Azobenzene-containing linker, protein-coupled drug containing linker, and pharmaceutical use thereof

Abstract

The present disclosure relates to an azobenzene-containing linker represented by formula I, a protein-coupled drug containing the linker, and a use of the linker, and also relates to a pharmaceutical composition containing the protein-coupled drug, and further relates to a use of the protein-coupled drug for treating and / or preventing diseases.

Description

Azobenzene-containing linker, linker-containing protein-coupled drug, and medical use thereof

[0001] This application is based on the application with CN application number 202311603188.4 and application date November 28, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present disclosure belongs to the field of medicinal chemistry, and specifically relates to azobenzene-containing linkers, linker-containing protein-conjugated drugs, pharmaceutical compositions containing protein-conjugated drugs, and uses of these protein-conjugated drugs for treating and / or preventing diseases. Background Art

[0003] Antibody-drug conjugates (ADCs) are composed of antibodies and effector molecules covalently coupled via linkers. They combine the strong targeting of antibodies with the high activity of effector molecules and are known as "biological missiles" for the precise delivery of effector molecules (Signal Transduction and Targeted Therapy, 2022, 7(1):93).

[0004] In essence, all ADCs on the market are macromolecular prodrugs, and their targeting efficiency is increased 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 between 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.

[0005] 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 in immunology, 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 at 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.

[0006] On the other hand, antibiotic-resistant Staphylococcus aureus remains a public health concern, and approximately 40% of bloodstream infections in the United States are caused by methicillin-resistant Staphylococcus aureus (MRSA). Few FDA-approved treatment options are available for MRSA bloodstream infections, and vancomycin remains the antibiotic of choice. Despite appropriate antibiotic treatment, the mortality rate of S. aureus bloodstream infections remains approximately 18%, prompting research into combinations / combinations that could improve treatment efficacy. 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.

[0007] Therefore, new linkers need to be developed to solve the above problems.

[0008] Public content

[0009] The present disclosure 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 disclosure can be specifically recognized and reduced by azoreductase to 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.

[0010] Linker

[0011] The present disclosure provides a compound represented by formula I, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs,

[0012] in: represents a linker used to couple the azobenzene group to the targeting compound,

[0013] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0014] R2 and R3 are each independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

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

[0016] q is 0, 1, 2, or 3;

[0017] R is H, C 1-4 alkyl, wherein r1 and r2 are each independently an integer between 1 and 12;

[0018] Z is hydroxy, fluorine, chlorine, bromine, iodine or Wherein s is 0, 1, 2, 3 or 4, R5 is hydrogen, fluorine, chlorine, bromine, iodine, C 1-4 Alkyl, nitro or C 1-4 Alkoxy.

[0019] In certain embodiments, in the compound of Formula I, The definitions of B, V, L, and W are as follows:

[0020] B is or H, wherein r is an integer between 1 and 4; or

[0021] B is selected from The condition is that V, L, W1, W2, and W3 do not exist at the same time;

[0022] 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;

[0023] L is -(CH2CH2O) k -(CH2) l -,-CHR1-,-(CH2) m -, or absent, wherein R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, 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;

[0024] 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(O2)NH-, -NHS(O2)- or absent; W2 is selected from -(CH2) 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(O2)- or absent; wherein 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.

[0025] In certain embodiments, W in the compound of Formula I is selected from -NH-CH2-C(O)-NH-, -NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R10 )-C(O)-NH-, -C(O)-NH-(CH2) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH2) 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.

[0026] In certain embodiments, in the compound of Formula I:

[0027] B is

[0028] In certain embodiments, in the compound of Formula I:

[0029] B is

[0030] In certain embodiments, in the compound of Formula I:

[0031] B is

[0032] In certain embodiments, in the compound of Formula I:

[0033] B is

[0034] In certain embodiments, in the compound of Formula I:

[0035] B is

[0036] In certain embodiments, in the compound of Formula I:

[0037] B is

[0038] In certain embodiments, in the compound of Formula I:

[0039] B is

[0040] 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.

[0041] In certain embodiments, B is V is By introducing a cyclohexane group proximal to 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.

[0042] In certain embodiments, B is V is On the basis of the above, the introduction of oxygen atoms into the cyclohexane group can further increase the stability of the maleimide-derived thioether and increase the water solubility of the linker (Sci Rep. 2016 Aug 9:6:30835.).

[0043] In certain embodiments, B is V is wherein i is defined as described in the present disclosure, 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).

[0044] In certain embodiments, B is V is By introducing an electron-withdrawing benzene ring near the maleimide end, the maleimide is induced to spontaneously hydrolyze and open the ring, forming a stable ring-opening coupling form (Antibodies (Basel). 2017 Nov 28; 6(4): 20.)

[0045] 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.

[0046] In certain embodiments, in the compound of formula I, L is -(CH2CH2O) k -(CH2) l -, -CHR1- or -(CH2) m-, wherein k, l, R1, and 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, 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.

[0047] In certain embodiments, in the compound of formula I, W is -C(O)-NH-(CH2) 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 this disclosure.

[0048] In certain embodiments, in the compound of formula I, -VLW- is -(CH2) m -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-(CH2CH2O) k -(CH2) l-C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-CHR1-C(O)-NH-(CH2) o -NH-C(O)-, or -(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, where R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, m, o, i, k, l, and n are as defined in the present disclosure.

[0049] 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.

[0050] 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.

[0051] In certain embodiments, in the compound of Formula I, -VLW- is -(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)2-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)4-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)6-CH2-CH2-C(O)-NH-(CH2)2-NH -C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)8-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)3-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(C H2)2-C(O)NH-CH[(CH2)4-NHC(O)-(CH2CH2O)8-CH3]-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)7-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-.

[0052] In certain embodiments, in the compound of formula I, Ar is phenyl, pyridyl, pyrimidinyl, furanyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound of formula I, Ar is phenyl, furanyl, imidazolyl or thienyl. In certain embodiments, in the compound of formula I, Ar is phenyl.

[0053] In certain embodiments, in the compound of formula I, R is H or C 1-4 In certain embodiments, in the compound of formula I, R is H, methyl, ethyl, n-propyl, or n-butyl. In certain embodiments, in the compound of formula I, R is H, methyl, ethyl, or n-propyl. In certain embodiments, in the compound of formula I, R is H, methyl, or ethyl. In certain embodiments, in the compound of formula I, R is H. In certain embodiments, in the compound of formula I, R is methyl.

[0054] In certain embodiments, in the compound of formula I, R2 and R3 are each independently hydrogen, C 1-4In certain embodiments, in the compound shown in Formula I, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound shown in Formula I, R2 and R3 are each independently hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound shown in Formula I, R2 and R3 are each independently hydrogen.

[0055] In certain embodiments, in the compound of formula I, q is 0, 1 or 2, preferably 0 or 1.

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

[0057] In certain embodiments, in the compound of formula I, Z is hydroxy, fluorine, chlorine, bromine, iodine or wherein s is 0, 1, 2, 3, or 4; and R 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, or tert-butoxy. In certain embodiments, s is 0, 1, or 2, preferably 0 or 1. In certain embodiments, R is hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, or n-propyl. In certain embodiments, R is nitro, methoxy, ethoxy, or n-propoxy.

[0058] 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.

[0059] In certain embodiments, Z is Wherein R5 is hydrogen, fluorine, chlorine, bromine, iodine, C 1-4 Alkyl, nitro or C 1-4 In certain embodiments, R5 is alkoxy, s is 0, 1, 2, 3, or 4. In certain embodiments, s is 0. In certain embodiments, s is 1. In certain embodiments, s is 2. In certain embodiments, s is 3. In certain embodiments, s is 4. In certain embodiments, R5 is hydrogen. In certain embodiments, R5 is fluorine. In certain embodiments, R5 is chlorine. In certain embodiments, R5 is bromine. In certain embodiments, R5 is iodine. In certain embodiments, R5 is C 1-4In certain embodiments, R5 is nitro. In certain embodiments, R5 is C 1-4 In certain embodiments, R5 is methyl. In certain embodiments, R5 is ethyl. In certain embodiments, R5 is propyl. In certain embodiments, R5 is methoxy. In certain embodiments, R5 is ethoxy. In certain embodiments, R5 is propoxy.

[0060] In certain embodiments, the compound of formula I has the structure shown in I-1,

[0061] wherein B, V, L, W, R, and Z are as defined in the present disclosure.

[0062] In certain embodiments, in the compound of formula I, B is selected from V, L, W1, W2, and W3 do not exist; R is wherein R1 and R2 are as defined herein. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, or 6. In certain embodiments, R1 and R2 are each independently 1, 2, 3, or 4.

[0063] In certain embodiments, the compound of Formula I is selected from:

[0064] The linker provided by the present disclosure contains an azobenzene structure, which can be specifically recognized by azoreductase and reduce the azobenzene structure, resulting in the cleavage of the azo bond.

[0065] The present disclosure also provides uses of the compound represented by Formula I, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs in the preparation of antibody-drug conjugates.

[0066] Linker-drug conjugates

[0067] The present disclosure also provides compounds represented by Formula II, their geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs.

[0068] in: represents a linker, used to couple the right azobenzene group to the targeting compound,

[0069] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0070] R2 and R3 are each independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0071] p is 0, 1, 2, or 3;

[0072] q is 0, 1, 2, or 3;

[0073] R is H, C 1-4 alkyl, wherein r1 and r2 are each independently an integer between 1 and 12;

[0074] D is 0 or 1;

[0075] 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, such as a Toll-like receptor agonist (such as a TLR7 agonist) or a STING agonist, an antibacterial drug,

[0076] For example, rifampicin and its analogs (e.g., Rifalogue, KRM-1657);

[0077] 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.

[0078] In certain embodiments, in the compound represented by Formula II, The definitions of B, V, L, and W are as follows:

[0079] B is or H, wherein r is 1-4; or

[0080] B is selected from The condition is that V, L, W1, W2, and W3 do not exist at the same time;

[0081] 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;

[0082] L is -(CH2CH2O) k -(CH2) l -,-CHR1-,-(CH2) m -, or absent, wherein R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, 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;

[0083] 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(O2)NH-, -NHS(O2)- or absent; W2 is selected from -(CH2) 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(O2)- or absent; wherein 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.

[0084] In certain embodiments, in the compound of formula II: W is selected from -NH-CH2-C(O)-NH-, -NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH2) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH2) 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 this disclosure.

[0085] In certain embodiments, in the compound of formula II: B is

[0086] In certain embodiments, in the compound of formula II: B is

[0087] In certain embodiments, in the compound of Formula II:

[0088] B is

[0089] In certain embodiments, in the compound of Formula II:

[0090] B is

[0091] In certain embodiments, in the compound of Formula II:

[0092] B is

[0093] In certain embodiments, in the compound of Formula II:

[0094] B is

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

[0096] B is

[0097] 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.

[0098] 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.

[0099] In certain embodiments, in the compound of formula II, L is -(CH2CH2O) k -(CH2) l -, -CHR1- or -(CH2) m -, wherein k, l, R1, and 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, 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.

[0100] In certain embodiments, in the compound of formula II, W is -C(O)-NH-(CH2) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R11 and R 12 is as defined in the present disclosure. 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 are each independently hydrogen. In certain embodiments, R 11 and R 12 Each independently is C 1-6 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.

[0101] In certain embodiments, in the compound of formula II, -VLW- is -(CH2) m -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-CHR1-C(O)-NH-(CH2) o -NH-C(O)-, or -(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, where R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, m, o, i, k, l, and n are as defined in the present disclosure.

[0102] 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.

[0103] 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.

[0104] In certain embodiments, in the compound of Formula II, -VLW- is -(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)2-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)4-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)6-CH2-CH2-C(O)-NH-(CH2)2-N H-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)8-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)3-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(C H2)2-C(O)NH-CH[(CH2)4-NHC(O)-(CH2CH2O)8-CH3]-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)7-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-.

[0105] In certain embodiments, in the compound shown in formula II, Ar is phenyl, pyridyl, pyrimidinyl, furyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound shown in formula II, Ar is phenyl, furyl, imidazolyl or thienyl. In certain embodiments, in the compound shown in formula II, Ar is phenyl.

[0106] In certain embodiments, in the compound of formula II, R is H or C 1-4In certain embodiments, in the compound of formula II, R is H, methyl, ethyl, n-propyl or n-butyl. In certain embodiments, in the compound of formula II, R is H, methyl, ethyl or n-propyl. In certain embodiments, in the compound of formula II, R is H, methyl or ethyl. In certain embodiments, in the compound of formula II, R is H. In certain embodiments, in the compound of formula II, R is methyl.

[0107] In certain embodiments, in the compound of formula II, R2 and R3 are each independently hydrogen, C 1-4 In certain embodiments, in the compound shown in formula II, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound shown in formula II, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound shown in formula II, R2 and R3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound shown in formula II, R2 and R3 are each independently hydrogen.

[0108] In certain embodiments, in the compound of Formula II, q is 0, 1 or 2, preferably 0 or 1.

[0109] In certain embodiments, in the compound of Formula II, p is 0, 1 or 2, preferably 0 or 1.

[0110] In certain embodiments, in the compound of 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).

[0111] In certain embodiments, in the compound of Formula II, C is monomethyl auristatin E (MMAE), rifampicin or Rifalogue.

[0112] In certain embodiments, in the compound of Formula II, C is monomethyl auristatin E (MMAE) or Rifalogue.

[0113] 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 a Rifalogue.

[0114] In certain embodiments, in the compound of formula II, C is a MMAF derivative having the structure

[0115] In certain embodiments, the compound of Formula II has the structure of Formula II-1,

[0116] wherein B, V, L, W, R, C, and D are as defined in the present disclosure.

[0117] In certain embodiments, in the compound of formula II, B is selected from V, L, W1, W2, and W3 do not exist; R is wherein R1 and R2 are as defined herein. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, R1 and R2 are each independently 1, 2, 3, 4, 5, or 6. In certain embodiments, R1 and R2 are each independently 1, 2, 3, or 4.

[0118] In certain embodiments, the compound of Formula II is selected from:

[0119] The present disclosure 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-drug conjugates.

[0120] Protein-conjugated drugs

[0121] The present disclosure also provides compounds represented by Formula III or Formula IV, their geometric or optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs.

[0122] Wherein, A represents the targeted compound;

[0123] represents a linker used to couple the azobenzene group to the targeting compound;

[0124] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0125] R2 and R3 are each independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0126] p is 0, 1, 2, or 3;

[0127] q is 0, 1, 2, or 3;

[0128] R is H, C 1-4 alkyl, wherein r1 and r2 are each independently an integer between 1 and 12;

[0129] R' is Where k2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0130] B is selected from

[0131] D is 0 or 1;

[0132] 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; 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 molecule, peptide or nucleotide, an immunomodulator, 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;

[0133] E is a number between 1 and 20.

[0134] In certain embodiments, in the compound represented by Formula III, A represents a targeting compound;

[0135] represents a linker used to couple the azobenzene group to the targeting compound;

[0136] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0137] R2 and R3 are each independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0138] p is 0, 1, 2, or 3;

[0139] q is 0, 1, 2, or 3;

[0140] R is H, C 1-4 alkyl, wherein r1 and r2 are each independently an integer between 1 and 12;

[0141] D is 0 or 1;

[0142] 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; 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 molecule, peptide or nucleotide, an immunomodulator, 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;

[0143] E is a number between 1 and 20.

[0144] In certain embodiments, in the compound represented by formula III, The definitions of B1, V, L, and W are as follows:

[0145] B1 is Where k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0146] 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;

[0147] L is -(CH2CH2O) k -(CH2) l -,-CHR1-,-(CH2) m -, or absent, wherein R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, 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;

[0148] 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(O2)NH-, -NHS(O2)- or absent; W2 is selected from -(CH2) 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(O2)- or absent; wherein 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.

[0149] In certain embodiments, in the compound represented by Formula III, A is a targeting compound selected from proteins, antibodies, polypeptides, enzymes and small molecules.

[0150] In certain embodiments, in the compound represented by Formula III, A is coupled to the site ** of the B1 group through the S atom or N atom in the targeting compound molecule, or A is coupled to the site ** of the B1 group through the reaction of the carbonyl group in the targeting compound molecule with the hydroxylamine group in the B1 group to form an oxime bond.

[0151] In certain embodiments, in the compound of formula III described in the present disclosure, W is selected from -NH-CH2-C(O)-NH-, -NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10)-C(O)-NH-, -C(O)-NH-(CH2) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH2) 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 this disclosure.

[0152] In certain embodiments, in the compound of formula III, B1 is wherein k1 is as defined in the present disclosure.

[0153] In certain embodiments, in the compound of formula III, B1 is wherein k1 is as defined in the present disclosure.

[0154] In certain embodiments, in the compound of formula III, k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, in the compound of formula III, k1 is 1, 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, in the compound of formula III, k1 is 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, in the compound of formula III, k1 is 4, 5, 6, 7, or 8. In certain embodiments, in the compound of formula III, k1 is 9 or 10.

[0155] 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.

[0156] 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.

[0157] In certain embodiments, in the compound of formula III, L is -(CH2CH2O) k -(CH2) l -, -CHR1- or -(CH2) m -, wherein k, l, R1, and 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, 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.

[0158] In certain embodiments, in the compound of formula III, W is -C(O)-NH-(CH2) o -NH-C(O)- or -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-,o、R 11 and R 12is as defined in the present disclosure. 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 are each independently hydrogen. In certain embodiments, R 11 and R 12 Each independently is C 1-6 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.

[0159] In certain embodiments, in the compound of formula III, -VLW- is -(CH2) m -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-CHR1-C(O)-NH-(CH2) o -NH-C(O)-, or -(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, where R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, m, o, i, k, l, and n are as defined in the present disclosure.

[0160] 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.

[0161] 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.

[0162] In certain embodiments, in the compound of formula III described in the present disclosure, -VLW- is -(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)2-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)4-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)6-CH2-CH2-C(O)-NH-(CH2) 2-NH-C(O)-,-(CH2)2-C(O)NH-(CH2CH2O)8-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-,-(CH2CH2O)3-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-,- (CH2)2-C(O)NH-CH[(CH2)4-NHC(O)-(CH2CH2O)8-CH3]-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)7-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-.

[0163] In certain embodiments, in the compound shown in formula III, Ar is phenyl, pyridyl, pyrimidinyl, furyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound shown in formula II, Ar is phenyl, furyl, imidazolyl or thienyl. In certain embodiments, in the compound shown in formula III, Ar is phenyl.

[0164] In certain embodiments, in the compound of formula III, R is H or C 1-4In certain embodiments, in the compound of formula III, R is H, methyl, ethyl, n-propyl, or n-butyl. In certain embodiments, in the compound of formula III, R is H, methyl, ethyl, or n-propyl. In certain embodiments, in the compound of formula III, R is H, methyl, or ethyl. In certain embodiments, in the compound of formula III, R is H. In certain embodiments, in the compound of formula III, R is methyl.

[0165] In certain embodiments, in the compound of formula III, R2 and R3 are each independently hydrogen, C 1-4 In certain embodiments, in the compound shown in formula III, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound shown in formula III, R2 and R3 are each independently hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound shown in formula III, R2 and R3 are each independently hydrogen.

[0166] In certain embodiments, in the compound represented by formula III, q is 0, 1 or 2, preferably 0 or 1.

[0167] In certain embodiments, in the compound represented by formula III, p is 0, 1 or 2, preferably 0 or 1.

[0168] In certain embodiments, in the compound of Formula III, C is selected from the group consisting of 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).

[0169] In certain embodiments, in the compound of formula III, C is monomethyl auristatin E (MMAE), rifampicin or Rifalogue.

[0170] In certain embodiments, in the compound of formula III, C is monomethyl auristatin E (MMAE) or Rifalogue.

[0171] In certain embodiments, in the compound of Formula III, C is SN-38, Exatecan, Doxorubicin, a TLR7 agonist with CAS 1821304-87-3, lapatinib, MMAE, an MMAF derivative, or Rifalogue.

[0172] In certain embodiments, in the compound of formula IV, C is rifampicin or Rifalogue.

[0173] In certain embodiments, in the compound of formula IV, C is a Rifalogue.

[0174] In certain embodiments, in the compound represented by formula III, C is a MMAF derivative having the structure

[0175] 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.

[0176] In certain embodiments, in the compound represented by Formula III, A contains an antibody fragment or a substitute or variant thereof, a protein ligand or a protein scaffold.

[0177] In certain embodiments, in the compound of Formula III, A is a monoclonal antibody, bispecific antibody, or multispecific antibody with a sulfhydryl or amino group as a coupling site, or a monoclonal antibody, bispecific antibody, or multispecific antibody with a site-directed mutation or modification with a sulfhydryl or amino group as a coupling site, or a monoclonal antibody, bispecific antibody, or multispecific antibody with a site-directed mutation with a carbonyl group in acetylphenylalanine (pAF) as a coupling site.

[0178] In certain embodiments, in the compound of 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.

[0179] In certain embodiments, in the compound of formula III, A is trastuzumab.

[0180] 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.

[0181] In certain embodiments, in the compound represented by formula III, A is an antibacterial antibody.

[0182] In certain embodiments, in the compound represented by Formula III, A is an antibody subjected to site-directed mutation of a non-natural amino acid, comprising:

[0183] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0184] (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 more 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 VH;

[0185] (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 more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR2 contained in the VH; and

[0186] (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 more 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;

[0187] and / or

[0188] (b) the following three light chain variable region (VL) CDRs:

[0189] (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 more 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;

[0190] (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 more 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

[0191] (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 more 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.

[0192] In certain embodiments, the substitutions described in any one of (i)-(vi) are conservative substitutions. 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 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.

[0193] In certain embodiments, in the compound represented by Formula III, A is an antibacterial antibody, which comprises:

[0194] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0195] (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 more 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 VH;

[0196] (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 more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR2 contained in the VH; and

[0197] (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 more 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;

[0198] and / or

[0199] (b) the following three light chain variable region (VL) CDRs:

[0200] (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 more 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;

[0201] (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 more 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

[0202] (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 more 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.

[0203] In certain embodiments, the substitutions described in any one of (i) to (vi) are conservative substitutions. 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 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.

[0204] 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, or about 8.

[0205] In certain embodiments, the compound of formula III has a structure shown in formula III-1, formula III-2, or formula III-3.

[0206] Wherein: A, k1, V, L, W, R, C, D, and E are defined as described in the present disclosure.

[0207] In certain embodiments, in the compound of formula III-1, A is coupled to site # via an S atom in the targeting compound molecule. In certain embodiments, in the compound of formula III-2, A is coupled to site ## via an N atom in the targeting compound. In certain embodiments, in the compound of formula III-3, A is coupled to site ### via a carbonyl group in the targeting compound.

[0208] In certain embodiments, the compound of formula III is selected from:

[0209] in, represents A, A and E are as defined in this disclosure, is a protein, preferably human serum albumin, which is linked to the # site via an S atom in the albumin molecule. In certain embodiments, in the compound of formula III, A is an antibody, preferably a monoclonal antibody (MAB), a bispecific antibody, or a multispecific antibody, further preferably trastuzumab, an antibody with site-directed mutagenesis of non-natural amino acids, and an antibacterial antibody. Preferably, A is an antibody, which is linked to the # site via an S atom in the antibody molecule, or to the ## site via a carbonyl group in the antibody molecule, or to the ### site via an N atom in the antibody molecule. In certain embodiments, in the compound of formula III, E is approximately 4.

[0210] In certain embodiments, in the compound represented by Formula IV,

[0211] A represents the targeted compound;

[0212] Ar is a five-membered or six-membered aryl or heteroaryl group;

[0213] R2 and R3 are each independently hydrogen, C 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano;

[0214] p is 0, 1, 2, or 3;

[0215] q is 0, 1, 2, or 3;

[0216] R' is Where k2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0217] B is selected from

[0218] D is 0 or 1;

[0219] 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; 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 molecule, peptide or nucleotide, an immunomodulator, 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;

[0220] E is a number between 1 and 20.

[0221] In certain embodiments, in the compound represented by Formula IV, A is a targeting compound selected from proteins, antibodies, polypeptides, enzymes and small molecules.

[0222] In certain embodiments, in the compound of Formula IV, A is coupled to the site ** of the R' group via the N atom in the targeting compound molecule.

[0223] In certain embodiments, in the compound shown in formula IV, Ar is phenyl, pyridyl, pyrimidinyl, furyl, imidazolyl, pyrrolyl, thiazolyl, pyrazolyl or thienyl. In certain embodiments, in the compound shown in formula IV, Ar is phenyl, furyl, imidazolyl or thienyl. In certain embodiments, in the compound shown in formula IV, Ar is phenyl.

[0224] In certain embodiments, in the compound of formula IV, R2 and R3 are each independently hydrogen, C 1-4 In certain embodiments, in the compound shown in formula IV, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound shown in formula IV, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In certain embodiments, in the compound shown in formula IV, R2 and R3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano. In certain embodiments, in the compound shown in formula IV, R2 and R3 are each independently hydrogen.

[0225] In certain embodiments, in the compound represented by Formula IV, q is 0, 1 or 2, preferably 0 or 1.

[0226] In certain embodiments, in the compound of formula IV, p is 0, 1 or 2, preferably 0 or 1.

[0227] In certain embodiments, in the compound of Formula IV, C is selected from the group consisting of 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).

[0228] In certain embodiments, in the compound of formula IV, C is monomethyl auristatin E (MMAE), rifampicin or Rifalogue.

[0229] In certain embodiments, in the compound of formula IV, C is monomethyl auristatin E (MMAE) or Rifalogue.

[0230] In certain embodiments, in the compound of formula IV, C is rifampicin or Rifalogue.

[0231] In certain embodiments, in the compound of formula IV, C is a Rifalogue.

[0232] In certain embodiments, in the compound of Formula IV, C is SN-38, Exatecan, Doxorubicin, a TLR7 agonist with CAS 1821304-87-3, lapatinib, MMAE, an MMAF derivative, or Rifalogue.

[0233] In certain embodiments, in the compound represented by formula IV, C is a MMAF derivative having the structure

[0234] 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.

[0235] In certain embodiments, in the compound represented by Formula IV, A contains an antibody fragment or a substitute or variant thereof, a protein ligand or a protein scaffold.

[0236] 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-directedly mutated or modified with an amino group or a thiol group as a coupling site.

[0237] 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.

[0238] In certain embodiments, in the compound of formula IV, A is an antibacterial antibody.

[0239] In certain embodiments, in the compound of formula IV, A is trastuzumab.

[0240] 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.

[0241] In certain embodiments, in the compound of formula IV, A is an antibody subjected to site-directed mutation of a non-natural amino acid, comprising:

[0242] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0243] (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 more 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 VH;

[0244] (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 more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR2 contained in the VH; and

[0245] (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 more 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;

[0246] and / or

[0247] (b) the following three light chain variable region (VL) CDRs:

[0248] (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 more 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;

[0249] (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 more 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

[0250] (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 more 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.

[0251] In certain embodiments, the substitutions described in any one of (i)-(vi) are conservative substitutions. 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 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.

[0252] In certain embodiments, in the compound represented by Formula IV, A is an antibacterial antibody, which comprises:

[0253] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0254] (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 more 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 VH;

[0255] (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 more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR2 contained in the VH; and

[0256] (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 more 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;

[0257] and / or

[0258] (b) the following three light chain variable region (VL) CDRs:

[0259] (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 more 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;

[0260] (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 more 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

[0261] (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 more 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.

[0262] In certain embodiments, the substitutions described in any one of (i) to (vi) are conservative substitutions. 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 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.

[0263] 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, or about 8.

[0264] In certain embodiments, the compound of formula IV has the structure shown in formula IV-1,

[0265] Wherein, B, R', C, D, E, and A are as defined in the present disclosure,

[0266] In certain embodiments, k2 in the compound of formula IV is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, k2 in the compound of formula IV is 1, 2, 3, 4, 5, 6, 7 or 8.

[0267] In certain embodiments, the compound of formula IV is:

[0268] in, represents A, and A and E are as defined in the present disclosure. In certain embodiments, A is trastuzumab and E is about 3.

[0269] 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 mutagenesis 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.

[0270] The present disclosure 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.

[0271] In certain embodiments, the compound of Formula III or Formula IV, its geometric isomers, optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs are present in an effective amount in the pharmaceutical composition.

[0272] In certain embodiments, the pharmaceutical composition further comprises additional drugs, including one or more other antibiotics (e.g., antibiotics that can be used for MRSA infection) such as vancomycin, trimethoprim-sulfamethoxazole, tetracycline, doxycycline / minocycline, clindamycin, cephalosporins (e.g., cephalexin), nafcillin, fidaxomicin, linezolid, etc., and / or any other suitable antibiotics.

[0273] The present disclosure also provides the use of a 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 medicament 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.

[0274] The present disclosure also provides compounds represented by Formula III or Formula IV, their geometric isomers, optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs, and their use in drugs for treating diseases or conditions or alleviating the severity of the diseases or conditions, wherein the diseases or conditions are selected from tumors, infectious diseases, hematological diseases, metabolic diseases, and inflammation.

[0275] The present disclosure also provides a method for treating a disease or condition or alleviating the severity of the disease or condition, comprising administering an effective amount of a compound of Formula III or Formula IV, its geometric isomers, optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs to a subject in need thereof, wherein the disease or condition is selected from tumors, infectious diseases, hematological diseases, metabolic diseases, and inflammation.

[0276] The present disclosure also provides a pharmaceutical composition for treating a disease or condition or alleviating the severity of the disease or condition, comprising a compound represented by Formula III or Formula IV, 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.

[0277] 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.

[0278] 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.

[0279] 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 infection, heart, brain or skin infection, gastrointestinal infection, bacterial meningitis, and abscesses in any organ (such as muscle, liver, meninges, or lung).

[0280] 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.

[0281] In certain embodiments, the infectious disease is a Staphylococcus aureus infection of the tissue surrounding a prosthetic joint.

[0282] In certain embodiments, the infectious disease is a Staphylococcus aureus infection of the catheter and / or tissue surrounding the catheter.

[0283] In certain embodiments, the infectious disease is a Staphylococcus aureus infection of the foreign body and / or the tissue surrounding the foreign body.

[0284] In certain embodiments, the infectious disease is tuberculosis.

[0285] In the present disclosure, the rifampicin analog is Rifalogue, and its structural formula is:

[0286] 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, rifamycins are used in combination with first-line antibiotics to improve outcomes, typically for prosthetic or foreign body device infections.

[0287] The ADC comprising a rifampicin analog described in the present disclosure (e.g., a compound represented by Formula III or Formula IV, a geometric isomer, an optical isomer, a pharmaceutically acceptable salt, a hydrate, a solvate or a polymorph thereof, wherein C is rifampicin or an analog thereof, and other substituents are as described in any embodiment of the present disclosure, such as a compound represented by Formula III-16) can be used to prevent or treat bacterial growth and / or bacterial infection in a subject.

[0288] In certain embodiments, the bacteria is a Gram-positive bacteria (Gram-positive bacteria are responsible for bacterial infections).

[0289] In certain embodiments, the bacteria is a penicillin-resistant bacteria (penicillin-resistant bacteria are the cause of bacterial infections).

[0290] In certain embodiments, the bacteria is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) bacteria (MRSA bacteria are a cause of bacterial infections).

[0291] In certain embodiments, the bacteria are methicillin-sensitive Staphylococcus aureus (MSSA) bacteria (MSSA bacteria are responsible for bacterial infections).

[0292] 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).

[0293] In certain embodiments, the bacterium is Chlamydia trachomatis resistant, eg, to azithromycin (Chlamydia trachomatis resistant, eg, to azithromycin is a cause of bacterial infection).

[0294] 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).

[0295] The present disclosure 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 a prosthetic joint, infection associated with a catheter, or infection associated with an implant in a subject, comprising administering to the subject an effective therapeutic amount of an antibody-drug conjugate comprising a rifampicin analog (e.g., a compound of Formula III or Formula IV, a geometric isomer, optical isomer, pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, wherein C is rifampicin or its analog, and other substituents are as described in any embodiment of the present disclosure, for example, a compound of Formula III-16).

[0296] The present disclosure also provides a method for preventing or treating intracellular bacterial infection in a subject, comprising administering to the subject an effective therapeutic amount of an antibody-drug conjugate comprising a rifampicin analog (e.g., a compound of Formula III or Formula IV, a geometric isomer, optical isomer, pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof, wherein C is rifampicin or an analog thereof, and other substituents are as described in any embodiment of the present disclosure, for example, a compound of Formula III-16).

[0297] In certain embodiments, the present disclosure provides a method of treating a subject in need thereof, comprising administering an ADC comprising a rifampicin analog (e.g., a compound of Formula III or Formula IV, a geometric isomer, an optical isomer, a pharmaceutically acceptable salt, a hydrate, a solvate, or a polymorph thereof, wherein C is rifampicin or its analog, and other substituents are as described in any embodiment of the present disclosure, for example, a compound of Formula III-16), for treating and / or preventing a bacterial infection in the subject, and / or a disease or disorder or condition associated with a staphylococcal infection (e.g., a Staphylococcus aureus infection), and / or alleviating at least one symptom associated with such a disease or disorder or condition. The disease, disorder, or condition can 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 conjugates disclosed herein can be used to treat and / or prevent S. aureus infection of tissue surrounding the prosthetic joint.

[0298] 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.

[0299] 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.

[0300] In certain embodiments, the subject suffers from tuberculosis, and the antibody-drug conjugates disclosed herein can be used to treat tuberculosis.

[0301] 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 disclosure can be administered in combination with vancomycin and can be used to prevent or treat bacterial infections in subjects.

[0302] definition

[0303] 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.

[0304] As used herein, the term "cytotoxin" refers to molecules that are toxic to cancer cells upon release thereof. Toxins of particular interest in the present disclosure include methyl auristatin E (MMAE), auristatin, maytansinoids or their derivatives (e.g., maytansinoids, DM1, DM3, DM4), calicheamicin, duocarmycin, doxorubicin, camptothecin, or PBD-type cytotoxins.

[0305] 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 generally a site that can be coupled through the sulfhydryl or lysine amine groups of the cysteine ​​on the antibody, and the toxin coupling end of the linker is generally 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 a coupled linker, 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 a leaving group of a sulfhydryl reactive group, a leaving group of an amine reactive group).

[0306] As used herein, the terms "antibody-drug conjugate" or "ADC" or "antibody-drug conjugate" have the same meaning and are products formed by coupling multiple molecules (usually 1-8) of cytotoxins to an antibody molecule via a linker. An antibody conjugated to one or more cytotoxins. The antibody is typically a monoclonal antibody selective for a specific antigen of a cancer.

[0307] As used herein, the term "about" is 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."

[0308] 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, lung adenocarcinoma, and lung squamous cell carcinoma; peritoneal cancer; liver cancer; gastric cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urethral cancer; hepatocellular carcinoma; 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.

[0309] 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).

[0310] As used herein, the term "salt" refers to salts that retain the biological effectiveness and properties of a compound, but are biologically or otherwise undesirable for use in pharmaceuticals. In many cases, the compounds disclosed herein are capable of forming acid and / or base salts due to the presence of amino and / or carboxyl groups or similar groups. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived 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, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic 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 isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, as described in WO 87 / 05297, Johnston et al., published September 11, 1987 (incorporated herein by reference in its entirety).

[0311] As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclic group of 5 to 14 carbon atoms having a conjugated π electron system, a single ring or two or more fused rings. The "aryl" preferably has 5 to 10, 5 to 8, or 5 to 6 carbon atoms. Typical examples of "aryl" include, but are not limited to, phenyl, naphthyl, anthracenyl, and the like.

[0312] 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" group preferably has 5-10, 5-8, or 5-6 ring members. Typical examples of "heteroaryl" include, but are not limited to, furyl, imidazolyl, thienyl, triazolyl, indolyl, tetrazolyl, pyridinyl, pteridinyl, pyrimidinyl, triazolyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, and the like.

[0313] As used herein, the term "aroheterocyclyl" refers to a cyclic group having two or more fused rings in which 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.

[0314] As used herein, the term "heteroaryloheterocyclyl" refers to a cyclic group having two or more fused rings in which 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.

[0315] As used herein, the term "heterocyclyl" refers to a saturated or partially unsaturated cyclic hydrocarbon group having a monocyclic, bicyclic, or multiple fused rings (including fused, bridged, and spirocyclic) of 3 to 12 ring members, wherein at least one ring member is a heteroatom selected from nitrogen, oxygen, or sulfur. The "heterocyclyl" preferably has 3-10, 3-8, 5-8, 3-6, or 5-6 ring members. Typical examples of "heterocyclyl" include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperazinyl, thiazinyl, piperidinyl, and morpholinyl.

[0316] Abbreviations / acronyms

[0317] ADC (antibody-drug conjugate): antibody-drug conjugate;

[0318] AcOH (Acetic acid): glacial acetic acid;

[0319] Boc (t-Butyloxy carbonyl): tert-Butyloxycarbonyl;

[0320] Cys(Cysteine): L-cysteine;

[0321] DAR (Drug to antibody ratio): drug / antibody molar ratio;

[0322] DCM (Dichloromethane): dichloromethane;

[0323] DIPEA (N,N-Diisopropylethylamine): diisopropylethylamine;

[0324] DMAC (Dimethylacetamide): N,N-dimethylacetamide;

[0325] DMF (N,N-Dimethylformamide): N,N-dimethylformamide;

[0326] DMSO (Dimethyl Sulphoxide): dimethyl sulfoxide;

[0327] Diox(1,4-Dioxane): 1,4-dioxane;

[0328] DIAD (Diisopropylazodicarboxylate): diisopropyl azodicarboxylate;

[0329] DIC (Diisopropylcarbodiimide): diisopropylcarbodiimide;

[0330] EA (Ethyl acetate): ethyl acetate;

[0331] EtOH (Ethanol): ethanol;

[0332] EDTA (Ethylenediamine tetraacetic acid): ethylenediaminetetraacetic acid;

[0333] EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride): 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0334] FITC (fluorescein isothiocyanate): fluorescein isothiocyanate;

[0335] HOBT (1-Hydroxybenzotriazole): 1-Hydroxybenzotriazole;

[0336] HCl (hydrogen chloride): hydrogen chloride; HER2 (Human epidermal growth factor receptor 2): human epidermal growth factor receptor 2;

[0337] MAB (Monoclonal Antibody): Monoclonal Antibody

[0338] MMAE (Monomethyl auristatin E): Monomethyl auristatin E;

[0339] NAC (N-Acetyl-L-cysteine): N-acetylcysteine;

[0340] NADPH (nicotinamide adenine dinucleotide phosphate): reduced coenzyme II;

[0341] NH2NH2 (hydrazine): hydrazine;

[0342] NHS(N-Hydroxy succinimide): N-hydroxysulfosuccinimide;

[0343] PPh3 (Triphenylphosphine): triphenylphosphine;

[0344] TCEP (Tris(2-carboxyethyl)phosphine): tris(2-carboxyethyl)phosphine;

[0345] THF (Tetrahydrofuran): Tetrahydrofuran;

[0346] TFA (Trifluoroacetic acid): trifluoroacetic acid;

[0347] PE (Petroleum ether): petroleum ether;

[0348] VC-ADC (Herceptin-VC-MMAE): A cathepsin-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 methods in the prior art, wherein the DAR is approximately 4.

[0349] When the compound name used in this article is inconsistent with the chemical structural formula, the chemical structural formula shall prevail.

[0350] The pharmaceutical compositions described herein comprise a compound of Formula III or Formula IV, or a salt or solvate thereof, and a conventional pharmaceutical carrier or excipient. The pharmaceutical compositions may be administered, for example, orally or parenterally, such as by intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

[0351] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic effect, for example, to achieve a reduction in the symptoms associated with the disease being treated.

[0352] It should also be noted that the dosage and method of use of the disclosed compounds depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, potency of the compound, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 100 mg / kg body weight / day.

[0353] The linker provided herein contains an azophenyl fragment and exhibits excellent enzymatic cleavage performance. The antibody-drug conjugate (ADC) containing the linker provided herein exhibits excellent structural stability, ideal enzymatic drug release, and hypoxia dependence, resulting in relatively ideal in vitro and in vivo efficacy and safety.

[0354] In certain specific embodiments, the synthetic route of the ADC described in the present disclosure is as follows:

[0355] Among them: C, E, Definitions are as described in the present disclosure, X represents -VLW-, and the definitions of V, L, and W are as described in the present disclosure.

[0356] Using p-nitrobenzyl alcohol (1) as the starting material, the unstable intermediate p-aminobenzyl alcohol is obtained under the reduction effect of zinc powder, and further oxidized with ferric chloride to obtain intermediate (2). Under the action of acetic acid, it reacts with 4-aminobenzoic acid to prepare compound (3). Under the action of EDCI, HOBT, and DIPEA, it undergoes amide condensation to prepare compound (4). 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) reacts 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).

[0357] In certain specific embodiments, another synthetic route of the ADC described in the present disclosure is as follows:

[0358] Among them: E, Definitions are as described in the present disclosure, X represents -VLW-, and the definitions of V, L, and W are as described in the present disclosure.

[0359] Using p-aminobenzoic acid (11) as the starting material, compound (12) is oxidized with ferric chloride, which is then reacted with compound (13) in the presence of acetic acid to produce compound (14). Compound (15) is then obtained through amide condensation in the presence of EDCI, HOBT, and DIPEA. The Boc protecting group is removed with trifluoroacetic acid to produce compound (16), which is then further subjected to substitution reaction to produce compound (17). Compound (17) is then coupled with an antibody to produce the final ADC product (18).

[0360] In certain specific embodiments, another synthetic route of ADC described in the present disclosure is as follows:

[0361] Among them: E, Definitions As described in this disclosure,

[0362] Compound (19) was used as the starting material and reacted with compound (5) in the presence of EDCI, HOBT, and DIPEA to produce compound (20). The Fmoc protecting group was removed by piperidine to produce compound (21), which was then reacted with compound (22) to produce compound I-6. Compound I-6 was reacted with di(p-nitrophenyl) carbonate to produce compound I-12, which was then reacted with active compound C to produce the corresponding ADC loading (23), which was further coupled with an antibody to produce the final ADC product (24).

[0363] In certain specific embodiments, another synthetic route of ADC described in the present disclosure is as follows:

[0364] Among them: C, E, Definitions are as described in the present disclosure, X represents -VLW-, and the definitions of V, L, and W are as described in the present disclosure.

[0365] Compound (26) was prepared from 2-hydroxyisoindoline-1,3-dione and compound (25) under the action of DIAD and PPh3. The tert-butyloxy protecting group was removed by hydrochloric acid to obtain compound (27). Compound (28) was prepared by amide condensation with compound (5) under the action of DIC. 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). Compound (31) was obtained by removal of NH2NH2, and further coupled with an antibody to obtain the final ADC product (32).

[0366] In certain specific embodiments, another synthetic route of ADC described in the present disclosure is as follows:

[0367] wherein: C, E, and k1 are as defined in the present disclosure; X represents -VLW-; and V, L, and W are as defined in the present disclosure.

[0368] Compound (33) and compound (5) were used as starting materials to prepare compound (34) under the action of DIC, which was then chlorinated with cyanuric chloride to obtain compound (35), which was then reacted with active compound C to obtain compound (36). The antibody was coupled with compound (37) to obtain conjugate (38), which was further coupled with the antibody via click chemistry to obtain the final ADC product (39).

[0369] In certain specific embodiments, the synthesis route of the albumin conjugates described in the present disclosure is as follows:

[0370] Wherein: X, C, and E are as defined in this disclosure,

[0371] Compound (9) is coupled with albumin to prepare conjugate (40). BRIEF DESCRIPTION OF THE DRAWINGS

[0372] Figure 1 shows the DAR and degree of polymerization test results of compound III-2.

[0373] Figure 2 shows the DAR and degree of polymerization test results of compounds III-1, 3, 4, 5 and VC-ADC.

[0374] FIG3 shows the results of the stability evaluation of Compound III-2 in plasma.

[0375] Figure 4 shows the results of the study on the binding affinity of compound III-2 to HER2 antigen. The results show (Figure 4) that compound III-2 and Herceptin have similar binding affinity to HER2 antigen, EC 50 The values ​​were 0.14 μg / mL and 0.042 μg / mL, respectively.

[0376] Figure 5 shows the results of a binding study of compound III-2 on cell lines. The results showed that both Herceptin and compound III-2 exhibited strong fluorescence in HER2-positive SKOV3 cells (right panel in Figure 5) and weak fluorescence in HER2-negative MCF-7 cells (left panel in Figure 5), indicating that compound III-2 specifically targets the HER2 antigen on tumor cells.

[0377] Figure 6 shows the results of the endocytosis study of compound III-2 in cell lines. The results showed that the internalization rate of compound III-2 was slightly higher than that of Herceptin, 34.1% vs. 22.1% at 8 hours and 41.7% vs. 31.1% at 24 hours.

[0378] Figure 7 shows the binding and endocytosis imaging results of compound III-2 in cell lines. The results show that Herceptin and compound III-2 were incubated with cells at 4°C. Under these 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 colocalized with the lysosomal signal (Pearson correlation coefficient = 0.87), indicating that compound III-2 was internalized and transported to lysosomes.

[0379] Figure 8 shows the results of the cell proliferation inhibitory activity study of compound III-2 on HER2 antigen positive and negative cell lines. The results showed that under hypoxic conditions, compound III-2 showed potent cell proliferation inhibitory activity on NCI-N87, BT-474 and SKBR3 cell lines, EC 50The 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).

[0380] Figure 9 shows the results of the hypoxia-dependent in vitro cytotoxicity study of compound III-2. The results show that compound III-2 exhibits oxygen concentration-dependent cell proliferation inhibitory activity, which increases with decreasing oxygen concentration. The EC values ​​of compound III-2 on SKOV3 cells were 0.1% and 2.0%, respectively. 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.

[0381] Figure 10 shows the results of the bystander effect study of compound III-2. 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.

[0382] Figure 11 shows the results of the study on the proliferation inhibition activity of compound III-2 on normal cell lines. 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%).

[0383] Figure 12 shows the results of a drug release study of compound III-2 under the action of azoreductase. The results show that the release of MMAE shows a dual dependence on azoreductase concentration and oxygen concentration. Under hypoxic conditions (0.1% O2), the release of MMAE increases 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 MMAE release amounts were 100%, 74.3% and 27.49%, respectively. Under normoxic conditions (20% O2), after incubation of substrate 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 disclosure can be cleaved and release toxins under the combined action of hypoxic conditions and azoreductase, while the release of toxins is very small when only azoreductase is used.

[0384] Figure 13 shows the results of a study on the effects of oxidizing, reducing, and salt substances on the stability of compound II-2. The results show that 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.

[0385] Figure 14 shows the results of a metabolite study of compound III-2 in cells. The results show that when 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 hypoxia. More importantly, when compound III-2 was co-cultured with SKOV3 cells under normoxic conditions, Cys-II-2 was the major metabolite, and its release was time-dependent. However, the amount of Cys-II-2 in cells under hypoxia was very low. This result indicates that under normoxic conditions, Cys-II-2 is released after the degradation of the antibody of compound III-2 and is not further cleaved by azoreductase.

[0386] Figure 15 shows the results of the cytotoxicity study of Cys-II-2. The results show that the cytotoxic activity of Cys-II-2 is reduced by more than ten to several hundred times compared to MMAE.

[0387] Figure 16 shows the results of the study on the inhibition of tubulin by Cys-II-2. The results show that the tubulin inhibitory activity of Cys-II-2 is lower than that of MMAE and is concentration-dependent.

[0388] Figure 17 shows the results of the cell cycle arrest study of compound III-2. The results showed that compound III-2 caused G2 / M phase arrest in 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%). In contrast, under hypoxia, after treatment with the same concentration of compound III-2, the proportion of cells in the G2 / M phase remained at 71.12%-87.22%.

[0389] Figure 18 shows the results of a study on the induction of apoptosis by compound III-2. The results show that under normoxic conditions (20% O2), compound III-2 has almost no effect on apoptosis. In contrast, under hypoxic conditions (0.1% O2), compound III-2 induces apoptosis in more than half of the cells. The apoptosis results indicate that compound III-2 has good hypoxia selectivity.

[0390] Figure 19 shows the distribution of compound III-2 labeled with DyLight-680 dye in mice. 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. Six hours after administration, a strong fluorescence signal could be clearly observed at the tumor site 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 shows that compound III-2 has 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.

[0391] Figure 20 shows the drug distribution and metabolism of compound III-2. The results show that high concentrations of the prodrug Cys-II-2 were detected in the blood and peripheral tissues 6 hours after administration of compound III-2. In addition, the results show that Cys-II-2 can be rapidly cleared from normal tissues, with a half-life of only 26 hours in the blood, which will further reduce its toxicity and side effects. After 96 hours of administration, Cys-II-2 is almost undetectable in the heart, liver, spleen, and lungs. The peak concentration and area under the curve of the toxin MMAE in tumors are 293 times and 941 times that of the peripheral blood, respectively, further confirming the excellent targeting of compound III-2. The area under the curve of MMAE in the heart, liver, spleen, and lungs account for 11%, 47%, 5%, and 7% of the area under the curve of the tumor, respectively. The results indicate that Cys-II-2 in peripheral tissues, except the liver, cannot be metabolized to MMAE, while Cys-II-2 in tumors can effectively release MMAE.

[0392] Figure 21 shows the anti-tumor efficacy of compound III-2 in NCI-N87 tumor-bearing mice. The results showed that in the NCI-N87 tumor xenograft model, compound III-2 significantly inhibited tumor growth at a 5 mg / kg dose compared to the control group, with a tumor inhibition rate of 90.97%. This result demonstrates that compound III-2 can achieve an anti-tumor effect nearly equivalent to that of traditional VC-ADCs. More importantly, tumors completely regressed in all mice in the 10 mg / kg dose group, demonstrating the excellent anti-tumor therapeutic potential of compound III-2.

[0393] Figure 22 shows the anti-tumor effect of III-2 in JIMT-1 tumor-bearing mice. The results showed that in the JIMT-1 xenograft model, at a dose of 12 mg / kg, compound III-2 still exhibited a tumor-suppressing effect. Although the tumor did not completely disappear, the inhibition rate reached 62.36%.

[0394] Figure 23 shows the anti-tumor effect of compound III-2 in combination with Herceptin, bevacizumab, sunitinib, or docetaxel in NCI-N87 tumor-bearing mice. The results showed that the compound III-2 combined with sunitinib group showed a stronger anti-tumor effect (p < 0.01), which was superior to compound III-2 and other combination groups. In the combined administration group, only 3 mg / kg of III-2 was able to achieve almost complete tumor regression, with a tumor inhibition rate of 98.93%. The above results indicate that the excellent tumor inhibition effect of combined administration may be due to 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.

[0395] Figure 24 shows the results of a maximum tolerated dose study of compound III-2. The results showed that the MTD of compound III-2 was >200 mg / kg, while mice in the VC-ADC and T-DM1 groups experienced sustained weight loss (>20%) and severe adverse reactions from the start of dosing. For example, all mice in the VC-ADC group died after dosing at 120 mg / kg.

[0396] Figure 25 shows the results of hematological and histopathological studies of Compound III-2 in BALB / C mice. The results showed no significant differences in biochemical, blood routine, and histopathological blood markers between the Compound III-2-treated and control groups. However, in the VC-ADC-treated group, changes in blood biochemistry reflected the adverse effects of the VC-ADC on the liver, such as increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK). The side effects of the VC-ADC were also reflected in hematological changes, including increases in lymphocyte count (LYMPH), monocyte count (MONO), and neutrophil count (NEUT).

[0397] FIG26 shows that compound III-16 has excellent anti-Staphylococcus aureus effect.

[0398] Figure 27 shows that compound III-16 has a bacteriostatic effect comparable to that of free Rifalogue.

[0399] FIG28 shows that compound III-16 has a significant therapeutic effect in a mouse model of bacterial infection. DETAILED DESCRIPTION

[0400] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0401] Example 1: Preparation of III-1

[0402] 1. Preparation of (4-nitrosophenyl)methanol

[0403] 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) with stirring. Then, slowly add 15 mL of NH₄Cl (3.14 g, 58.77 mmol) in water. After the addition is complete, stir at room temperature for 90 minutes until the p-nitrobenzyl alcohol reacts substantially completely. After filtering to remove insoluble solids, cool the filtrate to -5°C. Then, slowly add a solution of FeCl₃ (58.77 mmol) in 30 mL of methanol and 15 mL of water with stirring. After the addition is complete, continue stirring at low temperature for 1 hour. After the reaction is complete, filter to remove insoluble matter from the reaction solution. Add the same volume of saturated saline solution to the filtrate, and extract with DCM. Combine the organic phases and wash three more times with saturated saline solution. Dry the organic phase over anhydrous Na₂SO₄ and concentrate under reduced pressure to obtain the 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] + .

[0404] 2) Preparation of (E)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzoic acid

[0405] (4-Nitrosophenyl)methanol (2.7 g, 19.69 mmol) was dissolved in 50 mL of AcOH. After the solid was completely dissolved, p-aminobenzoic acid (2.7 g, 19.69 mmol) was added. The reaction was stirred at room temperature for 36 h, during which insoluble matter slowly precipitated. After the reaction was completed, the insoluble solid precipitated during the reaction was separated by filtration, and the filter cake was washed with DCM to obtain the desired 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] +.

[0406] 3. Preparation of tert-butyl(E)-(2-(4-((4-(hydroxymethyl)phenyl)diazenyl)benzamido)ethyl)carbamate

[0407] (E)-4-((4-(Hydroxymethyl)phenyl)diazenyl)benzoic acid (2.5 g, 9.76 mmol) was dissolved in 40 mL of DMF. After complete dissolution of the solid, 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 sequentially, and the mixture was reacted at room temperature for 5 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting residue was dispersed in DCM, and the insoluble material was separated by filtration. The filter cake was washed with a 1:1 v / v mixture of PE:EA to obtain the desired 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] + .

[0408] Preparation of (E)-N-(2-aminoethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide

[0409] 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. TFA (2 mL) was added dropwise with stirring. The mixture became clear after addition. The mixture was allowed to react at room temperature for 5 h. After completion of the reaction, the solvent was removed under reduced pressure. The resulting oil was re-dissolved in EA, and then PE was slowly added dropwise to precipitate the insoluble material. The mixture was sonicated and filtered, and the filter cake was washed with ether to obtain the desired 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] + .

[0410] 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

[0411] (E)-N-(2-aminoethyl)-4-((4-(hydroxymethyl)phenyl)diazenyl)benzamide (237.82 mg, 0.6 mmol) was dissolved in 10 mL of DMF. 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 with stirring. The mixture was stirred at room temperature for 24 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain an oil. Diethyl ether was added to the oil and sonicated 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] + .

[0412] 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)

[0413] (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 sequentially with stirring. The mixture was stirred at room temperature overnight. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting residue was dispersed with diethyl ether and sonicated. The precipitated insoluble solid was separated by filtration, and the filter cake was washed with diethyl ether several times to obtain the desired 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] + .

[0414] 7) Preparation of Compound II-1

[0415] (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 flask. Then, 5 mL of anhydrous DMF was added and stirred at room temperature for complete dissolution. After complete dissolution of the solid, DIPEA (18.1 μL, 0.14 mmol) was added dropwise, and the reaction was allowed to react at room temperature for 24 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the crude product, which was purified by column chromatography to obtain the desired product as a white powdery solid (63 mg, 75.4% yield). HR-MS(ESI)m / z:1193.6604[M+H] + ;1215.6453[M+Na] + .

[0416] 8) Synthesis of Compound III-1

[0417] The method described in the literature (Int J Mol Sci. 2017, 18(9): e1860.) was used to couple compound II-1 with the anti-HER2 antibody trastuzumab (purchased from Roche Pharmaceuticals) to obtain the target antibody-drug conjugate III-1, wherein: (MAB) indicates the anti-HER2 antibody trastuzumab with a drug-antibody conjugate ratio (DAR) E of approximately 4.

[0418] Example 2: Preparation of Compounds III-2, III-3, III-4, and III-5

[0419] 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:

[0420] Compounds I-2, I-3, I-4, and I-5 were prepared.

[0421] 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 N6O8 609.27,found 609.24; ESI-MS m / z(M+Na) + calculated for C 30 H 36 N6NaO8 631.25,found 631.23.

[0422] I-3:1 H-NMR(400MHz,DMSO-d6):δ8.69(t,J=5.5Hz,1H),8.05(m,4H),7.92(m,4H),

[0423] 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-MS m / z(M+H) + calculated for C 38 H 53 N6O 12 785.37,found 785.36;ESI-MS m / z(M+Na) + calculated for C 38 H 52 N6NaO 12 807.35,found 807.35.

[0424] 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] + .

[0425] 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] + .

[0426] 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, and I-11.

[0427] 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] + .

[0428] 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] + .

[0429] 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] + .

[0430] 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] + .

[0431] 3) Referring to the preparation method of compound II-1 in Example 1, compound I-8, I-9, I-10, I-11 were used to replace compound I-7 to prepare compounds II-2, II-3, II-4, and II-5.

[0432] II-2: 1 H NMR (600MHz, CDCl3-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 for C 70 H 102 N 11 O 16 1352.7506,found 1352.7502;ESI-HRMS m / z(M+Na) + calculated for C 70 H 101 N 11 NaO 16 1374.7325,found 1374.7327.

[0433] II-3: 1 H NMR(600MHz,CDCl3-d6)11.01(br,1H),8.04(br,3H),7.91(d,J=6.0Hz,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.

[0434] II-4: 1H NMR(600MHz,CDCl3-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 20 1528.8555,found 1528.8539;ESI-HRMS m / z(M+Na) + calculated for C 78 H 117 N 11 NaO 20 1550.8374,found 1550.8363.

[0435] II-5: 1 H NMR(600MHz,CDCl3-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),4.80-4.68(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 O22 1616.9079, found 1616.9067; ESI-HRMS m / z(M+Na) + calculated for C 82 H 125 N 11 NaO 22 1638.8898,found 1638.8886.

[0436] 4) Referring to the preparation method of compound III-1 in Example 1, compound II-2, II-3, II-4, and II-5 were used to replace compound II-1 to prepare compound III-2, III-3, III-4, and III-5.

[0437] 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.

[0438] Example 3: Preparation of Compound III-6

[0439] 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

[0440] (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 resulting residue was purified by silica gel column chromatography to obtain the target product as an orange-yellow solid powder (730 mg, 79.25% yield). 1 H-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] + .

[0441] 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

[0442] (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, and piperidine (0.8 mL) was added. The mixture was reacted at room temperature for 1 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the target product as an orange-yellow viscous oil (328 mg, 92.8% yield). 1 H-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] + .

[0443] 3) Preparation of Compound I-6

[0444] (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. 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (190 mg, 0.73 mmol) was added with stirring. The mixture was stirred at room temperature for 24 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain an orange-yellow solid. The crude residue was further purified by silica gel column chromatography to obtain the desired product as an orange-yellow oil (330 mg, 69.3% yield). 1H-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] + .

[0445] 4) Preparation of Compound I-12

[0446] 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 sequentially with stirring. The mixture was stirred at room temperature for 36 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting orange-yellow oily residue was purified by silica gel column chromatography to obtain the desired 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] + .

[0447] 5) Preparation of Compound II-6

[0448] 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 flask. 10 mL of anhydrous DMF was then added and stirred at room temperature to completely dissolve the mixture. After the solid was completely dissolved, DIPEA (18.1 μL, 0.14 mmol) was added dropwise and allowed to react at room temperature for 24 hours. After the reaction was complete, the solvent was removed by concentration under reduced pressure to obtain the crude product, which was then purified by column chromatography to obtain the desired product as a white powdery solid (92 mg, 76.6% yield). HR-MS (ESI) m / z: 1715.9727 [M+H] + ;1738.9583[M+Na] + .

[0449] 6) Synthesis of Compound III-6

[0450] 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 conjugate ratio (DAR) E of approximately 4.

[0451] Example 4: Preparation of Compounds III-7, III-8, III-9, III-10, III-11, III-12, and III-13

[0452] 1) Preparation of compounds II-7, II-8, II-9, II-10, II-11, II-12, and II-13

[0453] 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.

[0454] II-7: 1H NMR(600MHz,DMSO-d6):δ7.996(d,J=8.4Hz,2H),7.925-7.828(m,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 FN8O 20 1291.53,found 1291.54.

[0455] II-8: 1 H NMR(600MHz,DMSO-d6):δ8.706(br,1H),8.211(d,J=8.4Hz,1H),8.064-8.037(m,4H),7.944(d,J=7.2Hz,4H),7.753(d,J=10.8Hz,1H),7.658(d,J=7.8Hz,2H),7.305(s,1H),6.992(s,2H),5.435(dd,J=22.2Hz,16.8Hz,2H),5.31-5.221(m,5H),3.620-3.574(m,5H),3.463(d,J=2.4Hz,4H),3.355-3.336(m,4H),3.258(dd,J=11.4Hz,5.4Hz,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=7.2Hz,3H).ESI-HRMS m / z(M+H) + calculated for C 55 H 57 FN9O13 1070.3982,found 1070.4094.

[0456] II-9: 1 HNMR(600MHz,DMSO-d6):δ13.991(s,1H),13.231(s,1H),8.674(s,1H),8.027(d,J=7.8Hz,4H),7.905(d,J=7.8Hz,2H),7.857(t,J=7.2Hz,4H),7.571-7.526(m,3H),7.040(d,J=7.8Hz,1H),6.991(s,2H),5.442(s,1H),5.229(s,1H),5.078(dd,J=28.2Hz,13.2Hz,2H),4.891(d,J=28.2Hz,2H),4.758(d,J=5.4Hz,1H),4.590(d,J=4.8Hz,2H),4.185(d,J=6.0Hz,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-HRMSm / z(M+H) + calculated for C 58 H 64 N7O 20 1178.4128,found 1178.4195.

[0457] 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,found 1215.3850.

[0458] II-11: 1 H 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),6.156(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.

[0459] 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.

[0460] II-12: 1 H NMR (600MHz, DMSO-d6): δ7.996(d,J=8.4Hz,2H),7.925-7.828(m,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),2.5 08-2.476(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 FN8O 20 1291.53,found 1291.54.

[0461] II-13: 1H NMR (600MHz, DMSO-d6): δ7.996(d,J=8.4Hz,2H),7.925-7.828(m,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),2.5 08-2.476(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 FN9O 19 1334.56,found 1334.56.

[0462] 2) Preparation of compounds III-7, III-8, III-9, III-10, III-11, III-12, and III-13

[0463] Referring to the preparation method of compound III-1 in Example 1, compound 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.

[0464] 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.

[0465] Example 5: Preparation of Compound III-14

[0466] 1) Preparation of 4-nitrosobenzoic acid

[0467] Potassium peroxymonosulfonate (17.93 g, 29.17 mmol) was dissolved in 150 mL of water. A solution of p-aminobenzoic acid (2.0 g, 14.58 mmol) in DCM (20 mL) was slowly added with stirring. The suspension was stirred at room temperature for 3 hours, during which time the reaction system gradually changed from white to yellow. After completion of the reaction, the mixture was filtered, and the yellow filter cake was washed several times with pure water and dried under reduced pressure to obtain the desired 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).

[0468] 2. Preparation of (E)-4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzoic acid

[0469] 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 proceeded, an orange-yellow precipitate gradually formed, and the color of the reaction mixture changed from green to orange-yellow. After the reaction was completed, the insoluble material was filtered off, and the filter cake was washed with AcOH and DCM. Purification was performed by beating with DCM to obtain the desired product as an orange-yellow powdery 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] + .

[0470] 3) Preparation of tert-butyl(E)-(2-(4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzamido)ethyl)carbamate

[0471] (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. EDCI (373.8 mg, 1.95 mmol), HOBT (176 mg, 1.30 mmol), and DIPEA (226 μL, 1.3 mmol) were added sequentially. The mixture was allowed to react at room temperature for 45 minutes, followed by the addition of mono-Boc-ethylenediamine (312 mg, 1.95 mmol). The reaction was stirred at room temperature overnight. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the desired product as an orange-yellow solid powder (400 mg, 68.3% yield). 1 H-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] + .

[0472] 4) Preparation of (E)-N-(2-aminoethyl)-4-((4-methyl-2-oxo-2H-chromen-7-yl)diazenyl)benzamide

[0473] 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. TFA (1 mL) was added dropwise with stirring. The mixture became clear after the addition. The mixture was allowed to react at room temperature for 1 h. After completion of the reaction, the solvent was removed under reduced pressure to obtain an orange-red solid residue. The desired product was purified by silica gel column chromatography to obtain an orange solid powder (140 mg, 94.8% yield). 1H-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] + .

[0474] 5) Preparation of Compound II-14

[0475] (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 with stirring. (131.21 mg, 0.308 mmol) and DIPEA (39.8 mg, 0.308 mmol) were stirred at room temperature overnight. After the reaction was complete, the solvent was removed by concentration under reduced pressure to obtain a crude oil. Further purification by silica gel column chromatography afforded the desired product as 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] + .

[0476] 6) Preparation of Compound III-14

[0477] 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.

[0478] The DAR value E of compound III-14 is about 4, It is the anti-HER2 antibody trastuzumab.

[0479] Example 6: Preparation of Compound III-15

[0480] 1) Preparation of tert-butyl 3-(2-(2-(2-((1,3-dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)propanoate

[0481] 2-Hydroxy-isoindole-1,3-dione (1.27 g, 7.81 mmol) was added to a 100 mL eggplant-shaped flask. 30 mL of THF was added and the mixture was cooled to 0°C in an ice bath. After the 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 mixture. The reaction mixture was transferred to room temperature and continued for 1 hour. The solvent was removed by concentrating under pressure, and the sample was mixed and purified by column chromatography to obtain the desired product 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] + .

[0482] 2) Preparation of 3-(2-(2-(2-((1,3-dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)propanoic acid

[0483] Tert-butyl 3-(2-(2-(2-((1,3-dioxoisoindolin-2-yl)oxy)ethoxy)ethoxy)ethoxy)propanoate (1.136 g, 2.68 mmol) was added to a 50 mL eggplant-shaped flask. 10 mL of 4N HCl in dioxane was added and stirred at room temperature. After reacting for 1 hour, the solvent was removed by pressure concentration. The sample was stirred and purified by column chromatography to obtain the title compound as a white solid (0.41 g, 42% yield). 1 H-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] - .

[0484] 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)

[0485] 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 flask, and 4 mL of anhydrous DMF was added. After complete dissolution, NHS (0.055 g, 0.48 mmol) and DIC (0.076 g, 0.6 mmol) were added to the reaction solution, 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 reaction was allowed to react at room temperature for 15 min. The solvent was removed by pressure concentration, and the desired product was purified by column chromatography to obtain a yellow solid (0.071 g, 25%), which was used directly in the next step.

[0486] 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)

[0487] (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 flask, and 3 mL of anhydrous DMF was added. After the reaction starting materials were completely dissolved, di(p-nitrophenyl) carbonate and DIPEA were added sequentially, and the mixture was allowed to react at room temperature for 4 h. The solvent was removed by vacuum concentration, and the crude product was mixed and purified by column chromatography to obtain the desired product as a yellow solid (0.08 g, 90% yield), which was used directly in the next reaction.

[0488] 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)

[0489] (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 flask, and 2 mL of anhydrous DMF was added. After the starting compound was completely dissolved, the MMAF derivative, HOBT, and DIPEA were added to the reaction mixture in sequence, and the mixture was 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 used directly in the next reaction.

[0490] Synthesis steps of MMAF derivatives:

[0491] 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

[0492] MMAF (300 mg, 0.39 mmol) was dissolved in 5 mL of DMF. 9-Fluorenylmethyl chloroformate (50 mg, 0.195 mmol) and sodium bicarbonate (13 mg, 0.156 mmol) were added sequentially with stirring. The mixture was stirred at room temperature overnight. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The desired product was purified by column chromatography as a white solid powder (200 mg, 57% yield). MS (ESI) m / z: 953.55 [M+H] + .

[0493] 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

[0494] ((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. DIPEA (27 mg, 0.21 mmol) was added to the solution with stirring. The mixture was stirred at room temperature for 2 h. After completion of the reaction, the solvent was removed by concentration under pressure. 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] + .

[0495] 3) Preparation of MMAF derivatives

[0496] (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 while stirring. The mixture was stirred for 20 minutes before the reaction was complete. The solvent was removed by concentration under pressure and the target product was purified by column chromatography to obtain a white solid powder (39 mg, 56% yield). MS (ESI) m / z: 788.54 [M+H] + .

[0497] 6) Preparation of Compound II-15

[0498] Compound I-15 (20 mg, 0.014 mmol) was added to a 10 mL eggplant-shaped flask, and 1 mL of anhydrous DCM was added. The reaction solution was cooled to 0°C in an ice bath. Hydrazine hydrate (0.001 g, 0.021 mL) was diluted with 0.5 mL of anhydrous DCM and slowly added dropwise to the reaction solution. The reaction was allowed to react overnight, resulting in the precipitation of an insoluble solid. The reaction solution was extracted with DCM several times and washed with saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate overnight, and concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative thin-layer chromatography to yield the desired 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.

[0499] 7) Synthesis of Compound III-15

[0500] The method described in the literature (PNAS.111(5):1766-71.) was used to couple the II-15 compound with an anti-HER2 site-directed mutagenesis antibody to obtain the target antibody-conjugated drug III-15, wherein the anti-HER2 site-directed mutagenesis antibody contains an unnatural amino acid acetylphenylalanine (pAF). 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.

[0501] The sequence of the anti-HER2 site-directed mutagenesis antibody (Mol Cancer Ther (2020) 19 (9): 1833-1843.) is as follows:

[0502] Heavy chain:

[0503] Light chain:

[0504] In the sequence, "U" stands for acetylphenylalanine.

[0505] Example 7: Preparation of III-16

[0506] 1) Preparation of Compound I-16

[0507] Cyanuric chloride was dissolved in 200 μL of anhydrous DMF and stirred at room temperature for 2 hours, resulting in the formation of a large amount of white precipitate. (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) was dissolved in 2 mL of anhydrous DCM and added to the reaction flask. The mixture was stirred at room temperature for 2 days. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the crude product, which was purified by column chromatography to obtain the desired product as a yellow solid (30 mg, 30% yield). This product was used directly in the next reaction.

[0508] 2) Preparation of Compound II-16

[0509] Compound I-16 (25 mg, 0.034 mmol) and the effector molecule Rifalogue (28 mg, 0.03 mmol) were added to a 1.5 mL EP tube and dissolved in 400 μL of anhydrous DMF. 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 completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the crude product, which was purified by column chromatography to obtain the desired product as a black solid (10 mg, 20% yield). HR-MS (ESI) m / z: 1626.792 [M+H] + .

[0510] 3) Preparation of antibacterial antibodies modified with azide groups

[0511] The antibacterial antibody BD37 (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 -3After stirring overnight at room temperature, the coupled product was replaced in PBS to prepare the azide-modified antibacterial antibody BD37, wherein the drug-antibody coupling ratio (DAR) E was about 4. It is the antibacterial antibody BD37.

[0512] The sequence of the antibacterial antibody BD37 is:

[0513] Heavy chain:

[0514] Light chain:

[0515] 4) Preparation of Compound III-16

[0516] The antibacterial antibody BD37 (2.5 mg, 1.67 × 10 -5 mmol) was added to a 2 mL EP tube, and compound II-16 (0.41 mg, 2.5×10 -4 mmol) of DMA solution. Then, CuSO4 (0.13 mg, 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, stirred at room temperature for 5 hours, and then the coupling product was replaced with PBS to prepare the target antibody-drug conjugate III-16, wherein the drug-antibody coupling ratio (DAR) E was about 2, It is the antibacterial antibody BD37.

[0517] Example 8: Preparation of Compound III-17

[0518] 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 with PBS to prepare the target antibody conjugate III-17, wherein the drug-antibody coupling ratio (DAR) E was 1. Stands for albumin.

[0519] Example 9: Preparation of Compound III-18

[0520] 1) Preparation of (E)-1-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)but-3-yn-1-ol

[0521] The compound 1-(4-aminophenyl)but-3-yn-1-ol (0.3 g, 1.86 mmol) was added to a 50 mL eggplant-shaped flask. 12 mL of concentrated hydrochloric acid and 6 mL of water were added. The mixture was cooled to 0°C in an ice bath. NaNO2 (0.152 g, 2.2 mmol) was added and stirred for 1 hour. N,N-dimethylaniline (0.27 g, 2.2 mmol) was dissolved in 2 mL of glacial acetic acid and added to the reaction mixture. The reaction mixture was allowed to react overnight. The solvent was removed by pressure concentration, the sample was mixed, and purified by column chromatography to obtain a brownish-red solid (0.44 g, 80% yield). ESI-MS m / z (M+H) + calculated for C 18 H 19 N3O 294.15,found 294.16.

[0522] 2) Preparation of (E)-1-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate

[0523] (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 flask, and 3 mL of anhydrous DMF was added. After the reaction starting materials were completely dissolved, di(p-nitrophenyl) carbonate and DIPEA were added sequentially, and the mixture was allowed to react at room temperature for 4 h. The solvent was removed by vacuum concentration, and the crude product was mixed and purified by column chromatography to obtain the desired product as a yellow solid (0.42 g, 67% yield). ESI-MS m / z (M+H) + calculated for C 25 H 22 N4O5 459.16,found 459.17.

[0524] 3) Preparation of Compound II-17

[0525] 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 flask. 10 mL of anhydrous DMF was then added and stirred at room temperature for complete dissolution. After complete dissolution of the solid, DIPEA (50 μL, 0.288 mmol) was added dropwise, and the mixture was allowed to react at room temperature for 24 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the crude product, which was then purified by column chromatography to obtain the desired product as a white powdery solid (200 mg, 80% yield). HR-MS (ESI) m / z: 1037.64 [M+H] + .

[0526] 4) Preparation of the Azide-Modified Anti-HER2 Antibody Trastuzumab

[0527] 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 coupled product was replaced in PBS to prepare the azide-modified anti-HER2 antibody trastuzumab, wherein the drug-antibody coupling ratio (DAR) E was about 4. It is the anti-HER2 antibody trastuzumab.

[0528] 5) Preparation of Compound III-18

[0529] The anti-HER2 antibody trastuzumab modified with an azide group (2.5 mg, 1.67×10 -5 mmol) was added to a 2 mL EP tube, and compound II-17 (0.26 mg, 2.5×10 -4 mmol) of DMA solution. Then, CuSO4 (0.13 mg, 5.01×10 -4 mmol), THPTA (0.87mg, 2.0×10 -3mmol) and sodium ascorbate (1.49 mg, 7.51×10 -3 mmol) aqueous solution was added to the reaction system, stirred at room temperature for 5 hours, and then the coupling product was replaced with 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.

[0530] Example 10: Quality Control Study of ADC Represented by Compound III-2

[0531] This example evaluates the DAR and degree of polymerization of the ADC shown in compound III-2 (Figure 1). The main peaks displayed by hydrophobic interaction chromatography of compound III-2 represent 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 a monomer (purity>95%). Liquid chromatography-mass spectrometry (UPLC-Q-TOF-MSLC-MS) results also verified that each antibody molecule of compound III-2 is coupled with 4.0 small molecule drugs.

[0532] Example 11: Quality Control Study of Compounds III-1, III-3, III-4, III-5 and VC-ADC

[0533] This example evaluated the DAR and degree of polymerization (DOP) of III-1, III-3, III-4, III-5, and Herceptin-valine-citruline-MMAE (VC-ADC) (Figure 2). Hydrophobic interaction chromatography (HIC) results showed that the DARs of III-1, III-3, III-4, III-5, and VC-ADC were approximately 4.0. Size exclusion chromatography (SEC) revealed that III-1, III-3, III-4, III-5, and VC-ADC were almost entirely monomeric (>95% purity).

[0534] Example 12: Plasma stability study of compound III-2

[0535] 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 the sampling was completed, 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 show (Figure 3) that compound III-2 exhibits excellent plasma stability, with the total amount of MMAE released in human plasma within 7 days being <1%.

[0536] Example 13: Antigen binding study of compound III-2

[0537] 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 humidified chamber 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 H2SO4 was added to stop color development. The absorbance of each well at 450 nM was monitored on a microplate reader. The results showed (Figure 4) that compound III-2 and Herceptin had comparable binding affinities to HER2 antigen, and EC 50 The values ​​were 0.14 μg / mL and 0.042 μg / mL, respectively.

[0538] Example 14: Study on the binding ability of compound III-2 on cell lines

[0539] This example evaluated 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 use. Compound III-2 (5 μg / mL), Herceptin (5 μg / mL), or a blank control PBS solution were added to the cells and incubated at 4°C for 30 minutes. The cells were then washed twice with FACS solution and incubated with a PE-labeled goat anti-human IgG Fc secondary antibody for 30 minutes at 4°C. After washing three times with FACS solution, the samples were detected on a FACSCalibur. The results (Figure 5) show that both Herceptin and Compound III-2 exhibited strong fluorescence in HER2-positive SKOV3 cells (right panel in Figure 5) and weak fluorescence in HER2-negative MCF-7 cells (left panel in Figure 5), indicating that Compound III-2 specifically targets the HER2 antigen on tumor cells.

[0540] Example 15: Internalization study of compound III-2 in cell lines

[0541] This example evaluated the internalization properties of compound III-2 in 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. Compound III-2- and Herceptin-treated samples were then divided into control and experimental groups. After washing twice with pre-chilled FACS solution, the experimental groups were transferred to a 37°C incubator and incubated for 8 or 24 hours, while the control group was incubated at 4°C for the same time points. All samples were collected at the designated time points, washed three times with FACS solution, and then incubated with a PE-conjugated 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 measured by flow cytometry. The internalization rate was calculated using the rMFI formula: internalization percentage (%) = [100 - (rMFI of experimental group / rMFI of control group)] × 100%. The results showed ( FIG. 6 ) that the internalization rate of compound III-2 was slightly higher than that of Herceptin, 34.1% versus 22.1% at 8 h and 41.7% versus 31.1% at 24 h.

[0542] Example 16: Compound III-2 binding and endocytosis imaging studies

[0543] This example evaluated the binding and endocytosis properties of Compound III-2 in the SKOV3 cell line.

[0544] Compound III-2 was fluorescently labeled with FITC NHS ester (purchased from Innochem) according to the manufacturer's instructions. Specifically, compound III-2 was reacted with the fluorescent dye (FITC NHS ester) in NaHCO3 buffer (pH = 8.3) for 3 hours with stirring to label the antibody with the dye. The resulting FITC-III-2 was then replaced with PBS buffer for later use.

[0545] SKOV3 cells were seeded in eight-chamber 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 both 4°C and 37°C. SKOV3 cells incubated at 4°C were used for compound III-2 binding assays, while cells incubated at 37°C were used to study compound III-2 internalization. At the designated time points, cells were washed with PBS and stained for nuclei with DAPI and lysosomes with LysoTracker Red. After two PBS washes, fluorescence imaging was performed on a confocal laser scanning microscope. The results (Figure 7) show that Herceptin and compound III-2 bind to the antigen on the cell membrane under these conditions when incubated with cells at 4°C, but are not internalized. After 24 hours of incubation at 37°C, the intracellular protein signal colocalizes with the lysosomal signal (Pearson correlation coefficient = 0.87), indicating that compound III-2 is internalized and transported to lysosomes.

[0546] Example 17: In vitro cytotoxicity study of compound III-2

[0547] This example evaluates the in vitro cytotoxicity of test drugs, including compound III-2, trastuzumab (Herceptin), herceptin-valine-citruline-MMAE (VC-ADC), and the toxin MMAE. The cell lines tested include the HER2 antigen-positive breast cancer cell line BT474, the herceptin-resistant BT474 cell line (BT474-HDR), the HER2 antigen-positive ovarian cancer cell line SKOV3, the HER2 antigen-positive gastric cancer cell line NCI-N87, the HER2 antigen-weakly positive breast cancer cell line MCF-7, the HER2 antigen-negative breast cancer cell line MDA-MB-231, mouse embryonic fibroblasts NIH3T3, human renal epithelial cells 293T, and human normal liver cells LO2 (all purchased from ATCC).

[0548] The reagents, instruments and consumables used in the experiment are described in the following table:

[0549] The test process is as follows:

[0550] ① Cell thawing

[0551] Thaw the vial containing target cells by gentle agitation in a 37°C water bath;

[0552] After the contents were thawed, the vials were removed from the water bath and decontaminated by immersion or spraying with 70% ethanol;

[0553] The contents of the vial were transferred to a centrifuge tube containing 9 mL of complete medium DMEM and centrifuged (200 g; 5 min);

[0554] Resuspend the cells in culture medium, pellet and distribute them onto a 75 cm 2 in culture flasks;

[0555] Incubate the culture at 37°C with 5% CO2 The cells were cultured in a CO2 incubator (48R, #CO48312044) with an oxygen concentration of 20% and a carbon dioxide concentration of 5%.

[0556] ② Expand cells

[0557] The cells were passaged three times a week in a culture medium containing 10% FBS (heat inactivated) and 1% penicillin / streptomycin solution (Penicillin-Streptomycin) at a ratio of 1:4;

[0558] For passaged cells, first rinse adherent cells with a trypsin / EDTA solution (3 mL). Then, add trypsin / EDTA (3 mL, T75 flask) and swirl to evenly coat the cells. Incubate the culture at 37°C until the cells detach. Verify detachment under a microscope, inactivate the trypsin by adding an equal volume of cell culture medium, collect the detached cells, centrifuge at 200 g for 5 minutes, and resuspend in fresh culture medium.

[0559] ③Cell inoculation

[0560] Collect cells and count the cell number;

[0561] 100 μL of cell suspension with adjusted density was added to the designated 96-well cell culture plate. The final cell density was approximately 5,000-10,000 cells / well.

[0562] The cells were covered with a lid and placed in a 37°C 5% CO2 incubator for incubation for 24 h with an oxygen concentration of 20%.

[0563] ④Prepare compounds

[0564] The compound solutions were serially diluted at a ratio of 1:3, with the initial maximum concentrations of test drug III-2 and VC-ADC being approximately 400 μg / mL, and the initial concentration of MMAE being 400 nM;

[0565] ④ Co-incubation of drugs and cells

[0566] Remove the culture medium from the 96-well plates seeded with cells, add 150 μL of fresh culture medium to each well, and then add 50 μL of the prepared test drug to the 96-well plate. Incubate the drug and cells in a 37°C, 5% CO2, 0.1% O2 incubator for 24 hours. After that, wash away the drug with fresh culture medium, and incubate the cells in a 37°C, 5% CO2, 20% O2 incubator for an additional 72 or 120 hours.

[0567] ⑤Read the plate

[0568] Remove the plate from the incubator and equilibrate at room temperature for 15 minutes;

[0569] CellTiter Glo reagent was incubated at 37°C before the experiment;

[0570] Dilute the CellTiter-Glo reagent and PBS at a volume ratio of 3:7 to prepare the detection solution, and add the diluted detection solution to the wells to be tested;

[0571] The plate was then placed at room temperature with shaking for 8 min, allowed to rest for 2 min, and then read on an EnSpire reader for signal detection.

[0572] ⑥Data analysis

[0573] The survival percentage is expressed as follows:

[0574] %Viability=100×(Sample / HC)

[0575] HC refers to cells not treated with the test drug.

[0576] The cytotoxicity results showed (Figure 8, Table 1) that all cell lines were highly sensitive to MMAE, with EC 50 Compound III-2 showed potent cell proliferation inhibitory activity on NCI-N87, BT-474 and SKBR3 cell lines under hypoxic conditions, EC 50 The values ​​were 0.07 nM, 0.026 nM and 0.015 nM respectively (Figure 8), while compound III-2 had lower inhibitory activity against HER2-negative cell lines (MCF-7, MDA-MB-231) (EC 50The above results confirmed that under hypoxic conditions, compound III-2 has the same proliferation inhibitory activity as the toxin MMAE only on HER2-positive cell lines, but has weaker activity on HER2-negative cell lines.

[0577] Table 1 Cell proliferation inhibitory activity of compound III-2 under hypoxic conditions (0.1% O2)

[0578] Example 18: Hypoxia-dependent cytotoxicity study of compound III-2

[0579] In this example, hypoxia-activated compound III-2 was tested for its hypoxia-dependent in vitro cytotoxicity. The cell lines tested were the Herceptin-resistant, HER2-positive breast cancer cell line BT474-HDR and the HER2-positive ovarian cancer cell line SKOV3. The culture process was as described in Example 14, with oxygen concentrations in the cell culture incubator set to 0.1%, 1.0%, 5.0%, and 20.0%, respectively. Cytotoxicity results (Figure 9, Table 2) show that the cytotoxic activity of compound III-2 increases with decreasing oxygen concentration. The EC values ​​of compound III-2 on SKOV3 cells were significantly higher at oxygen concentrations of 20%, 5%, 1%, and 0.1%. 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. This result indicates that compound III-2 has potent anti-proliferation activity under hypoxic conditions (O2 <1%), but weak activity under normoxic conditions. In contrast, the activity of traditional VC-ADCs did not show oxygen concentration dependence, and they had potent anti-proliferation activity under both normoxic and hypoxic conditions. This result indicates that traditional ADCs have the risk of off-target toxicity (Figure 9, Table 2).

[0580] Table 2 Oxygen concentration-dependent cytotoxicity of compound III-2 and VC-ADC

[0581] Example 19: Study on the Bystander Effect of Compound III-2

[0582] In this example, a bystander effect study was conducted on the hypoxia-activated type III-2. The HER2 antigen-positive ovarian cancer cell line SKOV3 and the HER2 antigen-weakly positive breast cancer cell line MCF-7 were seeded in a 1:1 cell density mixture in a well plate. 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 show (Figure 10, Table 3) that 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 (Figure 3). Under hypoxic conditions, compound III-2 maintains high activity in the co-culture model, EC 50 =0.053 nM. This result indicates 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).

[0583] Table 3 Cell proliferation inhibitory activity of compound III-2 in co-culture cell model

[0584] Example 20: Cytotoxicity study of compound III-2 on normal cells

[0585] In this example, the cytotoxicity of the hypoxia-activated compound III-2 on normal cells was studied. The culture process of the tested cell lines, mouse embryonic fibroblasts NIH3T3, human renal epithelial cells 293T, and human normal liver cells LO2, was as described in Example 14. At the highest tested concentration (666nM) of normal cells NIH3T3 and 293T, compound III-2 showed only weak inhibitory activity, with a maximum inhibition rate of <50% (Figure 11, Table 4). However, traditional VC-ADCs still show high off-target toxicity (EC 50 <10 nM, maximum inhibition rate >90%, Figure 11, Table 4).

[0586] Table 4 Cell proliferation inhibitory activity of compound III-2 on normal cell lines

[0587] Example 21: MMAE Release Study of Compound II-2

[0588] This example evaluated 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 of MPA-II-2 was carried out according to the literature (Y. Wang, S. Fan, W. Zhong, X. Zhou, S. Li, Int. J. Mol. Sci. 2017, 18, e1860). Under 0.1% O₂, 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. The normoxic group (20% O2) was treated with human NADPH-CYP reductase (10 μL, 18.8 mg / mL), and other factors were the same as the hypoxic 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.

[0589] The results (Figure 12) show that the amount of MMAE released is dually dependent on the azoreductase concentration and oxygen concentration. Under hypoxic conditions (0.1% O2), the amount of MMAE released increases with increasing azoreductase concentration. After incubation of the substrate with azoreductase (0.4 mg / mL, 0.1 mg / mL, and 0.05 mg / mL) for 24 hours, the MMAE release amounts were 100%, 74.3%, and 27.49%, respectively. Under normoxic conditions (20% O2), only 14.99% of MMAE was released after incubation of the substrate with 0.1 mg / mL azoreductase for 24 hours. This result indicates that the azoreductase-cleavable linker provided by the present disclosure can be cleaved and release the toxin under the combined action of hypoxia and azoreductase, while the toxin release amount is very small when the azoreductase alone is activated (Figure 12).

[0590] Example 22: Effects of Oxidizing Properties, Reducing Properties, and Salts on the Stability of Compound II-2

[0591] This example evaluates the effects of oxidizing, reducing, and salt properties 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 the reference (Y. Wang, S. Fan, W. Zhong, X. Zhou, S. Li, Int. J. Mo. l Sci. 2017, 18, e1860.). NAC-II-2 was incubated with Ary, Cys, NaCl, KCl, MgCl2, CaCl2, H2O2, NaClO, Arg, Glu, Ser, Vc, Glucose, and NADPH in a 20% O2 environment for 24 hours, while NAC-II-2 was incubated with azoreductase (NTR) in a 0.1% O2 environment for 24 hours. All samples were incubated for 24 hours and quenched with methanol, and the amount of MMAE released was measured by LC-MS / MS. The results (Figure 13) show that 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.

[0592] Example 23: Study on the metabolites of compound III-2 in cells

[0593] This example evaluated the metabolite forms of compound III-2 in cells under normoxic (20% O2) and hypoxic (0.1% O2) conditions. 5SKOV3 cells (100 cells / well) were seeded in 12-well plates and 100 nM of compound III-2 was added to the cells. Compound III-2 and cells were co-incubated under 20% or 0.1% O2 conditions for 6, 17, or 24 hours. At the designated time points, cells were harvested and washed three times with cold PBS. Methanol was added to the samples, and the cells were incubated at -20°C for 2 hours to completely disrupt the cells. The samples were then centrifuged at 13,000 g for 20 minutes. 50 μL of supernatant was collected from each sample, dried under vacuum, and reconstituted with 100 μL of methanol. LC-MS / MS was used to analyze the MMAE and Cys-II-2 content in the samples. The results showed that MMAE was detected in SKOV3 cells when compound III-2 was co-cultured with cells under hypoxia, and the amount of MMAE released was time-dependent, directly demonstrating that compound III-2 can be cleaved and released from tumor cells under hypoxic conditions (Figure 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 indicates 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 (Figure 14).

[0594] Example 24: Cytotoxicity study of Cys-II-2

[0595] 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. After BT-474 and SKBR3 cells were seeded in 96-well plates, they were incubated in an incubator for 24 hours. Equally diluted MMAE and Cys-II-2 were added to the cells, respectively. After the cells and drugs were incubated for 24 hours in an oxygen content of 20% O2 environment, the drugs were washed away with culture medium, and SKBR3 continued to be cultured in an incubator for 3 days, and BT474 continued to be cultured for 5 days. Cell viability was detected using the CellTiter-Glo assay kit. The results show (Figure 15) that the cytotoxic activity of Cys-II-2 was reduced by more than ten to hundreds of times compared to MMAE.

[0596] Example 25: Study on the Inhibition of Tubulin by Cys-II-2

[0597] Tubulin inhibition experiments were performed using a tubulin polymerization assay kit (BK011P, Cytoskeleton, USA). 5 μL of MMAE, Cys-II-2, or DMSO at different concentrations was 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 a microplate reader. The results showed (Figure 16) that the tubulin inhibitory activity of Cys-II-2 was reduced compared to that of MMAE and was concentration-dependent.

[0598] Example 26: Cell cycle arrest study of compound III-2

[0599] 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. After the hypoxia group was cultured in a 0.1% O2 environment for 1 day, the cells were transferred to a 20% O2 environment and cultured for another day. The normoxic group was cultured in a 20% O2 environment for 2 days after administration. All cells in each well were collected into a 1.5 mL EP tube and fixed with 70% cold ethanol. Before sending the sample, after washing once with cold PBS, the sample was stained with propidium iodide solution in the dark for 30 minutes at 37 ° C and then analyzed using FACSCalibur. The cycle arrest results showed (Figure 17) 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 0.3 nM, 1 nM and 3 nM, 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%.

[0600] Example 27: Study on the apoptosis induced by compound III-2

[0601] This example evaluated the effect of III-2 on apoptosis of SKOV3 cells. 5Cells / 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. After the hypoxia group was cultured in a 0.1% O2 environment for 1 day, the cells were transferred to a 20% O2 oxygen concentration environment and continued to be cultured for two days. The normoxia group was cultured in a 20% O2 oxygen concentration environment for three days after administration. All cells in each well were collected in a 1.5 mL EP tube, washed once with cold PBS, and the sample was stained with Annexin V-FITC and propidium iodide solution in the dark for 20 minutes at room temperature and then analyzed using FACSCalibur. The results show (Figure 18) that under normoxic conditions (20% O2), compound III-2 had almost no effect on cell apoptosis. In contrast, under hypoxic conditions (0.1% O2), compound III-2 induced more than half of the cell apoptosis. The cell apoptosis results show that compound III-2 has good hypoxia selectivity.

[0602] Example 28: Distribution of Compound III-2 Labeled with DyLight-680 Dye in Mice

[0603] This example evaluated the distribution of Compound III-2 in mice.

[0604] DyLight 680-Herceptin and DyLight 680-III-2 were prepared using DyLight 680NHS ester dye (purchased from Thermo Fisher Scientific) according to the manufacturer's instructions. Specifically, Herceptin or compound III-2 in NaHCO3 buffer (pH = 8.3) was stirred and reacted with the fluorescent dye (DyLight 680NHS ester) for 3 hours to label the antibody with the dye. The resulting DyLight 680-Herceptin and DyLight 680-III-2 were then exchanged into PBS buffer for later use.

[0605] SKOV3 ovarian cancer-bearing mice (purchased from Yikang (Beijing) Pharmaceutical Technology Co., Ltd.) were injected with 0.2 mL of PBS, DyLight 680-Herceptin, and DyLight 680-III-2 via tail vein injection. The mice were then imaged using the Maestro in vivo fluorescence imaging system at time points (6, 24, 30, 48, 72, 120, 216, and 360 hours). The results (Figure 19) show that after tail vein injection of DyLight 680-labeled DyLight 680-Herceptin (25 mg / kg) and DyLight 680-III-2 (25 mg / kg), strong fluorescence signals were clearly observed in the tumors 6 hours after administration. This fluorescence was maintained for approximately 9 days, and the fluorescence signal finally disappeared on the 15th day after administration. The fluorescence signal intensity curves for DyLight 680-III-2 were nearly identical to those for DyLight 680-Herceptin, indicating that compound III-2 and Herceptin share similar metabolic properties. Furthermore, the fluorescence intensity at the tumor site was significantly higher than that at other normal tissues (Figure 19). These imaging results demonstrate that compound III-2 exhibits excellent tumor targeting and a long half-life in vivo.

[0606] Example 29: Drug distribution and metabolism studies of compound III-2

[0607] This example evaluated the drug distribution and metabolism of Compound III-2. SKOV3 ovarian cancer-bearing mice were divided equally into six groups. After a single tail vein injection of Compound III-2 (25 mg / kg), mice were euthanized at 6, 24, 72, 96, 168, and 192 hours, and blood, heart, liver, spleen, lung, and tumor samples were collected. Blood and tissue samples were stored at -80°C for further analysis. Results (Figure 20, Table 5) showed that high concentrations of the prodrug, Cys-II-2, were detected in the blood and peripheral tissues 6 hours after administration of Compound III-2. Furthermore, the results demonstrated that Cys-II-2 is rapidly cleared from normal tissues, with a blood half-life of only 26 hours, further minimizing its toxicity and side effects. After 96 hours of administration, Cys-II-2 was virtually undetectable in the heart, liver, spleen, and lungs. The peak concentration and area under the curve (AOC) of the toxin MMAE in tumors were 293 times and 941 times higher than those in peripheral blood, respectively, further confirming the excellent targeting of compound III-2. The AOCs of MMAE in the heart, liver, spleen, and lung accounted for 11%, 47%, 5%, and 7% of the AOC of the tumor, respectively. These results demonstrate that Cys-II-2 in peripheral tissues, with the exception of the liver, cannot be metabolized to MMAE, whereas Cys-II-2 in tumors can effectively release MMAE (Table 6).

[0608] Table 5 Cys-II-2 concentration in various tissues

[0609] Note: BQL means beyond detection limit.

[0610] Table 6 Concentration of MMAE in various tissues

[0611] Note: BQL means beyond detection limit.

[0612] Example 30: Pharmacodynamic study of compound III-2 in NCI-N87 tumor-bearing mice

[0613] This example evaluated 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. Tumor size was measured with a caliper twice a week. The tumor volume was calculated as 1 / 2 × length × width × width (mm). When the tumor volume of any mouse exceeded 2000mm, 3The mice were euthanized. The results (Figure 21) show that in the NCI-N87 tumor xenograft model, compared to the control group, the 5 mg / kg dose of compound III-2 significantly inhibited tumor growth, with a tumor inhibition rate of 90.97%. This result indicates that compound III-2 can achieve an anti-tumor effect almost equivalent to that of traditional VC-ADCs. More importantly, all mice in the 10 mg / kg dose group completely regressed their tumors, demonstrating the excellent anti-tumor therapeutic potential of compound III-2.

[0614] Example 31: Pharmacodynamic study of compound III-2 in JIMT-1 tumor-bearing mice

[0615] This example evaluated the anti-tumor effect of compound III-2 on JIMT-1 tumor-bearing mice (purchased from Hangzhou Tsinghua Kerui Biotechnology Co., Ltd.). To evaluate the anti-tumor effect of compound III-2 in JIMT-1 tumor-bearing mice with low HER2 antigen expression, when the tumor volume grew to 150 mm 3 Mice were randomly divided into two groups: a PBS group and a III-2 group. Compound III-2 was administered once weekly via tail vein injection for a total of four doses. Tumor size was measured twice weekly using a caliper. The results (Figure 22) show that compound III-2 exhibited tumor inhibition in the JIMT-1 xenograft model at a dose of 12 mg / kg. Although tumors did not completely regress, the inhibition rate reached 62.36%.

[0616] Example 32: Pharmacodynamic study of compound III-2 in combination with Herceptin, bevacizumab, sunitinib or docetaxel in NCI-N87 tumor-bearing mice

[0617] This example evaluated the antitumor efficacy of compound III-2 in combination with Herceptin, bevacizumab, sunitinib or docetaxel in 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 grew to 100 mm 3At the same time, PBS, Herceptin, III-2 and docetaxel were administered once a week by tail vein injection for a total of 3 times. Among them, docetaxel was administered 6 hours in advance. Sunitinib was administered by gavage once a day for three consecutive weeks. The results showed (Figure 23) that the group administered with III-2 combined with sunitinib 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 of III-2 could almost achieve the effect of complete tumor regression, with a tumor inhibition rate of 98.93%. The above results indicate that the excellent tumor inhibition effect of combined administration may be due to the fact that the angiogenesis inhibitor sunitinib can expand the range of tumor tissue hypoxia and enhance the degree of hypoxia in tumor tissue, thereby further improving the efficacy of III-2.

[0618] Example 33: Study on the Maximum Tolerated Dose of Compound III-2

[0619] This example evaluated the tolerability and adverse reactions of healthy female CD-1 mice (purchased from Hangzhou Tsinghua Kerui Biotechnology Co., Ltd.) to Compound III-2. Female CD-1 mice, aged 7-9 weeks, were acclimated for at least 3 days prior to dosing. Each dose group consisted of 3 mice. Healthy female CD-1 mice were administered a single tail vein injection of 120, 160, 200, and 240 mg / kg of Compound III-2. In addition, the marketed drug T-DM1 and the traditional ADC drug VC-ADC were used as controls. Following tail vein administration, the mice were monitored daily for weight changes and behavior. The results (Figure 24) showed that the maximum tolerated dose (MTD) of Compound III-2 was >200 mg / kg. However, mice in the VC-ADC and T-DM1 groups experienced sustained weight loss (>20%) and exhibited severe adverse reactions from the start of dosing. For example, all mice in the VC-ADC group died after receiving 120 mg / kg.

[0620] Example 34: Hematological and histopathological studies of compound III-2 in BALB / C mice

[0621] This example evaluated the hematological and histopathological effects of compound III-2 in BALB / C mice (purchased from Hangzhou Tsinghua Kerui Biotechnology Co., Ltd.). Female BALB / C mice (n=3 / group) were administered III-2 (60 mg / kg), VC-ADC (60 mg / kg), and PBS via tail vein injection. Following administration, mouse body weight was monitored every three days, and mice were euthanized on day 7 for whole blood hematology and histopathology analysis. Results (Figure 25) showed no significant differences in biochemical, blood routine, and histopathological markers between the III-2-treated and control groups. However, in the VC-ADC-treated group, changes in blood biochemistry reflected adverse effects of the VC-ADC on the liver, such as increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK). VC-ADC side effects were also reflected in hematological changes, including increases in lymphocyte count (LYMPH), monocyte count (MONO), and neutrophil count (NEUT).

[0622] Example 35: Antibacterial Study of Compound III-16 on Staphylococcus aureus

[0623] This example evaluated the antibacterial effect of compound III-16 against Staphylococcus aureus in macrophages. 37.5 μg / mL of compound III-16 and 150 μg / mL of an antibacterial antibody (whose heavy and light chain sequences are shown in SEQ ID NOs: 3 and 4, respectively) were added to a well plate and incubated with macrophages infected with Staphylococcus aureus for 24 hours. The colonies were counted and photographed. The results show (Figure 26) that compound III-16 has excellent anti-Staphylococcus aureus activity.

[0624] Example 36: Antibacterial Study of Compound III-16 on Free Staphylococcus aureus

[0625] This example evaluates the antibacterial effect of compound III-16 on free Staphylococcus aureus. Take 5-10 μL of the Staphylococcus aureus USA300 culture liquid frozen at -80°C and streak it on a Brain Heart Infusion solid culture dish, and culture it in a biochemical incubator at 37°C overnight. Select monoclonal USA300 in the culture dish, add an appropriate amount of Brain Heart Infusion culture medium, activate it in a bacterial shaking incubator for 16-18 hours, and then centrifuge it at 4000 rpm for 5 minutes to collect the bacteria. Add 1×PBS and mix well, and dilute the bacteria to a density of about 1×10 -3 / mL. Add the diluted bacterial solution to a 96-well plate, 100 μL per well. Add the gradient dilutions of III-16 and Rifalogue to the bacterial solution in the 96-well plate, incubate in a 37°C biochemical incubator for 16-18 hours, and measure the OD value using a microplate reader. 600nm The results showed (Figure 27) that compound III-16 had an antibacterial activity against free USA300 (MIC 90 About 15.7nM) and the antibacterial activity of Rifalogue (MIC 90 Without being limited by theory, it is speculated that the activity of compound III-16 against free bacteria may be due to the fact that azoreductase secreted by bacteria to the extracellular space cleaves the linker in III-16, thereby releasing the rifalogue to produce an antibacterial effect.

[0626] Example 37: Pharmacodynamic study of compound III-16 in a sepsis model

[0627] This example evaluates the therapeutic effect of compound III-16 on sepsis model mice. Take 5-10 μL of the Staphylococcus aureus USA300 bacterial solution frozen at -80°C and streak it on the Brain Heart Infusion solid culture dish, and culture it in a biochemical incubator at 37°C overnight. Select the monoclonal USA300 in the culture dish, add an appropriate amount of Brain Heart Infusion culture medium, activate it in a bacterial shaking incubator at 37°C for 16-18h, and then centrifuge it at 4000rpm for 5min to collect the bacteria. Add 1×PBS and mix well, and dilute the bacteria to an OD of 600nm =5 / mL. 100 μL of diluted USA300 was injected into the mice by tail vein injection. 24 hours after the mice were infected, the mice were divided into groups and then dosed. 4 days after infection, the mice were dissected to obtain tissues such as kidneys and hearts. After tissue grinding, the grinding fluid of the kidney and heart tissue of the mice was taken for plate counting to evaluate the antibacterial effect of compound III-16 in mice. The results showed (Figure 28) that the high-dose group of compound III-16 (60 mg / kg) showed excellent antibacterial effect in the kidneys, and the colony counts in the mouse kidneys were reduced to the detection limit. The high-dose group of compound III-16 showed significant statistical differences in kidney colony counts compared with the control group, the antibacterial antibody BD37+Rifalogue combination group (60 mg / kg BD37 and 60 mg / kg compound III-16 carried Rifalogue mixed administration), and the Vancomycin group (110 mg / kg) (One-Way ANOVA: **** P<0.0001, *** P<0.005, ** P<0.05).

[0628] The above experimental results demonstrate that the ADCs provided herein (e.g., ADC shown in III-2) can be rapidly recognized and cleaved by azoreductase, efficiently releasing MMAE, and exhibit anti-tumor effects in both in vitro and in vivo activity evaluations. Furthermore, the ADCs provided herein (e.g., ADC shown in III-16) exhibit excellent antibacterial effects.

[0629] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure and not to limit it. Although the present disclosure has been described in detail with reference to the preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present disclosure can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solutions of the present disclosure, which should all be included in the scope of the technical solutions requested for protection in the present disclosure.

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; R2 and R3 are each 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, Wherein r1 and r2 are each independently an integer between 1 and 12; Z is hydroxy, fluorine, chlorine, bromine, iodine or Where s is 0, 1, 2, 3 or 4, R5 is hydrogen, fluorine, chlorine, bromine, iodine, C 1-4 Alkyl, nitro or C 1-4 Alkoxy; Preferably, middle: B is or H, wherein r is an integer between 1 and 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 -(CH2CH2O) k -(CH2) l -,-CHR1-,-(CH2) m -, or absent, wherein R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, 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(O2)NH-, -NHS(O2)- or absent; W2 is selected from -(CH2) 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(O2)- or not present, wherein 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 of claim 1, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein W is selected from -NH-CH2-C(O)-NH-, -NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH2) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH2) 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, wherein: 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, characterized in that: 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 -(CH2CH2O) k -(CH2) l -, -CHR1- or -(CH2) m -, wherein k, l, R1, m are defined as in claim 1; iii) W is -C(O)-NH-(CH2) 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 -(CH2) m -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-CHR1-C(O)-NH-(CH2) o -NH-C(O)-, or -(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, where R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, m, o, i, k, l, n are as defined 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, -VLW- is -(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)2-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)4-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)6-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)8-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)3-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2 -C(O)NH-CH[(CH2)4-NHC(O)-(CH2CH2O)8-CH3]-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)7-(CH2)2-C(O)-NH-(CH2)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, characterized in that 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) R2 and R3 are each independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; Preferably, R2 and R3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; Preferably, R2 and R3 are each independently 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 wherein s is 0, 1, 2, 3 or 4; R5 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 wherein r1 and r2 are defined as in claim 1; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5 or 6; Preferably, r1 and r2 are each independently 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; R2 and R3 are each 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, Wherein r1 and r2 are each independently 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 -(CH2CH2O) k -(CH2) l -,-CHR1-,-(CH2) m -, or absent, wherein R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, 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(O2)NH-, -NHS(O2)- or absent; W2 is selected from -(CH2) 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(O2)- or not present; wherein 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-CH2-C(O)-NH-, -NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH2) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH2) 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, characterized in that 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 -(CH2CH2O) k -(CH2) l -, -CHR1- or -(CH2) m -, wherein k, l, R1, m are defined as in claim 9; iii) W is -C(O)-NH-(CH2) 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 -(CH2) m -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-CHR1-C(O)-NH-(CH2) o -NH-C(O)-, or -(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, where R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, m, o, i, k, l, n are as defined 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, -VLW- is -(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)2-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)4-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)6-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)8-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)3-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2 -C(O)NH-CH[(CH2)4-NHC(O)-(CH2CH2O)8-CH3]-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)7-(CH2)2-C(O)-NH-(CH2)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, characterized in that 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) R2 and R3 are each independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; Preferably, R2 and R3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; Preferably, R2 and R3 are each independently 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 wherein r1 and r2 are defined as in claim 9; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, r1 and r2 are each independently 1, 2, 3, 4, 5 or 6; Preferably, r1 and r2 are each independently 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; R2 and R3 are each 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, Wherein r1 and r2 are each independently an integer between 1 and 12; R' is Where k2 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 B1 is Where k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; 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 -(CH2CH2O) k -(CH2) l -,-CHR1-,-(CH2) m -, or absent, wherein R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, 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(O2)NH-, -NHS(O2)- or absent; W2 is selected from -(CH2) 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(O2)- or not present; wherein 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 the site ** of the B1 group through the S atom or N atom in the targeting compound molecule, or A is coupled to the site ** of the B1 group through the reaction of the carbonyl group in the targeting compound molecule with the hydroxylamine group in the B1 group to form an oxime bond, 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 isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula III, wherein W is selected from -NH-CH2-C(O)-NH-, -NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -C(O)-NH-CH2-C(O)-NH-, -C(O)-NH-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, -NH-CH(R 10 )-C(O)-NH-, -C(O)-NH-(CH2) o -NH-C(O)-, -C(O)-NH-[CH(R 11 )CH(R 12 )] o -NH-C(O)-, -NH-(CH2) 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 B1 is Preferably, B1 is wherein k1 is defined as in claim 17; Preferably, k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; further preferably, k1 is 1, 2, 3, 4, 5, 6, 7 or 8; even more preferably, k1 is 2, 3, 4, 5, 6, 7 or 8.

20. The compound according to any one of claims 17 to 19, its geometric or optical isomers, pharmaceutically acceptable salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula III, characterized in that: 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 -(CH2CH2O) k -(CH2) l -, -CHR1- or -(CH2) m -, wherein k, l, R1, m are defined as in claim 17; iii) W is -C(O)-NH-(CH2) 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 -(CH2) m -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, -(CH2) i -C(O)-NH-CHR1-C(O)-NH-(CH2) o -NH-C(O)-, or -(CH2CH2O) k -(CH2) l -C(O)-NH-(CH2) o -NH-C(O)-, where R1 is -(CH2) n -NHC(O)-(CH2CH2O) k -CH3, m, o, i, k, l, n are as defined 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, -VLW- is -(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)2-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)4-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)6-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2-C(O)NH-(CH2CH2O)8-CH2-CH2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)3-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2)2 -C(O)NH-CH[(CH2)4-NHC(O)-(CH2CH2O)8-CH3]-C(O)-NH-(CH2)2-NH-C(O)-, -(CH2CH2O)7-(CH2)2-C(O)-NH-(CH2)2-NH-C(O)-.

21. The compound according to any one of claims 17 to 20, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula III, characterized in that: 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) R2 and R3 are each independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; Preferably, R2 and R3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; Preferably, R2 and R3 are each independently 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: The definitions of A, k1, V, L, W, R, C, D, and E are as described in any one of claims 17 to 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, its geometric isomers, optical isomers, salts, hydrates, solvates or polymorphs, wherein the compound is a compound of formula IV, characterized in that: 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) R2 and R3 are each independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxy, nitro or cyano; Preferably, R2 and R3 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; Preferably, R2 and R3 are each independently fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; Preferably, R2 and R3 are each independently 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 antibacterial antibody, 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, k2 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, k2 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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