Linker Containing Arylnitro, Antibody-Drug Conjugate Containing Linker and Use of Linker

Abstract

The present application relates to a linker containing arylnitro, an antibody-drug conjugate the linker and use of the linker, as well as a pharmaceutical composition comprising the antibody-drug conjugate and use of the antibody-drug conjugate for treatment and/or prevention of a disease.

##STR00001##

Claims

1. A compound represented by Formula I or a salt thereof, ##STR00167## wherein: R.sub.1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.2 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.3 is fluorine, chlorine, bromine, iodine or ##STR00168## R.sub.4 is ##STR00169## and in ##STR00170## the two carbonyl groups are located on the same side of the CC double bond, which is a cis structure, R.sub.11 is a C.sub.1-6 linear or branched alkyl, and R.sub.11 is optionally mono- or multi-substituted by one or more substituents selected from the group consisting of: halogen and C.sub.1-4 alkoxy; X is oxygen atom (O), nitrogen atom (N) or sulfur atom (S); Ar is aryl, heteroaryl, aryl-fused-heterocyclyl or heteroaryl-fused-heterocyclyl; R.sub.5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; R.sub.6 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; R.sub.7 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.8 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.9 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; q is 0, 1, 2 or 3; p is 0, 1, 2 or 3; a is 0, 1, 2, 3 or 4; L is (CH.sub.2).sub.iO(CH.sub.2).sub.j, (CH.sub.2).sub.iO(CH.sub.2).sub.jC(O), (CH.sub.2).sub.iC(O)NH(CH.sub.2).sub.j, (CH.sub.2CH.sub.2O).sub.i(CH.sub.2).sub.jC(O), (CH.sub.2).sub.i(CH.sub.2CH.sub.2O).sub.jC(O), (OCH.sub.2CH.sub.2).sub.i, (CH.sub.2).sub.m, (CH.sub.2).sub.kC(O)NH(CH.sub.2CH.sub.2O).sub.g(CH.sub.2).sub.hC(O), (CH.sub.2).sub.rC(O), (CH.sub.2).sub.bC(O)NHCH[(CH.sub.2).sub.dNHC(O)(CH.sub.2CH.sub.2O).sub.e(CH.sub.2).sub.fCH.sub.3], ##STR00171## W represents a linking group, and is NHCH.sub.2C(O)NH, NHCH.sub.2CH.sub.2C(O)NH, C(O)NH, NHC(O), O, S, NH, N(CH.sub.3), C(O) or NHCH(R.sub.10)C(O)NH; R.sub.10 is H, CH.sub.3, C.sub.3H.sub.6, CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2C.sub.6H.sub.5, C.sub.8NH.sub.6, CH.sub.2C.sub.6H.sub.4OH, CH.sub.2COOH, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2COOH, (CH.sub.2).sub.4NH.sub.2, (CH.sub.2).sub.2CONH.sub.2, (CH.sub.2)SCH.sub.3, CH.sub.2OH, CH(CH.sub.3)OH or CH.sub.2SH; m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; each i is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; each j is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; r is 1, 2, 3, 4, 5, 6, 7, 8 or 9; k is 1, 2, 3, 4, 5 or 6; g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; h is 1, 2, 3, 4, 5 or 6; b is 1, 2, 3, 4, 5 or 6; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6.

2. The compound or a salt thereof according to claim 1, wherein the compound has a structure represented by Formula la, ##STR00172## wherein: R.sub.1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.2 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.3 is fluorine, chlorine, bromine, iodine or ##STR00173## R.sub.4 is ##STR00174## and in ##STR00175## the two carbonyl groups are located on the same side of the CC double bond, which is a cis structure, R.sub.11 is a C.sub.1-6 linear or branched alkyl, and R.sub.11 is optionally mono- or multi-substituted by one or more substituents selected from the group consisting of: halogen and C.sub.1-4 alkoxy; X is oxygen atom (O), nitrogen atom (N) or sulfur atom (S); Ar is aryl, heteroaryl, aryl-fused-heterocyclyl or heteroaryl-fused-heterocyclyl; R.sub.5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; R.sub.6 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; R.sub.7 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.8 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.9 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; R.sub.10 is H, CH.sub.3, C.sub.3H.sub.6, CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2C.sub.6H.sub.5, C.sub.8NH.sub.6, CH.sub.2C.sub.6H.sub.4OH, CH.sub.2COOH, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2COOH, (CH.sub.2).sub.4NH.sub.2, (CH.sub.2).sub.2CONH.sub.2, (CH.sub.2)SCH.sub.3, CH.sub.2OH, CH(CH.sub.3)OH or CH.sub.2SH; q is 0, 1, 2 or 3; p is 0, 1, 2 or 3; a is 0, 1, 2, 3 or 4; L is (CH.sub.2).sub.iO(CH.sub.2).sub.j, (CH.sub.2).sub.iO(CH.sub.2).sub.jC(O), (CH.sub.2).sub.iC(O)NH(CH.sub.2).sub.j, (CH.sub.2CH.sub.2O).sub.i(CH.sub.2).sub.jC(O), (CH.sub.2).sub.i(CH.sub.2CH.sub.2O).sub.jC(O), (OCH.sub.2CH.sub.2).sub.i, (CH.sub.2).sub.m, (CH.sub.2).sub.rC(O), (CH.sub.2).sub.kC(O)NH(CH.sub.2CH.sub.2O).sub.g(CH.sub.2).sub.hC(O), (CH.sub.2).sub.bC(O)NHCH[(CH.sub.2).sub.dNHC(O)(CH.sub.2CH.sub.2O).sub.e(CH.sub.2).sub.fCH.sub.3], ##STR00176## m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; r is 1, 2, 3, 4, 5, 6, 7, 8 or 9; each i is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; each j is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; k is 1, 2, 3, 4, 5 or 6; g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; h is 1, 2, 3, 4, 5 or 6; b is 1, 2, 3, 4, 5 or 6; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6.

3. (canceled)

4. (canceled)

5. The compound or a salt thereof according to claim 1, wherein the compound has a structure represented by Formula I-1, Formula I-2, Formula I-3 or Formula I-4, ##STR00177## wherein, R.sub.4, R.sub.10 and L are defined as described in claim 1; or the compound has a structure represented by Formula I-5, Formula I-6, Formula I-7 or Formula I-8, ##STR00178## wherein, R.sub.10 and L are defined as described in claim 1; or the compound represented by Formula I or Formula la has a structure represented by Formula I-9, Formula I-10, Formula I-11 or Formula I-12, ##STR00179## wherein, L is defined as described in claim 1.

6. (canceled)

7. A compound represented by Formula II or a salt thereof, ##STR00180## wherein: R.sub.1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.2 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.4 is ##STR00181## and in ##STR00182## the two carbonyl groups are located on the same side of the CC double bond, which is a cis structure, R.sub.11 is a C.sub.1-6 linear or branched alkyl, and R.sub.11 is optionally mono- or multi-substituted by one or more substituents selected from the group consisting of: halogen and C.sub.1-4 alkoxy; X is oxygen atom (O), nitrogen atom (N) or sulfur atom (S); Ar is aryl, heteroaryl, aryl-fused-heterocyclyl or heteroaryl-fused-heterocyclyl; R.sub.5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; R.sub.6 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; R.sub.7 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.8 is hydrogen, methyl, ethyl, n-propyl or isopropyl; q is 0, 1, 2 or 3; p is 0, 1, 2 or 3; L is (CH.sub.2).sub.iO(CH.sub.2).sub.j, (CH.sub.2).sub.iO(CH.sub.2).sub.jC(O), (CH.sub.2).sub.iC(O)NH(CH.sub.2).sub.j, (CH.sub.2CH.sub.2O).sub.i(CH.sub.2).sub.jC(O), (CH.sub.2).sub.i(CH.sub.2CH.sub.2O).sub.jC(O), (OCH.sub.2CH.sub.2).sub.i, (CH.sub.2).sub.m, (CH.sub.2).sub.rC(O), (CH.sub.2).sub.kC(O)NH(CH.sub.2CH.sub.2O).sub.g(CH.sub.2).sub.hC(O), (CH.sub.2).sub.bC(O)NHCH[(CH.sub.2).sub.dNHC(O)(CH.sub.2CH.sub.2O).sub.e(CH.sub.2).sub.fCH.sub.3], ##STR00183## W represents a linking group, and is NHCH.sub.2C(O)NH, NHCH.sub.2CH.sub.2C(O)NH, C(O)NH, NHC(O), O, S, NH, N(CH.sub.3), C(O) or NHCH(R.sub.10)C(O)NH, further preferably NHCH.sub.2C(O)NH, C(O)NH, NHC(O) or O; R.sub.10 is H, CH.sub.3, C.sub.3H.sub.6, CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2C.sub.6H.sub.5, C.sub.8NH.sub.6, CH.sub.2C.sub.6H.sub.4OH, CH.sub.2COOH, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2COOH, (CH.sub.2).sub.4NH.sub.2, (CH.sub.2).sub.2CONH.sub.2, (CH.sub.2)SCH.sub.3, CH.sub.2OH, CH(CH.sub.3)OH or CH.sub.2SH; m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; each i is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; each j is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; r is 1, 2, 3, 4, 5, 6, 7, 8 or 9; k is 1, 2, 3, 4, 5 or 6; g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; h is 1, 2, 3, 4, 5 or 6; b is 1, 2, 3, 4, 5 or 6; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6; B is an active compound, selected from the group consisting of drug, cytotoxin, detection reagent, diagnostic reagent and targeting carrier; B is coupled to the site* or site** through a N atom or O atom in the active compound molecule; t is 0 or 1.

8. The compound or a salt thereof according to claim 7, wherein the compound has a structure represented by Formula IIa, ##STR00184## wherein: R.sub.1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.2 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.4 is ##STR00185## and in ##STR00186## the two carbonyl groups are located on the same side of the CC double bond, which is a cis structure, R.sub.11 is a C.sub.1-6 linear or branched alkyl, and R.sub.11 is optionally mono- or multi-substituted by one or more substituents selected from the group consisting of: halogen and C.sub.1-4 alkoxy; X is oxygen atom (O), nitrogen atom (N) or sulfur atom (S); Ar is aryl, heteroaryl, aryl-fused-heterocyclyl or heteroaryl-fused-heterocyclyl; R.sub.5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; R.sub.6 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; R.sub.7 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.8 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.10 is H, CH.sub.3, C.sub.3H.sub.6, CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2C.sub.6H.sub.5, C.sub.8NH.sub.6, CH.sub.2C.sub.6H.sub.4OH, CH.sub.2COOH, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2COOH, (CH.sub.2).sub.4NH.sub.2, (CH.sub.2).sub.2CONH.sub.2, (CH.sub.2)SCH.sub.3, CH.sub.2OH, CH(CH.sub.3)OH or CH.sub.2SH; q is 0, 1, 2 or 3; p is 0, 1, 2 or 3; L is (CH.sub.2).sub.iO(CH.sub.2).sub.j, (CH.sub.2).sub.iO(CH.sub.2).sub.jC(O), (CH.sub.2).sub.iC(O)NH(CH.sub.2).sub.j, (CH.sub.2CH.sub.2O).sub.i(CH.sub.2).sub.jC(O), (CH.sub.2).sub.i(CH.sub.2CH.sub.2O).sub.jC(O), (OCH.sub.2CH.sub.2).sub.i, (CH.sub.2).sub.m, (CH.sub.2).sub.rC(O), (CH.sub.2).sub.kC(O)NH(CH.sub.2CH.sub.2O).sub.g(CH.sub.2).sub.hC(O), (CH.sub.2).sub.bC(O)NHCH[(CH.sub.2).sub.dNHC(O)(CH.sub.2CH.sub.2O).sub.e(CH.sub.2).sub.fCH.sub.3], ##STR00187## m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; r is 1, 2, 3, 4, 5, 6, 7, 8 or 9; each i is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; each j is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; k is 1, 2, 3, 4, 5 or 6; g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; h is 1, 2, 3, 4, 5 or 6; b is 1, 2, 3, 4, 5 or 6; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6; B is an active compound, selected from the group consisting of drug, cytotoxin, detection reagent, diagnostic reagent and targeting carrier; B is coupled to the site* or site** through a N atom or O atom in the active compound molecule; t is 0 or 1.

9. (canceled)

10. (canceled)

11. The compound or a salt thereof according to claim 7, wherein the compound has a structure represented by Formula II-1, Formula II-2, Formula II-3 or Formula II-4, ##STR00188## wherein, R.sub.4, R.sub.10, L and B are defined as described in claim 7; or the compound has a structure represented by Formula II-5, Formula II-6, Formula II-7 or Formula II-8, ##STR00189## wherein, R.sub.10, L and B are defined as described in claim 7; or the compound has a structure represented by Formula II-9, Formula II-10, Formula II-11, Formula II-12 or Formula II-13, ##STR00190## ##STR00191## wherein, R.sub.10 and B are defined as described in claim 7; or the compound represented by Formula II or Formula IIa has a structure represented by Formula II-14, Formula II-15, Formula II-16, Formula II-17 or Formula II-18, ##STR00192## wherein, B is defined as described in claim 7.

12. (canceled)

13. A compound represented by Formula III or Formula III or a salt thereof, ##STR00193## wherein: R.sub.1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.2 is hydrogen, methyl, ethyl, n-propyl or isopropyl; X is oxygen atom (O), nitrogen atom (N) or sulfur atom (S); Ar is aryl, heteroaryl, aryl-fused-heterocyclyl or heteroaryl-fused-heterocyclyl; R.sub.5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; R.sub.6 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; R.sub.7 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.8 is hydrogen, methyl, ethyl, n-propyl or isopropyl; q is 0, 1, 2 or 3; p is 0, 1, 2 or 3; L is (CH.sub.2).sub.iO(CH.sub.2).sub.j, (CH.sub.2).sub.iO(CH.sub.2).sub.jC(O), (CH.sub.2).sub.iC(O)NH(CH.sub.2).sub.j, (CH.sub.2CH.sub.2O).sub.i(CH.sub.2).sub.jC(O), (CH.sub.2).sub.i(CH.sub.2CH.sub.2O).sub.jC(O), (OCH.sub.2CH.sub.2).sub.i, (CH.sub.2).sub.m, (CH.sub.2).sub.rC(O), (CH.sub.2).sub.kC(O)NH(CH.sub.2CH.sub.2O).sub.g(CH.sub.2).sub.hC(O), (CH.sub.2).sub.bC(O)NHCH[(CH.sub.2).sub.dNHC(O)(CH.sub.2CH.sub.2O).sub.e(CH.sub.2).sub.fCH.sub.3], ##STR00194## W represents a linking group, and is NHCH.sub.2C(O)NH, NHCH.sub.2CH.sub.2C(O)NH, C(O)NH, NHC(O), O, S, NH, N(CH.sub.3), C(O) or NHCH(R.sub.10)C(O)NH, further preferably NHCH.sub.2C(O)NH, C(O)NH, NHC(O) or O; R.sub.10 is H, CH.sub.3, C.sub.3H.sub.6, CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2C.sub.6H.sub.5, C.sub.8NH.sub.6, CH.sub.2C.sub.6H.sub.4OH, CH.sub.2COOH, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2COOH, (CH.sub.2).sub.4NH.sub.2, (CH.sub.2).sub.2CONH.sub.2, (CH.sub.2)SCH.sub.3, CH.sub.2OH, CH(CH.sub.3)OH or CH.sub.2SH, preferably; m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; each i is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; each j is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; r is 1, 2, 3, 4, 5, 6, 7, 8 or 9; k is 1, 2, 3, 4, 5 or 6; g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; h is 1, 2, 3, 4, 5 or 6; b is 1, 2, 3, 4, 5 or 6; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6; B is an active compound, selected from the group consisting of drug, cytotoxin, detection reagent, diagnostic reagent and targeting carrier; B is coupled to the site* or site** through a N atom or O atom in the active compound molecule; t is 0 or 1; A is a targeting compound, selected from the group consisting of: protein, antibody, polypeptide, enzyme and small molecule; n is a number between 0.5 and 8.5.

14. The compound or a salt thereof according to claim 13, wherein the compound has a structure represented by Formula IIIa, ##STR00195## wherein: R.sub.1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.2 is hydrogen, methyl, ethyl, n-propyl or isopropyl; X is oxygen atom (O), nitrogen atom (N) or sulfur atom (S); Ar is aryl, heteroaryl, aryl-fused-heterocyclyl or heteroaryl-fused-heterocyclyl; R.sub.5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, iodine, hydroxyl or cyano; R.sub.6 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; R.sub.7 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.8 is hydrogen, methyl, ethyl, n-propyl or isopropyl; R.sub.10 is H, CH.sub.3, C.sub.3H.sub.6, CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2C.sub.6H.sub.5, C.sub.8NH.sub.6, CH.sub.2C.sub.6H.sub.4OH, CH.sub.2COOH, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2COOH, (CH.sub.2).sub.4NH.sub.2, (CH.sub.2).sub.2CONH.sub.2, (CH.sub.2)SCH.sub.3, CH.sub.2OH, CH(CH.sub.3)OH or CH.sub.2SH; q is 0, 1, 2 or 3; p is 0, 1, 2 or 3; L is (CH.sub.2).sub.iO(CH.sub.2).sub.j, (CH.sub.2).sub.iO(CH.sub.2).sub.jC(O), (CH.sub.2).sub.iC(O)NH(CH.sub.2).sub.j, (CH.sub.2CH.sub.2O).sub.i(CH.sub.2).sub.jC(O), (CH.sub.2).sub.i(CH.sub.2CH.sub.2O).sub.jC(O), (OCH.sub.2CH.sub.2).sub.i, (CH.sub.2).sub.m, (CH.sub.2).sub.rC(O), (CH.sub.2).sub.kC(O)NH(CH.sub.2CH.sub.2O).sub.g(CH.sub.2).sub.hC(O), (CH.sub.2).sub.bC(O)NHCH[(CH.sub.2).sub.dNHC(O)(CH.sub.2CH.sub.2O).sub.e(CH.sub.2).sub.fCH.sub.3], ##STR00196## m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; r is 1, 2, 3, 4, 5, 6, 7, 8 or 9; each i is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; each j is independently 1, 2, 3, 4, 5, 6, 7, 8 or 9; k is 1, 2, 3, 4, 5 or 6; g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; h is 1, 2, 3, 4, 5 or 6; b is 1, 2, 3, 4, 5 or 6; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6; B is an active compound, selected from the group consisting of drug, cytotoxin, detection reagent, diagnostic reagent and targeting carrier; B is coupled to the site* or site** through a N atom or O atom in the active compound molecule; t is 0 or 1; A is a targeting compound, selected from the group consisting of: protein, antibody, polypeptide, enzyme and small molecule; n is a number between 0.5 and 8.5.

15. (canceled)

16. (canceled)

17. The compound or a salt thereof according to claim 13, wherein the compound has a structure represented by Formula III-1, Formula III-2, Formula III-3 or Formula III-4, ##STR00197## wherein, A, R.sub.10, L, B and n are defined as described in claim 13; or the compound has a structure represented by Formula III-5, Formula III-6, Formula III-7, Formula III-8 or Formula III-9, ##STR00198## wherein, A, B, R.sub.10 and n are defined as described in claim 13; or the compound represented by Formula III or Formula IIIa has a structure represented by Formula III-10, Formula III-11, Formula III-12, Formula III-13 or Formula III-14, ##STR00199## wherein, A, B and n are defined as described in claim 13.

18. A pharmaceutical composition comprising at least one of the compound or a salt thereof according to claim 13, and one or more pharmaceutically acceptable carriers or excipients.

19. (canceled)

20. A method for diagnosis, prevention or treatment of a disease or condition or alleviation of a severity of the disease or condition, the method comprising administering to a patient in need of such treatment an effective amount of the compound or a salt thereof according to claim 13, wherein the disease or condition is selected from the group consisting of tumor, infectious disease, hematological disease, metabolic disease, and inflammation.

21. (canceled)

22. The compound or a salt thereof according to claim 1, wherein the compound is selected from the group consisting of: ##STR00200## ##STR00201## ##STR00202##

23. The compound or a salt thereof according to claim 7, wherein the compound is selected from the group consisting of: ##STR00203## ##STR00204## ##STR00205## ##STR00206## ##STR00207##

24. The compound or a salt thereof according to claim 13, wherein the compound is selected from the group consisting of: ##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212## wherein, MAB is a monoclonal antibody, and n is defined as described in claim 13.

25. The compound or a salt thereof according to claim 13, wherein B is selected from the group consisting of: auristatin, monomethyl-auristatin E (MMAE), maytansine or derivatives thereof (such as maytansinoids, DM1, DM3, DM4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxin and derivatives thereof.

26. The compound or a salt thereof according to claim 13, wherein A is selected from the group consisting of: anti-HER2 humanized monoclonal antibody mil40, trastuzumab (HERCEPTIN), pertuzumab (PERJETA), cetuximab (ERBITUX), panitumumab (VECTIBIX), rituximab (RITUXAN), alemtuzumab (CAMPATH), ibritumomab (ZEVALIN), tositumomab (BEXXAR), ofatumumab (ARZERRA), bevacizumab (AVASTIN), ipilimumab (YERVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), Avelumab (Bavencio), Atezolizumab (Tecentriq), durvalumab (Imfinzi), sacituzumab, rovalpituzumab, and biological analogues thereof, and A is coupled to the site # through a S atom in the targeting compound molecule.

27. The method according to claim 20, the tumor is selected from the group consisting of cancer, lymphoma, lymphoid tumor, blastoma, sarcoma and leukemia.

28. The method according to claim 27, the cancer is selected from the group consisting of: breast cancer; squamous cell carcinoma; lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of lung and squamous cell carcinoma of lung; peritoneal cancer; liver cancer; gastric cancer; gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urethral cancer; hepatocellular tumor; breast cancer; intestinal cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma; multiple myeloma and B-cell lymphoma; brain cancer; gallbladder cancer; esophageal cancer; cholangiocarcinoma; head and neck cancer and related metastatic tumor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0678] FIG. 1 shows the stability evaluation results of ADC-1 and NAC-L01-MMEA in plasma.

[0679] FIG. 2 shows the degradation curve of ADC-1 over time under hypoxic environment, wherein Hypoxia represents a condition in which there is not enough oxygen (oxygen concentration is 0.1%), Oxygen represents normoxia; E represents nitroreductase (Human NADPH CYP-reducates), N represents the reducing substance NADPH.

[0680] FIG. 3 shows the release curves of ADC-1 toxin MMAE under different conditions, wherein Hypoxia represents a condition in which where is not enough oxygen (oxygen concentration is 0.1%), Oxygen represents normoxia; E represents nitroreductase (Human NADPH CYP-reducates), N represents the reducing substance NADPH.

[0681] FIG. 4 shows the detection results of MMEA contents in extracts of NCI-N87 and BT-474 cells treated with ADC-1 after 24 hours of administration, in which: A1 represents the detected chromatographic peak of toxin MMAE, A2 represents the quantitative standard curve; B1 represents the detected chromatographic peak of MMAE in NCI-N87 cells, B2 represents the blank control corresponding to B1; C1 represents the detected chromatographic peak of MMAE in BT-474 cells, and C2 represents the blank control corresponding to C1. The results show that the HPLC retention time of the extracts of NCI-N87 and BT-474 cells treated with ADC-1 is very close to the retention time of the MMAE standard, which proves that under the condition of 0.1% 02 partial pressure, ADC-1 can successfully release the carried cytotoxin MMAE in the in vitro cultured NCI-N87 and BT-474 cells, and then induce tumor cell apoptosis through the released cytotoxin.

[0682] FIG. 5 shows the curves of tumor volume over time in the xenograft model of human breast cancer cell line BT-474 after administration. The results show that ADC-1 shows better in vivo tumor inhibitory activity than the naked antibody, part of the tumors of the test animals in the medium-dose administration group (3 mg/kg) disappeared, and the tumor disappeared continuously after the drug withdraw.

[0683] FIG. 6 shows the anti-tumor effect of ADC-1 in the xenograft model of human breast cancer cell line BT-474 under different dosages. The results show that the tumor disappearance of the test animals shows a significant dose-efficacy dependence relationship, the tumors of the test animals in the ADC-1 low- and medium-dose administration groups (0.75 mg/kg, 1.5 mg/kg, 3.0 mg/kg) are inhibited and partially disappeared, while the tumors of the test animals in the high-dose group (6.0 mg/kg) basically completely disappeared and persistently passed away.

[0684] FIG. 7 shows the curves of body weight change of animals in the xenograft model of human breast cancer cell line BT-474 after receiving ADC-1 treatment. The results show that the test animals in the ADC-1 treatment groups show no significant body weight loss during the treatment process and the observation period of drug withdrawal, indicating that ADC-1 has preliminary safety at the therapeutic doses.

[0685] FIG. 8 shows the anti-tumor effect of ADC-1 in the xenograft model of human gastric cancer cell line NCI-N87 under different dosages. The results show that ADC-1 also shows a significant dose-efficacy dependence relationship in the xenograft model of human gastric cancer cell line NCI-N87.

[0686] FIGS. 9 and 10 show the curves of tumor volume over time in the xenograft model of human gastric cancer cell line NCI-N87 after administration. The results show that compared with the vehicle blank control group and the naked antibody positive control group at the same dose (5 mg/kg), ADC-1 shows therapeutic advantages with significant differences (P(Vehicle VS ADC)=0.0178, P (mAb VS ADC)=0.0028). In addition, compared with single administration, the combined administration group has the best tumor-inhibitory activity. The drug efficacy of the combined administration group is significantly better than that of the ADC-1 single administration test group (P=0.0097), but it shows no significant therapeutic advantage (P=0.6430) in comparison with chemotherapeutic drugs.

[0687] FIG. 11 shows the curves of body weight change of the animals in the xenograft model of human gastric cancer cell line NCI-N87 after receiving ADC-1 treatment. The results show that the body weights of the animals in all ADC-1 test groups increase steadily, while the chemotherapeutic drug (Doxetaxel) shows a significant weight loss (P(Vehicle VS Doxetaxel)=0.0422), which reflects to a certain extent that ADC-1 is safer than the chemotherapy drug.

[0688] FIG. 12 shows the curves of body weight change of CD-1 mice after receiving drug treatment at different dosages. The results show that the test animals in the ADC-1 administration groups show no obvious adverse reactions, body weight loss and death at the doses of 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg and 160 mg/kg, indicating that ADC-1 has a very high therapeutic safety window, and will not cause obvious intolerance in the test animals when it is administered at therapeutic doses or even larger doses.

SPECIFIC MODELS FOR CARRYING OUT THE DISCLOSURE

[0689] The embodiments of the present application will be described in detail below in combination with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If specific conditions are not indicated in the examples, they shall be carried out in accordance with conventional conditions or conditions recommended by the manufacturers. The reagents or instruments used without giving manufacturers are all conventional products that were commercially available.

Example 1: Preparation of ADC-1

1) Preparation of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (MCOH)

[0690] ##STR00096##

[0691] Maleic anhydride (4.86 g, 49.55 mmol) was added into a 500 mL three-necked flask, dissolved with acetic acid (150 mL), after the dissolution was completed, 6-aminohexanoic acid (5.0 g, 38.11 mmol) was added, the resulting reaction solution was heated to 120 C. and refluxed for 6 hours. After the reaction was completed, the reaction solution was cooled to room temperature, poured into distilled water, and extracted with an appropriate amount of ethyl acetate for several times. The organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na.sub.2SO.sub.4 overnight, concentrated, and purified by chromatography to obtain a white powdery solid (5.92 g, 74% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 11.98 (br, 1H), 7.01 (s, 2H), 3.39 (t, J=7.3 Hz, 2H), 2.17 (t, J=7.3 Hz, 2H), 1.51-1.44 (m, 2H), 1.24-1.17 (m, 2H). MS (ESI) m/z: 210.0 [MH].sup..

2) Preparation of (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidin-1-yl ester (MCOSu)

[0692] ##STR00097##

[0693] MCOH (4.66 g, 22 mmol) was added into a 250 mL three-necked flask, dissolved with THF (100 mL), after the dissolution was completed, NHS (5.08 g, 44 mmol) and 2,4,6,-trimethylpyridine (10.65 g, 88 mmol) were added in sequence. After the addition was completed, the three-necked flask was transferred to a low temperature reaction tank at 5 C. and the resulting mixture was cooled down at stirring; trifluoroacetic anhydride (TFAA, 9.24 g, 44 mmol) was slowly added dropwise. After the addition was completed, the three-necked flask was transferred to room temperature and the reaction was continued for 1 hour. The solvent was removed by evaporation under reduced pressure, and the residue obtained was re-dissolved with EA (200 mL), and washed with IN hydrochloric acid, saturated NaHCO.sub.3 and saturated NaCl for 3 times. The concentrated crude product was further purified by column chromatography to obtain a viscous oil, which was placed at a low temperature and became a white lumpy solid (5.03 g, 69% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 6.69 (s, 2H), 3.53 (t, J=7.3 Hz, 2H), 2.84 (s, J=3.6 Hz, 2H), 2.61 (t, J=7.3 Hz, 2H), 1.78 (m, 2H), 1.63 (m, 2H), 1.45-1.37 (m, 2H). MS (ESI) m/z: 309.4 [M+H].sup.+; 331.2 [M+Na].sup.+.

3) Preparation of ethyl methylaminoacetate hydrochloride

[0694] ##STR00098##

[0695] SOCl.sub.2 (107 mL, 1.48 mol) was slowly added dropwise to a 500 mL eggplant-shaped flask containing 0 C. ethanol solution, sarcosine (120 g, 1.35 mol) was added in batches under stirring, continuously stirred for 30 minutes, then slowly heated to reflux, and the reaction was continued for 2 hours. The reaction solution was cooled to room temperature, then the solvent was removed by evaporation under reduced pressure to obtain a white crystalline solid, which showed basically no raw material remained in TLC detection (close to a quantitative reaction) and was directed used in the next step without further purification (Procedure of Methyl sarcosine hydrochloride; Meng F, ect. Mol Cancer Ther, 2012, 11(3): 740-751.).

4) Preparation of ethyl (formyl-methyl-amino)acetate

[0696] ##STR00099##

[0697] Ethyl methylaminoacetate hydrochloride (188 g, 1.35 mol) was added into a 1000 mL three-necked flask, and dissolved by adding ethanol (250 mL), and then anhydrous K.sub.2CO.sub.3 (186 g, 1.35 mol) and ethyl formate (163 mL, 2.0 mol) were respectively added to the reaction solution. The resulting reaction solution was heated and refluxed for 3 hours, then filtered to remove insoluble salts, the filtrate was concentrated to obtain a crude target product as a pale yellow oil, which was not further purified and directly used in the next reaction (Procedure of Methyl 2-(N-methylformamido)acetate; Meng F, ect. Mol Cancer Ther, 2012, 11(3): 740-751.).

5) Preparation of (E)-2-carbethoxy-2-(formyl-methyl-amino)vinyl alcohol sodium salt

[0698] ##STR00100##

[0699] Ethyl (formyl-methyl-amino)acetate (31.4 g, 0.24 mol) was added to a 500 mL three-necked flask, and dissolved with ethyl formate (100 mL), the three-necked flask was placed in a low temperature reaction tank to be cooled to 0 C., then NaH (11.5 g, 0.36 mol) was added in batches. The reaction was carried out for 1 hour, then the reaction solution was slowly heated to room temperature, and then the reaction was continued under stirring overnight. The reaction solution was concentrated, a yellow sticky substance like chewing gum was obtained, which was not further purified and directly used in the next step of reaction (Procedure of Sodium 3-methoxy-2-(N-methylformamido)-3-oxoprop-1-en-1-olate; Meng F, ect. Mol Cancer Ther, 2012, 11 (3): 740-751.).

6) Preparation of ethyl (E)-2-(formyl-methyl-amino)-3-hydroxyacrylate

[0700] ##STR00101##

[0701] (E)-2-carbethoxy-2-(formyl-methyl-amino)vinyl alcohol sodium salt (43.3 g, 0.24 mol) was added into a 500 mL three-necked flask and dissolved with ethanol (150 mL), concentrated hydrochloric acid (66 mL) was added, and the resulting reaction solution was heated and refluxed for 2 hours. The reaction solution was filtered to remove insoluble substance, and the filtrate was concentrated to obtain about 40 g of a crude product as a black oil, which was not further purified and directly used in the next reaction (Procedure of Ethyl 3-hydroxy-2-(methylamino)acrylate; Meng F, ect. Mol Cancer Ther, 2012, 11(3): 740-751.).

7) Preparation of ethyl 2-amino-3-methyl-3H-imidazole-4-carboxylate

[0702] ##STR00102##

[0703] Ethyl (E)-2-(formyl-methyl-amino)-3-hydroxyacrylate (65.3 g, 0.45 mol) was added to a 500 mL three-necked flask and dissolved with 10% acetic acid (250 mL), and sodium acetate (111 g, 1.35 mol) and cyanamide (37.8 g, 0.9 mol) were added. The resulting reaction solution was heated and refluxed for 2 hours. The reaction solution was concentrated under reduced pressure to remove acetic acid, and NaHCO.sub.3 solid was added to the resulting filtrate to adjust the pH to about 8. The aqueous phase was extracted with EA for several times, the organic phases were combined and washed with saturated NaCl for 3 times, dried over Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure, and the obtained crude product was purified by column chromatography to obtain an orange solid powder (15 g, the total yield of the above reactions was 19%). .sup.1H-NMR (400 MHz, DMSO-d6) 7.28 (s, 1H), 6.20 (s, 1H), 4.15 (q, J=7.0 Hz, 2H), 3.52 (s, 3H), 1.23 (t, J=7.0 Hz, 3H). MS (EI) m/z: 170.3 [M+H].sup.+; 192.1 [M+Na].sup.+.

8) Preparation of ethyl 3-methyl-2-nitro-3H-imidazole-4-carboxylate

[0704] ##STR00103##

[0705] Sodium nitrite (42 g, 0.6 mol) was added to a 1000 mL three-necked flask, dissolved with distilled water (250 mL) and cooled to 5 C., then ethyl 2-amino-3-methyl-3H-imidazole-4-carboxylate (8.5 g, 50 mol) in acetic acid solution (70 mL) was added dropwise, after the addition was completed, the resulting reaction solution was slowly heated to room temperature and the reaction was continued for 16 hours. The reaction solution was extracted with DCM for several times, then the organic phases were combined, washed with saturated NaCl for 3 times, and dried over Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure, and the obtained crude product was purified by column chromatography to obtain a pale yellow solid powder (6.25 g, 63% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.75 (s, 1H), 4.40 (q, J=7.0 Hz, 2H), 4.30 (s, 3H), 1.41 (t, J=7.0 Hz, 3H). MS (ESI) m/z: 200.2 [M+H].sup.+; 222.1 [M+Na].sup.+.

9) Preparation of 3-methyl-2-nitro-3H-imidazole-4-carboxylic acid

[0706] ##STR00104##

[0707] Ethyl 3-methyl-2-nitro-3H-imidazole-4-carboxylate (6.6 g, 33 mol) was added into a 250 mL three-necked flask, dissolved with an aqueous ethanol solution (120 mL), and NaOH (4 g, 100 mmol) was added. The reaction was carried out under stirring at room temperature for 16 hours, then concentrated HCl was added to adjust the pH to about 1. The reaction solution was extracted with EA for several times, the organic phases were combined, washed with saturated NaCl for 3 times, and dried over Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure to obtain a pale yellow solid powder (5.56 g, 97% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 13.83 (br, 1H), 7.74 (s, 1H), 4.18 (s, 1H). MS (ESI) m/z: 172.2 [M+H].sup.+; 169.9 [MH].sup..

10) Preparation of (3-methyl-2-nitro-3H-imidazol-4-yl)methanol

[0708] ##STR00105##

[0709] 3-Methyl-2-nitro-3H-imidazole-4-carboxylic acid (1.0 g, 5.8 mol) was added into a 250 mL three-necked flask, and dissolved with anhydrous THF (100 mL), then triethylamine (1.3 mL, 9.3 mmol) was added, and then the three-necked flask was placed in a low temperature reaction tank to be cooled to 40 C., isobutyl chloroformate (1.2 mL, 9.3 mmol) was slowly added dropwise, after the addition was completed, the reaction solution was warmed to room temperature, and the reaction was continued under stirring for 1 hour. NaBH.sub.4 (4 eq) and THF-H.sub.2O (3:1, 5 mL) were added to the reaction solution. After the reaction was completed, the reaction solution was filtered and concentrated, extracted with DCM for several times, the organic phases were combined, washed with saturated NaCl for 3 times, and dried over Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure, and the obtained crude product was purified by column chromatography to obtain a pale yellow solid powder (0.76 g, 69% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 7.12 (s, 1H), 5.51 (t, J=5.4 Hz, 1H), 4.54 (d, J=5.4 Hz, 2H), 3.92 (s, 3H). MS (ESI) m/z: 158.4 [M+H].sup.+.

11) Preparation of 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole

[0710] ##STR00106##

[0711] (3-Methyl-2-nitro-3H-imidazol-4-yl)methanol (0.5 g, 3.2 mol) was added into a 50 mL eggplant-shaped flask, and dissolved with anhydrous THF (10 mL), then DIPEA (0.67 mL, 3.8 mmol) and mesyl chloride (0.30 mL, 3.8 mmol) were added. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was diluted with EA and washed twice with 1 mol/L hydrochloric acid. The organic phase was dried with anhydrous Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure to obtain a yellow solid powder (0.45 g, 81% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 7.30 (s, 1H), 5.03 (s, 2H), 3.94 (s, 3H). MS (ESI) m/z: 176.2 [M+H].sup.+.

12) Preparation of 2-amino-4-(hydroxymethyl)phenol

[0712] ##STR00107##

[0713] 4-Hydroxy-3-nitrobenzaldehyde (0.55 g, 3.3 mmol) was added into a 100 mL three-necked flask, and dissolved with methanol (10 mL), then a catalytic amount of Pd/C was added; the air in the reaction solution was replaced with hydrogen gas, the reaction was carried out under stirring at room temperature for 4 hours in hydrogen environment. After the reaction was completed, the insoluble substance was removed by filtration, and the obtained filtrate was concentrated and purified by column chromatography to obtain a brown solid powder (0.38 g, 84% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 8.83 (s, 1H), 6.56-6.65 (m, 2H), 6.32 (dd, 1H), 4.82 (t, J=5.7 Hz, 1H), 4.47 (br, 2H), 4.25 (d, J=5.7 Hz, 2H). MS (ESI) m/z: 140.2 [M+H].sup.+; 138.0 [MH].sup..

13) Preparation of tert-butyl [(2-hydroxy-5-hydroxymethyl-phenylcarbamoyl)-methyl]-carbamate

[0714] ##STR00108##

[0715] N-Boc-glycine (0.40 g, 2.28 mmol) was added into a 50 mL eggplant-shaped flask, and dissolved with DMF (15 mL), EDCI (0.53 g, 2.74 mmol), HOBt (0.37 g, 2.74 mmol) and DIPEA (0.44 g, 3.42 mmol) were added in sequence. The reaction was carried out under stirring at room temperature for 90 minutes, then 2-amino-4-(hydroxymethyl)phenol (0.32 g, 2.28 mmol) was added, and then the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was poured into 10 times the volume of water, and extracted with EA for several times, the organic phases were combined and washed with saturated NaCl for 3 times, and dried over Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure, and the obtained crude product was purified by column chromatography to obtain an orange solid powder (0.57 g, 84% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.81 (s, 1H), 8.98 (s, 1H), 7.92 (s, 1H), 7.32 (s, 1H), 6.85 (d, J=8.2 Hz, 1H), 6.79 (d, J=8.2 Hz, 1H), 5.00 (br, 1H), 4.35 (s, 2H), 3.72 (s, 2H), 1.40 (s, 9H). MS (ESI) m/z: 297.2 [M+H].sup.+; 319.1 [M+Na].sup.+.

14) Preparation of tert-butyl {[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenylcarbamoyl]methyl}-carbamate

[0716] ##STR00109##

[0717] Tert-butyl [(2-Hydroxy-5-hydroxymethyl-phenylcarbamoyl)-methyl]-carbamate (0.10 g, 0.34 mmol) was added to a 50 mL eggplant-shaped flask, and dissolved with anhydrous DMF (5 mL), then 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole (70 mg, 0.39 mmol) and CsCO.sub.3 (165 mg, 0.51 mmol) were added in sequence. The reaction was carried out under stirring at room temperature for 4 hours, then the reaction solution was poured into distilled water (40 mL), and extracted with EA for several times, the organic phases were combined and washed for 3 times with saturated NaCl and dried over Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure, and the obtained crude product was purified by column chromatography to obtain a yellow solid powder (0.12 g, 81% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 8.92 (s, 1H), 8.01 (s, 1H), 7.37 (s, 1H), 7.32 (br, 1H), 7.22 (d, J=8.4 Hz, 1H), 7.03 (d, J=8.4 Hz, 1H), 5.30 (s, 2H), 5.15 (t, 1H), 4.41 (d, J=5.6 Hz, 2H), 4.00 (s, 3H), 3.71 (d, J=5.9 Hz, 2H), 1.34 (s, 9H). MS (ESI) m/z: 436.4 [M+H].sup.+; 458.4 [M+Na].sup.+.

15) Preparation of 2-amino-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide

[0718] ##STR00110##

[0719] Tert-butyl{[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenylcarbamoyl]methyl}-carbamate (1.44 g, 3.3 mmol) was added to a 50 mL eggplant-shaped flask, and dissolved with DCM (10 mL), then TFA (3.3 mL) was added. The reaction was carried out under stirring at room temperature for 2 hours, then the solvent was removed by evaporation under reduced pressure to obtain a crude target product (1.13 g, 99% yield), which was directly used in the next reaction without further purification. MS (ESI) m/z: 336.2 [M+H].sup.+.

16) Preparation of 2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexylamino]-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide

[0720] ##STR00111##

[0721] 2-Amino-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide (1.0 g, 2.2 mmol, crude product) was added to a 50 mL eggplant-shaped flask, and dissolved with DMF (15 mL), then an excess of DIPEA (1.0 g, 8.0 mmol) was added, and stirred at room temperature for 10 minutes, and then the intermediate MCOSu (0.68 g, 2.2 mmol) was added, the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a pale yellow solid (0.85 g, 73% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 8.95 (s, 1H), 8.33 (t, J=5.9 Hz, 1H), 7.97 (s, 1H), 7.38 (s, 1H), 7.20 (d, J=8.4 Hz, 1H), 7.03 (d, J=8.4 Hz, 1H), 6.99 (s, 2H), 5.27 (s, 2H), 5.15 (t, J=5.8 Hz, 1H), 4.39 (d, J=5.6 Hz, 2H), 3.83 (d, J=5.6 Hz, 2H). 1.99 (t, J=12.0 Hz, 2H), 1.43 (m, 4H), 1.14 (m, 2H). MS (ESI) m/z: 529.3 [M+H].sup.+; 551.3 [M+Na].sup.+; 527.5 [MH].sup..

17) Preparation of carbonic acid 3-{2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexylamino]-acetylamino}-4-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-benzyl ester 4-nitrophenyl ester (L01)

[0722] ##STR00112##

[0723] The intermediate 2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexylamino]-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide (200 mg, 1 eq) was used as raw material, and dissolved with anhydrous DMF (20 mL), then bis(p-nitrophenyl) carbonate (2 eq) and DIPEA (2 eq) were added in sequence, and then the reaction was carried out under stirring at room temperature for 12 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was dispersed and slurried in diethyl ether to obtain a crude product, which was further purified by column chromatography to obtain a target product as a white solid powder (91% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.06 (s, 1H), 8.33 (m, 1H), 8.31 (dt, J=9.0 Hz, 2H), 8.16 (s, 1H), 7.57 (dt, J=9.0 Hz, 2H), 7.41 (s, 1H), 7.30 (d, J=8.7 Hz, 1H), 7.22 (dd, J=8.4 Hz, 1H), 6.99 (s, 2H), 5.33 (s, 2H), 5.24 (s, 2H), 3.99 (s, 3H), 3.86 (d, J=5.6 Hz, 2H), 3.32 (m, 2H), 2.00 (t, J=7.4 Hz, 1H), 1.43 (m, 4H), 1.14 (m, 2H). MS (ESI) m/z: 694.2 [M+H]+; 716.3 [M+Na]+; 692.0 [MH].sup..

18) Preparation of L01-MMAE Conjugate

[0724] ##STR00113##

[0725] The linker L01 (53 mg, 0.06 mmol) was added to a 10 mL eggplant-shaped flask, and dissolved in anhydrous DMF (3 mL), then HOBt (9.4 mg, 0.06 mmol), MMAE (50 mg, 0.07 mmol, purchased from Concortis Biosystems) and DIPEA (18.2 L) were added in sequence, and the reaction was carried out under stirring at room temperature for 18 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was further purified by column chromatography to obtain a target product as a white solid powder (75% yield). HRMS (ESI) m/z: 1272.6876 [M+H].sup.+; 1294.6697 [M+Na].sup.+; 636.8487 [M+2H].sup.2+.

19) Preparation of ADC-1

[0726] ##STR00114##

[0727] By using the method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.), the L01-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. Co., Ltd., a biosimilar of Herceptin) to obtain a target antibody-drug conjugate ADC-1, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 2: Preparation of ADC-2

1) Preparation of 4-[(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-methyl]-cyclohexanecarboxylic acid

[0728] ##STR00115##

[0729] By using a method similar to that for the synthesis of MCOH, trans-4-(aminomethyl)cyclohexanecarboxylic acid (7.86 g, 50.0 mmol) and maleic anhydride (4.90 g, 50.0 mmol) were added to a 500 mL three-necked flask, dissolved with DMF (250 mL), then heated to 120 C. and refluxed for 6 hours. After the reaction was completed, the reaction solution was cooled to room temperature, poured into distilled water, and extracted with an appropriate amount of ethyl acetate for several times. The organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na.sub.2SO.sub.4 overnight. The solvent was removed by concentration to obtain a white solid powder (9.96 g, 84% yield), which was used directly in the next reaction.

2) Preparation of 2,5-dioxo-pyrrolidin-1-yl 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexane carboxylate (SMCC)

[0730] ##STR00116##

[0731] N-hydroxysuccinimide (23.0 g, 200 mmol) was added into a 1000 mL three-necked flask, dissolved in DMF (250 mL), then stirred at 0 C. for 30 minutes, and trifluoroacetic anhydride (27.8 mL, 200 mmol) was added dropwise. The reaction mixture was stirred for 10 minutes, trimethylpyridine (26.4 mL, 200 mmol) was added dropwise, and stirred for 10 min, then 4-[(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-methyl]-cyclohexanecarboxylic acid (13.1 g, 55 mmol) was added, continuously reacted at 0 C. for 2 hours, then slowly warmed to room temperature and continuously reacted for 18 hours. After the reaction was completed, chloroform (300 mL) and hydrochloric acid solution (1 mol/L, 250 mL) were added to the reaction solution, and then the resulting mixture was extracted with DCM. The organic layer obtained by the extraction was washed twice with hydrochloric acid solution (1 mol/L) (2250 mL), dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude yellow solid, which was slurried in diethyl ether (3200 mL) to obtain a white powdery solid (15 g, 90% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 6.71 (s, 1H), 3.39 (d, J=7.0 Hz, 2H), 2.82 (d, J=7.3 Hz, 4H), 2.58 (m, 1H), 2.15 (m, 2H), 1.80 (m, 2H), 1.56 (m, 1H), 1.54 (m, 2H), 1.06 (m, 2H). MS (ESI) m/z: 352.6 [M+NH.sub.4].sup.+; 357.4 [M+Na].sup.+.

3) Preparation of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)]-cyclohexanecarboxylic acid {[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenylcarbamoyl]-methyl}amide

[0732] ##STR00117##

[0733] By referring to the method for preparation of 2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexylamino]-N-[5-hydroxylmethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide as described in step 16) of Example 1, a pale yellow solid powder (61% yield) was obtained. .sup.1H-NMR (400 MHz, DMSO-d6) 9.06 (s, 1H), 8.34 (br, 1H), 8.31 (dt, J=9.0 Hz, 2H), 8.16 (s, 1H), 7.57 (dt, J=9.0 Hz, 2H), 7.40 (s, 1H), 7.30 (d, J=8.7 Hz, 1H), 7.22 (dd, J=8.4 Hz, 1H), 6.99 (s, 2H), 5.33 (s, 2H), 5.24 (s, 2H), 3.99 (s, 1H), 3.86 (d, J=5.6 Hz, 2H), 3.32 (m, 2H), 2.00 (t, J=7.4 Hz, 2H), 1.43 (m, 4H), 1.15 (m, 2H). MS (ESI) m/z: 694.2 [M+H]+; 716.3 [M+Na].sup.+; 692.0 [MH].sup..

4) Preparation of carbonic acid 3-(2-{[4-[(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)cyclohexanecarbonyl]-amino}-acetylamino)-4-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)benzylester 4-nitro-phenyl ester (L02)

[0734] ##STR00118##

[0735] The intermediate 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)]-cyclohexanecarboxylic acid {[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenylcarbamoyl]-methyl}amide was used as raw material, and a preparation method similar to that of L01 was used for synthesis of the target product. A pale yellow solid powder (90% yield) was obtained. .sup.1H-NMR (400 MHz, DMSO-d6) 9.01 (s, 1H), 8.31 (dt, J=9.3 Hz, 2H), 8.22 (t, J=5.7 Hz, 1H), 8.17 (s, 1H), 7.56 (dt, J=9.3 Hz, 2H), 7.39 (s, 1H), 7.30 (d, J=8.7 Hz, 2H), 7.21 (dd, J=6.6 Hz, 1H), 7.01 (s, 2H), 5.32 (s, 2H), 5.24 (s, 2H), 3.99 (s, 3H), 3.84 (d, J=5.6 Hz, 2H), 3.23 (d, J=7.0 Hz, 2H), 2.04 (tt, J=11.8 Hz, 1H), 1.63 (m, 2H), 1.48 (m, 1H), 1.18 (m, 2H), 0.86 (qd, 2H). MS (ESI) m/z: 720.3 [M+H].sup.+; 737.6 [M+NH.sub.4].sup.+; 742.8 [M+Na].sup.+.

5) Preparation of L02-MMAE Conjugate

[0736] ##STR00119##

[0737] Carbonic acid 3-(2-{[4-[(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)cyclohexanecarbonyl]-amino}-acetylamino)-4-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)benzyl ester 4-nitro-phenyl ester (L02) was used as raw material, and a preparation method similar to that of L01-MMAE conjugate was used for synthesis of the target product. The target product was a white solid powder (72% yield). HRMS (ESI) m/z: 1298.7041 [M+H].sup.+; 1320.6851 [M+Na].sup.+.

6) Preparation of ADC-2

[0738] ##STR00120##

[0739] By using the method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.), the L02-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 to obtain a target antibody-drug conjugate ADC-2, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 3: Preparation of ADC-3

1) Preparation of tert-butyl {[5-hydroxymethyl-2-(4-nitro-benzyloxy)phenylcarbamoyl]methyl}carbamate

[0740] ##STR00121##

[0741] Tert-butyl [(2-Hydroxy-5-hydroxymethyl-phenylcarbamoyl)-methyl]-carbamate (0.15 g, 0.50 mmol) was added to a 50 mL eggplant-shaped flask and dissolved in anhydrous DMF (5 mL), then p-nitrobenzyl bromide (0.13 mg, 0.60 mmol) and CsCO.sub.3 (0.26 mg, 0.8 mmol) were added in sequence. The reaction was carried out under stirring at room temperature for 5 hours, then the reaction solution was poured into distilled water (40 mL) to precipitate solid insoluble substances. The resulting mixture containing the solid insoluble substances was extracted with EA for several times, the organic phases were combined and washed for 3 times with saturated NaCl and dried over Na.sub.2SO.sub.4. The solvent was removed by evaporation under reduced pressure to obtain a pale yellow solid powder (0.12 g, 95% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.11 (s, 1H), 8.27 (d, J=8.7 Hz, 2H), 8.02 (s, 1H), 7.77 (d, J=8.7 Hz, 2H), 7.38 (s, 1H), 7.04 (d, J=8.4 Hz, 1H), 6.98 (dd, J=8.4 Hz, 1H), 5.36 (s, 2H), 4.39 (s, 2H), 3.76 (d, J=5.9 Hz, 2H), 1.32 (s, 9H). MS (ESI) m/z: 432.2 [M+H].sup.+; 454.4 [M+Na].sup.+.

2) Preparation of 2-amino-N-[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenyl]-acetamide

[0742] ##STR00122##

[0743] Tert-butyl {[5-hydroxymethyl-2-(4-nitro-benzyloxy)phenylcarbamoyl]methyl}carbamate (0.15 g, crude product) was added into a 10 mL eggplant-shaped flask, and dissolved in EA (1 mL), then 2 mol/L HCl in ethyl acetate solution (0.3 mL) was added. The reaction was carried out under stirring at room temperature for 2 hours, then the reaction solution was directly filtered with suction to obtain a crude product as a pale pink powdery solid, which was further slurried in EA to be purified to obtain the target product (0.1 g, 95% yield). H-NMR (400 MHz, DMSO-d6) 9.87 (s, 1H), 8.25 (t, J=4.4 Hz, 2H), 7.79 (t, J=8.6 Hz, 2H), 7.03 (m, 2H), 5.38 (s, 1H), 4.40 (s, 2H), 3.81 (m, 4H). MS (ESI) m/z: 332.12 [M+H].sup.+.

3) Preparation of 6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoic acid {[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenylcarbamoyl]-methyl}amide

[0744] ##STR00123##

[0745] 2-Amino-N-[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenyl]-acetamide (0.5 g, 1.36 mmol) was added into a 50 mL eggplant-shaped flask and dissolved in DMF (15 mL), then an excess of DIPEA (0.88 g, 6.8 mmol) was added and stirred at room temperature for 10 minutes, and then the intermediate MCOSu (0.46 g, 1.5 mmol) was added, the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a reddish brown solid (0.23 g, 43% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.9 (s, 1H), 8.36 (t, 1H), 8.25 (d, J=4.4 Hz, 2H), 7.98 (s, 1H), 7.78 (d, J=8.7 Hz, 2H), 7.04 (d, J=8.4 Hz, 1H), 7.01 (s, 2H), 6.98 (d, 2H), 5.34 (s, 2H), 5.12 (t, J=5.8 Hz, 1H), 4.39 (d, J=5.9 Hz, 2H), 3.89 (d, J=5.6 Hz, 2H). 3.33 (t, J=8.4 Hz, 2H), 2.06 (t, J=7.6 Hz, 2H), 1.43 (m, 4H), 1.13 (m, 2H). MS (ESI) m/z: 525.5 [M+H].sup.+; 547.5 [M+Na].sup.+.

[0746] 4) Preparation of carbonic acid 3-{2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoyl-amino]-acetylamino}-4-(4-nitro-benzyloxy)benzyl ester 4-nitro-phenyl ester (L03)

##STR00124##

[0747] 6-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoic acid {[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenylcarbamoyl]-methyl}amide was used as raw material, and a preparation method similar to that of L01 was used for synthesis of the target product. A pale yellow solid powder was obtained (63% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.21 (s, 1H), 8.37 (t, J=5.5 Hz, 1H), 8.31 (dt, J=9.2 Hz, 2H), 8.27 (dt, J=9.0 Hz, 2H), 8.15 (s, 1H), 7.79 (d, J=8.7 Hz, 2H), 7.57 (dt, J=9.2 Hz, 2H), 7.18 (dd, J=8.4 Hz, 1H), 7.13 (d, J=8.4 Hz, 1H), 6.98 (s, 2H), 5.40 (s, 2H), 5.22 (s, 2H), 3.91 (d, J=5.6 Hz, 2H), 3.31 (m, 2H), 2.07 (t, J=7.4 Hz, 2H), 1.43 (m, 4H), 1.14 (m, 2H). MS (ESI) m/z: 690.4 [M+H].sup.+; 712.5 [M+Na].sup.+.

5) Preparation of L03-MMAE Conjugate

[0748] ##STR00125##

[0749] Carbonic acid 3-{2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoylamino]-acetylamino}-4-(4-nitro-benzyloxy)benzyl ester 4-nitro-phenyl ester was used as raw material, and a preparation method similar to that of the L01-MMAE conjugate was used for synthesis of the target product. The target product was a white solid powder (72% yield). HRMS (ESI) m/z: 1268.6921 [M+H].sup.+; 1290.6636 [M+Na].sup.+.

6) Preparation of ADC-3

[0750] ##STR00126##

[0751] By using the method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.), the L03-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 to obtain the target antibody-drug conjugate ADC-3, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 4: Preparation of ADC-4

1) Preparation of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexanecarboxylic acid {[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenylcarbamoyl]-methyl}-amide

[0752] ##STR00127##

[0753] 2-Amino-N-[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenyl]-acetamide was used as raw material, the intermediate MCOSu was replaced with SMCC, and the method for preparation of 6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoic acid {[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenylcarbamoyl]-methyl}amide as described in the step 3) of Example 3 was used for synthesis of the target product. The target product was a pale yellow solid powder (61% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.02 (s, 1H), 8.33 (t, J=5.6 Hz, 1H), 8.25 (d, J=8.7 Hz, 2H), 8.02 (s, 1H), 7.76 (d, J=8.7 Hz, 2H), 7.03 (d, J=8.4 Hz, 1H), 7.00 (s, 2H), 6.97 (dd, J=8.1 Hz, 1H), 5.34 (s, 2H), 5.11 (t, J=5.8 Hz, 1H), 4.38 (d, J=5.6 Hz, 2H), 3.87 (d, J=5.6 Hz, 2H). 3.19 (d, J=7.0 Hz, 2H), 2.08 (tt, J=12.1 Hz, 1H), 1.68 (d, J=12.32 Hz, 2H), 1.55 (d, J=12.9 Hz, 2H), 1.45 (m, 1H), 1.22 (qd, 2H), 0.78 (qd, 2H). MS (ESI) m/z: 551.5 [M+H].sup.+; 573.5 [M+Na].sup.+.

2) Preparation of carbonic acid 3-(2-{[4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexanecarbonyl]-amino}-acetylamino)-4-(4-nitrobenzyloxy)benzyl ester 4-nitro-phenyl ester (L04)

[0754] ##STR00128##

[0755] The intermediate 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexanecarboxylic acid {[5-hydroxymethyl-2-(4-nitro-benzyloxy)-phenylcarbamoyl]-methyl}-amide was used as raw material, and a preparation method similar to that of L01 was used for synthesis of the target product. The target product was a pale yellow solid powder (76% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.14 (s, 1H), 8.31 (dt, 2H), 8.27 (dt, 2H), 8.19 (s, 1H), 7.78 (d, J=8.7 Hz, 2H), 7.56 (dt, J=9.2 Hz, 2H), 7.16 (m, 2H), 7.00 (s, 2H), 5.40 (s, 2H), 5.22 (s, 2H), 3.90 (d, J=5.6 Hz, 2H), 3.19 (d, J=7.0 Hz, 2H), 2.08 (tt, J=12.0 Hz, 1H), 1.68 (d, J=13.2 Hz, 2H), 1.53 (d, J=12.9 Hz, 2H), 1.44 (m, 1H), 1.23 (m, 1H), 0.80 (qd, 2H). MS (ESI) m/z: 716.4 [M+H].sup.+; 738.4 [M+Na].sup.+.

3) Preparation of L04-MMAE Conjugate

[0756] ##STR00129##

[0757] Carbonic acid 3-(2-{[4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexanecarbonyl]-amino}-acetylamino)-4-(4-nitrobenzyloxy)benzyl ester 4-nitro-phenyl ester was used as raw material, and a preparation method similar to that of L01-MMAE was used for synthesis of the target product. The target product was a pale yellow solid powder (72% yield). HRMS (ESI) m/z: 1294.6976 [M+H].sup.+; 1316.6980 [M+Na].sup.+.

4) Synthesis of ADC-4

[0758] ##STR00130##

[0759] By using the method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.), the L04-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 to obtain the target antibody-drug conjugate ADC-4, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 5: Preparation of ADC-5

1) Preparation of (5-nitrofuran-2-yl)methanol

[0760] ##STR00131##

[0761] 5-Nitro-furan-2-carboxylic acid (0.91 g, 5.8 mmol) was dissolved in anhydrous THF (20 mL), and placed in a low temperature reaction tank at 40 C. and cooled down after the dissolution was completed. Triethylamine (1.3 mL) and isobutyl chloroformate (1.2 mL) were slowly added dropwise, respectively, after the addition was completed, the reaction was continued for 30 minutes, the reaction solution was warmed slowly to 10 C., and the reaction was continued for 1 hour. NaBH.sub.4 (1.1 g) was added in batches, and then a small amount of THF-H.sub.2O (3:1, 1.2 mL) mixed solvent was added dropwise. After the reaction was completed, an appropriate amount of THF was added to the reaction solution, then THF was removed by suction filtration, the aqueous phase was extracted with EA for several times, the organic phases were combined, washed with saturated NaCl for 3 times, dried over anhydrous Na.sub.2SO.sub.4, and concentrated under reduced pressure to obtain a crude product, which was further purified by column chromatography to obtain a white solid powder (0.42 g, 51% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.30 (d, 1H), 6.56 (d, 1H), 4.73 (s, 2H), 2.28 (m, 1H). MS (ESI) m/z: 161.0 [M+NH.sub.4]f; 142.9[MH].sup..

2) 2-Chloromethyl-5-nitro-furan

[0762] ##STR00132##

[0763] The reactant (5-nitrofuran-2-yl)methanol (0.35 g, 2.44 mmol) was dissolved in anhydrous THF (10 mL). After the dissolution was completed, DIPEA (0.51 mL, 2.93 mmol) and mesyl chloride (0.34 g, 2.93 mmol) were slowly added dropwise, respectively. After the dropwise addition was completed, the reaction was continued for 30 minutes. The reaction solution was concentrated under reduced pressure, a crude product was obtained as a yellow oil, which was further purified by column chromatography to obtain a yellow oil product (0.32 g, 81% yield). H-NMR (400 MHz, CDCl.sub.3) 7.28 (d, 1H), 6.62 (d, 1H), 4.60 (s, 2H). MS (ESI) m/z: 324.4 [2M+H].sup.+.

3) Preparation of carbonic acid 3-{2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoylamino]-acetylamino}-4-(5-nitro-furan-2-yl-methoxy)-benzyl ester 4-nitro-phenyl ester (L05)

[0764] ##STR00133##

[0765] By referring to the method for preparation of 2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexylamino]-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide as described in the step 16) of Example 1, the raw material 6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoic acid {5-hydroxymethyl-2-(5-nitro-furan-2-yl-methoxy)-phenylcarbamoyl]-methyl}-amide was prepared. Then, 6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoic acid {5-hydroxymethyl-2-(5-nitro-furan-2-yl-methoxy)-phenylcarbamoyl]-methyl}-amide was used as raw material, and a preparation method similar to that of L01 was used to obtain the target linker product (L05) as a white solid powder (73% yield). H-NMR (400 MHz, DMSO-d6) 9.06 (s, 1H), 8.33 (m, 1H), 8.31 (dt, J=9.0 Hz, 2H), 8.16 (s, 1H), 7.57 (dt, J=9.0 Hz, 2H), 7.30 (d, J=8.7 Hz, 1H), 7.30 (d, 1H), 7.22 (dd, J=8.4 Hz, 1H), 6.99 (s, 2H), 6.68 (d, 1H), 5.33 (s, 2H), 5.24 (s, 2H), 3.86 (d, J=5.6 Hz, 2H), 3.32 (m, 2H), 2.00 (t, J=7.4 Hz, 1H), 1.43 (m, 4H), 1.14 (m, 2H). MS (ESI) m/z: 680.2 [M+H].sup.+; 702.1 [M+Na].sup.+.

4) Preparation of L05-MMAE Conjugate

[0766] ##STR00134##

[0767] Carbonic acid 3-{2-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexanoylamino]-acetylamino}-4-(5-nitro-furan-2-yl-methoxy)-benzyl ester 4-nitro-phenyl ester was used as raw material, and a preparation method similar to that of L01-MMAE was used for synthesis of the target product. The target product was a white solid powder (69% yield). HRMS (ESI) m/z: 1258.6811 [M+H].sup.+; 1280.6422 [M+Na].sup.+.

5) Preparation of ADC-5

[0768] ##STR00135##

[0769] By using the method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.), the L05-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 to obtain the target antibody-drug conjugate ADC-5, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 6: Preparation of ADC-6

1) Preparation of (Z)-6-(3-carboxyacrylamido)hexanoic acid

[0770] ##STR00136##

[0771] 6-Aminohexanoic acid (3.94 g, 30 mM) was dissolved in acetic acid (100 mL) under stirring, maleic anhydride (2.94 g, 30 mM) was added and stirred at room temperature, the reaction solution became clear and transparent; the reaction was continued under stirring at room temperature for one hour, a white insoluble substance was precipitated out, then the reaction was continued for 10 hours, the reaction solution was filtered and the filter cake was washed with acetonitrile to obtain a white powdery solid, the white powdery solid was weighed as 5.7 g and had a yield of 82%. H-NMR (400M, DMSO-d6) 15.08 (br, 1H), 11.92 (br, 1H), 9.12 (t, 1H), 6.40 (d, J=12.6 Hz, 1H), 6.25 (d, J=12.6 Hz, 1H), 3.16 (t, 2H), 2.20 (t, 2H), 1.54-1.43 (m, 4H), 1.29 (m, 2H). MS m/z 228.2 ([MH].sup.).

2) Preparation of (Z)-4-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-4-oxo-but-2-enoic acid

[0772] ##STR00137##

[0773] (Z)-6-(3-carboxyacrylamido)hexanoic acid (4.00 g, 17.45 mM, 1 eq) was dissolved in DMF (70 mL) under stirring, then N-hydroxysuccinimide (NHS; 8.03 g, 69.80 mM, 4 eq) was added and stirred at room temperature, and then placed in a 5 C. cold trap to be cooled for 20 minutes, then 2,4,6-trimethylpyridine (9.31 mL, 69.80 mM, 4 eq) was added and continuously stirred for 20 minutes at 5 C., trifluoroacetic anhydride (9.80 mL, 4 eq) was slowly added dropwise within half an hour. After the addition was completed, the reaction solution was dropped into 1 mol/L HCl and extracted with chloroform for 3 to 4 times. The organic phases were combined, washed with water for 3 times, washed with 1 mol/L HCl for 2 times, washed with saturated brine for 3 times, dried over anhydrous magnesium sulfate; and then the organic phase was concentrated to obtain the target product as a white solid, the white solid was dried, and weighed as 5.11 g, which had a yield of 89.74%. .sup.1H-NMR (400M, DMSO-d6) 15.13 (1H), 9.11 (t, 1H), 6.40 (d, J=12.6 Hz, 1H), 6.25 (d, J=12.6 Hz, 1H), 3.17 (q, 2H), 2.81 (s, 4H), 2.76 (t, 2H), 1.639 (m, 2H), 1.50 (m, 2H), 1.38 (m, 2H). MS m/z 325.4 ([MH].sup.); 327.5 ([M+H].sup.+).

3) Preparation of 2,5-dioxopyrrolidin-1-yl (Z)-6-(4-methoxy-4-oxo-but-2-enamido)hexanoate

[0774] ##STR00138##

[0775] (Z)-4-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-4-oxo-but-2-enoic acid (1.50 g, 4.6 mM, 1 eq) was dissolved in DMF (25 mL) under stirring, anhydrous potassium carbonate (1.27 g, 9.2 mM, 2 eq) was added, then iodomethane (1.3 g, 9.2 mM, 2 eq) was added dropwise, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the insoluble substance was removed by filtration, the solvent was removed by concentration under reduced pressure, the residue was dissolved in the added ethyl acetate, the resulting mixture was filtered, the filtrate was washed with saturated brine for 3 times, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and re-dissolved in ethyl acetate, then the resulting solution was mixed with silica gel and purified by column chromatography to obtain the target product as a white solid, the white solid was dried, and weighed as 1.1 g, which had a yield of 66%. .sup.1H-NMR (400M, CDCl.sub.3) 8.21 (br, 1H), 6.35 (d, J=12.6 Hz, 1H), 6.13 (d, J=12.6 Hz, 1H), 3.80 (s, 3H), 3.35 (q, 2H), 2.84 (s, 4H), 2.63 (t, 2H), 1.80 (m, 2H), 1.63 (m, 2H), 1.49 (m, 2H). MS m/z 341.2 ([M+H].sup.+); 363.2 ([M+Na].sup.+).

4) Preparation of methyl (S)-4-[(6-{[2-({5-(hydroxymethyl)-2-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]phenyl}amino)-(methyl)-2-oxoethyl]amino}hexyl)amino]-4-oxo-but-2-enate

[0776] ##STR00139##

[0777] 2-Amino-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide (crude product, 134 mg, 0.4 mM, 1 eq) was added to a 50 mL eggplant-shaped flask, dissolved in DMF (10 mL), then an excess of DIPEA (103 mg, 0.8 mM, 2 eq) was added and stirred at room temperature for 10 minutes, 2,5-dioxopyrrolidin-1-yl (Z)-6-(4-methoxy-4-oxo-but-2-enamido)hexanoate (136 mg, 0.4 mM, 1 eq) was added, and the reaction was carried out under stirring at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain an orange-yellow viscous oil (crude product, 300 mg, >100% yield). The resulting oil was used directly in the next reaction. MS m/z 561.22 ([M+H].sup.+); 583.20 ([M+Na].sup.+).

5) Preparation of methyl ((S)-4-{(2-{[2-({2-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]-5-{[(4-nitrophenoxy)carbonyl]oxymethyl}phenyl}amino)-2-oxoethylamino]hexyl}amino)-4-oxo-but-2-enate (L06)

[0778] ##STR00140##

[0779] According to the method described in step 17) of Example 1, methyl (S)-4-[(6-{[2-({5-(hydroxymethyl)-2-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]phenyl}amino)-(methyl)-2-oxoethyl]amino}hexyl)amino]-4-oxo-but-2-enate (crude product, 300 mg, 0.535 mM, 1 eq) was dissolved in DMF (10 mL), bis(p-nitrophenyl) carbonate (2 eq) and DIPEA (2 eq) were added in sequence, and the reaction was carried out under stirring for 12 hours at room temperature. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by column chromatography to obtain the target product as a white solid powder (275 mg, 71% yield). .sup.1H-NMR (400M, 400M, DMSO-d6) 9.06 (s, 1H), 8.36-8.30 (m, 3H), 8.20-8.16 (m, 2H), 7.57 (dt, 2H), 7.41 (s, 1H), 7.30 (m, 1H), 7.22 (dd, 1H), 6.27 (d, J=12.6 Hz, 1H), 6.21 (d, J=12.6 Hz, 1H), 5.33 (s, 2H), 5.24 (s, 2H), 3.99 (s, 3H), 3.87 (d, 2H), 3.62 (s, 3H), 3.04 (m, 2H), 2.01 (m, 2H), 1.41 (m, 4H), 1.23 (m, 2H). MS m/z 726.23 ([M+H].sup.+); 748.22 ([M+Na].sup.+).

6) Preparation of L06-MMAE

[0780] ##STR00141##

[0781] According to the method described in step 18) of Example 1, L06 (60.6 mg, 0.084 mmol) was added to a 10 mL eggplant-shaped flask, dissolved in anhydrous DMF (3 mL), and HOBt (9.46 mg, 0.07 mmol), MMAE (50 mg, 0.07 mmol, purchased from Concortis Biosystems) and DIPEA (18.1 mg) were added in sequence, and the reaction was carried out under stirring at room temperature for 18 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was further purified by column chromatography to obtain the target product as a white solid powder (87 mg; 95% yield). HRMS (ESI) m/z: 1304.7137 [M+H].sup.+; 1326.6957 [M+Na].sup.+.

7) Preparation of ADC-6

[0782] ##STR00142##

[0783] According to the method described in step 19) of Example 1, the L06-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd., a biosimilar of Herceptin) to obtain the target antibody-drug conjugate ADC-6, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 7: Preparation of ADC-7

1) Preparation of 3-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)-N-{2-[2-(3-{[2-({5-(hydroxylmethyl)-2-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]phenyl}amino)-2-oxoethyl]amino}-3-oxo-propoxy)ethoxy]ethyl}propionamide

[0784] ##STR00143##

[0785] 2-Amino-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide (200 mg, 0.596 mmol) was added to a 25 mL eggplant-shaped flask, dissolved in DMF (5 mL), then an excess of DIPEA (154 mg, 1.193 mmol) was added, and stirred at room temperature for 10 minutes, then intermediate PEG2 (0.68 g, 2.2 mmol) was added, and the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a pale yellow viscous oil (crude product, 380 mg, >100% yield). The resulting oil was used directly in the next reaction. MS m/z 646.24 ([M+H].sup.+); 668.22 ([M+Na].sup.+).

2) Preparation of carbonic acid 3-[16-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,14-dioxo-7,10-dioxa-3,13-dialkylamide]-4-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]benzyl ester 4-nitrophenyl ester (L07)

[0786] ##STR00144##

[0787] According to the method described in step 17) of Example 1, 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{2-[2-(3-{[2-({5-(hydroxymethyl)-2-[(1-methyl-2-nitro-1H-imidazol-5-yl) methoxy]phenyl}amino)-2-oxo-ethyl]amino}-3-oxo-propoxy)ethoxy]ethyl}propionamide (crude product, 350 mg, 0.542 mM, 1 eq) was dissolved in DMF (15 mL), bis(p-nitrophenyl) carbonate (2 eq) and DIPEA (2 eq) were added in sequence, and the reaction was carried out under stirring at room temperature for 12 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by column chromatography to obtain the target product as a pale yellow solid powder (180 mg, 0.22 mM, 41% yield). .sup.1H-NMR (400M, 400M, DMSO-d6) 9.08 (s, 1H), 8.40 (m, 1H), 8.31 (dt, 2H), 8.12 (s, 1H), 8.02 (t, 1H), 7.56 (dt, 2H), 7.41 (s, 1H), 7.30 (m, 1H), 7.23 (dd, 1H), 7.00 (s, 2H), 5.33 (s, 2H), 5.24 (s, 2H), 3.99 (s, 3H), 3.89 (d, 2H), 3.65-3.45 (m, 10H), 3.14 (m, 4H), 2.30 (m, 4H). MS m/z 811.24 ([M+H].sup.+); 833.22 ([M+Na].sup.+).

3) Preparation of L07-MMAE

[0788] ##STR00145##

[0789] According to the method described in step 18) of Example 1, L07 (68.1 mg, 0.084 mmol) was added to a 10 mL eggplant-shaped flask, dissolved in anhydrous DMF (5 mL), then HOBt (9.46 mg, 0.07 mmol), MMAE (50 mg, 0.07 mmol, purchased from Concortis Biosystems) and DIPEA (18.1 mg) were added in sequence, and the reaction was carried out under stirring at room temperature for 18 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was further purified by column chromatography to obtain the target product as a white solid powder (76 mg, 78% yield). HRMS (ESI) m/z: 1389.7299 [M+H].sup.+; 1411.7091 [M+Na].sup.+.

4) Preparation of ADC-7

[0790] ##STR00146##

[0791] According to the method described in step 19) of Example 1, the L07-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd., a biosimilar of Herceptin) to obtain the target antibody-drug conjugate ADC-7, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 8: Preparation of ADC-8

1) Preparation of 1-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionylamino]-N-(2-{[5-(hydroxymethyl)-2-(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl]amino}-2-oxoethyl-3,6,9,12-tetraoxa-pentadecane-15-amide

[0792] ##STR00147##

[0793] 2-Amino-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide (134 mg, 0.4 mmol) was added to a 25 mL eggplant-shaped flask, dissolved in DMF (10 mL), an excess of DIPEA (103 mg, 1.193 mmol) was added and stirred at room temperature for 10 minutes, then the intermediate PEG4 (205 mg, 0.4 mmol) was added, and the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a pale yellow viscous oil (crude product, 400 mg, >100% yield). The resulting oil was used directly in the next reaction. MS m/z 734.30 ([M+H].sup.+); 756.28 ([M+Na].sup.+).

2) Preparation of carbonic acid 3-[22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,20-dioxo-7,10,13,16-tetraoxa-3,19-diaza-dibenzamide]-4-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]-benzyl ester 4-nitrophenyl ester (L08)

[0794] ##STR00148##

[0795] According to the method described in step 17) of Example 1, 1-[3-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)propionylamino]-N-(2-{[5-(hydroxymethyl)-2-(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl]amino}-2-oxoethyl-3,6,9,12-tetraoxa-pentadecane-15-amide (crude product, 0.4 mM, 1 eq) was dissolved in DMF (10 mL), bis(p-nitrophenyl) carbonate (2 eq) and DIPEA (2 eq) were added in sequence, and the reaction was carried out under stirring at room temperature for 12 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, the resulting residue was purified by column chromatography to obtain the target product as a yellow oil (200 mg, 0.22 mM, 56% yield). .sup.1H-NMR (400M, 400M, DMSO-d6) 9.08 (s, 1H), 8.40 (m, 1H), 8.31 (dt, 2H), 8.12 (s, 1H), 8.02 (t, 1H), 7.56 (dt, 2H), 7.41 (s, 1H), 7.30 (m, 1H), 7.23 (dd, 1H), 7.00 (s, 2H), 5.33 (s, 2H), 5.24 (s, 2H), 3.99 (s, 3H), 3.89 (d, 2H), 3.65-3.45 (m, 18H), 3.14 (m, 4H), 2.30 (m, 4H). MS m/z 899.31 ([M+H].sup.+); 921.29 ([M+Na].sup.+).

3) Preparation of L08-MMAE

[0796] ##STR00149##

[0797] According to the method described in step 18) of Example 1, L08 (75.5 mg, 0.084 mmol) was added to a 10 mL eggplant-shaped flask, dissolved in anhydrous DMF (5 mL), then HOBt (9.46 mg, 0.07 mmol), MMAE (50 mg, 0.07 mmol, purchased from Concortis Biosystems) and DIPEA (18.1 mg) were added in sequence, and the reaction was carried out under stirring at room temperature for 18 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was further purified by column chromatography to obtain the target product as a white solid powder (50.7 mg; 49% yield). HRMS (ESI) m/z: 739.3961 [M+2H].sup.2+; 750.3850[M+H+Na].sup.2+; 761.3793 [M+2Na].sup.2+.

4) Preparation of ADC-8

[0798] ##STR00150##

[0799] According to the method described in step 19) of Example 1, the L08-MMAE conjugate was coupled to anti-HER humanized monoclonal antibody mil40 (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd., a biosimilar of Herceptin) to obtain the target antibody-drug conjugate ADC-8, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 9: Preparation of ADC-9

1) Preparation of 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(2-((5-(hydroxymethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)amino)-2-oxoethyl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide

[0800] ##STR00151##

[0801] 2-Amino-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide (134 mg, 0.4 mmol) was added to a 25 mL eggplant-shaped flask, dissolved in DMF (10 mL), an excess of DIPEA (103 mg, 1.193 mmol) was added and stirred for 10 minutes at room temperature, then the intermediate PEG6 (240.64 mg, 0.4 mmol) was added, and the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a pale yellow viscous oil (crude product, 400 mg, >100% yield). The resulting oil was used directly in the next reaction. MS m/z 822.36 ([M+H].sup.+); 844.33 ([M+Na].sup.+).

2) Preparation of carbonic acid 3-[28-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,26-dioxo-7,10,13,16,19,22-hexaoxa-3,25-amide]-4-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]benzyl ester 4-nitrophenyl ester (L09)

[0802] ##STR00152##

[0803] According to the method described in step 17) of Example 1, 1-(3-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)propanamido)-N-(2-((5-(hydroxymethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)amino)-2-oxoethyl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide (crude product, 329 mg, 0.4 mM, 1 eq) was dissolved in DMF (10 mL), bis(p-nitrophenyl) carbonate (2 eq) and DIPEA were added (2 eq) in sequence, and the reaction was carried out under stirring at room temperature for 12 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, the residue was purified by column chromatography to obtain the target product as a yellow oil (130 mg, 33% yield). .sup.1H-NMR (400M, 400M, DMSO-d6) 9.08 (s, 1H), 8.40 (m, 1H), 8.31 (dt, 2H), 8.12 (s, 1H), 8.02 (t, 1H), 7.56 (dt, 2H), 7.41 (s, 1H), 7.30 (m, 1H), 7.23 (dd, 1H), 7.00 (s, 2H), 5.33 (s, 2H), 5.24 (s, 2H), 3.99 (s, 3H), 3.89 (d, 2H), 3.65-3.45 (m, 26H), 3.14 (m, 4H), 2.30 (m, 4H). MS m/z 987.36 ([M+H].sup.+); 1009.34 ([M+Na].sup.+).

3) Preparation of L09-MMAE

[0804] ##STR00153##

[0805] According to the method described in step 18) of Example 1, L09 (82.9 mg, 0.084 mmol) was added to a 10 mL eggplant-shaped flask, dissolved in anhydrous DMF (5 mL), HOBt (9.46 mg, 0.07 mmol), MMAE (50 mg, 0.07 mmol, purchased from Concortis Biosystems) and DIPEA (18.1 mg) were added in sequence, and the reaction was carried out under stirring at room temperature for 18 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was further purified by column chromatography to obtain the target product as a white solid powder (42 mg; 38% yield). HRMS (ESI) m/z: 1587.8142 [M+Na].sup.+; 783.4223 [M+2H].sup.2+.

4) Preparation of ADC-9

[0806] ##STR00154##

[0807] According to the method described in step 19) of Example 1, the L09-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd., a biosimilar of Herceptin) to obtain the target antibody-drug conjugate ADC-9, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 10: Preparation of ADC-10

[0808] 1) Preparation of 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(2-((5-(hydroxymethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)amino)-2-oxoethyl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide

##STR00155##

[0809] 2-Amino-N-[5-hydroxymethyl-2-(3-methyl-2-nitro-3H-imidazol-4-yl-methoxy)-phenyl]-acetamide (134 mg, 0.4 mmol) was added to a 25 mL eggplant-shaped flask, dissolved in DMF (10 mL), an excess of DIPEA (103 mg, 1.193 mmol) was added and stirred for 10 minutes at room temperature, then the intermediate PEG8 (276 mg, 0.4 mmol) was added, and the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a pale yellow viscous oil (crude product, 410 mg, >100% yield). The resulting oil was used directly in the next reaction. MS m/z 910.41 ([M+H].sup.+); 932.39 ([M+Na].sup.+).

2) Preparation of carbonic acid 3-[34-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,32-dioxo-7,10,13,16,19,22,25,28-octaoxa-3,31-diaza-triazaamido]-4-[(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy] benzyl ester 4-nitrophenyl ester (L10)

[0810] ##STR00156##

[0811] According to the method as described in the step 17) of Example 1, 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(2-((5-(hydroxymethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)amino)-2-oxoethyl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (410 mg, 0.45 mM) was dissolved in DMF (10 mL), bis(p-nitrophenyl) carbonate (2 eq) and DIPEA (2 eq) were added in sequence, and the reaction was carried out under stirring at room temperature for 12 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, the residue was purified by column chromatography to obtain the target product as a yellow oil (280 mg, 58% yield). .sup.1H-NMR (400M, 400M, DMSO-d6) 9.07 (s, 1H), 8.41 (m, 1H), 8.31 (dt, 2H), 8.13 (s, 1H), 8.04 (t, 1H), 7.57 (dt, 2H), 7.41 (s, 1H), 7.31 (m, 1H), 7.23 (dd, 1H), 7.00 (s, 2H), 5.34 (s, 2H), 5.24 (s, 2H), 3.99 (s, 3H), 3.90 (d, 2H), 3.65-3.46 (m, 32H), 3.15 (m, 4H), 2.31 (m, 4H). MS m/z 1075.40 ([M+H].sup.+); 1097.38 ([M+Na].sup.+).

3) Preparation of L10-MMAE

[0812] ##STR00157##

[0813] According to the method described in step 18) of Example 1, L09 (90.3 mg, 0.084 mmol) was added to a 10 mL eggplant-shaped flask, dissolved in anhydrous DMF (5 mL), then HOBt (9.46 mg, 0.07 mmol), MMAE (50 mg, 0.07 mmol, purchased from Concortis Biosystems) and DIPEA (18.1 mg) were added in sequence, and the reaction was carried out under stirring at room temperature for 18 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was further purified by column chromatography to obtain the target product as a white solid powder (72 mg; 62% yield). HRMS (ESI) m/z: 827.4484 [M+2H].sup.2+; 838.4369 [M+H+Na].sup.+; 849.4286 [M+2Na].sup.2+.

4) Preparation of ADC-10

[0814] ##STR00158##

[0815] According to the method described in step 19) of Example 1, the L10-MMAE conjugate was coupled to anti-HER2 humanized monoclonal antibody mil40 (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd., which was a biosimilar of Herceptin) to obtain the target antibody-drug conjugate ADC-10, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 11: Preparation of Antibody-Linker-(Toxin Substitute) Conjugate

[0816] 1) The preparation route and structure of the toxin substitute were as follows:

##STR00159##

[0817] Preparation of Boc-Val-An:

##STR00160##

[0818] N-Boc-valine (Boc-Val, 2.17 g, 10 mmol/L) was dissolved in anhydrous THF (30 mL), then aniline (0.93 g, 10 mmol/L) and DCC (2.39 g, 11 mmol/L) were added, the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the insoluble solid by-product dicyclohexylurea (DCU) was removed by filtration, and the obtained filtrate was concentrated under reduced pressure and further purified by column chromatography to obtain the target product as a white solid powder (2.2 g, 75% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 9.98 (s, 1H), 7.60 (d, J=7.6 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 7.04 (t, J=7.4 Hz, 1H), 6.90 (d, J=8.4 Hz, 1H), 3.91 (t, J=7.0 Hz, 1H), 1.96 (m, 1H), 1.39 (s, 9H), 0.88 (d, J=6.7 Hz, 6H). MS (ESI) m/z: 293.1 [M+H].sup.+; 315.2 [M+Na].sup.+.

[0819] {circle around (2)} Preparation of Val-An:

##STR00161##

[0820] The product Boc-Val-An (5.85 g, 20 mmol/L) obtained in step {circle around (1)} was dissolved in DCM (50 mL), then TFA (12.5 mL) was added to the reaction solution, and the reaction was carried out under stirring overnight at room temperature. After the reaction was completed, the reaction solution was concentrated under reduced pressure and further purified by column chromatography to obtain the target product as a pale yellow oily liquid (2.8 g, 88% yield). H-NMR (400 MHz, DMSO-d6) 9.84 (br, 1H), 7.63 (dd, J=8.7 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 7.03 (td, J=7.4 Hz, 1H), 3.10 (d, J=5.6 Hz, 1H), 1.93 (m, 1H), 0.88 (d, J=6.7 Hz, 6H). MS (ESI).sub.m/z: 193.1 [M+H].sup.+; 215.1 [M+Na].sup.+.

[0821] {circle around (3)} Preparation of Boc-Val-Val-An:

##STR00162##

[0822] N-methyl-N-Boc-valine (Val, 0.58 g, 2.5 mmol/L) was dissolved in DCM (10 mL), then EDCI (0.58 g, 3 mmol/L), HOBt (0.41 g, 3 mmol/L) and DIPEA (0.51 mL, 3.0 mmol/L) were added in sequence, and the reaction was carried out under stirring at room temperature for 1 hour, then Val-An (0.48 g, 2.5 mmol/L) was added, and the reaction was continued under stirring overnight at room temperature. After the reaction was completed, the solvent was removed by concentration, a crude product was obtained, the crude product was further dried to obtain a pale pink solid powder (0.43 g, 42% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 10.11 (d, 1H), 7.95 (d, J=8.3 Hz, 2H), 7.58 (d, J=7.6 Hz, 2H), 7.31 (t, J=8.0 Hz, 2H), 7.05 (t, J=7.4 Hz, 3H), 4.23 (t, J=9.4 Hz, 2H), 2.77 (s, 1H), 2.04 (m, 2H), 1.43 (s, 9H), 0.87-0.78 (m, 12H). MS (ESI).sub.m/z: 406.2 [M+H].sup.+; 428.3 [M+Na].sup.+.

[0823] {circle around (4)} Preparation of Val-Val-An:

##STR00163##

[0824] Boc-Val-Val-An (0.42 g, 1.04 mmol/L) obtained in step was dissolved in DCM (5 mL), then TFA (1.25 mL) was added to the reaction solution, and the reaction was carried out under stirring at room temperature for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the residue was re-dissolved with ethyl acetate and washed twice with saturated sodium bicarbonate, and then concentrated, the product was recrystallized from ethyl acetate to obtain the product as a white powdery solid (0.31 g, 99% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 10.16 (s, 1H), 8.03 (d, J=9.0 Hz, 1H), 7.59 (d, J=8.7 Hz, 2H), 7.30 (t, J=7.1 Hz, 2H), 7.04 (t, J=7.4 Hz, 1H), 4.39 (t, J=8.0 Hz, 1H), 2.69 (d, J=6.2 Hz, 1H), 2.20 (s, 3H), 2.03 (m, 1H), 1.76 (m, 1H), 0.89 (m, 12H). MS (ESI) m/z: 306.22 [M+H].sup.+; 328.20 [M+Na].sup.+.

2) Preparation of L01-(Toxin Substitute) Conjugate (LT-01)

[0825] ##STR00164##

[0826] By using the linker L01 as raw material, a L1-(toxin substitute) conjugate (LT-01) was synthesized by using a preparation method similar to that of the L01-MMAE conjugate. The target product was obtained as a white solid powder (81% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 10.00 (d, 1H), 8.99 (s, 1H), 8.32 (s, 1H), 8.08 (s, 1H), 7.94 (d, J=8.9 Hz, 1H), 7.57 (d, J=7.8 Hz, 2H), 7.39 (s, 1H), 7.29 (t, J=7.9 Hz, 2H), 7.24 (d, J=8.4 Hz, 1H), 7.11 (m, 2H), 7.04 (t, J=7.2 Hz, 1H), 6.98 (s, 2H), 5.30 (s, 2H), 5.04 (m, 2H), 4.29 (m, 1H), 4.17 (t, J=8.0 Hz, 1H), 3.98 (s, 3H), 3.84 (d, J=5.6 Hz, 2H), 3.39 (m, 2H), 2.85 (s, 3H), 2.07 (m, 1H), 1.99 (t, J=7.3 Hz, 2H), 1.43 (m, 4H), 1.14 (m, 1H), 1.09 (t, J=7.0 Hz, 2H), 0.89-0.78 (m, 12H). MS (ESI) m/z: 860.6 [M+H].sup.+; 882.6 [M+Na].sup.+; 898.6 [M+K].sup.+; 858.5 [MH].sup.; 904.4 [M+Cl].sup..

3) Preparation of Antibody-Linker-(Toxin Substitute) Conjugate

[0827] ##STR00165##

[0828] By using the method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.), the conjugate LT-01 was coupled to anti-HER2 humanized monoclonal antibody mil40 (hereinafter referred to as naked anti-mil40), and the target antibody-drug conjugate was obtained, in which MAB represented monoclonal antibody, and the drug to antibody ratio (DAR) n was about 4.

Example 12: Evaluation of In Vitro Cytotoxicity of ADC (DAR: About 4)

[0829] In this example, the ADCs prepared in Examples 1 to 10, toxin MMAE and naked anti-mil40 were evaluated in terms of in vitro cytotoxicity. The cell lines tested included antigen HER2-positive breast cancer cell lines BT-474 and HCC1954, antigen HER2-positive ovarian cancer cell line SK-OV-3, antigen HER2-positive gastric cancer cell line NCI-N87, antigen HER2-weakly positive breast cancer cell line MCF-7, and antigen HER2-negative breast cancer cell line MDA-MB-468 (the above cell lines were all purchased from ATCC).

[0830] The reagents, instruments and consumables used in the test were described in the following table:

TABLE-US-00001 Vendor Cat# Reagents and consumables RPMI 1640 Invitrogen A10491-01 FBS Invitrogen 10099 Penicillin-Streptomycin Invitrogen 15140 Mccoy's 5A Invitrogen 16600-082 DMEM Invitrogen 10569010 CellTiter Glo kit Promega G9243 Instruments Enspire PE 2300

[0831] The test process was as follows:

[0832] {circle around (1)} Cell Thawing

[0833] The vial containing the target cells was gently stirred in a 37 C. water bath to be thawed.

[0834] After the content was thawed, the vial was taken out from the water bath and decontaminated by immersion or spraying with 70% ethanol.

[0835] The content of the vial was transferred into a centrifuge tube containing 9 mL of complete medium (for cell lines BT-474 and MCF-7, DMEM medium was used; for cell lines NCI-N87, HCC1954 and MDA-MB-468, RPMI1640 medium was used; for cell line SK-OV-3, Mccoy's 5A medium was used; the media described below were the same as here), and centrifuged (200 g; 5 min).

[0836] The cells were re-suspended in the culture medium, settled and distributed into a culture flask with an area of 75 cm.sup.2.

[0837] The resulting culture was incubated in a Galaxy CO.sub.2 incubator (48R, #CO48312044) with 5% CO.sub.2 at 37 C., and the oxygen concentration in the incubator was 0.1%.

[0838] {circle around (2)} Expansion of Cells

[0839] The cells were passaged three times a week at a ratio of 1:4 in a medium containing 10% PBS (heat inactivated) and 1% penicillin-streptomycin solution.

[0840] For the passaged cells, the adherent cells were firstly rinsed with trypsin/EDTA solution (3 mL), then trypsin/EDTA (3 mL, T75 flask) was added, and spun to coat the cells evenly. The resulting culture was incubated at 37 C. until the cells were detached. It was observed by microscope to verify that the cells had been detached, then an equal volume of cell culture medium was added to inactivate trypsin, the detached cells were collected, and centrifuged at 200 g for 5 minutes, and then re-suspended in a fresh culture medium.

[0841] {circle around (3)} Preparation of Compound

[0842] The compound stock solution was diluted in series in a ratio of 1:3 to produce 10 diluted solutions (the compound stock solution was an L-His buffer salt solution with a concentration of about 2 mg/mL, which was diluted with PBS, and the initial maximum concentration at test point was about 500 g/mL);

[0843] 10 L of the compound solution was distributed into a 384-well plate.

[0844] {circle around (4)} Cell Seeding

[0845] The cells were harvested and counted to determine the number of cells;

[0846] 30 L of the cell suspension with an adjusted density was added to the specified 384-well cell culture plate; and the final cell density was about 1,000 cells/well;

[0847] the plate was covered with a lid, and placed in an incubator to perform incubation at 37 C., 5% CO.sub.2 and 0.1% O.sub.2 for 168 hours.

[0848] {circle around (5)} Reading Plate

[0849] After 168 hours, the plate was taken out from the incubator and equilibrated at room temperature for 15 minutes;

[0850] CellTiter Glo reagent was incubated at 37 C. before experiment;

[0851] 40 L of the CellTiter-Glo reagent was added to each well to be tested (the ratio of CellTiter-Glo reagent to medium was 1:1);

[0852] the plate was then placed at room temperature for 30 minutes, and then reading was carried out on the EnSpire reader for cell counting.

[0853] {circle around (6)} Data Analysis

[0854] The inhibition percentage was expressed as the following formula:

% Inhibition=100[1(SampleLC)/(HCLC)]

[0855] wherein, HC represented the reading of the well in which cells treated with only 0.1% DMSO, and LC represented the reading of the well containing only medium and no cell.

[0856] {circle around (7)} Experimental Results

[0857] The experimental results of the in vitro cytotoxicity of the ADCs prepared in Examples 1 to 5 were shown in Table 1.

TABLE-US-00002 TABLE 1 Experimental results of in vitro cytotoxicity Test cmpds (IC.sub.50 nM) Cell lines MMAE MAB ADC-1 ADC-2 ADC-3 ADC-4 ADC-5 BT-474 0.06 84.14 0.05 0.08 0.16 0.24 0.27 N87 0.23 28.52 0.20 0.36 0.33 0.75 0.71 SK-OV-3 0.54 40.00 1.41 2.51 11.14 16.69 3.68 HCC1954 0.07 239.81 0.00003 0.0007 0.18 0.41 0.002 MCF-7 3.81 4194 >1000 >1000 >1000 >1000 >1000 MDA-MB-468 0.13 1840 >1000 >1000 >1000 >1000 >1000

[0858] The results showed that through the in vitro activity test of the tested ADCs in hypoxic environment, it was verified that the ADCs prepared in Examples 1 to 5 all had an in vitro cytotoxicity significantly better than that of the corresponding MAB (naked anti-mil40).

[0859] The experimental results of in vitro cytotoxicity of the ADC-7, ADC-8 and ADC-10 prepared in Examples 7, 8 and 10 were shown in Table 1-1. The results showed that the ADC-7, ADC-8 and ADC-10 have very significant in vitro cytotoxicity to HER2-positive BT-474 (0.142 to 0.199 nmol/L). Compared with antigen HER2-negative MCF-7, the in vitro cytotoxicity of the ADC-7, ADC-8 and ADC-10 that contain PEG structure linkers to HER2-positive BT-474 is significantly increased by 502 to 704 times, indicating that this type of ADCs has a high antigen selectivity.

TABLE-US-00003 TABLE 1-1 In vitro cytotoxicity experiment of ADCs containing PEG structure linker Test IC.sub.50 (nM) ADCs BT-474(HER2.sup.+) MCF-7(HER2.sup.) ADC-7 0.199 >100 ADC-8 0.142 >100 ADC-10 0.193 >100

Example 13: Hypoxia-Dependent Cytotoxicity of ADC-1

[0860] In this example, the hypoxia-dependent in vitro cytotoxicity of hypoxia-activated ADC-1 was studied. The tested cell line was antigen HER2-positive breast cancer cell line BT-474. The cell culture process was as described in Example 12, in which the oxygen concentrations in the cell incubator were separately set to 0.1%, 1.0%, 5.0%, 10.0% and 20.0%. The experimental results were shown in Table 2. The results showed that compared with under normoxic condition (oxygen concentration of 20%), under hypoxic condition (oxygen concentration of 0.1%), the activity of the hypoxia-activated ADC-1 was increased by more than 10 times, while the corresponding MAB (naked anti-mil40) and the linker-toxin conjugate (L01-MMAE) was increased by nearly 2 times respectively.

TABLE-US-00004 TABLE 2 Cytotoxicity of ADC-1 to BT-474 cells in different oxygen environments Test cmpds (IC.sub.50 nM) O.sub.2 % MAB ADC-1 L01-MMAE 0.1% 0.6844 0.0215 3.1041 1.0% 0.2402 0.0233 3.1052 5.0% 0.8971 0.0463 4.3166 10.0% 1.3605 0.2179 8.9445 20.0% 1.0831 0.2497 5.3939

Example 14: Time-Dependent Cytotoxicity of ADC-1

[0861] In this example, the time-dependent in vitro cytotoxicity of hypoxia-activated ADC-1 was studied. The tested cell line was antigen HER2-positive breast cancer cell line BT-474. The cell culture process was as described in Example 12, in which the oxygen concentration in the incubator was 0.1%, and the incubation time was separately set to 6 h, 12 h, 24 h, 48 h, 72 h and 96 h. The results were shown in Table 3. The results showed that the in vitro cytotoxicity of hypoxia-activated ADC-1 was dependent on the hypoxia time, and the speed of taking effect was significantly faster than that of the corresponding MAB (naked anti-mil40).

TABLE-US-00005 TABLE 3 Cytotoxicity of ADC-1 to BT-474 cells at different hypoxia time Test IC.sub.50 nM cmpds 6 h 12 h 24 h 48 h 72 h 96 h MAB >1719.12 >1719.12 >1719.12 0.9785 0.7064 0.8578 ADC-1 >1494.89 >1494.89 0.8465 1.3550 0.2305 0.1284

Example 15: Stability of ADC-1 in Plasma In Vitro

[0862] In this example, the stability of ADC-1 and its corresponding linker-toxin conjugate L01-MMAE in in vitro plasma was studied, ADC with an average DAR value of about 4 was selected, and L01-MMAE was treated with NAC before the experiment to obtain a corresponding NAC-L01-MMAE. The preparation method of NAC-L01-MMAE was referred to Y. Wang, S. Fan, W. Zhong, X. Zhou, S. Li, Int. J. Mol. Sci. 2017, 18, e1860. In short, 125 L of aqueous solution of NAC (0.1 mmol/L) and 125 L of PBS buffer (pH=7.4, 30 mmol/L) were added to 900 L of water, the above mixed liquid was mixed well and 100 L of NAC in DMSO solution (10 mmol/L) was added, then incubation was carried out at 37 C. for 5 minutes, it was showed that all was converted to NAC-L01-MMAE by HPLC detection.

[0863] ADC-1 and NAC-L01-MMAE were diluted with PBS buffer (pH=7.4) to a concentration of 100 g/mL, and an equal volume of human plasma was added for dilution, the resulting mixture was mixed well and then incubated in a sterile cell incubator at 37 C., and the samples were taken at the specified time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d), and stored at 80 C. for short-term storage, and subjected to LC/MS analysis after the sampling was completed (the quality of off-target MMAE was quantified according to the standard curve method). The results showed that within 7 days, ADC-1 and NAC-L01-MMAE tested had no significant MMAE off-targeting (see FIG. 1). This example preliminarily confirmed that the type of arylnitro-based enzymatically cleavable ADC provided in the present application had ideal plasma stability.

Example 16: Evaluation of Enzymatic Drug-Release Performance of L01-MMAE Conjugate

[0864] 1. Reagents and materials: Human NADPH CYP-reducates (purchased from Cypex, #CYP004), NADPH (purchased from ARK), L01-MMAE conjugate, potassium phosphate buffer (100 mmol/L, pH=7.4), DMSO, cold MeOH, etc.

[0865] 2. Operation:

[0866] {circle around (1)} Preparation of stock solution of sample to be tested: the sample to be tested was the L01-MMAE conjugate, which was treated with NAC before the test to obtain the corresponding NAC-L01-MMAE. NAC-L01-MMAE was dissolved in DMSO to prepare an stock solution of sample to be tested with a concentration of 10 mmol/L;

[0867] {circle around (2)} preparation of cofactor (NADPH) solution: 50 mg of NADPH was dissolved in phosphate buffer (3 mL) to obtain a NADPH solution with a final concentration of 20 mmol/L;

[0868] {circle around (3)} a vial containing 390 L of potassium phosphate buffer (100 mmol/L, pH=7.4) was fed with nitrogen gas for deoxygenation, and then NADPH solution (75 L, 20 mM) and Human NADPH CYP-reducates (10 L, 3 mg/mL; original enzyme was diluted 3 times) were quickly added, the resulting mixture was incubated at 37 C. for 10 minutes; the stock solution (25 L, 10 mmol/L) of sample to be tested was added to the above incubation solution to initiate the reaction, and then incubated under hypoxia condition (initial concentration of sample to be tested was 0.5 mmol/L; DMSO accounted for 5%);

[0869] {circle around (4)} at the specified time points (t=0, 0.5 h, 1 h, 2 h, 4 h, 12 h, 24 h), samples of equal amount (50 L) were taken from the incubation solution, and 200 L of cold methanol was added to quench the reaction, and then the resulting incubation solution was centrifuged to remove proteins; the supernatant was taken and analyzed by HPLC to determine the release amount of the target drug;

[0870] {circle around (5)} each of the peaks of the incubation solution sample was measured by LC/MS to determine the molecular weight thereof as qualitative parameter (including substrate, target product, transition state substance after reduction).

[0871] 3. Experimental Results

[0872] In this example, the enzymatic drug-release performance of the novel linker that was based on the reduction of arylnitro to drive drug-release was studied. The reduction of arylnitro depended on NADPH-CYP reducates (E.C. 1.6.2.4), reduced NADPH (NADH), and hypoxic environment. In the hypoxic environment, when nitroreductase and reduced NADPH were present at the same time, the substrate (NAC-L01-MMAE) could be rapidly degraded, while the concentration of the corresponding blank group basically showed no significant change within 24 hour (see FIG. 2). At the same time, the release of toxin MMAE also showed dependence on hypoxia, reductase and NADPH (see FIG. 3).

Example 17: Evaluation of the Drug-Release Performance of ADC-1 in Cells In Vitro

[0873] In order to explore the mechanism of hypoxia-activated ADC-1 taking effect, the performance of ADC-1 to release the toxin MMAE in cells in vitro was further studied in this example. The tested cell lines were antigen HER2-positive breast cancer cell line BT-474 and antigen HER2-positive gastric cancer cell line NCI-N87 (purchased from ATCC).

[0874] Test method: BT-474 was taken as an example. BT-474 cells were cultured in DMEM medium containing 10% FBS, 1% PS and 0.01 mg/mL insulin at 37 C., 5% CO.sub.2, 95% relative humidity in a flask. When the cells reached 80% to 90% confluence, the cells were separated and inoculated. The BT-474 cells in number of about 2.010.sup.6 were inoculated in a T75 flask, cultured for 2 days at 37 C., 5% CO.sub.2 and 95% relative humidity, to reach confluence of 50% to 60%. The cells were also divided into a blank group and ADCs administration groups, and the administration groups each contained two replicates. The medium was replaced, the cells of the administration groups were separately re-cultured with 15 mL of the above-mentioned medium containing about 1500 ng of ADCs, while the cells of the blank control group were cultured with the above-mentioned medium containing no the tested ADCs. After the replacement treatment was completed, the cells were cultured under hypoxia condition (1% O.sub.2) for 12 h, 24 h and 48 h, respectively (Table 4 and FIG. 4). The medium was discarded, the cells were separated with trypsin/EDTA, a medium containing excess serum was added to inactivate trypsin, the cells were centrifuged at 120 g for 5 minutes, 10 mL of the corresponding medium was added and mixed. The cells were counted by using automatic cell counter (NexcelomCellometer VisionCell Profiler), centrifuged and washed with cold PBS solution twice. The cell pellets were extracted by adding 6 mL of cold methanol; then the suspension was kept at 20 C. for 30 minutes and centrifuged at 13000 g for 20 minutes. The supernatant was evaporated by blowing nitrogen gas. The resulting residue was re-dissolved in 600 L of methanol containing an internal standard (IS=100 nmol/L alprazolam), and the sample was analyzed by LC-MS/MS.

[0875] The experimental results confirmed that after 24 hours of administration, a large amount of intracellular MMAE was detected by LC/MS (see: FIG. 4 and Table 4), while no cytotoxin MMAE was detected in the corresponding blank group. The HPLC retention time of the extracts of NCI-N87 and BT-474 cells treated with ADC-1 was very close to the retention time of MMAE standard, which confirmed that under the condition of 0.1% 02 partial pressure, ADC-1 could successfully release the carried cytotoxin MMAE in NCI-N87 and BT-474 cells that were cultured in vitro, and then induce tumor cell apoptosis through the released cytotoxin (FIG. 4).

TABLE-US-00006 TABLE 4 Toxin detection results of ADC-1 group and control group in BT-474 and NCI-N87 cell lines Cell Group Parallel sample MMAE peak area (cps) NCI-N87 Blank 1 0.00E+00 2 0.00E+00 ADC-1 1 1.18E+05 2 1.13E+05 BT-474 Blank 1 0.00E+00 2 0.00E+00 ADC-1 1 1.62E+04 2 1.48E+04

Example 18: Evaluation of Efficacy of ADC-1 in SCID Mice Xenograft Model of BT-474 Human Breast Cancer Cell

[0876] In this example, a xenograft model of human breast cancer cell line BT-474 expressed by HER2 (purchased from ATCC) was used to evaluate the in vivo efficacy of the tested drug, in which the tested drug was ADC-1, and 4 dose groups were set, and the doses were 0.75 mg/kg, 1.5 mg/kg, 3 mg/kg and 6 mg/kg, respectively. At the same time, a naked antibody (mil40) positive control group and a vehicle (normal saline) blank control group were set up. The tested drug was dissolved in physiological saline and administered via tail vein injection.

[0877] BT-474 human breast cancer cells (purchased from ATCC) were cultured with DMEM medium containing inactivated 10% fetal bovine serum, 100 U/mL penicillin, 100 g/mL streptomycin, and 2 mmol/L glutamine in an incubator at 37 C., 5% CO.sub.2. The initial concentration of cultured cells was 110.sup.6 cells/ml, and the cells were divided into bottles for passage every 3 to 4 days after the cells were full. The cancer cells in the logarithmic growth phase were inoculated under skin of right lateral thorax of SCID mice (female, 6-8 weeks old, 18-22 g, purchased from Beijing Ankai Yibo Biotechnology Co., Ltd.), and when the tumor grew to a volume of 150 mm.sup.3, the modeling was successful. Then the administration was started, 6 mice in each dose group were administered at the specified dose once a week, the administration volume was 5 mL/kg, and the administration was performed 4 times in total. All mice were injected subcutaneously with estrogen (veterinary estradiol benzoate injection, 4 mg/2 mL, purchased from Sichuan Lansheng Pharmaceutical Co., Ltd.) on the day before the cancer cell inoculation until the end of experiment, 2 times per week, 40 g/20 L each time. The tumor volume was measured twice a week by measurement of the long and short diameters of tumor with vernier caliper. The volume calculation formula was: volume=0.5long diametershort diameter.sup.2. When the tumor volume was measured, the mice were weighed at the same time. The relationship between the change of mouse body weight and the time of administration was recorded. At the same time, the survival and health status of the mice, such as animal activity and eating during the administration period were observed.

[0878] The results showed that in the SCID mice xenograft model of BT-474 human breast cancer, ADC-1 showed better in vivo tumor inhibitory activity than the naked antibody, part of the tumors of the test animals in the medium-dose administration group (3 mg/kg) disappeared, and the tumors persistently passed away after drug withdrawal (FIG. 5); at the same time, in the 4 dose groups of ADC-1 (0.75 mg/kg, 1.5 mg/kg, 3.0 mg/kg, 6.0 mg/kg), the tumor disappearance of the test animals showed a significant dose-efficacy dependence relationship (FIG. 6), the tumors of the test animals in the ADC-1 low- and medium-dose administration groups (for example, 0.75 mg/kg, 1.5 mg/kg, 3.0 mg/kg) were inhibited and partially disappeared, while the tumors of the test animals in the high-dose group (for example, 6.0 mg/kg) basically disappeared completely and persistently passed away. In addition, the test animals in the ADC-1 treatment groups showed no significant body weight loss during the treatment process and the observation period of drug withdrawal, indicating that the ADC-1 has preliminary safety at therapeutic doses (FIG. 7). The above studies indicated that the arylnitro-based ADCs provided in the present application have a good therapeutic potentiality for solid tumors.

Example 19: Evaluation of Efficacy of ADC-1 in Nude Mice Xenograft Model of NCI-N87 Human Gastric Cancer Cell

[0879] In this example, a xenograft model of human gastric cancer cell line NCI-N87 expressed by HER2 (purchased from ATCC) was used to evaluate the efficacy of the tested drug, in which the tested drug was ADC-1, 3 dose groups were set, and the doses were 1 mg/kg, 2.5 mg/kg and 5 mg/kg, respectively. At the same time, a naked antibody (mil40) positive control group, a vehicle (normal saline) blank control group, a chemotherapeutic drug (medicinal Doxetaxel, purchased from Zhejiang Hisun Pharmaceutical Co., Ltd.) control group, and ADC-1/chemotherapeutic drug combined administration group were set. The tested drug was dissolved in physiological saline and administered via tail vein injection.

[0880] The NCI-N87 gastric adenocarcinoma cells (purchased from ATCC) were cultured with DMEM medium containing inactivated 10% fetal bovine serum, 100 U/mL penicillin, 100 g/mL streptomycin, and 2 mM glutamine in an incubator at 37 C. and 5% CO.sub.2. The initial concentration of the cultured cell was 110.sup.6 cells/ml, and the cells were divided into bottles for passage every 3 to 4 days after the cells were full. The cancer cells in the logarithmic growth phase at 510.sup.6 cells/0.1 mL were inoculated under skin of right lateral thorax of BALB/c nude mice (female, 6-8 weeks old, 18-22 g, purchased from Beijing Ankai Yibo Biotechnology Co., Ltd.). When the tumor grew to a volume of 150 mm.sup.3, the modeling was successful. Then the administration was started, 6 mice in each dose group were administered at the specified dose once a week, the administration volume was 5 mL/kg, and the administration was performed 4 times in total. The tumor volume was measured twice a week by measurement of the long and short diameters of tumor with vernier caliper. The volume calculation formula was: volume=0.5long diametershort diameter.sup.2. When the tumor volume was measured, the mice were weighed at the same time. The relationship between the change of mouse body weight and the time of administration was recorded. At the same time, the survival and health status of the mice, such as animal activity and eating during the administration period were observed.

[0881] The results showed that ADC-1 also showed a significant dose-dependent relationship in the xenograft model of human gastric cancer cell line NCI-N87 (FIG. 8). Compared with the vehicle blank group and the naked antibody positive control group at the same dose (5 mg/kg), ADC-1 showed a significantly different therapeutic advantage (P(Vehicle VS ADC)=0.0178, P(mAb VS ADC)=0.0028) (FIG. 9). In addition, compared with single administration, the combined administration group had the best tumor inhibitory activity; although the efficacy of the combined administration group was significantly better than the ADC-1 single administration group (P=0.0097), it had no significant therapeutic advantage in comparison with the chemotherapeutic drug (P=0.6430) (FIG. 10). The reason could be that the selected dose was not reasonable enough. However, in the course of administration and treatment, the animals of all ADC-1 administration groups had steady or slightly increased body weight, while those of the chemotherapeutic drug group (Doxetaxel) showed a significant weight loss (P(Vehicle VS Doxetaxel)=0.0422), which reflected to a certain extent that ADC-1 was safer than the chemotherapeutic drug (FIG. 11).

Example 20: In Vivo Safety Evaluation of ADC-1

[0882] In order to further confirm the safety of this type of ADC-1, the maximum tolerated dose (MTD) of ADC-1 in a normal CD-1 mouse model was further evaluated in this example. CD-1 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals in the experiment were raised according to the protocol approved by the Institutional Animal Care and Use Committee of Pharmaron Beijing. Each test dose group had 6 CD-1 mice (3 males and 3 females), aged 7-9 weeks, average weight of 22-40 g (male) and 20-35 g (female). ADC-1 or naked antibody mil40 or ADC-a was administered via tail vein injection, and five dose groups were set for each drug, in which the doses of ADC-1 were 10 mg, 20 mg, 40 mg, 80 mg and 160 mg, while the doses of mil40 and ADC-a were 10 mg, 20 mg, 40 mg, 80 mg and 120 mg. After administration, all test animals were monitored for body weight changes once a day, and animal behavior was observed beside cage, twice a day. The observation records included animal death or sudden death, the general health of animals and symptoms of drug toxicity. The detailed clinical observations included changes in skin, fur, eyes and mucous membranes, changes in respiratory system, circulatory system, autonomic and central nervous systems, body movements and behavior patterns of the animals. After the last observation, all surviving animals were euthanized by inhalation 90% to 100% carbon dioxide.

[0883] The structural formula of ADC-a was shown below (the preparation method of ADC-a referred to the relevant description in International Journal of Molecular Sciences, 2017, 18(9): 1860.).

##STR00166##

[0884] Referring to FIG. 12, the experimental results showed that similar to the naked antibody mil40, the test animals in the ADC-1 administration groups showed no obvious adverse reactions, body weight loss or death of animals at administration doses of 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg and 160 mg/kg, indicating that ADC-1 has a very high therapeutic safety window, and would not cause obvious intolerance in the test animals when it is administered at therapeutic doses or even larger doses. As a control, the traditional cathepsin cleavage dipeptide-type ADC-6 showed continuous weight loss during the first 6 days of administration at a dose of 80 mg/kg, and there were common adverse reactions such as hair loss and scab in the later stage of the test; and at a dose of 120 mg/kg of ADC-6, half of the test animals died within one week after administration. The experimental data of this example showed that the application potentiality of the enzymatically cleavable ADC containing arylnitro linker provided in the present application is better than that of the traditional enzymatically cleavable ADC containing dipeptide linker.

[0885] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than to limit them; although the present application has been described in detail with reference to the preferred examples, those of ordinary skill in the art should understand that: the specific implementation of the present application can be modified or some technical features can be equivalently replaced without departing from the spirit of the technical solution of the present application, and all of them shall be covered by the scope of the technical solution that are sought to be protected by the present application.