one or more embodiments, the AA is Glu-Arg.

[0162] In one or more embodiments, the AA is Gly-Gly-Phe-Gly.

[0163] In one or more embodiments, the AA is Gly-Gly-Glu-Gly.

[0164] In one or more embodiments, the FF' is wherein each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4; wherein * links AA.

[0165] In one or more embodiments, the W is (As ), wherein * is attached to M', ** is attached to AA, and 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.

[0166] In one or more embodiments, M' is R 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 -, 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.

[0167] In one or more embodiments, R is -(CH2) r- In one or more embodiments, R is -(CH2) r - r is 1-5. In one or more embodiments, R is -(CH2)3- In one or more embodiments, R is -CH2-.

[0168] In one or more embodiments, M' is

[0169] In one or more embodiments, M' is wherein * is attached to Abu. In one or more embodiments, R is -(CH2) r -.

[0170] In one or more embodiments, M' is wherein * is attached to Abu.

[0171] 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-1 or Formula II-1' or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof:

[0172] wherein,

[0173] AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly;

[0174] FF' is wherein each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4;

[0175] M' is R 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;

[0176] 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.

[0177] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly.

[0178] In one or more embodiments, AA is Glu-Arg.

[0179] In one or more embodiments, AA is Gly-Gly-Phe-Gly.

[0180] In one or more embodiments, AA is Gly-Gly-Glu-Gly.

[0181] In one or more embodiments, R is -(CH2) r -. In one or more embodiments, R is -(CH2) r-CH2-; r is 1-5. In one or more embodiments, R is -(CH2)3-. In one or more embodiments, R is -CH2-.

[0182] In one or more embodiments, M' is

[0183] In one or more embodiments, M' is wherein * is attached to Abu. In one or more embodiments, R is -(CH2) r -.

[0184] In one or more embodiments, M' is wherein * is attached to Abu.

[0185] In one or more embodiments, R F is F.

[0186] In one or more embodiments, z is 0.

[0187] In one or more embodiments, z is 1 or 2.

[0188] In one or more embodiments, FF' is wherein * is attached to AA.

[0189] In one or more embodiments, FF' is

[0190] In one or more embodiments, FF' is

[0191] In one or more embodiments, FF' is

[0192] In one or more embodiments, the compound has a structure according to Formula II-2, Formula II-2A, Formula II-3, Formula II-3A, Formula II-6, Formula II-6A, Formula II-7, or Formula II-7A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0193] wherein,

[0194] R is selected from the group consisting of: -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl, -O-(CH2)r -alkylene-, -(CH2) r -alkylene-, -(CH2) r -alkylene-, -(CH2) r -(C3-C8carbocyclyl)-, -(C3-C8carbocyclyl)-(CH2) r -alkylene-, -(CH2) r -(C3-C8heterocyclyl)-, -(C3-C8heterocyclyl)-(CH2) r -alkylene-, -(CH2) r C(O)NR m (CH2) r -alkylene-, -(CH2CH2O) r -alkylene-, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -alkylene-, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -alkylene-, -(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;

[0195] 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.

[0196] In one or more embodiments, the compound has the structure of Formula II-4, Formula II-4A, Formula II-5, Formula II-5A, Formula II-8, Formula II-8A, Formula II-9, or Formula II-9A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0197] 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. In one or more embodiments, n is 16.

[0198] In another aspect, the present application provides 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:

[0199] M'-W-AA-FF-D Formula III

[0200] wherein 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;

[0201] AA is a short peptide consisting of 2-5 amino acid residues;

[0202] FF is a spacer unit, or a self-immolative spacer unit;

[0203] M' is a precursor moiety of an antigen binding unit covalently bound moiety;

[0204] W is an optional extension unit.

[0205] In one or more embodiments, the AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly.

[0206] In one or more embodiments, the AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, and Glu-Gly-Gly-Phe-Gly.

[0207] In one or more embodiments, the AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, and Gly-Phe-Arg-Gly.

[0208] In one or more embodiments, the AA is Glu-Arg.

[0209] In one or more embodiments, the AA is Gly-Gly-Phe-Gly.

[0210] In one or more embodiments, the AA is Gly-Gly-Glu-Gly.

[0211] In one or more embodiments, FF is Among them, each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4; where * connects to AA and ** connects to D.

[0212] In one or more embodiments, W is (like ), where * connects M', ** connects AA, and 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.

[0213] In one or more embodiments, M' is Where * connects W, and R is 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-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.

[0214] In one or more embodiments, R is -(CH2) r - In one or more embodiments, R is -(CH2) r - and r is 1-5. In one or more embodiments, R is -(CH2)3-. In one or more embodiments, R is -CH2-.

[0215] In one or more embodiments, M' is wherein * is attached to W.

[0216] In one or more embodiments, M' is wherein * is attached to Abu. In one or more embodiments, R is -(CH2) r -.

[0217] In one or more embodiments, M' is wherein * is attached to Abu.

[0218] In one or more embodiments, the intermediate of the drug conjugate (such as an antibody drug conjugate) is a compound of Formula III-1 or Formula III-1', or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0219] 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;

[0220] AA is selected from the group consisting of Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly;

[0221] FF is wherein each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4; wherein * links AA, and ** links D;

[0222] M' is R is selected from the group consisting of: -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl, -O-(CH2) r -, arylene, -(CH2) r -, 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) rC(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;

[0223] 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.

[0224] In one or more embodiments, the AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, and Gly-Phe-Arg-Gly.

[0225] In one or more embodiments, AA is Glu-Arg.

[0226] In one or more implementations, AA is Gly-Gly-Phe-Gly.

[0227] In one or more embodiments, AA is Gly-Gly-Glu-Gly.

[0228] In one or more embodiments, R is -(CH2). r - In one or more embodiments, R is -(CH2). r -, r is 1-5. In one or more embodiments, R is -(CH2)3-. In one or more embodiments, R is -CH2-.

[0229] In one or more embodiments, M' is

[0230] In one or more embodiments, M' is Where * connects to Abu. In one or more embodiments, R is -(CH2). r -

[0231] In one or more embodiments, M' is wherein * is attached to Abu.

[0232] In one or more embodiments, the halogen is F.

[0233] In one or more embodiments, each R F is independently -CH3, F, -NO2, or -OCH3.

[0234] In one or more embodiments, z is 0.

[0235] In one or more embodiments, z is 1 or 2.

[0236] In one or more embodiments, FF is wherein * is attached to AA and ** is attached to D.

[0237] In one or more embodiments, FF is wherein * is attached to AA and ** is attached to D.

[0238] In one or more embodiments, FF is wherein * is attached to AA and ** is attached to D.

[0239] In one or more embodiments, FF is wherein * is attached to AA and ** is attached to D.

[0240] In one or more embodiments, FF is wherein * is attached to AA and ** is attached to D.

[0241] In one or more embodiments, AA-FF is wherein ** is attached to D.

[0242] In one or more embodiments, AA-FF is wherein ** is attached to D.

[0243] In one or more embodiments, AA-FF is wherein ** is attached to D.

[0244] In one or more embodiments, AA-FF is wherein ** is attached to D.

[0245] In one or more embodiments, AA-FF is wherein ** is attached to D.

[0246] In one or more embodiments, AA-FF is wherein ** is connected to D.

[0247] In one or more embodiments, the compound is a compound of Formula III-2, Formula III-2A, Formula III-7, or Formula III-7A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0248] wherein,

[0249] R 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 -, 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 mindependently H, C1-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0250] 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 an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease;

[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 is -(CH2) r - In one or more embodiments, R is -(CH2) r - and r is 1-5. In one or more embodiments, R is -(CH2) r - and r is 3.

[0253] In one or more embodiments, the compound is a compound of Formula III-3, Formula III-3A, Formula III-8, or Formula III-8A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0254] wherein,

[0255] 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 an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease;

[0256] 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.

[0257] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, a DNA topoisomerase inhibitor, or a DNA damage response (DDR) inhibitor.

[0258] In one or more embodiments, the tubulin inhibitor is selected from the group consisting of dolastatins, auristatins, maytansines.

[0259] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), beta-glucuronyl-monomethyl auristatin E (MMAU), monomethyl auristatin F (MMAF), auristatin F (AF). The structure of MMAU is as follows:

[0260] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, a pyrrolobenzodiazepine (PBD) derivative.

[0261] In one or more embodiments, D is a DNA topoisomerase inhibitor or salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-amino camptothecin, 9-nitro camptothecin, 10-hydroxy camptothecin, 9-chloro-10-hydroxy camptothecin, SN-38, a camptothecin derivative, 22-hydroxytriptolide, topotecan, lurtotecan, belotecan, exatecan, an exatecan derivative (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-beta-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.

[0262] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxy camptothecin, topotecan, belotecan, irinotecan, 22-hydroxytriptolide, exatecan, an exatecan derivative (e.g., Dxd), or a salt thereof.

[0263] In one or more embodiments, D is a Tubulysin, a taxane derivative, a leptomycine derivative, a CC-1065 and analogs thereof, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, doxorubicin, methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, or a phenylbutyric acid mustard derivative.

[0264] In one or more embodiments, D is a DNA damage response (DDR) inhibitor, for example a PARP inhibitor, an ATR inhibitor, an ATM inhibitor, a DNA-PK inhibitor, a CHK1 inhibitor, a WEE1 inhibitor, a POLQ inhibitor, a CDK12 inhibitor, a USP1 inhibitor, a PKMYT1 inhibitor, or a RAD51 inhibitor. In one or more embodiments, the PARP inhibitor is Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, Senaparib, Veliparib, A-966492, Saruparib, Venadaparib, Stenoparib, Nesuparib, Mefuparib, or analogs thereof. In one or more embodiments, the ATR inhibitor is Berzosertib, Gartisertib, Ceralasertib, Elimusertib, Camonsertib, Tuvusertib, or analogs thereof. In one or more embodiments, the CHK1 inhibitor is Prexasertib, Rabusertib, AZD7762, MK-8776, CHIR-124, PF-477736, CCT245737 (SRA737), GDC-0575 (ARRY-575), LY2880070, or analogs thereof. In one or more embodiments, the WEE1 inhibitor is Azenosertib (ZN-c3), Adavosertib, Debio 0123, IMP7068, or analogs thereof.

[0265] In one or more embodiments, D is

[0266] wherein

[0267] X 1 and X 2 each independently is:

[0268] H,

[0269] hydroxyl,

[0270] C1-C6 alkyl,

[0271] C1-C6alkyl substituted by one or more hydroxy, halogen, nitro or cyano,

[0272] C2-C6alkenyl,

[0273] C2-C6alkynyl,

[0274] C1-C6alkoxy,

[0275] C1-C6aminoalkoxy,

[0276] halogen,

[0277] nitro,

[0278] cyano,

[0279] mercapto,

[0280] alkylthio,

[0281] amino, amino substituted by an amino protecting group, C1-C6aminoalkyl optionally substituted in the amino moiety by an amino protecting group or C1-C6alkyl,

[0282] C1-C6aminoalkylamino optionally substituted in the amino moiety by an amino protecting group or C1-C6alkyl,

[0283] C1-C6alkyl attached to a heterocyclic ring, which is optionally substituted by one or more C1-C6alkyl, C1-C6alkoxy, amino, halogen, nitro or cyano,

[0284] C1-C6alkylamino attached to a heterocyclic ring, which is optionally substituted by C1-C6alkyl, C1-C6alkoxy, which amino is optionally substituted by an amino protecting group, halogen, nitro, cyano or a protecting group,

[0285] heterocyclyl substituted by amino, which is optionally substituted in the nitrogen atom of the heterocyclic moiety or in the amino moiety by a protecting group or one or more C1-C6alkyl,

[0286] heterocyclyl substituted by amino, which is optionally substituted in the nitrogen atom of the heterocyclic moiety or in the amino moiety by a protecting group or one or more C1-C6alkyl,

[0287] carbamoyl optionally substituted by a carbamoyl protecting group or C1-C6alkyl,

[0288] morpholin-1-yl, or

[0289] piperidin-1-yl;

[0290] 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;

[0291] X 3 is H, C1-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl;

[0292] X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8carbocyclyl, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -arylene-CH3, -arylene-(CH2) q -CH3, -(CH2) q -(C3-C8carbocyclyl)-CH3, -(C3-C8carbocyclyl)-(CH2) q -CH3, C3-C8heterocyclyl, -(CH2) q -(C3-C8heterocyclyl)-CH3, -(C3-C8heterocyclyl)-(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-C6alkyl, C3-C8carbocyclyl, phenyl, or benzyl; and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0293] ** is the point of attachment;

[0294] y is 0, 1 or 2;

[0295] Y is O, S or CR 1 R 2 wherein R 1 and R 2 are each independently H or C1-C6 alkyl;

[0296] s and t are each independently 0, 1 or 2, but not both 0.

[0297] In one or more embodiments, X 4 is H or C1-C6 alkyl.

[0298] In one or more embodiments, the heterocycle is azetidine, ethylenediazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole or pyridine.

[0299] In one or more embodiments, the amino protecting group is formyl, acetyl, trityl, t-butoxycarbonyl, benzyl or p-methoxybenzyloxycarbonyl.

[0300] 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 dioxole, such as wherein k is 1 or 2; ** is the point of attachment.

[0301] 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 dioxole, such as wherein k is 1 or 2; ** is the point of attachment.

[0302] In one or more embodiments, X 1 and X 2 are each -CH3.

[0303] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0304] In one or more embodiments, X 1 and X 2each F.

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

[0306] In one or more embodiments, X 1 and X 2 each independently is F or -CH3.

[0307] In one or more embodiments, X 1 is -CH3and X 2 is F.

[0308] In one or more embodiments, X 1 , X 2 together with the atoms to which they are attached form wherein k is 1.

[0309] In one or more embodiments, X 1 , X 2 together with the atoms to which they are attached form wherein k is 2.

[0310] In one or more embodiments, the compound is a compound of Formula III-4, Formula III-4A, Formula III-9, or Formula III-9A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0311] wherein

[0312] R 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 -, 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-C6 alkyl, C3-C8 carbocyclyl, phenyl, or benzyl; and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0313] 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.

[0314] In one or more embodiments, R is -(CH2) r - In one or more embodiments, R is -(CH2) r -, r is 1-5. In one or more embodiments, R is -(CH2) r -, r is 3.

[0315] In one or more embodiments, the compound is of Formula III-5, Formula III-5A, Formula III-10, or Formula III-10A:

[0316] wherein 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.

[0317] In one or more embodiments, n is 16.

[0318] In one or more embodiments, the compound has the structure of Formula III-6 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0319] In one or more embodiments, the compound is (54S,57S)-57-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[DE]pyrano[3',4':6,7]indolino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-5-guanidino-1-oxopentan-2-yl)carbamoyl)-54-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoic amide)-50,55-dioxa-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexaoxa-51,56-diazepane-60-oic acid, or a pharmaceutically acceptable salt or solvate thereof.

[0320] In one or more embodiments, the compound has the structure of Formula III-11 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0321] In another aspect, one or more embodiments of the present application 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 anticancer drugs. The pharmaceutical composition can be administered by any convenient route, for example, by infusion or bolus injection, absorbed through epithelial or mucocutaneous membranes (e.g., oral mucosa, rectal and intestinal membranes, etc.), and can be co-administered 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 patches to the skin), buccally, or via oral or nasal aerosol.

[0322] In one or more embodiments, the term "pharmaceutically acceptable carrier" refers generally to any type of nontoxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type, etc.

[0323] 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 petroleum, animal or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Antibacterial agents, such as benzyl alcohol or methyl parabens, antioxidants, chelating agents, and agents for the adjustment of tonicity can also be used as desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as glycerol. Oral formulations 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, the entire disclosure of which is hereby incorporated by reference into this application. 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 of preparation which can be used syringes, vials, etc. The formulations should be in dosages suitable for the chosen route of administration. The formulations can be packaged in ampules, in pre- filled syringes or in multi-dose vials made of glass or plastic.

[0324] In one or more 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 ampule or sachette indicating the precise quantity of active ingredient. Where the composition is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by subcutaneous injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0325] In one aspect, the present application provides the use of a drug conjugate in the manufacture of a medicament for the treatment of a disease, such as a cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

[0326] In one aspect, the present application provides uses of a drug conjugate in treating a disease, such as a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0327] In one aspect, the present application provides methods of treating a disease, such as a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease, comprising administering to a patient in need thereof an effective amount of a drug conjugate described herein.

[0328] 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.

[0329] In one or more embodiments, the disease is a cancer. In one or more embodiments, the drug conjugate is used in combination with other anti-cancer drugs.

[0330] In one aspect, one or more embodiments of the present application provide uses 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.

[0331] 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, piperizine, 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 a compound of the present application.

[0332] In one aspect, one or more embodiments of the present application provide methods of treating a disease, comprising administering to a patient an effective amount of a drug conjugate. An effective amount means that amount of an active pharmaceutical ingredient or pharmaceutical agent that results in a biological or pharmacological response of tissues, systems, animals, individuals, and humans that is being sought by a researcher, veterinarian, medical doctor, or other clinician, which includes treating a disease.

[0333] 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.

[0334] In one aspect, one or more embodiments of the present application provide a drug conjugate, such as an antibody drug conjugate, for use as a medicament.

[0335] In one aspect, one or more embodiments of the present application 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.

[0336] In one or more embodiments, the disease is 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, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glioma, hepatobiliary cancer, colorectal cancer, T-cell lymphoma, 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 bowel cancer, testicular embryonal carcinoma, placental choriocarcinoma, testicular cancer, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

[0337] In one aspect, one or more embodiments of the present application provide an article of manufacture comprising a drug conjugate or a pharmaceutical composition;

[0338] a container; and

[0339] a package insert, instructions, or a label indicating that the compound or composition is used for treating a disease, such as cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. BRIEF DESCRIPTION OF DRAWINGS

[0340] Figure 1 is the time course of enzymatic cleavage of 13 polypeptide substrates in human plasma (wherein the numbers on the vertical axis represent the average value of normalized fluorescence values);

[0341] Figures 2A to 2C are the results of the proliferation inhibition test of mAb1-ANK-VC, mAb1-ANK-ER, mAb1-ANK-GGFG and mAb1-ANK-EGCit and ADC1 on SK-BR-3 cells;

[0342] Figures 3A to 3C are the results of the proliferation inhibition test of mAb1-ANK-VC, mAb1-ANK-ER, mAb1-ANK-GGFG and mAb1-ANK-EGCit and ADC1 on MDA-MB-468 cells;

[0343] Figures 4A to 4C are the bystander killing effect of the metastatic supernatant of Her2+ cells SK-BR-3 (mAb1-ANK-VC, mAb1-ANK-ER, mAb1-ANK-GGFG and mAb1-ANK-EGCit and ADC1) on MDA-MB-468 cells;

[0344] Figures 5A to 5C are the bystander killing effect of the metastatic supernatant of Her2- cells MDA-MB-468 (mAb1-ANK-VC, mAb1-ANK-ER, mAb1-ANK-GGFG and mAb1-ANK-EGCit and ADC1) on MDA-MB-468 cells.

[0345] Figure 6 is the results of the in vitro cathepsin B enzyme cleavage efficiency test of 4 polypeptide substrates under different pH conditions;

[0346] Figure 7 is the results of the in vitro elastase enzyme cleavage efficiency test of 4 polypeptide substrates under different pH conditions;

[0347] Figures 8A to 8C are the results of the proliferation inhibition test of mAb1-ANK-GGEG, mAb1-ANK-GEFG, mAb1-ANK-EGGFG and mAb1-ANK-GGFG on SK-BR-3, N87, A549 cells. DETAILED DESCRIPTION

[0348] "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 an alkyl group having from 1 to 20 carbon atoms, for example, an alkyl group having 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.

[0349] "Carbocyclyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely 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 the group consisting of alkyl, halogen, haloalkyl, cyano, nitro, oxo, aryl, aralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl.

[0350] "Aryl" refers to an all-carbon monocyclic or fused-ring polycyclic ring system that is completely conjugated and generally has from 5 to 14 carbon atoms, for example, 6, 10, 12, 14 carbon atoms. The aryl group can be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxyl, carboxyl, aryl, aralkyl, amine, halogen, sulfonyl, sulfinyl, phosphonyl. Non-substituted aryl examples include, but are not limited to, phenyl, naphthyl, and anthracyl.

[0351] "Heterocyclyl" refers to a stable 3- to 14-membered aromatic or nonaromatic ring radical, which is either saturated, unsaturated, or aromatic, which consists of 2 to 8 (e.g., 2, 3, 4, 5, 6, 7, or 8) carbon atoms and from 1 to 6 (1, 2, 3, 4, 5, or 6) heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically indicated in the specification, the heterocyclyl radical 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 optionally be oxidized; the nitrogen atom can optionally be 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, octahydroindolizinyl, octahydroisoindolyl, 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, the heterocyclyl radical can be optionally substituted by one or more substituents selected from 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.

[0352] "Alkoxy" means a group of the formula -O-alkyl, wherein alkyl is alkyl as defined herein. Non- limiting examples of alkoxy include methoxy, ethoxy, n-propyloxy, 1-methylethoxy (isopropoxy), n-butyloxy, isobutyloxy, sec-butyloxy, t-butyloxy. The alkoxy group can be substituted or unsubstituted.

[0353] "Halo" means fluoro (F), chloro (CI), bromo (Br), or iodo (I). "Amino" means -NH2. "Cyano" means -CN. "Nitro" means -NO2. "Hydroxy" means -OH. "Carboxy" means -COOH. "Mercapto" means -SH. "Carbonyl" means C=O.

[0354] The term "linker" or "linker" refers to a chemical moiety or bond that links one end of an antibody binding unit to the other end of a drug, and can also be linked to other linkers before linking to a drug. The linker can include an antigen binding unit covalent binding moiety (M or M'), an extension unit (W), an amino acid unit (AA), and a spacer unit (FF).

[0355] As used herein, an "extension unit" is used to link the M or M' portion of a drug conjugate to an amino acid unit portion (AA). In one or more embodiments, the extension unit comprises a PEG unit (-(CH2CH20) n -). For example, a drug conjugate of the application can comprise a drug moiety linked to an amino acid unit portion (AA) via an extension unit (E) and a linker unit (L) as follows: (as ).

[0356] As used herein, a spacer unit can be "self-immolative" or "non-self-immolative." A "non-self-immolative spacer unit" is a unit in which part or all of the spacer unit remains bound to the drug moiety after enzymatic (e.g., proteolytic) cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Other combinations of peptide spacer regions that are susceptible to cleavage by sequence-specific enzymes are also contemplated. For example, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease will result in the release of the glycine-glycine-drug moiety from the remainder of the ADC. In one such embodiment, the glycine-glycine-drug moiety is then subjected to a separate hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

[0357] A "self-immolative spacer" allows for partial release of the drug moiety without the need for a separate hydrolysis step. In certain embodiments, the spacer of the linker comprises a p-aminobenzyl unit. In one such embodiment, the p-aminobenzyl alcohol is attached via an amide bond to an amino acid unit and creates a carbamate, methylcarbamate, or carbonate between the benzyl alcohol and the cytotoxic agent. See, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103. In one embodiment, the spacer is p-aminobenzyloxycarbonyl (PAB). Examples of self-immolative spacers also include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, e.g., US 2005 / 0256030 Al), such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetales. Spacers that cyclize upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc, 1972, 94, 5815); and 2-aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs substituted at the a-position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacers that can be used in ADCs.

[0358] An "antigen binding unit" can specifically bind to an antigen (e.g., a tumor antigen). A "tumor antigen" as described herein refers to a target substance expressed in a tumor cell. A tumor antigen can be used for the identification of a tumor cell, can be a potential indicator for a tumor therapy, or can be a target for a tumor therapy.

[0359] An "antigen binding unit" can be any of those known in the art, or those to become known, including peptides and non-peptides, such as antibodies (e.g., monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport molecules (transferrin), or other cell binding molecules or substances that can specifically bind to a target. These substances that bind to a target are antibodies, antibody fragments, cell specific ligands, or peptides, carbohydrates, natural or synthetic compounds, or pharmaceutically acceptable salts or solvates.

[0360] Many tumor antigens well known in the art and new tumor antigens can be identified by screening. Non-limiting examples of tumor antigens include: HER2, TROP-2, Nectin-4, B7H3, B7H4, CLDN18, BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD20, CD21, CD22, CD25, CD30, FcRH2, NCA, MDP, IL20R alpha, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ral, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, FR alpha.

[0361] Hormones and cytokines can be tumor antigens. Non-limiting examples include lymphokines, such as interleukin-2, interleukin-3, interleukin-4, interleukin-6, hormones, such as insulin, thyrotropin-releasing hormone, melanocyte-stimulating hormone, steroid hormones, such as androgens and estrogens; growth factors and colony-stimulating factors, such as epidermal growth factor, TGF-alpha, FGF, VEGF, G-CSF and GM-CSF (see Burgess, Immunology Today 5:155-158 (1984)), transferrin (see O'Keefe et al., J. Biol. Chem. 260:932-937 (1985)), and vitamins, such as folic acid.

[0362] “Polypeptide” refers to a molecule composed of two or more amino acid monomers linearly linked by amide bonds (also known as peptide bonds), and can include dipeptides, tripeptides, tetrapeptides, oligopeptides, and the like.

[0363] "Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as a- and β-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 (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (lie, 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), β-amino isobutyric acid (β-AiBA), citrulline (Cit), and β-alanine (β-Ala), among others.

[0364] It is understood by those skilled in the art that when an amino acid or polypeptide is referred to as a component of a molecule (e.g., 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 the moiety after its part group (e.g., one of the hydrogens of its amino group and / or the hydroxyl of its carboxyl group) is lost as it forms a covalent bond (e.g., an amide bond) with other parts of the molecule.

[0365] 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 that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are broadly grouped as follows: (1) acidic amino acids as aspartic acid, glutamic acid; (2) basic amino acids as lysine, arginine, histidine; (3) non-polar amino acids as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids as glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other families of amino acids include (i) serine and threonine, which belong to the aliphatic-hydroxyl family; (ii) asparagine and glutamine, which belong to the amide family; (iii) alanine, valine, leucine and isoleucine, which belong to the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which belong to 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.

[0366] 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 polypeptides outside of domains that form 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, or to disrupt other types of secondary structure characteristic of the parent sequence). Examples of secondary and tertiary structure of polypeptides recognized by the art 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).

[0367] The term "recombinant" with respect to a polypeptide or polynucleotide means a form of the polypeptide or polynucleotide that does not occur in nature, without limitation, embodiments can be generated by combining polynucleotides or polypeptides that normally do not occur together.

[0368] "Identity" or "identical" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Identity or similarity can be determined by comparing a position 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 identical or similar at that position. A degree of identity between sequences is a function of the number of matching or identical positions shared by the sequences. "At least 80% identical" is about 80% identical, about 81% identical, about 82% identical, about 83% identical, about 85% identical, about 86% identical, about 87% identical, about 88% identical, about 90% identical, about 91% identical, about 92% identical, about 94% identical, about 95% identical, about 98% identical, about 99% identical, or a range between any two of these values, inclusive, or any value therein. "At least 90% identical" is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or a range between any two of these values, inclusive, or any value therein.

[0369] "Antibody," "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 characteristic of an antigen binding. Antibodies and antigen binding fragments include, but are not limited to, a complementarity determining region (CDR) of a heavy 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. The number of amino acids for conservative amino acid substitutions of a 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 values) or any value therein. The number of amino acids for conservative amino acid substitutions of a heavy chain constant region, a light chain constant region, a heavy chain, or a 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 values) or any value therein.

[0370] The term "antigen binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a particular antigen.

[0371] The term "antibody" includes 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 refers to the type of constant domain or region in its heavy chains (e.g., IgG, IgM, IgA, IgG, or IgE). Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and the functional specificity conferred thereby is 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 through disulfide bonds in a "Y" configuration, with the light chain extending from the "Y" mouth and continuing around the heavy chain through the variable region.

[0372] Antibodies, antigen-binding fragments, or derivatives of the present disclosure include, but are not limited to, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab')2), single-chain antibodies (scFv).

[0373] Antibodies of the present disclosure can be derived from any animal, including but not limited to, fish, birds, and mammals. Preferably, the antibody is a human, murine, equine, rabbit, goat, camelid, llama, horse, or chicken antibody. In another embodiment, the variable region can be condricthoid in origin (e.g., from a shark).

[0374] 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 VK; the immunoglobulin l light chain variable region is Vl.

[0375] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in the sense of function. 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, placental transfer, Fc receptor binding, complement binding, etc. Conventionally, the numbering of the constant regions increases as they become more distal from the antigen-binding site or amino-terminal end 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.

[0376] The "heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment. 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 may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region may include a hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another embodiment, a portion of the heavy chain may include a chimeric hinge region partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0377] The “light chain constant region” comprises a portion of the amino acid sequence from the antibody light chain. Preferably, the light chain constant region contains at least one of a constant κ domain or a constant λ domain. A “light chain-heavy chain pair” refers to a set of light and heavy chains that can form dimers via disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0378] The term "antibody-drug conjugate" or "ADC" refers to one or more compounds linked to an antigen-binding unit (such as an antibody or its antigen-binding fragment), which may optionally be a therapeutic agent or a cytotoxic agent. In preferred embodiments, an ADC includes an antibody, a drug (e.g., a cytotoxic drug), and a connector capable of attaching or conjugating the drug to the antibody. Non-limiting examples of drugs that may be included in an ADC include mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclides, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine / threonine kinase inhibitors), and radiosensitizers.

[0379] The term "drug-antibody conjugation ratio" or "DAR" refers to the amount of drug (e.g., eczemab) attached to an antibody in an ADC. The DAR of an ADC can range from 1 to 10, but higher loadings (e.g., 20) are possible depending on the number of binding sites on the antibody. The term DAR may be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. In some embodiments, its 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, i.e., the average number of drugs bound to the antibody, or the average drug-antibody conjugation ratio, its value is selected from about 0 to about 10, or about 2 to about 8. In some embodiments, the drug-antibody conjugation ratio is about 7 to about 8. DAR values ​​may be represented herein as p.

[0380] The DAR value of ADC can be determined using ultraviolet-visible absorption spectroscopy (UV-Vis), high-performance liquid chromatography-hydrophobic chromatography (HPLC-HIC), high-performance liquid chromatography-reversed-phase chromatography (RP-HPLC), and liquid chromatography-mass spectrometry (LC-MS). These techniques are described in Ouyang, J. Methods Mol Biol, 2013, 1045: pp. 275-83.

[0381] Other chemical terms used in this article are used in accordance with the usual usage in the field, such as in The McGraw-Hill Dictionary of Chemical Terms (edited by Parker, S., McGraw-Hill, San Francisco (1985)).

[0382] All publications, patents, and patent applications cited in this document are incorporated herein by reference in their entirety for all purposes.

[0383] antibody portion

[0384] The antibody described in this invention can be any antibody suitable for preparing antibody-drug conjugates. It can have a complete antibody structure or include antibody fragments such as Fab, Fab', F(ab)'2, and Fv.

[0385] Antibodies can specifically bind to antigens, such as tumor-specific antigens. Tumor antigens can be used to identify tumor cells, serve as potential indicators for tumor treatment, or act as targets for tumor therapy. Therefore, the selection of specific antibodies mainly depends on the type of disease and the cells and tissues that are the target.

[0386] 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, TROP2, B7H3, FRalpha (FRα), Nectin-4, B7H4, CLDN18, BMPR1B, E16, STEAP1, O772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD22, CD25, FcRH2, NCA, MD P, IL20Rα, 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, etc.

[0387] In one or more embodiments, the antibody may be a humanized monoclonal antibody.

[0388] In one or more embodiments, the antibody is an anti-HER2 antibody that specifically targets human epidermal growth factor receptor 2, such as trastuzumab, pertuzumab, maggotuximab, dicetuzumab, zanidatamab (ZW25), anbenitamab (KN026), etc. The anti-HER2 antibody may be modified, such as by altering, adding, or deleting one or more amino acid sequences, to achieve a specific purpose, such as enhancing antibody-dependent cell-mediated cytotoxicity.

[0389] In one or more embodiments, the anti-HER2 antibody is trastuzumab.

[0390] The heavy and light chain sequences of trastuzumab are shown in the table below as SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0391] Antibodies can be prepared using conventional recombinant DNA techniques. Vectors and cell lines for antibody production can be selected, constructed, and cultured using techniques well-known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as *Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary information, are incorporated herein by reference.

[0392] In one or more embodiments, antibody-encoding DNA can be designed and synthesized according to the antibody amino acid sequence described herein using conventional methods, placed into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in a culture medium to produce monoclonal antibodies. In one or more embodiments, the antibody expression vector includes at least one promoter element, an antibody-encoding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters of long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as actin promoters. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, 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, mouse L cells, and CHO cells, etc.

[0393] In one or more embodiments, the inserted gene fragment must contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the aforementioned genes, and after culturing in a selective medium, successfully transfected cells grow in large numbers, producing the desired target protein. The antibody is then purified through one or more purification steps. Purification can be performed using conventional methods, such as first centrifuging the cell suspension and collecting 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.

[0394] In one or more embodiments of the present invention, the number of small molecule drugs bound to one antibody in an antibody-drug conjugate, i.e., the drug-binding number of the antibody, is called the drug-antibody conjugate ratio (DAR). In some embodiments, its 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, i.e., the average drug-binding number of the antibody, or the average drug-antibody conjugate ratio, its value is selected from about 0 to 10, or 2 to 8. In one or more embodiments, the drug-antibody conjugate ratio is about 7 to about 8.

[0395] Synthesis method

[0396] This invention also provides drug conjugates, such as antibody-drug conjugates, and methods for preparing intermediates. The drug conjugates, such as antibody-drug conjugates, and intermediates of this invention can be prepared using known formulations and methods. In some embodiments, the preparation methods are as follows.

[0397] Preparation method

[0398] Step 1: Compound of Formula 1-1 and compound of general formula 1-1' react under alkaline conditions to give compound of Formula 1-2;

[0399] Step 2: Remove the amino protecting group W from compound 1-2 1 Compounds of formula 1-3 were obtained;

[0400] Step 3: Compounds of Formula 1-3 and Formula 1-4 react under alkaline conditions to obtain compound 1-5;

[0401] Step 4: Compounds of Formulas 1-5 and Formula AA-FF 1 The reaction, in the presence of a condensing agent and under alkaline conditions, yields compounds of formulas 1-6;

[0402] Step 5: Compounds of Formula 1-6 and di(p-nitrobenzene) carbonate react under alkaline conditions to give compounds of Formula 1-7.

[0403] Step 1: The compound of Formula 1 and the compound of General Formula 1' react under alkaline conditions to give the compound of Formula 2;

[0404] Step 2: Remove the amino protecting group W from compound formula 2 1 Compound of formula 3 was obtained;

[0405] Step 3: Compounds 3 and 4 react under alkaline conditions to obtain compound 5;

[0406] Step 4: Compound of Formula 5 and Compound AA-FF 1 The compound of formula 6 was obtained by reacting in the presence of a condensing agent under alkaline conditions.

[0407] Step 5: The compound of Formula 6 and di(p-nitrobenzene) carbonate react under alkaline conditions to give the compound of Formula 7.

[0408] in,

[0409] W 1 It is an amino protecting group, for example, 9-fluorenemethyloxycarbonyl; W 2 It is the alcohol-terminal group of the active ester of a carboxylic acid, such as a succinimide group. Pentafluorophenyl or benzotriazole

[0410] M' is R is 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.

[0411] When M' is In this case, the fifth step above can synthesize compounds of formula A1-7 or A7.

[0412] Wherein, n, AA, and R are as described in Equation II-1, and FF 1 for FF2 is The asterisk (*) is connected to the abbreviation AA.

[0413] The alkaline conditions described above can be provided by reagents, including organic and inorganic bases. The organic bases include, but are 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 bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

[0414] The condensing agent mentioned above can be selected from N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole- N,N,N',N'-Tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazole-1-yl-oxytripyrrolylphosphonium hexafluorophosphate.

[0415] Preparation method

[0416] Compounds of general formulas 1-7 react with D under basic conditions in the presence of a condensing agent to give compounds of general formulas 1-8.

[0417] Compound of general formula 7 reacts with D under basic conditions in the presence of a condensing agent to give compound of general formula 8.

[0418] Where M' is R is 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.

[0419] In one or more embodiments, compounds of general formula A1-7 and D are reacted under basic conditions in the presence of a condensing agent to give compounds of general formula A1-8.

[0420] In one or more embodiments, compound of general formula A7 and D react in the presence of a condensing agent under basic conditions to give compound of general formula A8.

[0421] Wherein, n, AA, R, FF, and D are as described in Equation III-1, and FF 2 for The asterisk (*) is connected to the abbreviation AA.

[0422] The alkaline conditions described above can be provided by reagents, including organic and inorganic bases. The organic bases include, but are 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 bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

[0423] The condensing agent mentioned above can be selected from N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole- N,N,N',N'-Tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazole-1-yl-oxytripyrrolylphosphonium hexafluorophosphate.

[0424] Preparation method

[0425] Compounds of general formulas 1-8 and Abu were coupled under weakly acidic conditions to give compounds of general formulas 1-9.

[0426] Compound of general formula 8 and Abu were coupled under weakly acidic conditions to give compound of general formula 9.

[0427] Where M is Where * connects to Abu, and R is 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.

[0428] In one or more embodiments, compounds of general formula A1-8 and Abu are coupled under weakly acidic conditions to obtain compounds of general formula A1-9.

[0429] In one or more embodiments, compound of general formula A8 and Abu are coupled under weakly acidic conditions to obtain compound of general formula A9.

[0430] Wherein, n, AA, R, FF, D, and Abu are as described in Equation I-1.

[0431] The aforementioned weakly acidic conditions can be provided by reagents, including organic and inorganic acids. The organic acids include, but are 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, and propionic acid. The inorganic acids include, but are 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, and sodium bisulfite.

[0432] Drug conjugates can be purified using conventional methods, such as preparative high-performance liquid chromatography (prep-HPLC).

[0433] Example

[0434] In the following embodiments and throughout the patent application, the following abbreviations have the following meanings. Unless otherwise defined, these terms have their generally accepted meanings.

[0435] AMC = 7-amino-4-methylcoumarin

[0436] DCM = dichloromethane

[0437] DCC = N,N'-dicyclohexylcarbodiimide

[0438] DIPEA = N,N-diisopropylethylamine

[0439] DMF = N,N-dimethylformamide

[0440] TBSCl = tert-butyldimethylchlorosilane

[0441] DMAP = 4-Dimethylaminopyridine

[0442] EEDQ = 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

[0443] THF = Tetrahydrofuran

[0444] HATU = N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-ylurea hexaphosphate)

[0445] (PNP)₂CO = Di(p-nitrobenzene) carbonate

[0446] MTBE = Methyl tert-butyl ether

[0447] Fmoc-Glu(OtBu)-OH = fluorenemethyloxycarbonyl-L-glutamic acid 5-tert-butyl ester

[0448] HOBT = 1-Hydroxybenzotriazole

[0449] EDCI = 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0450] Me = methyl

[0451] MeOH = Methanol

[0452] TCEP = Tricarboxyethylphosphine

[0453] DTNB = 5,5'-dithiobis(2-nitrobenzoic acid)

[0454] NAC = N-acetylcysteine

[0455] LC-MS = Liquid Chromatography-Mass Spectrometry

[0456] TBAF = Tetrabutylammonium fluoride

[0457] Example 1. Structure, molecular weight, and purity of peptide substrates

[0458] The enzymatic stability of the amino acid-containing peptides in Table 1 at the small molecule level was evaluated. Thirteen Z-AA-AMC peptide substrates were synthesized, and their structures, molecular weights, and purities are shown in Table 1. AA represents dipeptides, tripeptides, or tetrapeptides, Z represents 2-aminobenzamide, and AMC (7-amino-4-methylcoumarin) is a fluorescent group used to measure peptidase activity; peptidase cleaves the amide bond of AMC, causing an increase in fluorescence. In Z-GFRG-A-AMC, amino acid residue R is in the native configuration L, and Z-GFRG-B-AMC is in configuration D (the amino group on amino acid residue R is racemic).

[0459] The peptide substrate can be synthesized using conventional methods. AMC is condensed with the first amino acid at the C-terminus of the short peptide structure via a mixed anhydride reaction.

[0460] Table 1. Structure, molecular weight, and purity of peptide substrates

[0461] Example 2. Stability of polypeptide substrates in human plasma

[0462] The peptide substrates were dissolved in DMSO to a concentration of 8 mM. Each peptide substrate was added to a microplate at a concentration of 3 μl / well. 100 μl of human plasma was added to each well, with 3 replicates. The plates were incubated at 37°C. Readings were taken at different time points using a microplate reader. The microplate reader was set to the detection parameters of AMC fluorophore (excitation wavelength 355 nm, emission wavelength 460 nm).

[0463] The results are shown in Figure 1 and Table 2. Z-FR-AMC, Z-ERR-AMC, and Z-GFRG-B-AMC showed high enzymatic cleavage efficiency but were unstable in human plasma.

[0464] Z-EGC-AMC, Z-GGFG-AMC, Z-ER-AMC, Z-KR-AMC, Z-VC-AMC, Z-DGC-AMC, and Z-GFRG-A-AMC exhibit good stability in human plasma.

[0465] Table 2. Enzyme digestion data of peptide substrates in human plasma Note: The numbers 0h, 6h, 24h, 49h, and 72h represent the normalized average fluorescence values; the number kcat represents the enzyme digestion efficiency after transformation.

[0466] Example 3. Stability of peptide substrates in PBS buffer

[0467] Dissolve the peptide substrates in DMSO to a concentration of 8 mM. Add 3 μl of each peptide substrate to each well of the microplate. Add 100 μl of PBS buffer (pH 7.2) to each well, repeat in triplicate, and incubate at 37°C. Take readings at different time points using a microplate reader. Set the microplate reader to the detection parameters of AMC fluorophore (excitation wavelength 355 nm, emission wavelength 460 nm).

[0468] As shown in Table 3, the three tripeptide substrates Z-FRN-AMC, Z-RRN-AMC, and Z-DGC-AMC are unstable and undergo self-cleavage in PBS buffer.

[0469] Table 3. Stability of peptide substrates in PBS buffer at pH 7.2 Note: The numbers 0h, 7h, 25h, 72h, 96h, and 121h represent the normalized average fluorescence values; the number kcat represents the enzyme digestion efficiency after transformation.

[0470] Example 4. Stability of peptide substrate in DAMI cell homogenate

[0471] DAMI cells (human megakaryocyte leukemia cells) (purchased from Beina Biotechnology) were resuspended in 0.32M sucrose solution, repeatedly pipetted with a 1ml syringe, centrifuged at 12000rpm for 1min, and the cell homogenate supernatant was collected.

[0472] The peptide substrates were dissolved in DMSO to a concentration of 8 mM. The following solutions were added to each microplate: 3 μl of each peptide substrate / well; 10 μl of DAMI cell homogenate supernatant / well; 11.6 μl of 1 M Na₂HPO₄ / well; 4.2 μl of 1 M citric acid / well; 1 μl of 0.1 M EDTA / well; 2 μl of 5 M NaCl / well; 1 μl of 0.5 M DTT / well; and 67.2 μl of pure water / well, for a final volume of 100 μl / well. The pH was set to 5.6. Triple replicates were used. The plates were incubated at 37°C. Readings were taken at different time points using a microplate reader set to the AMC fluorophore detection parameters (excitation wavelength 355 nm, emission wavelength 460 nm).

[0473] The peptide substrates were dissolved in DMSO to a concentration of 8 mM. The following solutions were added to each microplate: 3 μl of each peptide substrate per well; 10 μl of DAMI cell homogenate supernatant per well; 17.39 μl of 1 M Na₂HPO₄ per well; 1.31 μl of 1 M citric acid per well; 1 μl of 0.1 M EDTA per well; 2 μl of 5 M NaCl per well; 1 μl of 0.5 M DTT per well; and 64.3 μl of pure water per well, for a final volume of 100 μl per well. The pH was 7.2. Three replicates were performed, and the plates were incubated at 37°C. Readings were taken at different time points using a microplate reader. The detection parameters for AMC fluorophore were recorded (excitation wavelength 355 nm, emission wavelength 460 nm).

[0474] Table 4. Enzyme digestion data of peptide substrates in DAMI cell homogenate (pH 5.6) Note: The numbers 0h, 1h, 2h, 3h, 4h, and 5h represent the normalized average fluorescence values; the number kcat represents the enzyme digestion efficiency after transformation.

[0475] Table 5. Enzyme digestion data of peptide substrates in DAMI cell homogenate (pH 7.2) Note: The numbers 0h, 1h, 2h, 3h, 4h, and 5h represent the normalized average fluorescence values; the number kcat represents the enzyme digestion efficiency after transformation.

[0476] The results are shown in Tables 4 and 5. Z-ER-AMC exhibits completely different properties in DAMI cell homogenates under different pH conditions.

[0477] Example 5. Stability of peptide substrates in SK-BR-3, HCT-15, or MC-38 cell homogenates

[0478] SK-BR-3 cells (human breast adenocarcinoma cells), HCT-15 cells (human colorectal adenocarcinoma cells), and MC-38 cells (mouse colorectal cancer cells) (Beina Biotechnology) were resuspended in 0.32M sucrose solution, repeatedly pipetted with a 1ml syringe, centrifuged at 12000rpm for 1min, and the cell homogenate supernatant of each cell line was collected.

[0479] Dissolve the peptide substrates to 8 mM concentration using DMSO. Add the following to each microplate: 3 μl of each peptide substrate / well; 10 μl of SK-BR-3, HCT-15, or MC-38 cell homogenate supernatant / well; 9.36 μl of 1 M Na2HPO4 / well; 5.33 μl of 1 M citric acid / well; 1 μl of 0.1 M EDTA / well; 2 μl of 5 M NaCl / well; 1 μl of 0.5 M DTT / well; and 68.31 μl of pure water / well, for a final volume of 100 μl / well. Set the pH to 4.6. Apply three replicates to each well and incubate at 37°C. Read the values ​​at different time points using a microplate reader. Measure the AMC fluorophore detection parameters (excitation wavelength 355 nm, emission wavelength 460 nm).

[0480] Table 6. Enzymatic digestion data of peptide substrates in SK-BR-3 cell homogenates (pH 4.6) Note: The numbers 0min, 30min, 60min, 120min, 180min, and 210min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0481] Table 7. Enzyme digestion data of peptide substrates in HCT-15 cell homogenates (pH 4.6) Note: The numbers 0min, 30min, 60min, 120min, 150min, and 180min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0482] Table 8. Enzymatic digestion data of peptide substrates in MC-38 cell homogenates (pH 4.6) Note: The numbers 0min, 30min, 60min, 120min, 150min, and 180min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0483] The results are shown in Tables 6-8. Z-ER-AMC, Z-KR-AMC, and Z-RR-AMC showed high enzyme digestion efficiency in cell homogenates.

[0484] Example 6. Stability of polypeptide substrates in cathepsin B, cathepsin L, and cathepsin S

[0485] Dissolve the peptide substrates to 8 mM concentration using DMSO. Add the following to each microplate: 3 μl of each peptide substrate / well; 2 μl of cathepsin B (400 μg / ml, ACRO), cathepsin L (655 μg / ml, ACRO), or cathepsin S (1730 μg / ml, ACRO); 9.36 μl of 1 M Na2HPO4 / well; 5.33 μl of 1 M citrate / well; 1 μl of 0.1 M EDTA / well; 2 μl of 5 M NaCl / well; 0.5 M... DTT, 1 μl / well; pure water, 76.31 μl / well, final volume 100 μl / well, pH 4.6, 3 replicates, incubated at 37°C, readings were taken at different time points using a microplate reader, with the microplate reader parameters set to the detection parameters for AMC fluorescein (excitation wavelength 355 nm, emission wavelength 460 nm).

[0486] Table 9. Enzyme digestion data of polypeptide substrates in cathepsin B (pH 4.6) Note: The numbers 0min, 5min, 15min, 60min, 90min, 120min, and 150min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0487] Table 10. Enzyme digestion data of polypeptide substrates in cathepsin L (pH 4.6) Note: The numbers 0min, 5min, 15min, 30min, 60min, 120min, and 150min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0488] Table 11. Enzyme digestion data of polypeptide substrates in cathepsin S (pH 4.6) Note: The numbers 0min, 5min, 15min, 30min, 60min, 90min, 120min, and 150min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0489] The results are shown in Tables 9-11. Cathepsin B, cathepsin L, and cathepsin S showed high cleavage efficiency for the peptide substrates of Z-ER-AMC, Z-RR-AMC, Z-EGC-AMC, Z-GGFG-AMC, and Z-VC-AMC.

[0490] Example 7. Stability of polypeptide substrates in elastase

[0491] The peptide substrates were dissolved in DMSO to a concentration of 8 mM. The following solutions were added to each microplate: 3 μl of each peptide substrate / well; 10 μl of elastase (0.8 mg / ml, Sangon Biotech); 9.36 μl of 1 M Na₂HPO₄ / well; 5.33 μl of 1 M citric acid / well; 1 μl of 0.1 M EDTA / well; 2 μl of 5 M NaCl / well; 1 μl of 0.5 M DTT / well; and 68.31 μl of pure water / well, for a final volume of 100 μl / well. The pH was 4.6. Three replicates were prepared, and the plates were incubated at 37°C. Readings were taken at different time points using a microplate reader set to the AMC fluorophore detection parameters (excitation wavelength 355 nm, emission wavelength 460 nm).

[0492] Table 12. Enzymatic digestion data of polypeptide substrates in elastase (pH 4.6) Note: The numbers 0min, 5min, 15min, 30min, 60min, 90min, 120min, and 150min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0493] The peptide substrates were dissolved in DMSO to a concentration of 8 mM. The following solutions were added to each microplate: 3 μl of each peptide substrate / well; 10 μl of elastase (0.8 mg / ml, Sangon Biotech); 17.4 μl of 1 M Na₂HPO₄ / well; 1.3 μl of 1 M citric acid / well; 1 μl of 0.1 M EDTA / well; 2 μl of 5 M NaCl / well; 1 μl of 0.5 M DTT / well; and 64.3 μl of pure water, for a final volume of 100 μl / well. The pH was set to 7.2, with three replicates. The plates were incubated at 37°C. Readings were taken at different time points using a microplate reader, set to the AMC fluorophore detection parameters (excitation wavelength 355 nm, emission wavelength 460 nm).

[0494] Table 13. Enzymatic digestion data of polypeptide substrates in elastase (pH 7.2) Note: The numbers 0min, 5min, 15min, 30min, 60min, 90min, 120min, and 150min represent the normalized average fluorescence values; the number in kcat represents the enzyme digestion efficiency after transformation.

[0495] The following examples relate to the synthesis of antibody-drug conjugates and their intermediates.

[0496] Example 8. Synthesis of ANK-A4 and ANK-A5

[0497] The synthesis routes for ANK-A4 and ANK-A5 are as follows:

[0498] Synthesis operation:

[0499] 1) Synthesis of pentafluorophenyl ester (ANK-A1) of 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl acetylic acid.

[0500] Add 8.23 ​​g (0.045 mol) of pentafluorophenol to a flask, add 200 mL of dichloromethane (DCM), add 10.0 g (0.037 mol) of 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylic acid, stir, and add 15.38 g (0.075 mol) of N,N'-dicyclohexylcarbodiimide (DCC). React at room temperature. Monitor by TLC. After the reaction is complete, filter and wash with a small amount of DCM. Concentrate the solution to dryness, add 80 mL of n-heptane and slurry. Filter and wash with a small amount of n-heptane. Collect the filter cake, dry it, and obtain a white powder ANK-A1 (16.78 g, 100%).

[0501] 2) Synthesis of ANK-A2.

[0502] Add (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (7.596 g, 34.80 mmol) to a flask, add DCM (500 mL), stir, add N,N-diisopropylethylamine (DIPEA) (8.996 g, 69.60 mmol), and cool to approximately 0°C under nitrogen protection. Dissolve 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecanoic acid-N-succinimide ester (m-PEG15-NHS) (20.0 g, 23.20 mmol) in DCM (100 mL) and add it dropwise to the reaction solution. After the addition is complete, remove the cooling bath and react overnight at room temperature. Take a sample. LC-MS monitoring shows the PEG fragment reaction is complete. After the reaction is terminated, concentrate the DCM. Add ethyl acetate (200 mL), cool to approximately 0°C, add Na₂CO₃ (10%, 250 mL) solution, and extract and separate. Wash the organic phase once more with Na₂CO₃ (10%, 250 mL) solution. Combine the aqueous phases, cool to approximately 0°C, adjust the pH to 3-4 with dilute hydrochloric acid, add DCM (200 mL * 3), and extract. Confirm extraction complete by TLC. Concentrate to obtain an oily substance ANK-A2 (24.18 g, 100%).

[0503] 3) Synthesis of ANK-A3.

[0504] Add ANK-A2 (24.18 g, 23.20 mmol) to a flask, add DCM (120 mL), cool to approximately 0 °C under nitrogen protection, and add HCl / dioxane (75 mL, 301.6 mmol) dropwise. After the addition is complete, remove the cold bath and react overnight at room temperature. Take a sample. Monitor the reaction centrally by LC-MS. After the reaction is terminated, concentrate the DCM. Add n-heptane (100 mL), stir for 10 minutes, let stand, discard the n-heptane phase, and retain the oily substance in the flask. Dry to obtain the oily substance ANK-A3 (24.43 g, crude product).

[0505] 4) Synthesis of ANK-A4.

[0506] Add ANK-A3 (24.43 g, 23.20 mmol) to a flask, add DCM (125 mL), add DIPEA (8.995 g, 69.6 mmol), and add ANK-A1 (12.59 g, 29.0 mmol) under nitrogen protection. React at room temperature for 2-3 hours. Monitor the reaction centrally by LC-MS until complete. Add water (150 mL), cool to approximately 0°C, adjust pH to 3-4 with dilute hydrochloric acid, and extract. Extract the aqueous phase with DCM (200 mL * 3), and confirm complete extraction by TLC. Concentrate the extract, purify the crude product by column chromatography, and collect the product spot to obtain the oily substance ANK-A4 (26.22 g, 100%).

[0507] 5) Synthesis of 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline (ANK-A5).

[0508] Add p-aminobenzyl alcohol (70 g, 0.568 mol, 1.0 eq) to a flask, add N,N-dimethylformamide (DMF) (700 mL), stir, add tert-butyldimethylchlorosilane (TBSCl) (102.8 g, 0.682 mol), add 4-dimethylaminopyridine (DMAP) (7.0 g, 0.0568 mol), cool to about 0 °C under nitrogen protection, add DIPEA (147.1 g, 1.137 mol) dropwise, remove the cold bath after addition, and react overnight at room temperature. Monitor by TLC until the reaction is complete. Add the reaction solution to ice water (3 L) and stir for half an hour. Extract with ethyl acetate (1 L). Wash the organic phase once with saturated brine (1 L), dry, concentrate, and purify the crude product by column chromatography. Collect the product spot to obtain an oily substance ANK-A5 (121.8 g, 90.3%).

[0509] Example 9. Synthesis of ANK-VC

[0510] The synthesis roadmap for ANK-VC is as follows:

[0511] Synthesis operation

[0512] 1) Synthesis of ANK-VC-1.

[0513] Under nitrogen protection at 0-5℃, 2.25 g of N2-fluorenylmethoxycarbonyl-L-2,4-diaminobutyric acid was dissolved in 20 mL of DMF. 5 g of 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecanoic acid-N-succinimide ester and 10 mL of DMF were added. 1.9 mL of DIPEA was added dropwise while maintaining the temperature at 0-5℃. After 1 h of addition, the reaction was stirred at room temperature for 4 h. After the reaction was completed, DMF was removed under reduced pressure, and column chromatography (using dichloromethane and methanol as eluents) yielded 6.50 g of pure ANK-VC-1 as a pale yellow oily liquid.

[0514] 2) Synthesis of ANK-VC-2.

[0515] Under nitrogen protection at room temperature, 11 g of (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidovalerate and 5.5 g of p-aminobenzyl alcohol were dissolved in 2 L of dichloromethane and 1 L of methanol. With mechanical stirring, 17 g of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) was added in portions, and the reaction was carried out for 15 h in the dark. After the reaction was complete, the solvent was removed under reduced pressure to obtain a paste-like solid, which was transferred to a 3 L container. 2 L of methyl tert-butyl ether was added, and the mixture was stirred for 5 h. The mixture was filtered, dried under vacuum, and subjected to column chromatography (using dichloromethane and methanol as eluents) to obtain 11.9 g of a grayish-white solid, ANK-VC-2.

[0516] 3) Synthesis of ANK-VC-3.

[0517] Under nitrogen protection at room temperature, 90 mL of acetonitrile was added to 11.9 g of ANK-VC-2, and 18 mL of piperidine was added dropwise with stirring. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 h. After the reaction was complete, the solvent and piperidine were removed by vacuum distillation to obtain a white solid. 900 mL of methyl tert-butyl ether was added and the mixture was stirred. The solid was dried under vacuum and subjected to column chromatography (with dichloromethane and methanol as eluents) to obtain 7.5 g of white solid ANK-VC-3.

[0518] 4) Synthesis of ANK-VC-4.

[0519] Under a nitrogen atmosphere at 0℃, 6.55 g of ANK-VC-1 was dissolved in 46.5 mL of tetrahydrofuran (THF). 5.29 g of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-ylurea hexaphosphate (HATU)) and 0.76 mL of N,N-diisopropylethylamine (DIPEA) were added in portions. After stirring for 30 min, 6 mL of a DMF solution containing 1.76 g of ANK-VC-3 was added, and the reaction was maintained at low temperature for 1.5 h. After the reaction was complete, 200 mL of methyl tert-butyl ether was added and the mixture was stirred. The filtered solid was then slurried with 150 mL of a methanol / DMF mixture (8:1) as solvent, followed by 8-10 times the volume of methyl tert-butyl ether. The solvent was removed by filtration and vacuum distillation. Column chromatography (using dichloromethane and methanol as eluents) yielded solid ANK-VC-4.

[0520] 5) Synthesis of ANK-VC-5.

[0521] Under nitrogen protection at room temperature, 5.4 g of ANK-VC-4 was mixed with 190 mL of acetonitrile as solvent, and 8.1 mL of piperidine was added. The mixture was stirred for 3-4 h. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The solution was dissolved in DCM / MeOH by stirring, and then 500 mL of methyl tert-butyl ether was slowly added. The mixture was stirred for 1-2 h, filtered, and the crude product was obtained. Column chromatography (using dichloromethane and methanol as eluents) yielded a pale yellow solid, ANK-VC-5.

[0522] 6) Synthesis of ANK-VC-6.

[0523] Under a nitrogen atmosphere at 0-5℃, 0.68 g of 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acetylate pentafluorophenyl ester (ANK-A1) was dissolved in 20 mL of DMF and stirred until dissolved. Then, 1.31 g of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) and 0.57 mL of N,N-diisopropylethylamine were added. After stirring for 30 min, 2.82 g of ANK-VC-5 was added, and the reaction was carried out at low temperature for 2-3 h. After the reaction was complete, 160 mL of methyl tert-butyl ether was added, and the mixture was stirred at low temperature for 1 h. After filtration and drying, the crude product was subjected to column chromatography (using dichloromethane and methanol as eluents) to obtain a pale yellow solid, ANK-VC-6.

[0524] 7) Synthesis of ANK-VC-7.

[0525] 1.6 g of ANK-VC-6 was dissolved in 16 mL of DMF. Under a nitrogen atmosphere at 0 °C, 1.7 g of di(p-nitrobenzene) carbonate ((PNP)₂CO) was added and dissolved. Then, 0.11 mL of N,N-diisopropylethylamine (DIPEA) was added, and the reaction was maintained at low temperature for 4 h. After the reaction was completed, 80 mL of methyl tert-butyl ether was added dropwise at low temperature, and the mixture was stirred for 2 h. The mixture was then filtered, the filter cake was dried, and the crude product was subjected to column chromatography (with dichloromethane and methanol as eluents) to obtain a white solid, ANK-VC-7.

[0526] 8) Synthesis of ANK-VC.

[0527] Add 0.6g of ANK-VC-7 and 5ml of N,N-dimethylformamide to reaction flask R1, stir under nitrogen protection, and cool to 0-5℃. Simultaneously, add 0.18g of eczemab mesylate (CAS:169869-90-3) and 5ml of N,N-dimethylformamide to another reaction flask R2, and add 0.2mL of N,N-diisopropylethylamine (DIPEA) dropwise at 0-5℃, stirring until completely dissolved. Add the solution from reaction flask R2 dropwise to reaction flask R1, then wash reaction flask R2 with 2ml of N,N-dimethylformamide, and add the washings back to reaction flask R1. Weigh 90 mg of 1-hydroxybenzotriazole and add it to reaction flask R1. Stir at 0-5℃ for 10 min, then raise to room temperature and stir for 3 h. After the reaction is complete, add 80 mL of methyl tert-butyl ether dropwise at low temperature, slurry for 2 h, filter, and purify the crude product by preparative high performance liquid chromatography. Freeze-dry to obtain a white powder ANK-VC. LC-MS: [1 / 2M+H]+=970.

[0528] Example 10. Synthesis of ANK-ER

[0529] The synthesis route of ANK-ER is as follows:

[0530] Synthesis operation:

[0531] 1) Synthesis of ANK-ER-1

[0532] Add 70 g (0.108 mol) of Fmoc-Pbf-L-arginine to a flask, add DMF (350 mL), stir, add ANK-A5 (28.2 g, 0.119 mol), add HATU (49.19 g, 0.129 mol), and dropwise add DIPEA (20.86 g, 0.162 mol). After the addition is complete, react at room temperature for 2-3 hours, taking a sample for monitoring, indicating the reaction of the starting materials is complete. Add the reaction solution to ice water (2 L) and stir for half an hour. Filter and wash with water. Collect the solid, dissolve it in DCM (1 L), and separate the water. Wash the organic phase once with saturated brine (1 L). Dry and concentrate to obtain an orange oily substance ANK-ER-1 (117 g, crude product).

[0533] 2) Synthesis of ANK-ER-2

[0534] Add 117 g of crude ANK-ER-1 (0.1078 mol) to a flask, add 1 L of DCM, stir, add 78.9 g of diethylamine (1.078 mol), and stir overnight at room temperature. The reaction was monitored by LC-MS until complete. Concentrate to dryness, dilute with 50 mL of methyl tert-butyl ether (MTBE), and then add dropwise to 2 L of petroleum ether. A solid precipitates; after stirring for 1 hour, filter and wash with petroleum ether. Collect the solid and air dry. A gel-like compound, ANK-ER-2, is obtained.

[0535] 3) Synthesis of ANK-ER-3

[0536] ANK-ER-2 (5.0 g), fluorenemethyloxycarbonyl-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu(OtBu)-OH) (3.6 g), DMF (50 mL), and DIPEA (1.5 g) were added sequentially to the reaction flask, followed by HATU (3.5 g). The reaction mixture was stirred at room temperature (22 °C) for 4 h. After the reaction was completed, the reaction mixture was poured into ice water (400 mL), and then extracted twice with dichloromethane. The organic phases were combined, and saturated sodium chloride was added. After stirring until homogeneous, the organic phase was separated by standing. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain an oily substance, which was used directly in the next reaction without purification.

[0537] Dichloromethane (100 ml) was added to the crude product, followed by ethylenediamine (5.6 g). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction mixture was distilled under reduced pressure at 45 °C to remove most of the solvent, yielding a semi-oily substance. 3.6 g of ANK-ER-3 was obtained by column chromatography.

[0538] 4) Synthesis of ANK-ER-4

[0539] ANK-ER-3 (3.6 g), tetrabutylammonium fluoride (3.4 g), and THF (50 mL) were added sequentially to the reaction flask, and the reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, most of the solvent was removed by vacuum distillation to obtain a semi-oily substance. 2.3 g of ANK-ER-4 was obtained by column chromatography, with a yield of 74%.

[0540] 5) Synthesis of ANK-ER-5

[0541] ANK-A4 (3.0 g), ANK-ER-4 (2.3 g), DMF (30 mL), and DIPEA (1.5 g) were added sequentially to the reaction flask, followed by HATU (1.13 g). The reaction mixture was stirred at room temperature for 4 h. After the reaction was completed, the mixture was poured into ice water (400 mL), and then extracted twice with dichloromethane. The organic phases were combined, and saturated sodium chloride was added. After stirring and allowing to stand, the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, and after evaporating the solvent, an oily substance was obtained. This oily substance was purified by column chromatography to obtain 2.1 g of ANK-ER-5, with a yield of 43%.

[0542] 6) Synthesis of ANK-ER-6.

[0543] 0.52 g of ANK-ER-5 was dissolved in 16 mL of DMF. Under a nitrogen atmosphere at 0 °C, 0.43 g of di(p-nitrobenzene) carbonate ((PNP)₂CO) was added and dissolved. Then, 0.03 mL of N,N-diisopropylethylamine (DIPEA) was added, and the reaction was maintained at low temperature for 4 h. After the reaction was completed, 30 mL of methyl tert-butyl ether was added dropwise at low temperature, and the mixture was stirred for 2 h. The mixture was then filtered, the filter cake was dried, and the crude product was subjected to column chromatography (with dichloromethane and methanol as eluents) to obtain a white solid, ANK-ER-6.

[0544] 7) Synthesis of ANK-ER.

[0545] Add 0.57 g of ANK-ER-6 and 5 mL of N,N-dimethylformamide to reaction flask R1, stir under nitrogen protection, and cool to 0-5 °C. Simultaneously, add 0.19 g of eczemab mesylate (CAS: 169869-90-3) and 5 mL of N,N-dimethylformamide to another reaction flask R2, and add 0.11 mL of N,N-diisopropylethylamine (DIPEA) dropwise at 0-5 °C, stirring until completely dissolved. Add the solution from reaction flask R2 dropwise to reaction flask R1, then wash reaction flask R2 with 2 mL of N,N-dimethylformamide, and add the washings back to reaction flask R1. Weigh 100 mg of 1-hydroxybenzotriazole and add it to the reaction flask R1. Stir at 0-5℃ for 10 min, then raise to room temperature and stir for 3 h. After the reaction is complete, add 80 mL of methyl tert-butyl ether (MTBE) dropwise at low temperature, slurry for 2 h, filter, dry the filter cake, and use it directly for the next reaction without purification.

[0546] Under nitrogen protection at 0℃, the crude product was placed in a mixed solution of trifluoroacetic acid:triisopropylsilane:water (trifluoroacetic acid:triisopropylsilane:water = 95:3:2 volume ratio) and stirred for 3 h. After the reaction was completed, 90 mL of methyl tert-butyl ether was added dropwise, and the mixture was stirred for 1 h. The mixture was then filtered, the filter cake was dried, and the crude product was purified by preparative high-performance liquid chromatography (HPLC). The purified product was then lyophilized to obtain a white powder, ANK-ER. LC-MS: [1 / 2M+H]+ = 984.

[0547] Example 11. Synthesis of ANK-EGCit

[0548] The synthetic route for ANK-EGCit is as follows:

[0549] Synthesis operation:

[0550] 1) Synthesis of ANK-EGCit-1.

[0551] N-(9-fluorenylmethoxycarbonyl)-L-citrulline (35.0 g, 0.10 mol) was added to a flask, followed by DMF (350 mL). The mixture was stirred, and then ANK-A5 (26.3 g, 0.11 mol) and 1-hydroxybenzotriazole (HOBT) (15.0 g, 0.11 mol) were added. Under nitrogen protection, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (21.2 g, 0.11 mol) was added, and the reaction was carried out at room temperature for 2 hours. TLC was used to monitor the reaction until the reactants had reacted completely. The reaction solution was added to ice water (2000 mL), and a solid precipitated. The mixture was stirred for half an hour. The mixture was filtered, the filter cake was washed with water, the solid was collected, dried, and the crude product ANK-EGCit-1 was used directly for the next reaction.

[0552] 2) Synthesis of ANK-EGCit-2.

[0553] Add the crude ANK-EGCit-1 (60g) from the previous step to a flask, add DCM (600mL), and add diethylamine (71.1g, 0.10mol). React overnight at room temperature. Monitor the reaction by TLC until the starting material has reacted completely. Concentrate the solution to dryness, add MTBE (150mL), stir for half an hour, filter, wash the filter cake with a small amount of MTBE, and dry to obtain ANK-EGCit-2 (41.0g, crude product).

[0554] 3) Synthesis of ANK-EGCit-3.

[0555] Add 40.0 g crude ANK-EGCit-2 (0.1 mol) to a flask, add DMF (450 mL), stir to dissolve, add Fmoc-glycine (33.2 g, 0.11 mol), add HATU (46.3 g, 0.015 mol), and add DIPEA (19.7 g, 0.15 mol) dropwise under nitrogen protection. After the addition is complete, react at room temperature for 1.5 h. Take a sample for monitoring; once the reaction is complete, add the reaction solution to ice water (2000 mL) and stir for half an hour. Filter and wash with water. Collect the solid, dry to obtain ANK-EGCit-3 (73 g, crude).

[0556] 4) Synthesis of ANK-EGCit-4.

[0557] Add 73.0 g crude ANK-EGCit-3 (1.0 eq) to a flask, add 800 mL DCM, and add diethylamine (76.5 g, 1.05 mol). React overnight at room temperature. Monitor the reaction by TLC until the starting material has reacted completely. Concentrate the solution to dryness, add 150 mL MTBE dropwise, and stir for 30 min. Repeat twice. Filter, and wash with a small amount of MTBE. Collect the solid, dissolve it in DCM, mix with silica gel, and purify by column chromatography to obtain 25.4 g ANK-EGCit-4 (55.9% yield in four steps).

[0558] 5) Synthesis of ANK-EGCit-5.

[0559] Add ANK-EGCit-4 (15.4 g, 34.2 mmol) to a flask, add DMF (150 mL), stir to dissolve, add Fmoc-Glu(OtBu)-OH (16.0 g, 37.6 mmol), add HATU (15.5 g, 41.0 mmol), cool to 0℃ under nitrogen protection, add DIPEA (6.6 g, 51.7 mmol) dropwise, and react at room temperature for 1.5 hours after the addition is complete. Take a sample for monitoring, and the reaction is complete. Pour the reaction solution into 800 mL of ice water, stir for 30 min, filter, dry under vacuum, and dry overnight at 50℃ with forced air to obtain 33.1 g of crude product.

[0560] LC-MS analysis showed that most of ANK-EGCit-5 was converted to ANK-EGCit-6, accounting for approximately 80%. The crude product was dissolved in DCM and purified by column chromatography to obtain ANK-EGCit-6 (21.5 g, two-step yield approximately 83%).

[0561] 6) Synthesis of ANK-EGCit-7.

[0562] ANK-EGCit-6 (21.5 g, 28.9 mmol) was added to a flask, followed by DCM (200 mL) and diethylamine (3.90 g, 0.29 mol). The mixture was reacted overnight at room temperature. TLC was used to monitor the reaction until the starting material was fully reacted. The solution was concentrated to dryness, and MTBE (60 mL) was added and stirred for half an hour. The mixture was filtered, and this process was repeated twice. The filter cake was dissolved in DCM, mixed, and purified by column chromatography to obtain ANK-EGCit-7 (11.2 g, 74.3%).

[0563] 7) Synthesis of ANK-EGCit-8.

[0564] Add 3.0 g (2.7 mmol) of ANK-A4 to a flask, add 30 mL of DMF, stir to dissolve, add 1.7 g (3.2 mmol) of ANK-EGCit-7, add 1.1 g (3.0 mmol) of HATU, cool to 0 °C under nitrogen protection, and add 1.0 g (8.1 mmol) of DIPEA dropwise. After the addition is complete, react at room temperature for 2 hours. Take a sample for control, and the reaction of the starting material ANK-A4 is complete. Add the reaction solution to ice water (200 mL), and extract multiple times with DCM / MeOH (20:1, 100 mL) until TLC confirms that there is no product in the water. Concentrate at 45 °C to remove DMF, and purify the crude product by column chromatography to obtain a white foamy solid ANK-EGCit-8 (3.1 g, yield 71.1%).

[0565] 8) Synthesis of ANK-EGCit-9.

[0566] 0.52 g of ANK-EGCit-8 was dissolved in 16 mL of DMF. Under a nitrogen atmosphere at 0 °C, 0.50 g of di(p-nitrobenzene) carbonate ((PNP)₂CO) was added and dissolved. Then, 0.03 mL of N,N-diisopropylethylamine (DIPEA) was added, and the reaction was maintained at low temperature for 4 h. After the reaction was completed, 10 mL of methyl tert-butyl ether was added dropwise at low temperature, and the mixture was stirred for 2 h. The mixture was then filtered, the filter cake was dried, and the crude product was subjected to column chromatography (with dichloromethane and methanol as eluents) to obtain a white solid, ANK-EGCit-9.

[0567] 9) Synthesis of ANK-EGCit.

[0568] Add 0.57 g of ANK-EGCit-9 and 5 mL of N,N-dimethylformamide to reaction flask R1, stir under nitrogen protection, and cool to 0-5 °C. Simultaneously, add 0.19 g of eczemab mesylate (CAS: 169869-90-3) and 5 mL of N,N-dimethylformamide to another reaction flask R2, and add 0.11 mL of N,N-diisopropylethylamine (DIPEA) dropwise at 0-5 °C, stirring until completely dissolved. Add the solution from reaction flask R2 dropwise to reaction flask R1, then wash reaction flask R2 with 2 mL of N,N-dimethylformamide, and add the washings back to reaction flask R1. Weigh out 90 mg of 1-hydroxybenzotriazole and add it to reaction flask R1. Stir at 0-5℃ for 10 min, then raise to room temperature and stir for 3 h. After the reaction is complete, add 80 mL of methyl tert-butyl ether dropwise at low temperature, slurry for 2 h, filter, dry the filter cake, and use it directly in the next reaction without purification.

[0569] Under nitrogen protection at 0℃, the crude product was placed in a mixed solution of trifluoroacetic acid:triisopropylsilane:water (trifluoroacetic acid:triisopropylsilane:water = 95:3:2 volume ratio) and stirred for 3 h. After the reaction was completed, 90 mL of methyl tert-butyl ether was added dropwise, and the mixture was stirred for 1 h. The mixture was then filtered, the filter cake was dried, and the crude product was purified by preparative high-performance liquid chromatography (HPLC). The purified product was then lyophilized to obtain a white powder, ANK-EGCit. LC-MS: [1 / 2M+H]+ = 10¹³.

[0570] Example 12. Synthesis of ANK-GGFG

[0571] The synthesis route for ANK-GGFG is as follows:

[0572] Synthesis operation

[0573] 1) Synthesis of ANK-GGFG-2.

[0574] In a reaction flask, (((9H-fluorene-9-yl)methoxy)carboxyl)glycylglycyl-L-phenylalanylglycine (5.5 g), ANK-A5 (2.8 g), DMF (55 mL), and DIPEA (1.9 g) were added sequentially, followed by HATU (4.5 g). The reaction mixture was stirred at room temperature (22 °C) for 4 h. After the reaction was completed, the mixture was poured into ice water (400 mL), and a large amount of solid precipitated out. After filtering out the solid, n-butanol (50 mL) was added, and the mixture was heated to 60 °C until the solid completely dissolved. Then, ethyl acetate (150 mL) and saturated sodium chloride (200 mL) were added, stirred until homogeneous, and allowed to stand to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate, and after evaporating the solvent, an oily substance, ANK-GGFG-1, was obtained and used directly in the next reaction without purification.

[0575] 100 mL of tetrahydrofuran (THF) was added to the crude product, followed by 3 mL of ethylenediamine. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was distilled under reduced pressure at 45 °C to remove most of the solvent (but not to dryness). Then, 500 mL of n-heptane was added, and the mixture was stirred at room temperature for 2 hours. The resulting solid was filtered off, and 100 mL of methyl ether was added. The mixture was stirred for 30 minutes. The solid was filtered off and dried to obtain ANK-GGFG-2 (3.5 g, two-step yield: 64.5%). The obtained sample was used directly in the next reaction step.

[0576] 2) Synthesis of ANK-GGFG-3.

[0577] ANK-A4 (2.1 g), ANK-GGFG-2 (1.56 g), HATU (1.44 g), and DMF (40 mL) were added sequentially to the reaction flask, followed by DIPEA (0.73 g). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, water (460 mL) was added, and the mixture was stirred at room temperature for 2 hours, resulting in a viscous precipitate at the bottom of the reaction flask. The supernatant was discarded, and the viscous precipitate was purified by column chromatography to obtain ANK-GGFG-3 (2.7 g, yield 87%).

[0578] 3) Synthesis of ANK-GGFG-4.

[0579] ANK-GGFG-3 (2.6 g), glacial acetic acid (30 mL), tetrahydrofuran (10 mL), and water (10 mL) were added sequentially to a reaction flask and stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation at 60 °C to obtain an oily substance, which was then purified by column chromatography to obtain ANK-GGFG-4 (2.1 g, yield 87%).

[0580] 4) Synthesis of ANK-GGFG-5.

[0581] 0.66 g of ANK-GGFG-4 was dissolved in 6 mL of DMF. Under a nitrogen atmosphere at 0 °C, 0.52 g of di(p-nitrobenzene) carbonate ((PNP)₂CO) was added and dissolved. Then, 0.07 mL of N,N-diisopropylethylamine (DIPEA) was added, and the reaction was maintained at low temperature for 4 h. After the reaction was completed, 40 mL of methyl tert-butyl ether was added dropwise at low temperature, and the mixture was stirred for 2 h. The mixture was then filtered, the filter cake was dried, and the crude product was subjected to column chromatography (with dichloromethane and methanol as eluents) to obtain a white solid, ANK-GGFG-5.

[0582] 5) Synthesis of ANK-GFG.

[0583] Add 0.73g of ANK-GGFG-5 and 5mL of N,N-dimethylformamide to reaction flask R1, stir under nitrogen protection, and cool to 0-5℃. Simultaneously, add 0.23g of eczemab mesylate (CAS:169869-90-3) and 5mL of N,N-dimethylformamide to another reaction flask R2, and add 0.29mL of N,N-diisopropylethylamine (DIPEA) dropwise at 0-5℃, stirring until completely dissolved. Add the solution from reaction flask R2 dropwise to reaction flask R1, then wash reaction flask R2 with 2mL of N,N-dimethylformamide, and add the washings back to reaction flask R1. Weigh 100 mg of 1-hydroxybenzotriazole and add it to reaction flask R1. Stir at 0-5℃ for 10 min, then raise to room temperature and stir for 3 h. After the reaction is complete, add 80 mL of methyl tert-butyl ether dropwise at low temperature, slurry for 2 h, filter, dry the filter cake, and purify the crude product by preparative high performance liquid chromatography. Freeze-dry to obtain a white powder ANK-GGFG. LC-MS: [1 / 2M+H]+=1000.

[0584] Example 13. Preparation of ADC

[0585] 1. Reduction reaction

[0586] 100 mM EDTA was first added to antibody mAb1 (trastuzumab) to a final concentration of 2 mM. Based on the amount of antibody, 4 eq of tricarboxyethylphosphine (TCEP) was added, and the pH was adjusted to 7.5-8.0 with 1 M Tris-HCl. The reaction was carried out at 25°C with shaking for 2 hours. Excess TCEP was removed by ultrafiltration with 10 mM succinic acid. After ultrafiltration, the number of free sulfhydryl groups in the antibody was determined using the Ellman method, specifically by measuring the absorbance at 412 nm of the reaction product of free sulfhydryl groups and DTNB (5,5'-dithiobis(2-nitrobenzoic acid)).

[0587] 2. Coupling reaction

[0588] Depending on the amount of antibody, add 9 eq of small molecules (ANK-ER, ANK-VC, ANK-EGCit, or ANK-GGFG) and react at 37°C with shaking for 16 h. Then add 0.2 M NAC (N-acetylcysteine) to a final concentration of 2 mM and continue reacting for 15 min to terminate the reaction. After the reaction, replace the buffer with 10 mM succinate via ultrafiltration.

[0589] Four antibody-drug conjugates were ultimately obtained, all exhibiting good stability. The concentration of mAb1-ANK-VC was approximately 21.1 mg / mL, with a DAR value of approximately 5.89. The concentration of mAb1-ANK-ER was approximately 18.6 mg / mL, with a DAR value of approximately 7.22. The concentration of mAb1-ANK-GGFG was approximately 19.2 mg / mL, with a DAR value of approximately 7.05. The concentration of mAb1-ANK-EGCit was approximately 18.3 mg / mL, with a DAR value of approximately 7.01.

[0590] The structure of mAb1-ANK-ER is shown in Equation I-5:

[0591] The structure of mAb1-ANK-VC is shown in Equation IV-1:

[0592] The structure of mAb1-ANK-GGFG is shown in Equation IV-2:

[0593] The structure of mAb1-ANK-EGCit is shown in Equation IV-3:

[0594] Example 14. Inhibition of ADC proliferation in SK-BR-3 and MDA-MB-468 cells

[0595] The inhibitory effects of four ADCs on tumor cell proliferation were evaluated using the HER2-positive breast tumor cell line SK-BR-3 and the HER2-negative breast tumor cell line MDA-MB-468 (purchased from Beina Biotechnology).

[0596] ADC1 was used as a positive control, and its preparation method was the same as that described in WO2022253284A1 for the synthesis of ADC1.

[0597] Step 1: Pre-resuscitate SK-BR-3 (HER2+) and MDA-MB-468 (HER2-) cells. Once the cells have recovered to a relatively optimal state, experiments can be conducted. Adjust the cell density of SK-BR-3 and MDA-MB-468 cells to 1.5 × 10⁻⁶ using assay dilution buffer. 5 cells / mL and 1×10 5Cell suspension was added at a rate of 100 μL / mL to each well in rows 2-11 of a 96-well cell culture plate using a multichannel pipette. 100 μL of sterile PBS was added to each well in the non-experimental edge wells. The cell culture plate was incubated at 37°C in a 5% CO2 incubator for 3–5 hours or overnight. The four ADCs from Example 13 and the positive control ADC1 were serially diluted using assay dilution buffer to achieve an initial sample concentration of 13.33 nM. A total of nine 3-fold serial dilutions were performed, with the last dilution having a drug concentration of 0 nM. NC represents the negative killing control. 100 μL of water was added to each non-experimental well. 100 μL of the diluted ADC drug was added to each well, with three replicates for each concentration. The 96-well cell culture plate was then incubated at 37°C in a 5% CO2 incubator for 6 days.

[0598] Step 2: 15 min in advance, add DMSO to the NC negative control sample wells and treat for 10 min to create wells with complete cell killing. The cell viability of these wells is defined as 0%. Remove the supernatant from the culture plate, add 50 μL / well of DMEM medium containing 10% FBS, then add 50 μL / well of CellCounting-Lite 2.0 Luminescent Cell Viability Assay (Vazyme, LOT: 1101E710), mix well, incubate at room temperature in the dark for 5 min, and then read the absorbance using a SpectraMax M3 microplate reader.

[0599] As shown in Figures 2A-2C, for Her2+ cells SK-BR-3, mAb1-ANK-ER and mAb1-ANK-VC showed better killing effects than mAb1-ANK-GGFG and mAb1-ANK-EGCit, and mAb1-ANK-ER had a lower plateau.

[0600] The results are shown in Figures 3A-3C. mAb1-ANK-ER, mAb1-ANK-VC, mAb1-ANK-GGFG, and mAb1-ANK-EGCit showed virtually no killing effect on Her2-negative cells.

[0601] Example 15. Bystander effect of ADC

[0602] HER2-negative MDA-MB-468 cells were pre-coated at a density of 8000 cells / well. After culturing for 3-5 hours, the culture supernatant was spun dry. 150 μL of the supernatant cultured for 4 days in step 1 of Example 10 was transferred to the MDA-MB-468 cells and cultured for another 3 days in a 37°C, 5% CO2 incubator.

[0603] Add DMSO to the NC negative killing control sample wells 15 minutes in advance and treat for 10 minutes to obtain complete cell killing wells. The cell viability of these wells is defined as 0%.

[0604] Remove the supernatant from the culture plate, add 50 μl / well of DMEM medium containing 10% FBS, then add 50 μl / well of CellCounting-Lite 2.0 Luminescent Cell Viability Assay (Vazyme, LOT: 1101E710), mix well, incubate at room temperature in the dark for 5 min, and then read the absorbance using a SpectraMax M3 microplate reader.

[0605] As shown in Figures 4A-4C, for the transfer supernatant of Her2+ cells SK-BR-3, mAb1-ANK-ER and mAb1-ANK-VC showed better bystander killing effects than mAb1-ANK-GGFG and mAb1-ANK-EGCit.

[0606] As shown in Figures 5A-5C, mAb1-ANK-ER, mAb1-ANK-VC, mAb1-ANK-GGFG, and mAb1-ANK-EGCit did not exhibit bystander killing effects in the transfer supernatant of Her2-negative cells MDA-MB-468.

[0607] Example 16. Synthesis of A1-1 and A5-1

[0608] 1) Synthesis of compound A1-1

[0609] Take N2-fluorenylmethoxycarbonyl-L-2,4-diaminobutyric acid (Fmoc-Dab-OH, 8.58 g, 17.4 mmol), add it to 300 mL of dichloromethane and disperse it evenly. Cool to 0-5 °C, add N,N-diisopropylethylamine (DIPEA, 6.74 g, 52.2 mmol) dropwise, and stir to free it for 30 minutes. After freeing, add a solution of methoxy-pentadecanoethylene glycol-succinimide ester (m-PEG15-NHS, 15.09 g, 22.8 mmol) dissolved in 90 mL of dichloromethane, and slowly raise the temperature to 20-2 °C. The reaction was carried out at 5℃ for 3 hours (h); LCMS analysis showed the reaction was complete; the solvent was removed by concentration under reduced pressure, and the solution was redissolved in 600 mL of ethyl acetate. The organic phase was extracted three times with 10% sodium carbonate aqueous solution (450 mL each time, 1350 mL in total), and the combined aqueous phases were collected. The pH was adjusted to 2-3 with 3M hydrochloric acid solution at 0-5℃, and then extracted three times with dichloromethane (450 mL each time, 1350 mL in total), and the combined organic phases were collected. The solution was dried over anhydrous sodium sulfate, concentrated to remove the solvent, and 20.43 g of oil was obtained. HPLC purity: 97.14%; MS: [M+H] + =1087.86; for compound A1-1.

[0610] 2) Synthesis of compound A5-1

[0611] At 0-5℃, N-hydroxysuccinimide (3.09 g, 26.8 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylic acid (6.00 g, 22.4 mmol) were added to 300 mL of dichloromethane and stirred until dissolved. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 6.43 g, 33.5 mmol) was added in portions. The flask walls were rinsed with 100 mL of dichloromethane, and the reaction was stirred for 2 h. Samples were taken, and the reaction was monitored by LCMS until complete. 5% sodium bisulfate aqueous solution (250 mL each time, for a total of...) was used. The reaction mixture was extracted twice with 500 mL of water, allowed to stand, and separated. The lower organic phase was collected and extracted twice more with 200 mL of saturated sodium chloride aqueous solution (400 mL total). The lower organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding a yellow viscous substance. The yellow viscous substance was redissolved in 60 mL of dichloromethane, and while stirring, a total of 300 mL of n-heptane was slowly added dropwise. A white solid gradually precipitated out. The solid was filtered, washed with a 1:5 dichloromethane:n-heptane mixture, and the filter cake was collected and dried under vacuum at 35 °C to obtain 7.64 g of product with an HPLC purity of 97.24% and MS: [M+H]. + =366.19, which is compound A5-1.

[0612] Example 17. Synthesis of ANK-EGGFG

[0613] Synthesis operation

[0614] 1) Synthesis of compound A2-1

[0615] 1. CTC Resin Substitution Reaction

[0616] CTC Resin (CTC Resin specification: 1.307mmol / g) (13.8g, 18.0mmol) was loaded onto a solid-phase synthesis column and eluted three times with dichloromethane (100mL each time, 300mL total). The column was then swollen with 150mL of dichloromethane for 2 hours. The solution was dried under vacuum, and fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 16.1g, 54.0mmol) and DIPEA (7.67g, 59.mmol) dissolved in 150mL of dichloromethane were added. Add 4 mmol) of the mixed solution, open the nitrogen valve, and react for 2 h; after the reaction is complete, dry the mixture and wash once with 100 mL of dichloromethane; add the prepared blocking solution (methanol:DIPEA:dichloromethane = 25 mL:25 mL:50 mL); open the nitrogen valve and react for 2 h; dry the mixture and wash three times each with dichloromethane (150 mL each time, 450 mL in total) and N,N-dimethylformamide (DMF, 150 mL each time, 450 mL in total).

[0617] 2. Deprotection and condensation

[0618] Add 100 mL of deprotection solution (20% DMF solution of hexahydropyridine) to the solid-phase synthesis column, open the nitrogen valve, and react for 5 min; dry under vacuum, add 100 mL of deprotection solution (20% DMF solution of hexahydropyridine), open the nitrogen valve, and react for 5 min; take a sample, test for ninhydrin, and it turns blue-black; dry under vacuum, and wash three times each with dichloromethane (150 mL each time, 450 mL total) and DMF (150 mL each time, 450 mL total); add 100 mL of... A mixed solution of N-fluorenemethoxycarbonyl-L-phenylalanine (Fmoc-Phe-OH, 20.9 g, 54.0 mmol), HOBt (8.0 g, 59.4 mmol), and N,N'-diisopropylcarbodiimide (DIC, 9.3 mL, 59.4 mmol) dissolved in DMF was prepared, and the reaction was allowed to proceed for 2 hours with the nitrogen valve on. A sample was taken, and ninhydrin was tested; no color reaction was observed, indicating that the condensation was complete. The solution was then dried under vacuum and washed three times each with dichloromethane (150 mL each time, total 450 mL) and DMF (150 mL each time, total 450 mL). The deprotection and condensation processes were repeated, and Fmoc was added sequentially. c-Gly-OH (16.1 g, 54.0 mmol), Fmoc-Gly-OH (16.1 g, 54.0 mmol), and N-fluorenylmethoxycarbonyl-L-glutamic acid-5-tert-butyl ester (Fmoc-Glu(OtBu)-OH, 23.0 g, 54.0 mmol) were used. After the last amino acid was condensed, the resin was washed three times with DMF (150 mL each time, 450 mL in total) and then washed three times with methanol (150 mL each time, 450 mL in total). The resin was collected and transferred to a vacuum drying oven at 50 °C and dried to constant weight to obtain 28.7 g of resin.

[0619] 3. Cutting and curing:

[0620] Completely dried resin was added to 120 mL of cutting solution (20% trifluoroethanol in dichloromethane solution) under nitrogen protection at 0-5 °C. After stirring and dispersing evenly, the temperature was slowly raised to 20-25 °C and reacted for 3 h. After the reaction was completed, the mixture was filtered, washed with a small amount of freshly prepared cutting solution, and the filtrate was collected and concentrated to remove the solvent, yielding the crude product. The crude product was dissolved in a mixture of 40 mL dichloromethane and 20 mL methanol, and 300 mL methyl tert-butyl ether was gradually added dropwise. The mixture was stirred at 20-25 °C for 2 h. After filtration, the resin was washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 18.4 g of product. HPLC purity: 98.60%; MS: [M+H]. + =744.52,745.50; for compound A2-1.

[0621] 2) Synthesis of compound A2-2

[0622] Under nitrogen protection, A2-1 (6.02 g, 8.1 mmol) and p-aminobenzyl alcohol (1.99 g, 16.2 mmol) were placed in a reaction flask, and 180 mL of dichloromethane and 90 mL of methanol were added until completely dissolved. 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 3.99 g, 16.1 mmol) was added, and the mixture was reacted at 20-25 °C for 16 h in the dark. LC-MS monitoring showed the reaction was complete. The mixture was then rotary evaporated at 35 °C. After removing approximately two-thirds of the solvent, 400 mL of methyl tert-butyl ether was added and stirred for 2 hours. The mixture was filtered, washed with a small amount of methyl tert-butyl ether, and the filter cake was collected. The mixture was transferred to a reaction flask, and 40 mL of dichloromethane and 20 mL of methanol were added. After complete dissolution, 300 mL of methyl tert-butyl ether was gradually added dropwise, and the mixture was stirred for 2 hours. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected. The product was dried under vacuum at 45 °C to obtain 5.71 g of product. HPLC purity: 97.08%; MS: [M+H]. + =849.67,850.59; for compound A2-2.

[0623] 3) Synthesis of compound A2-3

[0624] Under nitrogen protection, 5.68 g (6.7 mmol) of A2-2 was added to 150 mL of acetonitrile and dispersed evenly. Then, 30 mL of diethylamine was added, and the mixture was stirred at 20-25 °C for 2 h. TLC monitoring showed that the reaction was complete, and the reaction was stopped. The mixture was concentrated to remove the diethylamine. Acetonitrile was added, and the mixture was rotary evaporated again. This process was repeated several times to remove residual diethylamine. Then, 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 4.24 g of product. HPLC purity: 96.10%; MS: [M+H]. + =627.44,628.46; for compound A2-3.

[0625] 4) Synthesis of compound B1-1

[0626] Under nitrogen protection at 0-5°C, A1-1 (3.73 g, 3.4 mmol) was placed in a reaction flask, and 30 mL of tetrahydrofuran was added and stirred until dissolved. N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 3.26 g, 10.2 mmol) and DIPEA (0.44 g, 2.9 mmol) were added, and the mixture was activated for 30 min. After activation, a solution of 10 mL tetrahydrofuran and 5 mL... The reaction was carried out in A2-3 (1.79 g, 2.9 mmol) in a DMF mixed solvent for 2 h; LCMS monitoring showed the reaction was complete. 200 mL of methyl tert-butyl ether was added and stirred, resulting in an oily precipitate. The supernatant was discarded, and a mixture of 90 mL of dichloromethane and 90 mL of methanol was added and stirred. A white insoluble substance was observed; this was removed by filtration, and the filtrate was concentrated to approximately one-third. 300 mL of methyl tert-butyl ether was slowly added to the concentrate, and a solid precipitated. This was filtered, the filter cake was collected, and dried to give 5.27 g of product. HPLC purity: 75.93%; MS: [1 / 2 M + H]. + =849.04; for compound B1-1.

[0627] 5) Synthesis of compound B1-2

[0628] Under nitrogen protection at 20-25℃, 5.27 g (3.1 mmol) of B1-1 was added to 30 mL of acetonitrile and dispersed evenly. Then, 15 mL of diethylamine was added, and the mixture was stirred for 2 h. TLC monitoring showed the reaction was complete, at which point the reaction was stopped. The mixture was concentrated to remove the diethylamine. Acetonitrile was added, and the mixture was concentrated again. This process was repeated several times to remove residual diethylamine. The product was then dissolved in a mixture of 15 mL dichloromethane and 15 mL methanol. 150 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 h until an oily precipitate formed. The supernatant was discarded. This stirring process was repeated once more. The product was then dissolved in a mixture of dichloromethane and methanol, transferred to a rotary evaporator, and concentrated to remove the solvent, yielding 4.43 g of product. HPLC purity: 90.02%; MS: [1 / 2 M + H]. + =737.87; for compound B1-2.

[0629] 6) Synthesis of compound B1-5

[0630] Under nitrogen protection at 20-25℃, 4.43 g (3.0 mmol) of B1-2 was dissolved completely in 20 mL of DMF. Then, 1.21 g (3.3 mmol) of A5-1 and 0.39 g (3.0 mmol) of DIPEA were added, and the reaction was allowed to proceed for 4 hours. LC-MS monitoring showed the reaction was complete. 160 mL of methyl tert-butyl ether was added and the mixture was stirred until an oily precipitate formed. The supernatant was discarded, and the mixture was stirred twice more with 80 mL of methyl tert-butyl ether each time (160 mL total). The mixture was concentrated at 35℃ to remove the solvent, yielding B1-3 with an HPLC purity of 72.29% and MS purity of [1 / 2 M + H]. + =862.99; used directly in the next reaction.

[0631] Under nitrogen protection, at 0-5℃, oily compound B1-3 was completely dissolved in 20 mL of DMF. p-Nitrophenol carbonate (4.57 g, 15.0 mmol) and DIPEA (0.23 g, 1.8 mmol) were added, and the mixture was stirred for 5 h. LC-MS monitoring showed the reaction was complete. 160 mL of methyl tert-butyl ether was added and the mixture was stirred until a solid precipitated. 10 mL of DMF was added to redissolve the compound, and then 80 mL of methyl tert-butyl ether was slowly added, resulting in another solid precipitating. The mixture was dried by rotary evaporation at 35℃ to remove residual solvent, yielding B1-4 with an HPLC purity of 67.35% and MS purity of [1 / 2 M + H]. + =945.48; directly used in the next reaction;

[0632] Under nitrogen protection at 20-25°C, 1.28 g (2.4 mmol) of exatecan mesylate was dispersed in 15 mL of DMF and could not be dissolved. While stirring, 1.24 g (9.6 mmol) of DIPEA was added, and the mixture was stirred until it gradually dissolved and set aside. In a separate reaction flask, 0.65 g (4.8 mmol) of B1-4 and HOBt obtained in the previous step was added and completely dissolved in 15 mL of DMF. The completely dissolved exatecan solution was then added, and nitrogen was displaced. The reaction was carried out at 30-35°C for 2 hours. LCMS monitoring showed that the reaction was complete. 180 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 hour. Solids gradually precipitated. The mixture was filtered, and the filter cake was transferred to a reaction flask. 25 mL of tetrahydrofuran and 5 mL of... Dissolve in DMF, add 180 mL of methyl tert-butyl ether again and stir for 1 h. Filter, wash with methyl tert-butyl ether, collect the filter cake, and dry at 35 °C to obtain 4.57 g of product. HPLC purity: 70.71%; MS: [1 / 2 M + H]. + =1093.63; [1 / 3M+H] + =729.58; for compound B1-5.

[0633] 7) Synthesis of compound ANK-EGGFG

[0634] Under nitrogen protection, at 0-5℃, dissolve 510 mg (0.23 mmol) of B1-5 in 5 mL of dichloromethane and 2 mL of DMF. Prepare 25 mL of a removal agent (trifluoroacetic acid:water:triisopropylsilane = 94:3:3, v / v) and cool to 0-5℃. Add the removal agent dropwise to the reaction system. After the addition is complete, raise the temperature to 20-25℃ and react for 1 h. LCMS monitoring shows the reaction is complete. Add 200 mL of a slurry solvent (methyl tert-butyl ether:n-heptane = 1:1, v / v). After solid precipitation, filter rapidly and wash with a large amount of slurry solvent. Dry under nitrogen to obtain crude ANK-EGGFG. Take 100 mg of the crude product and purify using high-performance preparative liquid chromatography (acetonitrile and water gradient elution). Lyophilize to obtain 19.4 mg of white solid powder. HPLC purity: 94.15%; MS: [1 / 3 M + H]. + =710.43; for compound ANK-EGGFG.

[0635] Example 18. Synthesis of ANK-GEFG

[0636] Synthesis operation

[0637] 1) Synthesis of compound A3-1

[0638] 1. CTC Resin substitution reaction:

[0639] CTC Resin (13.8 g, 18.0 mmol) was placed in a solid-phase synthesis column and washed three times with dichloromethane (100 mL each time, 300 mL total). The column was then swollen with 150 mL of dichloromethane for 2 h. The solution was dried under vacuum, and a mixed solution of Fmoc-Gly-OH (16.10 g, 54.0 mmol) and DIPEA (7.67 g, 59.4 mmol) dissolved in 150 mL of dichloromethane was added. The nitrogen valve was opened, and the reaction was allowed to proceed for 2 h. After the reaction was complete, the solution was dried under vacuum and washed once with 150 mL of dichloromethane. A blocking buffer (methanol:DIPEA:dichloromethane = 25 mL:25 mL:50 mL) was added, and the nitrogen valve was opened, and the reaction was allowed to proceed for 2 h. The solution was dried under vacuum and washed three times each with dichloromethane (200 mL each time, 600 mL total) and DMF (200 mL each time, 600 mL total).

[0640] 2. Deprotection and condensation:

[0641] Add 100 mL of deprotection solution (20% DMF solution of hexahydropyridine), open the nitrogen valve, and react for 5 min; dry under vacuum, add another 200 mL of deprotection solution (20% DMF solution of hexahydropyridine), open the nitrogen valve, and react for 5 min; take a sample, test for ninhydrin, which turns blue-black; dry under vacuum, and wash three times each with dichloromethane (150 mL each time, 450 mL total) and DMF (150 mL each time, 450 mL total); add 100 mL of... A mixed solution of Fmoc-Phe-OH (20.90 g, 54 mmol), HOBt (8.01 g, 59.4 mmol), and DIC (9.3 mL, 59.4 mmol) dissolved in DMF was prepared, and the nitrogen valve was opened. The reaction was allowed to proceed for 2 hours. A sample was taken, and ninhydrin was tested. No color reaction was observed, indicating the reaction was complete. The solution was then dried under vacuum and washed three times each with dichloromethane (150 mL each time, total 450 mL) and DMF (150 mL each time, total 450 mL). The deprotection and condensation processes were repeated. The procedure involved sequentially adding Fmoc-Glu(OtBu)-OH (23.0 g, 54.0 mmol) and Fmoc-Gly-OH (16.10 g, 54.0 mmol). After the last amino acid was fully condensed, the resin was washed three times with DMF (150 mL each time, 450 mL total) and then three times with methanol (150 mL each time, 450 mL total). The resin was collected and transferred to a vacuum drying oven at 50 °C and dried to constant weight, yielding 29.32 g of resin.

[0642] 3. Cutting and curing:

[0643] Completely dried resin was added to 120 mL of cutting solution (20% trifluoroethanol in dichloromethane solution) under nitrogen protection at 0-5 °C. After stirring and dispersing evenly, the temperature was slowly raised to 20-25 °C and reacted for 3 h. After the reaction was completed, the mixture was filtered, washed with a small amount of freshly prepared cutting solution, and the filtrate was collected and concentrated to remove the solvent, yielding the crude product. The crude product was dissolved in a mixture of 40 mL of dichloromethane and 20 mL of methanol, and 300 mL of methyl tert-butyl ether was gradually added dropwise. The mixture was stirred at 20-25 °C for 2 h. After filtration, the resin was washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 16.94 g of product. HPLC purity: 98.17%; MS: [M+H]. + =687.50,688.45; for compound A3-1.

[0644] 2) Synthesis of compound A3-2

[0645] Under nitrogen protection, A3-1 (5.22 g, 7.6 mmol) and p-aminobenzyl alcohol (1.87 g, 15.2 mmol) were placed in a reaction flask, and 150 mL of dichloromethane and 75 mL of methanol were added until completely dissolved. EEDQ (3.76 g, 15.2 mmol) was added, and the mixture was reacted at 20-25 °C for 16 h in the dark. LC-MS monitoring showed the reaction was complete. Approximately two-thirds of the solvent was removed by rotary evaporation at 35 °C, and 350 mL of methyl tert-butyl ether was added and the mixture was stirred for 2 h. The mixture was filtered, washed with a small amount of methyl tert-butyl ether, and the filter cake was collected. The mixture was transferred to a reaction flask, and 40 mL of dichloromethane and 20 mL of methanol were added until completely dissolved. Then, 300 mL of methyl tert-butyl ether was gradually added, and the mixture was stirred for 2 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected. The product was dried under vacuum at 45 °C to obtain 5.50 g of product. HPLC purity: 94.02%; MS: [M+H]. + =792.54,793.59; for compound A3-2.

[0646] 3) Synthesis of compound A3-3

[0647] Under nitrogen protection, 5.50 g (6.9 mmol) of A3-2 was added to 100 mL of acetonitrile and dispersed evenly. Then, 20 mL of diethylamine was added, and the mixture was stirred at 20-25 °C for 2 h. TLC monitoring showed that the reaction was complete, and the reaction was stopped. The mixture was concentrated to remove the diethylamine. Acetonitrile was added, and the mixture was rotary evaporated again. This process was repeated several times to remove residual diethylamine. Then, 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 3.86 g of product. HPLC purity: 95.90%; MS: [M+H]. + =570.43,571.45; for compound A3-3.

[0648] 4) Synthesis of compound C1-1

[0649] Under nitrogen protection at 0-5°C, add Al-1 (3.44 g, 3.2 mmol) to a reaction flask, add 24 mL of tetrahydrofuran and stir until dissolved. Add HATU (3.00 g, 7.9 mmol) and DIPEA (0.35 g, 2.7 mmol) and activate for 30 min. After activation, add a solution dissolved in 8 mL of tetrahydrofuran and 4 mL of... The reaction was carried out in A3-3 (1.50 g, 2.7 mmol) with a mixed DMF solvent for 2 h; LCMS monitoring showed the reaction was complete. A mixed solution of 120 mL methyl tert-butyl ether and 60 mL n-heptane was added and stirred, resulting in an oily precipitate. The supernatant was discarded, and another 100 mL methyl tert-butyl ether and 50 mL n-heptane were added and stirred again, discarding the supernatant. A mixed solution of 50 mL dichloromethane and 50 mL methanol was added and stirred, resulting in a white insoluble substance, which was filtered off. The filtrate was concentrated to about one-third. 200 mL methyl tert-butyl ether was slowly added to the concentrate, resulting in a precipitate that adhered to the walls; the supernatant was discarded, and the precipitate was redissolved in dichloromethane and methanol. The stirring process was repeated. After stirring, the product was transferred to a rotary evaporator and dried by rotary evaporation to obtain 4.53 g of product. HPLC purity: 81.23%; MS: [1 / 2 M + H]. + =820.48; for compound C1-1.

[0650] 5) Synthesis of compound C1-2

[0651] Under nitrogen protection at 20-25℃, 4.53 g (2.7 mmol) of Cl-1 was added to 40 mL of acetonitrile and dispersed evenly. Then, 10 mL of diethylamine was added, and the mixture was stirred for 2 h. TLC monitoring showed the reaction was complete, at which point the reaction was stopped. The mixture was concentrated to remove the diethylamine. Acetonitrile was added, and the mixture was rotary evaporated again. This process was repeated several times to remove residual diethylamine. The product was then dissolved in a mixture of 10 mL dichloromethane and 10 mL methanol. 100 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 h until an oily precipitate formed. The supernatant was discarded. This stirring process was repeated once more. The product was then dissolved in a mixture of dichloromethane and methanol, transferred to a rotary evaporator, and concentrated to remove the solvent, yielding 3.92 g of product. HPLC purity: 92.64%; MS: [1 / 2 M + H]. + =709.35; for compound C1-2.

[0652] 6) Synthesis of compound C1-5

[0653] Under nitrogen protection at 20-25℃, C1-2 (3.92 g, 2.8 mmol) was dissolved completely in 20 mL of DMF. Then, A5-1 (1.11 g, 3.0 mmol) and DIPEA (0.36 g, 2.8 mmol) were added, and the reaction was allowed to proceed for 4 h. LCMS monitoring showed the reaction was incomplete. DIPEA (0.36 g, 2.8 mmol) was added, and the reaction was continued for another 1 h. LCMS monitoring showed the reaction was complete. 200 mL of methyl tert-butyl ether was added and the mixture was stirred until an oily precipitate formed. The supernatant was discarded. 8 mL of DMF was added to reconstitute the precipitate, and then the mixture was stirred twice more with 80 mL of methyl tert-butyl ether. The solvent was removed by rotary evaporation at 35℃ to obtain C1-3. HPLC purity: 86.07%; MS: [1 / 2 M + H]. + =834.51; used directly in the next reaction.

[0654] Under nitrogen protection, at 0-5℃, the oily compound C1-3 was completely dissolved in 20 mL of DMF. p-Nitrophenol carbonate (4.21 g, 8.4 mmol) and DIPEA (0.22 g, 1.7 mmol) were added, and the mixture was stirred for 5 h. LC-MS monitoring showed the reaction was complete. 160 mL of methyl tert-butyl ether was added and the mixture was stirred until a wall-adhering solid-like substance precipitated. 10 mL of DMF was added to redissolve the solid, and then 80 mL of methyl tert-butyl ether was slowly added, again precipitating a wall-adhering solid-like substance. The solid was dried by rotary evaporation at 35℃ to remove residual solvent, yielding C1-4 with an HPLC purity of 82.49% and MS concentration of [1 / 2 M + H]. + =917.03; used directly for the next reaction;

[0655] Under nitrogen protection at 20-25°C, 1.18 g (2.2 mmol) of eczemacon mesylate was dispersed in 15 mL of DMF. Since it did not dissolve, DIPEA (1.14 g, 8.8 mmol) was added with stirring until it gradually dissolved. In a separate reaction flask, 0.60 g (4.4 mmol) of Cl-4 obtained in the previous step was added and completely dissolved in 15 mL of DMF. The completely dissolved eczemacon was then added, and the nitrogen atmosphere was displaced. The reaction was carried out at 30-35°C for 2 hours. LCMS monitoring showed that the reaction was complete. 180 mL of methyl tert-butyl ether was added and the mixture was stirred until a solid gradually precipitated. The mixture was filtered, and the filter cake was transferred to a reaction flask. 25 mL of tetrahydrofuran and 5 mL of... Dissolved in a mixed solvent of DMF, 200 mL of methyl tert-butyl ether was added again and stirred for 1 hour, allowing it to gradually solidify. The mixture was filtered, washed with methyl tert-butyl ether, and dried under vacuum at 35°C to obtain 3.97 g of product. HPLC purity: 74.48%; MS: [1 / 2 M + H]. + =1065.06; [1 / 3M+H] + =710.61; for compound C1-5.

[0656] 7) Synthesis of compound ANK-GEFG

[0657] Under nitrogen protection, at 0-5℃, C1-5 (520 mg, 0.24 mmol) was dissolved in 4 mL of dichloromethane and 2 mL of DMF. Separately, 25 mL of a removal agent (trifluoroacetic acid: water: triisopropylsilane = 94:3:3, v / v) was prepared and cooled to 0-5℃. The removal agent was added dropwise to the reaction system. After the addition was complete, the temperature was raised to 20-25℃ and the reaction was allowed to proceed for 1 h. LC-MS showed complete reaction. 200 mL of a slurry solvent (methyl tert-butyl ether: n-heptane = 1:1, v / v) was added. After solid precipitation, the mixture was rapidly filtered and washed with a large amount of slurry solvent. The mixture was then dried under nitrogen to obtain crude ANK-GEFG. 100 mg of the crude product was purified using high-performance preparative liquid chromatography (acetonitrile and water gradient elution), lyophilized, and yielded 22.1 mg of a white solid powder. HPLC purity: 94.11%; MS: [1 / 3 M + H]. + =691.40; for compound ANK-GEFG.

[0658] Example 19. Synthesis of ANK-GGEG

[0659] Synthesis operation

[0660] 1) Synthesis of compound A4-1

[0661] 1. CTC Resin substitution reaction:

[0662] CTC Resin (18.4 g, 24.0 mmol) was placed in a solid-phase synthesis column and washed three times with dichloromethane (150 mL each time, 450 mL total). The column was then swollen with 200 mL of dichloromethane for 2 h. The solution was dried under vacuum, and a mixed solution of Fmoc-Gly-OH (21.40 g, 72.0 mmol) and DIPEA (10.23 g, 79.2 mmol) dissolved in 250 mL of dichloromethane was added. The nitrogen valve was opened, and the reaction was allowed to proceed for 2 h. After the reaction was complete, the solution was dried under vacuum and washed once with 200 mL of dichloromethane. A blocking buffer (methanol:DIPEA:dichloromethane = 50 mL:50 mL:100 mL) was added, and the nitrogen valve was opened, and the reaction was allowed to proceed for 2 h. The solution was dried under vacuum and washed three times each with dichloromethane (150 mL each time, 450 mL total) and DMF (150 mL each time, 450 mL total).

[0663] 2. Deprotection and condensation:

[0664] Add 200 mL of deprotection solution (20% DMF solution of hexahydropyridine), open the nitrogen valve, and react for 5 min; dry under vacuum, add another 200 mL of deprotection solution (20% DMF solution of hexahydropyridine), open the nitrogen valve, and react for 5 min; take a sample, test for ninhydrin, which turns blue-black; dry under vacuum, and wash three times each with dichloromethane (200 mL each time, 600 mL total) and DMF (200 mL each time, 600 mL total); add 200 mL of... A mixed solution of Fmoc-Glu(OtBu)-OH (30.71 g, 72.0 mmol), HOBt (10.71 g, 79.2 mmol), and DIC (12.3 mL, 79.2 mmol) dissolved in DMF was prepared and reacted for 2 hours with the nitrogen valve turned on. A sample was taken and tested for ninhydrin; no color reaction was observed, indicating the reaction was complete. The solution was then dried and rinsed three times each with dichloromethane (200 mL each time, 600 mL total) and DMF (200 mL each time, 600 mL total). The deprotection and condensation operations were repeated, with Fmoc-Gly-OH (21.41 g, 72.0 mmol) and Fmoc-Gly-OH (21.41 g, 72.0 mmol) added sequentially. After the last amino acid was condensed, the resin was washed three times with DMF (200 mL each time, 600 mL total) and then three times with methanol (200 mL each time, 600 mL total). The resin was collected and transferred to a vacuum drying oven at 50 °C and dried to constant weight to obtain 32.49 g of resin.

[0665] 3. Cutting and curing:

[0666] Completely dried resin was added to 200 mL of a cutting solution (20% trifluoroethanol in dichloromethane solution) under nitrogen protection at 0-5 °C. After stirring and dispersing evenly, the temperature was slowly raised to 20-25 °C and reacted for 3 h. After the reaction was completed, the mixture was filtered, washed with a small amount of freshly prepared cutting solution, and the filtrate was collected and concentrated to remove the solvent, yielding the crude product. The crude product was dissolved in a mixture of 50 mL dichloromethane and 25 mL methanol, and 400 mL methyl tert-butyl ether was gradually added dropwise. The mixture was stirred at 20-25 °C for 2 h. After filtration, the resin was washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 17.83 g of product. HPLC purity: 99.04%; MS: [M+H]. + =597.30,598.33; for compound A4-1.

[0667] 2) Synthesis of compound A4-2

[0668] Under nitrogen protection, A4-1 (5.50 g, 9.2 mmol) and p-aminobenzyl alcohol (2.27 g, 18.4 mmol) were placed in a reaction flask, and 160 mL of dichloromethane and 80 mL of methanol were added until completely dissolved. EEDQ (4.56 g, 18.4 mmol) was added, and the mixture was reacted at 20-25 °C for 16 h in the dark. LC-MS analysis showed the reaction was complete. Approximately two-thirds of the solvent was removed by rotary evaporation at 35 °C, and 400 mL of methyl tert-butyl ether was added and stirred for 2 h. The mixture was filtered, washed with a small amount of methyl tert-butyl ether, and the filter cake was collected. The mixture was transferred to a reaction flask, and 100 mL of dichloromethane and 50 mL of methanol were added until completely dissolved. Then, 700 mL of methyl tert-butyl ether was gradually added and stirred for 2 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected. The product was dried under vacuum at 45 °C to obtain 5.20 g of product. HPLC purity: 97.33%; MS:

[0669] [M+H] + =702.45,703.49; for compound A4-2.

[0670] 3) Synthesis of compound A4-3

[0671] Under nitrogen protection, 5.20 g (7.4 mmol) of A4-2 was added to 100 mL of acetonitrile and dispersed evenly. Then, 20 mL of diethylamine was added, and the mixture was stirred at 20-25 °C for 2 h. TLC monitoring showed that the reaction was complete, and the reaction was stopped. The mixture was concentrated to remove the diethylamine. Acetonitrile was added, and the mixture was rotary evaporated again. This process was repeated several times to remove residual diethylamine. Then, 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to give 3.34 g of product. HPLC purity: 95.41%; MS: [M+H]. + =480.34,480.41; for compound A4-3.

[0672] 4) Synthesis of compound D1-1

[0673] Under nitrogen protection at 0-5℃, add Al-1 (4.41 g, 4.1 mmol) to a reaction flask, add 30 mL of tetrahydrofuran and stir until dissolved. Add HATU (3.85 g, 10.1 mmol) and DIPEA (0.44 g, 3.4 mmol) and activate for 30 min. After activation, add a solution dissolved in 10 mL of tetrahydrofuran and 8 mL of... The reaction was carried out in A4-3 (1.63 g, 3.4 mmol) of DMF mixed solvent for 2 h; LCMS monitoring showed the reaction was complete. 200 mL of methyl tert-butyl ether was added and stirred, resulting in an oily precipitate. The supernatant was discarded, and another 100 mL of methyl tert-butyl ether was added and stirred again, discarding the supernatant. A mixture of 60 mL of dichloromethane and 60 mL of methanol was added and stirred, resulting in a white insoluble substance, which was removed by filtration. The filtrate was collected and concentrated to one-third of its original volume. 300 mL of methyl tert-butyl ether was slowly added to the concentrate, and a precipitate formed sticking to the walls. The supernatant was discarded, and the precipitate was redissolved in dichloromethane and methanol, with the stirring process repeated. After stirring, the product was transferred to a rotary evaporator using a mixture of dichloromethane and methanol and dried by rotary evaporation to obtain 4.26 g of product. HPLC purity: 85.29%; MS: [1 / 2 M + H]. + =775.40; for compound D1-1.

[0674] 5) Synthesis of compound D1-2

[0675] Under nitrogen protection at 20-25℃, 4.26 g (2.7 mmol) of D1-1 was added to 40 mL of acetonitrile and dispersed evenly. Then, 10 mL of diethylamine was added, and the mixture was stirred for 2 h. TLC monitoring showed the reaction was complete, and the reaction was stopped. The mixture was concentrated to remove the diethylamine. Acetonitrile was added, and the mixture was rotary evaporated again. This process was repeated several times to remove residual diethylamine. The product was then dissolved in a mixture of 10 mL dichloromethane and 10 mL methanol. 100 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 h to precipitate a solid. The solid was filtered, and since it was hygroscopic during filtration, it was washed with methyl tert-butyl ether. The product was then reconstituted in a mixture of 10 mL dichloromethane and 10 mL methanol, and the stirring process was repeated once more. The mixture was then filtered, washed with methyl tert-butyl ether, and dried to obtain 3.02 g of product. HPLC purity: 87.81%; MS: [1 / 2 M + H]. + =664.17; for compound D1-2.

[0676] 6) Synthesis of compound D1-5

[0677] Under nitrogen protection at 20-25℃, D1-2 (2.90 g, 2.2 mmol) was dissolved completely in 20 mL of DMF, followed by A5-1 (0.88 g, 2.4 mmol) and DIPEA (0.29 g, 2.2 mmol). The reaction was allowed to proceed for 4 hours. LC-MS monitoring confirmed the reaction was complete. 150 mL of methyl tert-butyl ether was added and the mixture was stirred until a wall-adhering solid-like substance precipitated. The supernatant was discarded. 8 mL of DMF was added to redissolve the solid, and the mixture was stirred twice more with methyl tert-butyl ether (80 mL each time, 160 mL total). This resulted in the precipitation of another wall-adhering solid-like substance. Residual solvent was removed by rotary evaporation at 35℃ to obtain D1-3. HPLC purity: 89.71%; MS: [1 / 2 M + H]. + =789.44; directly used in the next reaction;

[0678] Under nitrogen protection, at 0-5℃, oily compound D1-3 was completely dissolved in 20 mL of DMF. p-Nitrophenol carbonate (3.33 g, 10.9 mmol) and DIPEA (0.17 g, 1.3 mmol) were added, and the mixture was stirred for 5 h. LC-MS monitoring showed the reaction was complete. 160 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 h, resulting in solid precipitation. The mixture was filtered, washed with a small amount of methyl tert-butyl ether, and the filter cake was collected. The cake was transferred to a rotary evaporator and dried at 35℃ to remove the solvent, yielding D1-4 with an HPLC purity of 81.84% and MS purity of [1 / 2 M + H]. + =871.93; used directly for the next reaction.

[0679] Under nitrogen protection at 20-25°C, 0.93 g (1.7 mmol) of eczemacon mesylate was dispersed in 15 mL of DMF. Since it did not dissolve, DIPEA (0.90 g, 7.0 mmol) was added with stirring until it gradually dissolved. In a separate reaction flask, D1-4 and HOBt (0.46 g, 3.4 mmol) obtained in the previous step were added and dissolved in 15 mL of DMF. The completely free eczemacon was then added, and the nitrogen atmosphere was displaced. The reaction was carried out at 30-35°C for 2 hours. LCMS monitoring showed that the reaction was complete. 200 mL of methyl tert-butyl ether was added and the mixture was stirred until a solid gradually precipitated. The mixture was filtered, and the filter cake was transferred to a reaction flask. 25 mL of tetrahydrofuran and 5 mL of... Dissolve in DMF, add 200 mL of methyl tert-butyl ether again and stir for 1 hour, allowing it to gradually solidify. Filter, wash with methyl tert-butyl ether, collect the filter cake and dry to obtain 3.42 g of product. HPLC purity: 83.47%; MS: [1 / 2 M + H]. + =1019.90; [1 / 3M+H] + =680.59; for compound D1-5.

[0680] 7) Synthesis of compound ANK-GGEG

[0681] Under nitrogen protection, at 0-5℃, dissolve 520 mg (0.24 mmol) of D1-5 in 4 mL of dichloromethane and 2 mL of DMF. Prepare 25 mL of a removal agent (trifluoroacetic acid:water:triisopropylsilane = 94:3:3, v / v) and cool to 0-5℃. Add the removal agent dropwise to the reaction system. After the addition is complete, raise the temperature to 20-25℃ and react for 1 h. LC-MS shows the reaction is complete. Add 200 mL of a slurry solvent (methyl tert-butyl ether:n-heptane = 1:1, v / v). After solid precipitation, filter rapidly and wash with a large amount of slurry solvent. Dry under nitrogen pressure to obtain crude ANK-GGEG. Take 100 mg of the crude product and purify it using high-performance preparative liquid chromatography (acetonitrile and water gradient elution). Lyophilize to obtain 30.6 mg of white solid powder. HPLC purity: 95.61%; MS: [1 / 3 M + H]. + =661.34; for compound ANK-GGEG.

[0682] Example 20. Synthesis of ANK-GGEG-Dxd

[0683] Synthesis operation

[0684] 1) Synthesis of compound A7-1

[0685] 1. CTC Resin substitution reaction:

[0686] CTC Resin (18.4 g, 24.0 mmol) was placed in a solid-phase synthesis column and washed three times with dichloromethane (200 mL each time, 600 mL total). The column was then swollen with 250 mL of dichloromethane for 2 h. The solution was dried under vacuum, and a mixed solution of Fmoc-Glu(OtBu)-OH (30.71 g, 72.0 mmol) and DIPEA (10.23 g, 79.2 mmol) dissolved in 250 mL of dichloromethane was added. The nitrogen valve was opened, and the reaction was allowed to proceed for 2 h. After the reaction was complete, the solution was dried under vacuum and washed once with 200 mL of dichloromethane. A blocking buffer (methanol:DIPEA:dichloromethane = 50 mL:50 mL:100 mL) was added, and the nitrogen valve was opened, and the reaction was allowed to proceed for 2 h. The solution was dried under vacuum and washed three times each with dichloromethane (200 mL each time, 600 mL total) and DMF (200 mL each time, 600 mL total).

[0687] 2. Deprotection and condensation:

[0688] Add 200 mL of deprotection solution (20% DMF solution of hexahydropyridine), open the nitrogen valve, and react for 5 min; dry under vacuum, add another 200 mL of deprotection solution (20% DMF solution of hexahydropyridine), open the nitrogen valve, and react for 5 min; take a sample, test for ninhydrin, which turns blue-black; dry under vacuum, and wash three times each with dichloromethane (200 mL each time, 600 mL total) and DMF (200 mL each time, 600 mL total); add 200 mL of... A mixed solution of Fmoc-Gly-OH (21.41 g, 72.0 mmol), HOBt (10.71 g, 79.2 mmol), and DIC (12.3 mL, 79.2 mmol) dissolved in DMF was prepared, and the nitrogen valve was opened. The reaction was allowed to proceed for 2 hours. A sample was taken, and ninhydrin was tested. No color reaction was observed, indicating that the reaction was complete. The sample was dried under vacuum and washed three times each with dichloromethane (200 mL each time, 600 mL total) and DMF (200 mL each time, 600 mL total). The deprotection and condensation operations were repeated. Fmoc-Gly-OH (21.41 g, 72.0 mmol) was added. After the last amino acid was condensed, the sample was washed three times with DMF (200 mL each time, 600 mL total) and then three times with methanol (200 mL each time, 600 mL total). The resin was collected and transferred to a vacuum drying oven at 50 °C. The resin was dried to constant weight, yielding 28.45 g of resin.

[0689] 3. Cutting and curing:

[0690] Completely dried resin was added to 200 mL of a cutting solution (20% trifluoroethanol in dichloromethane solution) under nitrogen protection at 0-5 °C. After stirring and dispersing evenly, the temperature was slowly raised to 20-25 °C and reacted for 3 h. After the reaction was completed, the mixture was filtered, washed with a small amount of freshly prepared cutting solution, and the filtrate was collected and concentrated to remove the solvent, yielding the crude product. The crude product was dissolved in a mixture of 50 mL dichloromethane and 25 mL methanol, and 400 mL methyl tert-butyl ether was gradually added dropwise. The mixture was stirred at 20-25 °C for 2 h. After filtration, the resin was washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 10.84 g of product. HPLC purity: 99.57%; MS: [M+H]. + =540.23; for compound A7-1.

[0691] 2) Synthesis of compound G1-1

[0692] Under nitrogen protection, fluorenemethoxycarbonyl-glycyl-glycine (Fmoc-Gly-Gly-OH, 9.40 g, 26.5 mmol) was placed in a reaction flask, and 240 mL of tetrahydrofuran and 80 mL of toluene were added. The mixture was stirred until dissolved. After complete dissolution, lead tetraacetate (14.7 g, 33.2 mmol) and 4 mL of pyridine were added. After the addition was complete, the temperature was raised to 75-80 °C, and the reaction was allowed to proceed for 2 h. TLC monitoring showed that the reaction was complete, and the reaction was stopped. The heating was turned off, and the mixture was cooled to room temperature. The insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure to obtain a yellow oily substance. Purification using silica gel column chromatography yielded 8.21 g of a white solid with an HPLC purity of 90.54%; MS: [M+H]. + =309.23 (no molecular ion peak, this is a fragment peak); this is compound G1-1.

[0693] 3) Synthesis of compound G1-2

[0694] Under nitrogen protection, 8.15 g (22.1 mmol) of G1-1 was placed in a reaction flask, and 120 mL of dichloromethane was added. After stirring until dissolved, benzyl glycolate (36.80 g, 221.7 mmol) and pyridine p-toluenesulfonate (PPTS, 570 mg, 2.21 mmol) were added. The mixture was heated to 45 °C and reacted for 18 h. The reaction was monitored by TLC and found to be complete. The product was purified by silica gel column chromatography to obtain 9.55 g of white solid. HPLC purity: 92.47%; MS: [M+H]. + =309.23 (no molecular ion peak, this is a fragment peak); this is compound G1-2.

[0695] 4) Synthesis of compound G1-4

[0696] Under nitrogen protection, 3.00 g (6.3 mmol) of G1-2 was placed in a reaction flask, 50 mL of acetonitrile was added, and the mixture was stirred until dissolved. Then, diethylamine (7.00 g, 95.7 mmol) was added, and the reaction was carried out at 20-25 °C for 2 h. The reaction was stopped when the reaction was complete by TLC monitoring. The solvent and diethylamine were removed by rotary evaporation at 35 °C, and acetonitrile (20 mL each time, 60 mL in total) was added to dissolve the mixture. The mixture was then rotary evaporated at 35 °C for 3 times to ensure that no diethylamine residue remained. A light yellow oily substance was obtained, which was the crude G1-3 product, and it was used directly for the next reaction.

[0697] Under nitrogen protection, A7-1 (3.07 g, 5.7 mmol) was placed in a reaction flask, and 15 mL of DMF was added and stirred until dissolved. The temperature was controlled at 0-5℃. HATU (3.61 g, 9.5 mmol) and DIPEA (1.64 g, 12.7 mmol) were added sequentially, and the mixture was activated at 0-5℃ for 30 min. After activation, the crude G1-3 obtained in the previous step was processed using 10 mL of [a specific solution / formula]. After dissolving DMF, it was added to the reaction system and reacted at 0-5℃ for 2 hours. LCMS monitoring showed the reaction was complete, at which point the reaction was stopped. 200 mL of ethyl acetate was added directly to the reaction solution, and after stirring to dissolve, the organic phase was extracted twice with 0.2 M hydrochloric acid solution (80 mL each time, 160 mL total). The organic phase was retained, and the aqueous phases were combined and extracted once with 40 mL of ethyl acetate. The combined organic phases were extracted twice with 3.0% sodium carbonate aqueous solution (80 mL each time, 160 mL total), and the organic phase was retained. Then, the organic phase was extracted twice with saturated sodium chloride aqueous solution (80 mL each time, 160 mL total), and the organic phase was retained. Anhydrous sodium sulfate was added for drying. Sodium sulfate was removed by filtration, and the filtrate was collected and purified using silica gel column chromatography to obtain 2.82 g of white solid. HPLC purity: 94.33%; MS: [M+H]. + =608.35 (no molecular ion peak, this is a fragment peak); this is compound G1-4.

[0698] 5) Synthesis of compound I1-2

[0699] Under nitrogen protection, G1-4 (2.86 g, 3.69 mmol) was placed in a reaction flask, 40 mL of acetonitrile was added, and after stirring until dissolved, diethylamine (4.05 g, 55.4 mmol) was added. The reaction was carried out at 20-25 °C for 1 h. The reaction was stopped when the reaction was complete by TLC monitoring. The solvent and diethylamine were removed by rotary evaporation at 35 °C, and acetonitrile (30 mL each time, 90 mL in total) was added to dissolve the residue. The reaction was then repeated at 35 °C for 3 times to ensure that no diethylamine residue remained. A light yellow oily substance was obtained, which was the crude I1-1 product, and it was used directly for the next reaction.

[0700] Under nitrogen protection, A1-1 (4.42 g, 4.1 mmol) was placed in a reaction flask, and 50 mL of dichloromethane was added and stirred until dissolved. The temperature was controlled at 0-5 °C. O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 1.82 g, 4.8 mmol) and DIPEA (716 mg, 5.5 mmol) were added sequentially, and the mixture was activated at 0-5 °C for 30 min. After activation, the crude I1-1 obtained in the previous step was used in 10 mL of... After dissolving N-methylpyrrolidone (NMP), it was added to the reaction system and reacted at 0-5℃ for 2 hours. LCMS monitoring showed the reaction was complete, at which point the reaction was stopped. 50 mL of dichloromethane and 10 mL of methanol were added directly to the reaction solution, and after stirring to dissolve, the organic phase was extracted twice with 0.2 M hydrochloric acid solution (80 mL each time, total 160 mL), retaining the organic phase. Then, the organic phase was extracted twice with 2.5% sodium carbonate aqueous solution (80 mL each time, total 160 mL), retaining the organic phase. Finally, the organic phase was extracted once with 0.2 M hydrochloric acid solution (80 mL), retaining the organic phase. Anhydrous sodium sulfate was added for drying. The sodium sulfate was removed by filtration, and the filtrate was collected and purified using silica gel column chromatography to obtain 2.52 g of product. HPLC purity: 86.79%; MS: [M / 2+H]. + =728.39 (no molecular ion peak, this is a fragment peak); this is compound I1-2.

[0701] 6) Synthesis of compound I1-5

[0702] Take I1-2 (1.10 g, 0.68 mmol) into a reaction flask, add 25 mL of mixed solvent (ethyl acetate: methanol = 1:1, v / v), stir until dissolved, add 0.1 g palladium on carbon, and replace with hydrogen gas three times; react at 20-25 °C for 16 h under hydrogen atmosphere; monitor with LCMS, the reaction is complete, and stop the reaction; filter to remove palladium on carbon, remove solvent by rotary evaporation to obtain I1-3, which can be used directly for the next reaction.

[0703] Under nitrogen protection, I1-3 obtained in the previous step was placed in a reaction flask, 20 mL of acetonitrile was added, and after stirring until dissolved, 4 mL of diethylamine was added. The reaction was carried out at 20-25 °C for 2 h. TLC monitoring showed that the reaction was complete, and the reaction was stopped. The solvent and diethylamine were removed by rotary evaporation at 35 °C, and acetonitrile (15 mL each time, 45 mL in total) was added to dissolve the residue. The reaction was then repeated at 35 °C for 3 times to ensure that no diethylamine remained. A light yellow oily substance was obtained, which was crude I1-4, and it was used directly for the next step of the reaction.

[0704] Under nitrogen protection, I1-4 obtained in the previous step was placed in a reaction flask, 8 mL of DMF was added, and the mixture was stirred until dissolved. A5-1 (744 mg, 2.04 mmol) and DIPEA (88 mg, 0.68 mmol) were then added, and the reaction was carried out at 20-25 °C for 2.5 h. LCMS monitoring showed the reaction was complete. 60 mL of methyl tert-butyl ether was added directly and stirred until a solid precipitated, which was then discarded. The crude product was dissolved in DMF (4 mL each time, 16 mL total), and then stirred with methyl tert-butyl ether (60 mL each time, 240 mL total). This process was repeated four times to ensure complete removal of A5-1. 1.34 g of product was obtained, with an HPLC purity of 90.46% and MS concentration of [M / 2+H]. + =741.40 (no molecular ion peak, this is a fragment peak); it is compound I1-5.

[0705] 7) Synthesis of compound I1-6

[0706] Under nitrogen protection at 20-25°C, 457 mg (0.86 mmol) of eczematidine mesylate was dispersed in 10 mL of DMF. Since it did not dissolve, DIPEA (222 mg, 1.72 mmol) was added with stirring and stirred until completely dissolved. After complete dissolution, the mixture was cooled to 0-5°C and set aside. Under nitrogen protection, 1.34 g (0.86 mmol) of I1-5 was placed in a reaction flask, and 15 mL of DMF was added. Dissolve DMF under stirring, maintaining the temperature at 0-5℃; add (2-oxime-cyanoethyl ethyl)-N,N-dimethyl-morpholinourea hexafluorophosphate (COMU, 405 mg, 0.95 mmol) and eczema solution sequentially, and react at 0-5℃ for 2 h; monitor by LCMS, and stop the reaction when it is complete; add 150 mL of methyl tert-butyl ether to the reaction solution with stirring, and a solid gradually precipitates out; after complete precipitation, discard the supernatant, add 90 mL of dichloromethane and 10 mL of methanol to the obtained solid, stir to dissolve, and extract the organic phase twice with 0.2 M hydrochloric acid solution (60 mL each time, 120 mL total), retain the organic phase, and dry with anhydrous sodium sulfate; filter, collect the filtrate, remove the solvent by rotary evaporation, and obtain 937 mg of product, HPLC purity: 89.83%; MS: [M+H] + =989.06; for compound I1-6.

[0707] 8) Synthesis of compound ANK-GGEG-Dxd

[0708] Under nitrogen protection, at 0-5℃, 360 mg (0.18 mmol) of I1-6 was dissolved in 1 mL of dichloromethane and 1 mL of DMF; 8 mL of trifluoroacetic acid cooled to 0-5℃ was added, and the reaction was allowed to proceed for 1 h; LCMS monitoring showed that the reaction was complete, and the reaction was stopped; at 0-5℃, 80 mL of a slurry solvent (methyl tert-butyl ether: n-heptane = 1:1, v / v) was added, and after solid precipitation, the mixture was rapidly filtered and washed with a large amount of slurry solvent, then dried under nitrogen pressure to obtain crude ANK-GGEG-Dxd; purification was performed using high-performance preparative liquid chromatography (acetonitrile and water gradient elution), followed by lyophilization to obtain 93 mg of white solid powder, HPLC purity: 93.34%; MS: [1 / 3 M + H] + =640.68; for compound ANK-GGEG-Dxd.

[0709] Example 21. Synthesis of Z-EGGFG-AMC

[0710] Synthesis operation

[0711] 1) Synthesis of compound E1-3

[0712] Under nitrogen protection, A2-2 (1.00 g, 1.3 mmol) and 7-amino-4-methylcoumarin (AMC, 353 mg, 2.0 mmol) were dissolved in 25 mL of a mixed solvent (tetrahydrofuran:water = 5:1, v / v) by stirring. EEDQ (1.00 g, 4.0 mmol) was added, and the mixture was reacted at 20-25 °C for 16 h. LC-MS showed complete reaction. 200 mL of methyl tert-butyl ether was added directly, and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to give 502 mg of a grayish-white solid. HPLC purity: 96.55%; MS: [M+H]. + =901.51; for compound E1-1.

[0713] Under nitrogen protection, 502 mg (0.56 mmol) of E1-1 was dispersed in 15 mL of acetonitrile, which was almost insoluble. 5 mL of diethylamine was added, and the mixture was reacted at 20-25 °C for 3 h. As the reaction proceeded, the solution gradually dissolved. LC-MS monitoring showed the reaction was complete. The solvent was removed by concentration, and acetonitrile was added and the mixture was repeatedly rotary evaporated to remove residual diethylamine. 10 mL of acetonitrile was added to redissolve the solution, followed by the addition of 60 mL of methyl tert-butyl ether and stirring for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 318 mg of a light brown solid. HPLC purity: 93.70%; MS: [M+H]. + =679.48; for compound E1-2.

[0714] Under nitrogen protection, E1-2 (318 mg, 0.47 mmol) was dissolved in 5 mL of DMF, and then benzooxycarbonyl succinimide (Cbz-NHS, 175 mg, 0.71 mmol) and DIPEA (61 mg, 0.47 mmol) were added. The mixture was stirred at 15-20 °C for 3 h; the reaction was complete as monitored by LCMS. 40 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to give 322 mg of light brown solid. HPLC purity: 97.51%; MS: [M+H]. + =813.55; for compound E1-3.

[0715] 2) Synthesis of compound Z-EGGFG-AMC

[0716] Under nitrogen protection, at 0-5℃, 322 mg (0.40 mmol) of E1-3 was directly added to 2 mL of a removal agent (trifluoroacetic acid:water:triisopropylsilane = 94:3:3, v / v); the reaction was slowly brought back to room temperature for 1 h; LCMS monitoring showed the reaction was complete; 8 mL of methyl tert-butyl ether was added to slurry, filtered, and washed with methyl tert-butyl ether. The filter cake was collected and dried to obtain crude Z-EGGFG-AMC; purification was performed using high-performance preparative liquid chromatography (acetonitrile and water gradient elution), and lyophilized to obtain 90 mg of white solid powder. HPLC purity: 97.75%; MS: [M+H] + =757.27; for compound Z-EGGEG-AMC.

[0717] Example 22. Synthesis of Z-GEFG-AMC

[0718] Synthesis operation

[0719] 1) Synthesis of compound E2-3

[0720] Under nitrogen protection, A3-2 (750 mg, 1.26 mmol) and AMC (330 mg, 1.89 mmol) were dissolved in 25 mL of a mixed solvent (tetrahydrofuran:water = 5:1, v / v) by stirring. EEDQ (935 mg, 3.78 mmol) was then added, and the mixture was reacted at 20-25 °C for 16 h. LC-MS showed complete reaction. 200 mL of methyl tert-butyl ether was added directly, and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to give 311 mg of a grayish-white solid. HPLC purity: 98.22%; MS: [M+H]. + =844.54; for compound E2-1.

[0721] Under nitrogen protection, 311 mg (0.37 mmol) of E2-1 was dispersed in 15 mL of acetonitrile, followed by 5 mL of diethylamine. The mixture was reacted at 20-25 °C for 3 h, gradually dissolving as the reaction proceeded. LC-MS monitoring showed the reaction was complete. The solvent was removed by concentration, and acetonitrile was added and the mixture was repeatedly rotary evaporated to remove residual diethylamine. The mixture was reconstituted with 10 mL of acetonitrile, and then 60 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 189 mg of a light brown solid. HPLC purity: 97.74%; MS: [M+H]. + =622.39; for compound E2-2.

[0722] Under nitrogen protection, E2-2 (189 mg, 0.30 mmol) was dissolved in 5 mL of DMF, and then Cbz-NHS (113 mg, 0.45 mmol) and DIPEA (39 mg, 0.30 mmol) were added. The mixture was stirred at 15-20 °C for 3 h. LC-MS monitoring showed that the reaction was complete. 40 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to give 166 mg of a light brown solid. HPLC purity: 98.12%; MS: [M+H]. + =756.50; for compound E2-3.

[0723] 2) Synthesis of compound Z-GEFG-AMC

[0724] Under nitrogen protection, at 0-5℃, 166 mg (0.22 mmol) of E2-3 was directly added to 2 mL of a removal agent (trifluoroacetic acid:water:triisopropylsilane = 94:3:3, v / v); the reaction was slowly brought back to room temperature for 1 h; LCMS monitoring showed the reaction was complete; 8 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 h, filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain crude Z-GEFG-AMC; purification was performed using high-performance preparative liquid chromatography (acetonitrile and water gradient elution), and lyophilized to obtain 29 mg of white solid powder, HPLC purity: 92.55%; MS: [M+H] + =700.17; for compound Z-GEFG-AMC.

[0725] Example 23. Synthesis of Z-GGEG-AMC

[0726] Synthesis operation

[0727] 1) Synthesis of compound E3-3

[0728] Under nitrogen protection, A4-2 (1.00 mg, 1.68 mmol) and AMC (440 mg, 2.51 mmol) were dissolved in 25 mL of a mixed solvent (tetrahydrofuran:water = 5:1, v / v) by stirring. EEDQ (1.24 g, 5.01 mmol) was added, and the mixture was reacted at 20-25 °C for 16 h. LC-MS showed complete reaction. 200 mL of methyl tert-butyl ether was added directly, and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to give 448 mg of a grayish-white solid. HPLC purity: 93.87%; MS: [M+H]. + =754.48; for compound E3-1.

[0729] Under nitrogen protection, 448 mg (0.59 mmol) of E3-1 was dispersed in 15 mL of acetonitrile, followed by 5 mL of diethylamine. The reaction was carried out at 20-25 °C for 3 h. As the reaction proceeded, the solid gradually dissolved. LCMS monitoring showed that the reaction was complete. The solvent was removed by concentration, and acetonitrile was added and the mixture was repeatedly rotary evaporated to remove residual diethylamine. The mixture was reconstituted with 10 mL of acetonitrile, and then 60 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 278 mg of a light brown solid. HPLC purity: 96.99%; MS: [M+H]. + =532.38; for compound E3-2.

[0730] Under nitrogen protection, E3-2 (278 mg, 0.52 mmol) was dissolved in 5 mL of DMF, and then Cbz-NHS (196 mg, 0.79 mmol) and DIPEA (67 mg, 0.52 mmol) were added. The mixture was stirred at 15-20 °C for 3 h. LC-MS monitoring showed that the reaction was complete. 40 mL of methyl tert-butyl ether was added and the mixture was slurried and dried to obtain 334 mg of a light brown solid. HPLC purity: 96.57%; MS: [M+H]. + =666.42; for compound E3-3.

[0731] 2) Synthesis of compound Z-GGEG-AMC

[0732] Under nitrogen protection, at 0-5℃, 166 mg (0.22 mmol) of E3-3 was directly added to 2 mL of a removal agent (trifluoroacetic acid:water:triisopropylsilane = 94:3:3, v / v); the reaction was slowly brought back to room temperature for 1 h; LCMS monitoring showed the reaction was complete; 8 mL of methyl tert-butyl ether was added and the mixture was stirred, resulting in solid precipitation. The solid was washed with methyl tert-butyl ether, the filter cake was collected and dried to obtain crude Z-GGEG-AMC; purification was performed using high-performance preparative liquid chromatography (acetonitrile and water gradient elution), and lyophilized to obtain 72 mg of white solid powder. HPLC purity: 99.18%; MS: [M+H] + =610.05; for compound Z-GGEG-AMC.

[0733] Example 24. Synthesis of Z-GGFG-AMC

[0734] Synthesis operation

[0735] 1) Synthesis of compound E4-1

[0736] Under nitrogen protection at 0-5℃, 1.36 g (4.57 mmol) of Fmoc-Gly-OH was placed in a reaction flask, 8 mL of tetrahydrofuran and 0.1 mL of DMF were added, and the mixture was stirred until dissolved. Then, 1.20 mL (14.07 mmol) of oxalyl chloride was added and the mixture was stirred for 1 h. A sample was taken, and the active intermediate was converted using methanol. The reaction was monitored by TLC and found to be complete. After removing the solvent by rotary evaporation, 8 mL of tetrahydrofuran was added to redissolve the intermediate, and the rotary evaporation was repeated to remove residual oxalyl chloride.

[0737] Under nitrogen protection, at 0-5℃, 5 mL of tetrahydrofuran was added to the intermediate obtained in the previous step and stirred to dissolve. AMC (0.40 g, 2.28 mmol) was completely dissolved in 5 mL of pyridine and added dropwise to the reaction system. After the addition was complete, the reaction was carried out at 0-5℃ for 2 h. TLC monitoring showed that the reaction was complete. 60 mL of methyl tert-butyl ether was added and the mixture was stirred until a grayish-white solid precipitated. The precipitate was filtered, washed with methyl tert-butyl ether, and the filter cake was collected. The filter cake was purified by column chromatography to give 500 mg of a white solid. HPLC purity: 94.69%; MS: [M+H]. + =455.11; for compound E4-1.

[0738] 2) Synthesis of compound E4-2

[0739] Under nitrogen protection, 500 mg (1.10 mmol) of E4-1 was dissolved in 15 mL of acetonitrile by stirring. Then, 5 mL of diethylamine was added, and the reaction was carried out at 20-25 °C for 3 h. LC-MS monitoring showed the reaction was complete. The solvent was removed by concentration, and acetonitrile was added and the mixture was repeatedly rotary evaporated to remove residual diethylamine. The mixture was reconstituted with 10 mL of acetonitrile, and then 55 mL of methyl tert-butyl ether was added and stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected and dried to obtain 218 mg of a light brown solid. HPLC purity: 95.28%; MS: [M+H]. + =233.11; for compound E4-2.

[0740] 3) Synthesis of compound Z-GGFG-AMC

[0741] Under nitrogen protection and at 0-5℃, N-(benzyloxycarbonyl)glycylglycyl-L-phenylalanine (Cbz-GGF-OH, 140 mg, 0.34 mmol) was dissolved in 5 mL of DMF by stirring. HATU (142 mg, 0.37 mmol) and DIPEA (132 mg, 1.02 mmol) were added, and the mixture was stirred and activated for 15 min. After activation, E4-2 dissolved in 3 mL of DMF was added. After the addition was complete, the mixture was slowly returned to room temperature and reacted for 16 h. LCMS monitoring showed that the reaction was complete. 60 mL of methyl tert-butyl ether was added and the mixture was stirred for 1 h. The mixture was filtered, washed with methyl tert-butyl ether, and the filter cake was collected to obtain crude Z-GGFG-AMC. Purification was performed using high-performance preparative liquid chromatography (acetonitrile and water gradient elution), followed by lyophilization to obtain 82 mg of white solid powder. HPLC purity: 99.53%; MS: [M+H]. + =628.22; for compound Z-GGFG-AMC.

[0742] Example 25. In vitro cathepsin B cleavage efficiency test of polypeptide substrate

[0743] The 7-amino-4-methylcoumarin (AMC) group can generate a fluorescent signal under excitation light. Its fluorescence is intramolecularly quenched when the amino group of AMC forms an amide bond with the carboxyl group of a peptide residue. Attaching the AMC group to a peptide substrate can be used to measure the activity of peptidases capable of cleaving the amide bond of that peptide substrate; the release of AMC will lead to an increase in fluorescence.

[0744] The lyophilized powders of Z-GGFG-AMC, Z-GGEG-AMC, Z-GEFG-AMC, and Z-EGGFG-AMC were dissolved in dimethyl sulfoxide (DMSO) to prepare 8 mM solutions. The in vitro cathepsin B cleavage efficiency of the peptide substrates under different pH conditions was investigated by adjusting the pH with citric acid and Na2HPO4. The experimental system is shown in the table below.

[0745] Table 14. In vitro cathepsin B cleavage efficiency test system for polypeptide substrates

[0746] Three experimental wells and two blank wells were set up. At 37℃, samples were taken out at 0 min, 5 min, 15 min, 30 min, 60 min, 90 min, 120 min, and 180 min, respectively, and the fluorescence intensity was measured using a microplate reader. The detection parameters were: excitation wavelength (Ex) 355 nm and emission wavelength (Em) 460 nm. The experimental results are shown in Figure 6.

[0747] Example 26. In vitro elastase cleavage efficiency test of polypeptide substrates

[0748] The lyophilized powders of Z-GGFG-AMC, Z-GGEG-AMC, Z-GEFG-AMC, and Z-EGGFG-AMC were dissolved in DMSO to prepare 8 mM solutions. The in vitro elastase cleavage efficiency of the peptide substrates under different pH conditions was investigated by adjusting the pH with citric acid and Na2HPO4. The experimental system is shown in the table below:

[0749] Table 15. In vitro elastase cleavage efficiency test system for peptide substrates

[0750] Three experimental wells and two blank wells were set up. At 37℃, samples were collected at 0 min, 5 min, 15 min, 30 min, 60 min, 90 min, 120 min, and 150 min, respectively, and then analyzed using a microplate reader with the following parameters: Ex 355 nm, Em 460 nm. The experimental results are shown in Figure 7.

[0751] Example 27. Preparation of ADC

[0752] 1. Antibody preparation

[0753] Following the method described by Wood et al., J Immunol., 145:3011 (1990), a monoclonal antibody mAb1 (trastuzumab) specifically binding to the extracellular region of HER2 was produced in CHO cells. The expression vector OptiCHO™ Antibody Express System (invitrogen) containing the antibody gene was constructed using conventional molecular biology methods, with CHO cells serving as the host cell.

[0754] 2. Reduction reaction

[0755] 100 mM EDTA was first added to antibody mAb1 (trastuzumab) to a final concentration of 2 mM. Based on the amount of antibody, 4.5 eq of tricarboxyethylphosphine (TCEP) was added, and the pH was adjusted to approximately 7.5 with 1 M Tris-HCl. The reaction was carried out at 25°C with shaking for 1.5 h. Excess TCEP was removed by ultrafiltration with 10 mM succinic acid. After ultrafiltration, the number of free sulfhydryl groups in the antibody was determined using the Ellman method, specifically by measuring the absorbance at 412 nm of the reaction product of free sulfhydryl groups and DTNB (5,5'-dithiobis(2-nitrobenzoic acid)).

[0756] 3. Coupling reaction

[0757] Depending on the amount of antibody and the content of free sulfhydryl groups, 9 eq of small molecules (ANK-GGEG, ANK-GEFG, ANK-EGGFG, or ANK-GGFG) were added, and the reaction was carried out at 37°C with shaking for 16 h. Then, 0.2 M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was terminated after 15 min. The reaction solution was then replaced by ultrafiltration with 10 mM succinic acid.

[0758] RP-HPLC analysis showed that the DAR value of mAb1-ANK-GGEG was approximately 8.06; the DAR value of mAb1-ANK-GEFG was approximately 8.07; and the DAR value of mAb1-ANK-EGGFG was approximately 8.09.

[0759] Example 28. Preparation of ADC

[0760] 1. Reduction reaction

[0761] Based on the amount of antibody, 5.0 molar equivalents of TCEP were added to the mAb1 antibody (trastuzumab), and the pH of the system was adjusted to 7.5 with 1M Tris base. The mixture was incubated at 25°C for 2 hours for reduction. TCEP was removed by ultrafiltration with 1×PBS (pH 7.4). The thiol antibody value was determined by absorbance measurement. The equivalent amount of free thiol groups was determined by measuring the absorbance at 412 nm, based on the reaction of the thio groups with DTNB.

[0762] 2. Coupling reaction

[0763] During the coupling reaction, based on the amount of antibody and the content of free sulfhydryl groups, 10 molar equivalents of ANK-GGEG-Dxd were added. After stirring at 37°C for 16 hours, 0.2M acetylcysteine ​​was added to a final concentration of 2mM, and the reaction was terminated by stirring for another 15 minutes. The solution was then ultrafiltered to 10mM succinate buffer. This yielded the ADC (mAb1-ANK-GGEG-Dxd).

[0764] Example 29. In vitro biological activity of ADC

[0765] The inhibitory effect of ADCs on tumor cell growth was evaluated using human breast cancer cell line SK-BR-3, human gastric cancer cell line N87, and human non-small cell lung cancer cell line A549 (all purchased from the Cell Bank of the Chinese Academy of Sciences).

[0766] SK-BR-3, N87, and A549 cells in good growth condition were harvested and their density adjusted to 50,000 cells / ml using cell culture medium. Cell dilution buffer was seeded at 100 μL / well into 96-well plates and incubated at 37°C with 5% CO2 for 2 hours. The ADCs (mAb1-ANK-GGEG, mAb1-ANK-GEFG, mAb1-ANK-EGGFG, or mAb1-ANK-GGFG) to be tested were diluted with cell culture medium to 2 μg / mL, and then further diluted down by 3-fold, resulting in 9 concentration gradients. 100 μL / well of each concentration was added to the cells, with 3 replicates per well. The 96-well plates were incubated at 37°C with 5% CO2 for 5 days. The culture medium was then discarded, and 100 μL / well of the mixture of cell culture medium and... The test reagent prepared by Reagent was allowed to stand for 10 minutes. The plate was then read using an ELISA reader, and the analysis was performed using SoftMax Pro software with 4-Parameter selection. The four-parameter curve y = (AD) / (1 + (x / C)^B) + D was obtained. The experimental results are shown in Figures 8A-8C.

[0767] Example 30. Acute toxicity test of ADC in mice

[0768] Sixteen healthy 5-6 week old female BalB / C mice were randomly divided into four groups of four. The ADC (mAb1-ANK-GGEG, mAb1-ANK-GEFG, mAb1-ANK-EGGFG, or mAb1-ANK-GGFG) was diluted to 20 mg / mL and administered intraperitoneally. Changes in mouse body weight were observed after administration. The experimental design is as follows:

[0769] Table 16. Dosing Regimen

[0770] The results showed that one mouse in group G4 died on the sixth day after administration, two mice died on the seventh day after administration, and the remaining 13 mice survived to the end of the observation period.

Claims

1. A drug conjugate having the structure shown in Formula I or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: Abu-[MW-AA-FF-D] p Formula I in, Abu stands for antigen-binding unit, and D stands for drug. Wherein, AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly; FF stands for spacer unit, or self-degradable spacer unit; M is the covalently bound portion of the antigen-binding unit; W is an optional extension unit; p is 1-10.

2. The drug conjugate of claim 1, wherein AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly.

3. The drug conjugate according to claim 1 or 2, wherein FF is... Among them, each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, 3, or 4, where * connects to AA and ** connects to D.

4. The drug conjugate according to any one of claims 1-3, wherein W is Where * connects M, ** connects AA, and n is an integer from 1 to 24.

5. The drug conjugate of claim 4, wherein W is 6. The drug conjugate according to any one of claims 1-5, wherein M is Where * connects to Abu, ** connects to W, and R is 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.

7. The drug conjugate of claim 6, wherein M is Where * connects to Abu, and ** connects to W.

8. A drug conjugate having a structure as shown in Formula I-1 or Formula I-1' or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, Abu stands for antigen-binding unit; D stands for drug. AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly; FF is Among them, each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, wherein * is connected to AA, ** is connected to D; z is 0, 1, 2, 3, or 4; M is Where * connects to Abu, and R is 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 from 1 to 10.

9. A drug conjugate having a structure as shown in Formula I-2, Formula I-2A, Formula I-7 or Formula I-7A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, Abu stands for antigen-binding unit; D stands for drug. R is 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 from 1 to 10.

10. A drug conjugate having a structure as shown in Formula I-3, Formula I-3A, Formula I-8 or Formula I-8A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, Abu represents the antigen-binding unit; D represents the drug; n is an integer from 1 to 24; p is from 1 to 10.

11. A drug conjugate having a structure as shown in Formula I-4, Formula I-4A, Formula I-9 or Formula I-9A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, Abu is the antigen-binding unit; n is an integer from 1 to 24; p is from 1 to 10.

12. The drug conjugate according to any one of claims 1-11, 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, CD25, CD30, FcRH2, NCA, MDP, IL20Rα, short proteoglycans (Brev) ican), 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.

13. The drug conjugate according to any one of claims 1-12, wherein the amino acid sequence of Abu comprises one or more cysteine ​​residues and is linked to other portions of the drug conjugate (such as M of formula I and formula I-1) via the sulfur atom of the cysteine ​​residue.

14. The drug conjugate according to any one of claims 1-13, wherein Abu is an antibody or an antigen-binding fragment thereof.

15. The drug conjugate according to any one of claims 1-14, wherein Abu is trastuzumab.

16. A drug conjugate having a structure as shown in Formula I-5, Formula I-5A, Formula I-6, Formula I-6A, Formula IV-2 or Formula IV-2A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, p is 7-9.

17. A compound having the structure shown in Formula II or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: M'-W-AA-FF' Formula II in, AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly; FF' represents a spacer unit, or a self-degrading spacer unit; M' is the precursor portion that covalently binds to the antigen-binding unit; W is an optional extension unit.

18. The compound of claim 17, wherein AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly.

19. The compound of claim 17 or 18, wherein FF' is Among them, each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4; where * is connected to AA.

20. The compound according to any one of claims 17-19, wherein W is Where * connects M', ** connects AA, and n is an integer from 1 to 24.

21. The compound of claim 20, wherein W is 22. The compound according to any one of claims 17-21, wherein M' is R is selected from: -(CH2) r -、-(CHR m )r-, C3-C8 carbon cyclic 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.

23. The compound of claim 22, wherein M' is 24. A compound having the structure shown in Formula II-1 or Formula II-1' or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly; FF' is Among them, each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen; z is 0, 1, 2, 3, or 4; M' is R is 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.

25. The compound of claim 24, wherein M' is 26. A compound having the structure shown in Formula II-2, Formula II-2A, Formula II-3, Formula II-3A, Formula II-6, Formula II-6A, Formula II-7 or Formula II-7A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, R is 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.

27. A compound having the structure shown in Formula II-4, Formula II-4A, Formula II-5, Formula II-5A, Formula II-8, Formula II-8A, Formula II-9 or Formula II-9A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, n is an integer from 1 to 24.

28. A compound having the structure shown in Formula III or a polymeric isomer thereof or a pharmaceutically acceptable salt or solvate thereof: M'-W-AA-FF-D Formula III in, D represents a drug; AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly; FF stands for spacer unit, or self-degradable spacer unit; M' is the precursor portion that covalently binds to the antigen-binding unit; W is an optional extension unit.

29. The compound of claim 28, wherein AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly.

30. The compound of claim 28 or 29, wherein FF is... Among them, each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, 3, or 4, where * connects to AA and ** connects to D.

31. The compound according to any one of claims 28-30, wherein W is Where * connects M', ** connects AA, and n is an integer from 1 to 24.

32. The compound of claim 31, wherein W is 33. The compound according to any one of claims 28-32, wherein M' is R is 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.

34. The compound of claim 33, wherein M' is 35. A compound having the structure of formula III-1 or III-1' or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, D represents a drug; AA is selected from Glu-Arg, Lys-Arg, Arg-Arg, Gly-Phe-Arg-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Gly-Glu-Phe-Gly, Glu-Gly-Gly-Phe-Gly; FF is Among them, each R F Independently, it is a C1-C6 alkyl, C1-C6 alkoxy, -NO2, or halogen, and z is 0, 1, 2, 3, or 4, wherein * connects to AA and ** connects to D; M' is R is 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.

36. The compound of claim 35, wherein M' is 37. A compound having the structure of formula III-2, formula III-2A, formula III-7 or formula III-7A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, D represents a drug; R is 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.

38. A compound having the structure of formula III-3, III-3A, III-8 or III-8A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, D represents the drug; n is an integer from 1 to 24.

39. A compound having the structure of formula III-4, III-4A, III-9 or III-9A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, R is 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.

40. A compound having the structure of formula III-5, formula III-5A, formula III-10 or formula III-10A, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: in, n is an integer from 1 to 24.

41. A compound having the structure of formula III-6 or III-11, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof:

42. The drug conjugate according to any one of claims 1-15, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

43. The drug conjugate according to any one of claims 4-15 or the compound according to any one of claims 20-27, 31-40, wherein n is 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.

44. The drug conjugate according to any one of claims 1-10, 12-15 or the compound according to any one of claims 28-38, 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.

45. The drug conjugate according to any one of claims 1-10, 12-15 or the compound according to any one of claims 28-38, wherein D is a microtubule inhibitor, a DNA damaging agent, a DNA topoisomerase inhibitor or a DNA damage response (DDR) inhibitor.

46. ​​The drug conjugate according to any one of claims 1-10, 12-15, or the compound according to any one of claims 28-38, wherein D is selected from MMAE, MMAU (beta-glucuronyl-monomethyl auristatin) E), MMAF, AF, calicheamicin derivatives, duocarmycin derivatives, atromycin derivatives PBD (pyrrolobenzodiazepine), talazoparib, Berzosertib, Gartisertib, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivatives SN-38, 22-hydroxyeclipticine, topotecan, letopotecan, belotetan, ixenonotecan, ixenonotecan derivatives, homosilatecan, 6,8-dibromo-2 -Methyl-3-[2-(D-pyranoxylosylamino)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-12,13-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-acrylamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acrylamide, or salts thereof.

47. A pharmaceutical composition comprising the drug conjugate of any one of claims 1-18, 48-52, and a pharmaceutically acceptable carrier, excipient, and / or excipient; or, optionally, other anticancer drugs.

48. Use of the drug conjugate of any one of claims 1-16, 42-46, or the pharmaceutical composition of claim 47 in the preparation of a medicament for treating diseases such as cancer, autoimmune diseases, inflammatory diseases, or infectious diseases.

49. Use of the compound of any one of claims 17-41, 43-46, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a pharmaceutical conjugate.

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