Novel thiol reducing agent, its production method and use

Abstract

The present disclosure relates to novel thiol reducing agents having formula (I), their preparation, and use in the preparation of antibodies with improved homogeneity and site-specific modification of thiol groups.

Description

Detailed Description of the Invention

[0001] [Title of invention] This application claims priority to PCT Application No. PCT / CN2022 / 113992 filed on August 22, 2022, PCT Application No. PCT / CN2022 / 119999 filed on September 20, 2022, PCT Application No. PCT / CN2022 / 119955 filed on September 20, 2022, and PCT Application No. PCT / CN2023 / 073070 filed on January 19, 2023. The contents of the prior PCT applications are considered part of this disclosure and are incorporated herein in their entireties.

[0002] [Technical field] The present disclosure relates to novel thiol reducing agents, methods and uses thereof, which may be used to modify antibodies.

[0003] [Background technology] The statements in this section provide background information related to the present disclosure and may not necessarily constitute prior art.

[0004] Antibody-drug conjugates (ADCs) are innovative biologics that combine a monoclonal antibody with a small molecule drug via a stable linker. ADCs ideally combine the specificity of antibodies with the potency of cytotoxic drugs by delivering potent cytotoxic drugs to antigen-expressing cells and enhancing targeted cytotoxic activity.

[0005] Typically, conjugation of antibodies to cytotoxic drugs involves conjugation to exposed residues such as lysine or reduction of disulfide bonds to expose free interchain cysteines in therapeutic IgG (immunoglobulin G) antibodies. Newer approaches to introduce conjugation sites into mAbs include site-specific glycan conjugation, cysteine engineering, the introduction of unnatural amino acids, and the attachment of short peptide tags to drug linkers. Antibodies typically contain 80 lysine residues, but fewer than 10 are chemically conjugable. Cysteine conjugation involves the reduction of four interchain disulfide bonds. These bonds can be reduced under specific conditions, resulting in the generation of two, four, six, or eight exposed sulfhydryl groups. Both Cys and Lys conjugation methods result in heterogeneous mixtures. (Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Aug; 9(8):872.)

[0006] The drug-antibody ratio (DAR), i.e., the number of drug molecules conjugated to a single ADC, is crucial for determining the efficacy of an ADC. The DAR varies widely and depends on other ADC variables. The DAR value also depends on the conjugation site and the use of light or heavy chain conjugation. The DAR value influences drug efficacy, with low drug loading resulting in decreased potency. On the other hand, increased drug loading can affect toxicity and pharmacokinetics ("Introduction to Antibody-Drug Conjugates". Antibodies (Basel). 2021 Dec; 10(4):42.). Establishing a robust conjugation process is always challenging because traditional nonspecific binding and conjugate distribution are significantly affected by factors such as pH, concentration, salt concentration, and cosolvents.

[0007] Many methods have been developed to improve the homogeneity of ADCs. For example, Genentech's THIOMAB technology was developed to improve the homogeneity of ADCs through antibody engineering by introducing cysteine residues into the primary antibody sequence, enabling site-specific conjugation. This improves product homogeneity ("Cysteine-Based Coupling: Challenges and Solutions". Bioconjug Chem. 2021 Aug 18;32(8):1525-1534.).

[0008] US20210040145 discloses a 14-amino acid peptide, Tub-tag, that can be used at the C-terminus of any POI to catalyze the addition of a variety of different tyrosine derivatives. By utilizing this enzyme, Tub-tag technology reuses tubulin tyrosine ligase to attach functional moieties to the C-terminus of antibodies, resulting in the uniform generation of antibody conjugates bearing DAR 2.

[0009] WO2018036438 discloses a method for producing ADCs using a technology called K-Lock, which can selectively react a designed linker-drug with four specific lysine residues on the Fab of an IgG antibody, producing up to 50% ADC products containing D2 (DAR value of approximately 2). Finally, pure D2 can be achieved through further purification.

[0010] However, these techniques involve protein engineering or enzyme catalysis and suffer from drawbacks such as low antibody expression levels, risk of immunogenicity, complex purification, and / or high costs.

[0011] Therefore, antibody-drug conjugates with increased homogeneity may offer advantages in terms of stability and reduced immunogenicity, and may also offer therapeutic benefits, such as better efficacy and lower toxicity. Therefore, new reducing agents and processes for preparing ADCs with high homogeneity are highly desirable and are the subject of long-term pursuit.

[0012] [Summary of the Invention] For the above purposes, provided herein are compounds having the following formula (I): [ka] or a salt, solvate, or stereoisomer thereof, X, Y and Z are independently 3 ) or PC(sp 2 ) is covalently bonded to the phosphorus atom via a PC bond.

[0013] X is of formula (II): [ka] L1 is selected from the group consisting of: -CH(R1)-, -C(CH3)(R1)-, -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, an aryl group optionally substituted with a group containing at least one coordination atom independently selected from N, O, and S. and heteroaryl groups optionally substituted with groups containing at least one coordination atom independently selected from O and S.

[0014] R1, R2 and R3 are independently H, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, a C1-C5 carboxyalkyl group, a C1-C5 hydroxylamine alkyl group, a C1-C5 N-hydroxyamido alkyl group, an aryl group or a heteroaryl group; or

[0015] R2 or R3 together with L2 form an optionally substituted 5- or 6-membered ring.

[0016] A is optionally present and is —C(O)— or —C(O)J—.

[0017] J is an organic group containing both an amino group or an imino group and a carbonyl group, and the amino group or the imino group may form an amide group with -C(O), and the carboxyl group may be covalently bonded to L2.

[0018] L2 is optionally present and acts as a transition metal chelator motif and is -N(R4)(R5) or hydroxy.

[0019] R4 and R5 are independently hydrogen, a C0-C5 hydroxyalkyl group, a C1-C5 alkyl group, a C1-C5 alkoxy group, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), an optionally substituted 5- to 6-membered heterocyclic group, an optionally substituted arylalkyl group, an optionally substituted arylalkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted heteroarylalkyl group, or R4 and R5 together form a 5- to 6-membered optionally substituted ring, and R4 or R5 together with R2 or R3 form a 5- to 6-membered optionally substituted ring.

[0020] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted arylalkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroarylalkyl.

[0021] R7 is a hydroxy group, a C1-C5 alkoxy group, or -NH(CH2CONH)n 3 It's OH.

[0022] n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0023] R4 and R5 cannot be simultaneously hydroxy.

[0024] Y is the same as X, Z is the same as X, or Y and Z are independently a 5- to 6-membered optionally substituted heterocyclic group, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, an aryl group, a C1-C5 carboxyalkyl group, a 5- to 6-membered optionally substituted cycloalkyl group, or [ka] , -C(O)Q is an ester group, an imide group, or an amide group; X, Y and Z are not simultaneously —CH2CH2C(O)OH.

[0025] In one aspect, provided herein is a composition comprising the compound described above and a transition metal ion.

[0026] In one aspect, provided herein are methods for preparing the compounds described above.

[0027] In one aspect, provided herein is the use of the above-described compound or the above-described composition in modifying the above-described antibody.

[0028] In some embodiments, the antibody is modified by selectively reducing the interchain S-S bonds of the antibody, and optionally, the antibody is modified by selectively reducing one of the interchain S-S bonds.

[0029] In some embodiments, provided herein is a use of the compound or composition in the preparation of an antibody having a thiol group site-specific modification, optionally wherein the antibody having a thiol group site-specific modification is an antibody-drug conjugate (ADC).

[0030] In one aspect, provided herein is a method for preparing an antibody having thiol group site-specific modification, characterized in that a thiol group(s) is reduced from an interchain disulfide bond of the antibody, the method comprising using the compound described above and a transition metal ion, or using the composition described above.

[0031] In one aspect, provided herein is a thiol group site-specifically modified antibody prepared by the above-described method.

[0032] In one aspect, provided herein is the use of a thiol group site-specifically modified antibody prepared by the above-described method in the manufacture of a therapeutic agent for the prevention, diagnosis, or treatment of a disease.

[0033] In one aspect, provided herein is a pharmaceutical composition comprising a thiol group site-specifically modified antibody prepared by the method described above and at least a pharmaceutically acceptable carrier.

[0034] In one aspect, provided herein is a method for preventing, diagnosing, or treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the above-described thiol group site-specifically modified antibody or the above-described pharmaceutical composition. [Brief explanation of the drawings]

[0035] A brief description of the drawings follows, which are presented for purposes of illustrating exemplary embodiments disclosed herein, and not for purposes of limiting the same. [Figure 1] Figure 1 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 34. HIC-HPLC stands for hydrophobic interaction chromatography-high performance liquid chromatography. [Figure 2] FIG. 2 shows the HIC-HPLC chromatogram of the sacituzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 35. [Figure 3] FIG. 3 shows the HIC-HPLC chromatogram of the belantamab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 36. [Figure 4] FIG. 4 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 1. [Figure 5] FIG. 5 shows the HIC-HPLC chromatogram of the sacituzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 2. [Figure 6] FIG. 6 shows the HIC-HPLC chromatogram of the belantamab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 3. [Figure 7] FIG. 7 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 37. [Figure 8] FIG. 8 shows the HIC-HPLC chromatogram of the sacituzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 38. [Figure 9] FIG. 9 shows the HIC-HPLC chromatogram of the belantamab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 39. [Figure 10] FIG. 10 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 4. [Figure 11] FIG. 11 shows the HIC-HPLC chromatogram of the sacituzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 5. [Figure 12] FIG. 12 shows the HIC-HPLC chromatogram of the belantamab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 6. [Figure 13] FIG. 13 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 40. [Figure 14] FIG. 14 shows the HIC-HPLC chromatogram of the sacituzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 41. [Figure 15] FIG. 15 shows the HIC-HPLC chromatogram of the belantamab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 42. [Figure 16]FIG. 16 shows the HIC-HPLC chromatogram of the sacituzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 8. [Figure 17] FIG. 17 shows the HIC-HPLC chromatogram of the belantamab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 9. [Figure 18] Figure 18A-H show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 43-50, where the reducing agents are TCEP-1, TCEP-2, TCEP-3, TCEP-4, TCEP-5, TCEP-6, TCEP-7, and TCEP-8. [Figure 19] Figure 19A-H show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 51-58, where the reducing agents are TCEP-9, TCEP-10, TCEP-15, TCEP-18, TCEP-19, TCEP-20, TCEP-21, and TCEP-23. [Figure 20] Figure 20A-G show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 59-65, where the reducing agents are TCEP-24, TCEP-25, TCEP-26, TCEP-28, TCEPA, TCEP-34, and TCEP-35. [Figure 21] FIG. 21 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 66, where the reducing agent is TCEP-37. [Figure 22] FIG. 22 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 11. [Figure 23] FIG. 23 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 12. [Figure 24]FIG. 24 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 67. [Figure 25] FIG. 25 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 68. [Figure 26] FIG. 26 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 69. [Figure 27] FIG. 27 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 70. [Figure 28] FIG. 28 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 71. [Figure 29] FIG. 29 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 72. [Figure 30] FIG. 30 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 73. [Figure 31] FIG. 31 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 74. [Figure 32] FIG. 32 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 75. [Figure 33] FIG. 33 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 76. [Figure 34] FIG. 34 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 77. [Figure 35]FIG. 35 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 78. [Figure 36] Figure 36A to 36C show HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 79-81. [Figure 37] FIG. 37 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 10. [Figure 38] Figure 38A-D show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 82-85, where the molar ratio of antibody to reducing agent was different. [Figure 39] FIG. 39 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 86. [Figure 40] FIG. 40 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 87. [Figure 41] FIG. 41 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 88. [Figure 42] FIG. 42 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 89. [Figure 43] FIG. 43 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 90. [Figure 44] FIG. 44 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 91. [Figure 45] FIG. 45 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 92. [Figure 46] FIG. 46 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 93. [Figure 47] FIG. 47 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 94. [Figure 48] FIG. 48 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 95. [Figure 49] FIG. 49 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 96. [Figure 50] FIG. 50 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 97. [Figure 51] FIG. 51 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 98. [Figure 52] Figure 52A to 52C show HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 99-101. [Figure 53] Figure 53A-C show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 102-104, where the incubation temperature in step (1) was different. [Figure 54] Figure 54A to 54D show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 105-108, where the incubation time in step (1) was different. [Figure 55] Figure 55A to 55E show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 109-113, where the incubation time in step (1) was different. [Figure 56] Figure 56 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 114, where the antibody is a recombinant antibody. [Figure 57] FIG. 57 shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG-N-DBCO-Cy] conjugate prepared in Example 115. [Figure 58] FIG. 58 shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide]1[MC-GGFG-DXd]6 conjugate prepared in Example 116. [Figure 59] FIG. 59 shows the HIC-HPLC chromatogram of the trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 conjugate prepared in Example 117. [Figure 60] Figure 60A shows an HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 118, and Figure 60B shows an HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 conjugate prepared in Examples 118-119. [Figure 61] FIG. 61 shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] conjugate prepared in Example 120. [Figure 62] Figure 62A shows an HIC-HPLC chromatogram of the trastuzumab-[maleimide]1 conjugate prepared in Example 121, and Figure 62B shows an HIC-HPLC chromatogram of the trastuzumab-[maleimide]1[MC-VC-PAB-MMAE]4 conjugate prepared in Examples 121-122. [Figure 63]Figure 63A shows an HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2 conjugate prepared in Example 123, and Figure 63B shows an HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 conjugate prepared in Examples 123-124. [Figure 64] Figure 64 shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] conjugate prepared in Example 125.

[0036] [Detailed explanation] The present disclosure is described in further detail below. This description is not intended to be a detailed catalog of all the different ways in which the present invention may be practiced or all the features that can be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments. Also, features described with respect to a particular embodiment may be omitted from that embodiment. Moreover, numerous variations and additions to the various embodiments proposed herein will be apparent to those skilled in the art in light of this disclosure without departing from the invention(s) presented herein. Accordingly, the following description is intended to illustrate some specific embodiments of the invention(s) herein, but is not intended to exhaustively identify all permutations, combinations, and variations thereof.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains.In carrying out the testing of this disclosure, methods and materials similar or equivalent to those described herein can be used, but preferred materials and methods are described herein.In describing and claiming this disclosure, the following terms are used:

[0038] reducing agent The present disclosure provides examples of compounds that can act as reducing agents in preparing antibodies with thiol group site-specific modifications.

[0039] Provided herein are compounds having the following formula (I): [ka] or a salt, solvate, or stereoisomer thereof, X, Y and Z are independently 3 ) or PC(sp 2 ) is covalently bonded to the phosphorus atom via a PC bond.

[0040] X is of formula (II): [ka] L1 is selected from the group consisting of: -CH(R1)-, -C(CH3)(R1)-, -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, an aryl group optionally independently substituted with a group containing at least a coordination atom, and a heteroaryl group optionally independently substituted with a group containing at least a coordination atom.

[0041] R1, R2 and R3 are independently H, a C1-C3 alkyl group, a C1-C3 hydroxyalkyl group, a C1-C3 carboxyalkyl group, a C1-C3 hydroxylamine alkyl group, a C1-C3 N-hydroxyamido alkyl group, an aryl group or a heteroaryl group; or R2 or R3 together with L2 form an optionally substituted 5- or 6-membered ring.

[0042] A is optionally present and is —C(O)— or —C(O)J—.

[0043] J is an organic group containing both an amino group or an imino group and a carbonyl group, and the amino group or the imino group may form an amide group with -C(O), and the carboxyl group may be covalently bonded to L2.

[0044] L2 is optionally present and acts as a transition metal chelator motif and is -N(R4)(R5) or hydroxy.

[0045] R4 and R5 are independently hydrogen, a C0-C5 hydroxyalkyl group, a C1-C5 alkyl group, a C1-C5 alkoxy group, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), an optionally substituted 5- to 6-membered heterocyclic group, an optionally substituted arylalkyl group, an optionally substituted arylalkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted heteroarylalkyl group, or R4 and R5 together form a 5- to 6-membered optionally substituted ring, and R4 or R5 together with R2 or R3 form a 5- to 6-membered optionally substituted ring.

[0046] R6 is hydrogen, amino, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, aryl, optionally substituted arylalkyl, C1-C3 N-hydroxyamidoalkyl, heteroaryl or heteroarylalkyl.

[0047] R7 is a hydroxy group, a C1-C3 alkoxy group, or -NH(CH2CONH)n 3 It's OH.

[0048] n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0049] R4 and R5 cannot be simultaneously hydroxy.

[0050] Y is the same as X, Z is the same as X, or Y and Z are independently a 5- to 6-membered optionally substituted heterocyclic group, a C1-C3 alkyl group, a C-C3 hydroxyalkyl group, an aryl group, a C1-C3 carboxyalkyl group, a 5- to 6-membered optionally substituted cycloalkyl group, or [ka] -C(O)Q is an ester group, an imide group, or an amide group; X, Y and Z are not simultaneously —CH2CH2C(O)OH.

[0051] The term "aryl group" refers to an aromatic or heteroaromatic group containing one or more rings and containing 3 to 14 carbon atoms, preferentially 6 to 10 carbon atoms. An exemplary aryl group is the phenyl group.

[0052] The term "aryl group" refers to an aromatic group in which one or more hydrogen atoms are independently selected from the group consisting of F, Cl, Br, I, hydroxy, carboxy, sulfonyl, amino, methoxy, ethoxy, N-hydroxyformamido, N-hydroxyacetamido, 4-pyridyl, 2-pyridyl, [ka] and the like.

[0053] The term "heteroaryl group" refers to an aromatic group in which one or several carbons, preferably one, two, three or four carbon atoms, are replaced by O, N, Si, Se, P or S, preferably O, S, N. Exemplary heteroaryl groups are imidazolyl, pyridyl, bipyridyl, quinolinyl, and isoquinolinyl groups.

[0054] The term "heteroaryl group" refers to a heteroaromatic group in which one or more hydrogen atoms are independently replaced with other groups, such as F, CI, Br, I, hydroxy, carboxy, amino, hydroxyalkyl groups, carboxyalkyl groups, N-hydroxyamidoalkyl groups, heteroaryl groups, and the like.

[0055] The term "coordinating atom" refers to an atom having an unshared pair of electrons, examples of which include N, O, S, P, F, Cl, Br, and I.

[0056] The term "C1-C5 alkyl group" refers to a linear or cyclic aliphatic hydrocarbon group of 1 to 3 carbon atoms. Exemplary alkyl groups include methyl, ethyl, n-propyl, and i-propyl.

[0057] The term "C0-C5 hydroxyalkyl group" refers to a hydroxy group or a C1-C5 alkyl group in which one or more hydrogen atoms are replaced with one, two, or three hydroxy groups. Exemplary C1-C5 hydroxyalkyl groups are hydroxymethyl, 2-hydroxyethyl, and 3-hydroxypropyl groups.

[0058] The term "C1-C5 carboxyalkyl group" refers to a C1-C5 alkyl group substituted with one, two, or three carboxy groups. Exemplary C1-C5 carboxyalkyl groups are -COOH, -CH2COOH, -CH2CH2COOH, -CH2(CH3)COOH.

[0059] The term "C1-C5 hydroxylamine alkyl group" refers to a C1-C5 alkyl group substituted with one, two, or three hydroxylamine groups. Exemplary C1-C5 hydroxylamine alkyl groups are -CH2NHOH, -CH2CH2NHOH.

[0060] The term "C1-C5N-hydroxyamidoalkyl group" refers to a C1-C5 carboxyalkyl group in which one, two, or three carboxyl groups are amide-linked to a hydroxylamine. Exemplary C1-C5N-N-hydroxyamidoalkyl groups are -C(O)NHOH, -CH2C(O)NHOH, -CH2CH2C(O)NHOH.

[0061] The term "heterocyclic group" refers to an aromatic or non-aromatic C5-C6 heterocyclic group consisting of one or two rings. 10 It refers to a cyclic group in which one or two of the ring carbon atoms are independently replaced with a heteroatom selected from the group of O, N, P, and S. Preferred heteroatoms are O, N, and S. Suitable heterocyclic compounds are also disclosed in The Handbook of Chemistry and Physics, 76* Edition, CRC Press, Inc., 1995-1996, pp. 2-25 to 2-26, which is incorporated herein by reference. Preferred non-aromatic heterocyclic compounds include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyranyl, dihydropyridyl, dihydropyridyl, tetrahydropyrinidinyl, dihydrothiopyranyl, a / epanyl, and fused systems formed by condensation with a phenyl group.

[0062] The term "arylalkyl group" refers to a linear, branched, or cyclic alkyl group that is bonded to at least one aryl group. Preferably, the linear or cyclic group contains 1 to 4 carbon atoms. Representative arylalkyl groups are -CH2C6H5, -CH2CH2C6H5, -CH2CH2CH2C6H5, -CH2(CH3)CH2C6H5, and -CH2(CH3)CH2CH2C6H5.

[0063] The term "heteroarylalkyl group" refers to a linear, branched, or cyclic alkyl group that is bonded to at least one heteroaryl group. Preferably, the linear or cyclic alkyl group contains 1 to 4 carbon atoms. Exemplary heteroarylalkyl groups are: [ka] is.

[0064] The term "C1-C5 alkoxy group" refers to an oxygen atom linked to a C1-C5 alkyl group. Exemplary C1-C5 alkoxy groups are -OCH3, -OCH2CH3, -OCH2(CH3)2, -OCH2CH2CH3.

[0065] The term "arylalkoxy group" refers to an aromatic group in which one or more hydrogen atoms have been replaced with an alkoxy group. Exemplary are phenyl-O-CH2-, phenyl-O-(CH2)2-, phenyl-O-(CH2)3-, phenyl-O-(CH2)4-, and phenyl-O-(CH2)5-.

[0066] The term "cycloalkyl group" refers to a 3-, 4-, 5-, or 6-membered saturated or non-aromatic unsaturated carbocyclic ring. Representative cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, and the like. Cycloalkyl groups can be unsubstituted or substituted with one or more groups including, but not limited to, carboxyl, sulfonyl, amino, hydroxy, -C(O)NHOH, -CHC(O)NHOH, -CHCHC(O)NHOH, -COOH, -CHCOOH, -CHCHCOOH, -CH(CH)COOH, F, Cl, Br, or I.

[0067] The term "halogen" refers to F, Cl, Br, or I.

[0068] The term "alkenyl" refers to a straight- or branched-chain unsaturated hydrocarbon containing 2 to 12 carbon atoms. An "alkenyl" group contains at least one double bond in the chain. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl, and hexenyl. Alkenyl groups can be unsubstituted or substituted, and can be straight-chain or branched.

[0069] The term "cyano group" refers to a substituent having a carbon atom attached to a nitrogen atom by a triple bond, for example, --CN.

[0070] In some embodiments, L1 is -CH(R1)-, -CH(R1)CH(R2)-, or -CH(R1)CH(R2)CH(R3)-. In some embodiments, L1 is -CH(R1)CH(R2)-.

[0071] In some embodiments, R1, R2, and R3 are independently H, methyl, isopropyl, hydroxymethyl, hydroxyethyl, carboxymethyl, carboxyethyl, N-hydroxyethylamide, phenyl, 2-pyridyl, 4-pyridyl, or 4-imidazole, and R2 forms a 5- to 6-membered optionally substituted ring with L2.

[0072] In some embodiments, L1 is -CH(R1)CH(R2)-, where R1 is H, and R2 forms a 5-6 membered optionally substituted ring with R4 of L2. In some embodiments, R2 forms a 5-6 membered optionally substituted ring with L2. [ka] In some embodiments, A is —C(O), L 2 is —N(R 4 )(R 5 ), and R 5 is hydroxy.

[0073] In some embodiments, L1 is -CH(R1)CH(R2)-, where R1 is a methyl group, an isopropyl group, a carboxyethyl group, or an N-hydroxyethylamide group, and R2 is H.

[0074] In some embodiments, L1 is -CH(R1)CH(R2)-, where R1 is a methyl group, an isopropyl group, a carboxyethyl group, or an N-hydroxyethylamide group, R2 is H, A is -C(O-), L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxy.

[0075] In some embodiments, L1 is -CH(R1)CH(R2)-, where R1 is H and R2 is a methyl group, a hydroxymethyl group, a hydroxyethyl group, a carboxyethyl group, a phenyl group, an N-hydroxyethylamide group, a 2-pyridyl group, a 4-pyridyl group, or a 4-imidazole group.

[0076] In some embodiments, L1 is -CH(R1)CH(R2)-, where R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyethylamide, 2-pyridyl, 4-pyridyl, or 4-imidazole, A is a -C(O) group, L2 is a -N(R4)(R5) group, R4 is hydrogen, an optionally substituted 5- to 6-membered heterocyclic group, and R5 is a hydroxy group. In some embodiments, R4 is [ka] is.

[0077] In some embodiments, L1 is -CH(R1)CH(R2)-, where R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyethylamido, 2-pyridyl, 4-pyridyl, or 4-imidazole, A is a -C(O) group, and L2 is a -N(R4)(R5) group, where R4 and R5 form a 5-6 membered, optionally substituted ring. In these embodiments, L2 is [ka] is.

[0078] In some embodiments, L1 is an optionally substituted phenyl group bonded to A at the ortho, meta, or para position, A is -C(O)-, L2 is -N(R4)(R5) or hydroxy, R4 is hydrogen, and R5 is hydroxy.

[0079] In some embodiments, L is a phenyl group optionally substituted at the ortho, meta, or para position with hydroxy, halogen, carboxyl, sulfonyl, amino, methoxy, or ethoxy. In these embodiments, A and L are absent. Halogen refers to F, Cl, Br, or I.

[0080] In some embodiments, L1 is [ka] In these embodiments, A and L2 are absent.

[0081] In some embodiments, L is an optionally substituted 4-pyridyl group or an optionally substituted 4-quinolyl group. [ka] In these embodiments, A and L2 are absent.

[0082] In some embodiments, L1 is -CH(R1)CH(R2)-, and R1 and R2 are independently H.

[0083] In some embodiments, A is -C(O), L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxy.

[0084] In some embodiments, L2 is -N(R4)(R5), where R4 is hydrogen, a C1-C5 alkyl group, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), an optionally substituted 5- to 6-membered heterocyclic group, an optionally substituted arylalkyl group, an optionally substituted aryl group, an optionally substituted heteroarylalkyl group, or R4 and R5 together form a 5- to 6-membered optionally substituted ring. R5 is hydroxy.

[0085] In some embodiments, L2 is -N(R4)(R5), where R4 is hydrogen, a C1-C5 alkyl group, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), an optionally substituted arylalkyl group, an optionally substituted aryl group, and R5 is hydroxy.

[0086] In some embodiments, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl group, C1-C5 carboxyalkyl group, aryl group, C1-C5 N-hydroxyamidoalkyl group, heteroaryl group, or heteroarylalkyl group.

[0087] In some embodiments, R6 is hydrogen, a C1-C5 alkyl, a C1-C5 hydroxyalkyl group, or a heteroarylalkyl group.

[0088] In some embodiments, R6 is hydrogen, a methyl group, a hydroxymethyl group, an amino group, a benzyl group, a carboxyethyl group, an N-hydroxyethylamide group, [ka] and optionally R6 is hydrogen.

[0089] In some embodiments, R7 is hydroxy, a C1-C5 alkoxy group, -NH(CH2CONH)n 3OH, and optionally R7 is a hydroxy or C1-C5 alkoxy group. In some embodiments, R7 is a hydroxy, methoxy group, -NH(CH2CONH)n 3 OH, and optionally R7 is a hydroxy or methoxy group.

[0090] In some embodiments, n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0091] In some embodiments, R4 is [ka] and hydrogen or -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydroxy, R6 is hydrogen, a methyl group, a hydroxymethyl group, or [ka] and R7 is hydroxy or -NH(CH2CONH)n 3 It's OH.

[0092] n 1 , n 2 and n 3 are independently 0.

[0093] In some embodiments, R4 is [ka] and hydrogen or —CH(R6)CO(R7), where R5 is hydroxy; R6 is hydrogen; R7 is hydroxy.

[0094] In some embodiments, L2 is -N(R4)(R5); R4 and R5 independently represent -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or an optionally substituted heteroarylalkyl group.

[0095] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroarylalkyl.

[0096] R7 is a hydroxy group, a C1-C5 alkoxy group, or -NH(CH2CONH)n 3 It's OH.

[0097] n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0098] In some embodiments, R4 and R5 are independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or a 6-membered heteroarylalkyl group.

[0099] R6 is hydrogen; R7 is a hydroxy group, a C1-C5 alkoxy group, or -NH(CH2CONH)n 3 It's OH.

[0100] n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0101] In some embodiments, R4 and R5 are independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or [ka] and R6 is hydrogen; R7 is hydroxy or -NH(CH2CONH)n 3 It's OH.

[0102] n 1 , n 2 and n 3 are independently 0.

[0103] In some embodiments, R4 and R5 are independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R6 is hydrogen; R7 is hydroxy or -NH(CH2CONH)n 3 It's OH.

[0104] n 1 , n 2 and n 3 are independently 0.

[0105] In some embodiments, L2 is -N(R4)(R5); R4 is hydrogen, a C0-C5 hydroxyalkyl group, a C1-C5 alkyl group, an optionally substituted C1-C5 alkoxy group, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), an optionally substituted arylalkyl group, an optionally substituted arylalkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted heteroarylalkyl group.

[0106] R5 is hydrogen; R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted arylalkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroarylalkyl.

[0107] R7 is a hydroxy group, a C1-C5 alkoxy group, or -NH(CH2CONH)n 3 It's OH.

[0108] n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0109] In some embodiments, R4 is hydrogen, a C0-C3 hydroxyalkyl group, a C1-C3 alkoxy group, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), a phenyl group substituted with carboxy, hydroxy, amino or halogen, a pyridyl group, an amino substituted with 2-methylpyridine, a benzyl group substituted with carboxy, hydroxy, amino or halogen, an arylalkoxy group, a pyridyl group substituted with carboxy, a bipyridyl group, [ka] is.

[0110] In some embodiments, R4 is hydrogen, hydroxy, methyl hydroxyl, ethyl hydroxyl, propyl hydroxyl, methoxy, ethoxy, [ka] and R5 is hydrogen.

[0111] In some embodiments, R4 is a hydroxy group, a methoxy group, or [ka] and R5 is hydrogen.

[0112] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), where R5 is hydrogen and R6 is hydrogen, amino, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, aryl, arylalkyl optionally substituted with hydroxyl, halogen, cyano or nitro, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroarylalkyl; R7 is a hydroxy group, a C1-C5 alkoxy group, or -NH(CH2CONH)n 3 It's OH.

[0113] n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0114] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), where R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl group, benzyl group, hydroxyl group, halogen, cyano group, benzyl group substituted with nitro group, halogen, carboxyethyl group, N-hydroxyethylamide group, [ka] and R7 is hydroxyl, -NH(CH2CONH)n 3 It's OH.

[0115] n 1 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0116] n2 is 0.

[0117] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl group, benzyl group, carboxyethyl group, [ka] N-hydroxyethylamide group, [ka] and R7 is hydroxyl or -NH(CH2CONH)n 3 It's OH.

[0118] n 1 is 0 or 2.

[0119] n 2 is either 0 or 1.

[0120] n 3 is 0.

[0121] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen. R7 is -NH(CH2CONH)n 3 OH. n 1 n 2 and n 3 is 0.

[0122] In some embodiments, A is -C(O)J-, where J is a peptide residue, and is a monoamino acid residue, a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, an aminopropionic acid, an aminobutyric acid, an aminovaleric acid, an amino acid, an aminoheptanoic acid, an aminooctanoic acid, or NH2(OCH2CH2O)n 4CH2COOH, n 4 is a number between 2 and 10.

[0123] The amino acid is selected from the group consisting of glycine (Gly), alanine (Ala), serine (Ser), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).

[0124] In some embodiments, J is a residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine, or asparagine.

[0125] In some embodiments, A is -C(O)J-, where J is a residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine, and L2 is -N(R4)(R5), where R4 is hydrogen and R5 is hydroxy.

[0126] In some embodiments, Y is the same as X.

[0127] In some embodiments, Z is the same as X.

[0128] In some embodiments, Y and Z independently represent: [ka] and Q is -NHOH, -NHCH2CH2SO3H, -N(CH2CH2OH)2, -NHCH2COOH, -NHCH(CH3)COOH, -NH(CH2CH2O)3CH3.

[0129] In some embodiments, the compound is selected from the group consisting of, but not limited to: [ka] JPEG2025527005000028.jpg168149 JPEG2025527005000029.jpg175149 JPEG2025527005000030.jpg192149 JPEG2025527005000031.jpg59149 The present disclosure provides compositions comprising the above compounds and transition metal ions.

[0130] In some embodiments, the transition metal ion is Zn 2+ , Cd 2+ , Hg 2+ , Ni 2+ , Co 2+ or a combination thereof. In some embodiments, the transition metal ion is Zn 2+ is.

[0131] In some embodiments, the molar ratio of the compound to the transition metal ion is 1:0.4 to 1:250, 1:0.4 to 1:200, 1:0.4 to 1:60, or 1:6 to 1:16.

[0132] The compounds having the above formula (I) can be prepared by the following steps.

[0133] By introducing a condensing agent under an inert atmosphere, at least one carboxyl group of the following formula III is converted to a transition metal chelating moiety: [ka] is bonded to the heteroatom of

[0134] [ka] where X' is [ka] is.

[0135] L1 is selected from the group consisting of -CH(R1)-, -C(CH3)(R1)-, -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, an aryl group optionally substituted with a group containing N, O, or S, and a heteroaryl group optionally substituted with a group containing O or S.

[0136] R1, R2 and R3 are independently H, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, a C1-C5 carboxyalkyl group, a C1-C5 hydroxylamine alkyl group, a C1-C5 N-hydroxyamido alkyl group, an aryl group or a heteroaryl group; or R2 or R3 together with L2 form an optionally substituted 5- or 6-membered ring.

[0137] A' is -COOH or -C(O)J-COOH.

[0138] J is an organic group containing both an amino group or an imino group and a carbonyl group, and the amino group or the imino group may form an amide group with -C(O), and the carboxyl group may be covalently bonded to L2.

[0139] L2 is optionally present and acts as a transition metal chelator motif and is -N(R4)(R5) or hydroxy.

[0140] R4 and R5 are independently hydrogen, a C0-C5 hydroxyalkyl group, a C1-C5 alkyl group, a C1-C5 alkoxy group, -(CH2)n 1 (OCH2CH2O)n 2CH(R6)CO(R7), an optionally substituted 5- to 6-membered heterocyclic group, an optionally substituted arylalkyl group, an optionally substituted arylalkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted heteroarylalkyl group, or R4 and R5 form a 5- to 6-membered optionally substituted ring, and R4 or R5 together with R2 or R3 form a 5- to 6-membered optionally substituted ring; R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted arylalkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroarylalkyl.

[0141] R7 is a hydroxy group, a C1-C5 alkoxy group, or -NH(CH2CONH)n 3 It's OH.

[0142] n 1 , n 2 and n 3 are independently the numbers 0, 1, 2, 3, or 4.

[0143] R4 and R5 cannot be simultaneously hydroxy.

[0144] Y' is the same as X', Z' is the same as X', or Y and Z are independently a 5- to 6-membered optionally substituted heterocyclic group, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, an aryl group, a C1-C5 carboxyalkyl group, a 5- to 6-membered optionally substituted cycloalkyl group, or [ka] -C(O)Q is an ester group, an imide group, or an amide group.

[0145] In some embodiments, the transition metal chelating moiety is selected from the group consisting of 2-phenoxyethylamine, phenylamine, benzylamine, 4-aminobenzene-1,2-diol, 5-amino-2-hydroxybenzoic acid, bis(pyridin-2-ylmethyl)amine, 5-amino-8-hydroxyquinoline, bis(pyridin-2-yl)methanamine, 4-aminophthalic acid, tert-butyl-L-tyrosinate, DL-3-(4-fluorophenyl)alanine, DL-4-cyanophenylalanine, DL-4-nitrophenylalanine, N-benzylhydroxylamine hydrochloride, N-phenylhydroxylamine, [ka] can be provided by

[0146] In some embodiments, the structure of formula III is [ka] is.

[0147] The term "condensing agent" refers to a condensation reaction reagent that aids in the covalent bonding of two molecules (functional groups) to form one molecule. Condensing agents include, but are not limited to, 1-hydroxybenzotriazole (HOBT), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0148] The term "inert atmosphere" refers to a chemically inert atmosphere such as nitrogen, carbon dioxide, helium, etc.

[0149] Use in producing antibodies having site-specific modification with thiol groups The compounds having formula (I) provided above have reducing properties and can reduce disulfide bonds in antibodies, and therefore, the compounds having formula (I) can act as reducing agents in the process of protein or antibody modification.

[0150] As used herein, the term "disulfide bond" refers to a covalent bond having the structure RSS-R'. Cysteine, an amino acid, contains a thiol group that can form a disulfide bond with, for example, a second thiol group from another cysteine residue. A disulfide bond is formed between the thiol groups of two cysteine residues present in each of two polypeptide chains, thereby forming an interchain crosslink or bond.

[0151] In some embodiments, the compound having formula (I) is capable of reducing the interchain S—S bonds of an antibody.

[0152] In some embodiments, the compound having formula (I) can selectively reduce one of the interchain S—S bonds, thus selectively modifying the antibody.

[0153] In some embodiments, the compound having formula (I) provided above acts as a reducing agent in the preparation of an antibody having a thiol group site-specific modification, optionally an antibody drug conjugate (ADC).

[0154] The antibody-drug conjugate mixture is produced by a conventional conjugation process or the bioconjugation process of the present disclosure. Generally, one antibody molecule belonging to the IgG1 or IgG4 subgroup has four interchain S-S bonds formed by two -SH groups. The antibody molecule undergoes partial or complete reduction of one or more interchain S-S bonds to form 2n (n is an integer selected from 1, 2, 3, or 4) reactive -SH groups, thereby allowing the number of agents (or drugs) conjugated to one antibody molecule to be 1, 2, 3, 4, 5, 6, 7, or 8. According to the number of drugs bound to a single antibody molecule, different conjugates containing different numbers and / or types of drug molecules are named as DO, D2, D1, D4, D6, D8, D1+D6, D1+D3, D2+D6, D2+D3, DO+D6, DO+D3, ADCs with DO+D4, ADCs with D2+D4, ADCs with D1+D4, ADCs with D2+D2, or ADCs with D1+D2. Thus, the "homogeneity" of an antibody-drug conjugate is used to describe the predominance of one particular type of antibody-drug conjugate (i.e., one type selected from DO, D1, D2, D4, D6, D8, D1+D6, D1+D3, D2+D6, D2+D3, DO+D6, or DO+D3 conjugates) in a given mixture of antibody-drug conjugates.

[0155] The drug-to-antibody ratio (DAR) of an ADC is the average number of drugs conjugated to each antibody. DAR significantly affects the potency of an ADC and is therefore an important characteristic for assessing the quality of the ADC. The distribution of DAR (D0, D2, D4, D6, D8) may reflect the homogeneity of the ADC.

[0156] Drug loading is expressed as the number of drug moieties per antibody molecule in an ADC molecule. In some antibody-drug conjugates, drug loading may be limited by the number of binding sites on the antibody. For example, when the attachment is a cysteine thiol, in certain exemplary embodiments described herein, drug loading ranges from 0 to 8 drug moieties per antibody.

[0157] As used herein, the term "D0" or "ADC with D0" refers to an ADC in which the number of drugs bound to a single antibody molecule is approximately zero.

[0158] As used herein, the term "D2" or "ADC with D2" refers to a DAR of about 2, which means that about two drug molecules (e.g., 1.5, 2.0, 2.5 molecules) are bound to a single antibody molecule. The drug molecules may be bound via a linker to -SH groups generated by reduction of disulfide bonds between the heavy and light chains or between the heavy and heavy chains.

[0159] As used herein, the term "D4" or "ADC with D4" refers to an ADC in which approximately four drug molecules (e.g., 3.5, 4.0, 4.5 molecules) are conjugated to one antibody molecule, where the drug molecules can be attached to four -SH groups generated by reduction of two inter-chain or intra-chain disulfide bonds.

[0160] As used herein, the term "D6" or "ADC with D6" refers to an ADC in which approximately six drug molecules (e.g., 5.5, 6.0, 6.5 molecules) are conjugated to one antibody molecule, where the drug molecules can be attached to six -SH groups generated by reduction of three disulfide bonds.

[0161] As used herein, the term "D8" or "ADC with D8" refers to an ADC in which approximately eight drug molecules (e.g., 7.5, 8.0, 8.5 molecules) are conjugated to one antibody molecule, where the drug molecules can be attached to the eight -SH groups generated by reduction of four disulfide bonds.

[0162] As used herein, the term "D1" or "ADC with D1" refers to an ADC in which one of the first thiol bridging groups carrying a first linker payload re-bridges two thiol groups of one single antibody molecule.

[0163] As used herein, the term "D3" or "ADC with D3" refers to an ADC in which three of the first thiol bridging groups carrying the first linker payload re-bridge six thiol groups of one single antibody molecule.

[0164] As used herein, the term "D1+D6" or "ADC with D1+D6" refers to an ADC in which one of the first thio bridging groups with a first linker payload re-bridges two thiol groups and six of the second linker payloads are attached to one antibody molecule, where the first and second linker payloads can be the same or different.

[0165] As used herein, the term "D1+D3" or "ADC with D1+D3" refers to an ADC in which one first thio bridging group with a first linker payload and three second thio bridging groups with a second linker payload re-bridge eight thiol groups of a single antibody molecule, where the first thio bridging group and the second thio bridging group can be the same or different, and the first linker payload and the second linker payload can be the same or different.

[0166] As used herein, the term "D2+D6" or "ADC having D2+D6" refers to an ADC in which two first linker payloads and six second linker payloads are attached to one antibody molecule, and the first and second linker payloads can be the same or different.

[0167] As used herein, the term "D2+D3" or "ADC with D2+D3" refers to an ADC in which two first linker payloads are attached to one antibody molecule and three second thio bridging groups with a second linker payload re-bridge the six thiol groups of the antibody, where the first and second linker payloads can be the same or different.

[0168] As used herein, the term "D0+D6" or "ADC with D0+D6" refers to an ADC in which one first thio bridging group re-bridging two thiol groups and six second linker payloads are attached to a single antibody molecule, or an ADC in which two end-capping reagents and six second linker payloads are attached to a single antibody molecule.

[0169] As used herein, the term "D0+D3" or "ADC with D0+D3" refers to an ADC in which one thio bridging group re-bridges two thiol groups and three of the second thio bridging groups re-bridge six thiol groups on a single antibody molecule, where the first and second thio bridging groups can be the same or different. In some embodiments, D0+D3" refers to an ADC in which two of the end-capping reagents react with two thiol groups and three of the second thio bridging groups bearing a linker payload re-bridge six thiol groups on a single antibody molecule.

[0170] As used herein, the term "D0+D4" or "ADC with D0+D4" refers to an ADC in which one of the first thio bridging groups re-bridges two thiol groups and four of the second linker payloads are attached to one antibody molecule, or an ADC in which two of the end-capping reagents and four of the second linker payloads are attached to one antibody molecule.

[0171] As used herein, the term "D1+D4" or "ADC with D1+D4" refers to an ADC in which one of the first thio bridging groups carrying a first linker payload re-bridges two thiol groups and four of the second linker payloads are attached to one antibody molecule.

[0172] As used herein, the term "D2+D4" or "ADC with D2+D4" refers to an ADC in which two first linker payloads and four second linker payloads are attached to one antibody molecule.

[0173] As used herein, the term "D2+D2" or "ADC with D2+D2" refers to an ADC in which two first linker payloads and two second linker payloads are attached to one antibody molecule.

[0174] As used herein, the term "ADC having D1+D2" or "D1+D4" refers to an ADC in which one of the first thio bridging groups with a first linker payload re-bridges two thiol groups and two of the second linker payloads are attached to one antibody molecule.

[0175] As used herein, the term "D4+D2" or "ADC with D4+D2" refers to an ADC in which four first linker payloads and two second linker payloads are attached to one antibody molecule.

[0176] As used herein, the term "D4+D4" or "ADC with D4+D4" refers to an ADC in which four of the first linker payloads and four of the second linker payloads are attached to one antibody molecule.

[0177] As used herein, the term "homogeneity of ADC with Dx" refers to the weight content of ADC with Dx in all ADCs produced by the method, where Dx can be D1, D2, D1+D6, D1+D3, D2+D6, D2+D3, D0+D6, D0+D3, D0+D4, D2+D4, D1+D4, D2+D2, or D1+D2.

[0178] As used herein, the term "about" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the term "about" preceding a numerical value indicates a value that is plus or minus a range of 50%, 30%, 15%, 10%, 5%, or 1%.

[0179] In some embodiments, a compound having formula (I) provided above or a composition provided above can be used to prepare an ADC with improved homogeneity.

[0180] In some embodiments, the disclosure provides use of a compound having Formula (I) or a composition provided above in the preparation of an ADC having D2, an ADC having D1, an ADC having D2+D6, an ADC having D2+D3, an ADC having D1+D6, an ADC having D1+D3, an ADC having D0+D6, an ADC having D0+D3, an ADC having D0+D4, an ADC having D2+D4, an ADC having D1+D4, an ADC having D2+D2, or an ADC having D1+D2.

[0181] In some embodiments, the ADC comprises a D2 content of at least 53% of the total combined weight of DO, D2, D4, D6, and D8. In some embodiments, the ADC comprises a D2 content of up to 55% of the total combined weight of DO, D2, D4, D6, and D8. In some embodiments, the ADC comprises a D2 content of up to 60%, 65%, 70%, 75%, 80%, 84%, 87%, 89%, 90%, 91%, 92%, or 95% of the total combined weight of DO, D2, D4, and D8.

[0182] In some embodiments, the homogeneity of the ADC with D1, the ADC with D2+D6, and the ADC with D0+D6 is up to 80%.

[0183] In some embodiments, the content of ADC with D2+D2 is generally up to 68% or 70%.

[0184] In some embodiments, the content of ADC with D1+D2 is generally up to 80% or 83%.

[0185] In some embodiments, the content of ADC with D0+D4 is generally at most 55%, 61%, or 65%.

[0186] In some embodiments, the content of ADC with D2+D4 is generally up to 70%, 75%, or even 78% or 80%.

[0187] In some embodiments, the content of ADC with D1+D4 is generally up to 60%, 65%, or 70%.

[0188] Method for preparing antibodies having site-specific modification with thiol groups The present disclosure also provides methods for preparing antibodies with site-specific modification of thiol groups, wherein thiol groups are reduced from interchain disulfide bonds within the antibody, the methods comprising using the compounds or salts, solvates, stereoisomers and transition metal ions described above, or the compositions described above.

[0189] In some embodiments, the number of thiol groups is 1, 2, 3, 4, 5, 6, 7, or 8.

[0190] In some embodiments, the number of thiol groups is two or eight.

[0191] In some embodiments, interchain disulfide bonds link the two upper heavy chains in the hinge region, or the heavy and light chains in the Fab region.

[0192] In some embodiments, interchain disulfide bonds link the two heavy chains in the hinge region and the heavy and light chains in the Fab region.

[0193] In some embodiments, site-specific modification does not refer to antibody techniques, enzymatic techniques, and glycosylation.

[0194] In some embodiments, the method comprises the steps of: (a) incubating the above-described compound, or a salt, solvate, or stereoisomer thereof, which acts as a first reducing agent, with the antibody in the presence of a transition metal ion in a first buffer system to selectively reduce interchain disulfide bonds within the antibody; or The compound described above acts as a first reducing agent and is incubated with the antibody in a first buffer system to selectively reduce the interchain disulfide bonds within the antibody. (b) introducing a metal chelator and modifying reagent 1 to react with the reduced thiol group obtained in step (a), where modifying reagent 1 is an end-capping reagent, a first linker payload, or a first thio-bridging reagent, and optionally, the first thio-bridging reagent has a first linker payload or a reactive group;

[0195] In some embodiments, when the first thio bridging reagent has a reactive group, step (b) comprises the steps of:

[0196] introducing a first thio bridging reagent having a metal chelator and a reactive group to re-crosslink the reduced thiol groups obtained in step (a), and then incubating the first linker payload in a first buffer system to react with the reactive group of the thio bridging group.

[0197] In some embodiments, the method further comprises the steps of: (c) Incubating the reaction product obtained in step (b) with a second reducing agent in a second buffer system to reduce interchain disulfide bonds in the reaction product and, optionally, introduce transition metal ions. (d) introducing the incubation product from step (c) and modifying reagent 2 and reacting with the reduced thiol group from step (c). Optionally, introducing a metal chelator. Here, modifying reagent 2 is a second linker payload or a second thio bridging reagent. Optionally, the second thio bridging reagent has a second linker payload or a reactive group.

[0198] In some embodiments, when the second thio bridging reagent has a reactive group, step (d) comprises:

[0199] introducing the reaction product obtained in step (c) and a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol group obtained from step (c), optionally introducing a metal chelator, and then incubating a second linker payload in a second buffer system to react with the reactive group of the thio bridging group.

[0200] In some embodiments, if transition metal ions are introduced in step (c), a metal chelator is introduced in step (d) to scavenge excess transition metal ions.

[0201] As used herein, the terms "bear," "bears," or "bearing" refer to "having" or "having."

[0202] In some embodiments, a first reducing agent selectively reduces one interchain disulfide bond of an antibody with a transition metal ion, and optionally, a second reducing agent reduces the remaining three interchain disulfide bonds without a transition metal ion, or a second reducing agent reduces one or two of the interchain disulfide bonds with a transition metal ion.Antibodies having thiol group site-specific modifications, such as ADCs having D1 or ADCs having D2, can be prepared by a method comprising steps (a) and (b). An antibody having a thiol group site-specific modification, such as an ADC having D1+D6, an ADC having D1+D3, an ADC having D2+D6, an ADC having D2+D3, an ADC having D0+D6, an ADC having D0+D3, an ADC having D0+D4, an ADC having D2+D4, an ADC having D1+D4, an ADC having D2+D2, or an ADC having D1+D2, can be prepared by a method comprising steps (a), (b), (c), and (d).

[0203] In some embodiments, salt refers to an acid addition salt or a base addition salt.

[0204] In some embodiments, acid addition salts may be formed using inorganic and organic acids. Inorganic acids from which salts are derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Organic acids from which salts are derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfasalicylic acid, etc.

[0205] In some embodiments, base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include those from Groups 1 and 2 of the periodic table. In one embodiment, salts are derived from lithium, sodium, potassium, calcium, magnesium, etc. Organic bases from which salts can be derived include, for example, primary amines, secondary amines, tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Specific organic amines include isopropylamine, benzathine, cholinamide, diethanolamine, diethylamine, lysine, meglumine, piperazine, tromethamine, etc.

[0206] In some embodiments, a first reducing agent and a transition metal ion are used together in the method for preparing an antibody having a thiol group site-specific modification. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:250. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:200. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:60. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion is 1:1 to 1:60. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion is 1:2 to 1:60. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion is 1:6 to 1:16. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion is 1:190, 1:1 to 1:180, 1:170, 1:160, 1:1 to 1:150, 1:140, 1:1 to 1:130, 1:120, 1:1 to 1:100, 1:1 to 1:80, or 1:1 to 1:70.

[0207] In some embodiments, the molar ratio of first reducing agent to antibody is 3:1, 0.5:1, or 3:1 to 1:1. In some embodiments, the molar ratio of first reducing agent to antibody is 2:1 to 1:1. In some embodiments, the molar ratio of first reducing agent to antibody is 1:1, 1.5:1, 1.8:1, 2:1, 2.5:1, 2.8:1, or 3:1.

[0208] In some embodiments, the concentration of the first reducing agent is not particularly limited, as long as the concentrations of the transition metal ion and the antibody are increased or decreased at an equal rate. In some embodiments of the present application, the concentration of the first reducing agent is 0.01 mM to 0.2 mM. In some embodiments of the present application, the concentration of the first reducing agent is 0.02 mM to 0.15 mM. In some embodiments of the present application, the concentration of the first reducing agent is 0.05 mM to 0.1 mM. In some embodiments of the present application, the concentration of the first reducing agent is 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.10 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, or 0.20 mM.

[0209] In some embodiments, there is no particular limit to the concentration of the transition metal ion in step (a), so long as the concentrations of the first reducing agent and the antibody are increased or decreased in equal proportions.

[0210] In some embodiments of the present application, there is no particular limitation on the concentration of the antibody in step (a), as long as the concentrations of the first reducing agent and the transition metal ion are increased or decreased at an equal rate.

[0211] The reducing agent selectively reduces disulfide bonds in the first buffer system, and the first buffer system and the second buffer system are independently selected from the group consisting of HEPES buffer, histidine buffer, PBS, PB, MES buffer, BES buffer, MOPS buffer, Bis-Tris buffer, acetate buffer, DIPSO buffer, MOPSO buffer, TES buffer, ACES buffer, MOBS buffer, TAPSO buffer, ADA buffer, PIPES buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer, or Tris buffer.

[0212] As used herein, the term "HEPES buffer" refers to "4-hydroxyethylpiperazine ethanesulfonic acid buffer."

[0213] As used herein, the term "PBS" refers to "phosphate buffered saline."

[0214] As used herein, the term "PB'" refers to "phosphate buffer."

[0215] As used herein, the term "MES buffer" refers to 2-(N-morpholino)ethanesulfonic acid buffer.

[0216] As used herein, the term "BES buffer" refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer.

[0217] As used herein, the term "MOPS buffer" refers to 3-morpholinopropanesulfonic acid buffer.

[0218] As used herein, the term "bis-tris buffer" refers to bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane buffer.

[0219] As used herein, the term "DIPSO buffer" refers to 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer.

[0220] As used herein, the term "MOPSO buffer" refers to 3-(N-morpholino)-2-hydroxy-1-propanesulfonic acid buffer.

[0221] As used herein, the term "TES buffer" refers to 2-[tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid buffer.

[0222] As used herein, the term "ACES buffer" refers to N-(carbamoylmethyl)taurine buffer.

[0223] As used herein, the term "MOBS buffer" refers to 4-(N-morpholino)butyric acid buffer.

[0224] As used herein, the term "TAPSO buffer" refers to 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid buffer.

[0225] As used herein, the term "ADA buffer" refers to N-(carbamoylmethyl)iminodiacetic acid buffer.

[0226] As used herein, the term "PIPES buffer" refers to piperazine-1,4-bisethanesulfonic acid buffer.

[0227] As used herein, the term "BTP buffer" refers to a bis-tris-propane buffer.

[0228] As used herein, the term "HEPSO buffer" refers to N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid buffer.

[0229] As used herein, the term "POPSO buffer" refers to piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) buffer.

[0230] As used herein, the term "EPPS buffer" refers to 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer.

[0231] As used herein, the term "Tris buffer" refers to tris(hydroxymethyl)aminomethane buffer.

[0232] In some embodiments, the first buffer system and the second buffer system are independently selected from the group consisting of Bis-Tris buffer, PIPES buffer, MOPS buffer, BES buffer, HEPES buffer, ADA buffer, PB buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer, or MES buffer.

[0233] In some embodiments, the first buffer system and the second buffer system are MES buffer.

[0234] In some embodiments, the concentrations of the first buffer system and the second buffer system are between 10 and 100 mM (mmol / L).

[0235] In some embodiments, the pH values of the first and second buffer systems are between 5.5 and 8.0. In some embodiments, the pH values of the buffer systems are between 5.8 and 8.0. In some embodiments, the pH values of the first and second buffer systems are between 6.0 and 7.4. In some embodiments, the pH values of the first and second buffer systems are between 6.7 and 7.4. In some embodiments, the pH values of the first and second buffer systems are 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, or 7.4.

[0236] In some embodiments, the first buffer system and the second buffer system are MES buffers, and the MES buffers have a pH value of 5.8 to 6.7.

[0237] The term "transition metal ions" refers to elements from groups 4 to 12, justified by their typical chemical properties: a wide range of complex ions in various oxidation states, colored complexes, and catalytic properties as elements or ions (or both). Sc and Y from group 3 are also generally recognized as transition metals.

[0238] In some embodiments, the transition metal ion is Zn 2+ , Cd 2+ , Hg 2+ , Ni2+ , Co 2+ or a combination thereof.

[0239] In some embodiments, the transition metal ion is Zn 2+ is.

[0240] In some embodiments, the salt of the transition metal ion is not particularly limited, as long as the transition metal ion is soluble in the reaction solution and free transition metal ions are released into the reaction solution. In some embodiments, the salt of the transition metal ion is a chloride, nitrate, sulfate, acetate, iodide, bromide, formate, or tetrafluoroborate.

[0241] In some embodiments, Zn 2+ The salt is ZnCl 2+ , Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, or zinc tetrafluoroborate. In some embodiments of the present application, Zn 2+ The salt is ZnCl2.

[0242] Those skilled in the art will understand that the incubation temperature and incubation time in step (a) depend on the specific antibody to be conjugated. In some embodiments, the incubation temperature in step (a) is 0°C to 37°C, 0°C to 25°C, or 0°C to 15°C, and the incubation time is 0.2 hours to 24 hours. Optionally, the incubation temperature in step (a) is 0°C to 10°C, and the incubation time is 2 hours to 16 hours.

[0243] In some embodiments, the incubation temperature in step (a) is 0° C. to 15° C., 0° C. to 10° C., 0° C. to 8° C., or 0° C. to 6° C. In some embodiments, the incubation temperature in step (a) is 4° C., 8° C., 12° C., 15° C., 18° C., 24° C., 30° C., 35° C., or 37° C.

[0244] In some embodiments, the incubation time in step (a) is 0.5 to 24 hours, 0.5 to 20 hours, 0.5 to 16 hours, 0.5 to 12 hours, 0.5 to 8 hours, or 0.5 to 6 hours. In some embodiments, the incubation time is 0.25 hours, 0.3 hours, 0.5 hours, 0.7 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0245] In some embodiments, the molar ratio of the first reducing agent to the antibody is 2:1 to 3:1, and the incubation time in step (a) is 0.5 to 9 hours. In some embodiments, the molar ratio of the first reducing agent to the antibody is 2.8:1 to 3:1, and the incubation time in step (a) is 1 to 9 hours. In some embodiments, the molar ratio of the first reducing agent to the antibody is 2.2:1, 2.4:1, 2.6:1, 2:8.1, or 3:1, and the incubation time in step (a) is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 9.5 hours.

[0246] In some embodiments, the incubation temperature in step (a) is 0°C to 25°C, and the incubation time in step (a) is 0.5 hours to 24 hours. In some embodiments, the incubation temperature in step (a) is 0°C to 15°C, and the incubation time in step (a) is 0.5 hours to 24 hours. In some embodiments, the incubation temperature in step (a) is 0°C to 10°C, and the incubation time in step (a) is 2 hours to 16 hours.

[0247] In some embodiments, in step (c), the second reducing agent is not particularly limited as long as it can reduce the interchain disulfide bond in the antibody. In some embodiments, the second reducing agent is the same as the first reducing agent. In some embodiments, the second reducing agent is TCEP, tris(3-hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT). In some embodiments, the second reducing agent is TCEP.

[0248] In some embodiments, the second reducing agent used in step (c) is not particularly limited as long as it is capable of reducing interchain disulfide bonds within the antibody. In some embodiments of the present application, the molar ratio of second reducing agent to antibody is 3:1 to 20:1, 3:1 to 10:1, 4:1 to 10:1, 5:1 to 9:1, 6:1 to 9:1, or 6:1 to 8:1. In some embodiments, the molar ratio of second reducing agent to antibody is 20:3.

[0249] In some embodiments, the incubation time of the second reducing agent in step (c) is 0.5 hours to 24 hours, or 5 hours to 20 hours. In some embodiments, the incubation time of the second reducing agent in step (c) is 6 hours to 18 hours, 8 hours to 18 hours, 8 hours to 15 hours, or 8 hours to 12 hours. In some embodiments, the incubation time of the second reducing agent in step (c) is 8 hours to 12 hours.

[0250] In some embodiments, in step (c), two interchain disulfide bonds are selectively reduced by introducing a transition metal ion. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 20:1, and / or the incubation time is 1 hour to 24 hours. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05, 1:0.08, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20. In some embodiments, in step (c), the molar ratio of the second reducing agent to the antibody is 2.5:1, 3:1, 5:1, 7:1, 9:1, 11:1, 13:1, 15:1, 17:1, 19:1, or 20:1. In some embodiments, in step (c), the incubation time is 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 3:1 to 15:1, and / or the incubation time is 1 hour to 12 hours. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 15:1, and / or the incubation time is 12 hours to 24 hours.

[0251] In some embodiments, in step (c), one interchain disulfide bond is selectively reduced by introducing a transition metal ion. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.5 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 to 24 hours. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.5, 1:1, 1:4, 1:8, 1:12, 1:24, 1:30, 1:40, 1:50, 1:50, 1:70, 1:80, 1:90, or 1:100. In some embodiments, in step (c), the molar ratio of second reducing agent to antibody is 0.8:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, or 2.5:1. In some embodiments, in step (c), the incubation time is 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours. In some embodiments, in step (c), the molar ratio of second reducing agent to transition metal ion is 1:0.5 to 1:100 and / or the molar ratio of second reducing agent to antibody is 0.8:1 to 2:1, or the incubation time is 0.5 hours to 24 hours. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.5 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 2:1 to 2.5:1, or the incubation time is 1 hour to 9 hours.

[0252] In some embodiments, the incubation temperature of the second reducing agent in step (c) is 0° C. to 37° C., or 5° C. to 30° C. In some embodiments, the incubation temperature of the second reducing agent in step (c) is 10° C. to 30° C., 15° C. to 30° C., 20° C. to 30° C., or 25° C. to 30° C. In some embodiments, the incubation temperature of the second reducing agent in step (c) is 25° C.

[0253] In some embodiments, the reaction temperature with the reduced thiol group in step (b) and step (d) is 4° C. to 40° C., 10° C. to 40° C., 10° C. to 35° C., 10° C. to 30° C., 10° C. to 25° C., 15° C. to 35° C., 20° C. to 30° C., 4° C. to 37° C., 20° C. to 30° C., or 20° C. to 25° C. In some embodiments, the reaction temperature with the reduced thiol group in step (b) and step (d) is 24° C.

[0254] In some embodiments, the reaction time with the reduced thiol group in step (b) and step (d) is 0.5 hours to 6 hours, 0.5 hours to 5 hours, 0.5 hours to 4 hours, 0.5 hours to 3 hours, 0.5 hours to 2 hours, or 0.5 hours to 1 hour. In some embodiments, the reaction time with the reduced thiol group in step (b) and step (d) is 0.5 hours, 1 hour, 2 hours, or 3 hours.

[0255] In some embodiments, the reaction temperature and reaction time with the reduced thiol group in step (b) and step (d) are independent.

[0256] In some embodiments, in steps (b) and (d), the reaction temperature with the reactive group is 10° C. to 37° C., 20° C. to 30° C., 10° C. to 30° C., 15° C. to 30° C., or 25° C. to 30° C. In some embodiments, in steps (b) and (d), the reaction temperature with the reactive group is 25° C.

[0257] In some embodiments, the reaction time with the reactive group in step (b) and step (d) is 2 to 12 hours, 2 to 10 hours, 4 to 10 hours, 6 to 10 hours, or 8 to 10 hours.

[0258] In some embodiments, in steps (b) and (d), the reaction time with the reactive group is 8 hours.

[0259] In some embodiments, the reaction temperature and reaction time with the reactive group in step (b) and step (d) are independent.

[0260] In some embodiments, the metal chelator is capable of capturing excess transition metal ions in step (b). In some embodiments, the specificity of the metal chelator is not important, so long as it is capable of capturing excess transition metal ions and does not affect the reduction of disulfide bonds in the antibody. In some embodiments, the metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), citric acid (CA), tartaric acid (TA), gluconic acid (GA), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA).

[0261] In some embodiments, the metal chelator is selected from the group consisting of EDTA, NTA, or DTPA. In some embodiments, the metal chelator is EDTA.

[0262] In some embodiments, in step (b), the molar ratio of metal chelator to antibody is 1:1 to 100:1, 10:1 to 100:1, 20:1 to 100:1, 20:1 to 80:1, 20:1 to 70:1, 30:1 to 60:1, 40:1 to 50:1, 35:1 to 60:1, or 40:1 to 55:1.

[0263] In some embodiments, in step (d), the molar ratio of metal chelator to antibody is 1:1 to 100:1, 1:1 to 60:1, 1:1 to 50:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 8:1, 1:1 to 6:1, 1:1 to 5:1, 2:1 to 8:1, or 2:1 to 6:1.

[0264] In some embodiments, the excess metal chelator and the complex of the metal chelator with the transition metal ion are filtered by dialysis, ultrafiltration, or gel filtration.

[0265] In some embodiments, in step (b), Modification Reagent 1 is in excess relative to the amount of antibody.

[0266] In some embodiments, in step (c), the molar ratio of the first reducing agent to the antibody is 5:1, 1:1, 2:1, 1:1, 1.5:1, 1:1, 1.2:1, 1:1, 1.1:1, or 1:1. In some embodiments, in step (b), the molar ratio of the first Thiobriguet reagent to the antibody is 1.05:1.

[0267] In some embodiments, in step (b), when the first linker payload reacts with a reduced thiol group, the molar ratio of first linker payload to antibody is 5:1, 1:1, 2:1, 10:1, 3:1, 10:1, 2:1, 9:1, 5:1, or 7:1. In some embodiments, when the first linker payload reacts with a reduced thiol group in step (b), the molar ratio of first linker payload to antibody is 5:1.

[0268] In some embodiments, in step (b), when the first linker payload reacts with the reactive group in the first thio bridging reagent, the molar ratio of the first linker payload to the antibody is 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, or 2:1 to 1:1. In some embodiments, in step (b), the molar ratio of the first linker payload to the antibody is 5:3.

[0269] In some embodiments, in step (d), modifying reagent 2 is in excess relative to the amount of antibody.

[0270] In some embodiments, in step (d), the molar ratio of second thio bridging reagent to antibody is 5:1 to 1:1, 5:1 to 3:1, 4:1 to 3:1, 4:1 to 3.2:1, or 4:1 to 3.5:1. In some embodiments, in step (b), the molar ratio of second thio bridging reagent to antibody is 5:1, 4.5:1, 4:1, 3.8:1, 3.5:1, or 3.2:1.

[0271] In some embodiments, when the second linker payload reacts with a reduced thiol group in step (d), the molar ratio of second linker payload to antibody is 20:1 to 2:1, 20:1 to 6:1, 18:1 to 8:1, 16:1 to 8:1, 14:1 to 8:1, or 12:1 to 10:1. In some embodiments, when the second linker payload reacts with a reduced thiol group in step (d), the molar ratio of second linker payload to antibody is 35:3.

[0272] In some embodiments, in step (d), when a second linker payload reacts with a reactive group in a second thio bridging reagent, the molar ratio of second linker payload to antibody is 10:1 to 1:1, 10:1 to 2:1, 10:1 to 3:1, 9:1 to 3:1, 8:1 to 3:1, 7:1 to 3:1, 6:1 to 3:1, 5:1 to 3:1, or 4:1 to 3:1.

[0273] In some embodiments, the method further comprises the steps of: Optionally, introducing a compound containing at least one thiol group to consume excess first linker payload in step (b) and / or second linker payload in step (d). A step of purifying and recovering the antibody obtained by the thiol group site-specific modification in step (b) and / or step (d).

[0274] In some embodiments, the compound that consumes the excess first linker payload and / or second linker payload is not particularly limited, as long as the compound contains at least one thiol group. In some embodiments, the compound is cysteine.

[0275] In step (b) and / or step (d), the resulting antibodies having thiol group site-specific modifications can be purified and recovered to increase the content of antibodies having thiol group site-specific modifications. In some embodiments, the resulting antibodies having thiol group site-specific modifications are purified by a desalting column, size exclusion chromatography, ultrafiltration, dialysis, and / or similar methods. In some embodiments, the resulting antibodies having thiol group site-specific modifications are purified by a desalting column. If necessary, further concentration (e.g., D2) may be applied using hydrophobic interaction chromatography (HIC).

[0276] In some embodiments, the antibody is not particularly limited. Depending on the antigen associated with the disease, one skilled in the art can select an appropriate antibody useful in the bioconjugation process of the present application. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody, or a multispecific antibody.

[0277] As used herein, the term "antibody" refers to an immunoglobulin that binds to a specific antigen. Naturally occurring, intact antibodies are composed of two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, and each light chain consists of a variable region and a constant region. Heavy chains in vertebrates are classified into one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM.

[0278] In some embodiments, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof.

[0279] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody made by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. The definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0280] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising amino acid residues of the heavy chain variable region (HVR) of non-human origin and amino acid residues of the FRs of human origin. In one embodiment, a humanized antibody comprises substantially all or at least one, typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0281] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0282] As used herein, the term "hinge region" refers to an antibody heavy chain molecule comprising the portion connecting the CH1 and CH2 domains. The hinge region comprises approximately 25 amino acid residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0283] As used herein, the term "Fab fragment" refers to the structural region of an antibody that can bind to an antigen. It consists of an intact light chain (variable and constant regions) and a portion of the heavy chain structure (variable and constant region fragments), with the light and heavy chains linked by disulfide bonds. Fab fragments are obtained by protease digestion of a full-length antibody. Papain degrades human immunoglobulin G into two Fab fragments and one Fc fragment. Pepsin degrades IgG into F(ab')2 and pFc' fragments. The F(ab')2 fragments are further reduced to form two Fab' fragments.

[0284] As used herein, the term "Fc region" refers to a monomeric, dimeric, or heterodimeric protein having at least an immunoglobulin CH2 and CH3 domain. The CH2 and CH3 domains may form at least a portion of the dimeric region of a protein / molecule (e.g., an antibody).

[0285] In some embodiments, an antibody refers to an immunoglobulin, a molecule that contains an antigen-binding site that immunospecifically binds to an antigen. In some embodiments, the antibody class is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the antibody class is IgG.

[0286] In some embodiments, the class of the antibody is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is IgG1 or IgG4.

[0287] In some embodiments, the antibody is wild-type. As used herein, the term "wild-type" refers to naturally occurring and free of mutations.

[0288] In some embodiments, the antibody is an engineered antibody with two amino acid substitutions of the two interchain cysteines that form one interchain disulfide bond in the hinge region.

[0289] In some embodiments, the amino acid substitutions are selected from cysteine to alanine, leucine, arginine, lysine, asparagine, methionine, aspartic acid, phenylalanine, praline, glutamine, serine, glutamic acid, threonine, glycine, tryptophan, histidine, tyrosine, isoleucine, or valine, respectively.

[0290] In some embodiments, the amino acid substitution is selected from cysteine to asparagine, glutamine, serine, threonine, or tyrosine, respectively.

[0291] In some embodiments, the amino acid substitution is selected from cysteine to serine.

[0292] In some embodiments, the antibody comprises at least one mutation in the Fc region, hi some embodiments, the at least one mutation modulates effector function or reduces or eliminates Fc-g receptor binding.

[0293] In some embodiments, the one or more mutations stabilize the antibody and / or increase half-life. In some examples, the one or more mutations modulate Fc receptor interactions, reducing or eliminating Fc effector functions such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In further examples, the one or more mutations modulate glycosylation.

[0294] In some embodiments, the one or more mutations are located in the Fc region. In some examples, the Fc region comprises mutations at residue positions L234, L235, or a combination thereof. In some examples, the mutations comprise L234 and L235. In some examples, the mutations comprise L234A and L235A. In some examples, the residue positions are relative to IgG1.

[0295] In some embodiments, the Fc region comprises mutations at residue positions L234, L235, D265, N21, K46, L52, or P53, or a combination thereof. In some examples, the mutations comprise L234 and L235 in combination with mutations at residue positions K46, L52, or P53. In some examples, the residue positions are relative to IgG1.

[0296] In some embodiments, the Fc region comprises mutations at L234, L235, and K46. In some examples, the Fc region comprises mutations at L234, L235, and L52. In some examples, the Fc region comprises mutations at L234, L235, and P53. In some examples, the Fc region comprises mutations at D265 and N21. In some examples, residue positions are relative to IgG1.

[0297] In some examples, the Fc region comprises L234A, L235A, D265A, N21G, K46G, L52R, or P53G, or a combination thereof. In some examples, the Fc region comprises L234A and L235A in combination with K46G, L52R, or P53G. In some examples, the Fc region comprises L234A, L235A, and K46G. In some examples, the Fc region comprises L234A, L235A, and L52R. In some examples, the Fc region comprises L234A, L235A, and P53G. In some examples, the Fc region comprises D265A and N21G. In some examples, residue positions are relative to IgG1.

[0298] In some embodiments, the Fc region comprises mutations at residue positions L233, L234, D264, N20, K45, L51, or P52. In some examples, the Fc region comprises mutations at L233 and L234 in combination with mutations at residue positions K45, L51, or P52. In some examples, the Fc region comprises mutations at L233, L234, and K45. In some examples, the Fc region comprises mutations at L233, L234, and L51. In some examples, the Fc region comprises mutations at L233, L234, and K45. In some examples, the Fc region comprises mutations at L233, L234, and P52. In some examples, the Fc region comprises mutations at D264 and N20. In some examples, positions equivalent to residues L233, L234, D264, N20, K45, L51, or P52 in an IgG1, IgG2, IgG3, or IgG4 framework are contemplated.

[0299] In some embodiments, the Fc region comprises L233A, L234A, D264A, N20G, K45G, L51R, or P52G. In some examples, the Fc region comprises L233A and L234A. In some examples, the Fc region comprises L233A and L234A in combination with K45G, L51R, or P52G. In some examples, the Fc region comprises L233A, L234A, and K45G. In some examples, the Fc region comprises L233A, L234A, and L51R. In some examples, the Fc region comprises L233A, L234A, and K45G. In some examples, the Fc region comprises L233A, L234A, and P52G. In some examples, the Fc region comprises D264A and N20G. In some instances, residue positions are relative to IgG1.

[0300] In some embodiments, the human IgG constant region is selected from the group consisting of, for example, Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2):181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan.Enzyme Regul., 48:152-164; Alegre et al., 1992 J Immunol, 148:3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11. Amino acid modifications are made to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0301] In some embodiments, the IgG1, IgG2, IgG3, or IgG4 antibody is a human or humanized antibody. IgG1, IgG2, IgG3, or IgG4 information can be obtained from NCBI or UniProt (https: / / www.uniprot.org / ).

[0302] In some embodiments, the antibody is a bispecific antibody. In some embodiments of the present application, the antibody is an IgG1-like bispecific antibody.

[0303] In some embodiments, one skilled in the art can select an appropriate method for preparing bispecific antibodies. In some embodiments of the present application, bispecific antibodies can be synthesized using a variety of techniques, including knobs-into-hole technology (Ridgway JBB, Presta LG, Paul C. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization[J]. Protein Engineering(7):617(2023-08-11).), format chain exchange (FORCE) technology, common light chain format technology (De Nardis C, Hendriks LJA, Poirier E, et al. A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1[J]. Journal of Biological Chemistry, 2017:jbc.M117.793497.), controlled Fab arm exchange technology (Yanakieva De, Pekar L, Evers A, et al. Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities[J]. MABS, 2022, 14(1), e2018960), CrossMAb technology (Klein C, Schaefer W, Regula J T. The use of CrossMAb technology for the generation of bi- and multispecific antibodies[J].MABS, 2016, 8(6), P1010-P1020.), or a combination thereof.

[0304] As used herein, the term "knobs-into-holes" is used in its broadest sense and encompasses a variety of situations, such as the CH1 domain of one heavy chain having a knob mutation and the CH1 domain of the other heavy chain having a hole mutation, the CH2 domain of one heavy chain having a knob mutation and the CH2 domain of the other heavy chain having a hole mutation, and / or the CH3 domain of one heavy chain having a knob mutation and the CH3 domain of the other heavy chain having a hole mutation. For example, "knobs-into-holes" can generally refer to modifications within the interface between two antibody heavy chains in the CH3 domain. i) In the CH3 domain of one heavy chain (first CH3 domain), an amino acid residue is replaced with another amino acid residue having a large side chain, thereby forming a protrusion ("knob") at the interface of the first CH3 domain. ii) In the CH3 domain of the other heavy chain (the second CH3 domain), an amino acid residue is replaced with another amino acid residue having a smaller side chain, thereby forming a cavity ("hole") in the interface of the second CH3 domain into which the knob of the first CH3 domain can be positioned.

[0305] In some embodiments, the antibody is selected from any one of a cytotoxic antibody, an inhibitor of cell proliferation, a modulator of cell activation and interaction, a modulator of the human immune system, a neutralizing antigen, an antibody immunologically specific for a viral antigen, or an antibody immunologically specific for a microbial antigen.

[0306] In some embodiments, the antibody may be a target-specific antibody. In some embodiments, the antibody is selected from the group consisting of, but not limited to, an anti-HER2 antibody, an anti-FAP antibody, an anti-OX-40 antibody, an anti-41BB antibody, an anti-angiogenic factor 2 antibody, an anti-IL-4Rα antibody, an anti-BCMA antibody, an anti-Blys antibody, an anti-BTNO2 antibody, an anti-C5 antibody, an anti-CD122 antibody, an anti-CD13 antibody, an anti-CD133 antibody, an anti-CD137 antibody, an anti-CD138 antibody, an anti-CD16a antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD ... antibody, an anti-CD133 antibody, an anti-CD137 antibody, an anti-CD138 antibody, an anti-CD16a antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD13 antibody, an anti-CD13 antibody, an anti-CD13 antibody, an anti-CD13 antibody, an anti-CD13 antibody, an anti-CD13 antibody, an anti-CD1 body, anti-CD27 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD47 antibody, anti-CD8 antibody, anti-CD79 antibody, anti-CEA antibody, anti-CGPR / CGRPR antibody, anti-CSPGs antibody, anti-CTLA4 antibody, anti-CTLA-4 domain antibody, anti-DLL-4 antibody, anti-EGFR antibody, anti-EpCAM antibody, anti-factor IXa antibody, anti-factor X antibody, anti-GITR antibody, Anti-GP130 antibody, anti-Her3 antibody, anti-HSG antibody, anti-ICOS antibody, anti-IGF1 antibody, anti-IGF1 / 2 antibody, anti-IGF-1R antibody, anti-IGF2 antibody, anti-IGFR antibody, anti-IL-1 antibody, anti-IL-12 antibody , anti-IL-12p40 antibody, anti-IL-13 antibody, anti-IL-17A antibody, anti-IL-1β antibody, anti-IL-23 antibody, anti-IL-5 antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-Lag-3 antibody, anti-LAG3 antibody, anti MAG antibody, anti-Met antibody, anti-NgR antibody, anti-NogoA antibody, anti-OMGp antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PDGFR antibody, anti-PDL-1 antibody, anti-PSMA antibody, anti-RGMA antibody, anti-RGMB The antibody may be an anti-SARS-CoV-2 antibody, an anti-Te38 antibody, an anti-TIM-3 antibody, an anti-TNF antibody, an anti-TNFα antibody, an anti-TROP-2 antibody, an anti-TWEAK antibody, an anti-VEGF antibody, or an anti-VEGFR antibody.

[0307] In some embodiments, the antibodies are target specific and target HER2 (human epidermal growth factor receptor 2), TROP2 (TACSTD2, tumor-associated calcium signaling factor 2), BCMA (TNFRSF17, TNF receptor superfamily member 17).

[0308] In some embodiments, the antibody can be trastuzumab, sacituzumab, belantamab, risankizumab, eptanumab, teprotumab, polatuzumab, tafasitamab, rovelizumab, romosozumab, dostallimub, enfortum, or ublituximab. In some embodiments, the antibody is trastuzumab, sacituzumab, or belantamab.

[0309] In some embodiments, the antibodies are commercially available or are produced by any method known to one of skill in the art.

[0310] In some embodiments, the first thio bridging reagent and the second thio bridging reagent independently comprise at least two substituents that allow for re-crosslinking of thiol groups.

[0311] In some embodiments, the first thio bridging reagent and the second thio bridging reagent are each selected from the group consisting of, but not limited to: [ka]

[0312] In some embodiments, the reactive groups include azide and / or dibenzocyclooctyne (DBCO), respectively.

[0313] In some embodiments, the thio bridging reagent and the reactive group are attached by an alkyl group or polyethylene glycol (PEG).

[0314] In some embodiments, the first thio bridging reagent having a reactive group and the second thio bridging reagent having a reactive group are each selected from the group consisting of, but not limited to: [ka] wherein n is 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3; optionally, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0315] In some embodiments, the first thio bridging reagent having a reactive group and the second thio bridging reagent having a reactive group are dibromomaleimide-PEG4-N3 having the formula: [ka]

[0316] In some embodiments, the method is characterized in that the linkers of the first linker-payload and the second linker-payload are selected from any one of a group that can be linked at one end to a reduced thiol group of an antibody or a reactive group of a thio bridging reagent and at the other end to a payload.

[0317] As used herein, the term "linker" refers to a substituted molecule that contains at least two substituents, one of which can be covalently attached to a drug molecule and the other of which can be covalently attached to a reactive group on an antibody or a thiobridging reagent.

[0318] In some embodiments, when the first linker payload and / or the second linker payload react with a reduced thiol group, the linker of the first linker payload and the linker of the second linker payload, respectively, comprise a cleavable linker or a non-cleavable linker. Cleavable linkers can be chemically labile or enzyme-labile. Due to their high plasma stability and the selectivity and efficiency of intracellular cleavage, enzyme-labile linkers are widely selected as potential cleavable linkers for ADCs. In some embodiments, the enzyme-labile linker comprises a structure comprising -maleimidocaproyl-(-MC-), -maleimidocaproyl-peptide moiety-(-MC-peptide moiety-), -p-aminobenzyl alcohol-(-PAB-), or -peptide moiety-. In some embodiments, the peptide moiety is a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

[0319] In some embodiments, the dipeptide can be, but is not limited to, valine-alanine (VA), valine-citrulline (VC), alanine-asparagine (AD), alanine-phenylalanine (AF), phenylalanine-lysine (FK), alanine-lysine (AK), alanine-valine (AV), valine-lysine (VK), lysine-lysine (KK), phenylalanine-citrulline (FC), leucine-citrulline (LC), isoleucine-citrulline (IC), tryptophan-citrulline (WC), or phenylalanine-alanine (FA).

[0320] In some embodiments, the tripeptide can be, but is not limited to, alanine-alanine-asparagine (AAD), glycine-valine-citrulline (GVC), glycine-glycine-glycine (GGG), phenylalanine-phenylalanine-lysine (FFK), glutamic acid-valine-citrulline (EVC), or glycine-phenylalanine-lysine (GFK).

[0321] In some embodiments, the tetrapeptide can be, but is not limited to, glycine-glycine-phenylalanine-glycine (GGFG).

[0322] In some embodiments, without limitation, when the first linker payload and / or the second linker payload react with a reduced thiol group, the linkers of the first linker payload and the second linker payload can be MC-VA-PAB, MC-VC-PAB, MC-AD-PAB, MC-AF-PAB, MC-FK-PAB, MC-AK-PAB, MC-AV-PAB, MC-VK-PAB, MC-KK-PAB, MC-FC-PAB, MC-LC-PAB, MC-IC-PAB, MC-WC-PAB, or MC-FA-PAB, respectively. In some embodiments, without limitation, when the first linker payload and / or the second linker payload react with a reduced thiol group, the linker of the first linker payload and the linker of the second linker payload can be any of MC-AAD-PAB, MC-GVC-PAB, MC-GGG-PAB, MC-FFK-PAB, MC-EVC-PAB, or MC-GFK-PAB, respectively.

[0323] In some embodiments, the linker comprises a maleimide containing drug, an organic chloride containing drug, an organic bromide containing drug, an organic iodide containing drug, and / or a vinylpyrimidine containing drug.

[0324] In some embodiments, when the first linker payload and / or the second linker payload reacts with a reactive group in a thio bridging reagent, the linker of the first linker payload and / or the second linker payload further comprises azide and / or dibenzocyclooctyne (DBCO). In some embodiments, when the linker of the first linker payload and / or the second linker payload comprises azide, the reactive group of the thio bridging group comprises DBCO. In some embodiments, when the linker of the first linker payload and / or the second linker payload comprises DBCO, the reactive group of the thio bridging group comprises azide.

[0325] In some embodiments, when the first linker payload and / or the second linker payload react with a reactive group in a thio bridging reagent, the linkers of the first linker payload and the second linker payload, respectively, are selected from the group consisting of:

[0326] [ka] wherein n is 0 to 20, 0 to 18, 0 to 15, 0 to 13, 0 to 10, 0 to 7, 0 to 5, or 0 to 3, and m is 0 to 20, 0 to 18, 0 to 15, 0 to 13, 0 to 10, 0 to 7, 0 to 5, or 0 to 3. Optionally, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0327] As used herein, the term "end-capping reagent" refers to a compound that does not carry a drug and contains at least one substituent that is capable of covalently binding to an antibody.

[0328] In some embodiments, the end-capping reagent is a cleavable or non-cleavable linker, hi some embodiments, the end-capping reagent is (2-aminoethyl)maleimide.

[0329] In some embodiments, there are no specific restrictions on the payload, as long as it contains at least one substituent that allows for connection from the payload to the linker.

[0330] As used herein, the term "payload" refers to any cytotoxic molecule having at least one substituent or moiety that can be attached to a linker structure. The payload can kill cancer cells and / or inhibit the growth, proliferation, or metastasis of cancer cells, thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder.

[0331] In some embodiments, the payload is a cytotoxic drug, cytokine, nucleic acid, radionuclide, kinase, or derivative thereof. In some embodiments, the payload includes, but is not limited to, a topoisomerase inhibitor and a tubulin inhibitor. In some embodiments, the payload is, but is not limited to, an anti-cancer drug, an anti-viral agent, or an anti-bacterial agent.

[0332] In some embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0333] Representative payloads include monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), monomethyl auristatin EF (MMAF), calicheamicin (CLM), martancillin (DM1), maytansinoids, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxins, quinoline alkaloids, Dxd, doxorubicin hydrochloride, methotrexate, erlotinib, bortezomib, fulvestrant, sunitib, imatinib mesylate, letrozole, finasunate, oxaliplatin, carboplatin, cisplatin, finasunate, fluorouracil, rapamycin, leucovorin, lapatinib, lonafamib, sorafenib, gefitinib, camptothecin, topotecan, bryostatin, and adzealand. and cyclophosphamide, doxorubicin, vincristine, prednisone or prednisolone, mechlorethamine, chlorambucil, other alkylating agents such as ifosfamide, antimetabolites such as azathioprine and mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, etoposide phosphate, epipodophyllotoxin, actinomycin, daunorubicin, valrubicin, idarubicin, edrecolomab, epirubicin, bleomycin, plicamycin, or mitomycin, and salts thereof.

[0334] In some embodiments, the payload is deruxtecan (DXd), cyanine 3 (Cy3), MMAE, MMAD, or MMAF. In some embodiments of the present application, the payload is MMAE, DXd, or Cy3.

[0335] The linker-payload is a chemical moiety that is synthesized by linking a linker and a payload. Depending on the desired payload and the selected linker, those skilled in the art can select an appropriate method for coupling them. For example, several conventional coupling methods, such as amine coupling, can be used to form the desired linker-payload containing a reactive group for covalently binding to an antibody. In this disclosure, drug-maleimide conjugates (i.e., maleimide-linked drugs) are used as examples of payloads with reactive groups. Maleimide is the most common reactive group that can be coupled to thiol groups in ADC preparation. In addition, organic chlorides, bromides, and iodides are also frequently used.

[0336] The linker payload can be any physically active compound or any compound used in the diagnosis, prevention, or treatment of disease. In some embodiments, when the first linker payload and / or the second linker payload react with a reduced thiol group, the first linker payload and / or the second linker payload are MC-VC-PAB-MMAE, MC-VC-PAB-MMAD, and MC-VC-PAB-MMAF, respectively.

[0337] In some embodiments, the first thio bridging reagent with the first linker payload and the second thio bridging reagent with the second linker payload each have the following formula: [ka] where Q is selected from the group consisting of:

[0338] [ka] S is selected from a cleavable linker or a non-cleavable linker, including but not limited to, S is selected from the group consisting of:

[0339] [ka] wherein n is 0 to 20 and m is 0 to 20, and optionally, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0340] T is the payload.

[0341] In some embodiments, without limitation, the first thio bridging reagent having a first linker payload and the second thio bridging reagent having a second linker payload are each selected from the group consisting of:

[0342] [ka]

[0343] In some embodiments, the payload of a first thio bridging reagent having a first linker payload and the payload of a second thio bridging reagent having a second linker payload are different or the same. In some embodiments, the linker of a first thio bridging reagent having a first linker payload and the linker of a second thio bridging reagent having a second linker payload can be different or the same. In some embodiments, the thio bridging reagent of a first thio bridging reagent having a first linker payload and the thio bridging reagent of a second thio bridging reagent having a second linker payload can be different or the same.

[0344] In some embodiments, the method of preparing an ADC having D2 comprises the steps of: (a1) incubating a first reducing agent and a compound according to the present application acting as an antibody in the presence of an effective amount of a transition metal ion in a first buffer system to selectively reduce interchain disulfide bonds with the antibody; or Incubating a composition according to the present application and an antibody in a first buffer system, wherein a compound according to the present application acts as a first reducing agent to selectively reduce interchain disulfide bonds in the antibody.

[0345] (b1) An excess of a metal chelator and an excess of a first linker payload are introduced to react with the reduced thiol groups obtained in step (a1).

[0346] In some embodiments, in step (b1), the reaction temperature is 15° C. to 25° C., and in step (b1), the reaction time is 0.5 hours to 2 hours.

[0347] In some embodiments, the homogeneity of the ADC with D2 is up to 53%, 55%, 60%, 65%, 70%, 75%, 80%, 84%, 87%, 89%, 90%, 91%, 92%, or 94%.

[0348] In some embodiments, an ADC prepared by the method of preparing an ADC with D2 comprises D0 and D4 in a content of less than 50%, 40%, 35%, 30%, 25%, 23%, 22%, or 21% of the total weight of D0, D2, D4, D6, and D8. In some embodiments, an ADC prepared by the method of preparing an ADC with D2 comprises D0 and D4 in a content of less than 20% of the total weight of D0, D2, D4, D6, and D8.

[0349] In some embodiments, the method of preparing an ADC having D2+D6 comprises the steps of: (c2) incubating the reaction product obtained in step (b1) and a second reducing agent in a second buffer system to reduce interchain disulfide bonds in the reaction product obtained in step (b1). (d2) introducing the incubation product obtained in step (c2) and an excess amount of a second linker payload to react with the reduced thiol group resulting from step (c2);

[0350] In some embodiments, the homogeneity of the ADC with D2+D6 is generally up to 75%, 80%, or even 90%.

[0351] In some embodiments, the method of preparing an ADC having D2+D3 comprises the steps of: (d3) introducing the incubation product obtained in step (c2) and an excess amount of a second thio bridging reagent having a second linker payload to react with the reduced thiol group generated from (c2);

[0352] In some embodiments, the method for preparing an ADC having D2+D3 comprises the steps of: (d3`) Introducing the incubation product obtained in step (c2) and a second thiobridge reagent having an excess amount of a reactive group to re-crosslink the reduced thiol group resulting from step (c2), and then incubating an excess amount of a second linker payload in a second buffer system to react with the reactive group of the thiobridge group.

[0353] In some embodiments, the method of preparing an ADC having D1 comprises the steps of: (b4) introducing an excess of a metal chelator and an excess of a first thio bridging reagent having a first linker payload to react with the reduced thiol groups obtained in step (a1);

[0354] In some embodiments, the method of preparing an ADC having D1 comprises the steps of: (b4`) introducing an excess amount of a metal chelator and a first thio bridging reagent having a reactive group to re-crosslink the reduced thiol groups obtained in step (a1), and then incubating an excess amount of a first linker payload in a first buffer system to react with the reactive group of the thio bridging group.

[0355] In some embodiments, the homogeneity of the ADC with D1 is up to 75%, 80%, 85%, or even 90%.

[0356] In some embodiments, the method of preparing an ADC having D1+D6 comprises the steps of: (c5) Incubating the reaction product obtained in step (b4) or step (b4') and a second reducing agent in a second buffer system to reduce interchain disulfide bonds in the reaction product obtained in step (b4) or step (b4'). (d5) introducing the incubation product obtained in step (c5) and an excess of a second linker payload to react with the reduced thiol group resulting from step (c5).

[0357] In some embodiments, the method of preparing an ADC having D1+D3 comprises the steps of: (d6) introducing the incubation product obtained in step (c5) and an excess amount of a second thio bridging reagent having a second linker payload to react with the reduced thiol group generated from (c5);

[0358] In some embodiments, the method for preparing an ADC having D1+D3 comprises the steps of: (d6`) Introducing the incubation product obtained in step (c5) and an excess amount of a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol group resulting from step (c5), and then incubating an excess amount of a second linker payload in a second buffer system to react with the reactive group of the thio bridging group.

[0359] In some embodiments, the method of preparing an ADC having D0+D6 comprises the steps of: (b7) introducing an excess of a metal chelating agent and an excess of a first thio bridging reagent to react with the reduced thiol groups obtained in step (a1); (c7) incubating the reaction product obtained in step (b7) and a second reducing agent in a second buffer system to reduce interchain disulfide bonds in the reaction product obtained in step (b7). (d7) introducing the incubation product obtained in step (c7) and an excess of a second linker payload to react with the reduced thiol group resulting from step (c7);

[0360] In some embodiments, the homogeneity of the ADC with D0+D6 is up to 75%, 80%, 85%, or even 90%.

[0361] In some embodiments, a method of preparing an ADC having D0+D3 comprises the steps of: (d8) introducing the incubation product obtained in step (c7) and an excess amount of a second thio bridging reagent having a second linker payload to react with the reduced thiol group generated from (c7);

[0362] In some embodiments, the method for preparing an ADC having D0+D3 comprises the steps of: (d8`) Introducing the incubation product obtained in step (c7) and an excess amount of a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol group resulting from step (c7), and then incubating an excess amount of a second linker payload in a second buffer system to react with the reactive group of the thio bridging group.

[0363] In some embodiments, in the site-specifically modified antibody (D1+D4 ADC, D1+D2 ADC) prepared by the method comprising steps (a), (b), (c), and (d), Modifying Reagent 1 is a first thio-bridge reagent having a first linker payload, and Modifying Reagent 2 is a second linker payload, while a transition metal ion is introduced in step (c).

[0364] In some embodiments, in the site-specifically modified antibody (ADC having D1+D4, ADC having D1+D2) prepared by the method comprising steps (a), (b), (c), and (d), Modifying Reagent 1 is a first thio-bridge reagent having a first linker payload, and Modifying Reagent 2 is a second linker payload, while a transition metal ion is introduced in step (c).

[0365] In some embodiments, in the site-specifically modified antibody (ADC having D2+D4, ADC having D2+D2) prepared by the method comprising steps (a), (b), (c), and (d), Modifying Reagent 1 is a first thio-bridge reagent having a reactive group that reacts with a first linker payload, and Modifying Reagent 2 is a second linker payload, while a transition metal ion is introduced in step (c).

[0366] In some embodiments, in a site-specifically modified antibody (ADC having D0+D4) prepared by a method comprising steps (a), (b), (c), and (d), Modifying Reagent 1 is a first linker payload and Modifying Reagent 2 is a second linker payload, while a transition metal ion is introduced in step (c).

[0367] Various analytical methods can be used to measure the yield and isomeric mixture of ADCs. In some embodiments, the analytical method is HIC-HPLC. HIC-HPLC can separate ADCs of antibodies loaded with various numbers of drugs. The drug loading level can be determined, for example, based on the ratio of absorbance at 250 nm to 280 nm. For example, the drug absorbs at 250 nm and the antibody absorbs at 280 nm. Therefore, the 250 / 280 ratio increases with drug loading.

[0368] Compared to ADCs produced by conventional conjugation processes, the ADCs of the present invention using the bioconjugation process described herein do not require protein engineering or ligases, and offer improved homogeneity, simplified operation, and reduced cost.

[0369] The process of selectively reducing one of the four interchain disulfide bonds on an IgG antibody to produce an ADC with a homogeneous D2 is based on the native interchain disulfide bond and requires only a novel reducing agent and transition metal ion, without requiring any protein engineering or enzyme catalysis. Therefore, compared with conventional ADC preparation processes, the disclosed process is less complicated and dramatically improves the homogeneity of the resulting antibody-drug conjugate.

[0370] In some embodiments, the method of preparing an ADC having D1 comprises the steps of: (A1) Incubate the first reducing agent (0.02 mM) and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours).

[0371] (B1) EDTA (0.6 mM) and an excess of dibromomaleimide-PEG4-N3 (0.013 mM) are introduced and reacted with the reduced thiol groups obtained in step (A1). The reaction temperature is 24°C, the reaction time is 3 hours, and the product is recovered using a desalting column.

[0372] (C1) The product of step (B1) and DBCO-Cy3 (0.02 mM) are incubated in MES (20 mM, pH 6.7), the reaction temperature is 25°C, and the reaction time is 8 hours.

[0373] In some embodiments, the method of preparing an ADC having D0+D6 comprises the steps of: (A2) Incubate the first reducing agent (0.02 mM) and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl2 (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (B2) EDTA (0.6 mM) and an excess of dibromomaleimide (0.013 mM) are introduced and reacted with the reduced thiol groups obtained in step (A2). The reaction temperature is 24°C, the reaction time is 3 hours, and the product is recovered using a desalting column. (C2) The product of step (B2) and TCEP (0.08 mM) are incubated in MES (20 mM, pH 6.7), the reaction temperature is 25°C, and the reaction time is 12 hours. (D2) In step (C2), a second linker payload (MC-GGFG-DXd, 0.14 mM) is introduced, and the reaction mixture is left standing at 24°C for 1 hour, after which the ADC obtained in D0+D6 is recovered using a desalting column.

[0374] In some embodiments, the method of preparing an ADC having D2+D6 comprises the steps of: (A3) Incubate the first reducing agent (0.02 mM) and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl2 (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (B3) EDTA (0.6 mM) and excess MC-MMAF (0.06 mM) are introduced to react with the reduced thiol group generated from step (A3), the reaction temperature is 24°C, the reaction time is 1 hour, and then the product is recovered using a desalting column. (C3) The product of step (B3) and TCEP (0.08 mM) are incubated in MES (20 mM, pH 6.7), the reaction temperature is 25°C, and the reaction time is 8 hours. (D2) In step (C3), a second linker payload (MC-GGFG-DXd, 0.14 mM) is introduced, and the reaction mixture is left at 24 °C for 1 hour. The bi-payload ADC obtained in D2+D6 is then recovered using a desalting column.

[0375] In some embodiments, the method of preparing an ADC having D2 comprises the steps of: (A4) Incubate the first reducing agent (0.02 mM) and transtuzumab (0.012 mM) in the presence of ZnCl (0.24 mM) in MES buffer (pH 6.7, 20 mM) at 4°C for 4 hours. (B4)Zn 2+ EDTA (0.6 mM) was added to capture the . (C4) MC-VC-PAB-MMAE (0.06 mM) is introduced and reacted with the reduced thiol group obtained in step (1), the reaction temperature is 24°C, and the reaction time is 1 hour. (D4) Introduce cysteine (0.08 mM) to consume excess MC-VC-PAB-MMAE. (E4) The resulting ADC is purified using a desalting column.

[0376] Antibodies with thiol group site-specific modification The present application provides antibodies having thiol group site-specific modifications prepared by the methods of the present application.

[0377] In some embodiments, an antibody having a thiol group site-specific modification is conjugated with Modifying Reagent 1 and / or Modifying Reagent 2.

[0378] In some embodiments, Modification Reagent 1 and / or Modification 2 are covalently attached to reduced thiol groups in the hinge region of the antibody.

[0379] In some embodiments, modification reagent 1 and / or modification 2 are covalently attached to reduced thiol groups in the Fab region of the antibody.

[0380] In some embodiments, an antibody having a thiol group site-specific modification is coupled with Modifying Reagent 1 to form an ADC having D2 or D1. In some embodiments, an antibody having a thiol group site-specific modification is coupled with Modifying Reagent 1 and Modifying Reagent 2 to form an ADC having D2+D6, an ADC having D2+D3, an ADC having D1+D6, an ADC having D1+D3, an ADC having D0+D6, an ADC having D0+D3, an ADC having D0+D4, an ADC having D2+D4, an ADC having D1+D4, an ADC having D2+D2, or an ADC having D1+D2.

[0381] In some embodiments, the ADC having D2 is trastuzumab-[MC-VC-PAB-MMAE]2, sacituzumab-[MC-VC-PAB-MMAE]2, or belantamab-[MC-VC-PAB-MMAE]2.

[0382] In some embodiments, the ADC having D1 is trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1.

[0383] In some embodiments, the ADC having D0+D6 is trastuzumab-[maleimide]1[MC-GGFG-DXd]6.

[0384] In some embodiments, the ADC having D2+D6 is trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6.

[0385] In some embodiments, the ADC is trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2, trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2, trastuzumab-[maleimide]1[MC-VC-PAB-MMAE]4, trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4, or trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4.

[0386] Use of antibodies with thiol group site-specific modifications The present disclosure provides the use of an antibody having a thiol group site-specific modification according to the present application in the manufacture of a therapeutic agent for preventing, diagnosing, or treating a disease.

[0387] As used herein, the term "treating any disease" refers to alleviating or ameliorating the disease (i.e., delaying or preventing the onset of the disease or at least one of its clinical symptoms), or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease (including those not discernible to the patient). In the case of cancer, "treating" refers to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or a combination thereof. In the case of tumors, "treating" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, delaying the development of tumors, or a combination thereof.

[0388] As used herein, the term "prevention of any disease" refers to the prophylactic treatment of the disease or the delay in the onset or progression of the disease.

[0389] In some embodiments, the disease is a tumor or cancer, hi some embodiments, the disease is an autoimmune disease, or the like.

[0390] In some embodiments, cancer includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia or lymphoid malignancies, etc. More specific examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0391] Pharmaceutical Composition The present application also provides a pharmaceutical composition comprising a thiol group site-specifically modified antibody prepared by the above method and at least a pharmaceutically acceptable carrier.

[0392] The pharmaceutical compositions provided herein can be formulated by any method known in the art, for example, the pharmaceutical compositions provided herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0393] Pharmaceutical compositions are formulated to be compatible with the intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0394] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, ingredients known in the art, or various combinations thereof.

[0395] Suitable ingredients include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars or cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody or antigen-binding fragment thereof and a conjugate provided herein reduces oxidation of the antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in one embodiment, a pharmaceutical composition is provided comprising one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine.

[0396] In some embodiments, the pharmaceutical composition may be a liquid solution, suspension, or emulsion. In some aspects, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for forming a liquid solution, suspension, or emulsion. Injectable preparations include sterile and / or non-caseating solutions ready for injection, sterile dry soluble preparations such as lyophilized powders ready for combination with a solvent immediately before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble preparations ready for combination with a vehicle immediately before use, and sterile and / or non-caseating emulsions. The solution may be aqueous or non-aqueous.

[0397] In some embodiments, the pharmaceutical composition is used in combination with other therapeutic agents. The other therapeutic agents are not particularly limited as long as they can reduce the side effects of the pharmaceutical composition or enhance the efficacy of the pharmaceutical composition. Examples of other therapeutic agents include anti-cancer agents, anti-autoimmune disease agents, anti-emetic agents, and anti-allergic agents.

[0398] In some embodiments, anticancer drugs include erlotinib, bortezomib, fulvestrant, sunitinib, imatinib, mesylate, letrozole, platins such as finasunate, oxaliplatin, carboplatin, and cisplatin, finasunate, fluorouracil, rapamycin, leucovorin, lapatinib, lonafamib, sorafenib, gefitinib, capmutotecin, topotecan, bryostatin, adzelesin, anthracyclines, carzelesin, bizelesin, dolastatins, auristatins, duocarmycins, eleutherobin, taxols such as paclitaxel or docetaxel, cyclosporin, doxorubicin, cyclosporin ... Other alkylating agents such as rubicin, vincristine, prednisone or prednisolone, mechlorethamine, chlorambucil and ifosfamide, antimetabolites such as azathioprine or mercaptopurine, other microtubule inhibitors (vinca alkaloids such as vincristine, vinblastine, vinorelbine and vindesine and taxanes), podophyllotoxins (etoposide, teniposide, etoposide phosphate, epipodophyllotoxin), topoisomerase inhibitors, actinomycin, daunorubicin, barbicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin and other cytotoxins.

[0399] In some embodiments, anti-autoimmune disease agents may include, but are not limited to, ibuprofen, loxoprofen, naproxen, diclofenac, indomethacin, meloxicam, lornoxicam, nabumetone, celecoxib, paracetamol, glucocorticoids, azathioprine, cyclophosphamide, and the like.

[0400] In some embodiments, patients may experience nausea during and after administration of the ADC of the present application. Therefore, antiemetic drugs can be administered to prevent nausea (upper respiratory tract inflammation) and vomiting. Antiemetic drugs include, but are not limited to, aprepitant, ondansetron, granisetron hydrochloride, lorazepam, dexamethasone, prochlorperazine, casopitant, etc.

[0401] In some embodiments, patients may experience allergic reactions during and after administration of the ADC of the present application. Therefore, to minimize the risk of allergic reactions, anti-allergic agents can be administered. Anti-allergic agents include dexamethasone, beclomethasone, hydrocortisone, prednisolone, methylprednisolone, hydroxyzine, cyproheptadine, bronchodilators, terbutaline, etc.

[0402] The present disclosure provides a method for preventing, diagnosing, or treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a thiol group site-specifically modified antibody prepared by the above-described method or a pharmaceutical composition according to the present application.

[0403] As used herein, the term "subject" refers to a mammal, a primate (e.g., a human, male or female), a dog, a rabbit, a guinea pig, a pig, a rat, or a mouse. In one embodiment, the subject is a primate. In yet another embodiment, the subject is a human.

[0404] As used herein, a "therapeutically effective amount" refers to an amount of an antibody having a thiol group site-specific modification, such as an ADC of the present application, that elicits a biological or medical response in a subject, such as symptom improvement, symptom alleviation, delay or slow progression of a disease, or disease prevention. The therapeutically effective amount will vary depending on the type and severity of the symptoms to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition. In some embodiments, the therapeutically effective amount is based on a variety of factors, including the type of disease, age, weight, sex, medical condition of the patient, severity of the condition, route of administration, and the particular antibody used. In some embodiments, the therapeutically effective amount can vary widely but can be routinely determined using standard methods. In some embodiments, the therapeutically effective amount can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values.

[0405] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the present application described herein are obvious and may be made using suitable equivalents without departing from the scope of the present disclosure or the embodiments disclosed herein. Now, having described the present disclosure in detail, the same will be more clearly understood by reference to the following examples. Furthermore, unless otherwise specified, the reagents and solvents described herein are readily available from commercial suppliers. [Example]

[0406] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0407] Reagents and manufacturers Trastuzumab is commercially available from Roche. Sacituzumab and belantamab are commercially available from MedChemExpress. TCEP is commercially available from Bidepharm. EDTA is commercially available from Aladdin. DMA (dimethylacetamide) is commercially available from Aldrich Sigma. MC-VC-PAB-MMAE is commercially available from Levena biopharma. MC-GGFG-DXd is commercially available from Levena. Dibromomaleimide is commercially available from Aladdin. Desalting columns (type: 40K, 0.5 mL, REF: 87766, lot SJ251704) are commercially available from Thermo Scientific.

[0408] Reagents used in the examples include, but are not limited to, 1-hydroxybenzotriazole (HOBT), dimethylacetamide (DMA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N,N-diisopropylethylamine (DIPEA), ethyl acetate (EtOAc), N,N-dimethylformamide (DMF), bicyclic amidine (DBU), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI), trifluoroacetic acid (TFA), dichloromethane (DCM), and tert-butylchlorodiphenylsilane (TBDPSCl), which are commercially available.

[0409] Synthetic Procedure A for Compounds According to the Present Application To a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), HATU (190 mg, 0.5 mmol, 1 eq) was added, followed by DIPEA (2.0 mmol, 4.0 eq) under a N2 atmosphere. The mixture was stirred for 30 min, and then the amine reagent (0.5 mmol, depending on the compound structure) was added. The reaction was stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product.

[0410] Synthesis procedure A-1 for compounds according to the present application The product prepared in Synthetic Procedure A was dissolved in DCM (3 mL) and TFA (0.3 mL) was added. The mixture was stirred for 1 hour, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was taken up in distilled water and washed twice with EtOAc. The aqueous layer was lyophilized to give the corresponding product.

[0411] Synthesis procedure B for compounds according to the present application To a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL) was added HOBt (67.5 mg, 0.5 mmol, 1 eq) and EDCI (95.5 mg, 0.5 mmol, 1.0 eq), followed by DIPEA (2.0 mmol, 4.0 eq) under a N2 atmosphere. The mixture was stirred for 30 min, and then the amine reagent (0.5 mmol, depending on the compound structure) was added. The reaction was stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the corresponding product.

[0412] Synthesis procedure B-1 for compounds according to the present application The product prepared in Synthesis Procedure B was dissolved in DCM (3 mL) and TFA (0.3 mL) was added. The mixture was stirred for 1 hour, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was taken up in distilled water and washed twice with EtOAc. The aqueous layer was lyophilized to give the corresponding product.

[0413] Example 1: Synthesis of TCEP-NO [ka] Compound 1: O-tritylhydroxylamine (NH2-O-Trt, 176 μmol, 48.5 mg, 1 eq), EDC (176 μmol, 33.7 mg, 1 eq), and HOBt (352 μmol, 53.8 mg, 2 eq) were dissolved in 1.5 mL of degassed DMF under an inert atmosphere. This solution was added to TCEP (528 μmol, 150 mg, 3 eq) dissolved in 1.5 mL of degassed DMF containing DIPEA (704 μmol, 123 μmol, 4 eq) under an inert atmosphere. The reaction was stirred at room temperature for 60 min. The DMF was then removed in vacuo, and CH3COOH (0.1 N, 5 mL) and EtOAc (2 mL) were added to the residue. The resulting mixture was stirred for 5 minutes and filtered to give compound 2 as a white solid (61.2 mg, 12.2%), which was not further purified. EtOAc (2 mL), 5% TFA, and 5% TIPS were added. The reaction was continued for another 2 hours. H2O (5 mL) and EtOAc (10 mL) were then introduced. The aqueous phase was washed twice with EtOAc (10 mL) and concentrated to give TCEP-NO (13 mg). Compound 2 (also known as TCEP-19-int1) was analyzed by MS [MH]. - =506.2, C 28 H 30 The calculated exact mass of NO6P is 507.18. 1 H NMR (400 MHz, DMSO-d6): δ 7.40-7.15 (m, 15H), 2.48-2.38 (m, 4H), 2.22 (s, 1H), 2.12-1.52 (m, 7H). For TCEP-NO, MS [MH] - =263.94, C9H 16 The calculated exact mass of NO6P is 265.07. 1 H-NMR (400 MHz, Deuterium Oxide): δ 2.93-2.85 (m, 4H), 2.73-2.56 (m, 8H).

[0414] Example 2: Synthesis of TCEP-3NO [ka] Compound 1: O-tritylhydroxylamine (NH2-O-Trt, 528 μmol, 145.5 mg, 3 eq), EDC (528 μmol, 101 mg, 3 eq), and HOBt (880 μmol, 134.5 mg, 5 eq) were dissolved in 4 mL of degassed DMF under an inert atmosphere. This solution was added to TCEP (176 μmol, 50 mg, 1 eq) dissolved in 4 mL of degassed DMF containing DIPEA (704 μmol, 123 μl, 4 eq) under an inert atmosphere. The reaction was stirred at room temperature for 60 min. The DMF was then removed in vacuo, and the residue was added with CH3COOH (0.1 N, 5 mL) and EtOAc (5 mL). The resulting mixture was stirred for 5 min and filtered to give compound 3 as a white solid, which, without further purification, was added with EtOAc (2 mL), 5% TFA, and 5% TIPS. The reaction was continued for an additional 2 h. Then, HO (5 ml) and EtOAc (10 ml) were added. The aqueous phase was washed twice with EtOAc (10 ml) and concentrated to give TCEP-NO (32 mg). For TCEP-3NO, MS [M+H] + =295.87, C9H 18 The calculated exact mass of N3O6P is 295.09. 1 H NMR (400 MHz, Deuterium Oxide):δ 3.08 - 2.38 (m, 10H), 2.24-2.17 (m, 5.6 Hz, 2H).

[0415] Example 3: Synthesis of TCEP-CO [ka] Compound 4 (176 μmol, 43.2 mg, 1 eq; di-tert-butyl iminodiacetate, Bidepharm), EDC (176 μmol, 33.7 mg, 1 eq), and HOBt (352 μmol, 53.8 mg, 2 eq) were dissolved in 1.5 mL of degassed DMF under an inert atmosphere. This solution was added to TCEP (528 μmol, 150 mg, 3 eq) dissolved in 1.5 mL of degassed DMF containing DIPEA (704 μmol, 123 μl, 4 eq) under an inert atmosphere. The reaction was stirred at room temperature for 60 min. The DMF was then removed in vacuo, and the residue was treated with CH3COOH (0.1 N, 5 mL) and EtOAc (2 mL). The resulting mixture was stirred for 5 min and filtered to give compound 5 as a white solid, which, without further purification, was added to EtOAc (2 mL), 5% TFA, and 5% TIPS. The reaction was continued for an additional 2 h. Then, HO (5 ml) and EtOAc (10 ml) were added. The aqueous phase was washed twice with EtOAc (10 ml) and concentrated to give TCEP-CO (8 mg). For TCEP-CO, MS [M−H] - =363.86, C9H 16 The calculated exact mass of NO6P is 365.09. 1 H NMR (400 MHz, Deuterium Oxide):δ 4.28 (s, 2H), 4.13 (s, 2H), 2.97 (dt, J = 20.0, 6.4 Hz, 2H), 2.83 (dt, J = 18.3, 6.9 Hz, 4H), 2.57-2.48 (m, 6H).

[0416] Example 4: Synthesis of TCEP-1 [ka]

[0417] 1. TCEP-1-int2 To a solution of TCEP-1-int1 (3.0 g, 10.0 mmol, 1.0 eq, Fmoc-Glycine, Bidepharm) in DMF (30 mL) was added HOBt (1.64 g, 12.0 mmol, 1.2 eq) and EDCI (2.32 g, 12 mmol, 1.2 eq), followed by DIPEA (4.4 mL, 25.2 mmol, 2.5 eq) under a N atmosphere. The mixture was stirred for 30 min, and then compound 1 O-tritylhydroxylamine (2.78 g, 10 mmol, 1.0 eq, Bidepharm) was added. The reaction mixture was stirred at room temperature for 1 h and poured into ice water. The precipitate was collected by filtration and washed with water. The filter cake was dried under vacuum to give the crude product TCEP-1-int2 (4.5 g, 80% yield, white solid), which was used directly in the next step without further purification.

[0418] 2. TCEP-1-int3 To a solution of TCEP-1-int2 (4.5 g, 8.1 mmol, 1.0 eq) in DMF (25 mL) was added DBU (5 mL). The resulting mixture was stirred at room temperature for 0.5 h, and LCMS confirmed the reaction was complete. The mixture was poured into ice water, extracted with EtOAc, dried under vacuum, and purified by flash column chromatography (EtOAc / petroleum ether = 0-50%) to give the product TCEP-1-int3 (2.0 g, 77% yield, white solid).

[0419] 3. TCEP-1 TCEP-1 was synthesized in the same manner as in Synthesis Procedure B-1, using TCEP-1-int3 as the amine reagent, to obtain TCEP-1 (45.1 mg, 28% yield) as a white solid. MS [MH] - =321.15, C 11 H 19 The calculated exact mass of N2O7P is 322.25. 1 H-NMR (400 MHz, Deuterium Oxide): δ 3.99 (s, 0.64H), 3.87 (s, 1.34 H), 2.96 - 2.81 (m, 6H), 2.63-2.56 (m, 6H).

[0420] Example 5: Synthesis of TCEP-2 [ka] TCEP-2 was synthesized in the same manner as in Synthesis Procedure B-1, using compound 5 (tert-butyl glycinate, Bidepharm) as the amine reagent, and TCEP-2 (52.3 mg, 34% yield) was obtained as a white solid. MS [MH] - =306.18, C 11 H 18 The calculated exact mass of NO7P is 307.24. 1 H NMR (400 MHz, Deuterium Oxide): δ 3.99 (s, 2H), 3.00 - 2.76 (m, 6H), 2.60 (dtd, J = 14.0, 7.0, 3.8 Hz, 6H).

[0421] Example 6: Synthesis of TCEP-3 [ka] TCEP-3 was synthesized using Procedure B using compound 6 (DL-phenylalanine, Adamas) as the amine reagent to give TCEP-3 (65.0 mg, 33% yield) as a white solid. MS [MH] - =396.24, C 18 H 24 The calculated exact mass of NO7P is 397.13. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.43 - 7.25 (m, 5H), 4.71 (dd, J = 10.3, 4.8 Hz, 1H), 3.31 (dd, J = 13.9, 4.8 Hz, 1H), 2.91 (dd, J = 13.9, 10.3 Hz, 1H), 2.86 - 2.59 (m, 6H), 2.53 - 2.27 (m, 6H).

[0422] Example 7: Synthesis of TCEP-4 [ka]

[0423] 1. TCEP-4-int1 To a solution of ethanolamine (610 mg, 10 mmol, 1.0 eq, Adams) in DMC (20 mL) was added imidazole (15 mmol, 1.5 eq) followed by TBDPSCl (10 mmol, 1.0 eq, Adams) at 0 °C. The mixture was stirred at room temperature for 2 h. TLC showed the reaction was complete, and the reaction mixture was washed with water and brine, and the organic layer was dried over NaSO and filtered. The filtrate was concentrated to give the crude product, which was used directly in the next step without further purification.

[0424] 2. TCEP-4 TCEP-4 was synthesized in the same manner as in Synthesis Procedure B-1, using TCEP-4-int1 as the amine reagent, to obtain TCEP-4 (60.0 mg, 40.8% yield) as a white solid. MS [MH] - =292.25, C 11 H 20 The calculated exact mass of NO6P is 293.10. 1 H NMR (400 MHz, Deuterium Oxide):δ 4.23 (t, J = 5.4 Hz, 1H), 3.64 (t, J = 5.4 Hz, 1H), 3.48 (t, J = 5.3 Hz, 1H), 3.32 (t, J = 5.5 Hz, 1H), 2.98 - 2.78 (m, 6H), 2.60 (dp, J = 13.4, 6.8 Hz, 6H).

[0425] Example 8: Synthesis of TCEP-5 [ka] TCEP-5 was synthesized in a similar manner to Procedure B using 2-phenoxy-ethylamine (Bidepharm) as the amine reagent to give TCEP-5 (105.0 mg, 56.8%) as a white solid. MS [MH] - =368.24, C 17 H 24The calculated exact mass of NO6P is 369.13. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.34 (dd, J = 8.5, 7.2 Hz, 2H), 7.00 (dd, J = 19.2, 7.7 Hz, 3H), 4.14 (t, J = 5.1 Hz, 2H), 3.56 (t, J = 5.1 Hz, 2H), 2.78 (ddt, J = 41.6, 20.1, 6.9 Hz, 6H), 2.60 - 2.43 (m, 6H).

[0426] Example 9: Synthesis of TCEP-6 [ka]

[0427] 1. TCEP-6-int1 To a solution of N-methylhydroxylamine hydrochloride (830 mg, 10 mmol, 1.0 eq, Adams) in DMC (20 mL) was added imidazole (15 mmol, 1.5 eq) followed by TBDPSCl (10 mmol, 1.0 eq, Adams) at 0 °C. The mixture was stirred at room temperature for 2 h. TLC showed the reaction was complete, and the reaction mixture was washed with water and brine, and the organic layer was dried over NaSO and filtered. The filtrate was concentrated to give the crude product, which was used directly in the next step without further purification.

[0428] 2. TCEP-6 TCEP-6 was synthesized in the same manner as in Procedure A-1, using TCEP-6-int1 as the amine reagent, to obtain TCEP-6 (13.0 mg, 9.3% yield) as a white solid. MS [M+H] + =280.22, C 10 H 18 The calculated exact mass of NO6P is 279.09. 1 H NMR (400 MHz, Deuterium Oxide):δ 3.15 (s, 3H), 3.01 - 2.80 (m, 4H), 2.64-2.45 (m, 6H), 2.17-2.08 (m, 2H).

[0429] Example 10: Synthesis of TCEP-7 [ka] TCEP-7 was synthesized in a similar manner to Procedure B using (phenylamine, Adamas) as the amine reagent to give TCEP-7 (73.0 mg, 45.0% yield) as a white solid. MS [MH] - =324.21, C 15 H 20 The calculated exact mass of NO5P is 325.11. 1 H NMR (400 MHz, Deuterium Oxide): δ 7.42 (d, J = 4.3 Hz, 4H), 7.25 (p, J = 4.5 Hz, 1H), 2.96 (ddt, J = 32.1, 18.3, 6.9 Hz, 6H), 2.75 - 2.50 (m, 6H).

[0430] Example 11: Synthesis of TCEP-8 [ka] TCEP-8 was synthesized similarly to Procedure B using benzylamine (Adamas) as the amine reagent to give TCEP-8 (85.6 mg, 50.5% yield) as a white solid. MS [MH] - =338.23, C 16 H 22 The calculated exact mass of NO5P is 339.12. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.43 - 7.27 (m, 5H), 4.36 (s, 2H), 2.93 - 2.77 (m, 6H), 2.63 - 2.45 (m, 6H).

[0431] Example 12: Synthesis of TCEP-9 [ka] TCEP-9 was synthesized in a similar manner to Procedure A using 4-aminobenzene-1,2-diol (Bidepharm) as the amine reagent to give TCEP-9 (63.8 mg, 35.7% yield) as a white solid. MS [MH] - =356.20, C 15 H 20 The calculated exact mass of NO7P is 357.10. 1 H NMR (400 MHz, Deuterium Oxide):δ 6.97 (d, J = 2.4 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.78 (dd, J = 8.5, 2.5 Hz, 1H), 2.92 (ddt, J = 17.8, 10.0, 7.0 Hz, 6H), 2.61 (dq, J = 13.9, 6.7 Hz, 6H).

[0432] Example 13: Synthesis of TCEP-10 [ka] TCEP-10 was synthesized in a similar manner to Procedure A using 5-amino-2-hydroxybenzoic acid (Bidepharm) as the amine reagent to give TCEP-10 (53.7 mg, 27.9% yield) as a white solid. MS [MH] - =384.20, C 16 H 20 The calculated exact mass of NO8P is 385.09. 1 H NMR (400 MHz, Deuterium Oxide) δ 7.76 (d, J = 2.7 Hz, 1H), 7.41 (dd, J = 8.9, 2.7 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 3.00 - 2.83 (m, 6H), 2.61 (dq, J = 13.9, 6.7 Hz, 6H).

[0433] Example 14: Synthesis of TCEP-11 [ka] TCEP-11 TCEP-11 was synthesized in the same manner as in Procedure B using bis(pyridin-2-ylmethyl)amine (Shanghai Acmec Biochemical Co., Ltd.) as the amine reagent to give TCEP-11 (10.5 mg, 4.9% yield) as a brown solid. MS [M+H] + =432.24, C 21 H 26 The calculated exact mass of N3O5P is 431.16. 1 H NMR (400 MHz, Deuterium Oxide) δ 8.82 - 8.68 (m, 2H), 8.58 - 8.36 (m, 2H), 8.01 - 7.81 (m, 4H), 5.34 (s, 1H), 5.29 (s, 1H), 5.04 (d, J = 2.9 Hz, 2H), 4.48 (s, 1H), 3.16 (dt, J = 19.2, 6.5 Hz, 1H), 2.95 - 2.79 (m, 3H), 2.73 - 2.48 (m, 5H), 2.20 (ddt, J = 36.1, 11.6, 7.5 Hz, 2H).

[0434] Example 15: Synthesis of TCEP-12 [ka] TCEP-12 TCEP-12 was synthesized in a similar manner to Procedure A using (5-amino-8-hydroxyquinoline, Bidepharm) as the amine reagent to give TCEP-12 (33.2 mg, 16.9% yield) as a white solid. MS [MH] - =391.24, C 18 H 21 The calculated exact mass of N2O6P is 392.11. 1H NMR (400 MHz, Deuterium Oxide) δ 9.02 - 8.96 (m, 2H), 8.05 - 7.98 (m, 1H), 7.84 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 2.90 - 2.80 (m, 2H), 2.67 - 2.56 (m, 6H), 2.26 - 2.17 (m, 4H).

[0435] Example 16: Synthesis of TCEP-15 [ka] TCEP-15 TCEP-15 was synthesized in a similar manner to Procedure B using bis(pyridin-2-yl)methanamine (Bidepharm) as the amine reagent to give TCEP-15 (21.7 mg, 10.4% yield) as a white solid. MS [M+H] + =418.26, C 20 H 24 The calculated exact mass of N3O5P is 417.15. 1 H NMR (400 MHz, Deuterium Oxide) δ 8.69 (td, J = 6.2, 1.6 Hz, 2H), 8.37 (dtd, J = 15.8, 7.9, 1.7 Hz, 2H), 7.93 - 7.79 (m, 4H), 3.09 - 2.72 (m, 6H), 2.70 - 2.52 (m, 6H).

[0436] Example 17: Synthesis of TCEP-18 [ka]

[0437] 1. TCEP-18-int1 Phenylphosphine (110 mg, 1.0 mmol, Adamas) was dissolved in acetonitrile (5 ml, degassed) in a flame-dried round-bottom flask under N2(g). Potassium hydroxide (10 N, 10 μl) was added to this mixture, and the resulting solution was cooled to 0 °C. tert-Butyl acrylate (0.44 ml, 3.0 mmol, Adamas) was added. After complete addition of tert-butyl acrylate, the reaction was heated to 50 °C and stirred for 8 h. The reaction mixture was taken up in EtOAc (10 mL) and washed with brine (2 × 5 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc / petroleum ether = 0–20% (v / v)) to give the product TCEP-18-int1 as a clear liquid (254 mg, 69.4%).

[0438] 2. TCEP-18 A solution of TCEP-18-int1 (254 mg, 0.69 mmol) in HCl / 1,4-dioxane (4 M, Adams) was stirred at room temperature under a N atmosphere for 2 h. LCMS showed the reaction was complete, and the mixture was concentrated to remove 1,4-dioxane. The resulting residue was taken up in water and lyophilized to give TCEP-18 (152.7 mg, 88.2%) as a white solid. MS [MH] - =253.19, C 12 H 15 The calculated exact mass of O4P is 254.07. 1 H NMR (400 MHz, DMSO-d6): δ7.74 (dd, J = 10.9, 7.3 Hz, 2H), 7.62 - 7.49 (m, 3H), 2.46-2.35 (m, 2H), 2.33 - 2.00 (m, 6H).

[0439] Example 18: Synthesis of TCEP-19 [ka] To a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 eq) in DMF (3 mL) was added HATU (380 mg, 1.0 mmol, 2.5 eq), followed by DIPEA (174 μL, 1.0 mmol, 2.5 eq) under a N atmosphere at 0 °C. The mixture was stirred for 30 min, and tert-butyl glycinate (1 mmol, Adams) was added. The reaction was stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the protected product. The product was dissolved in DCM (3 mL), and TFA (0.5 mL) was added. The mixture was stirred for 1 h, and LCMS indicated the reaction was complete. The mixture was concentrated, and the residue was taken up with distilled water (10 mL) and washed with EtOAc (2 × 5 mL). The aqueous layer was lyophilized to give TCEP-19 (10.2 mg, 6.8%) as a brown solid. MS[M+H] + =380.24, C 13 H 22 The calculated exact mass of N3O8P is 379.31. 1 H-NMR (400 MHz, Deuterium Oxide): δ 3.99 (s, 4H), 2.93-2.84 (m, 4H), 2.76 - 2.53 (m, 8H).

[0440] Example 19: Synthesis of TCEP-20 [ka] To a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 eq) in DMF (3 mL) was added HATU (380 mg, 1.0 mmol, 2.5 eq), followed by DIPEA (174 μL, 1.0 mmol, 2.5 eq) under a N atmosphere at 0 °C. The mixture was stirred for 30 min, and sodium 3-aminopropane-1-sulfonate (1 mmol, Adamas) was added. The reaction was stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the protected product. The product was dissolved in DCM (3 mL), and TFA (0.5 mL) was added. The mixture was stirred for 1 h, and LCMS indicated the reaction was complete. The mixture was concentrated, and the residue was taken up in distilled water (10 mL) and washed with EtOAc (2 * 5 mL). The aqueous layer was lyophilized to give TCEP-20 (23.7 mg, 11.7%) as a brown solid. MS [MH] - =506.22, C 15 H 30 N3O 10 The calculated exact mass of PS2 is 507.51. 1 H NMR (400 MHz, Deuterium Oxide):δ 3.27 (t, J = 6.8 Hz, 4H), 2.91 - 2.85 (m, 4H), 2.80-2.71 (m, 4H), 2.69-2.61 (m, 2H), 2.58-2.49 (m, 6H), 1.94 - 1.85 (m, 4H).

[0441] Example 20: Synthesis of TCEP-21 [ka] To a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 eq) in DMF (3 mL) was added HATU (380 mg, 1.0 mmol, 2.5 eq), followed by DIPEA (174 μL, 1.0 mmol, 2.5 eq) under a N atmosphere at 0 °C. The mixture was stirred for 30 min, and diethanolamine (1 mmol, Bidepharm) was added. The reaction was stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the protected product. The product was dissolved in DCM (3 mL), and TFA (0.5 mL) was added. The mixture was stirred for 1 h, and LCMS indicated the reaction was complete. The mixture was concentrated, and the residue was taken up with distilled water (10 mL) and washed with EtOAc (2 × 5 mL). The aqueous layer was lyophilized to give TCEP-20 (13.8 mg, 7.8% yield) as a brown solid. MS[M+H] + =440.27, C 17 H 34 The calculated exact mass of N3O8P is 439.45. 1 H NMR (400 MHz, Deuterium Oxide):δ 4.50 - 4.28 (m, 4H), 3.78 (t, J = 5.2 Hz, 4H), 3.38 (t, J = 5.1 Hz, 4H), 3.17 (q, J = 5.2 Hz, 4H), 2.96-2.80 (m, 4H), 2.68-2.51 (m, 8H).

[0442] Example 21: Synthesis of TCEP-23 [ka]

[0443] 1. TCEP-23-int1 To a solution of 2-(aminooxy)tetrahydro-2H-pyran (1.17 g, 10 mmol, 2.0 eq, Bidepharm) in DMF (15 mL) was added DIPEA (3.5 mL, 20 mmol, 4 eq), followed by 2-(bromomethyl)pyridine hydrobromide (1.3 g, 5.0 mmol, 1.0 eq, Adamas). The mixture was stirred at 50 °C for 16 h. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL). The organic layer was washed with brine (30 mL), dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash column chromatography to give TCEP-23-int1 (800 mg, 80%) as a colorless oil.

[0444] 2. TCEP-23 To a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL) was added HATU (190 mg, 0.5 mmol, 1 eq), followed by DIPEA (2.0 mmol, 4.0 eq) under a N2 atmosphere. The mixture was stirred for 30 min, and TCEP-23-int1 (100 mg, 0.5 mmol, 1.0 eq) was added. The reaction was stirred at room temperature for 4 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product. The product was dissolved in HCl / 1,4-dioxane (3 mL). The mixture was stirred for 1 h, and LCMS indicated the reaction was complete. The mixture was concentrated, and the residue was taken up in distilled water and lyophilized to give TCEP-23 (17.9 mg, 10.0%) as a white solid. MS [M+H] + =357.19, C 15 H 21 The calculated exact mass of N2O6P is 356.31. 1 H-NMR (400 MHz, Deuterium Oxide):δ 8.73 (dd, J = 6.3, 1.7 Hz, 1H), 8.59 (td, J = 7.9, 1.6 Hz, 1H), 8.02 (dd, J = 6.4, 3.4 Hz, 2H), 5.21 (s, 2H), 3.21 - 2.97 (m, 2H), 2.95 - 2.80 (m, 4H), 2.69-2.56 (m, 6H).

[0445] Example 22: Synthesis of TCEP-24 [ka] TCEP-24 TCEP-24 was synthesized similarly to Procedure A using benzylamine 4-aminophthalic acid (Adamas) as the amine reagent to give TCEP-24 (21.5 mg, 10.4% yield) as a white solid. MS [MH] - =412.22, C 17 H 20 The calculated exact mass of NO9P is 413.09. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.87 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 2.1 Hz, 1H), 7.71 - 7.64 (m, 1H), 3.08-3.00 (m, 2H), 2.96-2.86 (m, 4H), 2.70-2.59 (m, 6H).

[0446] Example 23: Synthesis of TCEP-25 [ka] 1. TCEP-25-int1 To a solution of 2-pyridinecarboxaldehyde (1.0 g, 10 mmol, 1.0 eq, Adamas) and tert-butyl glycinate (1.3 g, 10.0 mmol, 1.0 eq) in MeOH (25 mL) was added Pd / C (150 mg) and two drops of AcOH. The mixture was degassed three times and purged with H2, then stirred at room temperature under an H2 atmosphere for 16 h. The reaction mixture was filtered through a Celite pad, and the filtrate was purified by flash column chromatography to give TCEP-25-int1 (1.6 g, 72.0%) as a yellow oil.

[0447] 2. TCEP-25 To a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL) was added HATU (190 mg, 0.5 mmol, 1 eq), followed by DIPEA (2.0 mmol, 4.0 eq) under a N2 atmosphere. The mixture was stirred for 30 min, and TCEP-25-int1 (111 mg, 0.5 mmol, 1.0 eq) was added. The reaction was stirred at room temperature for 4 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product. The product was dissolved in HCl / 1,4-dioxane (3 mL). The mixture was stirred for 1 h, and LCMS indicated the reaction was complete. The mixture was concentrated, and the residue was taken up in distilled water and lyophilized to give TCEP-25 (51.3 mg, 17.2% yield) as a white solid. MS [M+H] + =399.25, C 17 H 23 The calculated exact mass of N2O7P is 398.12. 1 H NMR (400 MHz, Deuterium Oxide):δ 8.60 (dd, J = 5.9, 1.6 Hz, 1H), 8.45 (td, J = 8.0, 1.6 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.87 (ddd, J = 7.5, 5.9, 1.3 Hz, 1H), 4.88 (s, 2H), 4.39 (s, 2H), 2.84 - 2.68 (m, 6H), 2.51-2.41 (m, 6H).

[0448] Example 24: Synthesis of TCEP-26 [ka]

[0449] 1. TCEP-26-int1 To a solution of Fmoc-iminodiacetic acid (1.8 g, 5.0 mmol, 1.0 eq, Bidepharm) in DMF (30 mL) was added HATU (4.3 g, 11.0 mmol, 2.2 eq), followed by DIPEA (2.0 mmol, 4.0 eq) under a N atmosphere. The mixture was stirred for 30 min, and O-tritylhydroxylamine (3.0 g, 11.0 mmol, 2.2 eq) was added. The reaction was stirred at room temperature for 4 h. The reaction mixture was poured into water (200 mL). The precipitate was collected by filtration, and the filter cake was dried under vacuum to give TCEP-23-int1 (4.0 g, 92.0%) as a white solid.

[0450] 2. TCEP-26-int2 To a solution of TCEP-26-int1 (2.0 g, 2.3 mmol, 1.0 eq) in DMF (10 mL) was added DBU (2 mL). The mixture was stirred at room temperature for 1 h, poured into ice-water (100 mL), and extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash column chromatography (EtOAc / petroleum ether = 0-50%, v / v) to give TCEP-26-int2 (1.2 g, 80%).

[0451] 3. TCEP-26 The compound was synthesized using Procedure A-1, using TCEP-26-int2 as the amine reagent, to give TCEP-26 (31.5 mg, 21.3% yield) as a white solid. MS [M+H] + =396.17, C 13 H 22 The calculated exact mass of N3O9P is 395.11. 1 H NMR (400 MHz, Deuterium Oxide) δ 4.13 (s, 2H), 3.97 (s, 2H), 2.85-2.77(m, 4H), 2.54-2.47(m,6H), 2.18-2.08(m,2H).

[0452] Example 25: Synthesis of TCEP-28 [ka]

[0453] 1. TCEP-28-int1 To a solution of O-tritylhydroxylamine (1.4 g, 5.0 mmol, 1.0 eq) in DMF (15 mL) was added DIPEA (1.7 mL, 10 mmol, 2 eq), followed by tert-butyl bromoacetate (1.0 g, 5.0 mmol, 1.0 eq, Adams). The mixture was stirred at 50 °C for 16 h. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL*3). The organic layer was washed with brine (30 mL), dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash column chromatography to give TCEP-28-int1 (1.4 g, 70%) as a white solid.

[0454] 2. TCEP-28-int2 To a solution of TCEP-28-int1 (1.4 g, 3.6 mmol, 1.0 eq) in DCM (15 mL) was added TFA (1.5 mL). The mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated and purified by flash column chromatography to give TCEP-28-int2 (380 mg, 71.8%) as a colorless oil.

[0455] 3. TCEP-28 To a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), HATU (190 mg, 0.5 mmol, 1 eq) was added, followed by DIPEA (2.0 mmol, 4.0 eq) under a N2 atmosphere. The mixture was stirred for 30 min, and TCEP-28-int2 (73.5 mg, 0.5 mmol, 1.0 eq) was added. The reaction was stirred at room temperature for 2 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product. The product was dissolved in HCl / 1,4-dioxane (3 mL, Adamas). The mixture was stirred for 1 h, and LCMS indicated the reaction was complete. The mixture was concentrated, and the residue was taken up in distilled water and lyophilized to give TCEP-28 (43.6 mg, 27.1% yield) as a white solid. MS [MH] = 322.16, C 11 H 18The calculated exact mass of NO8P is 323.08. 1 H NMR (400 MHz, Deuterium Oxide): δ 4.36 (s, 2H), 2.86-2.77 (m, 6H), 2.56-2.48 (m, 6H).

[0456] Example 26: Synthesis of TCEPA [ka] TCEPA was synthesized in the same manner as in Procedure A-1, using 4-methoxybenzylamine as the amine reagent, to obtain TCEPA (13.5 mg, 11%). MS [M+H] + =250.18, C9H 16 The calculated exact mass of NO5P is 249.08. 1 H NMR (400 MHz, Deuterium Oxide) δ 2.85-2.70 (m, 4H), 2.61-2.43 (m, 6H), 2.15-2.06 (m, 2H).

[0457] Example 27: Synthesis of TCEP-30 [ka] TCEP-30 was synthesized in the same manner as in Procedure A-1 using tert-butyl L-tyrosinate (Adamas) as the amine reagent, and TCEP-30 (25.3 mg, 12.25% yield) was obtained as a white solid. MS [M+H] + =414.23, C 18 H 24 The calculated exact mass of NO8P is 413.12. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.24-7.14 (m, 2H), 6.91-6.82 (m, 2H), 4.67 (dd, J = 10.3, 4.7 Hz, 1H), 3.26 (dd, J = 14.0, 4.7 Hz, 1H), 2.92-2.64 (m, 7H), 2.57-2.30 (m, 6H).

[0458] Example 28: Synthesis of TCEP-31 [ka] TCEP-31 was synthesized in a similar manner to Procedure A using (DL-3-(4-fluorophenyl)alanine, Bidepharm) as the amine reagent to give TCEP-31 (27.1 mg, 13.10% yield) as a white solid. MS [M+H] + =416.01, C 18 H 23 The calculated exact mass of NO7P is 415.12. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.36 - 7.26 (m, 2H), 7.12 (t, J = 8.8 Hz, 2H), 4.70 (dd, J = 10.1, 4.8 Hz, 1H), 3.31 (dd, J = 14.0, 4.9 Hz, 1H), 2.95 (dd, J = 14.0, 10.1 Hz, 1H), 2.90 - 2.65 (m, 6H), 2.56-2.40 (m, 6H).

[0459] Example 29: Synthesis of TCEP-32 [ka] TCEP-32 was synthesized in a similar manner to Procedure A using (DL-4-cyanophenylalanine, Bidepharm) as the amine reagent to give TCEP-32 (18.5 mg, 8.76% yield) as a white solid. MS [M+H] + =423.24, C 19 H 23 The calculated exact mass of N2O7P is 422.12. 1H NMR (400 MHz, Deuterium Oxide):δ 7.77 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 4.77 - 4.71 (m, 1H), 3.42 (dd, J = 14.0, 5.0 Hz, 1H), 3.05 (dd, J = 14.0, 10.0 Hz, 1H), 2.91 - 2.70 (m, 6H), 2.68 - 2.39 (m, 6H).

[0460] Example 30: Synthesis of TCEP-33 [ka] TCEP-33 was synthesized similarly to Procedure A using (DL-4-nitrophenylalanine, Bidepharm) as the amine reagent to give TCEP-33 (20.7 mg, 9.37% yield) as a white solid. MS [M+H] + =443.24, C 19 H 23 The calculated exact mass of N2O9P is 442.11. 1 H NMR (400 MHz, Deuterium Oxide):δ 8.22 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 4.84-4.80 (m, 1H), 3.47 (dd, J = 14.0, 4.8 Hz, 1H), 3.11 (dd, J = 13.9, 10.3 Hz, 1H), 2.91-2.73 (m, 6H), 2.58-2.38 (m, 6H).

[0461] Example 31: Synthesis of TCEP-34 [ka] TCEP-34 was synthesized in a similar manner to Procedure A using N-benzylhydroxylamine hydrochloride (Bidepharm) as the amine reagent to give TCEP-34 (15.7 mg, 8.85% yield) as a white solid. MS [M+H] +=356.05, C 16 H 22 The calculated exact mass of NO6P is 355.12. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.55-7.34 (m, 5H), 4.84 (s, 2H), 3.14 (dt, J = 19.8, 6.9 Hz, 2H), 2.92 (dt, J = 18.2, 7.0 Hz, 4H), 2.62 (dq, J = 14.5, 7.3 Hz, 6H).

[0462] Example 32: Synthesis of TCEP-35 [ka] TCEP-35 was synthesized in a similar manner to Procedure A using N-phenylhydroxylamine (Bidepharm) as the amine reagent to give TCEP-35 (17.1 mg, 10.0% yield) as a white solid. MS [M+H] + =341.97, C 15 The calculated exact mass of H2NO6P is 341.10. 1 H NMR (400 MHz, Deuterium Oxide):δ 7.57-6.88 (m, 5H), 3.27-3.22 (m, 1H), 2.92-2.78 (m, 3H), 2.65-2.53 (m, 6H), 2.29-1.98 (m, 2H).

[0463] Example 33: Synthesis of TCEP-37 [ka]

[0464] 1. TCEP-37-int1 To a solution of 2,4-dimethoxybenzaldehyde (1.66 g, 10.0 mmol, 1.0 eq, Adamas) in MeOH (25 mL) was added O-methylhydroxylamine hydrochloride (1.66 g, 20.0 mmol, 2.0 eq, Bidepharm). The resulting mixture was stirred at room temperature for 16 h, and LCMS confirmed the reaction was complete. The mixture was concentrated, and the residue was taken up in AcOH (20 mL), and NaBH3CN was added and stirred at room temperature for 5 h. LCMS showed the reaction was complete. The reaction mixture was concentrated, and the residue was poured into ice-water (200 mL) and extracted with EtOAc (50 mL*3). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and purified by flash column (EtOAc / petroleum ether=0-50%) to give the product TCEP-37-int1 (N-(2,4-dimethoxybenzyl)-O-methylhydroxylamine, 1.5 g, 76.1%, colorless oil).

[0465] 2. TCEP-37 TCEP-37 was synthesized in the same manner as in Synthesis Procedure A-1, using TCEP-73-int1 as the amine reagent, to obtain TCEP-37 (12.8 mg, 9.14% yield) as a white solid. MS [M+H] + =280.18, C 10 H 18 The calculated exact mass of NO6P is 279.09. 1 H NMR (400 MHz, Deuterium Oxide): δ (s, 3H), 2.89 (dt, J = 18.4, 7.1 Hz, 4H), 2.72 (dd, J = 18.1, 6.6 Hz, 2H), 2.61 (dq, J = 13.9, 6.7 Hz, 6H).

[0466] Examples 34-66: Preparation of ADCs with D2 The ADC is prepared in a one-pot reaction: (1) ZnCl2 (0.24 mM) and a reducing agent (0.02 mM) were added to a solution of monoclonal antibody (0.012 mM, MES buffer, pH 6.7, 20 mM), and the reaction mixture was left standing at 4°C for 4, 8, and 12 hours. (2) Zn 2+ EDTA (0.6 mM) was added to capture the . (3) MC-VC-PAB-MMAE (0.06 mM) in DMA was introduced, and the reaction was continued at 24 °C for 1 h. (4) Cysteine (0.08 mM) was added to remove excess MC-VC-PAB-MMAE. (5) The reaction mixture was purified using a desalting column.

[0467] The monoclonal antibody and reducing agent, the molar ratio of antibody to reducing agent, and the incubation time used in step (1) are as follows: On the other hand, a MOPS buffer was used as the buffer, and in Example 40, the pH value was set to 7.4. [Table 1] JPEG2025527005000080.jpg28149

[0468] Examples 67-81 and Comparative Example 10: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates with varying molar ratios of ZnCl2 and reducing agent The preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to the preparation of the ADC having D2 in Example 34, except that the dosage of ZnCl2 in step (1) is adjusted. The dosage of ZnCl2 and the molar ratio of ZnCl2 to the reducing agent are as follows: [Table 2] "E" stands for Example, and "C" stands for Comparative Example.

[0469] Examples 82-85: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates with varying molar ratios of antibody to reducing agent The preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to the preparation of the ADC having D2 in Example 34, except that the dose of the antibody in step (1) or the incubation time in step (1) is adjusted. The dose of the antibody and the molar ratio of the antibody to the reducing agent are as follows: [Table 3]

[0470] Examples 86-101: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates in different buffers The preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to the preparation of the ADC in Example 34, except that the buffer is adjusted as follows: [Table 4] All buffers are commercially available from Macklin.

[0471] Examples 102 to 113: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates with varying incubation temperatures or times in step (1) The preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to the preparation of the ADC in Example 34, except that the incubation temperature or time in step (1) is adjusted as follows: [Table 5]

[0472] Example 114: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates with artificial antibodies The preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate was similar to the preparation of the ADC in Example 34, but an artificial antibody was used.

[0473] This artificial antibody is a variant of trastuzumab (procured from Biointron) in which the disulfide bond between the heavy and light chains has been replaced with serine instead of cysteine.

[0474] Example 115: Preparation of trastuzumab-[maleimide-PEG-N-DBCO-Cy] (ADC with D1) 1. Synthesis of dibromomaleimide-PEG4-N3 [ka] To a solution of 3,4-dibromomaleimide (127 mg, 0.5 mmol, 1 eq) and N-methylmorpholine (0.22 mL, 2 mmol, 4 eq) in THF (3.5 mL) was added chloromethyl chloroformate (0.18 mL, 2 mmol, 4 eq) and the mixture was stirred at room temperature for 20 min. DCM (10 mL) was then added, the organic phase was washed with HO, dried over MgSO, and the solvent was removed in vacuo to give the title product 1 (139 mg, 0.4 mmol, 80%).

[0475] A solution of azido-PEG4-amine (105 mg, 0.4 mmol, 1 eq, Xi'an Confluore Biological Technology Co., Ltd) in dichloromethane (2 mL) was added to a stirred solution of product 1 (139 mg, 0.4 mmol, 1 eq) in dichloromethane (2 mL).

[0476] After 30 min, dichloromethane (6 mL) was added, and the solution was washed with 0.68 M acetate buffer pH 5 (10 mL), water (1 mL), and dried over MgSO. After concentration in vacuo, purification by column chromatography (100% EtOAc as mobile phase) afforded dibromomaleimide-PEG-N3 (title product 2) as a pale yellow oil (150 mg, 0.3 mmol, 75%).

[0477] 2. Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (1) Incubate the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) are introduced and reacted with the reduced thiol group obtained in step (1). The reaction temperature is set to 24°C and the reaction time is set to 3 hours. The product is then recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1. (3) Trastuzumab-maleimide-PEG4-N3 and DBCO-Cy3 (0.02 mM) are incubated in MES (20 mM, pH 6.7), and after 8 hours at 25°C, trastuzumab-maleimide-PEG4-N3-DBCO-Cy3 is recovered using a desalting column.

[0478] Example 116: Preparation of trastuzumab-[maleimide]l[MC-GGFG-DXd]6 (ADC with D0+D6) (1) Incubate the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and dibromomaleimide (0.013 mM) are introduced and reacted with the reduced thiol group obtained in step (1). The reaction temperature is set to 24°C and the reaction time is set to 3 hours. The product is then recovered using a desalting column to obtain trastuzumab-[maleimide] 1. (3) Trastuzumab-[maleimide] 1 and TCEP (0.08 mM) were introduced into MES (20 mM, pH 6.7), the reaction temperature was 25 °C, and the reaction time was 12 h. (4) MC-GGFG-DXd (0.14 mM) was added to the solution obtained in step (3), and the reaction mixture was allowed to stand at 24°C for 1 hour. Trastuzumab-[maleimide]1[MC-GGFG-DXd]6 was then recovered using a desalting column.

[0479] Example 117: Preparation of trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 (ADC with D2+D6) (1) Incubate the first reducing agent TCEP-NO (0.02 mM) prepared according to Example 1 and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and excess MC-MMAF (0.06 mM) are introduced to react with the reduced thiol groups generated from step (1), the reaction temperature is 24°C, and the reaction time is 1 hour. Then, the product is recovered using a desalting column to obtain trastuzumab-[MC-MMAF]2. (3) Trastuzumab-[MC-MMAF]2 and TCEP (0.08 mM) were introduced into MES (20 mM, pH 6.7), the reaction temperature was 25 °C, and the reaction time was 8 h. (4) MC-GGFG-DXd (0.14 mM) was added to the solution obtained in step (3), and the reaction mixture was allowed to stand at 24°C for 1 hour. The resulting trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 was then recovered using a desalting column.

[0480] Examples 118-119: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 (ADC with D2+D2) (1) Incubate the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl (0.12 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.048 mM) are introduced to react with the reduced thiol group obtained in step (1), the reaction temperature is 24°C, the reaction time is 1 hour, and the product is recovered using a desalting column to obtain trastuzumab-[MC-VC-PAB-MMAE]2. (3) ZnCl (1.2 mM), the second reducing agent TCEP-3 (0.0144 mM) / TCEP-6 (0.0216 mM), and the product of step (2) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4°C for 4 hours. (4) Zn by introducing EDTA (3 mM) 2+ was captured, and MC-GGFG-DXd (0.1 mM) was introduced to react with the reduced thiol group obtained in step (3), the reaction temperature was 24°C, and the reaction time was 1 hour. (5) The reaction mixture was purified using a desalting column.

[0481] Example 120: Preparation of trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] (ADC with D+D) (1) Incubate the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) are introduced to react with the reduced thiol groups obtained in step (1). The reaction temperature is set to 25°C and the reaction time is set to 1 hour. The product is then recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1. (3) Trastuzumab-[maleimide-PEG4-N3]1 from step (2) and DBCO-Cy3 (0.02 mM) are incubated in MES (20 mM, pH 6.7), and after a reaction time of 6 hours at 25°C, trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 is recovered using a desalting column. (4) ZnCl2 (1.2 mM), the second reducing agent TCEP-3 (0.0144 mM), and the product prepared from step (3) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 4 hours. (5) Zn was introduced by adding EDTA (3 mM). 2+ , and MC-VC-PAB-MMAE (0.1 mM) was reacted with the reduced thiol group obtained in step (4), the reaction temperature was 25°C, and the reaction time was 1 hour. (6) The reaction mixture was purified using a desalting column.

[0482] Examples 121-122: Preparation of trastuzumab-[maleimide]1[MC-VC-PAB-MMAE]4 (ADC with D0+D4) (1) Incubate the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and dibromomaleimide (0.013 mM) are introduced and reacted with the reduced thiol group obtained in step (1). The reaction temperature is set to 24°C and the reaction time is set to 3 hours. The product is then recovered using a desalting column to obtain trastuzumab-[maleimide]. (3) ZnCl2 (0.36 mM), the second reducing agent TCEP-3 (0.036 mM) or TCEP-6 (0.048 mM), and the product of step (2) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4°C for 16 hours. (4) Zn was introduced by introducing EDTA (0.6 mM). 2+ , and MC-VC-PAB-MMAE (0.1 mM) was reacted with the reduced thiol group obtained in step (4), the reaction temperature was 24°C, and the reaction time was 1 hour. (5) The reaction mixture was purified using a desalting column.

[0483] Examples 123-124: Preparation of trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 (ADC with D2+D4) (1) Incubate the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and MC-GGFG-DXd (0.072 mM) were introduced to react with the reduced thiol group obtained in step (1), the reaction temperature was 24°C, the reaction time was 1 hour, and the product was recovered using a desalting column to obtain trastuzumab-[MC-GGFG-DXd]2. (3) ZnCl2 (0.36 mM), the second reducing agent TCEP-3 (0.036 mM) or TCEP-6 (0.048 mM), and the product of step (2) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4°C for 16 hours. (4) Zn was introduced by introducing EDTA (0.6 mM). 2+ , and MC-VC-PAB-MMAE (0.1 mM) was reacted with the reduced thiol group obtained in step (4), the reaction temperature was 24°C, and the reaction time was 1 hour. (5) The reaction mixture was purified using a desalting column.

[0484] Example 125: Preparation of trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] (ADC with D1+D4) (1) Incubate the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl (0.24 mM) (incubation temperature: 4°C, incubation time: 4 hours). (2) EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) are introduced and reacted with the reduced thiol group obtained in step (1). The reaction temperature is set to 25°C and the reaction time is set to 6 hours. The product is then recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]. (3) Trastuzumab-maleimide-PEG4-N3 and DBCO-Cy3 (0.02 mM) are incubated in MES (20 mM, pH 6.7), and after 6 hours at 25°C, trastuzumab-maleimide-PEG4-N3-DBCO-Cy3 is recovered using a desalting column. (4) ZnCl2 (0.36 mM), the second reducing agent TCEP-3 (0.0408 mM) / ), and the product of step (3) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 16 hours. (5) Zn was introduced by introducing EDTA (0.6 mM). 2+ , and MC-VC-PAB-MMAE (0.1 mM) was reacted with the reduced thiol group obtained in step (4), the reaction temperature was 24°C, and the reaction time was 1 hour. (6) The reaction mixture was purified using a desalting column.

[0485] Comparative Examples 1-9: Preparation of ADCs without transition metal ions ADC with D2 was prepared as follows: (1) TCEP-NO, TCEP-3NO, or TCEP-CO (0.02 mM) was added to a solution of monoclonal antibody (0.012 mM, MES buffer, pH 6.7, 20 mM), and the reaction mixture was allowed to stand at 4°C for 4, 8, or 12 hours, respectively. (2) MC-VC-PAB-MMAE (0.06 mM) in DMA was introduced, and the reaction was continued at 24 °C for 1 h. (3) Cysteine (0.08 mM) was added to remove excess MC-VC-PAB-MMAE. (4) The reaction mixture was subjected to purification using a desalting column.

[0486] The monoclonal antibodies and reducing agents used were as follows: [Table 6]

[0487] Comparative Example 11: Preparation of ADC using TCEP (1) TCEP (0.02 mM) was added to a solution of trastuzumab (0.012 mM, MES buffer, pH 6.7, 20 mM), and the reaction mixture was allowed to stand at 4°C for 4 hours. (2) MC-VC-PAB-MMAE (0.06 mM) in DMA was introduced, and the reaction was continued at 24 °C for 30 min. (3) Cysteine (0.08 mM) was added to remove excess MC-VC-PAB-MMAE. (4) The reaction mixture was subjected to purification using a desalting column.

[0488] Comparative Example 12: Preparation of ADC using TCEP (1) ZnCl2 (0.24 mM) and TCEP (0.02 mM) were added to a solution of trastuzumab (0.012 mM, MES buffer, pH 6.7, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 4 hours. (2) Zn 2+ EDTA (0.6 mM) was added to capture the . (3) MC-VC-PAB-MMAE (0.06 mM) in DMA was introduced, and the reaction was continued at 24 °C for 30 min. (4) Cysteine (0.08 mM) was added to remove excess MC-VC-PAB-MMAE. (5) The reaction mixture was subjected to purification using a desalting column.

[0489] Homogeneity Assay The drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC (Agilent 1200) with a TSK gel Butyl-NPR column (4.6 mm IDX 3.5 cm) (procured from Tosoh Biosciences) at a flow rate of 0.5 mL / min and 30 °C. Solvent A was 1.5 M (NH4)2SO4 and 50 mM potassium phosphate pH 7. Solvent B was 75% v / v 50 mM potassium phosphate pH 7 and 25% v / v isopropanol. The washing procedure was as follows: [Table 7]

[0490] The results of Examples 34 to 36 and Comparative Examples 1 to 3 are shown in Table 1. Chromatograms are shown in Figures 1 to 6.

[0491] As shown in Table 1, the ADCs prepared by TCEP-NO in Examples 34-36 and Comparative Examples 1-3 had different D2 and D4 ratios, indicating that MC-VC-PAB-MMAE was successfully linked to trastuzumab, sacituzumab, or belantamab. TCEP-NO may be used as a reducing agent in antibody modification and ADC preparation. In contrast to C1-C3, the ADCs prepared by TCEP-NO in Examples 34-36 had significantly increased D2 ratios. This suggests that TCEP-NO significantly increased the D2 ratio of Zn. 2+ These results indicate that TCEP-NO has reductive selectivity in the presence of D2, and that it may be useful for preparing ADCs bearing D2. [Table 8] "E" stands for Example. "C" stands for Comparative Example.

[0492] The results of Examples 37 to 39 and Comparative Examples 4 to 6 are shown in Table 2, and the chromatograms are shown in FIGS.

[0493] As shown in Table 2, the ADCs of Examples 37-39 and C4-C6 prepared with TCEP-3NO had different D2 and D4 ratios, indicating that MC-VC-PAB-MMAE was successfully linked to trastuzumab, sacituzumab, or belantamab. TCEP-3NO may be used as a reducing agent in antibody modification and ADC preparation. In contrast to C4-C6, the ADCs of Examples 37-39 prepared with TCEP-3NO significantly increased the D2 ratio. This suggests that TCEP-3NO significantly increased the D2 ratio of Zn. 2+ These results indicate that TCEP-3NO has reductive selectivity in the presence of D2, suggesting that it may be useful for preparing ADCs bearing D2. [Table 9]

[0494] The results of Examples 40 to 42 and Comparative Examples 7 to 9 are shown in Table 3, and the chromatograms are shown in Figures 13 to 17.

[0495] As shown in Table 3, the ADCs of Examples 40-42 and C7-C9 prepared by TCEP-CO had different D2 and D4 ratios, indicating that MC-VC-PAB-MMAE was successfully linked to trastuzumab, sacituzumab, or belantamab. TCEP-CO may be used as a reducing agent in antibody modification and ADC preparation. In contrast to C7-C9, the ADCs of Examples 40-42 prepared by TCEP-CO significantly increased the D2 ratio. This suggests that TCEP-CO significantly increased the D2 ratio of Zn. 2+ These results indicate that TCEP-CO has reductive selectivity in the presence of D2, and that it may be useful for preparing ADCs bearing D2. [Table 10]

[0496] As a negative control, the conjugate formation process was performed using the same steps without adding transition metal ions in step (a) (see Tables 1-3). This disclosure successfully demonstrated that the combination of transition metal ions and the novel reducing agent resulted in higher D2 levels in the resulting ADC. Furthermore, this new process was confirmed to produce ADC products with high Fc and / or Fab preferences. Using the process of this disclosure to prepare antibody-drug conjugates dramatically improved the homogeneity of the antibody-drug conjugates.

[0497] As can be seen from the results of Examples 43-66 and Comparative Examples 11-12 shown in Table 4 and Figures 18 to 23, the compounds of the present invention have the following properties: 2+ Compared with conventional methods using TCEP without D2, the homogeneity of D2-containing ADCs was improved, and the selective reduction ability of TCEP-6 was the most excellent, with a D2 content of up to 94.25%. Meanwhile, the selective reduction ability of TCEP-NO, TCEP-3NO, TCEP-CO, TCEP-1, TCEP-19, TCEP-20, TCEP-21, TCEP-23, TCEP-24, TCEP-26, TCEP-28, TCEP-34, TCEP-35, and TCEP-37 was also excellent, with D2 contents of up to 84%, 87%, and even 90% or 93%. [Table 11]

[0498] The results of Examples 67 to 81 and Comparative Example 10 are shown in Table 5, and the chromatograms are shown in Figures 24 to 37. As shown in the results in Table 5, the content of D2 increases by adding transition metal ions. The D2 ratio is 2+ The D2 ratio increases as the Zn / TCEP-NO molar ratio increases from 0.4 to 6. After that, the D2 ratio reaches a plateau. 2+ When the molar ratio of D2 to TCEP-NO was 200:1 and up to 250:1, the content of D2 was Zn 2+ This is lower than when the molar ratio of TCEP-NO / TCEP-NO is 2:1 to 125:1. This is because the transition metal ions, especially Zn 2+This indicates that the / TCEP-NO ratio plays an important role in determining the D2 ratio and reduction selectivity. [Table 12]

[0499] The results for Examples 82-85 are shown in Table 6, and the chromatograms are shown in Figure 38. As shown in Table 6 and the results for Examples 46, 49, 53, and 54, when the antibody / TCEP-NO molar ratio was 1:0.9 to 1:3.0, the D2 content of the ADC reached 55%, 60%, 70%, 75%, and even 80%, 85%, and 90%. When the antibody / TCEP-NO molar ratio was 1:2 or 1:2.5, the reduction time was shortened to 1 hour, and the D2 content reached 80% or more. [Table 13]

[0500] The results for Examples 86-101 are shown in Table 7, and the chromatograms are shown in Figures 39-52. As shown by the results in Table 7, different buffers dramatically affect the reduction rate and selectivity. The buffers in Examples 86-101 are useful for improving the D2 content of ADCs. [Table 14]

[0501] The results of Examples 102 to 113 are shown in Table 8, and the chromatograms are shown in Figures 53 to 55. As shown in Table 8, when the reduction temperature was 4 to 37°C and the reduction time was 0.25 to 6 hours, the content of ADC having D2 was up to 80%. As the reduction time in step (1) increased from 0.25 to 1 hour, the content of D2 increased and reached a plateau after 1 hour, indicating that the reaction rate was very fast. [Table 15]

[0502] The results of Example 114 are shown in Table 9, and the chromatogram is shown in Figure 56. As shown in Table 9, the content of D2 prepared by the engineered antibody was as high as 96%. These results indicated that this method can be applied to antibodies with simple mutations and has even better reduction selectivity in some mutant antibodies. [Table 16]

[0503] As shown in Table 10 and Figure 57, the content of ADCs with D1 was generally up to 83%. As shown in Table 11 and Figure 58, the results show that the content of ADCs with D0+D6 was generally up to 84.68%. As shown in Table 12 and Figure 59, the results show that the content of ADCs with D2+D6 was generally up to 81.31%. These results demonstrate that the method of the present application can modify antibodies with site specificity and prepare different types of ADCs with improved homogeneity. [Table 17] [Table 18] [Table 19]

[0504] In step (3) of Examples 118-119, one of the interchain disulfide bonds of the ADC with D2 was reduced. As shown in Table 13 and Figure 60, the results showed that the content of ADC with D2+D2 was generally up to 68% or 70%, indicating that this method step is useful for site-specifically modifying antibodies with D2+D2 and improving homogeneity. [Table 20]

[0505] As shown in Table 14 and Figure 61, the results showed that the content of ADC with D1+D2 was generally up to 80% or 83%, indicating that the process steps are beneficial for site-specifically modifying antibodies with D1+D2 and improving homogeneity. [Table 21]

[0506] In step (3) of Examples 121-122, two of the interchain disulfide bonds of the ADCs bearing D2 were reduced. As shown in Table 15 and Figure 62, the results showed that the content of ADCs bearing D0+D4 was generally up to 55% or 61%, indicating that this method step is beneficial for site-specifically modifying antibodies bearing D0+D4 and improving homogeneity. [Table 22]

[0507] As shown in Table 16 and Figure 63, the results showed that the content of ADCs with D2+D4 generally reached 70%, 75%, and even 78% or 80%, indicating that the steps of this method are beneficial for site-specifically modifying antibodies with D2+D4 and improving homogeneity. [Table 23] As shown in Table 17 and Figure 64, the results showed that the content of ADC with D1+D4 generally reached 60%, 65%, and even 70%, indicating that the steps of this method are beneficial for site-specifically modifying antibodies with D1+D4 and improving homogeneity. [Table 24]

[0508] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents may occur that are presently unforeseen or conceivable to applicant or those skilled in the art. Accordingly, the appended claims, as filed and as they may be amended, are intended to embrace all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. Formula (I): 【Chemical 1】 or a salt, solvate or stereoisomer thereof, X, Y and Z are independently P-C(sp ３ ) or PC (sp ２ ) is covalently bonded to the phosphorus atom via a P-C bond, X is a group represented by formula (II): 【Chemistry 2】 and L １ is -CH(R １ ) -, -C(CH ３ ) (R １ ) -, -CH(R １ ) CH(R ２ ) -, -CH(R １ ) CH(R ２ ) CH(R ３ )-, an aryl group optionally substituted with a group containing at least one coordinating atom selected from N, O, and S, and a heteroaryl group optionally substituted with a group containing at least one coordinating atom selected from O and S; R １ , R ２ and R ３ are independently H, C １ -C ５ Alkyl group, C １ -C ５ Hydroxyalkyl group, C １ -C ５ Carboxyalkyl group, C １ -C ５ Hydroxylamine alkyl group, C １ -C ５ an N-hydroxyamidoalkyl group, an aryl group, or a heteroaryl group; R ２ or R ３ Is, L ２ and forming an optionally substituted 5- or 6-membered ring, A is optionally present and is —C(O)— or —C(O)J—; J is an organic group containing both an amino group or an imino group and a carbonyl group, and the amino group or the imino group forms an amide group with —C(O), and the carboxyl group forms an amide group with L ２ may be covalently bonded to L ２ is optionally present and acts as a transition metal chelator motif, -N(R ４ ) (R ５ ) or hydroxy, R ４ and R ５ are independently hydrogen, C ０ -C ５ Hydroxyalkyl group, C １ -C ５ Alkyl group, C １ -C ５ Alkoxy group, -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ), an optionally substituted 5- to 6-membered heterocyclic group, an optionally substituted arylalkyl group, an optionally substituted arylalkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted heteroarylalkyl group, or R ４ and R ５ form an optionally substituted 5- or 6-membered ring, and R ４ or R ５ is R ２ or R ３ and forming an optionally substituted 5- or 6-membered ring with R ６ is hydrogen, amino, C １ -C ５ Alkyl, C １ -C ５ Hydroxyalkyl group, C １ -C ５ a carboxyalkyl group, an aryl group, an optionally substituted arylalkyl group, C １ -C ５ an N-hydroxyamidoalkyl group, a heteroaryl group, or a heteroarylalkyl group; R ７ is hydroxy, C １ -C ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ are independently 0, 1, 2, 3, or 4; R ４ and R ５ is not hydroxy at the same time, Y is the same as X, Z is the same as X, or Y and Z are independently an optionally substituted 5- to 6-membered heterocyclic group, C １ -C ５ Alkyl group, C １ -C ５ Hydroxyalkyl group, aryl group, C １ -C ５ a carboxyalkyl group, an optionally substituted 5- or 6-membered cycloalkyl group, or 【Chemistry 3】 -C(O)Q is an ester group, an imide group, or an amide group; X, Y and Z are simultaneously —CH ２ CH ２ A compound or a salt, solvate or stereoisomer thereof, characterized in that it is not C(O)OH.

2. L １ -CH(R １ ) CH(R ２ ) - and R １ and R ２ are independently H, methyl, isopropyl, hydroxymethyl, hydroxyethyl, carboxymethyl, carboxyethyl, N-hydroxyethylamide, phenyl, 2-pyridyl, 4-pyridyl, or 4-imidazole, or R ２ L ２ The compound according to claim 1, wherein the compound forms an optionally substituted 5- or 6-membered ring with

3. L １ -CH(R １ ) CH(R ２ ) - and R １ is H, R ２ L ２ R ４ and forming an optionally substituted 5- or 6-membered ring with L ２ -N (R ４ ) (R ５ ) and R ５ 3. The compound according to claim 1 or 2, characterized in that is hydroxy.

4. L １ is an optionally substituted phenyl group attached to A at the ortho, meta, or para position, A is —C(O)—, L ２ -N (R ４ ) (R ５ ) or hydroxy, R ４ is hydrogen, and R ５ 3. The compound according to claim 1 or 2, characterized in that is hydroxy.

5. L １ 5. The compound according to claim 4, wherein is a phenyl group substituted with a hydroxy group or a carboxy group at the ortho or meta position, and the phenyl group is bonded to A at the para position.

6. L １ is a phenyl group optionally substituted at the ortho, meta, or para position with hydroxy, halogen, carboxy, sulfonyl, amino, methoxy, or ethoxy; A and L ２ 3. The compound according to claim 1 or 2, characterized in that: is absent.

7. L １ is an optionally substituted 4-pyridyl group or an optionally substituted 4-quinolyl group, and A and L ２ 3. The compound according to claim 1 or 2, characterized in that: is absent.

8. L １ teeth, 【Chemistry 4】 【Chemistry 5】 The compound according to claim 7, characterized in that:

9. L １ is -CH(R １ ) CH(R ２ ) - and R １ is a methyl group, an isopropyl group, a carboxyethyl group, or an N-hydroxyethylamide group, and R ２ 3. The compound according to claim 1 or 2, characterized in that: is H.

10. L １ is -CH(R １ ) CH(R ２ ) - and R １ is H, R ２ is a methyl group, a hydroxymethyl group, a hydroxyethyl group, a carboxyethyl group, a phenyl group, an N-hydroxyethylamide group, a 2-pyridyl group, a 4-pyridyl group, or a 4-imidazole group.

11. L １ is -CH(R １ ) CH(R ２ ) - and R １ is a methyl group, an isopropyl group, a carboxyethyl group, or an N-hydroxyethylamide group, R ２ is H, A is —C(O)—, L ２ HA-N(R ４ ) (R ５ ) and R ４ is hydrogen, and R ５ 3. The compound according to claim 1 or 2, characterized in that is hydroxy.

12. L １ is -CH(R １ ) CH(R ２ ) - and R １ is H, R ２ is a methyl group, a hydroxymethyl group, a hydroxyethyl group, a carboxyethyl group, a phenyl group, an N-hydroxyethylamide group, a 2-pyridyl group, a 4-pyridyl group, or a 4-imidazole group, A is —C(O)—, L ２ HA-N(R ４ ) (R ５ ) and R ４ is hydrogen or an optionally substituted 5- or 6-membered saturated heterocyclic group, R ５ 3. The compound according to claim 1 or 2, characterized in that is hydroxy.

13. R ４ teeth 【Chemistry 6】 The compound according to claim 12, characterized in that:

14. L １ is -CH(R １ ) CH(R ２ ) - and R １ is H, R ２ is a methyl group, a hydroxymethyl group, a hydroxyethyl group, a carboxyethyl group, a phenyl group, an N-hydroxyethylamide group, a 2-pyridyl group, a 4-pyridyl group, or a 4-imidazole group, A is —C(O)—, L ２ HA-N(R ４ ) (R ５ ) and R ４ and R ５ The compound according to claim 1 or 2, wherein: forms an optionally substituted 5- or 6-membered ring.

15. L ２ teeth 【Chemistry 7】 15. The compound according to claim 14, wherein

16. L １ is -CH(R １ ) CH(R ２ ) - and R １ and R ２ The compound of claim 1 , wherein:

17. L ２ HA-N(R ４ ) (R ５ ) and R ４ is hydrogen, C １ -C ５ Alkyl group, -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ), an optionally substituted 5- to 6-membered saturated heterocyclic group, an optionally substituted arylalkyl group, an optionally substituted aryl group, an optionally substituted heteroarylalkyl group, or R ４ and R ５ form an optionally substituted 5- or 6-membered ring, R ５ is hydroxy, R ６ is hydrogen, amino, C １ -C ５ Alkyl, C １ - ５ Hydroxyalkyl group, C １ - ５ Carboxyalkyl group, aryl group, C １ - ５ an N-hydroxyamidoalkyl group, a heteroaryl group, or a heteroarylalkyl group; R ７ is a hydroxy group, C １ -C ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ 17. The compound according to claim 1 or 16, wherein each of is independently 0, 1, 2, 3, or 4.

18. R ４ is hydrogen, methyl group, -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ), a 5-6 membered saturated heterocyclic group containing a heteroatom N or O, a benzyl group, a benzyl group substituted with hydroxy on the phenyl ring, a phenyl group optionally substituted with hydroxy, halogen or carboxy group, a heteroarylalkyl group containing a heteroatom N, or R ４ and R ５ forms a 5- to 6-membered ring, R ５ is hydroxy, R ６ is hydrogen, C １ -C ５ Alkyl, C １ -C ５ a hydroxyalkyl group, or a heteroarylalkyl group, R ７ is a hydroxy group, C １ -C ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ 18. The compound according to claim 1 or 17, wherein: are independently 0, 1, 2, 3, or 4.

19. R ４ teeth 【Chemistry 8】 Hydrogen or -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ) and R ５ is hydroxy, R ６ is a hydrogen atom, a methyl group, a hydroxymethyl group, or 【Chemistry 9】 and R ７ is hydroxy or -NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ The compound of claim 18, wherein: is independently 0.

20. L ２ HA-N(R ４ ) (R ５ ) and R ４ and R ５ are independently -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ) or an optionally substituted heteroarylalkyl group, R ６ is hydrogen, amino, C １ -C ５ Alkyl, C １ -C ５ Hydroxyalkyl group, C １ -C ５ Carboxyalkyl group, aryl group, C １ -C ５ an N-hydroxyamidoalkyl group, a heteroaryl group, or a heteroarylalkyl group; R ７ is hydroxy, C １ -C ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ 17. The compound according to claim 1 or 16, wherein each of is independently 0, 1, 2, 3, or 4.

21. R ４ and R ５ are independently -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ) or a 6-membered heteroarylalkyl group; R ６ is hydrogen, R ７ is hydroxy, C １ -C ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ 21. The compound according to claim 20, wherein each independently represents a value of 0, 1, 2, 3, or 4.

22. R ４ and R ５ are independently -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ )or 【Chemistry 10】 and R ６ is hydrogen, R ７ is hydroxy or -NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ 22. The compound according to claim 21, wherein is 0.

23. L ２ HA-N(R ４ ) (R ５ ) and R ４ is hydrogen, C ０ -C ５ Hydroxyalkyl group, C １ -C ５ alkyl group, optionally substituted C １ -C ５ Alkoxy group, -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ), an optionally substituted arylalkyl group, an optionally substituted arylalkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted heteroarylalkyl group; R ５ is hydrogen, R ６ is hydrogen, amino, C １ -C ５ Alkyl, C １ - ５ Hydroxyalkyl group, C １ - ５ a carboxyalkyl group, an aryl group, an optionally substituted arylalkyl group, C １ - ５ an N-hydroxyamidoalkyl group, a heteroaryl group, or a heteroarylalkyl group; R ７ is hydroxy, C １ - ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ 17. The compound according to claim 1 or 16, wherein each of is independently 0, 1, 2, 3, or 4.

24. R ４ is hydrogen, C ０ -C ３ Hydroxyalkyl group, C １ -C ３ Alkoxy group, -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ), a phenyl group substituted with carboxy, hydroxy, amino, or halogen, a pyridyl group, an amino substituted with 2-methylpyridine, a benzyl group substituted with carboxy, hydroxy, amino, or halogen, an arylalkoxy group, a pyridyl group substituted with carboxy, a bipyridyl group, 【Chemistry 11】 and R ５ is hydrogen, R ６ is hydrogen, amino, C １ - ３ Alkyl, C １ - ３ Hydroxyalkyl group, C １ - ３ a carboxyalkyl group, an aryl group, an arylalkyl group which may be substituted with a hydroxy group, a halogen, a cyano group or a nitro group; １ - ５ an N-hydroxyamidoalkyl group, a heteroaryl group, or a heteroarylalkyl group; R ７ is hydroxy, C １ - ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ 24. The compound of claim 23, wherein each of is independently 0, 1, 2, 3, or 4.

25. R ４ is hydrogen, hydroxyl group, ethylhydroxyl group, methoxy group, 【Chemistry 12】 and R ５ 25. The compound of claim 24, wherein is hydrogen.

26. R ４ Ha-(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ) and R ５ is hydrogen, R ６ represents hydrogen, amino, methyl, a hydroxymethyl group, a carboxyethyl group, a benzyl group, —OH, F, —CN, or —NO ２ a benzyl group substituted with, an N-hydroxyethylamide group; 【Chemistry 13】 and R ７ is hydroxy, -NH(CH ２ CONH)n ３ OH, n １ and n ３ are independently 0, 1, 2, 3, or 4; n ２ 24. The compound according to claim 23, wherein is 0.

27. R ４ Ha-(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ) and R ５ is hydrogen, R ６ represents hydrogen, amino, methyl, hydroxymethyl group, benzyl group, carboxyethyl group, 【Chemistry 14】 an N-hydroxyethylamide group, a heteroaryl group, or a heteroarylalkyl group; R ７ is hydroxy, -NH(CH ２ CONH)n ３ OH, n １ is 0 or 2, n ２ is 0 or 1, n ３ 24. The compound according to claim 23, wherein is 0.

28. J is a monoamino acid residue, a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, an aminopropionic acid, an aminobutyric acid, an aminovaleric acid, an amino acid, an aminoheptanoic acid, an aminooctanoic acid, or —NH(OCH ２ CH ２ O)n ４ CH ２ is a peptide residue containing COOH, n ４ is a number between 2 and 10, 2. The compound of claim 1, wherein the amino acid is selected from the group consisting of glycine (Gly), alanine (Ala), serine (Ser), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).

29. 29. The compound of claim 28, wherein J is a residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine.

30. A is -C(O)J-, J is a histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine, or asparagine residue; L ２ HA-N(R ４ ) (R ５ ) and R ４ is hydrogen, and R ５ 30. The compound according to claim 28 or 29, characterized in that is hydroxy.

31. Y and Z are independently 【Chemistry 15】 and Q is -NHOH, -NHCH ２ CH ２ SO ３ H, —N(CH ２ CH ２ OH) ２ , -NHCH ２ COOH, -NHCH(CH ３ )COOH, -NH(CH ２ CH ２ O) ３ CH ３ The compound according to claim 1, characterized in that:

32. [Catalog 16] 【change】 【change】 【change】 【change】 The compound according to claim 1, characterized in that it is selected from the group consisting of:

33. A composition comprising the compound according to any one of claims 1 to 32 and a transition metal ion.

34. The transition metal ion is Zn ２＋ , Cd ２＋ , Hg ２＋ , Ni ２＋ , Co ２＋ or a combination thereof, and optionally the transition metal ion is selected from the group consisting of Zn ２＋ 34. The composition of claim 33, wherein:

35. 35. The composition according to claim 33 or 34, characterized in that the molar ratio of the compound according to any one of claims 1 to 32 to the transition metal ion is from 1:0.4 to 1:250, and optionally the molar ratio of the compound according to any one of claims 1 to 32 to the transition metal ion is from 1:0.4 to 1:

200.

36. 35. The composition according to claim 33 or 34, characterized in that the molar ratio of the compound according to any one of claims 1 to 32 to the transition metal ion is 1:0.4 to 1:60 or 1:6 to 1:

16.

37. A method for preparing a compound according to any one of claims 1 to 32, comprising the steps of: By introducing a condensing agent under an inert atmosphere, at least one carboxy group of the following formula III is converted to a transition metal chelator moiety: 【Chemistry 17】 The method of claim 1, wherein the heteroatom is bonded to the heteroatom of 【Chemistry 18】 In the formula, X′ is 【Chemistry 19】 and L １ is -CH(R １ ) -, -CH(R １ ) CH(R ２ ) -, -CH(R １ ) CH(R ２ ) CH(R ３ )-, an aryl group optionally independently substituted with a group containing at least one coordinating atom selected from N, O, and S, and a heteroaryl group optionally independently substituted with a group containing at least one coordinating atom selected from O and S; R １ , R ２ and R ３ are independently H, C １ -C ５ Alkyl group, C １ -C ５ Hydroxyalkyl group, C １ -C ５ Carboxyalkyl group, C １ -C ５ Hydroxyamine alkyl group, C １ -C ５ an N-hydroxyamidoalkyl group, an aryl group, or a heteroaryl group; R ２ or R ３ Is, L ２ and forming an optionally substituted 5- or 6-membered ring with A' is -COOH or -C(O)J-COOH; J is an organic group containing both an amino group or an imino group and a carbonyl group, and the amino group or the imino group forms an amide group with —C(O), and the carboxy group forms an amide group with L ２ may be covalently bonded to L ２ is optionally present and acts as a transition metal chelator motif, -N(R ４ ) (R ５ ) or hydroxy, R ４ and R ５ are independently hydrogen, C ０ -C ５ Hydroxyalkyl group, C １ -C ５ Alkyl group, C １ -C ５ Alkoxy group, -(CH ２ ) n １ (OCH ２ CH ２ O)n ２ CH (R ６ ) CO(R ７ ), an optionally substituted 5- to 6-membered saturated heterocyclic group, an optionally substituted arylalkyl group, an optionally substituted arylalkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted heteroarylalkyl group, or R ４ and R ５ form an optionally substituted 5- or 6-membered ring, or R ４ or R ５ is R ２ or R ３ and forming an optionally substituted 5- or 6-membered ring with R ６ is hydrogen, amino, C １ -C ５ Alkyl, C １ -C ５ Hydroxyalkyl group, C １ -C ５ a carboxyalkyl group, an aryl group, an optionally substituted arylalkyl group, C １ -C ５ an N-hydroxyamidoalkyl group, a heteroaryl group, or a heteroarylalkyl group; R ７ is a hydroxy group, C １ -C ５ Alkoxy group, —NH(CH ２ CONH)n ３ OH, n １ , n ２ and n ３ are independently 0, 1, 2, 3, or 4; R ４ and R ５ is not hydroxy at the same time, Y' is the same as X'; Z' is the same as X', or Y' and Z' are independently an optionally substituted 5- to 6-membered saturated heterocyclic group, C １ -C ５ Alkyl group, C １ -C ５ Hydroxyalkyl group, aryl group, C １ -C ５ a carboxyalkyl group, an optionally substituted 5- or 6-membered cycloalkyl group, or 【Chemistry 20】 and -C(O)Q is an ester group, an imide group, or an amide group.

38. The transition metal chelator moiety may be selected from the group consisting of 2-phenoxyethylamine, phenylamine, benzylamine, 4-aminobenzene-1,2-diol, 5-amino-2-hydroxybenzoic acid, bis(pyridin-2-ylmethyl)amine, 5-amino-8-hydroxyquinoline, bis(pyridin-2-yl)methanamine, 4-aminophthalic acid, tert-butyl-L-tyrosinate, DL-3-(4-fluorophenyl)alanine, DL-4-cyanophenylalanine, DL-4-nitrophenylalanine, N-benzylhydroxylamine hydrochloride, N-phenylhydroxylamine, 【Chemical 21】 38. The method of claim 37, characterized in that it can be provided by:

39. The structure of formula III 【Chemical 22】 39. The method according to claim 37 or 38, characterized in that:

40. Use of a compound according to any one of claims 1 to 32 or a composition according to any one of claims 33 to 36 in antibody modification.

41. 41. The use according to claim 40, wherein the antibody is modified by selectively reducing the interchain S-S bonds, optionally wherein the antibody is modified by selectively reducing one of the interchain S-S bonds.

42. The use according to claim 40 or 41, wherein in the preparation of an antibody having a thiol group site-specific modification, optionally the antibody having a thiol group site-specific modification is an antibody-drug conjugate (ADC), more optionally the ADC is an ADC having D2, an ADC having D1, an ADC having D2+D6, an ADC having D2+D3, an ADC having D1+D6, an ADC having D1+D3, an ADC having D0+D6, an ADC having D0+D3, an ADC having D0+D4, an ADC having D2+D4, an ADC having D1+D4, an ADC having D2+D2, or an ADC having D1+D2.

43. A method for preparing an antibody having a thiol group site-specific modification, characterized in that a thiol group is reduced from an interchain disulfide bond within the antibody, the method comprising using a compound according to any one of claims 1 to 32, or a salt, solvate, or stereoisomer thereof, and a transition metal ion, or using a composition according to any one of claims 33 to 36.

44. 44. The method of claim 43, wherein the number of thiol groups is 1, 2, 3, 4, 5, 6, 7 or 8.

45. 45. The method of claim 44, wherein interchain disulfide bonds link the two upper heavy chains in the hinge region, or the heavy chains and the light chains in the Fab region.

46. 45. The method of claim 44, wherein interchain disulfide bonds link the two heavy chains in the hinge region and the heavy chains with the light chains in the Fab region.

47. 44. The method of claim 43, comprising the steps of: (a) incubating the compound according to any one of claims 1 to 32, or a salt, solvate or stereoisomer thereof, acting as a first reducing agent in the presence of a transition metal ion, with an antibody in a first buffer system to selectively reduce interchain disulfide bonds in the antibody, or incubating the composition according to any one of claims 33 to 36, with the compound according to any one of claims 1 to 32 acting as a first reducing agent, and the antibody in a first buffer system to selectively reduce interchain disulfide bonds of the antibody; (b) introducing a metal chelator and modifying reagent 1 to react with the reduced thiol group resulting from step (a), wherein modifying reagent 1 is an end-capping reagent, a first linker payload, or a first thio bridging reagent, and optionally, the first thio bridging reagent has a first linker payload or a reactive group.

48. 48. The method of claim 47, further comprising the steps of: (c) incubating the reaction product obtained in step (b) and a second reducing agent in a second buffer system to reduce interchain disulfide bonds in the reaction product and, optionally, introduce transition metal ions. (d) introducing a modifying reagent 2 for reacting with the incubation product obtained in step (c) and the reduced thiol group obtained in step (c), and optionally introducing a metal chelator, wherein the modifying reagent 2 is a second linker payload or a second thio bridging reagent, and optionally the second thio bridging reagent has a second linker payload or a reactive group.

49. 49. The method of claim 47 or 48, wherein the first thio bridging reagent and the second thio bridging reagent independently comprise at least two substituents that allow for re-crosslinking of thiol groups.

50. The first thio bridging reagent and the second thio bridging reagent are each independently 【Chemical 23】 49. The method of claim 47 or 48, characterized in that it is selected from the group consisting of:

51. 49. The method of claim 47 or 48, wherein the reactive groups independently comprise azide and / or dibenzocyclooctyne (DBCO).

52. 48. The method of claim 47, wherein the molar ratio of the first reducing agent to the transition metal ion is from 1:0.4 to 1:250, optionally the molar ratio of the first reducing agent to the transition metal ion is from 1:0.4 to 1:60, further optionally the molar ratio of the first reducing agent to the transition metal ion is from 1:6 to 1:16, and most optionally the molar ratio of the first reducing agent to the transition metal ion is 1:

12.

53. 48. The method of claim 47, wherein the molar ratio of the first reducing agent to the antibody is between 3:1 and 0.5:1, optionally the molar ratio of the first reducing agent to the antibody is between 3:1 and 1:1, and further optionally the molar ratio of the first reducing agent to the antibody is between 2:1 and 1:

1.

54. 48. The method of claim 47, wherein the first buffer system and the second buffer system are independently selected from the group consisting of HEPES buffer, histidine buffer, PBS, PB, MES buffer, BES buffer, MOPS buffer, Bis-Tris buffer, acetate buffer, DIPSO buffer, MOPSO buffer, TES buffer, ACES buffer, MOBS buffer, TAPSO buffer, IPES buffer, ADA buffer, PIPES buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer, or Tris buffer.

55. 55. The method of claim 54, wherein the first buffer system and the second buffer system are independently selected from the group consisting of PB, Bis-Tris buffer, MOPS buffer, HEPES buffer, BES buffer, PIPES buffer, MES buffer, ADA buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, or TAPSO buffer, and optionally, the first buffer system and the second buffer system are MES buffer.

56. 55. The method of claim 54, wherein the concentrations of the first buffer system and the second buffer system are between 10 mM and 100 mM.

57. 55. The method of claim 54, wherein the pH values of the first buffer system and the second buffer system are between 5.5 and 8, the pH values of the first buffer system and the second buffer system are between 6.0 and 7.4, and the pH values of the first buffer system and the second buffer system are between 6.7 and 7.

4.

58. The transition metal ion is Zn ２＋ , Cd ２＋ , Hg ２＋ , Ni ２＋ , Co ２＋ or a combination thereof, and optionally, the transition metal ion is selected from the group consisting of Zn ２＋ 48. The method of claim 47, wherein:

59. 48. The method of claim 47, wherein in step (a), the incubation temperature is 0°C to 37°C, 0°C to 25°C, or 0°C to 15°C, and the incubation time is 0.2 hours to 24 hours, and optionally, in step (a), the incubation temperature is 0°C to 10°C, and the incubation time is 2 hours to 16 hours.

60. 54. The method of claim 53, wherein the molar ratio of the first reducing agent to the antibody is between 2.8:1 and 3:1, and the incubation time is between 1 hour and 9 hours.

61. 49. The method of claim 48, wherein in step (c), the molar ratio of the second reducing agent to the transition metal ion is from 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is from 2.5:1 to 20:1, and / or the incubation time is from 1 hour to 24 hours.

62. 49. The method of claim 48, wherein in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.4 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 h to 24 h.

63. 48. The method of claim 47, wherein, if the first thio bridging reagent has a reactive group, step (b) comprises introducing a first thio bridging reagent having a metal chelator and a reactive group to re-crosslink the reduced thiol group obtained from step (a), and then incubating the first linker payload in a first buffer system to react with the reactive group of the thio bridging group.

64. If the second thio bridging reagent has a reactive group, step (d) may be replaced by the following steps: introducing the incubation product obtained in step (c) and a second thiobridge reagent having a reactive group to re-crosslink the reduced thiol group obtained from step (c), optionally introducing a metal chelator, and then incubating a second linker payload in a second buffer system to react with the reactive group of the thiobridge group.

49. The method of claim 48, comprising:

65. 48. The method of claim 47, comprising the steps of: (a1) Incubating the compound according to any one of claims 1 to 32, which acts as a first reducing agent, and an antibody in the presence of an effective amount of a transition metal ion in a first buffer system, thereby selectively reducing interchain disulfide bonds with the antibody, or incubating the composition according to any one of claims 33 to 36, wherein the compound according to any one of claims 1 to 32 acts as a first reducing agent and the antibody in the first buffer system selectively reduces interchain disulfide bonds within the antibody. (b1) introducing an excess of a metal chelator and an excess of a first linker payload to react with the reduced thiol groups obtained in step (a1).

66. The method of claim 65, wherein the antibody having thiol group site-specific modification is an ADC having D2.

67. 66. The method of claim 65, comprising the steps of: (c2) incubating the reaction product obtained from (b1) and a second reducing agent in a second buffer system to reduce interchain disulfide bonds in the reaction product obtained in (b1). (d2) introducing the incubation product obtained in step (c2) and an excess amount of a second linker payload to react with the reduced thiol group obtained from step (c2);

68. The method of claim 67, wherein the antibody having thiol group site-specific modification is an ADC having D2+D6.

69. 49. The method according to claim 47 or 48, characterized in that it comprises the following steps: Optionally, introducing a compound containing at least one thiol group to consume excess first linker payload in step (b) and / or second linker payload in step (d), and purifying and recovering the resulting antibody having thiol group site-specific modification in step (b) and / or step (d).

70. 44. The method of claim 43, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody or a multispecific antibody, optionally wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody or an antigen-binding portion thereof, further optionally wherein the antibody is an IgG1 or an IgG4.

71. 71. The method of claim 70, wherein the antibody is an engineered antibody having two amino acid substitutions of the two interchain cysteines that form one interchain disulfide bond in the hinge region, and optionally the amino acid substitutions are selected from the following: cysteine to alanine, leucine, arginine, lysine, asparagine, methionine, aspartic acid, phenylalanine, praline, glutamine, serine, glutamic acid, threonine, glycine, tryptophan, histidine, tyrosine, isoleucine, or valine, respectively, and further optionally the amino acid substitution is selected from the following: cysteine to serine.

72. The method of claim 47 or 48, wherein the linkers of the first linker-payload and the second linker-payload are selected from any one of a group capable of being bound at one end to a reduced thiol group of an antibody or a reactive group of a thio bridging reagent and at the other end to a payload.

73. 49. The method of claim 47 or 48, wherein the payload is selected from any one of those comprising at least one substituent that allows attachment of the payload to a linker, and optionally the payload is a cytotoxic drug, a cytokine, a nucleic acid, a radionuclide, a kinase or a derivative thereof.

74. An antibody having thiol group site-specific modification prepared by the method according to any one of claims 43 to 73.

75. An antibody having a thiol group site-specific modification as described in claim 74, characterized in that the antibody having a thiol group site-specific modification is bound to modification reagent 1 and / or modification reagent 2.

76. The antibody having a thiol group site-specific modification according to claim 74 or 75, characterized in that the antibody having a thiol group site-specific modification is an ADC having D2, an ADC having D1, an ADC having D2+D6, an ADC having D2+D3, an ADC having D1+D6, an ADC having D1+D3, an ADC having D0+D6, an ADC having D0+D3, an ADC having D2+D2, an ADC having D2+D4, an ADC having D1+D2, an ADC having D1+D4, or an ADC having D0+D4.

77. Use of an antibody having a thiol group site-specific modification according to any one of claims 74 to 76 in the manufacture of a therapeutic agent for the prevention, diagnosis or treatment of a disease.

78. A pharmaceutical composition comprising an antibody having a thiol group site modification according to any one of claims 74 to 76, and at least one pharmaceutically acceptable carrier.

79. A method for preventing, diagnosing, or treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody having a thiol group site-specific modification according to any one of claims 74 to 76 or a pharmaceutical composition according to claim 78.

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