Connexon containing azobenzene, protein coupling medicine containing connexon and medical application of protein coupling medicine

Abstract

The invention relates to an azobenzene-containing connexon as shown in a formula I, a protein coupling medicine containing the connexon and application of the connexon, further relates to a pharmaceutical composition containing the protein coupling medicine, and further relates to application of the protein coupling medicine for treating and / or preventing diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a linker containing azobenzene, a protein-coupled drug containing the linker, a pharmaceutical composition containing the protein-coupled drug, and the use of these protein-coupled drugs for treating and / or preventing diseases. Background Art

[0002] Antibody-drug conjugate (ADC) is composed of antibodies and effector molecules covalently coupled via linkers. It combines the strong targeting ability of antibodies with the high activity of effector molecules and is known as a "biological missile" for the precise delivery of effector molecules (Signal Transduction and Targeted Therapy, 2022, 7(1):93).

[0003] In essence, all ADCs on the market are macromolecular prodrugs, and their targeting efficiency is improved by more than 100 times compared with small molecule drugs (Clinical Cancer Research, 2015, 21 (22): 5131-5138). The linker is a key factor affecting the therapeutic index of ADC. At present, two recognized linker design principles, namely high stability during blood circulation and effective release of effector molecules in the lesion site, have achieved differential recognition of blood and lesion sites to a certain extent (Nature Reviews Clinical Oncology, 2021, 18 (6): 327-344; Chemical Society Reviews, 2019, 48 (16): 4361-4374). Therefore, efficient cleavage enzymes widely distributed in human tissues, such as cathepsins and glucuronidases, are often considered for the design of linkers for macromolecular conjugates (Cancer Res, 2017, 77 (24): 7027-7037; Bioconjugate chemistry, 2006, 17 (3): 831-840). However, the above-mentioned lytic enzyme-responsive ADCs cannot achieve specific drug release at the lesion site and are prone to off-target toxicity and other related problems.

[0004] Specifically, ADC has been widely used in the field of anti-tumor. However, clinical studies have shown that only less than 0.1% of ADC drugs injected into the body can accumulate in the lesions, and the remaining more than 99% of ADCs are off-target (Clinical cancer research, 2011, 17 (20): 6389-6397; The AAPS journal, 2015, 17: 525-534). Therefore, ADCs targeted to normal tissues (such as liver and lungs) will inevitably lead to nonspecific release of effector molecules (such as MMAE) under the action of cleavage enzymes, resulting in serious off-target toxicity (such as bone marrow suppression and hepatotoxicity), thereby limiting the increase in effective clinical doses (Acta Pharmaceutica Sinica B, 2020, 10 (9): 1589-1600; Current opinion inimmunology, 2016, 40: 14-23; Pharmacology & Therapeutics, 2019, 200: 110-125). Obviously, the existing linker technology does not truly achieve the specific release of effector molecules to the lesion site, but only relies on the targeting effect of antibodies, which will inevitably lead to off-target effector molecules and limit the advantages of ADC.

[0005] On the other hand, antibiotic-resistant Staphylococcus aureus remains a public health problem, and approximately 40% of bloodstream infections in the United States are caused by methicillin-resistant Staphylococcus aureus (MRSA). There are few FDA-approved treatment options for MRSA bloodstream infections, and vancomycin remains the antibiotic of choice. Despite appropriate antibiotic treatment, the mortality rate of Staphylococcus aureus bloodstream infections is still approximately 18%, which has prompted research on combinations / combinations that can improve treatment outcomes. ADC technology has been relatively rarely used in the antibacterial field. Reported antimicrobial conjugates rely on host cells rather than the lesion microenvironment to release antibiotics to exert their antibacterial effects (Nature 527.7578 (2015): 323-328), which to some extent limits the application of ADC technology in the antibacterial field.

[0006] Therefore, new linkers need to be developed to solve the above problems. Summary of the invention

[0007] The present invention provides a linker containing azobenzene, and also provides a protein-coupled drug containing the linker. Studies have shown that azoreductase is a type of reductase widely distributed in hypoxic tumors (Chemical Communications, 2019, 55 (22): 3235-3238; Chemical Communications, 2019, 55 (87): 13172-13175) and bacteria (Crit. Rev. Microbiol. 18: 175-190; Curr. Prot. Pept. Sci. 7: 101-111), which is rarely distributed in normal tissues. The protein-coupled drug provided by the present invention can be specifically recognized by azoreductase and reduce the azobenzene structure, causing the breakage of the azo bond, and specifically releasing the drug at the lesion site to achieve the effect of treating the disease.

[0008] Linker

[0009] The present invention provides a compound represented by formula I, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs.

[0010]

[0011] in: represents a linker used to couple the azobenzene group to the targeting compound,

[0012] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0013] R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0014] p is 0, 1, 2 or 3;

[0015] q is 0, 1, 2, or 3;

[0016] R is H, C 1-4 alkyl, where r 1 and r 2 Each independently is an integer between 1 and 12;

[0017] Z is hydroxy, fluorine, chlorine, bromine, iodine or Where s is 0, 1, 2, 3 or 4, R 5 For hydrogen, fluorine, chlorine, bromine, iodine, C 1-4 Alkyl, nitro or C 1-4 Alkoxy.

[0018] In certain embodiments, in the compound of formula I, The definitions of B, V, L, and W are as follows:

[0019] B is or H, wherein r is 1-4; or

[0020] B is selected from The condition is that V, L, W1, W2, and W3 do not exist at the same time;

[0021] V

[0022]

[0023] or not present, wherein each i is independently an integer between 0 and 6, and each j is independently an integer between 0 and 8;

[0024] L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -,-(CH 2 ) m -, or not present, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, and m is an integer between 0 and 30;

[0025] W is -W1-W2-W3-, wherein W1 is connected to L, W3 is connected to Ar, and W1 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )- or not present; W2 is selected from -(CH 2 ) o -,-[CH(R 11 )CH(R 12 )] o - or not present; W3 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O2 )NH-, -NHS(O 2 )-or not present; where o is an integer between 0 and 6, each R 11 are independently selected from hydrogen or C 1-6 Alkyl, R 12 Selected from hydrogen or C 1-6 alkyl.

[0026] In certain embodiments, in the compound of Formula I, W is selected from -NH-CH 2 -C(O)-NH-, -NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH 2 -C(O)-NH-, -C(O)-NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH 2 ) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH 2 ) o -NH-C(O)-, -NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, where R 10 is hydrogen, methyl or ethyl, o, R 11 and R 12 The definition as in claim 1.

[0027] In certain embodiments, in the compound of formula I:

[0028] B is

[0029] In certain embodiments, in the compound of formula I:

[0030] B is

[0031] In certain embodiments, in the compound of formula I:

[0032] B is

[0033] In certain embodiments, in the compound of formula I:

[0034] B is

[0035] In certain embodiments, in the compound of formula I:

[0036] B is

[0037] In certain embodiments, in the compound of formula I:

[0038] B is

[0039] In certain embodiments, in the compound of formula I:

[0040] B is

[0041] In certain embodiments, in the compound of formula I, V is or absent, wherein i is as defined herein. In certain embodiments, i is 1, 2, 3, 4, 5, or 6. In certain embodiments, i is 1. In certain embodiments, i is 2. In certain embodiments, i is 3. In certain embodiments, i is 4. In certain embodiments, i is 5. In certain embodiments, i is 6.

[0042] In certain embodiments, B is V By introducing a cyclohexane group near the maleimide end, the stability of maleimide-derived thioethers can be enhanced (Eur. J. Biochem. 101, 395-399 (1979)), and the off-target effects caused by the reverse Michael addition can be reduced.

[0043] In certain embodiments, B is V On the basis of the above, by introducing oxygen atoms into the cyclohexane group, the stability of the maleimide-derived thioether can be further increased, and the water solubility of the linker can be increased (Sci Rep. 2016 Aug 9:6:30835.).

[0044] In certain embodiments, B is V wherein i is defined as described in the present invention, and an amino group is introduced at the proximal end of maleimide to induce spontaneous hydrolysis and ring opening of maleimide to form a stable ring-opening coupling form (Bioconjugate Chem. 2015, 26, 1, 145-152).

[0045] In certain embodiments, B is V By introducing an electron-withdrawing benzene ring near the maleimide end, the maleimide is induced to spontaneously hydrolyze and open the ring to form a stable open-ring coupling form (Antibodies (Basel). 2017 Nov 28; 6(4): 20.)

[0046] In certain embodiments, in the compound of formula I, j is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula I, j is 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula I, j is 9 or 10.

[0047] In certain embodiments, in the compound of Formula I, L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -or-(CH 2 ) m -, where k, l, R 1, m are as defined herein. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, k is 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5 or 6. In certain embodiments, l is 1, 2, 3 or 4. In certain embodiments, l is 1. In certain embodiments, l is 2. In certain embodiments, l is 3. In certain embodiments, l is 4. In certain embodiments, l is 5. In certain embodiments, l is 6. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, n is 1, 2, 3, 4, 5, or 6. In certain embodiments, n is 1, 2, 3, or 4. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5. In certain embodiments, n is 6. In certain embodiments, m is an integer between 1 and 20. In certain embodiments, m is an integer between 1 and 15. In certain embodiments, m is an integer between 1 and 10. In certain embodiments, m is an integer between 1 and 8. In certain embodiments, m is an integer between 1 and 6. In certain embodiments, m is an integer between 1 and 4.

[0048] In certain embodiments, in the compound of Formula I, W is -C(O)-NH-(CH 2 ) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R 11 and R 12 The definition of is as described in the present invention.

[0049] In certain embodiments, in the compound of Formula I, -VLW- is -(CH 2 ) m -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-(CH 2CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-CHR 1 -C(O)-NH-(CH 2 ) o -NH-C(O)-, or -(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , m, o, i, k, l, and n are defined as described in the present invention.

[0050] In certain embodiments, in the compound of formula I, each i is independently 1, 2, 3 or 4; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each m is independently 1, 2, 3 or 4; each o is independently 1, 2, 3 or 4; each l is independently 1, 2, 3 or 4; and n is 1, 2, 3, 4, 5, 6, 7 or 8.

[0051] In certain embodiments, in the compound of formula I, each i is independently 1, 2 or 3; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; each m is independently 1, 2 or 3; each o is independently 1, 2 or 3; each l is independently 1, 2 or 3; and n is 1, 2, 3, 4 or 5.

[0052] In certain embodiments, in the compound of Formula I, -VLW- is -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O)2 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 4 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 6 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 8 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 CH 2 O) 3 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 ) 2 -C(O)NH-CH[(CH 2 ) 4 -NHC(O)-(CH 2 CH 2 O) 8 -CH 3 ]-C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 CH 2 O) 7 -(CH 2 ) 2 -C(O)-NH-(CH2 ) 2 -NH-C(O)-.

[0053] In certain embodiments, in the compound shown in formula I, Ar is phenyl, pyridyl, pyrimidyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound shown in formula I, Ar is phenyl, furanyl, imidazolyl or thienyl. In certain embodiments, in the compound shown in formula I, Ar is phenyl.

[0054] In certain embodiments, in the compound of formula I, R is H or C 1-4 In certain embodiments, in the compound shown in Formula I, R is H, methyl, ethyl, n-propyl or n-butyl. In certain embodiments, in the compound shown in Formula I, R is H, methyl, ethyl or n-propyl. In certain embodiments, in the compound shown in Formula I, R is H, methyl or ethyl. In certain embodiments, in the compound shown in Formula I, R is H. In certain embodiments, in the compound shown in Formula I, R is methyl.

[0055] In certain embodiments, in the compound of Formula I, R 2 and R 3 are independently hydrogen, C 1-4 In certain embodiments, in the compound of formula I, R 2 and R 3 Each is independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound of formula I, R 2 and R 3 Each is independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound of formula I, R 2 and R 3 are each independently hydrogen.

[0056] In certain embodiments, in the compound of formula I, q is 0, 1 or 2, preferably 0 or 1.

[0057] In certain embodiments, in the compound of formula I, p is 0, 1 or 2, preferably 0 or 1.

[0058] In certain embodiments, in the compound of formula I, Z is hydroxy, fluorine, chlorine, bromine, iodine or Where s is 0, 1, 2, 3 or 4; R 5is hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, nitro, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy. In certain embodiments, s is 0, 1 or 2, preferably 0 or 1. In certain embodiments, R 5 is hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl or n-propyl. 5 It is nitro, methoxy, ethoxy or n-propoxy.

[0059] In certain embodiments, in the compound of formula I, Z is chlorine, OH or In certain embodiments, in the compound of formula I, Z is OH or In certain embodiments, in the compound of formula I, Z is In certain embodiments, in the compound of formula I, Z is chlorine. In certain embodiments, in the compound of formula I, Z is OH.

[0060] In certain embodiments, Z is Where R 5 For hydrogen, fluorine, chlorine, bromine, iodine, C 1-4 Alkyl, nitro or C 1-4 In some embodiments, s is 0, 1, 2, 3, or 4. In some embodiments, s is 0. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 C 1-4 In certain embodiments, R 5 In certain embodiments, R 5 C 1-4 In certain embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 In certain embodiments, R 5 It is a propoxy group.

[0061] In certain embodiments, the compound of formula I has the structure shown in I-1,

[0062]

[0063] Wherein B, V, L, W, R and Z are defined as described in the present invention.

[0064] In certain embodiments, in the compound of formula I, B is selected from V, L, W1, W2, and W3 do not exist; R is where r 1 and r 2 The definition of is as described in the present invention. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5 or 6. In certain embodiments, r 1 and r 2 Each independently is 1, 2, 3 or 4.

[0065] In certain embodiments, the compound of formula I is selected from:

[0066]

[0067]

[0068]

[0069]

[0070] The linker provided by the present invention contains an azobenzene structure, which can be specifically recognized by azoreductase and reduce the azobenzene structure, thereby causing the cleavage of the azo bond.

[0071] The present invention also provides the use of the compound represented by formula I, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs in the preparation of antibody-drug conjugates.

[0072] Linker-drug conjugates

[0073] The present invention also provides a compound represented by formula II, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs,

[0074]

[0075] in: represents a linker, used to couple the right azobenzene group to the targeting compound,

[0076] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0077] R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0078] p is 0, 1, 2 or 3;

[0079] q is 0, 1, 2, or 3;

[0080] R is H, C 1-4 alkyl, where r 1 and r 2 Each independently is an integer between 1 and 12;

[0081] D is 0 or 1;

[0082] C is an active compound selected from a drug, a cytotoxin, a detection reagent, a diagnostic reagent or a targeting vector; preferably, C is an antitumor drug, an antiviral drug, an antibiotic, an antibacterial drug or an immunomodulator drug; further preferably, C is an antitumor drug, such as a cytotoxin, a microtubule inhibitor, a DNA alkylating agent, a DNA chimera, an RNA polymerase inhibitor, a kinase inhibitor, a topoisomerase inhibitor, a spindle kinesin inhibitor, an antimetabolite drug and other small molecules, peptides or nucleotides, an immunomodulator drug, such as a Toll-like receptor agonist (such as a TLR7 agonist) or a STING agonist, an antibacterial drug, such as rifampicin and its analogs (such as Rifalogue, KRM-1657);

[0083] C is coupled to the carbonyl group (i.e., site *) or the C atom at the L position through the N atom or O atom in the active compound molecule.

[0084] In certain embodiments, in the compound represented by Formula II, The definitions of B, V, L, and W are as follows:

[0085] B is or H, wherein r is 1-4; or

[0086] B is selected from The condition is that V, L, W1, W2, and W3 do not exist at the same time;

[0087] V

[0088]

[0089] or not present, wherein each i is independently an integer between 0 and 6, and each j is independently an integer between 0 and 8;

[0090] L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -,-(CH 2 ) m -, or not present, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, and m is an integer between 0 and 30;

[0091] W is -W1-W2-W3-, wherein W1 is connected to L, W3 is connected to Ar, and W1 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )- or not present; W2 is selected from -(CH 2 ) o -,-[CH(R 11 )CH(R 12 )] o - or not present; W3 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )-or not present; where o is an integer between 0 and 6, each R 11 are independently selected from hydrogen or C1-6 Alkyl, R 12 Selected from hydrogen or C 1-6 alkyl.

[0092] In certain embodiments, in the compound of formula II: W is selected from -NH-CH 2 -C(O)-NH-, -NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH 2 -C(O)-NH-, -C(O)-NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH 2 ) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH 2 ) o -NH-C(O)-, -NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, where R 10 is H, methyl or ethyl, o, R 11 and R 12 The definition of is as described in the present invention.

[0093] In certain embodiments, in the compound of formula II: B is

[0094] In certain embodiments, in the compound of formula II: B is

[0095] In certain embodiments, in the compound represented by Formula II:

[0096] B is

[0097] In certain embodiments, in the compound represented by Formula II:

[0098] B is

[0099] In certain embodiments, in the compound represented by Formula II:

[0100] B is

[0101] In certain embodiments, in the compound represented by Formula II:

[0102] B is

[0103] In certain embodiments, in the compound represented by Formula II:

[0104] B is

[0105] In certain embodiments, in the compound of formula II, V is or absent, wherein i is as defined herein. In certain embodiments, i is 1, 2, 3, 4, 5, or 6. In certain embodiments, i is 1. In certain embodiments, i is 2. In certain embodiments, i is 3. In certain embodiments, i is 4. In certain embodiments, i is 5. In certain embodiments, i is 6.

[0106] In certain embodiments, in the compound of formula II, j is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula II, j is 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula II, j is 9 or 10.

[0107] In certain embodiments, in the compound of Formula II, L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -or-(CH 2 ) m -, where k, l, R 1, m are as defined herein. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, k is 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5 or 6. In certain embodiments, l is 1, 2, 3 or 4. In certain embodiments, l is 1. In certain embodiments, l is 2. In certain embodiments, l is 3. In certain embodiments, l is 4. In certain embodiments, l is 5. In certain embodiments, l is 6. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, n is 1, 2, 3, 4, 5, or 6. In certain embodiments, n is 1, 2, 3, or 4. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5. In certain embodiments, n is 6. In certain embodiments, m is an integer between 1 and 20. In certain embodiments, m is an integer between 1 and 15. In certain embodiments, m is an integer between 1 and 10. In certain embodiments, m is an integer between 1 and 8. In certain embodiments, m is an integer between 1 and 6. In certain embodiments, m is an integer between 1 and 4.

[0108] In certain embodiments, in the compound of Formula II, W is -C(O)-NH-(CH 2 ) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R 11 and R 12 is as defined herein. In certain embodiments, o is an integer between 0 and 4. In certain embodiments, o is 1, 2, 3, or 4. In certain embodiments, o is 1. In certain embodiments, o is 2. In certain embodiments, o is 3. In certain embodiments, o is 4. In certain embodiments, R 11 and R 12 Each is independently hydrogen. In certain embodiments, R 11 and R 12 Each independently is C1-6 In certain embodiments, R 11 and R 12 Each independently is C 1-4 In certain embodiments, R 11 and R 12 Each is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl. 11 and R 12 Each is independently methyl, ethyl or n-propyl.

[0109] In certain embodiments, in the compound of formula II, -VLW- is -(CH 2 ) m -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-CHR 1 -C(O)-NH-(CH 2 ) o -NH-C(O)-, or -(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , m, o, i, k, l, and n are defined as described in the present invention.

[0110] In certain embodiments, in the compound of formula II, each i is independently 1, 2, 3 or 4; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each m is independently 1, 2, 3 or 4; each o is independently 1, 2, 3 or 4; each l is independently 1, 2, 3 or 4; and n is 1, 2, 3, 4, 5, 6, 7 or 8.

[0111] In certain embodiments, in the compound of formula II, each i is independently 1, 2 or 3; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; each m is independently 1, 2 or 3; each o is independently 1, 2 or 3; each l is independently 1, 2 or 3; and n is 1, 2, 3, 4 or 5.

[0112] In certain embodiments, in the compound of formula II, -VLW- is -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 2 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 4 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 6 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O)8 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 CH 2 O) 3 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-CH[(CH 2 ) 4 -NHC(O)-(CH 2 CH 2 O) 8 -CH 3 ]-C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 CH 2 O) 7 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-.

[0113] In certain embodiments, in the compound shown in formula II, Ar is phenyl, pyridyl, pyrimidyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound shown in formula II, Ar is phenyl, furanyl, imidazolyl or thienyl. In certain embodiments, in the compound shown in formula II, Ar is phenyl.

[0114] In certain embodiments, in the compound of formula II, R is H or C 1-4 In certain embodiments, in the compound shown in formula II, R is H, methyl, ethyl, n-propyl or n-butyl. In certain embodiments, in the compound shown in formula II, R is H, methyl, ethyl or n-propyl. In certain embodiments, in the compound shown in formula II, R is H, methyl or ethyl. In certain embodiments, in the compound shown in formula II, R is H. In certain embodiments, in the compound shown in formula II, R is methyl.

[0115] In certain embodiments, in the compound of Formula II, R 2 and R 3 are independently hydrogen, C 1-4In certain embodiments, in the compound of formula II, R 2 and R 3 Each is independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound of formula II, R 2 and R 3 Each is independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound of formula II, R 2 and R 3 are each independently hydrogen.

[0116] In certain embodiments, in the compound represented by formula II, q is 0, 1 or 2, preferably 0 or 1.

[0117] In certain embodiments, in the compound of formula II, p is 0, 1 or 2, preferably 0 or 1.

[0118] In certain embodiments, in the compound shown in Formula II, C is selected from: auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and derivatives thereof, maytansine or its derivatives (e.g., maytansine-like, DM1, DM3, DM4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins and derivatives thereof, SN-38, Exatecan, Doxorubicin, TLR7 agonist with CAS 1821304-87-3, lapatinib, rifampicin and its analogs (e.g., Rifalogue, KRM-1657).

[0119] In certain embodiments, in the compound of formula II, C is monomethyl auristatin E (MMAE), rifampicin or Rifalogue.

[0120] In certain embodiments, in the compound of formula II, C is monomethyl auristatin E (MMAE) or Rifalogue.

[0121] In certain embodiments, in the compound of Formula II, C is SN-38, Exatecan, Doxorubicin, a TLR7 agonist with CAS 1821304-87-3, Lapatinib, MMAE, an MMAF derivative or Rifalogue.

[0122] In certain embodiments, in the compound of formula II, C is a MMAF derivative having the structure

[0123]

[0124] In certain embodiments, the compound of formula II has the structure of formula II-1,

[0125]

[0126] Wherein, the definitions of B, V, L, W, R, C and D are as described in the present invention.

[0127] In certain embodiments, in the compound of formula II, B is selected from V, L, W1, W2, and W3 do not exist; R is where r 1 and r 2 The definition of is as described in the present invention. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, r 1 and r 2 Each is independently 1, 2, 3, 4, 5 or 6. In certain embodiments, r 1 and r 2 Each independently is 1, 2, 3 or 4.

[0128] In certain embodiments, the compound of formula II is selected from:

[0129]

[0130]

[0131]

[0132]

[0133] The present invention also provides the use of the compound represented by formula II, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs in the preparation of antibody-coupled drugs.

[0134] Protein-conjugated drugs

[0135] The present invention also provides a compound represented by formula III or formula IV, a geometric or optical isomer, a pharmaceutically acceptable salt, a hydrate, a solvate or a polymorph thereof,

[0136]

[0137] Wherein, A represents the targeted compound;

[0138] represents a linker used to couple the azobenzene group to the targeting compound;

[0139] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0140] R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0141] p is 0, 1, 2 or 3;

[0142] q is 0, 1, 2, or 3;

[0143] R is H, C 1-4 alkyl, where r 1 and r 2 Each independently is an integer between 1 and 12;

[0144] R' is where k 2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0145] B is selected from

[0146] D is 0 or 1;

[0147] C is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors; preferably, C is an antitumor drug, an antiviral drug, an antibiotic, an antibacterial drug or an immunomodulator; further preferably, C is an antitumor drug, such as a cytotoxin, a microtubule inhibitor, a DNA alkylating agent, a DNA chimera, an RNA polymerase inhibitor, a kinase inhibitor, a topoisomerase inhibitor, a spindle kinesin inhibitor, an antimetabolite drug or other small molecules, peptides or nucleotides, an immunomodulator drug, such as a Toll-like receptor agonist (such as a TLR7 agonist) or a STING agonist, an antibacterial drug, such as rifampicin and its analogs (such as Rifalogue, KRM-1657); C is coupled to the carbonyl group (i.e., site *) or the C atom at the L position through the N atom or O atom in the active compound molecule;

[0148] E is a number between 1 and 20.

[0149] In certain embodiments, in the compound represented by formula III, A represents a targeting compound;

[0150] represents a linker used to couple the azobenzene group to the targeting compound;

[0151] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0152] R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0153] p is 0, 1, 2 or 3;

[0154] q is 0, 1, 2, or 3;

[0155] R is H, C 1-4 alkyl, where r 1 and r 2 Each independently is an integer between 1 and 12;

[0156] D is 0 or 1;

[0157] C is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors; preferably, C is an antitumor drug, an antiviral drug, an antibiotic, an antibacterial drug or an immunomodulator; further preferably, C is an antitumor drug, such as a cytotoxin, a microtubule inhibitor, a DNA alkylating agent, a DNA chimera, an RNA polymerase inhibitor, a kinase inhibitor, a topoisomerase inhibitor, a spindle kinesin inhibitor, an antimetabolite drug or other small molecules, peptides or nucleotides, an immunomodulator drug, such as a Toll-like receptor agonist (such as a TLR7 agonist) or a STING agonist, an antibacterial drug, such as rifampicin and its analogs (such as Rifalogue, KRM-1657); C is coupled to the carbonyl group (i.e., site *) or the C atom at the L position through the N atom or O atom in the active compound molecule;

[0158] E is a number between 1 and 20.

[0159] In certain embodiments, in the compound represented by formula III, Medium B 1 , V, L, and W are defined as follows:

[0160] B 1 for where k 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0161] V

[0162]

[0163] or not present, wherein each i is independently an integer between 0 and 6, and each j is independently an integer between 0 and 8;

[0164] L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -,-(CH 2 ) m -, or not present, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH2 O) k -CH 3 , each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, and m is an integer between 0 and 30;

[0165] W is -W1-W2-W3-, wherein W1 is connected to L, W3 is connected to Ar, and W1 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )- or not present; W2 is selected from -(CH 2 ) o -,-[CH(R 11 )CH(R 12 )] o - or not present; W3 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )-or not present; where o is an integer between 0 and 6, each R 11 are independently selected from hydrogen or C 1-6 Alkyl, R 12 Selected from hydrogen or C 1-6 alkyl.

[0166] In certain embodiments, in the compound represented by formula III, A is a targeting compound selected from proteins, antibodies, polypeptides, enzymes and small molecules.

[0167] In certain embodiments, in the compound of formula III, A is coupled to B via an S atom or a N atom in the targeting compound molecule. 1 The site of the group, or A targets the carbonyl group in the compound molecule and B 1 The hydroxylamine group in the group reacts to form an oxime bond to couple to B 1 The site of the group is **.

[0168] In certain embodiments, in the compound of formula III of the present invention: W is selected from -NH-CH 2 -C(O)-NH-, -NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH 2 -C(O)-NH-, -C(O)-NH-CH 2 -CH 2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH 2 ) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH 2 ) o -NH-C(O)-, -NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, where R 10 is hydrogen, methyl or ethyl, o, R 11 and R 12 The definition of is as described in the present invention.

[0169] In certain embodiments, in the compound of formula III, B 1 for where k 1 The definition of is as described in the present invention.

[0170] In certain embodiments, in the compound of formula III, B 1 for where k 1 The definition of is as described in the present invention.

[0171] In certain embodiments, in the compound of formula III, k 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, in the compound of formula III, k 1 is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula III, k 1 is 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula III, k 1 is 4, 5, 6, 7 or 8. In certain embodiments, in the compound represented by formula III, k 1 is 9 or 10.

[0172] In certain embodiments, in the compound of formula III, V is or absent, wherein i is as defined herein. In certain embodiments, i is 1, 2, 3, 4, 5, or 6. In certain embodiments, i is 1. In certain embodiments, i is 2. In certain embodiments, i is 3. In certain embodiments, i is 4. In certain embodiments, i is 5. In certain embodiments, i is 6.

[0173] In certain embodiments, in the compound of formula III, j is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula III, j is 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, in the compound of formula III, j is 9 or 10.

[0174] In certain embodiments, in the compound of formula III, L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -or-(CH 2 ) m -, where k, l, R 1, m are as defined herein. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, k is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, k is 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5, 6, 7 or 8. In certain embodiments, l is 1, 2, 3, 4, 5 or 6. In certain embodiments, l is 1, 2, 3 or 4. In certain embodiments, l is 1. In certain embodiments, l is 2. In certain embodiments, l is 3. In certain embodiments, l is 4. In certain embodiments, l is 5. In certain embodiments, l is 6. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, n is 1, 2, 3, 4, 5, or 6. In certain embodiments, n is 1, 2, 3, or 4. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5. In certain embodiments, n is 6. In certain embodiments, m is an integer between 1 and 20. In certain embodiments, m is an integer between 1 and 15. In certain embodiments, m is an integer between 1 and 10. In certain embodiments, m is an integer between 1 and 8. In certain embodiments, m is an integer between 1 and 6. In certain embodiments, m is an integer between 1 and 4.

[0175] In certain embodiments, in the compound of formula III, W is -C(O)-NH-(CH 2 ) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R 11 and R 12 is as defined herein. In certain embodiments, o is an integer between 0 and 4. In certain embodiments, o is 1, 2, 3, or 4. In certain embodiments, o is 1. In certain embodiments, o is 2. In certain embodiments, o is 3. In certain embodiments, o is 4. In certain embodiments, R 11 and R 12 Each is independently hydrogen. In certain embodiments, R 11 and R 12 Each independently is C1-6 In certain embodiments, R 11 and R 12 Each independently is C 1-4 In certain embodiments, R 11 and R 12 Each is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl. 11 and R 12 Each is independently methyl, ethyl or n-propyl.

[0176] In certain embodiments, in the compound of formula III, -VLW- is -(CH 2 ) m -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-CHR 1 -C(O)-NH-(CH 2 ) o -NH-C(O)-, or -(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , m, o, i, k, l, and n are defined as described in the present invention.

[0177] In certain embodiments, in the compound of formula III, each i is independently 1, 2, 3 or 4; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each m is independently 1, 2, 3 or 4; each o is independently 1, 2, 3 or 4; each l is independently 1, 2, 3 or 4; and n is 1, 2, 3, 4, 5, 6, 7 or 8.

[0178] In certain embodiments, in the compound of formula III, each i is independently 1, 2 or 3; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; each m is independently 1, 2 or 3; each o is independently 1, 2 or 3; each l is independently 1, 2 or 3; and n is 1, 2, 3, 4 or 5.

[0179] In certain embodiments, in the compound of formula III of the present invention, -VLW- is -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 2 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 4 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 6 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O)8 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 CH 2 O) 3 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-CH[(CH 2 ) 4 -NHC(O)-(CH 2 CH 2 O) 8 -CH 3 ]-C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 CH 2 O) 7 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-.

[0180] In certain embodiments, in the compound shown in formula III, Ar is phenyl, pyridyl, pyrimidyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound shown in formula II, Ar is phenyl, furanyl, imidazolyl or thienyl. In certain embodiments, in the compound shown in formula III, Ar is phenyl.

[0181] In certain embodiments, in the compound of formula III, R is H or C 1-4 In certain embodiments, in the compound shown in formula III, R is H, methyl, ethyl, n-propyl or n-butyl. In certain embodiments, in the compound shown in formula III, R is H, methyl, ethyl or n-propyl. In certain embodiments, in the compound shown in formula III, R is H, methyl or ethyl. In certain embodiments, in the compound shown in formula III, R is H. In certain embodiments, in the compound shown in formula III, R is methyl.

[0182] In certain embodiments, in the compound of formula III, R 2 and R 3 are independently hydrogen, C 1-4In certain embodiments, in the compound of formula III, R 2 and R 3 Each is independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound of formula III, R 2 and R 3 Each is independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound of formula III, R 2 and R 3 are each independently hydrogen.

[0183] In certain embodiments, in the compound represented by formula III, q is 0, 1 or 2, preferably 0 or 1.

[0184] In certain embodiments, in the compound represented by formula III, p is 0, 1 or 2, preferably 0 or 1.

[0185] In certain embodiments, in the compound of formula III, C is selected from: auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and derivatives thereof, maytansine or its derivatives (e.g., maytansine-like, DM1, DM3, DM4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins and derivatives thereof, SN-38, Exatecan, Doxorubicin, TLR7 agonist with CAS 1821304-87-3, lapatinib, rifampicin and analogs thereof (e.g., Rifalogue, KRM-1657).

[0186] In certain embodiments, in the compound of formula III, C is monomethyl auristatin E (MMAE), rifampicin or Rifalogue.

[0187] In certain embodiments, in the compound of formula III, C is monomethyl auristatin E (MMAE) or Rifalogue.

[0188] In certain embodiments, in the compound represented by formula III, C is SN-38, Exatecan, Doxorubicin, a TLR7 agonist with CAS number 1821304-87-3, Lapatinib, MMAE, an MMAF derivative or Rifalogue.

[0189] In certain embodiments, in the compound represented by formula III, C is a MMAF derivative having the structure

[0190]

[0191] In certain embodiments, in the compound represented by formula III, A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody or a multispecific antibody.

[0192] In certain embodiments, in the compound represented by Formula III, A contains an antibody fragment or a substitute or a variant thereof, a protein ligand or a protein scaffold.

[0193] In certain embodiments, in the compound of Formula III, A is a monoclonal antibody, bispecific antibody or multispecific antibody with a thiol or amino group as a coupling site, or a monoclonal antibody, bispecific antibody or multispecific antibody with a thiol or amino group as a coupling site that is site-directed mutated or modified, or a monoclonal antibody, bispecific antibody or multispecific antibody with a carbonyl group in acetylphenylalanine (pAF) as a coupling site that is site-directed mutated.

[0194] In certain embodiments, in the compound shown in Formula III, A is selected from: anti-HER2 humanized monoclonal antibody mil40, trastuzumab (HERCEPTIN), pertuzumab (PERJETA), cetuximab (ERBITUX), panitumumab (VECTIBIX), rituximab (RITUXAN), alemtuzumab (CAMPATH), ibritumomab tiuxetan (ZEVALIN), tositumomab (BEXXAR), ofatumumab (ARZERRA), bevacizumab (AVASTIN), ipilimumab (YERVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi), sacituzumab, rovalvpituzumab, and biosimilars thereof, and antibacterial antibodies.

[0195] In certain embodiments, in the compound represented by formula III, A is trastuzumab.

[0196] In certain embodiments, in the compound of formula III, A is albumin. In certain embodiments, in the compound of formula III, A is human serum albumin.

[0197] In certain embodiments, in the compound represented by formula III, A is an antibody for site-directed mutation of a non-natural amino acid, comprising:

[0198] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0199] (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH;

[0200] (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and

[0201] (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;

[0202] and / or

[0203] (b) the following three light chain variable region (VL) CDRs:

[0204] (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VL;

[0205] (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and

[0206] (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL.

[0207] In certain embodiments, the substitution described in any one of (i)-(vi) is a conservative substitution. In certain embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems. In certain embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 2.

[0208] In certain embodiments, in the compound represented by formula III, A is an antibacterial antibody, which comprises:

[0209] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0210] (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH;

[0211] (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and

[0212] (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;

[0213] and / or

[0214] (b) the following three light chain variable region (VL) CDRs:

[0215] (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL;

[0216] (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and

[0217] (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL.

[0218] In certain embodiments, the substitution described in any one of (i)-(vi) is a conservative substitution. In certain embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems. In certain embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:4.

[0219] In certain embodiments, in the compound of formula III, E is a number between 1 and 15. In certain embodiments, in the compound of formula III, E is a number between 1 and 10. In certain embodiments, in the compound of formula III, E is a number between 1 and 8, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8.

[0220] In certain embodiments, the compound of formula III has a structure shown in formula III-1, formula III-2 or formula III-3,

[0221]

[0222]

[0223] Where: A, k 1 , V, L, W, R, C, D, and E are defined as described in the present invention.

[0224] In certain embodiments, in the compound of formula III-1, A is coupled to site # through the S atom in the targeting compound molecule. In certain embodiments, in the compound of formula III-2, A is coupled to site ## through the N atom in the targeting compound. In certain embodiments, in the compound of formula III-3, A is coupled to site ### through the carbonyl group in the targeting compound.

[0225] In certain embodiments, the compound of formula III is selected from:

[0226]

[0227]

[0228]

[0229]

[0230] in, represents A, A and E are as defined in the present invention, It is a protein, preferably human serum albumin, which is connected to the # site through the S atom in the albumin molecule. In certain embodiments, in the compound shown in the above formula III, A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody or a multispecific antibody, and further preferably trastuzumab, an antibody with site-directed mutation of non-natural amino acids and an antibacterial antibody, preferably, A is an antibody, which is connected to the # site through the S atom in the antibody molecule or to the ## site through the carbonyl group in the antibody molecule or to the ### site through the N atom in the antibody molecule. In certain embodiments, in the compound shown in the above formula III, E is about 4.

[0231] In certain embodiments, in the compound represented by formula IV,

[0232] A represents the targeted compound;

[0233] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0234] R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0235] p is 0, 1, 2 or 3;

[0236] q is 0, 1, 2, or 3;

[0237] R' is where k 2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0238] B is selected from

[0239] D is 0 or 1;

[0240] C is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors; preferably, C is an antitumor drug, an antiviral drug, an antibiotic, an antibacterial drug or an immunomodulator; further preferably, C is an antitumor drug, such as a cytotoxin, a microtubule inhibitor, a DNA alkylating agent, a DNA chimera, an RNA polymerase inhibitor, a kinase inhibitor, a topoisomerase inhibitor, a spindle kinesin inhibitor, an antimetabolite drug or other small molecules, peptides or nucleotides, an immunomodulator drug, such as a Toll-like receptor agonist (such as a TLR7 agonist) or a STING agonist, an antibacterial drug, such as rifampicin and its analogs (such as Rifalogue, KRM-1657); C is coupled to the carbonyl group (i.e., site *) or the C atom at the L position through the N atom or O atom in the active compound molecule;

[0241] E is a number between 1 and 20.

[0242] In certain embodiments, in the compound represented by formula IV, A is a targeting compound selected from proteins, antibodies, polypeptides, enzymes and small molecules.

[0243] In certain embodiments, in the compound of formula IV, A is coupled to the site ** of the R' group through the N atom in the targeting compound molecule.

[0244] In certain embodiments, in the compound shown in formula IV, Ar is phenyl, pyridyl, pyrimidyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound shown in formula IV, Ar is phenyl, furanyl, imidazolyl or thienyl. In certain embodiments, in the compound shown in formula IV, Ar is phenyl.

[0245] In certain embodiments, in the compound of formula IV, R 2 and R 3 are independently hydrogen, C 1-4 In certain embodiments, in the compound of formula IV, R 2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound of formula IV, R 2 and R 3 Each is independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound of formula IV, R 2 and R 3 are each independently hydrogen.

[0246] In certain embodiments, in the compound represented by formula IV, q is 0, 1 or 2, preferably 0 or 1.

[0247] In certain embodiments, in the compound represented by formula IV, p is 0, 1 or 2, preferably 0 or 1.

[0248] In certain embodiments, in the compound of formula IV, C is selected from: auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and derivatives thereof, maytansine or its derivatives (e.g., maytansine-like, DM1, DM3, DM4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins and derivatives thereof, SN-38, Exatecan, Doxorubicin, TLR7 agonist with CAS 1821304-87-3, lapatinib, rifampicin and analogs thereof (e.g., Rifalogue, KRM-1657).

[0249] In certain embodiments, in the compound of formula IV, C is monomethyl auristatin E (MMAE), rifampicin or Rifalogue.

[0250] In certain embodiments, in the compound of formula IV, C is monomethyl auristatin E (MMAE) or Rifalogue.

[0251] In certain embodiments, in the compound represented by formula IV, C is SN-38, Exatecan, Doxorubicin, a TLR7 agonist with CAS number 1821304-87-3, Lapatinib, MMAE, an MMAF derivative or Rifalogue.

[0252] In certain embodiments, in the compound represented by formula IV, C is a MMAF derivative having the structure

[0253]

[0254] In certain embodiments, in the compound represented by formula IV, A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody or a multispecific antibody.

[0255] In certain embodiments, in the compound represented by Formula IV, A contains an antibody fragment or a substitute or a variant thereof, a protein ligand or a protein scaffold.

[0256] In certain embodiments, in the compound of formula IV, A is a monoclonal antibody, bispecific antibody or multispecific antibody with an amino group or a thiol group as a coupling site, or a monoclonal antibody, bispecific antibody or multispecific antibody that is site-directed mutated or modified with an amino group or a thiol group as a coupling site.

[0257] In certain embodiments, in the compound of formula IV, A is selected from: anti-HER2 humanized monoclonal antibody mil40, trastuzumab (HERCEPTIN), pertuzumab (PERJETA), cetuximab (ERBITUX), panitumumab (VECTIBIX), rituximab (RITUXAN), alemtuzumab (CAMPATH), ibritumomab tiuxetan (ZEVALIN), tositumomab (BEXXAR), ofatumumab (ARZERRA), bevacizumab (AVASTIN), ipilimumab (YERVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi), sacituzumab, rovalvpituzumab, and biosimilars thereof, and antibacterial antibodies.

[0258] In certain embodiments, in the compound represented by formula IV, A is trastuzumab.

[0259] In certain embodiments, in the compound of formula IV, A is albumin. In certain embodiments, in the compound of formula III, A is human serum albumin.

[0260] In certain embodiments, in the compound of formula IV, A is an antibody for site-directed mutation of a non-natural amino acid, comprising:

[0261] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0262] (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH;

[0263] (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and

[0264] (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;

[0265] and / or

[0266] (b) the following three light chain variable region (VL) CDRs:

[0267] (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VL;

[0268] (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and

[0269] (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL.

[0270] In certain embodiments, the substitution described in any one of (i)-(vi) is a conservative substitution. In certain embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems. In certain embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 2.

[0271] In certain embodiments, in the compound represented by formula IV, A is an antibacterial antibody, which comprises:

[0272] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0273] (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH;

[0274] (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and

[0275] (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;

[0276] and / or

[0277] (b) the following three light chain variable region (VL) CDRs:

[0278] (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL;

[0279] (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and

[0280] (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL.

[0281] In certain embodiments, the substitution described in any one of (i)-(vi) is a conservative substitution. In certain embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems. In certain embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:4.

[0282] In certain embodiments, in the compound of formula IV, E is a number between 1 and 15. In certain embodiments, in the compound of formula IV, E is a number between 1 and 10. In certain embodiments, in the compound of formula IV, E is a number between 1 and 8, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8.

[0283] In certain embodiments, the compound of formula IV has the structure of formula IV-1,

[0284]

[0285] Wherein, B, R', C, D, E, and A are defined as described in the present invention,

[0286] In certain embodiments, in the compound of Formula IV, k 2 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, k in the compound of formula IV 2 is 1, 2, 3, 4, 5, 6, 7, or 8.

[0287] In certain embodiments, the compound of formula IV is:

[0288]

[0289] in, represents A, and A and E are as defined herein. In certain embodiments, A is trastuzumab, and E is about 3.

[0290] In certain embodiments, in the above compounds, A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody or a multispecific antibody, further preferably trastuzumab, an antibody with site-directed mutation of non-natural amino acids and an antibacterial antibody, preferably, A is an antibody, which is connected to the ### site through the N atom in the antibody molecule.

[0291] The present invention also provides a pharmaceutical composition comprising a compound represented by Formula III or Formula IV, its geometric isomers, optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0292] In certain embodiments, the pharmaceutical composition further comprises additional drugs. The additional drugs include one or more other antibiotics (e.g., antibiotics that can be used for MRSA infection) such as vancomycin, cotrimoxazole, tetracycline, doxycycline / minocycline, clindamycin, cephalosporins (e.g., cephalexin), nafcillin, fidaxomicin, linezolid, etc., and / or any other suitable antibiotics.

[0293] The present invention also provides the use of the compound represented by Formula III or Formula IV, its geometric isomers, optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs in the preparation of a drug for treating a disease or condition or alleviating the severity of the disease or condition, wherein the disease or condition is selected from tumors, infectious diseases, hematological diseases, metabolic diseases, and inflammation.

[0294] In certain embodiments, the tumor is selected from the group consisting of a carcinoma, a lymphoma, a lymphoid tumor, a blastoma, a sarcoma, and a leukemia.

[0295] In certain embodiments, the cancer is selected from the group consisting of breast cancer (e.g., HER2-positive breast cancer); squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung; peritoneal cancer; liver cancer; stomach cancer; gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urethral cancer; hepatoma; breast cancer; intestinal cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; kidney cancer or renal 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; bile duct cancer; head and neck cancer and related metastases.

[0296] In certain embodiments, the infectious disease includes bacterial infections such as infections caused by Staphylococcus aureus, Mycobacterium tuberculosis, Enterococcus faecium, Acinetobacter baumannii, Clostridium difficile, Streptococcus pneumoniae, Pseudomonas aeruginosa, sepsis caused by infection, tuberculosis or bacterial eye infections, heart, brain or skin infections, gastrointestinal infections, bacterial meningitis, and abscesses in any organ (such as muscle, liver, meninges, or lungs).

[0297] In certain embodiments, the infectious disease is cellulitis, bacteremia, skin necrosis, eyelid infection, eye infection, neonatal conjunctivitis, osteomyelitis, impetigo, ecthyma, scalded skin syndrome, food poisoning, pneumonia, surgical infection, urinary tract infection, burn infection, meningitis, endocarditis, sepsis, toxic shock syndrome, or septic arthritis, tuberculosis, infection associated with a prosthetic joint, infection associated with a catheter, or infection associated with an implant.

[0298] In certain embodiments, the infectious disease is a Staphylococcus aureus infection of tissue surrounding a prosthetic joint.

[0299] In certain embodiments, the infectious disease is a Staphylococcus aureus infection of the catheter and / or tissue surrounding the catheter.

[0300] In certain embodiments, the infectious disease is a Staphylococcus aureus infection of the foreign body and / or the tissue surrounding the foreign body.

[0301] In certain embodiments, the infectious disease is tuberculosis.

[0302] In the present invention, the rifampicin analog is Rifalogue, and its structural formula is:

[0303] Rifampicin (i.e., rifamycin) antibiotics inhibit bacterial RNA polymerase (RNAP) and have potent activity against Staphylococcus aureus. However, monotherapy with this class of antibiotics may result in the selection of resistant individuals during treatment. Therefore, rifamycin antibiotics may be used in combination with first-line antibiotics to improve outcomes, typically for prosthetic or foreign device infections.

[0304] The ADC comprising a rifampicin analogue described in the present invention (eg, a compound represented by formula III-16) can be used to prevent or treat bacterial growth and / or bacterial infection in a subject.

[0305] In certain embodiments, the bacteria is a gram-positive bacteria (gram-positive bacteria are responsible for bacterial infections).

[0306] In certain embodiments, the bacteria is a penicillin-resistant bacteria (penicillin-resistant bacteria are responsible for bacterial infections).

[0307] In certain embodiments, the bacteria is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) bacteria (MRSA bacteria are responsible for bacterial infections).

[0308] In certain embodiments, the bacteria are methicillin-sensitive Staphylococcus aureus (MSSA) bacteria (MSSA bacteria are responsible for bacterial infections).

[0309] In certain embodiments, the bacterium is vancomycin-resistant Staphylococcus aureus (VRSA) bacteria (VRSA bacteria are the cause of bacterial infection). In some embodiments, the bacterium is multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis) (multidrug-resistant Mycobacterium tuberculosis is the cause of bacterial infection).

[0310] In certain embodiments, the bacterium is Chlamydia trachomatis resistant to, for example, azithromycin (Chlamydia trachomatis resistant to, for example, azithromycin is the cause of bacterial infection).

[0311] In certain embodiments, the bacterium is Clostridium difficile resistant to, for example, metronidazole, vancomycin, and / or fidaxomicin ( Clostridium difficile resistant to, for example, metronidazole, vancomycin, and / or fidaxomicin is a cause of bacterial infection).

[0312] The present invention provides a method for preventing or treating cellulitis, bacteremia, skin necrosis, eyelid infection, eye infection, neonatal conjunctivitis, osteomyelitis, impetigo, ecthyma, scalded skin syndrome, food poisoning, pneumonia, surgical infection, urinary tract infection, burn infection, meningitis, endocarditis, sepsis, toxic shock syndrome, septic arthritis, mastitis, infection associated with prosthetic joints, infection associated with catheters, or infection associated with implants in a subject, which comprises administering to the subject an effective therapeutic amount of an antibody-drug conjugate comprising a rifampicin analog (e.g., a compound shown in Formula III-16).

[0313] The present invention also provides a method for preventing or treating intracellular bacterial infection in a subject, which comprises administering to the subject an effective therapeutic amount of an antibody-drug conjugate comprising a rilifampicin analog (eg, a compound shown in Formula III-16).

[0314] In certain embodiments, the present invention provides a method of treatment comprising administering to a subject in need thereof an ADC comprising a rifampicin analog (e.g., a compound shown in Formula III-16) for treating and / or preventing bacterial infection in the subject, and / or a disease or condition associated with a staphylococcal infection (e.g., a Staphylococcus aureus infection), and / or alleviating at least one symptom associated with the disease or condition. The disease, condition, or condition may be cellulitis, bacteremia, skin necrosis, eyelid infection, eye infection, neonatal conjunctivitis, osteomyelitis, impetigo, ecthyma, scalded skin syndrome, food poisoning, pneumonia, surgical infection, urinary tract infection, burn infection, meningitis, endocarditis, sepsis, toxic shock syndrome, and septic arthritis. In some embodiments, the subject has a prosthetic joint, and the antibody-drug conjugate disclosed in the present invention can be used to treat and / or prevent Staphylococcus aureus infection of tissue surrounding the prosthetic joint.

[0315] In certain embodiments, the subject has a catheter, and the antibody-drug conjugates disclosed herein can be used to treat and / or prevent S. aureus infection of the catheter and / or tissue surrounding the catheter.

[0316] In certain embodiments, the subject has an implanted foreign body, and the antibody-drug conjugates disclosed herein can be used to treat and / or prevent S. aureus infection of the foreign body and / or tissue surrounding the foreign body.

[0317] In certain embodiments, the subject suffers from tuberculosis, and the antibody-drug conjugates disclosed herein can be used to treat tuberculosis.

[0318] In some embodiments, the ADC comprising a rifampicin analog can be administered in combination with one or more other antibiotics (e.g., antibiotics that can be used for MRSA infections) such as vancomycin, cotrimoxazole, tetracycline, doxycycline / minocycline, clindamycin, cephalosporins (e.g., cephalexin), nafcillin, fidaxomicin, linezolid, etc., and / or any other suitable antibiotic. In some embodiments, the ADC comprising a rifamycin analog described in the present invention can be administered in combination with vancomycin and can be used to prevent or treat bacterial infections in subjects.

[0319] definition

[0320] As used herein, the term "antibody" is a common immunoglobulin, a Y-shaped protein used by the immune system to identify and neutralize foreign objects (such as bacteria and viruses). Antibodies can specifically recognize unique parts of foreign targets (called antigens) because each tip of the Y-shaped protein antibody contains a site that can specifically recognize the antigen. After the antibody binds to the specific antigen, it can mediate a variety of related biological effects. Antibodies are composed of two identical heavy chains and two identical light chains, and the chains are connected by disulfide bonds formed by the sulfhydryl groups in the cysteine ​​residues. "Monoclonal antibodies" are single-specific antibodies, all of which are composed of the same immune cells that are cloned as the only parental cells, so all antibody molecules are identical.

[0321] As used herein, the term "cytotoxin" refers to molecules that are toxic to cancer cells after release in the cancer cells. Toxins of particular interest in the present invention include methyl auristatin E (MMAE), auristatin, maytansinoid or its derivatives (e.g., maytansinoids, DM1, DM3, DM4), calicheamicin, duocarmycin, doxorubicin, camptothecin or PBD-type cytotoxins.

[0322] As used herein, the term "linker" is a molecule with two reactive ends, one end of which can be coupled to an antibody, and the other end is used to couple to an active compound, such as a cytotoxin. The antibody coupling end of the linker is usually a site that can be coupled through the sulfhydryl group of the cysteine ​​or the lysine amine group on the antibody, and the coupling end of the toxin of the linker is usually an active site that can be coupled through the sulfhydryl, amino, carboxyl or hydroxyl groups on the toxin molecule. When the term linker is used to describe the linker in a coupled form, since the linker has reacted with one or both of the antibody and the cytotoxin to form a covalent bond, it may no longer include one or two reactive terminal reaction sites (such as the leaving group of the sulfhydryl reactive group, the leaving group of the amine reactive group).

[0323] As used herein, the term "antibody-drug conjugate" or "ADC" or "antibody-drug conjugate" has the same meaning, which is a product formed by coupling multiple molecules (usually 1-8) of cytotoxins to antibody molecules through a linker. An antibody conjugated to one or more cytotoxins. The antibody is usually a monoclonal antibody that is selective for a specific antigen of a cancer.

[0324] As used herein, the term "about" may be understood to mean within + / -20%, + / -18%, + / -15%, + / -12%, + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1%, + / -0.5%, + / -0.4%, + / -0.3%, + / -0.2%, + / -0.1% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term "about".

[0325] The types of tumor diseases that the antibody-drug conjugates described herein are concerned about include, but are not limited to, cancer, breast cancer, lymphoma, lymphoid tumors, blastoma, sarcoma and leukemia. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung; peritoneal cancer; liver cancer; gastric cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urethral cancer; hepatoma; breast cancer, including, for example, HER2-positive breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma; myeloma and B-cell lymphoma; brain cancer; head and neck cancer and related metastases.

[0326] Infectious diseases of interest for the antibody-drug conjugates described herein include, but are not limited to, bacterial infections such as Staphylococcus aureus pneumonia, Enterococcus faecium, Acinetobacter baumannii, Clostridium difficile, Streptococcus pneumoniae, Pseudomonas aeruginosa, infections causing sepsis, tuberculosis or bacterial eye infections, heart, brain or skin infections, gastrointestinal infections, bacterial meningitis, and abscesses in any organ (such as muscle, liver, meninges, or lungs).

[0327] As used herein, the term "salt" refers to salts that retain the biological effectiveness and properties of a compound, which are not suitable for use in medicine in biological or other aspects. In many cases, the compounds disclosed herein are able to form acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or similar groups. Pharmaceutically acceptable acid addition salts can be composed of inorganic acids and organic acids. Inorganic acids that can be derived to form salts include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids that can be derived to form salts include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Pharmaceutically acceptable base addition salts can be composed of inorganic bases and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; ammonium, potassium, sodium, calcium, and magnesium salts are particularly preferred. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, as described in WO87 / 05297, Johnston et al., published September 11, 1987 (incorporated herein in its entirety by reference).

[0328] As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclic group of 5-14 carbon atoms having a single ring or two or more fused rings with a conjugated π electron system. The "aryl" preferably has 5-10, 5-8 or 5-6 carbon atoms. Typical examples of "aryl" include, but are not limited to, phenyl, naphthyl, anthracenyl, etc.

[0329] As used herein, the term "heteroaryl" refers to an aryl group as defined herein, wherein at least one ring member is a heteroatom selected from nitrogen, oxygen or sulfur. The "heteroaryl" preferably has 5-10, 5-8 or 5-6 ring members. Typical examples of "heteroaryl" include, but are not limited to, furanyl, imidazolyl, thienyl, triazolyl, indolyl, tetrazolyl, pyridinyl, pteridinyl, pyrimidinyl, triazolyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, etc.

[0330] As used herein, the term "aromatic heterocyclyl" refers to a cyclic group having two or more fused rings wherein two or more carbon atoms are common to two adjacent rings, wherein at least one ring is aryl as defined herein and at least one ring is heterocyclyl.

[0331] As used herein, the term "heteroaroheterocyclyl" refers to a cyclic group having two or more fused rings wherein two or more carbon atoms are common to two adjacent rings, wherein at least one ring is heteroaryl as defined herein and at least one ring is heterocyclyl.

[0332] In this article, the term "heterocyclic group" refers to a saturated or partially unsaturated cyclic hydrocarbon group having a monocyclic or bicyclic or multiple fused rings (including fused, bridged and spirocyclic) of 3 to 12 ring members, and at least one ring member is a heteroatom selected from nitrogen, oxygen or sulfur. The "heterocyclic group" preferably has 3-10, 3-8, 5-8, 3-6 or 5-6 ring members. Typical examples of "heterocyclic group" include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperazinyl, thiazinyl, piperidinyl and morpholinyl, etc.

[0333] Abbreviations / acronyms

[0334] ADC (antibody-drug conjugate): antibody-drug conjugate;

[0335] AcOH(Acetic acid): glacial acetic acid;

[0336] Boc (t-Butyloxy carbonyl): tert-Butyloxycarbonyl;

[0337] Cys(Cysteine): L-cysteine;

[0338] DAR (Drug to antibody ratio): drug / antibody molar ratio;

[0339] DCM (Dichloromethane): dichloromethane;

[0340] DIPEA (N,N-Diisopropylethylamine): diisopropylethylamine;

[0341] DMAC (Dimethylacetamide): N,N-dimethylacetamide;

[0342] DMF (N,N-Dimethylformamide): N,N-dimethylformamide;

[0343] DMSO (Dimethyl Sulphoxide): dimethyl sulfoxide;

[0344] Diox(1,4-Dioxane): 1,4-dioxane;

[0345] DIAD (Diisopropylazodicarboxylate): diisopropyl azodicarboxylate;

[0346] DIC (Diisopropylcarbodiimide): diisopropylcarbodiimide;

[0347] EA (Ethyl acetate): ethyl acetate;

[0348] EtOH (Ethanol): ethanol;

[0349] EDTA (Ethylenediamine tetraacetic acid): ethylenediaminetetraacetic acid;

[0350] EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride): 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0351] FITC (fluorescein isothiocyanate): fluorescein isothiocyanate;

[0352] HOBT (1-Hydroxybenzotriazole): 1-Hydroxybenzotriazole;

[0353] HCl (hydrogen chloride): hydrogen chloride; HER2 (Human epidermal growth factor receptor 2): human epidermal growth factor receptor 2;

[0354] MAB (Monoclonal Antibody): Monoclonal antibody

[0355] MMAE (Monomethyl auristatin E): Monomethyl auristatin E;

[0356] NAC (N-Acetyl-L-cysteine): N-acetylcysteine;

[0357] NADPH (nicotinamide adenine dinucleotide phosphate): reduced coenzyme II;

[0358] NH 2 NH 2 (hydrazine): hydrazine;

[0359] NHS(N-Hydroxy succinimide): N-hydroxysulfosuccinimide;

[0360] PPh 3 (Triphenylphosphine): Triphenylphosphine;

[0361] TCEP (Tris(2-carboxyethyl)phosphine): tris(2-carboxyethyl)phosphine;

[0362] THF (Tetrahydrofuran): Tetrahydrofuran;

[0363] TFA (Trifluoroacetic acid): trifluoroacetic acid;

[0364] PE (Petroleum ether): petroleum ether;

[0365] VC-ADC (Herceptin-VC-MMAE): A tissue protease cleavage-dependent antibody-drug conjugate with MMAE as the effector molecule, which can be obtained by conjugating Herceptin with VC-MMAE (commercially available) according to the method in the prior art, wherein the DAR is about 4.

[0366] When the compound name used in this article is inconsistent with the chemical structural formula, the chemical structural formula shall prevail.

[0367] The pharmaceutical composition described herein comprises a compound represented by formula III of the present invention, or a salt or solvate thereof, and a conventional pharmaceutical carrier or excipient. The pharmaceutical composition can be administered, for example, orally or parenterally, such as by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, and the like.

[0368] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic effect, for example, an amount to achieve a reduction in symptoms associated with the disease being treated.

[0369] It should also be noted that the dosage and method of use of the compounds of the present invention depend on many factors, including the patient's age, weight, gender, natural health, nutritional status, activity strength of the compound, time of administration, metabolic rate, severity of the disease, and the subjective judgment of the treating physician. The preferred dosage is between 0.01-100 mg / kg body weight / day.

[0370] The linker provided by the present invention contains an azobenzene fragment and has good enzymatic cleavage performance. The antibody-drug conjugate (ADC) containing the linker provided by the present invention has good structural stability, ideal enzymatic drug release performance, hypoxia dependence, and relatively ideal in vitro and in vivo efficacy and safety.

[0371] In certain specific embodiments, the synthetic route of the ADC described in the present invention is as follows:

[0372]

[0373] Among them: C, E, The definitions are as described in the present invention, X represents -VLW-, and the definitions of V, L and W are as described in the present invention.

[0374] Using p-nitrobenzyl alcohol (1) as the starting material, the unstable intermediate p-aminobenzyl alcohol is obtained under the reduction of zinc powder, and further oxidized by ferric chloride to obtain intermediate (2), which is reacted with 4-aminobenzoic acid under the action of acetic acid to prepare compound (3), which is condensed with amide under the action of EDCI, HOBT and DIPEA to prepare compound (4), and the Boc protecting group is removed by trifluoroacetic acid to prepare compound (5), and further substitution reaction is performed to prepare compound (7). Compound (7) is reacted with di(p-nitrophenyl) carbonate to obtain compound (8), which is then reacted with active compound C to prepare the corresponding ADC loading (9), which is further coupled with an antibody to obtain the final ADC product (10).

[0375] In certain specific embodiments, another synthetic route of the ADC described in the present invention is as follows:

[0376]

[0377] Among them: E, The definitions are as described in the present invention, X represents -VLW-, and the definitions of V, L and W are as described in the present invention.

[0378] Using p-aminobenzoic acid (11) as the starting material, compound (12) is obtained by oxidation with ferric chloride, and compound (13) is reacted with acetic acid to obtain compound (14), which is then condensed with EDCI, HOBT and DIPEA to obtain compound (15), and the Boc protecting group is removed with trifluoroacetic acid to obtain compound (16), which is then further subjected to substitution reaction to obtain compound (17). Compound (17) is coupled with an antibody to obtain the final ADC product (18).

[0379] In certain specific embodiments, another synthetic route of ADC described in the present invention is as follows:

[0380]

[0381] Among them: E, Definitions As described in the present invention,

[0382] Compound (19) is used as a starting material, and is reacted with compound (5) in the presence of EDCI, HOBT, and DIPEA to obtain compound (20), which is then removed from the Fmoc protecting group by piperidine to obtain compound (21), which is then reacted with compound (22) to obtain compound I-6. Compound I-6 is reacted with di(p-nitrophenyl) carbonate to obtain compound I-12, which is then reacted with active compound C to obtain the corresponding ADC loading (23), which is then coupled with an antibody to obtain the final ADC product (24).

[0383] In certain specific embodiments, another synthetic route of ADC described in the present invention is as follows:

[0384]

[0385] Among them: C, E, The definitions are as described in the present invention, X represents -VLW-, and the definitions of V, L and W are as described in the present invention.

[0386] Using 2-hydroxyisoindoline-1,3-dione and compound (25) as starting materials, in the presence of DIAD and PPh 3 Compound (26) was prepared under the action of HCl to remove the tert-butyloxy protecting group to obtain compound (27), which was then subjected to amide condensation with compound (5) under the action of DIC to obtain compound (28). Compound (28) was reacted with di(p-nitrophenyl) carbonate to obtain compound (29), which was then reacted with active compound C to obtain compound (30), which was then reacted with NH 2 NH 2 The compound (31) is removed and further coupled with an antibody to obtain the final ADC product (32).

[0387] In certain specific embodiments, another synthetic route of ADC described in the present invention is as follows:

[0388]

[0389] Where: C, E, k 1 The definitions are as described in the present invention, X represents -VLW-, and the definitions of V, L and W are as described in the present invention.

[0390] Compound (33) and compound (5) are used as starting materials, and compound (34) is prepared under the action of DIC, and compound (35) is prepared by chlorination with cyanuric chloride, and then reacted with active compound C to prepare compound (36). Antibody and compound (37) are coupled to prepare conjugate (38), and compound (38) and antibody are further coupled by click chemistry reaction to obtain the final ADC product (39).

[0391] In certain specific embodiments, the synthesis route of the albumin conjugate of the present invention is as follows:

[0392]

[0393] Wherein: X, C, and E are defined as described in the present invention,

[0394] Compound (9) is coupled with albumin to prepare conjugate (40). BRIEF DESCRIPTION OF THE DRAWINGS

[0395] Figure 1 The DAR and polymerization degree test results of compound III-2 are shown.

[0396] Figure 2 The DAR and polymerization degree test results of compounds III-1, 3, 4, 5 and VC-ADC are shown.

[0397] Figure 3 The results of stability evaluation of compound III-2 in plasma are shown.

[0398] Figure 4 The results of the study on the binding ability of compound III-2 to HER2 antigen are shown. The results show that ( Figure 4 ), compound III-2 and Herceptin have comparable binding affinity to HER2 antigen, EC 50 The values ​​were 0.14 μg / mL and 0.042 μg / mL respectively.

[0399] Figure 5 The results of the binding study of compound III-2 on cell lines are shown. The results showed that Herceptin and compound III-2 both showed strong fluorescence in HER2-positive SKOV3 cells ( Figure 5 In the right figure), weak fluorescence was observed in HER2-negative MCF-7 cells ( Figure 5 ), indicating that compound III-2 specifically targets the HER2 antigen of tumor cells.

[0400] Figure 6 The results of the endocytosis study of compound III-2 on cell lines are shown. The results show that the internalization rate of compound III-2 is slightly higher than that of Herceptin, 34.1% vs. 22.1% at 8h and 41.7% vs. 31.1% at 24h.

[0401] Figure 7The binding and endocytosis imaging results of compound III-2 on cell lines are shown. The results show that Herceptin and compound III-2 were incubated with cells at 4°C, under which conditions the antibody bound to the antigen on the cell membrane surface but was not internalized. After incubation at 37°C for 24 hours, the intracellular protein signal co-localized with the lysosomal signal (Pearson correlation coefficient = 0.87), indicating that compound III-2 was internalized and transported to the lysosome.

[0402] Figure 8 The results of the study on the cell proliferation inhibition activity of compound III-2 on HER2 antigen-positive and negative cell lines were shown. The results showed that compound III-2 showed strong cell proliferation inhibition activity on NCI-N87, BT-474 and SKBR3 cell lines under hypoxic conditions, EC 50 The values ​​were 0.07nM, 0.026nM and 0.015nM, respectively, while the activity against HER2-negative cell lines (MCF-7, MDA-MB-231) was lower (EC 50 >100nM).

[0403] Fig. 9 The results of the hypoxia-dependent in vitro cytotoxicity study of compound III-2 are shown. The results show that compound III-2 exhibits oxygen concentration-dependent cell proliferation inhibitory activity, and the proliferation inhibitory activity increases with the decrease of oxygen concentration. Under oxygen concentrations of 20%, 5%, 1% and 0.1%, the EC of compound III-2 on SKOV3 cells 50 The values ​​were 66.8nM, 3.74nM, 0.079nM, and 0.063nM, respectively, and the EC values ​​on BT474-HDR cells were 50 The values ​​are 109.1nM, 9.12nM, 0.57nM, and 0.19nM respectively.

[0404] Fig.10 The results of the bystander effect study of compound III-2 are shown. Under normoxic conditions, the cytotoxic activity of compound III-2 in the co-culture model was reduced to the μM level (EC 50 ≈0.1μM), which indicates that compound III-2 has high safety in normal tissues. Under hypoxic conditions, compound III-2 maintains high activity in the co-culture model, EC 50 =0.053 nM. The above results indicate that compound III-2 has a bystander effect in tumor tissues and also has a killing effect on tumor cells with low HER2 expression.

[0405] Fig.11The results of the study on the proliferation inhibition activity of compound III-2 on normal cell lines are shown. At the highest tested concentration (666nM) of normal cells NIH3T3 and 293T, compound III-2 only showed weak inhibitory activity, with a maximum inhibition rate of <50%. However, traditional VC-ADC still showed high off-target toxicity (EC 50 <10 nM, maximum inhibition rate >90%).

[0406] Fig.12 The results of the drug release study of compound III-2 under the action of azoreductase are shown. The results show that the release amount of MMAE is dually dependent on the concentration of azoreductase and oxygen concentration. Under hypoxic conditions (0.1% O 2 ), the amount of MMAE released increased with the increase of azoreductase concentration. After incubation of substrate and azoreductase (0.4 mg / mL, 0.1 mg / mL and 0.05 mg / mL) for 24 hours, the amount of MMAE released was 100%, 74.3% and 27.49%, respectively. Under normoxic conditions (20% O 2 ), after the substrate was incubated with 0.1 mg / mL azoreductase for 24 hours, only 14.99% MMAE was released. This result shows that the linker based on azoreductase cleavage provided by the present invention can be cleaved and release toxins under the combined action of hypoxic conditions and azoreductase, while the amount of toxin released is very small under the action of azoreductase alone.

[0407] Fig.13 The results of the study on the effects of oxidative, reductive and salt substances on the stability of compound II-2 were shown. The results showed that NAC-II-2 could release MMAE via azoreductase only under hypoxic conditions, and showed excellent stability when co-incubated with other redox-related substances, such as Cys, NADPH, and NaClO.

[0408] Fig.14 The results of the metabolite study of compound III-2 in cells are shown. The results show that compound III-2 was co-cultured with SKOV3 cells under hypoxia, MMAE was detected in SKOV3 cells, and the release of MMAE was time-dependent. This result directly indicates that compound III-2 can be cleaved and release MMAE in tumor cells under hypoxic conditions. More importantly, compound III-2 was co-cultured with SKOV3 cells under normoxic conditions, Cys-II-2 was the main metabolite and its release was time-dependent, while the amount of Cys-II-2 in cells under hypoxia was very low. This result indicates that under normoxic conditions, the antibody of compound III-2 is degraded and released Cys-II-2 will not be further cleaved by azoreductase.

[0409] Fig.15The results of the cytotoxicity study of Cys-II-2 are shown. The results show that the cytotoxic activity of Cys-II-2 is reduced by more than ten to hundreds of times compared with MMAE.

[0410] Fig.16 The results of the study on the inhibition of tubulin by Cys-II-2 are shown. The results show that the tubulin inhibitory activity of Cys-II-2 is lower than that of MMAE and is concentration-dependent.

[0411] Fig.17 The results of the cell cycle arrest study of compound III-2 are shown. The results showed that compound III-2 caused G2 / M phase arrest of tumor cells with hypoxia selectivity. Under normoxic conditions, after treatment with compound III-2 at concentrations of 0.3nM, 1nM and 3nM, the proportion of G2 / M phase cells was similar to that of the untreated group (17.50%). On the contrary, under hypoxia, after treatment with the same concentration of compound III-2, the proportion of G2 / M phase cells was maintained at 71.12%-87.22%.

[0412] Fig.18 The results of the study on the induction of cell apoptosis by compound III-2 are shown. The results show that under normoxic conditions (20% O 2 ) had little effect on cell apoptosis. In contrast, under hypoxic conditions (0.1% O 2 ) Compound III-2 induced apoptosis in more than half of the cells. The apoptosis results showed that compound III-2 had good hypoxia selectivity.

[0413] Fig.19 The distribution of compound III-2 labeled with DyLight-680 dye in mice is shown. The results showed that DyLight 680-Herceptin (25 mg / kg) and DyLight 680-III-2 (25 mg / kg) labeled with DyLight 680 were injected into mice by tail vein injection. A strong fluorescence signal could be clearly observed at the tumor site 6 hours after administration, and the fluorescence at the tumor site was clearly maintained for about 9 days. The fluorescence signal finally disappeared on the 15th day after administration. From the fluorescence signal intensity change curve, it was found that the DyLight 680-III-2 curve was almost consistent with the change curve of DyLight 680-Herceptin. The result showed that compound III-2 had similar metabolic properties to Herceptin. In addition, the fluorescence intensity at the tumor site was significantly higher than that of other normal tissues. The above results show that compound III-2 has excellent tumor targeting and a long half-life in vivo.

[0414] Fig. 20The drug distribution and metabolism of compound III-2 were shown. The results showed that high concentrations of prodrug Cys-II-2 were detected in the blood and peripheral tissues 6 hours after administration of compound III-2. In addition, the results showed that Cys-II-2 can be quickly cleared in normal tissues, and the half-life of Cys-II-2 in the blood is only 26 hours, which will further reduce its toxicity and side effects. After 96 hours of administration, Cys-II-2 was almost undetectable in the heart, liver, spleen, and lungs. The peak concentration and drug-time curve area of ​​the toxin MMAE in the tumor were 293 times and 941 times that of the peripheral blood, respectively, which further confirmed that compound III-2 has excellent targeting. The drug-time curve area of ​​MMAE in the heart, liver, spleen, and lungs accounted for 11%, 47%, 5%, and 7% of the drug-time curve area of ​​the tumor, respectively. The results show that Cys-II-2 in peripheral tissues, except for the liver, cannot be metabolized into MMAE, while Cys-II-2 in tumors can effectively release MMAE.

[0415] Fig.21 The anti-tumor effect of compound III-2 on NCI-N87 tumor-bearing mice was shown. The results showed that in the NCI-N87 tumor xenograft model, compared with the control group, the compound III-2 group at a dose of 5 mg / kg could significantly inhibit tumor growth, with a tumor inhibition rate of 90.97%. This result shows that compound III-2 can achieve almost the same anti-tumor effect as traditional VC-ADC. More importantly, all mouse tumors in the 10 mg / kg dose group completely regressed, indicating that compound III-2 has excellent anti-tumor therapeutic potential.

[0416] Fig. 22 The results showed that compound III-2 had an anti-tumor effect on JIMT-1 tumor-bearing mice. The results showed that in the JIMT-1 xenograft model, compound III-2 still showed an anti-tumor effect at a dose of 12 mg / kg. Although the tumor did not completely disappear, the inhibition rate reached 62.36%.

[0417] Fig.23 The anti-tumor effect of compound III-2 combined with Herceptin, bevacizumab, sunitinib or docetaxel on NCI-N87 tumor-bearing mice was shown. The results showed that the compound III-2 combined with sunitinib administration group showed a stronger anti-tumor effect (p<0.01), which was better than compound III-2 and other combination groups. In the combined administration group, only 3mg / kg III-2 can almost achieve the effect of complete tumor regression, with a tumor inhibition rate of 98.93%. The above results show that the excellent tumor inhibition effect of combined administration may come from the fact that angiogenesis inhibitors can expand the range of tumor tissue hypoxia and enhance the degree of hypoxia in tumor tissue, thereby further improving the efficacy of compound III-2.

[0418] Fig.24 The results of the maximum tolerated dose study of compound III-2 are shown. The results show that the MTD of compound III-2 is >200 mg / kg, while the mice in the VC-ADC group and the T-DM1 group experienced continuous weight loss (>20%) and severe adverse reactions since the start of administration. For example, all mice in the VC-ADC group died after being administered 120 mg / kg.

[0419] Fig.25 The results of hematological and histopathological studies of compound III-2 on BALB / C mice were shown. The results showed that there were no significant differences in blood markers of biochemistry, blood routine and histopathology between the compound III-2 administration group and the control group, while in the VC-ADC treatment group, the changes in blood biochemistry reflected the side effects of VC-ADC on the liver, such as increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK). The side effects of VC-ADC were also reflected in hematological changes, including increases in lymphocyte counts (LYMPH), monocyte counts (MONO), and neutrophil counts (NEUT).

[0420] Fig.26 It was shown that compound III-16 had excellent anti-Staphylococcus aureus effect. DETAILED DESCRIPTION

[0421] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0422] Example 1: Preparation of III-1

[0423] 1) Preparation of (4-nitrosophenyl)methanol

[0424]

[0425] Dissolve p-nitrobenzyl alcohol (6.0 g, 39.18 mmol) in 60 mL of methanol. After the solid is completely dissolved, add zinc powder (7.69 g, 117.54 mmol) under stirring, and then slowly add NH 4Cl (3.14 g, 58.77 mmol) aqueous solution (15 mL). After the addition, the reaction was stirred at room temperature for 90 minutes until the nitrobenzyl alcohol raw material was basically reacted. After filtering to remove the insoluble solid, the filtrate was cooled to -5 °C. Then, FeCl was slowly added dropwise under stirring. 3 (58.77mmol) solution (30mL methanol + 15mL water). After the addition was completed, the reaction was continued to stir at low temperature for 1h. After the reaction was completed, the insoluble matter in the reaction solution was filtered out, and the same volume of saturated saline solution was added to the filtrate, and then extracted with DCM. The organic phases were combined and washed with saturated saline solution for 3 times. The organic phase was washed with anhydrous Na 2 SO 4 After drying, the residue was concentrated under reduced pressure to obtain a crude product as a yellow residue. The target product was purified by silica gel column chromatography to obtain a yellow-green solid powder (3.0 g, 55.8% yield). 1 H-NMR (400MHz, DMSO-d6): δ7.92(d,J=8.12Hz,2H),7.66(d,J=8.12Hz,2H),5.56(t,J=5.64Hz,1H),4.63(d,J=5.64Hz,2H).MS(ESI)m / z:138.06[M+H] + .

[0426] 2) Preparation of (E)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzoic acid

[0427]

[0428] Dissolve (4-nitrosophenyl)methanol (2.7 g, 19.69 mmol) in 50 mL of AcOH, and add p-aminobenzoic acid (2.7 g, 19.69 mmol) after the solid is completely dissolved. Stir and react for 36 h at room temperature. Insoluble matter slowly precipitates during the reaction. After the reaction is completed, separate the insoluble solid precipitated during the reaction by suction filtration, and wash the filter cake with DCM to obtain the target product as an orange-yellow solid powder (3.3 g, 65.4% yield). 1 H-NMR (400MHz, DMSO-d6): δ13.19(br,1H),8.15(dt,2H),7.97(dt,2H),7.92 (dt,2H),7.56(d,2H),5.46(br,1H),4.63(s,2H).MS(ESI)m / z:257.09[M+H] + .

[0429] 3. Preparation of tert-butyl(E)-(2-(4-((4-(hydroxymethyl)phenyl)diazenyl)benzamido)ethyl)carbamate

[0430]

[0431] (E)-4-((4-(Hydroxymethyl)phenyl)diazenyl)benzoic acid (2.5 g, 9.76 mmol) was dissolved in 40 mL of DMF. After the solid was completely dissolved, mono-Boc ethylenediamine (2.03 g, 12.68 mmol), EDCI (2.43 g, 12.68 mmol), and HOBT (1.72 g, 12.68 mmol) were added in sequence, and the mixture was reacted at room temperature for 5 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the obtained residue was dispersed with DCM, and the insoluble matter was separated by filtration. The filter cake was washed with a PE:EA=1:1 v / v mixed solution to obtain the target product as a brown solid (3.15 g, 81.0% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.66(t,1H),8.04(d,2H),7.95-7.90(m,4H),7.55(d,2H),6.97(t,1 H),5.42(t,1H),4.62(d,2H),3.32(q,2H),3.12(q,2H),1.38(s,9H).MS(ESI)m / z:399.19[M+H] + ; 421.18[M+Na] + .

[0432] 4) Preparation of (E)-N-(2-aminoethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide

[0433]

[0434] Tert-butyl (E)-((2-(4-(4-(hydroxymethyl)phenyl)diazenyl)benzamido)ethyl)carbamate (0.95 g, 2.38 mmol) was dissolved in 10 mL of DCM, and TFA (2 mL) was added dropwise under stirring. After the addition, the mixture solution became clear, and the mixture was reacted at room temperature for 5 h. After the reaction was completed, the solvent was removed under reduced pressure, the obtained oil was redissolved with EA, and then PE was slowly added dropwise to precipitate insoluble matter. The mixture was filtered after ultrasonication, and the filter cake was washed with ether to obtain the target product as an orange-yellow solid powder (0.9 g, 95.4% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.83(t,1H),8.09(d,2H),7.98(m,2H),7.92(m,2H),7.56(d,2H) ,5.55(s,1H),4.62(d,2H),3.54(q,2H),3.45(m,2H),3.03(q,2H).MS(ESI)m / z:299.15[M+H] + ; 321.13[M+Na] + .

[0435] 5) Preparation of (E)-N-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide (I-1, (E)-N-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide

[0436]

[0437] (E)-N-(2-aminoethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide (237.82 mg, 0.6 mmol) was dissolved in 10 mL of DMF, and 2,5-dihydropyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (239.6 mg, 0.9 mmol) and DIPEA (155 mg, 1.2 mmol) were added under stirring, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain an oily substance. Ether was added to the oily substance and ultrasonicated to precipitate an insoluble solid. After filtering off the filtrate, the filter cake was further purified by silica gel column chromatography to obtain the target product as an orange-yellow solid powder (216 mg, 80% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.65(t,1H),8.11(t,1H),8.04(m,6H),7.55(d,2H),7.00(s,2H),5.38( t,1H),4.62(d,2H),3.62(t,2H),3.30(s,2H),3.21(m,2H),2.35(m,2H).MS(ESI)m / z:450.15[M+H] + ; 472.15[M+Na] + .

[0438] 6) Preparation of (E)-4-((4-((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl)carbamoyl)phenyl)diazenyl)benzyl(4-nitrophenyl)carbonate (I-7, (E)-4-((4-((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl)carbamoyl)phenyl)diazenyl)benzyl(4-nitrophenyl)carbonate

[0439]

[0440] (E)-N-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)ethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide (I-1) (45 mg, 0.1 mmol) was dissolved in 15 mL of DMF, and p-nitrophenol carbonate (60.91 mg, 0.2 mmol) and DIPEA (19.4 mg, 0.15 mmol) were added in sequence under stirring. The mixture was stirred at room temperature for overnight reaction. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The obtained residue was dispersed with ether and ultrasonicated. The precipitated insoluble solid was separated by filtration, and the filter cake was washed with ether for multiple times to obtain the target product as an orange-yellow solid powder (54 mg, 88% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.69(t,1H),8.39(m,0.5H),8.33(dt,2H),8.14(t,1H),8.05(m,2H),7.98(m,4H),7.75(m,0.5H),7.7 2(d,2H),7.61(dt,2H),7.01(s,2H),5.44(s,2H),3.62(t,2H),3.30(q,2H),3.20(q,2H),2.35(t,2H).MS(ESI)m / z:615.18[M+H] + ; 637.17[M+Na] + .

[0441] 7) Preparation of Compound II-1

[0442]

[0443] (E)-4-((4-((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)ethyl)carbamoyl)phenyl)diazenyl)benzyl(4-nitrophenyl)carbonate (I-7) (43.0 mg, 0.07 mmol), monomethyl auristatin E (MMAE) (50.0 mg, 0.07 mmol), and HOBT (9.46 mg, 0.07 mmol) were added to a 10 mL eggplant-shaped bottle, and then 5 mL of anhydrous DMF was added and stirred at room temperature to completely dissolve. After the solid was completely dissolved, DIPEA (18.1 μL, 0.14 mmol) was added dropwise and reacted at room temperature for 24 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain the target product as a white powdery solid (63 mg, 75.4% yield). HR-MS(ESI)m / z:1193.6604[M+H] + ; 1215.6453[M+Na] +.

[0444] 8) Synthesis of Compound III-1

[0445]

[0446] The method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.) was used to couple compound II-1 with anti-HER2 antibody trastuzumab (purchased from Roche Pharmaceuticals) to obtain the target antibody-coupled drug III-1, wherein: (MAB) indicates the anti-HER2 antibody trastuzumab, with a drug-antibody coupling ratio (DAR) E of about 4.

[0447] Example 2: Preparation of Compounds III-2, III-3, III-4, III-5

[0448] 1) Referring to the preparation method of compound I-1 in Example 1, 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate was replaced by the following compound:

[0449]

[0450] Compounds I-2, I-3, I-4, and I-5 were prepared.

[0451]

[0452] I-2: 1 H-NMR (400MHz, DMSO-d6): δ8.68(t,1H),8.04(m,4H),7.93(m,4H),7.56(d,2H),7.00(s,2H),5.42(t,J=5.8Hz,1H),4.62(d,J=5 .8Hz,2H),3.60(t,J=6.7Hz,4H),3.45(m,4H),3.37(m,2H),3.31(m,2H),3.29(m,2H),3.13(q,J=6.0Hz,2H),2.32(m,4H).ESI-MS m / z(M+H) + calculated for C 30 H 37 N 6 O 8 609.27, found 609.24; ESI-MS m / z(M+Na) +calculated for C 30 H 36 N 6 NaO 8 631.25,found 631.23.

[0453]

[0454] I-3: 1 H-NMR(400MHz,DMSO-d6):δ8.69(t,J=5.5Hz,1H),8.05(m,4H),7.92(m,4H),

[0455] 7.55(d,J=8.7Hz,2H),7.00(s,2H),5.42(t,J=5.7Hz,1H),4.62(d,J=5.7Hz,2H),3.61(m,4H),3.46(m,20H),3.39(m,2H),3.31(m,2H),3.22(m,4H),2.32(m,4H).ESI-MSm / z(M+H) + calculated for C 38 H 53 N 6 O 12 785.37,found 785.36;ESI-MS m / z(M+Na) + calculated for C 38 H 52 N 6 NaO 12 807.35,found 807.35.

[0456]

[0457] I-4: 1 H-NMR(400MHz,DMSO-d6):δ8.69(t,1H),8.05(m,4H),7.92(m,4H),7.55(d,2H),7.00(s,2H),5.42(t,1H),4.62(d,2H),3.61(m,4H),3.46(m,20H),3.39(m,2H),3.31(m,2H),3.22(m,4H),2.32(m,4H).MS(ESI)m / z:785.36[M+H] + ;807.35[M+Na] + .

[0458]

[0459] I-5: 1 H-NMR (400MHz, DMSO-d6): δ8.68(t,1H),8.04(m,4H),7.92(m,4H),7.55(d,2H),7.00(s,2H),5.42(t,1H),4.62( d,2H),3.61(m,4H),3.46(m,28H),3.36(m,2H),3.26(m,2H),3.14(m,4H),2.32(m,4H).MS(ESI)m / z:873.42[M+H] + ; 895.40[M+Na] + .

[0460] 2) Referring to the preparation method of compound I-7 in Example 1, compound I-2, I-3, I-4, I-5 were used to replace compound I-1 to prepare compounds I-8, I-9, I-10, I-11.

[0461]

[0462] I-8: 1 H-NMR (400MHz, DMSO-d6): δ8.70(t,1H),8.33(dt,2H),8.05(m,4H),7.95(m,4H),7.72(d,2H),7.61(d,2H),7.00(s,2H),5.44 (s,2H),3.59(q,4H),3.50(m,4H),3.35(m,2H),3.32(m,2H),3.26(t,2H),3.13(q,2H),2.32(m,4H).MS(ESI)m / z:774.27[M+H] + ; 796.26[M+Na] + .

[0463]

[0464] I-9: 1 H-NMR (400MHz, DMSO-d6): δ8.70(t,1H),8.41-8.33(m,3H),8.04(m,4H),7.96(m,4H),7.74(m,3H),7.61(dt,2H),7.00(s,2 H),5.44(s,2H),3.59(q,4H),3.47(m,14H),3.38(m,2H),3.26(t,2H),3.13(q,2H),2.33(m,4H).MS(ESI)m / z:862.31[M+H]+ ; 884.29[M+Na] + .

[0465]

[0466] I-10: 1 H-NMR (400MHz, DMSO-d6): δ8.70(t,1H),8.34(dt,2H),8.05(m,4H),7.96(m,4H),7.72(d,2H),7.61(dt,2H),7.00(s,2H) ,5.44(s,2H),3.59(q,4H),3.47(m,22H),3.36(m,2H),3.26(t,2H),3.14(q,2H),2.33(m,4H).MS(ESI)m / z:950.38[M+H] + ; 972.36[M+Na] + .

[0467]

[0468] I-11: 1 H-NMR (400MHz, DMSO-d6): δ8.70(t,1H),8.34(dt,2H),8.07-7.73(m,8H),7.72(d,2H),7.61(dt,2H),7.00(s,2H),5. 44(s,2H),3.59(q,4H),3.49(m,30H),3.36(m,2H),3.26(t,2H),3.14(q,2H),2.33(m,4H).MS(ESI)m / z:1038.43[M+H] + ; 1060.41[M+Na] + .

[0469] 3) Referring to the preparation method of compound II-1 in Example 1, replace compound I-7 with compounds I-8, I-9, I-10, and I-11 to prepare compounds II-2, II-3, II-4, and II-5.

[0470]

[0471] II-2: 1 H NMR (600 MHz, CDCl 3-d6)7.98-7.91(m,6H),7.59-7.50(m,3H),7.38-7.31(m,4H),7.24(t,J=6.0Hz,1H),7.10(s,1H),6.83(br,1H),6.66(s,1H),6.54(d,J=12.0Hz,1H),5.31(d,J=12.0Hz,1H),5.18(d,J=12.0Hz,1H),4.71-4.67(m,2H),4.25-4.05(m,5H),3.83(t,J=12.0Hz,3H),3.71(t,J=6.0Hz,2H),3.61-3.50(m,12H),3.41-3.30(m,10H),2.99-2.94(m,5H),2.58-2.55(m,2H),2.49(t,J=6.0Hz,2H),2.05-1.94(m,4H),1.28-1.21(m,8H),1.03-0.99(m,4H),0.96(d,J=6.0Hz,4H),0.88(t,J=12.0Hz,10H),0.82(t,J=6.0Hz,3H),0.76-0.69(m,3H).ESI-HRMS m / z(M+H) + calculated forC 70 H 102 N 11 O 16 1352.7506,found 1352.7502;ESI-HRMS m / z(M+Na) + calculated forC 70 H 101 N 11 NaO 16 1374.7325,found 1374.7327.

[0472]

[0473] II-3: 1 H NMR(600MHz,CDCl 3 -d6)11.01(br,1H),8.04(br,3H),7.91(d,J=6.0

[0474] Hz,5H),7.56-7.49(m,3H),7.31(br,4H),6.64(s,1H),5.31(d,J=12.0Hz,1H),5.18(d,J=12.0Hz,1H),4.92-4.69(m,4H),3.78-3.49(m,32H),3.40-3.30(m,11H),3.12-2.94(m,9H),1.50-1.47(m,14H),1.41(d,J=6.0Hz,11H),0.99-0.76(m,25H).ESI-HRMS m / z(M+H) + calculated for C 74 H 110 N 11 O 18 1440.8030,found 1440.8015;ESI-HRMS m / z(M+Na) + calculated for C 74 H 109 N 11 NaO 18 1462.7836,found 1462.7836.

[0475]

[0476] II-4: 1 H NMR(600MHz,CDCl 3 -d6)8.04-7.91(m,6H),7.56-7.50(m,3H),7.35-7.32(m,5H),7.26-7.24(m,1H),6.67(s,2H),5.30(d,J=12.0Hz,1H),5.19(d,J=12.0Hz,1H),4.93(br,1H),3.82(t,J=6.0Hz,2H),3.72-3.50(m,33H),3.39-3.31(m,10H),3.12-3.08(m,3H),3.01(br,2H),2.95(d,J=6.0Hz,2H),2.52-2.51(m,4H),1.48-1.40(m,15H),1.28-1.23(m,8H),1.00-0.77(m,25H).ESI-HRMS m / z(M+H) + calculated for C 78 H 118 N 11 O 201528.8555, found 1528.8539; ESI-HRMS m / z(M+Na) + calculated for C 78 H 117 N 11 NaO 20 1550.8374, found 1550.8363.

[0477]

[0478] II-5: 1 H NMR (600 MHz, CDCl 3 -d6)8.05-7.91(m,7H),7.56-7.50(m,3H),7.35-7.32(m,4H),7.26-7.24(m,1H) ,6.68(s,2H),5.31-5.27(m,1H),5.19(d,J=12.0Hz,1H),4.93(br,1H),4.80-4.6 8(m,2H),3.82(t,J=12.0Hz,2H),3.63-3.51(m,40H),3.40-3.31(m,11H),3.12-3 .08(m,8H),1.48-1.39(m,14H),1.29-1.24(m,9H),1.00-0.78(m,26H).ESI-HRMS m / z(M+H) + calculated for C 82 H 126 N 11 O 22 1616.9079, found 1616.9067; ESI-HRMSm / z(M+Na) + calculated for C 82 H 125 N 11 NaO 22 1638.8898, found 1638.8886.

[0479] 4) Referring to the preparation method of compound III-1 in Example 1, replace compound II-1 with compounds II-2, II-3, II-4, and II-5, respectively, to prepare compounds III-2, III-3, III-4, and III-5.

[0480]

[0481] The DAR values ​​E of compounds III-2, III-3, III-4, and III-5 are all about 4. It is the anti-HER2 antibody trastuzumab.

[0482] Example 3: Preparation of Compound III-6

[0483] 1) Preparation of (9H-fluoren-9-yl)methyl(S,E)-(38-(4-((4-(hydroxymethyl)phenyl)diazenyl)phenyl)-26,33,38-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,34,37-triazaoctatriacontan-32-yl)carbamate

[0484]

[0485] (S)-32-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatritriacontan-33-oic acid (810 mg, 1.06 mmol) and (E)-N-(2-aminoethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide (350 mg, 0.88 mmol) were dissolved in 20 mL of DMF, followed by the addition of EDCI (254 mg, 1.325 mmol), HOBT (119.31 mg, 0.88 mmol) and DIPEA (171.2 mg, 1.325 mmol). The mixture was reacted at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain the target product as an orange-yellow solid powder (730 mg, 79.25% yield). 1H-NMR (400MHz, DMSO-d6): δ8.65(t,1H),8.12(t,1H),8.04(d,2H),7.88(m,5H),7.73(m,3 H),7.56(d,2H),7.49(t,2H),7.40(m,3H),7.31(t,2H),5.43(t,1H),4.62(d,2H),4.20(m ,3H),3.91(q,1H),3.60(m,6H),3.47(m,24H),3.40(m,2H),3.24(m,3H),3.12(q,2H),3.0 0(t,2H),2.28(t,2H),1.63(m,1H),1.52(m,1H),1.35(m,4H).MS(ESI)m / z:1043.51[M+H] + ; 1065.49[M+Na] + .

[0486] 2) Preparation of (S,E)-N-(32-amino-26,33-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,34-diazahexatriacontan-36-yl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide

[0487]

[0488] (9H-fluoren-9-yl)methyl(S,E)-(38-(4-((4-(hydroxymethyl)phenyl)diazenyl)phenyl)-26,33,38-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,34,37-triazaoctatriacontan-32-yl)carbamate (450 mg, 0.43 mmol) was dissolved in 20 mL of DCM, piperidine (0.8 mL) was added, and the mixture was reacted at room temperature for 1 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography to give the target product as an orange-yellow viscous oil (328 mg, 92.8% yield). 1H-NMR (400MHz, DMSO-d6): δ8.77(t,1H),8.29(t,1H),8.05(m,2H),7.92(m,4H),7.84(t,1H),7. 55(d,2H),4.62(d,2H),3.58-3.29(m,40H),2.28(t,2H),1.54(m,6H).MS(ESI)m / z:821.45[M+H] + ; 843.43[M+Na] + .

[0489] 3) Preparation of Compound I-6

[0490]

[0491] (S,E)-N-(32-amino-26,33-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,34-diazahexatriacontan-36-yl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide (400 mg, 0.49 mmol) was dissolved in 20 mL of DMF, and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (190 mg, 0.73 mmol) was added under stirring, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain an orange-yellow solid. The crude product residue was further purified by silica gel column chromatography to obtain the target product as an orange-yellow oil (330 mg, 69.3% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.67(t,1H),8.09(m,4H),7.92(m,4H),7.82(t,1H),7.54(d,2H),7.00(s,2H),5.43(t,1H),4.62(d,2H), 3.59(m,8H),3.47(m,26H),3.23(d,3H),3.12(m,3H),2.99(br,4H),2.41(t,2H),2.28(t,2H),1.16(t,6H).MS(ESI)m / z:972.48[M+H] + ; 994.46[M+Na] + .

[0492] 4) Preparation of Compound I-12

[0493]

[0494] Compound I-6 (300 mg, 0.31 mmol) was dissolved in 15 mL of DMF, and di(p-nitrophenyl) carbonate (187.76 mg, 0.62 mmol) and DIPEA (59.8 mg, 0.46 mmol) were added in sequence under stirring. The mixture was stirred at room temperature for 36 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The obtained orange-yellow oily residue was purified by silica gel column chromatography to obtain the target product as an orange-yellow oil (200 mg, 56.7% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.71(t,1H),8.34(d,2H),8.15(d,1H),8.08(m,2H),7.94(t,4H),7.72(d,2H),7.61(d,2H),7.00(s,2H), 5.44(s,2H),3.62-3.46(m,42H),2.98(q,2H),2.41(t,2H),2.28(t,2H),1.59-1.41(m,4H),1.12(m,2H).MS(ESI)m / z:1137.48[M+H] + ; 1159.46[M+Na] + .

[0495] 5) Preparation of Compound II-6

[0496]

[0497] Compound I-12 (79.6 mg, 0.07 mmol), MMAE (50.0 mg, 0.07 mmol), and HOBT (9.46 mg, 0.07 mmol) were added to a 50 mL eggplant-shaped bottle, and then 10 mL of anhydrous DMF was added and stirred at room temperature to completely dissolve. After the solid was completely dissolved, DIPEA (18.1 μL, 0.14 mmol) was added dropwise and reacted at room temperature for 24 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain the target product as a white powdery solid (92 mg, 76.6% yield). HR-MS (ESI) m / z: 1715.9727 [M+H] + ; 1738.9583[M+Na] + .

[0498] 6) Synthesis of Compound III-6

[0499]

[0500] The method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.) was used to couple compound II-6 with the anti-HER2 antibody trastuzumab to obtain the target antibody-coupled drug III-6, wherein: (MAB) indicates the anti-HER2 antibody trastuzumab, with a drug-antibody coupling ratio (DAR) E of about 4.

[0501] Example 4: Preparation of Compounds III-7, III-8, III-9, III-10, III-11, III-12, III-13

[0502] 1) Preparation of compounds II-7, II-8, II-9, II-10, II-11, II-12, and II-13

[0503] Referring to the preparation method of compound II-1 in Example 1, compound I-8 was used to replace compound I-7, and SN-38, Exatecan, Doxorubicin, Lapatinib or TLR7 agonist with CAS number 1821304-87-3 was used to replace MMAE to prepare compounds II-7, II-8, II-9, II-10, and II-11.

[0504]

[0505] II-7: 1 H NMR (600 MHz, DMSO-d6): δ7.996 (d, J=8.4 Hz, 2H), 7.925-7.828 (m,

[0506] 6H),7.610(s,1H),7.530(d,J=8.4 Hz,3H),7.400(br,1H),6.683-6.671(m,2H),6.434-6.317(m,2H),5.672(d,J=16.2 Hz,1H),5.352-5.143(m,6H),3.796(t,J=7.2 Hz,2H),3.721(t,J=5.4 Hz,3H),3.616-3.485(m,42H),3.378-3.370(m,2H),3.126-3.095(m,2H),2.508-2.476(m,5H),2.346(s,3H),1.924(d,J=6.6 Hz,2H),1.333-1.255(m,4H),1.062(t,J=7.2 Hz,3H).ESI-HRMS m / z(M+H) +calculated for C 65 H 79 FN 8 O 20 1291.53,found1291.54.

[0507]

[0508] II-8: 1 H NMR(600 MHz,DMSO-d6):δ8.706(br,1H),8.211(d,J=8.4 Hz,1H),

[0509] 8.064-8.037(m,4H),7.944(d,J=7.2 Hz,4H),7.753(d,J=10.8 Hz,1H),7.658(d,J=7.8 Hz,2H),7.305(s,1H),6.992(s,2H),5.435(dd,J=22.2 Hz,16.8 Hz,2H),5.31-5.221(m,5H),3.620-3.574(m,5H),3.463(d,J=2.4 Hz,4H),3.355-3.336(m,4H),3.258(dd,J=11.4 Hz,5.4 Hz,3H),3.151-3.117(m,4H),2.361-2.317(m,7H),1.895-1.810(m,2H),1.301-1.261(m,5H),0.865(t,J=7.2 Hz,3H).ESI-HRMS m / z(M+H) + calculated forC 55 H 57 FN 9 O 13 1070.3982,found 1070.4094.

[0510]

[0511] II-9: 1H NMR(600 MHz,DMSO-d6):δ13.991(s,1H),13.231(s,1H),8.674(s,1H),8.027(d,J=7.8 Hz,4H),7.905(d,J=7.8 Hz,2H),7.857(t,J=7.2 Hz,4H),7.571-7.526(m,3H),7.040(d,J=7.8 Hz,1H),6.991(s,2H),5.442(s,1H),5.229(s,1H),5.078(dd,J=28.2 Hz,13.2 Hz,2H),4.891(d,J=28.2 Hz,2H),4.758(d,J=5.4 Hz,1H),4.590(d,J=4.8 Hz,2H),4.185(d,J=6.0 Hz,1H),3.945(s,3H),3.760(br,1H),3.618-3.574(m,5H),3.462(s,6H),3.256(d,J=5.4Hz,2H),3.138(d,J=5.4Hz,3H),2.942(dd,J=51.0Hz,18.0Hz,2H),2.337-2.317(m,4H),2.214(d,J=13.2Hz,1H),2.101(d,J=9.6Hz,1H),1.890(t,J=10.8Hz,1H),1.511(d,J=10.2Hz,1H),1.288-1.26(m,4H),1.145(d,J=5.4Hz,3H),1.091(t,J=7.2Hz,2H).ESI-HRMS m / z(M+H) + calculated for C 58 H 64 N 7 O 20 1178.4128,found 1178.4195.

[0512]

[0513] II-10: 1H NMR(600MHz,DMSO-d6):δ9.930-9.863(m,1H),8.738-8.563(m,3H),8.039-7.811(m,11H),7.731-7.586(m,3H),7.494-7.458(m,1H),7.342-7.270(m,3H),7.203-7.171(m,1H),7.092(d,J=3.0Hz,1H),6.991(s,1H),6.614(br,1H),5.309-5.256(m,4H),4.708-4.681(m,1H),4.031(q,J=7.2Hz,1H),3.804(br,2H),3.625-3.578(m,4H),3.495-3.438(m,7H),3.265(q,J=6.0Hz,1H),3.141(dd,J=11.4Hz,5.4Hz,2H),3.059-2.893(m,3H),2.337(dd,J=12.6Hz,6.0Hz,4H),1.992(s,1H),1.177(t,J=6.6Hz,1H).ESI-HRMS m / z(M+H) + calculated for C 60 H 61 ClFN 10 O 13 S1215.3735,found1215.3850.

[0514]

[0515] II-11: 1H NMR (600MHz, DMSO-d6): δ12.428(s,1H),9.137(s,1H),8.719(s,1H),8.072-7.921(m,9H),7.719(s,2H),7 .586-7.575(m,2H),7.421(s,1H),7.034-6.994(m,3H),6.820(d,J=5.4Hz,1H),6.692-6.681(m,1H),6.15 6(s,1H),5.217-5.193(d,J=14.4Hz,4H),4.035-4.024(m,1H),3.866(s,3H),3.608-3.464(m,17H),3.264 -3.142(m,5H),2.341(br,9H),1.990(s,1H),1.642(br,2H),1.361-1.175(m,8H),0.896(br,3H).ESI-HRMS m / z(M+H) + calculated for C 55 H 70 N 13 O 10 1072.5290, found 1072.5399.

[0516] Referring to the preparation method of compound II-1 in Example 1, compound I-11 was used to replace compound I-7, and SN-38 or Exatecan was used to replace MMAE to prepare compounds II-12 and II-13.

[0517]

[0518] II-12: 1 H NMR (600MHz, DMSO-d6): δ7.996 (d, J=8.4Hz, 2H), 7.925-7.828 (m,

[0519] 6H),7.610(s,1H),7.530(d,J=8.4Hz,3H),7.400(br,1H),6.683-6.671(m,2H),6.434-6.317(m,2H),5.672(d,J=16.2Hz,1H),5.352-5.143(m,6H),3.796(t,J=7.2Hz,2H),3.721(t,J=5.4Hz,3H),3.616-3.485(m,42H),3.378-3.370(m,2H),3.126-3.095(m,2H),2.508-2.476(m,5H),2.346(s,3H),1.924(d,J=6.6Hz,2H),1.333-1.255(m,4H),1.062(t,J=7.2Hz,3H).ESI-HRMS m / z(M+H) + calculated for C 65 H 79 FN 8 O 20 1291.53,found1291.54.

[0520]

[0521] II-13: 1 H NMR(600MHz,DMSO-d6):δ7.996(d,J=8.4Hz,2H),7.925-7.828(m,

[0522] 6H),7.610(s,1H),7.530(d,J=8.4Hz,3H),7.400(br,1H),6.683-6.671(m,2H),6.434-6.317(m,2H),5.672(d,J=16.2Hz,2H),5.352-5.143(m,6H),3.796(t,J=7.2Hz,2H),3.721(t,J=5.4Hz,3H),3.616-3.545(m,42H),3.378-3.370(m,2H),3.126-3.095(m,2H),2.508-2.476(m,5H),2.346(s,3H),1.924(d,J=6.6Hz,2H),1.333-1.255(m,4H),1.062(t,J=7.2Hz,3H).ESI-HRMS m / z(M+H) + calculated for C 67 H 81 FN 9 O19 1334.56, found1334.56.

[0523] 2) Preparation of compounds III-7, III-8, III-9, III-10, III-11, III-12, III-13

[0524] Referring to the preparation method of compound III-1 in Example 1, compounds II-7, II-8, II-9, II-10, II-11, II-12, and II-13 were used to replace compound II-1 to prepare compounds III-7, III-8, III-9, III-10, III-11, III-12, and III-13.

[0525]

[0526]

[0527] The DAR values ​​E of compounds III-7, III-8, III-9, III-10, III-11, III-12, and III-13 are all about 4. It is the anti-HER2 antibody trastuzumab.

[0528] Example 5: Preparation of Compound III-14

[0529] 1) Preparation of 4-nitrosobenzoic acid

[0530]

[0531] Potassium peroxymonosulfonate (17.93 g, 29.17 mmol) was dissolved in 150 mL of water, and a DCM solution (20 mL) of p-aminobenzoic acid (2.0 g, 14.58 mmol) was slowly added under stirring. After the suspension was stirred at room temperature for 3 hours, the reaction system gradually changed from white to yellow. After the reaction was completed, the yellow filter cake was filtered and washed with pure water for several times, and dried under reduced pressure to obtain the target product as a yellow powdery solid (2.13 g, 96.67% yield). 1 H-NMR (400MHz, DMSO-d6): δ13.59(s,1H),8.26(dt,2H),8.04(dt,2H).

[0532] 2) Preparation of (E)-4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzoic acid

[0533]

[0534] 7-amino-4-methyl-2H-chromen-2-one (0.58 g, 3.31 mmol) and 4-nitrosobenzoic acid (0.5 g, 3.31 mmol) were dissolved in glacial acetic acid (50 mL). The mixture was stirred at room temperature overnight. As the reaction continued, an orange-yellow precipitate gradually precipitated, and the reaction also changed from green to orange-yellow. After the reaction was completed, the insoluble matter was filtered off, and the filter cake was washed with AcOH and DCM, and purified by beating with DCM to obtain the target product as an orange-yellow powder solid (465 mg, 45.6% yield). 1 H-NMR (400MHz, DMSO-d6): δ13.34(s,1H),8.17(dt,2H),8.03(m,3H),7.93(dd,1H),7.87(d,1H),6.55(d,1H),2.51(s,3H).MS(ESI)m / z:309.08[M+H] + .

[0535] 3) Preparation of tert-butyl(E)-(2-(4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzamido)ethyl)carbamate

[0536]

[0537] (E)-4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzoic acid (400 g, 6.62 mmol) was dissolved in 20 mL of DMF. After all the solution was dissolved, EDCI (373.8 mg, 1.95 mmol), HOBT (176 mg, 1.30 mmol) and DIPEA (226 μL, 1.3 mmol) were added in sequence. After the mixture was reacted at room temperature for 45 minutes, mono-Boc-ethylenediamine (312 mg, 1.95 mmol) was added and stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography to obtain the target product as an orange-yellow solid powder (400 mg, 68.3% yield). 1H-NMR (400MHz, DMSO-d6): δ8.69(t,1H),8.05(m,5H),7.93(dd,1H),7.87(d,1H),6.97(t,1H) ,6.55(d,1H),3.33(m,2H),3.13(q,2H),2.51(s,3H),1.38(s,9H).MS(ESI)m / z:451.19[M+H] + ; 473.17[M+Na] + .

[0538] 4) Preparation of (E)-N-(2-aminoethyl)-4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzamide

[0539]

[0540] Tert-butyl(E)-(2-(4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzamido)ethyl)carbamate (150 mg, 0.33 mmol) was dissolved in 20 mL of DCM, and TFA (1 mL) was added dropwise under stirring. After the addition, the mixture became clear and the mixture was reacted at room temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure to obtain an orange-red solid residue. The target product was purified by silica gel column chromatography to obtain an orange solid powder (140 mg, 94.8% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.84(t,1H),8.05(m,5H),7.93(dd,1H),7.87(d,1H),6.96(t,1H) ,6.56(d,1H),3.51(q,2H),3.16(s,2H),2.98(t,2H),2.51(s,3H).MS(ESI)m / z:351.14[M+H] + ; 373.12[M+Na] + .

[0541] 5) Preparation of Compound II-14

[0542]

[0543] (E)-N-(2-aminoethyl)-4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzamide (115 mg, 0.257 mmol) was dissolved in 15 mL of DMF and added under stirring. (131.21 mg, 0.308 mmol) and DIPEA (39.8 mg, 0.308 mmol), the mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a crude oil. The target product was further purified by silica gel column chromatography to obtain an orange-yellow solid powder (147 mg, 86.6% yield). 1 H-NMR (400MHz, DMSO-d6): δ8.72(t,1H),8.03(m,7H),7.93(dd,1H),7.86(d,1H),7.00(s,2H),6.55(d,1H),3.71(t,2H),3.58 (m,2H),3.48(m,2H),3.36(m,2H),3.25(q,2H),3.14(m,4H),2.92(t,2H),2.81(s,3H),2.33(m,4H).MS(ESI)m / z:361.24[M+H] + ; 683.22[M+Na] + .

[0544] 6) Preparation of Compound III-14

[0545] Referring to the preparation method of compound III-1 in Example 1, compound II-14 was used to replace compound II-1 to prepare compound III-14.

[0546]

[0547] The DAR value E of compound III-14 is about 4, It is the anti-HER2 antibody trastuzumab.

[0548] Example 6: Preparation of Compound III-15

[0549] 1) Preparation of tert-butyl 3-(2-(2-(2-((1,3-dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)propanoate

[0550]

[0551] 2-Hydroxy-isoindole-1,3-dione (1.27 g, 7.81 mmol) was added to a 100 mL eggplant-shaped bottle, 30 mL THF was added, and the mixture was cooled to 0°C in an ice bath. After 2-Hydroxy-isoindole-1,3-dione was completely dissolved, tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (2 g, 7.1 mmol) and triphenylphosphine (2.04 g, 7.81 mmol) were added to the reaction solution in sequence. The reaction was transferred to room temperature and the reaction was continued for 1 h. The solvent was removed by pressure concentration, the sample was stirred, and the target product was purified by column chromatography as a white solid (2.0 g, 61% yield). 1 H-NMR (600MHz, DMSO-d6): δ7.864(s,4H),4.275-4.261(m,2H),3.731-3.716(m,2H),3.560(t,J=6.6Hz,2H), 3.511-3.495(m,2H),3.436-3.408(m,6H),2.399(t,J=6.6Hz,2H),1.387(s,9H).MS(ESI)m / z:446.18[M+Na] + .

[0552] 2) Preparation of 3-(2-(2-(2-((1,3-dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)propanoic acid

[0553]

[0554] Add tert-butyl 3-(2-(2-(2-((1,3-dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)propanoate (1.136 g, 2.68 mmol) into a 50 mL eggplant-shaped bottle, add 10 mL of 4N HCl in dioxane solution, and stir at room temperature. After reacting for 1 hour, concentrate under pressure to remove the solvent, stir the sample, and purify by column chromatography to obtain the target compound as a white solid (0.41 g, 42% yield). 1H-NMR (600MHz, DMSO-d6): δ12.152(s,1H),7.872-7.871(m,4H),4.282-4.268(m,2H),3.738-3.722( m,2H),3.580(t,J=6.6Hz,2H),3.447-3.409(m,6H),2.433(t,J=6.0Hz,2H).MS(ESI)m / z:366.12[MH] - .

[0555] 3) Preparation of (E)-N-(1-((1,3-dioxoisoindolin-2-yl)oxy)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide(I-13)

[0556]

[0557] 3-(2-(2-(2-((1,3-dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)propanoic acid (0.16 g, 0.44 mmol) was added to a 50 mL eggplant-shaped bottle, and 4 mL of anhydrous DMF was added thereto. After the compound was completely dissolved, NHS (0.055 g, 0.48 mmol) and DIC (0.076 g, 0.6 mmol) were added to the reaction solution in sequence, and the mixture was stirred at room temperature overnight. (E)-N-(2-aminoethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide and DIPEA were added to the reaction solution, and the mixture was reacted at room temperature for 15 min. The solvent was removed by pressure concentration, and the target product was purified by column chromatography as a yellow solid (0.071 g, 25%), which was directly used in the next step.

[0558] 4) Preparation of (E)-4-((4-((1-((1,3-dioxoisoindolin-2-yl)oxy)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)carbamoyl)phenyl)diazenyl)benzyl(4-nitrophenyl)carbonate(I-14)

[0559]

[0560] (E)-N-(1-((1,3-dioxoisoindolin-2-yl)oxy)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide(I-13)(0.07 g, 0.11 mmol) was added to a 25 mL eggplant-shaped bottle, and 3 mL of anhydrous DMF was added thereto. After the reaction raw materials were completely dissolved, di(p-nitrophenyl) carbonate and DIPEA were added thereto in sequence, and the reaction was carried out at room temperature for 4 h. The solvent was removed by pressure vacuum concentration, and the crude product was mixed and purified by column chromatography to obtain the target product as a yellow solid (0.08 g, 90% yield), which was directly used in the next step reaction.

[0561] 5)4-((E)-(4-((1-((1,3-dioxoisoindolin-2-yl)oxy)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)carbamoyl)phe nyl)diazenyl)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-((3-hydroxypropyl)amino))-1-oxo Preparation of -3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate(I-15)

[0562]

[0563] (E)-4-((4-((1-((1,3-dioxoisoindolin-2-yl)oxy)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)carbamoyl)phenyl)diazenyl)benzyl(4-nitrophenyl)carbonate(I-14)(0.034 g, 0.042 mmol) was added to a 25 mL eggplant-shaped bottle, and 2 mL of anhydrous DMF was added thereto. After the compound raw material was completely dissolved, MMAF derivative, HOBT and DIPEA were added to the reaction solution in sequence, and stirred at room temperature overnight. The solvent was removed by pressure concentration, and the sample was stirred. The crude product was purified by column chromatography to obtain the target compound as a yellow solid (0.01 g, 17% yield), which was directly used in the next step reaction.

[0564] Synthesis steps of MMAF derivatives:

[0565] 1)((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-methylbutanamido Preparation of )-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0566]

[0567] MMAF (300 mg, 0.39 mmol) was dissolved in 5 mL of DMF, and 9-fluorenylmethyl chloroformate (50 mg, 0.195 mmol) and sodium bicarbonate (13 mg, 0.156 mmol) were added in sequence under stirring. The mixture was stirred at room temperature for overnight reaction. After the reaction was completed, the solvent was removed by vacuum concentration. The target product was purified by column chromatography as a white solid powder (200 mg, 57% yield). MS (ESI) m / z: 953.55 [M+H] + .

[0568] 2)(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-((3-hydroxypropyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl Preparation of -3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0569]

[0570] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (100 mg, 0.105 mmol), 3-aminopropan-1-ol (10 mg, 0.126 mmol) and HATU (60 mg, 0.158 mmol) were dissolved in 5 mL of ultra-dry DMF, and DIPEA (27 mg, 0.21 mmol) was added to the solution under stirring. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed by pressure concentration. The target product was purified by column chromatography as a yellow solid powder (91 mg, 90% yield). MS (ESI) m / z: 1010.60 [M+H] + .

[0571] 3) Preparation of MMAF derivatives

[0572]

[0573] (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-((3-hydroxypropyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (90 mg, 0.089 mmol) was dissolved in 2 mL of ultra-dry DMF and cooled to 0°C in an ice-salt bath. 100 μL of hexahydropiperidine was added to the solution under stirring. The mixture was stirred for 20 min before the reaction was completed. The solvent was removed by concentration under pressure, and the target product was purified by column chromatography as a white solid powder (39 mg, 56% yield). MS (ESI) m / z: 788.54 [M+H] + .

[0574] 6) Preparation of Compound II-15

[0575]

[0576] Compound I-15 (20 mg, 0.014 mmol) was added to a 10 mL eggplant-shaped bottle, 1 mL of anhydrous DCM was added thereto, and the reaction solution was cooled to 0°C in an ice bath. Hydrazine hydrate (0.001 g, 0.021 mmol) was diluted to 0.5 mL of anhydrous DCM and slowly added dropwise to the reaction solution. The reaction was allowed to react overnight, and an insoluble solid precipitated. The reaction solution was extracted with DCM several times, washed with saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate overnight, concentrated under reduced pressure to remove the solvent, and the crude product was purified by thin layer preparative chromatography to obtain the target product as a reddish brown solid (11 mg, 59% yield). 1H-NMR (600MHz, DMSO-d6): δ8.679-8.670(m,1H),8.083-8.016(m,3H),7.975-7.870(m,4H),7.641-7.551(m,1H),7.245-7 .126(m,5H),5.274-5.160(m,1H),4.657-4.624(m,1H),4.491-4.395(m,2H),3.970(br,1H),3.803(t,J=8.4Hz,1H),3.621 -3.594(m,3H),3.518-3.454(m,10H),3.388-3.325(m,11H),3.263-3.171(m,9H),3.055-2.828(m,7H),2.333(t,J=6.6Hz, 2H),2.180-2.119(m,4H),1.531(t,J=6.6Hz,3H),1.346(s,4H),1.234(s,10H),1.042-0.749(m,26H).MS(ESI)m / z:ESI-MS m / z(M+H) + calculated for C 68 H 106 N 11 O 16 1332.77, found 1332.78.

[0577] 7) Synthesis of Compound III-15

[0578] The method described in the literature (PNAS.111(5):1766-71.) was used to couple the II-15 compound with the anti-HER2 site-directed mutagenesis antibody to obtain the target antibody-conjugated drug III-15, wherein the anti-HER2 site-directed mutagenesis antibody contains the non-natural amino acid acetylphenylalanine (pAF), and the carbonyl group on pAF reacts with the hydroxylamine group on the compound II-15 to form an oxime bond, resulting in site-specific coupling. In compound III-15, It is a site-directed mutagenesis antibody against HER2, and the drug-antibody coupling ratio (DAR) value E is about 2.

[0580]

[0581] The sequence of the anti-HER2 site-directed mutagenesis antibody (Mol Cancer Ther (2020) 19 (9): 1833-1843.) is as follows:

[0582] Heavy Chain:

[0583] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSUSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0584] Light chain:

[0585] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:2)

[0586] In the sequence, "U" represents acetylphenylalanine.

[0587] Example 7: Preparation of III-16

[0588] 1) Preparation of Compound I-16

[0589]

[0590] Dissolve cyanuric chloride in 200 μL of anhydrous DMF and stir at room temperature for 2 hours to generate a large amount of white precipitate. Dissolve (E)-N-(2-(4-((4-(hydroxymethyl)phenyl)diazenyl)benzamido)ethyl)-4,7,10,13,16,19,22,25-octaoxaoctacos-27-ynamide (100 mg, 0.14 mmol) in 2 mL of anhydrous DCM and add it to the reaction bottle and stir at room temperature for 2 days. After the reaction is completed, the solvent is removed by concentration under reduced pressure to obtain a crude product, which is purified by column chromatography to obtain the target product as a yellow solid (30 mg, 30% yield). The reaction product is directly used in the next step.

[0591] 2) Preparation of Compound II-16

[0592]

[0593] Compound I-16 (25 mg, 0.034 mmol) and effector molecule Rifalogue (28 mg, 0.03 mmol) were added to a 1.5 mL EP tube, and 400 μL of anhydrous DMF was added to dissolve it. DIPEA (19 mg, 0.15 mmol) was added dropwise to the reaction system, and the reaction was shaken at room temperature for 5 days. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain the target product as a black solid (10 mg, 20% yield). HR-MS (ESI) m / z: 1626.792 [M+H] + .

[0594] 3) Preparation of antibacterial antibodies modified with azidation groups

[0595]

[0596] Antibacterial antibodies (26.25 mg, 1.75 × 10 -4 mmol) was added into a 5 mL EP tube, and the compound 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (0.54 mg, 1.4 × 10 -3 After stirring overnight at room temperature, the coupling product was replaced in PBS to prepare an antibacterial antibody modified with an azide group, wherein the drug-antibody coupling ratio (DAR) E was about 4. For antibacterial antibodies.

[0597] The sequence of the antibacterial antibody is:

[0598] Heavy chain:

[0599] MKKNIAFLLASMFVFSIATNAYAEVQLVQSGAEVKKPGASVKVSCEASGYTLTSYDINWVRQATGQGPEWMGWMNANSGNTGYAQKFQGRVTLTGDTSISTAYMELSSLRSEDTAVYYCARSSILVRGALGRYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)

[0600] Light chain:

[0601] MKKNIAFLLASMFVFSIATNAYADIVMTQSPSILSASVGDRVTITCRASQTISGWLAWYQQKPAEAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFGIYYCQQYKSYSFNFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:4) 4) Preparation of Compound III-16

[0602]

[0603] The antibacterial antibody modified with an azide group (2.5 mg, 1.67×10 -5mmol) was added into a 2 mL EP tube, and compound II-16 (0.41 mg, 2.5×10 -4 mmol) of DMA solution. Then, CuSO 4 (0.13mg,5.01×10 -4 mmol), THPTA (0.87mg, 2.0×10 -3 mmol) and sodium ascorbate (1.49 mg, 7.51 × 10 - 3 mmol) aqueous solution was added to the reaction system, and the reaction was stirred at room temperature for 5 hours, and then the coupling product was replaced in PBS to prepare the target antibody-drug conjugate III-16, wherein the drug-antibody coupling ratio (DAR) E was about 2, For antibacterial antibodies.

[0604] Example 8: Preparation of Compound III-17

[0605]

[0606] Albumin (1 equivalent, purchased from Zhejiang Hisun Pharmaceutical Co., Ltd.) was added to a 10 mL reaction bottle, and a DMA solution of compound II-2 (10 equivalents) was added to the antibody. After stirring at room temperature overnight, the coupling product was replaced in PBS to prepare the target antibody conjugate III-17, wherein the drug-antibody coupling ratio (DAR) E was 1. Stands for albumin.

[0607] Example 9: Preparation of Compound III-18

[0608] 1) Preparation of (E)-1-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)but-3-yn-1-ol

[0609]

[0610] The compound 1-(4-aminophenyl)but-3-yn-1-ol (0.3 g, 1.86 mmol) was added to a 50 mL eggplant-shaped bottle, 12 mL of concentrated hydrochloric acid and 6 mL of water were added, and the temperature was cooled to 0 °C in an ice bath. NaNO 2(0.152g, 2.2mmol), stirred for 1h. N,N-dimethylaniline (0.27g, 2.2mmol) was dissolved in 2mL of glacial acetic acid and added to the reaction solution, and the reaction was allowed to proceed overnight. The solvent was removed by pressure concentration, the sample was stirred, and purified by column chromatography to obtain a brown-red solid (0.44g, 80% yield). ESI-MS m / z (M+H) + calculated for C 18 H 19 N 3 O 294.15, found 294.16.

[0611] 2) Preparation of (E)-1-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate

[0612]

[0613] (E)-1-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)but-3-yn-1-ol (0.4 g, 1.36 mmol) was added to a 25 mL eggplant-shaped bottle, and 3 mL of anhydrous DMF was added thereto. After the reaction raw materials were completely dissolved, di(p-nitrophenyl) carbonate and DIPEA were added thereto in sequence, and the reaction was carried out at room temperature for 4 h. The solvent was removed by pressure vacuum concentration, and the crude product was mixed and purified by column chromatography to obtain the target product as a yellow solid (0.42 g, 67% yield). ESI-MS m / z (M+H) + calculated for C 25 H 22 N 4 O 5 459.16, found 459.17.

[0614] 3) Preparation of Compound II-17

[0615]

[0616] Compound (E)-1-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate (110 mg, 0.24 mmol), MMAE (172.0 mg, 0.24 mmol), and HOBT (32.4 mg, 0.24 mmol) were added to a 50 mL eggplant-shaped bottle, and then 10 mL of anhydrous DMF was added and stirred at room temperature to completely dissolve it. After the solid was completely dissolved, DIPEA (50 μL, 0.288 mmol) was added dropwise and reacted at room temperature for 24 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain the target product as a white powdery solid (200 mg, 80% yield). HR-MS (ESI) m / z: 1037.64 [M+H] + .

[0617] 4) Preparation of anti-HER2 antibody trastuzumab modified with azidation group

[0618]

[0619] The anti-HER2 antibody trastuzumab (26.25 mg, 1.75 × 10 -4 mmol) was added into a 5 mL EP tube, and the compound 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (0.54 mg, 1.4 × 10 -3 After stirring overnight at room temperature, the coupling product was replaced in PBS to prepare an antibacterial antibody modified with an azide group, wherein the drug-antibody coupling ratio (DAR) E was about 4. It is the anti-HER2 antibody trastuzumab.

[0620] 5) Preparation of Compound III-18

[0621]

[0622] The anti-HER2 antibody trastuzumab modified with an azide group (2.5 mg, 1.67×10 -5 mmol) was added into a 2 mL EP tube, and compound II-17 (0.26 mg, 2.5×10 -4 mmol) of DMA solution. Then, CuSO 4 (0.13mg,5.01×10 -4mmol), THPTA (0.87mg, 2.0×10 -3 mmol) and sodium ascorbate (1.49 mg, 7.51 × 10 -3 mmol) aqueous solution was added to the reaction system, and the reaction was stirred at room temperature for 5 hours, and then the coupling product was replaced in PBS to prepare the target antibody-drug conjugate III-18, wherein the drug-antibody coupling ratio (DAR) E was about 3, It is the anti-HER2 antibody trastuzumab.

[0623] Example 10: Quality Control Study of ADC Represented by Compound III-2

[0624] This example evaluates the DAR and degree of polymerization ( Figure 1 ). The main peaks of the hydrophobic interaction chromatography of compound III-2 represent the coupling of 0, 2, 4 and 6 small molecule drugs, respectively, and the DAR of compound III-2 is about 4.0. Size exclusion chromatography shows that compound III-2 exists almost entirely in the form of monomers (purity>95%). The results of liquid chromatography-mass spectrometry (UPLC-Q-TOF-MSLC-MS) also verified that each antibody molecule of compound III-2 is coupled with 4.0 small molecule drugs.

[0625] Example 11: Quality Control Study of Compounds III-1, III-3, III-4, III-5 and VC-ADC This example evaluates the DAR and degree of polymerization (DAR) of III-1, III-3, III-4, III-5 and Herceptin-valine-citruline-MMAE (VC-ADC). Figure 2 ). The results of hydrophobic interaction chromatography showed that the DAR of III-1, III-3, III-4, III-5 and VC-ADC was about 4.0. Size exclusion chromatography showed that III-1, III-3, III-4, III-5 and VC-ADC existed almost entirely in the form of monomers (purity>95%).

[0626] Example 12: Plasma stability study of compound III-2

[0627] This example evaluates the stability of compound III-2 in 50% human plasma. Compound III-2 (25 μL, 0.0227 mM) was added to 2245 μL of 2-fold diluted human plasma to prepare a working solution. The working solution was incubated in a 37°C incubator and 50 μL was sampled at set time points (0, 3, 6, 24, 48, 72, 96, 120, 144, 168 h). The samples were quenched with cold acetonitrile before being frozen at -80°C. After sampling, all samples were thawed at room temperature, ultracentrifuged to remove proteins, and LC-MS / MS was used to detect the MMAE content in the supernatant. The results showed ( Figure 3 ), compound III-2 exhibited excellent plasma stability, with the total amount of MMAE released in human plasma within 7 days being <1%.

[0628] Example 13: Antigen binding study of compound III-2

[0629] This example evaluates the binding ability of formula III-2 to HER2 antigen. HER2 antigen (100 μL, 1 μg / mL) was coated on each well of a 96-well plate, and the 96-well plate was placed in a 4°C wet box overnight. 10% fetal bovine serum was added to the well plate and incubated at 37°C for 2 hours to block the protein binding sites in the well plate. A gradient dilution of compound III-2 or Herceptin was added to the well plate, incubated at 37°C for 1 hour, and then washed three times with PBS containing 0.2% Tween 20. Then, 100 μL of HRP-labeled goat anti-human IgG-Fc secondary antibody (Thermo Fisher Scientific, USA) was added to each well and incubated at room temperature for 45 minutes. After washing the well plate three times with PBS containing 0.2% Tween 20, TMB reagent was added to the well for color development, and then 2M H was added. 2 SO 4 To stop color development. Monitor the absorbance of each well at 450nM on the microplate reader. The results show ( Figure 4 ), compound III-2 and Herceptin have comparable binding affinity to HER2 antigen, EC 50 The values ​​were 0.14 μg / mL and 0.042 μg / mL respectively.

[0630] Example 14: Study on the binding ability of compound III-2 on cell lines

[0631] This example evaluates the binding ability of compound III-2 on the HER2-positive cell line SKOV3 and the HER2-negative cell line MCF-7. SKOV3 and MCF-7 cells were collected and counted using a cell counter. The cells were transferred to a 1.5 mL EP tube (approximately 5×10 5cells / sample). The cells were washed once with FACS solution (1×PBS containing 2% fetal bovine serum) for later use. Compound III-2 (5 μg / mL), Herceptin (5 μg / mL) or blank control PBS was added to the cells, respectively, and incubated at 4°C for 30 minutes. The cells were washed twice with FACS solution, and incubated with goat anti-human IgG Fc secondary antibody labeled with PE dye at 4°C for 30 minutes. After washing three times with FACS solution, the samples were detected on FACSCalibur. The results showed ( Figure 5 ), Herceptin and compound III-2 both showed strong fluorescence in HER2-positive SKOV3 cells ( Figure 5 In the right figure), weak fluorescence was shown in HER2-negative MCF-7 cells ( Figure 5 ), indicating that compound III-2 specifically targets the HER2 antigen of tumor cells.

[0632] Example 15: Internalization study of compound III-2 in cell lines

[0633] This example evaluates the endocytic properties of compound III-2 on the HER2-positive cell line SKOV3. SKOV3 cells were collected in 1.5 mL EP tubes and incubated with compound III-2 or Herceptin at 4°C for 1 hour. Then, the samples treated with compound III-2 and Herceptin were divided into a control group and an experimental group. After washing the samples twice with pre-cooled FACS solution, the experimental group was transferred to a 37°C incubator and incubated for 8 hours or 24 hours, while the control group was incubated at 4°C for the same time point. After sampling at the set time point, all samples were washed three times with FACS solution and then incubated with PE dye-labeled goat anti-human IgG Fc secondary antibody at 4°C for 30 minutes. After sampling, the mean fluorescence intensity (rMFI) of cell surface PE was detected by flow cytometry, and the formula for calculating the internalization rate using rMFI was: internalization percentage (%) = [100-(experimental group rMFI / control group rMFI)] × 100%. The results show ( Figure 6 ), the internalization rate of compound III-2 was slightly higher than that of Herceptin, 34.1% vs. 22.1% at 8 h, and 41.7% vs. 31.1% at 24 h.

[0634] Example 16: Compound III-2 binding and endocytosis imaging studies

[0635] This example evaluates the binding and endocytosis properties of compound III-2 on the SKOV3 cell line.

[0636] According to the manufacturer's instructions, compound III-2 was fluorescently labeled using FITC NHS ester (purchased from Innochem). Specifically, NaHCO 3 III-2 in a buffer solution (pH=8.3) was stirred and reacted with a fluorescent dye (FITC NHSester) for 3 hours to label the dye on the antibody, and the obtained FITC-III-2 was replaced in a PBS buffer solution for later use.

[0637] SKOV3 cells were seeded in eight chambers and incubated overnight in a cell culture incubator. After removing the culture medium, FITC-III-2 was added to the cells and incubated at 4°C and 37°C, respectively. SKOV3 cells incubated at 4°C were used for compound III-2 binding assays, while cells incubated at 37°C were used to study the endocytosis of III-2. After reaching the set time point, the cells were washed with PBS, and the nuclei were stained with DAPI and the lysosomes were stained with LysoTracker Red. After washing with PBS twice, fluorescence imaging was performed on a laser confocal microscope. The results show ( Figure 7 ), Herceptin and compound III-2 were incubated with cells at 4°C, under which the antibody bound to the antigen on the cell membrane surface but was not internalized. After incubation at 37°C for 24 hours, the intracellular protein signal co-localized with the lysosomal signal (Pearson correlation coefficient = 0.87), indicating that compound III-2 was internalized and transported to the lysosome.

[0638] Example 17: In vitro cytotoxicity study of compound III-2

[0639] This example evaluates the in vitro cytotoxicity of the test drugs such as compound III-2, trastuzumab (Herceptin), Herceptin-valine-citruline-MMAE (VC-ADC), and toxin MMAE. The cell lines tested include HER2 antigen-positive breast cancer cell line BT474, Herceptin-resistant BT474 cell line (BT474-HDR), HER2 antigen-positive ovarian cancer cell line SKOV3, HER2 antigen-positive gastric cancer cell line NCI-N87, HER2 antigen-weakly positive breast cancer cell line MCF-7, HER2 antigen-negative breast cancer cell line MDA-MB-231, mouse embryonic fibroblasts NIH3T3, human renal epithelial cells 293T, human normal liver cells LO 2 (The above cell lines were purchased from ATCC).

[0640] The reagents, instruments and consumables used in the experiment are described in the following table:

[0641]

[0642] The test process is as follows:

[0643] ① Thawing of cells

[0644] Thaw the vial containing target cells by gentle agitation in a 37°C water bath;

[0645] After the contents were thawed, the vials were removed from the water bath and decontaminated by immersion or spraying with 70% ethanol;

[0646] The vial contents were transferred to a centrifuge tube containing 9 mL of complete medium DMEM and centrifuged (200 g; 5 min);

[0647] Resuspend the cells in medium, pellet and distribute over an area of ​​75 cm 2 in the culture flask;

[0648] The cultures were incubated at 37°C with 5% CO 2 of CO 2 The cells were cultured in an incubator (48R, #CO48312044) with an oxygen concentration of 20% and a carbon dioxide concentration of 5%.

[0649] ② Expand cells

[0650] The cells were passaged three times a week in a medium containing 10% FBS (heat inactivated) and 1% penicillin / streptomycin solution (Penicillin-Streptomycin) at a ratio of 1:4;

[0651] For subculturing cells, first rinse adherent cells with trypsin / EDTA solution (3 mL). Then add trypsin / EDTA (3 mL, T75 bottle) and swirl to evenly coat the cells. Incubate the culture at 37°C until the cells detach. Verify that the cells have detached under a microscope, add an equal volume of cell culture medium to inactivate trypsin, collect the detached cells, centrifuge the cells at 200g for 5 minutes, and then resuspend in fresh culture medium.

[0652] ③Cell inoculation

[0653] Collect cells and count the number of cells;

[0654] Add 100 μL of cell suspension with adjusted density into the designated 96-well cell culture plate, and the final cell density is about 5000-10000 cells / well.

[0655] Cover the lid and place at 37°C with 5% CO 2 The cells were incubated in an incubator for 24 h with an oxygen concentration of 20%.

[0656] ④Prepare compounds

[0657] The compound solutions were serially diluted at a ratio of 1:3, and the initial maximum concentrations of the test drug III-2 and VC-ADC were approximately 400 μg / mL, and the initial concentration of MMAE was 400 nM;

[0658] ④Co-incubation of drugs and cells

[0659] The medium in the 96-well plate seeded with cells was removed, and 150 μL of fresh medium was added to each well. Then, 50 μL of the prepared test drug was added to the 96-well plate. Drugs and cells were incubated at 37°C and 5% CO 2 , 0.1% O 2 After incubation in the incubator for 24 h, the drug was washed away with fresh culture medium and the cells were placed at 37 °C, 5% CO 2 , 20% O 2 Incubate in the incubator for 72 h or 120 h.

[0660] ⑤Read the plate

[0661] Remove the plate from the incubator and equilibrate at room temperature for 15 min;

[0662] CellTiter Glo reagent was incubated at 37°C before the experiment;

[0663] Dilute the CellTiter-Glo reagent and PBS in a volume ratio of 3:7 to form a detection solution, and add the diluted detection solution to the wells to be tested;

[0664] The plate was then placed at room temperature with shaking for 8 min, left to rest for 2 min, and then read on an EnSpire reader for signal detection.

[0665] ⑥Data analysis

[0666] The survival percentage is expressed as follows:

[0667] %Viability=100×(Sample / HC)

[0668] HCs are cells that were not treated with the test drug.

[0669] Cytotoxicity results showed ( Figure 8 , Table 1), all cell lines were highly sensitive to MMAE, and their EC 50 The activity of compound III-2 in NCI-N87, BT-474 and SKBR3 cell lines was 0.01nM to 0.56nM under hypoxic conditions. 50The values ​​were 0.07nM, 0.026nM and 0.015nM ( Figure 8 ), while compound III-2 had lower inhibitory activity against HER2-negative cell lines (MCF-7, MDA-MB-231) (EC 50 >100nM). The above results confirmed that under hypoxic conditions, compound III-2 has the same proliferation inhibitory activity as the toxin MMAE only in HER2-positive cell lines, but has weaker activity in HER2-negative cell lines.

[0670] Table 1. Under hypoxic conditions (0.1% O 2 ), cell proliferation inhibitory activity of compound III-2

[0671]

[0672]

[0673] Example 18: Hypoxia-dependent cytotoxicity study of compound III-2

[0674] In this example, the hypoxia-activated compound III-2 was subjected to hypoxia-dependent in vitro cytotoxicity study. The culture process of the tested cell lines, the Herceptin-resistant HER2 antigen-positive breast cancer cell line BT474-HDR and the HER2 antigen-positive ovarian cancer cell line SKOV3, was as described in Example 14. The oxygen concentration in the cell culture incubator was set to 0.1%, 1.0%, 5.0% and 20.0%, respectively. The cytotoxicity results showed that ( Fig. 9 , Table 2), the cytotoxic activity of compound III-2 increased with the decrease of oxygen concentration. Under the conditions of oxygen concentration of 20%, 5%, 1% and 0.1%, the EC of compound III-2 on SKOV3 cells 50 The values ​​were 66.8nM, 3.74nM, 0.079nM, and 0.063nM, respectively, and the EC values ​​on BT474-HDR cells were 50 The values ​​were 109.1nM, 9.12nM, 0.57nM, and 0.19nM, respectively. The results showed that compound III-2 2 <1%), but weak activity under normoxic conditions. The activity of traditional VC-ADC did not show oxygen concentration dependence, and had strong cell proliferation inhibition activity regardless of normoxic or hypoxic conditions. This result indicates that traditional ADC has the risk of off-target toxicity ( Fig. 9 , Table 2).

[0675] Table 2 Oxygen concentration-dependent cytotoxicity of compound III-2 and VC-ADC

[0676]

[0677] Example 19: Study on the bystander effect of compound III-2

[0678] In this example, the bystander effect of hypoxia-activated type III-2 was studied. The HER2 antigen-positive ovarian cancer cell line SKOV3 and the HER2 antigen-weakly positive breast cancer cell line MCF-7 were inoculated in a well plate with a cell density of 1:1. The cell culture process was as described in Example 14, and the oxygen concentration in the cell culture incubator was set to 0.1% and 20.0%, respectively. The results showed that ( Fig.10 , Table 3), under normoxic conditions, the cytotoxic activity of compound III-2 in the co-culture model was reduced to μM level (EC 50 ≈0.1μM), which indicates that compound III-2 has high safety in normal tissues ( Figure 3 ). Under hypoxic conditions, compound III-2 maintained high activity in the co-culture model, EC 50 =0.053 nM. This result shows that compound III-2 has a bystander effect in tumor tissues and also has a killing effect on tumor cells with low HER2 expression ( Fig.10 , Table 3).

[0679] Table 3 Cell proliferation inhibitory activity of compound III-2 in co-culture cell model

[0680]

[0681] Example 20: Cytotoxicity study of compound III-2 on normal cells

[0682] In this example, the toxicity of the hypoxia-activated compound III-2 to normal cells was studied. The cell lines tested were mouse embryonic fibroblasts NIH3T3, human renal epithelial cells 293T and human normal liver cells LO 2 The culture process is as described in Example 14. At the highest tested concentration (666 nM) of normal cells NIH3T3 and 293T, compound III-2 showed only weak inhibitory activity, with a maximum inhibition rate of <50% ( Fig.11 , Table 4). However, traditional VC-ADCs still exhibit high off-target toxicity (EC 50 <10nM, maximum inhibition rate>90%, Fig.11 , Table 4).

[0683] Table 4 Cell proliferation inhibitory activity of compound III-2 on normal cell lines

[0684]

[0685] Example 21: MMAE release study of compound II-2

[0686] This example evaluates the release of SN-38 by compound II-2 under the action of azoreductase. Compound II-2 was reacted with 3-mercaptopropionic acid (MPA, 10 equivalents) to prepare a stock solution of MPA-II-2. The preparation method of MPA-II-2 was described in reference (Y. Wang, S. Fan, W. Zhong, X. Zhou, S. Li, Int. J. Mo. l Sci. 2017, 18, e1860.). At 0.1% O 2 MPA-II-2 (6 μL, 100 mM), NADPH (20 μL, 100 mL) and human NADPH-CYP reductase (5 μL, 10 μL or 40 μL, 18.8 mg / mL) were added to phosphate buffered saline containing 1% DMSO (v / v) and the mixture was incubated at 37°C. 2 ) were treated with human NADPH-CYP reductase (10 μL, 18.8 mg / mL), and other factors were consistent with the hypoxia group. Aliquots were collected at each set time point and quenched with acetonitrile before freezing at -80°C. After collecting the final aliquot, all samples were centrifuged to remove protein and analyzed by LC-MS / MS.

[0687] The results show that Fig.12 ), the release of MMAE showed a dual dependence on azoreductase concentration and oxygen concentration. Under hypoxic conditions (0.1% O 2 ), the amount of MMAE released increased with the increase of azoreductase concentration. After incubation of substrate and azoreductase (0.4 mg / mL, 0.1 mg / mL and 0.05 mg / mL) for 24 hours, the amount of MMAE released was 100%, 74.3% and 27.49%, respectively. Under normoxic conditions (20% O 2 ), after the substrate was incubated with 0.1 mg / mL azoreductase for 24 hours, only 14.99% of MMAE was released. This result shows that the linker based on azoreductase cleavage provided by the present invention can be cleaved and release toxins under the combined action of hypoxic conditions and azoreductase, while the amount of toxin released is very small when only azoreductase is used ( Fig.12 ).

[0688] Example 22: Effects of oxidizing properties, reducing properties, and salts on the stability of compound II-2

[0689] This example evaluates the effects of oxidative, reductive and salt substances on the stability of compound II-2. Compound II-2 was reacted with acetylcysteine ​​(NAC, 10 equivalents) to prepare a stock solution of NAC-II-2. The preparation method of NAC-II-2 was described in reference (Y. Wang, S. Fan, W. Zhong, X. Zhou, S. Li, Int. J. Mo. l Sci. 2017, 18, e1860.). NAC-II-2 was reacted with Ary, Cys, NaCl, KCl, MgCl 2 ,CaCl 2 ,H 2 O 2 ,NaClO,Arg,Glu,Ser,Vc,Glucose,NADPH in 20% O 2 Incubated for 24 h in the presence of 0.1% O 2 The samples were incubated for 24 hours under the same environment. After incubation for 24 hours, all samples were quenched with methanol and the release of MMAE in the samples was detected by LC-MS / MS. The results showed that ( Fig.13 ), NAC-II-2 can release MMAE via azoreductase only under hypoxic conditions and exhibits excellent stability when co-incubated with other redox-related substances, such as Cys, NADPH, and NaClO.

[0690] Example 23: Study on the metabolites of compound III-2 in cells

[0691] This example evaluates the activity of compound III-2 in normoxia (20% O 2 ), hypoxia (0.1% O 2 ) under the condition of , its metabolite form in cells. 5 Cells / well) were seeded in a 12-well plate, 100 nM compound III-2 was added to the cells, and compound III-2 and cells were incubated at an oxygen content of 20% or 0.1% O 2The cells were co-incubated under the same conditions for 6 hours, 17 hours or 24 hours. At the set time points, the cells were collected and washed three times with cold PBS. Methanol was added to the sample and placed at -20°C for 2 hours to completely break the cells, followed by centrifugation at 13,000 g for 20 minutes. 50 μL of supernatant was taken from each sample, and after vacuum drying, 100 μL of methanol was added to re-dissolve the sample, and LC-MS / MS was used to analyze the MMAE and Cys-II-2 content in the sample. The results showed that compound III-2 was co-cultured with SKOV3 cells under hypoxia, MMAE was detected in SKOV3 cells, and the amount of MMAE released was time-dependent. This result directly indicates that compound III-2 can be cleaved and release MMAE in tumor cells under hypoxic conditions ( Fig.14 More importantly, when compound III-2 was co-cultured with SKOV3 cells under normoxic conditions, Cys-II-2 was the main metabolite and its release was time-dependent, while the amount of Cys-II-2 in the cells was very low under hypoxic conditions. This result showed that under normoxic conditions, the antibody of compound III-2 was degraded and the released Cys-II-2 would not be further cleaved by azoreductase ( Fig.14 ).

[0692] Example 24: Cytotoxicity study of Cys-II-2

[0693] This example evaluates the toxicity of Cys-II-2 on BT474 and SKBR3 cells. Compound II-2 was reacted with L-cysteine ​​(Cys, 10 equivalents) to prepare a stock solution of Cys-II-2. BT-474 and SKBR3 cells were seeded in 96-well plates and incubated in an incubator for 24 hours. Equally diluted MMAE and Cys-II-2 were added to the cells, and the cells and drugs were incubated at 20% O 2 After 24 hours of incubation, the drug was washed out with culture medium, and SKBR3 was cultured in the incubator for 3 days, and BT474 was cultured for 5 days. Cell viability was detected using the CellTiter-Glo assay kit. The results showed ( Fig.15 ), the cytotoxic activity of Cys-II-2 was reduced by more than ten to hundreds of times compared with MMAE.

[0694] Example 25: Study on the inhibition of tubulin by Cys-II-2

[0695] Tubulin inhibition experiments were performed using a tubulin polymerization assay kit (BK011P, Cytoskeleton, USA). 5 μL of different concentrations of MMAE, Cys-II-2 or DMSO were added to a 96-well plate and incubated at 37°C for 1 min, followed by the addition of 50 μL of the prepared tubulin reaction solution. The fluorescence signal at 420 nM was immediately monitored and recorded on an ELISA reader. The results showed ( Fig.16 ), the tubulin inhibitory activity of Cys-II-2 was lower than that of MMAE and showed concentration dependence.

[0696] Example 26: Cell cycle arrest study of compound III-2

[0697] This example evaluates the cell cycle arrest of SKOV3 cells by compound III-2. 5 Cells / well) were seeded in 12-well plates and incubated for 24 hours. Compound III-2 at different concentrations (0.3, 1 and 3 nM) was added to the cells. 2 After 1 day of incubation, cells were transferred to 20% O 2 The normoxic group was kept in 20% O 2 The cells were cultured for 2 days. All cells in each well were collected into a 1.5 mL EP tube and fixed with 70% cold ethanol. Before sending the samples, they were washed once with cold PBS, stained with propidium iodide solution at 37°C for 30 minutes in the dark, and then analyzed using FACSCalibur. The cycle arrest results showed ( Fig.17 ), compound III-2 caused G2 / M phase arrest of tumor cells with hypoxia selectivity. Under normoxic conditions, after treatment with compound III-2 at concentrations of 0.3nM, 1nM and 3nM, the proportion of cells in the G2 / M phase was similar to that of the untreated group (17.50%). On the contrary, under hypoxia, after treatment with the same concentration of compound III-2, the proportion of cells in the G2 / M phase was maintained at 71.12%-87.22%.

[0698] Example 27: Study on the apoptosis induced by compound III-2

[0699] This example evaluates the effect of III-2 on apoptosis of SKOV3 cells. 5 Cells / well) were seeded in 12-well plates and incubated for 24 hours. Compound III-2 at different concentrations (0.3, 1 and 3 nM) was added to the cells. 2 After 1 day of incubation, cells were transferred to 20% O 2The normoxic group was cultured in 20% O 2 All cells in each well were collected into a 1.5 mL EP tube and washed once with cold PBS. The samples were stained with Annexin V-FITC and propidium iodide solution at room temperature for 20 minutes in the dark and then analyzed using FACSCalibur. The results showed ( Fig.18 ), under normoxic conditions (20% O 2 ) had little effect on cell apoptosis. In contrast, under hypoxic conditions (0.1% O 2 ) Compound III-2 induced apoptosis in more than half of the cells. The apoptosis results showed that compound III-2 had good hypoxia selectivity.

[0700] Example 28: Distribution of Compound III-2 Labeled with DyLight-680 Dye in Mice

[0701] This example evaluates the distribution of compound III-2 in mice.

[0702] DyLight 680-Herceptin and DyLight 680-III-2 were prepared using DyLight 680 NHS ester dye (purchased from ThermoFisher Scientific) according to the manufacturer's instructions. Specifically, NaHCO 3 Herceptin or compound III-2 in a buffer solution (pH=8.3) was stirred and reacted with a fluorescent dye (DyLight 680NHSester) for 3 hours to label the dye on the antibody, and then the obtained DyLight 680-Herceptin and DyLight 680-III-2 were replaced in a PBS buffer solution for use.

[0703] SKOV3 ovarian cancer-bearing mice (purchased from Yikang (Beijing) Pharmaceutical Technology Co., Ltd.) were injected with 0.2 mL PBS, DyLight 680-Herceptin and DyLight 680-III-2 by tail vein injection, and then the mice were imaged at time points (6h, 24h, 30h, 48h, 72h, 120h, 216h, 360h) using the Maestro in vivo fluorescence imaging system. The results showed ( Fig.19), DyLight 680-Herceptin (25 mg / kg) and DyLight 680-III-2 (25 mg / kg) labeled with DyLight 680 were injected into mice by tail vein injection. A strong fluorescence signal could be clearly observed in the tumor site 6 hours after administration, and the fluorescence of the tumor site was clearly maintained for about 9 days. The fluorescence signal finally disappeared on the 15th day after administration. From the fluorescence signal intensity change curve, it was found that the DyLight680-III-2 curve was almost consistent with the change curve of DyLight 680-Herceptin. This result shows that compound III-2 has similar metabolic properties to Herceptin. In addition, the fluorescence intensity of the tumor site is significantly higher than that of other normal tissues ( Fig.19 ). The above imaging results show that compound III-2 has excellent tumor targeting and a long half-life in vivo.

[0704] Example 29: Drug distribution and metabolism studies of compound III-2

[0705] This example evaluates the drug distribution and metabolism of compound III-2. SKOV3 ovarian cancer-bearing mice were divided into 6 groups. After a single dose of compound III-2 (25 mg / kg) was injected into the tail vein, the mice were euthanized at 6h, 24h, 72h, 96h, 168h, and 192h, and blood, heart, liver, spleen, lung, and tumor samples were collected. Blood and tissue samples were stored at -80°C for further analysis. The results showed ( Fig. 20 , Table 5), high concentrations of prodrug Cys-II-2 were detected in the blood and peripheral tissues 6 hours after administration of compound III-2. In addition, the results showed that Cys-II-2 can be quickly cleared in normal tissues, and the half-life of Cys-II-2 in the blood is only 26 hours, which will further reduce its toxicity and side effects. After 96 hours of administration, Cys-II-2 was almost undetectable in the heart, liver, spleen, and lungs. The peak concentration and drug-time curve area of ​​the toxin MMAE in the tumor are 293 times and 941 times that of the peripheral blood, respectively, which further confirms that compound III-2 has excellent targeting. The drug-time curve area of ​​MMAE in the heart, liver, spleen, and lungs accounted for 11%, 47%, 5%, and 7% of the tumor drug-time curve area, respectively. The results show that Cys-II-2 in peripheral tissues, except the liver, cannot be metabolized into MMAE, while Cys-II-2 in tumors can effectively release MMAE (Table 6).

[0706] Table 5 Concentration of Cys-II-2 in various tissues

[0707]

[0708] Note: BQL means beyond detection limit.

[0709] Table 6 Concentration of MMAE in various tissues

[0710]

[0711] Note: BQL means beyond detection limit.

[0712] Example 30: Pharmacodynamic study of compound III-2 in NCI-N87 tumor-bearing mice

[0713] This example evaluates the antitumor effect of compound III-2 on NCI-N87 tumor-bearing mice (purchased from Hangzhou Tsinghua Kerui Biotechnology Co., Ltd.). 3 At the time of the study, NCI-N87 tumor-bearing mice were randomly divided into 4 groups: PBS group, Herceptin group, III-2 group and VC-ADC group. The drugs were injected into the tail vein once a week for a total of 4 times. The tumor size was measured with a caliper twice a week, and the tumor volume was calculated as 1 / 2×length×width×width (mm). When the tumor volume of any mouse exceeded 2000mm 3 The mice were euthanized. Fig.21 ), in the NCI-N87 tumor xenograft model, compared with the control group, the compound III-2 group at a dose of 5 mg / kg can significantly inhibit tumor growth, with a tumor inhibition rate of 90.97%. This result shows that compound III-2 can achieve almost the same anti-tumor effect as traditional VC-ADC. More importantly, all mouse tumors in the 10 mg / kg dose group completely regressed, indicating that compound III-2 has excellent anti-tumor therapeutic potential.

[0714] Example 31: Pharmacodynamic study of compound III-2 in JIMT-1 tumor-bearing mice

[0715] This example evaluates the antitumor effect of compound III-2 on JIMT-1 tumor-bearing mice (purchased from Hangzhou Tsinghua Kerui Biotechnology Co., Ltd.). To evaluate the antitumor effect of compound III-2 in JIMT-1 tumor-bearing mice with low HER2 antigen expression, when the tumor volume grows to 150 mm 3 The mice were randomly divided into 2 groups: PBS group and III-2 group. The drug was injected into the tail vein once a week for a total of 4 times. The tumor size was measured with a caliper twice a week. The results showed that ( Fig. 22 ), in the JIMT-1 xenograft model, at a dose of 12 mg / kg, compound III-2 still showed an anti-tumor effect. Although the tumor did not completely disappear, the inhibition rate reached 62.36%.

[0716] Example 32: Pharmacodynamic study of compound III-2 in combination with Herceptin, bevacizumab, sunitinib or docetaxel in NCI-N87 tumor-bearing mice

[0717] This example evaluates the antitumor efficacy of compound III-2 in combination with Herceptin, bevacizumab, sunitinib or docetaxel on NCI-N87 tumor-bearing mice. The mice were randomly divided into 7 groups (PBS group, III-2 administration group (3 mg / kg), sunitinib administration group (54 mg / kg), III-2 (3 mg / kg) and Herceptin (12 mg / kg) combination group, III-2 (3 mg / kg) and bevacizumab (5 mg / kg) combination group, III-2 (3 mg / kg) and sunitinib (54 mg / kg) combination group, III-2 (3 mg / kg) and docetaxel (8 mg / kg) combination group). When the tumor volume grows to ~100 mm 3 PBS, Herceptin, III-2 and docetaxel were administered once a week by tail vein injection for a total of 3 times. Docetaxel was administered 6 hours in advance. Sunitinib was administered by gavage once a day for 3 consecutive weeks. The results showed that ( Fig.23 ), the III-2 combined with sunitinib group showed a stronger anti-tumor effect (p<0.01), which was better than III-2 and other combination groups. In the combined administration group, only 3mg / kg III-2 can almost achieve the effect of complete tumor regression, with a tumor inhibition rate of 98.93%. The above results show that the excellent tumor inhibition effect of combined administration may come from the fact that the angiogenesis inhibitor sunitinib can expand the range of tumor tissue hypoxia and strengthen the degree of tumor tissue hypoxia, thereby further improving the efficacy of III-2.

[0718] Example 33: Study on the maximum tolerated dose of compound III-2

[0719] This example evaluates the tolerance and adverse reactions of healthy female CD-1 mice (purchased from Hangzhou Tsinghua Core Biotechnology Co., Ltd.) to compound III-2. Female CD-1 mice aged 7-9 weeks were adapted to the environment for at least 3 days before administration, and each dose group consisted of 3 mice. Healthy female CD-1 mice were administered 120, 160, 200, and 240 mg / kg of compound III-2 by a single tail vein injection. At the same time, the marketed drug T-DM1 and the traditional ADC drug VC-ADC were used for control studies. After administration by tail vein injection, the weight changes and behavior of the mice were monitored every day. The results show ( Fig.24), the maximum tolerated dose (MTD) of compound III-2 was >200 mg / kg, while the mice in the VC-ADC group and the T-DM1 group experienced continuous weight loss (>20%) and exhibited severe adverse reactions since the start of administration. For example, all the mice in the VC-ADC group died after being administered 120 mg / kg.

[0720] Example 34: Hematological and histopathological studies of compound III-2 in BALB / C mice

[0721] This example evaluates the hematology and histopathology of compound III-2 on BALB / C mice (purchased from Hangzhou Tsinghua Kerui Biotechnology Co., Ltd.). Female BALB / C mice (n=3 / group) were administered with III-2 (60 mg / kg), VC-ADC (60 mg / kg) and PBS by tail vein injection. After administration, the weight of the mice was monitored every 3 days, and the mice were euthanized on the 7th day for whole blood hematology and histopathology analysis. The results showed ( Fig.25 ), there were no significant differences in blood markers of biochemistry, blood routine and histopathology between the III-2 administration group and the control group, while in the VC-ADC treatment group, the changes in blood biochemistry reflected the side effects of VC-ADC on the liver, such as increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK). The side effects of VC-ADC were also reflected in hematological changes, including increases in lymphocyte counts (LYMPH), monocyte counts (MONO), and neutrophil counts (NEUT).

[0722] Example 35: Antibacterial study of compound III-16 against Staphylococcus aureus

[0723] This example evaluates the antibacterial effect of compound III-16 on Staphylococcus aureus in macrophages. 37.5 μg / mL of compound III-16 and 150 μg / mL of antibacterial antibody (whose heavy chain and light chain sequences are shown in SEQ ID NO: 3 and 4, respectively) were added to the well plate and incubated with macrophages infected with Staphylococcus aureus for 24 hours, and then the colonies were counted and photographed. The results show ( Fig.26 ), compound III-16 has excellent anti-Staphylococcus aureus effect.

[0724] The above experimental results show that the ADC provided by the present invention (such as the ADC shown in III-2) can be quickly recognized and cleaved by azoreductase and efficiently release MMAE, and it exhibits anti-tumor effects in both in vivo and in vitro activity evaluations. In addition, the ADC provided by the present invention (such as the ADC shown in III-16) exhibits excellent antibacterial effects.

[0725] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A compound of formula I, or a geometric isomer, optical isomer, salt, hydrate, solvate or polymorph thereof, in represents a linker used to couple the azobenzene group to the targeting compound, Ar is a five-membered or six-membered aryl or heteroaryl group; R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; p is 0, 1, 2 or 3; q is 0, 1, 2, or 3; R is H, C 1-4 alkyl, where r 1 and r 2 Each independently is an integer between 1 and 12; Z is hydroxy, fluorine, chlorine, bromine, iodine or Where s is 0, 1, 2, 3 or 4, R 5 For hydrogen, fluorine, chlorine, bromine, iodine, C 1-4 Alkyl, nitro or C 1-4 Alkoxy; Preferably, middle: B is or H, wherein r is 1-4; or B is selected from The condition is that V, L, W1, W2, and W3 do not exist at the same time; V or not present, wherein each i is independently an integer between 0 and 6, and each j is independently an integer between 0 and 8; L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -,-(CH 2 ) m -, or not present, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, and m is an integer between 0 and 30; W is -W1-W2-W3-, wherein W1 is connected to L, W3 is connected to Ar, and W1 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )- or not present; W2 is selected from -(CH 2 ) o -,-[CH(R 11 )CH(R 12 )] o - or not present; W3 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )-or not present, where o is an integer between 0 and 6, and each R 11 are independently selected from hydrogen or C 1-6 Alkyl, R 12 Selected from hydrogen or C 1-6 alkyl.

2. The compound according to claim 1, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein W is selected from -NH-CH 2 -C(O)-NH-, -NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH 2 -C(O)-NH-, -C(O)-NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH 2 ) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH 2 ) o -NH-C(O)-, -NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, where R 10 is hydrogen, methyl or ethyl, o, R 11 and R 12 The definition as in claim 1.

3. The compound according to claim 1 or 2, its geometric isomer, optical isomer, salt, hydrate, solvate or polymorph, in: B is Preferably, B is Preferably, B is Preferably, 4. The compound according to any one of claims 1 to 3, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, It is characterized by the following Any one or more of i) to iii): i) V is or does not exist, wherein i is defined as in claim 1; ii) L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -or-(CH 2 ) m -, where k, l, R 1 , m is defined as in claim 1; iii) W is -C(O)-NH-(CH 2 ) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R 11 and R 12 The definition of is as in claim 1; Preferably, -VLW- is -(CH 2 ) m -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-CHR 1 -C(O)-NH-(CH 2 ) o -NH-C(O)-, or -(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , m, o, i, k, l, n are defined as in claim 1; Preferably, each i is independently 1, 2, 3 or 4; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each m is independently 1, 2, 3 or 4; each o is independently 1, 2, 3 or 4; each l is independently 1, 2, 3 or 4; n is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, each i is independently 1, 2 or 3; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; each m is independently 1, 2 or 3; each o is independently 1, 2 or 3; each l is independently 1, 2 or 3; n is 1, 2, 3, 4 or 5; Preferably, -V-L-W- is -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 2 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 4 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 6 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 8 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 CH 2 O) 3 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-CH[(CH 2 ) 4 -NHC(O)-(CH 2 CH 2 O) 8 -CH 3 ]-C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 CH 2 O) 7 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-。 5. The compound according to any one of claims 1 to 4, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, It is characterized by the following Any one or more of i) to iv): i) Ar is phenyl, pyridyl, pyrimidinyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl; Preferably, Ar is phenyl, furyl, imidazolyl or thienyl; Preferably, Ar is phenyl; ii) R is H or C 1-4 alkyl; Preferably, R is H, methyl, ethyl, n-propyl or n-butyl, preferably H, methyl, ethyl or n-propyl, more preferably H, methyl or ethyl, and even more preferably H; iii) R 2 and R 3 are independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R 2 and R 3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; preferably, R 2 and R 3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; preferably, R 2 and R 3 each independently is hydrogen; iv) q is 0, 1 or 2, preferably 0 or 1; p is 0, 1 or 2, preferably 0 or 1; v) Z is hydroxy, fluorine, chlorine, bromine, iodine or Where s is 0, 1, 2, 3 or 4; R 5 is hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, nitro, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy; Preferably, Z is chlorine, OH or Preferably OH or More preferably Preferably, the compound has the structure shown in I-1, Wherein, the definitions of B, V, L, W, R and Z are as described in claims 1-5.

6. The compound of claim 1, its geometric isomer, optical isomer, salt, hydrate, solvate or polymorph, wherein B is selected from V, L, W1, W2, and W3 do not exist; R is where r 1 and r 2 The definition of is as in claim 1; Preferably, r 1 and r 2 each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Preferably, r 1 and r 2 Each independently is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, r 1 and r 2 Each independently is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, r 1 and r 2 Each independently is 1, 2, 3, 4, 5 or 6; Preferably, r 1 and r 2 Each independently is 1, 2, 3 or 4.

7. The compound of claim 1, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein the compound is selected from:

8. Use of the compound according to any one of claims 1 to 7, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs in the preparation of antibody-drug conjugates.

9. A compound represented by formula II, or a geometric isomer, optical isomer, salt, hydrate, solvate or polymorph thereof, in: represents a linker, used to couple the right azobenzene group to the targeting compound, Ar is a five-membered or six-membered aryl or heteroaryl group; R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; p is 0, 1, 2 or 3; q is 0, 1, 2, or 3; R is H, C 1-4 alkyl, where r 1 and r 2 Each independently is an integer between 1 and 12; D is 0 or 1; C is an active compound selected from a drug, a cytotoxin, a detection reagent, a diagnostic reagent or a targeting vector; preferably, C is an antitumor drug, an antiviral drug, an antibiotic, an antibacterial drug or an immunomodulator drug; further preferably, C is an antitumor drug, such as a cytotoxin, a microtubule inhibitor, a DNA alkylating agent, a DNA chimera, an RNA polymerase inhibitor, a kinase inhibitor, a topoisomerase inhibitor, a spindle kinesin inhibitor, an antimetabolite drug and other small molecules, peptides or nucleotides, an immunomodulator drug, such as a Toll-like receptor agonist (such as a TLR7 agonist) or a STING agonist, an antibacterial drug, such as rifampicin and its analogs (such as Rifalogue, KRM-1657); C is coupled to the carbonyl group (i.e., site *) or the C atom at the L position through the N atom or O atom in the active compound molecule, Preferably, middle: B is or H, wherein r is 1-4; or B is selected from The condition is that V, L, W1, W2, and W3 do not exist at the same time; V or not present, wherein each i is independently an integer between 0 and 6, and each j is independently an integer between 0 and 8; L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -,-(CH 2 ) m -, or not present, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, and m is an integer between 0 and 30; W is -W1-W2-W3-, wherein W1 is connected to L, W3 is connected to Ar, and W1 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )- or not present; W2 is selected from -(CH 2 ) o -,-[CH(R 11 )CH(R 12 )] o - or not present; W3 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )-or not present; where o is an integer between 0 and 6, each R 11 are independently selected from hydrogen or C 1-6 Alkyl, R 12 Selected from hydrogen or C 1-6 alkyl.

10. The compound according to claim 9, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein W is selected from -NH-CH 2 -C(O)-NH-, -NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH 2 -C(O)-NH-, -C(O)-NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH 2 ) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH 2 ) o -NH-C(O)-, -NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, where R 10 is H, methyl or ethyl, o, R 11 and R 12 The definition as set forth in claim 9.

11. The compound according to claim 9 or 10, its geometric isomer, optical isomer, salt, hydrate, solvate or polymorph, wherein B is Preferably, B is Preferably, B is Preferably, 12. The compound according to any one of claims 9 to 11, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, It is characterized by the following Any one or more of i) to iii): i) V is or does not exist, wherein i is defined as in claim 9; ii) L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -or-(CH 2 ) m -, where k, l, R 1 , m is defined as in claim 9; iii) W is -C(O)-NH-(CH 2 ) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R 11 and R 12 The definition of is as in claim 9; Preferably, -VLW- is -(CH 2 ) m -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-CHR 1 -C(O)-NH-(CH 2 ) o -NH-C(O)-, or -(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , m, o, i, k, l, n are defined as in claim 9; Preferably, each i is independently 1, 2, 3 or 4; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each m is independently 1, 2, 3 or 4; each o is independently 1, 2, 3 or 4; each l is independently 1, 2, 3 or 4; n is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, each i is independently 1, 2 or 3; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; each m is independently 1, 2 or 3; each o is independently 1, 2 or 3; each l is independently 1, 2 or 3; n is 1, 2, 3, 4 or 5; Preferably, -V-L-W- is -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 2 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 4 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 6 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 8 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 CH 2 O) 3 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-CH[(CH 2 ) 4 -NHC(O)-(CH 2 CH 2 O) 8 -CH 3 ]-C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 CH 2 O) 7 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-。 13. The compound according to any one of claims 9 to 12, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, It is characterized by the following Any one or more of i) to v): i) Ar is phenyl, pyridyl, pyrimidinyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl; Preferably, Ar is phenyl, furyl, imidazolyl or thienyl; Preferably, Ar is phenyl; ii) R is H or C 1-4 alkyl; Preferably, R is H, methyl, ethyl, n-propyl or n-butyl, preferably H, methyl, ethyl or n-propyl, more preferably H, methyl or ethyl, and even more preferably H; iii) R 2 and R 3 are independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R 2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; preferably, R 2 and R 3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; preferably, R 2 and R 3 each independently is hydrogen; iv) q is 0, 1 or 2, preferably 0 or 1; p is 0, 1 or 2, preferably 0 or 1; v) C is selected from the group consisting of auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and their derivatives Maytansine or its derivatives (e.g., maytansine-like, DM1, DM3, DM4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins and their derivatives, SN-38, Exatecan, Doxorubicin, TLR7 agonist with CAS 1821304-87-3, lapatinib, rifampicin and its analogs (e.g., Rifalogue, KRM-1657), preferably monomethyl auristatin E (MMAE), SN-38, Exatecan, Doxorubicin, TLR7 with CAS 1821304-87-3 agonist, lapatinib, MMAF derivative or Rifalogue, more preferably monomethyl auristatin E (MMAE), rifampicin or Rifalogue; more preferably monomethyl auristatin E (MMAE) or Rifalogue; Preferably, the compound has the structure shown in Formula II-1, The definitions of B, V, L, W, R, C, and D are as described in claims 9-13.

14. The compound of claim 9, its geometric isomer, optical isomer, salt, hydrate, solvate or polymorph, wherein B is selected from V, L, W1, W2, and W3 do not exist; R is where r 1 and r 2 The definition of is as in claim 9; Preferably, r 1 and r 2 each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Preferably, r 1 and r 2 Each independently is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, r 1 and r 2 Each independently is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, r 1 and r 2 Each independently is 1, 2, 3, 4, 5 or 6; Preferably, r 1 and r 2 Each independently is 1, 2, 3 or 4.

15. The compound of claim 9, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein the compound is selected from:

16. Use of the compound according to any one of claims 9 to 15, its geometric isomer, optical isomer, salt, hydrate, solvate or polymorph in the preparation of an antibody-drug conjugate.

17. A compound of formula III or IV, or a geometric or optical isomer, a pharmaceutically acceptable salt, a hydrate, a solvate or a polymorph thereof, in, A represents the targeted compound; represents a linker used to couple the azobenzene group to the targeting compound; Ar is a five-membered or six-membered aryl or heteroaryl group; R 2 and R 3 are independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; p is 0, 1, 2 or 3; q is 0, 1, 2, or 3; R is H, C 1-4 alkyl, where r 1 and r 2 Each independently is an integer between 1 and 12; R' is where k 2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; B is selected from D is 0 or 1; C is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors; preferably, C is an antitumor drug, an antiviral drug, an antibiotic, an antibacterial drug or an immunomodulator; further preferably, C is an antitumor drug, such as a cytotoxin, a microtubule inhibitor, a DNA alkylating agent, a DNA chimera, an RNA polymerase inhibitor, a kinase inhibitor, a topoisomerase inhibitor, a spindle kinesin inhibitor, an antimetabolite drug or other small molecules, peptides or nucleotides, an immunomodulator drug, such as a Toll-like receptor agonist (such as a TLR7 agonist) or a STING agonist, an antibacterial drug, such as rifampicin and its analogs (such as Rifalogue, KRM-1657); C is coupled to the carbonyl group (i.e., site *) or the C atom at the L position through the N atom or O atom in the active compound molecule; E is a number between 1 and 20; Preferably, middle B 1 for where k 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; V is or not present, wherein each i is independently an integer between 0 and 6, and each j is independently an integer between 0 and 8; L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -,-(CH 2 ) m -, or not present, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , each k is independently an integer between 0 and 12, each l is independently an integer between 0 and 12, n is an integer between 0 and 12, and m is an integer between 0 and 30; W is -W1-W2-W3-, wherein W1 is connected to L, W3 is connected to Ar, and W1 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )- or not present; W2 is selected from -(CH 2 ) o -,-[CH(R 11 )CH(R 12 )] o - or not present; W3 is selected from -O-, -S-, -NR 11 -, -CONH-, -NHCO-, -S(O 2 )NH-, -NHS(O 2 )-or not present; where o is an integer between 0 and 6, each R 11 are independently selected from hydrogen or C 1-6 Alkyl, R 12 Selected from hydrogen or C 1-6 alkyl; Preferably, A is a targeting compound selected from proteins, antibodies, peptides, enzymes and small molecules; Preferably, A is coupled to B via an S atom or a N atom in the targeting compound molecule. 1 The site of the group, or A targets the carbonyl group in the compound molecule and B 1 The hydroxylamine group in the group reacts to form an oxime bond to couple to B 1 The site of the R' group is located at the site of the R' group, or A is coupled to the site of the R' group through the N atom in the targeting compound molecule.

18. The compound of claim 17, its geometric or optical isomer, pharmaceutically acceptable salt, hydrate, solvate or polymorph, wherein the compound is a compound of formula III, wherein W is selected from -NH-CH 2 -C(O)-NH-, -NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH 2 -C(O)-NH-, -C(O)-NH-CH 2 -CH 2 -C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH 2 ) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH 2 ) o -NH-C(O)-, -NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, where R 10 is hydrogen, methyl or ethyl, o, R 11 and R 12 The definition as set forth in claim 17.

19. The compound according to claim 17 or 18, or its geometric or optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula III, wherein B 1 for Preferably, B 1 for where k 1 The definition as in claim 17; Preferably, k 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; further preferably, k 1 is 1, 2, 3, 4, 5, 6, 7 or 8; more preferably, k 1 Is 2, 3, 4, 5, 6, 7 or 8.

20. The compound according to any one of claims 17 to 19, or its geometric or optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula III, It is characterized by the following Any one or more of i) to iii): i) V is or does not exist, wherein i is defined as in claim 17; ii) L is -(CH 2 CH 2 O) k -(CH 2 ) l -, -CHR 1 -or-(CH 2 ) m -, where k, l, R 1 , m is defined as in claim 17; iii) W is -C(O)-NH-(CH 2 ) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R 11 and R 12 The definition of as in claim 17; Preferably, -VLW- is -(CH 2 ) m -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, -(CH 2 ) i -C(O)-NH-CHR 1 -C(O)-NH-(CH 2 ) o -NH-C(O)-, or -(CH 2 CH 2 O) k -(CH 2 ) l -C(O)-NH-(CH 2 ) o -NH-C(O)-, where R 1 For -(CH 2 ) n -NHC(O)-(CH 2 CH 2 O) k -CH 3 , m, o, i, k, l, n are defined as in claim 17; Preferably, each i is independently 1, 2, 3 or 4; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each m is independently 1, 2, 3 or 4; each o is independently 1, 2, 3 or 4; each l is independently 1, 2, 3 or 4; n is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, each i is independently 1, 2 or 3; each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; each m is independently 1, 2 or 3; each o is independently 1, 2 or 3; each l is independently 1, 2 or 3; n is 1, 2, 3, 4 or 5; Preferably, -V-L-W- is -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 2 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 4 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 6 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-(CH 2 CH 2 O) 8 -CH 2 -CH 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 CH 2 O) 3 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-, -(CH 2 ) 2 -C(O)NH-CH[(CH 2 ) 4 -NHC(O)-(CH 2 CH 2 O) 8 -CH 3 ]-C(O)-NH-(CH 2 ) 2 -NH-C(O)-,-(CH 2 CH 2 O) 7 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -NH-C(O)-。 21. The compound according to any one of claims 17 to 20, or its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula III, It is characterized by the following Any one or more of i) to vii): i) Ar is phenyl, pyridyl, pyrimidinyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl; Preferably, Ar is phenyl, furyl, imidazolyl or thienyl; Preferably, Ar is phenyl; ii) R is H or C 1-4 alkyl; Preferably, R is H, methyl, ethyl, n-propyl or n-butyl, preferably H, methyl, ethyl or n-propyl, more preferably H, methyl or ethyl, and even more preferably H; iii) R 2 and R 3 are independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R 2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; preferably, R 2 and R 3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; preferably, R 2 and R 3 each independently is hydrogen; iv) q is 0, 1 or 2, preferably 0 or 1; p is 0, 1 or 2, preferably 0 or 1; v) C is selected from the group consisting of auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and their derivatives Maytansine or its derivatives (e.g., maytansine-like, DM1, DM3, DM4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins and their derivatives, SN-38, Exatecan, Doxorubicin, TLR7 agonist with CAS 1821304-87-3, lapatinib, rifampicin and its analogs (e.g., Rifalogue, KRM-1657), preferably SN-38, Exatecan, Doxorubicin, TLR7 with CAS 1821304-87-3 agonist, lapatinib, monomethyl auristatin E (MMAE), MMAF derivatives or Rifalogue, more preferably monomethyl auristatin E (MMAE), rifampicin or Rifalogue; more preferably monomethyl auristatin E (MMAE) or Rifalogue; vi) A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody or a multispecific antibody, or A contains an antibody fragment or a substitute or a variant thereof, a protein ligand or a protein scaffold; Preferably, A is a monoclonal antibody, bispecific antibody or multispecific antibody with a thiol or amino group as a coupling site, or a monoclonal antibody, bispecific antibody or multispecific antibody with a thiol or amino group as a coupling site subjected to site-directed mutation or modification, or a monoclonal antibody, bispecific antibody or multispecific antibody with a carbonyl group in acetylphenylalanine (pAF) as a coupling site subjected to site-directed mutation, Preferably, 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 tiuxetan (ZEVALIN), tositumomab (BEXXAR), ofatumumab (ARZERRA), bevacizumab (AVASTIN), ipilimumab (YERVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi), sacituzumab, rovalvpituzumab, and biosimilars thereof, and antibacterial antibodies, More preferably, A is trastuzumab, or A is albumin, preferably human serum albumin, or A is an antibody, which comprises: (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VL; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems; Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 2; or A is an antibody, which comprises: (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems; Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 4; vii) E is a number between 1 and 15, preferably a number between 1 and 10, and more preferably a number between 1 and 8, such as about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

22. The compound according to any one of claims 17 to 21, or its geometric isomers, optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs, wherein the compound has a structure shown in Formula III-1, Formula III-2 or Formula III-3, in: A.k 1 , V, L, W, R, C, D, E are defined as in any one of claims 17-21, Preferably, A is coupled to site # via an S atom in the targeting compound molecule, or is coupled to site ## via an N atom in the targeting compound, or is coupled to site ### via a carbonyl group in the targeting compound.

23. The compound of claim 17, or its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula IV, It is characterized by the following Any one or more of i) to vi) : i) Ar is phenyl, pyridyl, pyrimidinyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl; Preferably, Ar is phenyl, furyl, imidazolyl or thienyl; Preferably, Ar is phenyl; ii) R 2 and R 3 are independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R 2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; preferably, R 2 and R 3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; preferably, R 2 and R 3 each independently is hydrogen; iii) q is 0, 1 or 2, preferably 0 or 1; p is 0, 1 or 2, preferably 0 or 1; iv) C is selected from the group consisting of auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and their derivatives Maytansine or its derivatives (e.g., maytansine-like, DM1, DM3, DM4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins and their derivatives, SN-38, Exatecan, Doxorubicin, TLR7 agonist with CAS 1821304-87-3, lapatinib, rifampicin and its analogs (e.g., Rifalogue, KRM-1657), preferably SN-38, Exatecan, Doxorubicin, TLR7 with CAS 1821304-87-3 agonist, lapatinib, monomethyl auristatin E (MMAE), MMAF derivatives or Rifalogue, more preferably monomethyl auristatin E (MMAE), rifampicin or Rifalogue; more preferably monomethyl auristatin E (MMAE) or Rifalogue; v) A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody or a multispecific antibody, or A contains an antibody fragment or a substitute or a variant thereof, a protein ligand or a protein scaffold; Preferably, A is a monoclonal antibody, a bispecific antibody or a multispecific antibody with an amino group or a thiol group as a coupling site, or a monoclonal antibody, a bispecific antibody or a multispecific antibody with a site-directed mutation or modification with an amino group or a thiol group as a coupling site, Preferably, 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 tiuxetan (ZEVALIN), tositumomab (BEXXAR), ofatumumab (ARZERRA), bevacizumab (AVASTIN), ipilimumab (YERVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi), sacituzumab, rovalvpituzumab, and biosimilars thereof, and antibacterial antibodies, More preferably, A is trastuzumab, or A is albumin, preferably human serum albumin, or A is an antibody, which comprises: (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VL; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems; Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 2; or A is an antibody, which comprises: (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having the sequence of the CDR1 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having the sequence of the CDR2 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having the sequence of the CDR3 contained in the heavy chain as shown in SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 having the sequence of the CDR1 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having the sequence of the CDR2 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having the sequence of the CDR3 contained in the light chain as shown in SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems; Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 4; vi) E is a number between 1 and 15, preferably a number between 1 and 10, and more preferably a number between 1 and 8, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. Preferably, the compound has a structure shown in Formula IV-1, Among them, the definitions of B, R', C, D, E, and A are as mentioned above. Preferably, k 2 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, k 2 is 1, 2, 3, 4, 5, 6, 7, or 8.

24. The compound of claim 17, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein the compound is selected from: in, represents A, A and E as defined in claim 17, Preferably, A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody or a multispecific antibody, and more preferably trastuzumab, an antibody with site-directed mutation of non-natural amino acids, and an antibacterial antibody. Preferably, A is an antibody, which is linked to the # position through an S atom in the antibody molecule or to the ## position through a carbonyl group in the antibody molecule or to the ### position through an N atom in the antibody molecule. It is a protein, preferably human serum albumin, which is linked to the # position through the S atom in the albumin molecule.

25. A pharmaceutical composition comprising a compound according to any one of claims 17 to 24, a geometric isomer, an optical isomer, a pharmaceutically acceptable salt, a hydrate, a solvate or a polymorph thereof, and optionally one or more pharmaceutically acceptable carriers or excipients, Preferably, the pharmaceutical composition further contains an additional drug. Preferably, the additional medications include one or more other antibiotics (e.g., antibiotics useful for MRSA infections) such as vancomycin, cotrimoxazole, tetracycline, doxycycline / minocycline, clindamycin, cephalosporins (e.g., cephalexin), naficillin, fidaxomicin, linezolid, etc., and / or any other suitable antibiotics.

26. The use of a compound, a geometric isomer, an optical isomer, a pharmaceutically acceptable salt, a hydrate, a solvate or a polymorph thereof as claimed in any one of claims 17 to 24 in the preparation of a medicament for treating a disease or condition or alleviating the severity of the disease or condition, wherein the disease or condition is selected from a tumor, an infectious disease, a hematological disease, a metabolic disease, an inflammation, Preferably, the tumor is selected from the group consisting of cancer, lymphoma, lymphoid tumor, blastoma, sarcoma and leukemia, Preferably, the cancer is selected from the group consisting of: breast cancer (e.g., HER2-positive breast cancer); squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung; peritoneal cancer; liver cancer; stomach cancer; gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urethral cancer; hepatoma; breast cancer; intestinal cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; kidney cancer or renal 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; bile duct cancer; head and neck cancer and related metastases, Preferably, the infectious disease includes bacterial infection such as infection caused by Staphylococcus aureus, Mycobacterium tuberculosis, Enterococcus faecium, Acinetobacter baumannii, Clostridium difficile, Streptococcus pneumoniae, Pseudomonas aeruginosa, sepsis caused by infection, tuberculosis or bacterial eye infection, heart, brain or skin infection, gastrointestinal infection, bacterial meningitis, and abscess in any organ (such as muscle, liver, meninges, or lung). Preferably, the infectious disease is cellulitis, bacteremia, skin necrosis, eyelid infection, eye infection, neonatal conjunctivitis, osteomyelitis, impetigo, ecthyma, scalded skin syndrome, food poisoning, pneumonia, surgical infection, urinary tract infection, burn infection, meningitis, endocarditis, sepsis, toxic shock syndrome, or septic arthritis, tuberculosis, infection associated with a prosthetic joint, infection associated with a catheter, or infection associated with an implant, Preferably, the infectious disease is Staphylococcus aureus infection of tissues surrounding the prosthetic joint. Preferably, the infectious disease is Staphylococcus aureus infection of the catheter and / or tissue surrounding the catheter. Preferably, the infectious disease is Staphylococcus aureus infection of the foreign body and / or tissue surrounding the foreign body. Preferably, the infectious disease is tuberculosis.

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