Novel thiol reducing agents and methods and uses thereof

Abstract

The invention relates to a novel thiol reducing agent shown in a formula (I), a preparation method of the novel thiol reducing agent and application of the novel thiol reducing agent to preparation of an antibody with thiol group site specific modification and improved homogeneity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention 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 previous PCT applications are considered part of the present invention and are incorporated herein in their entirety. Technical Field

[0003] The present invention relates to a novel thiol reducing agent and a method and use thereof. The thiol reducing agent can be used for antibody modification. Background Art

[0004] The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.

[0005] Antibody-drug conjugates (ADCs) are innovative biopharmaceutical products in which monoclonal antibodies are linked to small molecule drugs using stable linkers. ADCs ideally combine the specificity of antibodies and the high potency of cytotoxic drugs by delivering potent cytotoxic drugs to cells expressing antigens, thereby enhancing their targeted cytotoxic activity.

[0006] In general, the conjugation of antibodies to cytotoxic agents typically involves conjugation to exposed residues (including lysine), or reduction of disulfide bonds to expose free interchain cysteines on therapeutic IgG (immunoglobulin G) antibodies. There are other newer methods to introduce conjugation sites into mAbs, such as site-specific glycan conjugation, cysteine ​​engineering, incorporation of non-natural amino acids, and coupling of short peptide tags to drug linkers. There are typically 80 lysine residues on antibodies; however, less than 10 residues can be chemically conjugated. Cysteine ​​conjugation ultimately achieves the reduction of four interchain disulfide bonds. These bonds are reduced under specific conditions, subsequently generating two, four, six, or eight exposed sulfhydryl groups. Both Cys and Lys conjugation methods produce heterogeneous mixtures. ("Advances and Limitations of Antibody Drug Conjugates for Cancer" Biomedicines. 2021 August; 9(8): 872.).

[0007] The drug-to-antibody ratio (DAR), or the number of drug molecules conjugated to a single ADC, is important in determining the efficacy of an ADC. DARs vary widely and depend on other ADC variables. DAR values ​​also depend on the conjugation site and the conjugated light or heavy chain used. DAR values ​​can affect drug effectiveness, as low drug loadings lead to reduced potency, while high drug loadings affect toxicity and pharmacokinetics (“Introduction to Antibody-Drug Conjugates.” Antibodies (Basel). 2021 Dec;10(4):42.). Establishing a robust conjugation process has always been challenging, as conventional nonspecific conjugation and distribution of conjugates are heavily influenced by factors such as pH, concentration, salt concentration, and cosolvents.

[0008] Many methods have been developed to improve the uniformity of ADCs. For example, Genentech's THIOMAB technology was developed based on improving the uniformity of ADCs through antibody engineering, by introducing cysteine ​​in the primary sequence of the antibody to achieve site-directed coupling to improve the uniformity of the product ("Cysteine-Based Coupling: Challenges and Solutions." Bioconjug Chem. 2021 Aug 18;32(8):1525-1534.).

[0009] US20210040145 discloses a 14-amino acid peptide Tub-tag for use at the C-terminus of any POI and catalyzes the addition of various tyrosine derivatives. Tub-tag technology utilizes this enzyme to reuse tubulin-tyrosine ligase to connect the functional part of the C-terminus of the antibody, thereby homogeneously generating DAR2-type antibody conjugates.

[0010] WO2018036438 discloses a method for generating ADC using K-Lock technology, which allows a carefully designed linker-drug to selectively react with four specific lysine residues on IgG antibody Fab to obtain an ADC product with a D2 (DAR value of about 2) of up to 50%. Finally, pure D2 can be obtained by further purification.

[0011] However, these technologies involve protein engineering and / or enzyme catalysis, and thus these technologies have some disadvantages, such as low antibody expression levels, risk of immunogenicity, complex purification processes, and / or high costs.

[0012] Therefore, antibody-drug conjugates with improved homogeneity can provide better stability and lower immunogenicity, and further bring therapeutic benefits, such as better efficacy and lower toxicity. Therefore, the development of new reducing agents and preparation processes for high-homogeneity ADCs is an urgent need and a long-term goal. Summary of the invention

[0013] For the above purposes, provided herein are compounds having the following formula (I):

[0014]

[0015] or a salt, solvate or stereoisomer thereof, characterized in that

[0016] X, Y and Z are each independently covalently linked to the phosphorus atom via a PC bond, where the PC bond is PC(sp 3 ) or PC(sp 2 );

[0017] X is formula (II):

[0018]

[0019] 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 and independently substituted by a group containing at least one coordinating atom selected from N, O and S, and a heteroaryl group optionally and independently substituted by a group containing at least one coordinating atom selected from O and S;

[0020] R1, R2 and R3 are each independently H, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, C1-C5 hydroxylaminealkyl, C1-C5 N-hydroxyamidealkyl, aryl or heteroaryl; or

[0021] R2 or R3 and L2 form a 5-6 membered optionally substituted ring;

[0022] A is optionally present and is -C(O)-, or -C(O)J-;

[0023] J is an organic group comprising an amino group or an imino group and a carbonyl group, wherein the amino group or the imino group forms an amide group with -C(O), and the carboxyl group is optionally covalently linked to L2;

[0024] L2 is optionally present, L2 is a transition metal chelator motif and is -N(R4)(R5) or hydroxyl;

[0025] R4 and R5 are each independently hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring, and R4 or R5 and R2 or R3 form a 5-6 membered optionally substituted ring;

[0026] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl;

[0027] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0028] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4;

[0029] R4 and R5 are not hydroxyl groups at the same time;

[0030] Y is the same as X,

[0031] Z is the same as X, or

[0032] Y and Z are each independently a 5-6 membered optionally substituted saturated heterocyclic group, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, an aryl group, a C1-C5 carboxyalkyl group, a 5-6 membered optionally substituted cycloalkyl group, or

[0033] -C(O)Q is an ester group, an imide group or an amide group,

[0034] X, Y and Z are not -CH2CH2C(O)OH at the same time.

[0035] In one aspect, provided herein is a composition comprising the above-described compound and a transition metal ion.

[0036] In one aspect, provided herein are methods for preparing the above-mentioned compounds.

[0037] In one aspect, the present invention provides use of the above-mentioned compound or the above-mentioned composition in antibody modification.

[0038] In some embodiments, the antibody is modified by selectively reducing interchain SS bonds within the antibody, optionally, the antibody is modified by selectively reducing one of the interchain SS bonds.

[0039] In some embodiments, provided herein is a use of the above-mentioned compound or the above-mentioned composition in preparing an antibody having a site-specific modification of a thiol group. Optionally, the antibody having a site-specific modification of a thiol group is an antibody-drug conjugate (ADC).

[0040] On the one hand, the present invention provides a method for preparing an antibody with site-specific modification of a thiol group, characterized in that the thiol group is reduced from the interchain disulfide bonds in the antibody, and the method comprises using the above-mentioned compound and transition metal ions or using the above-mentioned composition.

[0041] In one aspect, provided herein is an antibody having site-specific modification of a thiol group prepared by the above method.

[0042] In one aspect, the present invention provides use of an antibody having a site-specific modification of a thiol group prepared by the above method in preparing a therapeutic agent for preventing, diagnosing or treating a disease.

[0043] In one aspect, the present invention provides a pharmaceutical composition comprising an antibody having site-specific modification of a thiol group prepared by the above method and at least one pharmaceutically acceptable carrier.

[0044] In one aspect, the present invention provides 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-mentioned antibody having a site-specific modification of a thiol group, or the above-mentioned pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings are briefly described below and are intended to illustrate exemplary embodiments disclosed herein and not to limit the invention.

[0046] Figure 1 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 34 is shown. HIC-HPLC is an abbreviation for hydrophobic interaction chromatography-high performance liquid chromatography.

[0047] Figure 2 The HIC-HPLC chromatogram of the gosartuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 35 is shown.

[0048] Figure 3The HIC-HPLC chromatogram of the mabelantuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 36 is shown.

[0049] Figure 4 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 1 is shown.

[0050] Figure 5 The HIC-HPLC chromatogram of the gosartuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 2 is shown.

[0051] Figure 6 The HIC-HPLC chromatogram of the mabelantuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 3 is shown.

[0052] Figure 7 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 37 is shown.

[0053] Figure 8 The HIC-HPLC chromatogram of the gosartuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 38 is shown.

[0054] Fig. 9 The HIC-HPLC chromatogram of the mabelantuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 39 is shown.

[0055] Fig.10 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 4 is shown.

[0056] Fig.11 The HIC-HPLC chromatogram of the gosartuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 5 is shown.

[0057] Fig.12 The HIC-HPLC chromatogram of the Mabelantuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 6 is shown.

[0058] Fig.13 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 40 is shown.

[0059] Fig.14The HIC-HPLC chromatogram of the gosartuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 41 is shown.

[0060] Fig.15 The HIC-HPLC chromatogram of the mabelantuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 42 is shown.

[0061] Fig.16 The HIC-HPLC chromatogram of the gosartuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 8 is shown.

[0062] Fig.17 The HIC-HPLC chromatogram of the mabelantuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 9 is shown.

[0063] Fig.18 A to Fig.18 H shows the HIC-HPLC chromatogram of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 43 to 50, wherein the reducing agent is TCEP-1, TCEP-2, TCEP-3, TCEP-4, TCEP-5, TCEP-6, TCEP-7 and TCEP-8.

[0064] Fig.19 A to Fig.19 H shows the HIC-HPLC chromatogram of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 51 to 58, wherein the reducing agent is TCEP-9, TCEP-10, TCEP-15, TCEP-18, TCEP-19, TCEP-20, TCEP-21 and TCEP-23.

[0065] Fig. 20 A to Fig. 20 G shows the HIC-HPLC chromatogram of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 59 to 65, wherein the reducing agent is TCEP-24, TCEP-25, TCEP-26, TCEP-28, TCEPA, TCEP-34 and TCEP-35.

[0066] Fig.21 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 66 is shown, wherein the reducing agent is TCEP-37.

[0067] Fig. 22The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 11 is shown.

[0068] Fig.23 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 12 is shown.

[0069] Fig.24 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 67 is shown.

[0070] Fig.25 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 68 is shown.

[0071] Fig.26 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 69 is shown.

[0072] Fig. 27 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 70 is shown.

[0073] Fig.28 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 71 is shown.

[0074] Fig.29 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 72 is shown.

[0075] Fig.30 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 73 is shown.

[0076] Fig.31 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 74 is shown.

[0077] Fig.32 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 75 is shown.

[0078] Fig.33The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 76 is shown.

[0079] Fig.34 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 77 is shown.

[0080] Fig.35 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 78 is shown.

[0081] Fig.36 A to Fig.36 C shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 79 to 81.

[0082] Fig.37 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 10 is shown.

[0083] Fig.38 A to Fig.38 D shows the HIC-HPLC chromatogram of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 82 to 85, wherein the molar ratio of antibody to reducing agent was different.

[0084] Fig.39 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 86 is shown.

[0085] Fig.40 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 87 is shown.

[0086] Fig.41 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 88 is shown.

[0087] Fig.42 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 89 is shown.

[0088] Fig.43 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 90 is shown.

[0089] Fig.44 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 91 is shown.

[0090] Fig.45 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 92 is shown.

[0091] Fig.46 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 93 is shown.

[0092] Fig.47 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 94 is shown.

[0093] Fig.48 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 95 is shown.

[0094] Fig.49 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 96 is shown.

[0095] Fig.50 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 97 is shown.

[0096] Fig.51 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 98 is shown.

[0097] Fig.52 A to Fig.52 C shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Examples 99 to 101.

[0098] Fig.53 A to Fig.53 C shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 102 to 104, wherein the incubation temperature in step (1) was different.

[0099] Fig.54 A to Fig.54D shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 105 to 108, wherein the incubation time in step (1) was different.

[0100] Fig.55 A to Fig.55 E shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 109 to 113, wherein the incubation time in step (1) was different.

[0101] Fig.56 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 114 is shown, wherein the antibody is an engineered antibody.

[0102] Fig.57 The HIC-HPLC chromatogram of the trastuzumab-[Maleimide-PEG4-N3-DBCO-Cy3]1 conjugate prepared in Example 115 is shown.

[0103] Fig.58 The HIC-HPLC chromatogram of the trastuzumab-[maleimide]1[MC-GGFG-DXd]6 conjugate prepared in Example 116 is shown.

[0104] Fig.59 The HIC-HPLC chromatogram of the trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 conjugate prepared in Example 117 is shown.

[0105] Fig.60 A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 118; Fig.60 B shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 conjugate prepared in Examples 118 to 119.

[0106] Fig.61 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 conjugate prepared in Example 120 is shown.

[0107] Fig.62 A shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide] 1 conjugate prepared in Example 121; Fig.62B shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide]1[MC-VC-PAB-MMAE]4 conjugates of Examples 121 to 122.

[0108] Fig.63 A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2 conjugate prepared in Example 123; Fig.63 B shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 conjugate prepared in Examples 123 to 124.

[0109] Fig.64 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 conjugate prepared in Example 125 is shown. DETAILED DESCRIPTION

[0110] The present invention is explained in more detail below. This description is not an exhaustive list of all the different ways in which the present invention may be implemented, nor is it an exhaustive list of all the features that may be added to the present invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaust all permutations, combinations, and variations thereof.

[0111] Unless otherwise defined, all scientific and technological terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the detection practice of the present invention, preferred materials and methods are described herein. In describing and advocating the present invention, the following terms will be used.

[0112] reducing agent

[0113] The present invention provides examples of compounds that can be used as reducing agents in preparing antibodies with site-specific modification of thiol groups.

[0114] Provided herein is a compound having the following formula (I):

[0115]

[0116] or a salt, solvate or stereoisomer thereof, characterized in that

[0117] X, Y and Z are each independently covalently linked to the phosphorus atom via a PC bond, wherein the PC bond is PC(sp 3 ) or PC(sp 2 );

[0118] X is formula (II):

[0119]

[0120] L1 is selected from the group consisting of: -CH(R1)-, -C(CH3)(R1), -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, aryl groups each independently optionally substituted by a group containing at least one coordinating atom, and heteroaryl groups each independently optionally substituted by a group containing at least one coordinating atom;

[0121] R1, R2 and R3 are each independently H, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, C1-C3 hydroxylaminoalkyl, C1-C3 N-hydroxylamidealkyl, aryl or heteroaryl; or

[0122] R2 or R3 and L2 form a 5-6 membered optionally substituted ring;

[0123] A is optionally present and is -C(O)-, or -C(O)J-;

[0124] J is an organic group comprising an amino group or an imino group and a carbonyl group, wherein the amino group or the imino group forms an amide group with -C(O), and the carboxyl group is optionally covalently linked to L2;

[0125] L2 is optionally present, L2 serves as a transition metal chelator motif and is -N(R4)(R5) or hydroxyl;

[0126] R4 and R5 are each independently hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), an optionally substituted 5-6 membered saturated heterocyclyl, an optionally substituted aralkyl, an optionally substituted arylalkoxy, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring, and R4 or R5 and R2 or R3 form a 5-6 membered optionally substituted ring; R6 is hydrogen, amino, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C3 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl;

[0127] R7 is hydroxyl, C1-C3 alkoxy, -NH(CH2CONH)n 3 OH;

[0128] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4;

[0129] R4 and R5 are not hydroxyl groups at the same time;

[0130] Y is the same as X,

[0131] Z is the same as X, or

[0132] Y and Z are each independently a 5-6 membered optionally substituted saturated heterocyclic group, a C1-C3 alkyl group, a C1-C3 hydroxyalkyl group, an aryl group, a C1-C3 carboxyalkyl group, a 5-6 membered optionally substituted cycloalkyl group, or

[0133] -C(O)Q is an ester group, an imide group or an amide group,

[0134] X, Y and Z are not -CH2CH2C(O)OH at the same time.

[0135] The term "aryl" refers to an aromatic or heteroaromatic group consisting of one or several rings, which contains 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. An exemplary aryl group is phenyl.

[0136] The term "aryl" also refers to aromatic groups in which one or more H atoms are independently replaced by groups such as F, Cl, Br, I, hydroxy, carboxyl, sulfonyl, amino, methoxy or ethoxy, N-hydroxyformamide, N-hydroxyacetamide, 4-pyridyl, 2-pyridyl, The invention may be substituted by other groups such as .

[0137] The term "heteroaryl" refers to an aromatic group in which one or several carbon atoms, preferably one, two, three or four carbon atoms, are replaced by O, N, Si, Se, P or S, preferably by O, S, N. Exemplary heteroaryl groups are imidazolyl, pyridyl, bipyridyl, quinolyl, isoquinolyl.

[0138] The term "heteroaryl" also refers to heteroaromatic groups in which one or several H atoms are independently replaced by other groups such as F, Cl, Br, I, hydroxy, carboxyl, amino, hydroxyalkyl, carboxyalkyl, N-hydroxyamidoalkyl, heteroaryl, etc.

[0139] The term "coordinating atom" refers to an atom containing a lone pair of electrons, examples of which include N, O, S, P, F, Cl, Br, I.

[0140] The term "C1-C5 alkyl" refers to an aliphatic hydrocarbon group having 1 to 3 carbon atoms in the chain or ring. Exemplary alkyl groups include methyl, ethyl, n-propyl and isopropyl.

[0141] The term "C0-C5 hydroxyalkyl" refers to a hydroxyl group or a C1-C5 alkyl group in which one or several H atoms are substituted by one, two or three hydroxyl groups. Exemplary C1-C5 hydroxyalkyl groups are hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl.

[0142] The term "C1-C5 carboxyalkyl" refers to a C1-C5 alkyl substituted by one, two or three carboxyl groups. Exemplary C1-C5 carboxyalkyl groups are -COOH, -CH2COOH, -CH2CH2COOH, -CH2(CH3)COOH.

[0143] The term "C1-C5 hydroxylamine alkyl" refers to a C1-C5 alkyl substituted with one, two or three hydroxylamine groups. Exemplary C1-C5 hydroxylamine alkyls are -CH2NHOH, -CH2CH2NHOH.

[0144] The term "C1-C5 N-hydroxyamidoalkyl" refers to a C1-C5 carboxyalkyl group in which one, two or three carboxyl groups form an amide with hydroxylamine. Exemplary C1-C5 N-hydroxyamidoalkyl groups are -C(O)NHOH, -CH2C(O)NHOH, -CH2CH2C(O)NHOH.

[0145] The term "heterocyclyl" refers to an aromatic or non-aromatic C5-C 10 A ring wherein one or two of the ring carbon atoms are independently substituted with a heteroatom selected from O, N, P and S. Preferred heteroatoms are O, N and S. Suitable heterocyclic rings are also disclosed in The Handbook of Chemistry and Physics, 76th ed., CRC Press, Inc., 1995-1996, pp. 2-25 to 2-26, the disclosure of which is incorporated herein by reference. Preferred non-aromatic heterocycles include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxadioxolanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl and condensed systems obtained by condensation with phenyl.

[0146] The term "aralkyl" refers to a linear, branched or cyclic alkyl group connected to at least one aryl group. Preferably, the number of carbon atoms in the chain or ring is 1 to 4. Exemplary aralkyl groups are -CH2C6H5, -CH2CH2C6H5, -CH2CH2CH2C6H5, -CH2(CH3)CH2C6H5, -CH2(CH3)CH2CH2C6H5.

[0147] The term "heteroaralkyl" refers to a linear, branched or cyclic alkyl group attached to at least one heteroaryl group. Preferably, the number of carbon atoms in the chain or ring is 1 to 4. Exemplary heteroaralkyl groups are

[0148] The term "C1-C5 alkoxy" refers to an oxygen atom attached to a C1-C5 alkyl group. Exemplary C1-C5 alkoxy groups are -OCH3, -OCH2CH3, -OCH2(CH3)2, -OCH2CH2CH3.

[0149] The term "arylalkoxy" refers to an aromatic group in which one or several H atoms are replaced by alkoxy. Exemplary are phenyl-O-CH2-, phenyl-O-(CH2)2-, phenyl-O-(CH2)3-, phenyl-O-(CH2)4-, phenyl-O-(CH2)5-.

[0150] The term "cycloalkyl" refers to a 3-, 4-, 5- or 6-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl. Cycloalkyls may be unsubstituted or substituted with one or more groups including, but not limited to, carboxyl, sulfonyl, amino, hydroxyl, -C(O)NHOH, -CH2C(O)NHOH, -CH2CH2C(O)NHOH, -COOH, -CH2COOH, -CH2CH2COOH, -CH2(CH3)COOH, F, Cl, Br, I.

[0151] The term "halogen" refers to F, Cl, Br or I.

[0152] The term "alkenyl" refers to a straight or branched 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 may be uncoupled or coupled to another unsaturated group. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl. An alkenyl group may be unsubstituted or substituted and may be straight or branched.

[0153] The term "cyano" refers to a substituent in which a carbon atom is triple bonded to a nitrogen atom, for example -CN.

[0154] 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)-.

[0155] In some embodiments, R1, R2 and R3 are each independently H, methyl, isopropyl, hydroxymethyl, hydroxyethyl, carboxymethyl, carboxyethyl, N-hydroxyacetamido, phenyl, 2-pyridyl, 4-pyridyl or 4-imidazolyl, and R2 and L2 form a 5-6 membered optionally substituted ring.

[0156] In some embodiments, L1 is -CH(R1)CH(R2)-, R1 is H, and R2 and R4 of L2 form a 5-6 membered selectively substituted ring. In some embodiments, R2 and L2 form In these embodiments, A is -C(O)-, L2 is -N(R4)(R5), and R5 is hydroxy.

[0157] In some embodiments, L1 is -CH(R1)CH(R2)-, R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamido, and R2 is H.

[0158] In some embodiments, L1 is -CH(R1)CH(R2)-, R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamido, R2 is H, A is -C(O)-, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxyl.

[0159] In some embodiments, L1 is -CH(R1)CH(R2)-, R1 is H, and R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl, or 4-imidazolyl.

[0160] In some embodiments, L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-, L2 is -N(R4)(R5), R4 is hydrogen, an optionally substituted 5-6 membered saturated heterocyclic group, and R5 is hydroxyl. In some embodiments, R4 is

[0161] In some embodiments, L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-, L2 is -N(R4)(R5), and R4 and R5 form a 5-6 membered selectively substituted ring. In these examples, L2 is

[0162] In some embodiments, L1 is an optionally substituted phenyl group connected 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.

[0163] In some embodiments, L1 is phenyl optionally substituted by hydroxyl, halogen, carboxyl, sulfonyl, amino, methoxy or ethoxy at the ortho, meta or para positions. In some embodiments, A and L2 are absent. Halogen refers to F, Cl, Br or I.

[0164] In some embodiments, L1 is In these examples, A and L2 do not exist.

[0165] In some embodiments, L1 is optionally substituted 4-pyridyl or optionally substituted 4-quinolyl. In some embodiments, L1 is In some embodiments, A and L2 are absent.

[0166] In some embodiments, L1 is -CH(R1)CH(R2)-, and R1 and R2 are each independently H.

[0167] In some embodiments, A is -C(O)-, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxy.

[0168] In some embodiments, L2 is -N(R4)(R5), R4 is hydrogen, C1-C5 alkyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted aryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring; R5 is hydroxy.

[0169] In some embodiments, L2 is -N(R4)(R5), R4 is hydrogen, C1-C5 alkyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted aralkyl, optionally substituted aryl; R5 is hydroxyl.

[0170] In some embodiments, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidoalkyl, heteroaryl, or heteroaralkyl.

[0171] In some embodiments, R6 is hydrogen, C1-C5 alkyl, C1-C5 hydroxyalkyl, or heteroarylalkyl.

[0172] In some embodiments, R6 is hydrogen, methyl, hydroxymethyl, amino, benzyl, carboxyethyl, N-hydroxyacetamido, Optionally, R6 is hydrogen.

[0173] In some embodiments, R7 is hydroxy, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH, optionally, R7 is hydroxyl or C1-C5 alkoxy. In some embodiments, R7 is hydroxyl, methoxy, -NH(CH2CONH)n 3 OH, optionally, R7 is hydroxy or methoxy.

[0174] In some embodiments, n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0175] In some embodiments, R4 is Hydrogen or -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydroxyl,

[0176] R6 is hydrogen, methyl, hydroxymethyl or

[0177] R7 is hydroxyl or -NH(CH2CONH)n 3 OH;

[0178] n 1 、n 2 and n 3 Each independently is the number 0.

[0179] In some embodiments, R4 is Hydrogen or -CH(R6)CO(R7), R5 is hydroxyl,

[0180] R6 is hydrogen,

[0181] R7 is hydroxyl.

[0182] In some embodiments, L2 is -N(R4)(R5);

[0183] R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or an optionally substituted heteroarylalkyl group,

[0184] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl;

[0185] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0186] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0187] In some embodiments, R4 and R5 are independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or 6-membered heteroarylalkyl, R6 is hydrogen,

[0188] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0189] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0190] In some embodiments, R4 and R5 are independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or

[0191] R6 is hydrogen,

[0192] R7 is hydroxyl or -NH(CH2CONH)n 3 OH;

[0193] n 1 、n 2 and n 3 Each independently is the number 0.

[0194] In some embodiments, R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7),

[0195] R6 is hydrogen,

[0196] R7 is hydroxyl or -NH(CH2CONH)n 3 OH;

[0197] n1 、n 2 and n 3 Each independently is the number 0.

[0198] In some embodiments, L2 is -N(R4)(R5);

[0199] R4 is hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, optionally substituted C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl;

[0200] R5 is hydrogen,

[0201] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl;

[0202] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0203] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0204] In some embodiments, R4 is hydrogen, C0-C3 hydroxyalkyl, C1-C3 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), phenyl substituted by carboxyl, hydroxyl, amino, halogen, pyridyl, amino substituted by 2-methylpyridine, benzyl substituted by carboxyl, hydroxyl, amino or halogen, arylalkoxy, Substituted pyridinyl.

[0205] In some embodiments, R4 is hydrogen, hydroxy, methyl hydroxy, ethyl hydroxy, propyl hydroxy, methoxy, ethoxy,

[0206] and R5 is hydrogen.

[0207] In some embodiments, R4 is hydroxy, methoxy or and R5 is hydrogen.

[0208] In some embodiments, R4 is -(CH2)n 1(OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, aryl, aralkyl optionally substituted by hydroxy, halogen, cyano or nitro, C1-C5 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl;

[0209] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0210] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0211] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl, benzyl, benzyl substituted by hydroxy, halogen, cyano or nitro, halogen, carboxyethyl, N-hydroxyacetamido,

[0212] R7 is hydroxyl, -NH(CH2CONH)n 3 OH;

[0213] n 1 and n 3 Each is independently digitized 0, 1, 2, 3, 4,

[0214] n 2 Is the number 0.

[0215] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl, benzyl, carboxyethyl, N-hydroxyacetamide,

[0216] R7 is hydroxyl or -NH(CH2CONH)n 3 OH;

[0217] n 1 is the number 0 or 2,

[0218] n 2 is a number 0 or 1,

[0219] n 3 Is the number 0.

[0220] 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 the number 0.

[0221] In some embodiments, A is -C(O)J-, J is a peptide residue, including a single amino acid residue, a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, aminopropionic acid, aminobutyric acid, aminovaleric acid, amino acid, aminoheptanoic acid, aminooctanoic acid, or NH2(OCH2CH2O)n 4 CH2COOH,n 4 For numbers 2-10.

[0222] 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).

[0223] In some embodiments, J is the residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine, or asparagine.

[0224] In some embodiments, A is -C(O)J-, J is the residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxyl.

[0225] In some embodiments, Y and X are the same.

[0226] In some embodiments, Z and X are the same.

[0227] In some embodiments, Y and Z are each independently

[0228] Q is -NHOH, -NHCH2CH2SO3H, -N(CH2CH2OH)2, -NHCH2COOH, -NHCH(CH3)COOH, -NH(CH2CH2O)3CH3.

[0229] In some embodiments, the compound is selected from, but not limited to, the group consisting of:

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] The invention provides a composition comprising the above compound and transition metal ions.

[0236] 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+ .

[0237] 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.

[0238] The compound having the above formula (I) can be prepared according to the following steps:

[0239] Under an inert atmosphere, at least one carboxyl group in the following formula III is connected to the transition metal chelator portion by introducing a condensation reagent: On the heteroatom

[0240]

[0241] Among them, X' is

[0242] L1 is selected from the group consisting of: -CH(R1)-, -C(CH3)(R1), -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, aryl groups each independently selectively substituted by one or more groups containing N, O or S, and heteroaryl groups each independently selectively substituted by one or more groups containing O or S;

[0243] R1, R2 and R3 are each independently H, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, C1-C5 hydroxylaminealkyl, C1-C5 N-hydroxyamidealkyl, aryl or heteroaryl; or

[0244] R2 or R3 and L2 form a 5-6 membered selectively substituted ring;

[0245] A' is -COOH or -C(O)J-COOH;

[0246] J is an organic group comprising an amino group or an imino group and a carbonyl group, wherein the amino group or the imino group forms an amide group with -C(O), and the carboxyl group is optionally covalently linked to L2;

[0247] L2 is optionally present, L2 serves as a transition metal chelator motif and is -N(R4)(R5) or hydroxyl;

[0248] R4 and R5 are each independently hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring, and R4 or R5 and R2 or R3 form a 5-6 membered optionally substituted ring;

[0249] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl;

[0250] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0251] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4;

[0252] R4 and R5 are not hydroxyl groups at the same time;

[0253] Y' is the same as X';

[0254] Z' is the same as X', or

[0255] Y and Z are each independently a 5-6 membered optionally substituted saturated heterocyclic group, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, an aryl group, a C1-C5 carboxyalkyl group, a 5-6 membered optionally substituted cycloalkyl group, or

[0256] -C(O)Q is an ester group, an imide group or an amide group.

[0257] In some embodiments, the transition metal chelator portion can be 2-phenoxy-ethylamine, aniline, benzylamine, 4-aminobenzene-1,2-diol, 5-amino-2-hydroxybenzoic acid, bis(pyridin-2-ylmethyl)amine, 5-amino-8-hydroxyquinoline, bis(pyridin-2-yl)methylamine, 4-aminophthalic acid, L-tyrosine tert-butyl ester, DL-3-(4-fluorophenyl)alanine, DL-4-cyanophenylalanine, DL-4-nitrophenylalanine, N-benzylhydroxylamine hydrochloride, N-phenylhydroxylamine,

[0258] supply.

[0259] In some embodiments, the structure of formula (III) is

[0260]

[0261] The term "condensation reagent" refers to a condensation reaction reagent that helps two molecules (functional groups) to covalently combine to form a single molecule. Condensation reagents include, but are not limited to, 1-hydroxybenzotriazole (HOBT), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), o-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate and o-benzo(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0262] The term "inert atmosphere" refers to a chemically inactive atmosphere, such as nitrogen, carbon dioxide, helium.

[0263] Production and use of antibodies with site-specific modification of thiol groups

[0264] The compound of the above formula (I) has reducing properties and can reduce the disulfide bonds of antibodies. Therefore, the compound of formula (I) can be used as a reducing agent in the process of protein modification or antibody modification.

[0265] The term "disulfide bond" herein refers to a covalent bond having a RSS-R' structure. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond with a second thiol group (e.g., a thiol group from another cysteine ​​residue). A disulfide bond can be formed between the thiol groups of two cysteine ​​residues, respectively, on two polypeptide chains, thereby forming an interchain bridge or interchain bond.

[0266] In some embodiments, compounds of Formula (I) can reduce interchain SS bonds of antibodies.

[0267] In some embodiments, the compound of formula (I) can selectively reduce one of the interchain SS bonds, thereby selectively modifying the antibody.

[0268] In some embodiments, the compound having the above formula (I) can be used as a reducing agent to prepare an antibody having a site-specific modification of a thiol group. Optionally, the antibody having a site-specific modification of a thiol group is an antibody-drug conjugate (ADC).

[0269] By conventional coupling process or bioconjugation process of the present invention, a mixture of antibody drug conjugates is generated. Generally speaking, an antibody molecule belonging to IgG1 or IgG4 subclass has 4 interchain SS bonds, and each interchain SS bond is formed by two-SH groups. Antibody molecules can be partially or completely reduced to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive-SH groups of one or more interchain SS bonds, and therefore, the number of drugs (or payloads) coupled to a single antibody molecule is 1, 2, 3, 4, 5, 6, 7 or 8. According to the number of drugs coupled to a single antibody molecule, different conjugates containing different numbers of drug molecules are named D0, D2, D1, D4, D6, D8, D1+D6, D1+D3, D2+D6, D2+D3, D0+D6, D0+D3, D0+D4 type ADC, D2+D4 type ADC, D1+D4 type ADC, D2+D2 type ADC or D1+D2 type ADC. Thus, the "homogeneity" of an antibody drug conjugate is used to describe the predominant property of one particular type of antibody drug conjugate (i.e., one type selected from D0, D1, D2, D4, D6, D8, D1+D6, D1+D3, D2+D6, D2+D3, D0+D6, or D0+D3 conjugates) in a given antibody drug conjugate mixture.

[0270] The drug-to-antibody ratio (DAR) of an ADC is the average number of drugs attached to each antibody. DAR is a key attribute to measure the quality of an ADC because it can significantly affect ADC efficacy. The DAR distribution (D0, D2, D4, D6, D8) can reflect the uniformity of the ADC.

[0271] The drug loading is represented by the number of drug moieties per antibody in the molecule of the ADC. For some antibody drug conjugates, the drug loading may be limited by the number of attachment sites on the antibody. For example, when the attachment is a cysteine ​​thiol, in certain illustrative examples described herein, the drug loading may be 0 to 8 drug moieties per antibody.

[0272] The term "DO" or "DO-type ADC" herein refers to an ADC in which the amount of drug coupled to a single antibody molecule is approximately zero.

[0273] The term "D2" or "D2-type ADC" herein refers to a DAR of about 2, which means that about two drug molecules (e.g., 1.5, 2.0, 2.5 molecules) are coupled to a single antibody molecule. The drug molecule can be coupled to the -SH group generated by reducing the disulfide bond between the heavy chain and the light chain or between the heavy chain and the heavy chain via a linker.

[0274] The term "D4" or "D4-type ADC" herein refers to an ADC in which four drug molecules (e.g., 3.5, 4.0, 4.5 molecules) are coupled to one single antibody molecule, wherein the drug molecules can be coupled to four -SH groups generated by reducing two interchain disulfide bonds or intrachain disulfide bonds.

[0275] The term "D6" or "D6-type ADC" herein refers to an ADC in which six drug molecules (e.g., 5.5, 6.0, 6.5 molecules) are coupled to a single antibody molecule, wherein the drug molecules can be coupled to six -SH groups generated by reducing three disulfide bonds.

[0276] The term "D8" or "D8-type ADC" herein refers to an ADC in which eight drug molecules (e.g., 7.5, 8.0, 8.5 molecules) are coupled to one single antibody molecule, wherein the drug molecules can be coupled to eight -SH groups generated by reducing four disulfide bonds.

[0277] The term "D1-type ADC" or "D1" herein refers to an ADC in which one of the thiol bridging groups carrying a linker-payload rebridges two thiol groups of a single antibody molecule.

[0278] The term "D3" or "D3-type ADC" herein refers to an ADC in which three of the first sulfide-bridging groups carrying the first linker-payload rebridge the six thiol groups of a single antibody molecule.

[0279] The term "D1+D6" or "D1+D6 type ADC" herein refers to an ADC in which one of the first sulfide bridging groups carrying a first linker-payload rebridges two thiol groups, and six second linker-payloads are coupled to a single antibody molecule, wherein the first linker-payload and the second linker-payload can be the same or different.

[0280] The term "D1+D3" or "D1+D3 type ADC" herein refers to an ADC in which one of the first sulfide bridging groups carrying a first linker-payload and three of the second sulfide bridging groups carrying a second linker-payload re-bridge the eight thiol groups of a single antibody molecule, wherein the first linker-payload and the second linker-payload can be the same or different.

[0281] The term "D2+D6" or "D2+D6 type ADC" herein refers to an ADC in which two of the first linker-payloads and six of the second linker-payloads are coupled to a single antibody molecule, wherein the first linker-payload and the second linker-payload may be the same or different.

[0282] The term "D2+D3" or "D2+D3 type ADC" herein refers to an ADC in which two of the first linker-payloads are coupled to a single antibody molecule, and three of the second sulfide-bridging groups carrying the second linker-payload re-bridge the six thiol groups of the antibody, wherein the first linker-payload and the second linker-payload can be the same or different.

[0283] The term "D0+D6" or "D0+D6 type ADC" herein refers to an ADC in which one of the first sulfide bridge groups rebridges two thiol groups and six of the second linker-payloads are coupled to a single antibody molecule, or refers to an ADC in which two of the capping agents and six of the second linker-payloads are coupled to a single antibody molecule.

[0284] The term "D0+D3" or "D0+D3 type ADC" herein refers to an ADC in which one of the first sulfide bridging groups rebridges two thiol groups, and three of the second sulfide bridging groups carrying a linker-payload rebridge six thiol groups of a single antibody molecule, wherein the first sulfide bridging group and the second sulfide bridging group may be the same or different. In some embodiments, D0+D3 refers to an ADC in which two of the capping agents react with two thiol groups, and three of the second sulfide bridging groups carrying a linker-payload rebridge six thiol groups of a single antibody molecule.

[0285] The term "D0+D4" or "D0+D6 type ADC" herein refers to an ADC in which one of the first sulfide bridging groups rebridges two thiol groups and four of the second linker-payloads are coupled to a single antibody molecule, or refers to an ADC in which two of the capping agents and four of the second linker-payloads are coupled to a single antibody molecule.

[0286] The term "D1+D4" or "D1+D4 type ADC" herein refers to an ADC in which one of the first sulfide bridging groups carrying the first linker-payload rebridges two thiol groups and four of the second linker-payload are coupled to a single antibody molecule.

[0287] The term "D2+D4" or "D2+D4 type ADC" herein refers to an ADC in which two of the first linker-payloads and four of the second linker-payloads are coupled to a single antibody molecule.

[0288] The term "D2+D2" or "D2+D2-type ADC" herein refers to an ADC in which two of the first linker-payloads and two of the second linker-payloads are coupled to a single antibody molecule.

[0289] The term "D1+D2" or "D1+D2 type ADC" herein refers to an ADC in which one of the first sulfide bridging groups of the first linker-payload rebridges two thiol groups and the two second thiol groups of the second linker-payload are coupled to a single antibody molecule.

[0290] The term "D4+D2" or "D4+D2 type ADC" herein refers to an ADC in which four of the first linker-payloads and two of the second linker-payloads are coupled to a single antibody molecule.

[0291] The term "D4+D4" or "D4+D4 type ADC" herein refers to an ADC in which four of the first linker-payloads and four of the second linker-payloads are coupled to a single antibody molecule.

[0292] The term "homogeneity of Dx-type ADC" as used herein refers to the weight content of Dx-type ADC in all ADCs produced by the method, wherein 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.

[0293] The term "about" herein refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length by at most 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In a specific embodiment, the term "about" preceding a numerical value indicates a range of plus or minus 50%, 30%, 15%, 10%, 5%, or 1% of the value.

[0294] In some embodiments, the compound having the above formula (I) or the above composition can be used to prepare ADC with improved homogeneity.

[0295] In some embodiments, the present invention provides a compound having formula (I) or the above composition for use in the preparation of a D2-type ADC, a D1-type ADC, a D2+D6-type ADC, a D2+D3-type ADC, a D1+D6-type ADC, a D1+D3-type ADC, a D0+D6-type ADC, a D0+D3-type ADC, a D0+D4-type ADC, a D2+D4-type ADC, a D1+D4-type ADC, a D2+D2-type ADC or a D1+D2-type ADC.

[0296] In some embodiments, the ADC comprises D2 in an amount of at least 53% of the combined weight of D0, D2, D4, D6, and D8. In some embodiments, the ADC comprises D2 in an amount of up to 55% of the combined weight of D0, D2, D4, D6, and D8. In some embodiments, the ADC comprises D2 in an amount of up to 60%, 65%, 70%, 75%, 80%, 84%, 87%, 89%, 90%, 91%, 92%, or 95% of the combined weight of D0, D2, D4, and D8.

[0297] In some embodiments, the uniformity of the D1 type ADC, the D2+D6 type ADC, and the D0+D6 type ADC is as high as 80%.

[0298] In some embodiments, the D2+D2 ADC content is generally as high as 68% or 70%.

[0299] In some embodiments, the D1+D2 type ADC content is generally as high as 80% or 83%.

[0300] In some embodiments, the D0+D4 ADC content is generally as high as 55%, 61% or 65%.

[0301] In some embodiments, the D2+D4 ADC content is generally as high as 70%, 75%, or even 78% or 80%.

[0302] In some embodiments, the D1+D4 type ADC content is generally as high as 60%, 65%, or even 70%.

[0303] Method for preparing antibodies with site-specific modification of thiol groups

[0304] The present invention also provides a method for preparing the antibody with site-specific modification of thiol groups, reducing the thiol groups from the interchain disulfide bonds in the antibody, and the method comprises using the above-mentioned compound or its salt, solvate, stereoisomer and transition metal ion, or using the above-mentioned composition.

[0305] In some embodiments, the number of thiol groups is 1, 2, 3, 4, 5, 6, 7, or 8.

[0306] In some embodiments, the number of thiol groups is 2 or 8.

[0307] In some embodiments, the interchain disulfide bond connects the two upper heavy chains in the hinge region, or connects the heavy chain to the light chain in the Fab region.

[0308] In some embodiments, the interchain disulfide bonds connect the two heavy chains in the hinge region and connect the heavy chain to the light chain in the Fab region.

[0309] In some embodiments, the site-specific modification does not involve antibody technology, enzyme technology, and glycan modification.

[0310] In some embodiments, the method comprises the following steps:

[0311] (a) incubating the above compound or its salt, solvate, stereoisomer and the antibody as a first reducing agent in a first buffer system in the presence of the transition metal ion to selectively reduce the interchain disulfide bonds in the antibody; or

[0312] Incubating the composition and the antibody in the first buffer system to selectively reduce the interchain disulfide bonds in the antibody, wherein the compound is used as the first reducing agent;

[0313] (b) introducing a metal chelator and a modifying agent 1 to react with the reduced thiol group from step (a), wherein the modifying agent 1 is a capping agent, a first linker-payload or a first sulfide bridging agent, and optionally, the first sulfide bridging agent carries the first linker-payload or a reactive group.

[0314] In some embodiments, when the first sulfide bridging reagent carries the reactive group, the step (b) comprises the following steps:

[0315] The metal chelator and the first sulfide bridging reagent carrying the reactive group are introduced to re-bridge the reduced thiol group from step (a), and then the first linker-payload is incubated in the first buffer system to react with the reactive group of the sulfide bridging group.

[0316] In some embodiments, the method further comprises the following steps:

[0317] (c) incubating the reaction product from step (b) with a second reducing agent in a second buffer system to reduce the interchain disulfide bonds in the reaction product, and optionally, introducing the transition metal ion;

[0318] (d) introducing the incubation product from step (c) and a modification reagent 2 to react with the reduced thiol group from step (c), optionally introducing the metal chelator, wherein the modification reagent 2 is a second linker-payload or a second sulfide bridging reagent, optionally, the second sulfide bridging reagent carries the second linker-payload or a reactive group.

[0319] In some embodiments, when the second sulfide bridging reagent carries the reactive group, the step (d) comprises the following steps:

[0320] The reaction product from step (c) and the second sulfide bridging reagent carrying the reactive group are introduced to re-bridge the reduced thiol group from step (c), optionally, the metal chelator is introduced, and then the second linker-payload is incubated in the second buffer system to react with the reactive group of the sulfide bridging group.

[0321] In some embodiments, when the transition metal ion is introduced in step (c), the metal chelator is introduced in step (d) to capture excess transition metal ions.

[0322] The term "carry" herein means to have or possess.

[0323] In some embodiments, first, the first reducing agent selectively reduces one of the interchain disulfide bonds in the antibody using the transition metal ion, optionally, the second reducing agent reduces the remaining three interchain disulfide bonds without using the transition metal ion, or the second reducing agent reduces one or two of the interchain disulfide bonds using the transition metal ion. By the method comprising steps (a) and (b), an antibody with site-specific modification of a thiol group, such as a D1-type ADC or a D2-type ADC, can be prepared. By the method comprising steps (a), (b), (c) and (d), an antibody with site-specific modification of a thiol group, such as a D1+D6-type ADC, a D1+D3-type ADC, a D2+D6-type ADC, a D2+D3-type ADC, a D0+D6-type ADC, a D0+D3-type ADC, a D0+D4-type ADC, a D2+D4-type ADC, a D1+D4-type ADC, a D2+D2-type ADC or a D1+D2-type ADC can be prepared.

[0324] In some embodiments, the salt refers to an acid addition salt or a base addition salt.

[0325] In some embodiments, the acid addition salt can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Organic acids from which salts can be 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, sulfosalicylic acid, etc.

[0326] In some embodiments, the base addition salt can be formed with inorganic bases and organic bases. The inorganic base from which the salt can be derived includes Group 1 to Group 2 of the periodic table. In certain embodiments, the salt is derived from lithium, sodium, potassium, calcium, magnesium, etc. The organic base from which the salt can be derived includes, for example, primary amines, secondary amines and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc. Some organic amines include isopropylamine, dibenzylethylenediamine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

[0327] In some embodiments, the first reducing agent and the transition metal ion are used together in the method for preparing the antibody with site-specific modification of a thiol group. 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.

[0328] In some embodiments, the molar ratio of the first reducing agent to the antibody is 3: 1 or 0.5: 1 or 3: 1 to 1: 1. In some embodiments, the molar ratio of the first reducing agent to the antibody is 2: 1 to 1: 1. In some embodiments, the molar ratio of the first reducing agent to the antibody is 1: 1, 1.5: 1, 1.8: 1, 2: 1, 2.5: 1, 2.8: 1 or 3: 1.

[0329] In some embodiments, there is no specific limitation on the concentration of the first reducing agent, as long as the concentrations of the transition metal ion and the antibody are enlarged or reduced in equal proportion. In some embodiments of the present invention, the concentration of the first reducing agent is 0.01 mM to 0.2 mM. In some embodiments of the present invention, the concentration of the first reducing agent is 0.02 mM to 0.15 mM. In some embodiments of the present invention, the concentration of the first reducing agent is 0.05 mM to 0.1 mM. In some embodiments of the present invention, the concentration of the first reducing agent is 0.01mM, 0.02mM, 0.03mM, 0.04mM, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.11mM, 0.12mM, 0.13mM, 0.14mM, 0.15mM, 0.16mM, 0.17mM, 0.18mM, 0.19mM or 0.20mM.

[0330] In some embodiments, there is no specific limitation on the concentration of the transition metal ion in step (a), as long as the concentrations of the first reducing agent and the antibody are increased or decreased in equal proportions.

[0331] In some embodiments of the present invention, there is no specific 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 in equal proportions.

[0332] 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 and Tris buffer.

[0333] The term "HEPES buffer" herein refers to 4-hydroxyethylpiperazineethanesulfonic acid buffer.

[0334] The term "PBS" herein refers to phosphate buffered saline.

[0335] The term "PB" herein refers to phosphate buffered saline.

[0336] The term "MES buffer" herein refers to 2-(N-morpholino)ethanesulfonic acid buffer.

[0337] The term "BES buffer" herein refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer.

[0338] The term "MOPS buffer" herein refers to 3-morpholinepropanesulfonic acid buffer.

[0339] The term "Bis-Tris buffer" herein refers to bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane buffer.

[0340] The term "DIPSO buffer" herein refers to 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer.

[0341] The term "MOPSO buffer" herein refers to 3-(N-morpholino)-2-hydroxy-1-propanesulfonic acid buffer.

[0342] The term "TES buffer" herein refers to 2-[tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid buffer.

[0343] The term "ACES buffer" herein refers to N-(carbamoylmethyl)taurine buffer.

[0344] The term "MOBS buffer" herein refers to 4-(N-morpholino)butanesulfonic acid buffer.

[0345] The term "TAPSO buffer" herein refers to 3-[N-tris-(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid buffer.

[0346] The term "ADA buffer" herein refers to N-(carbamoylmethyl)iminodiacetic acid buffer.

[0347] The term "PIPES buffer" herein refers to piperazine-1,4-bisethanesulfonic acid buffer.

[0348] The term "BTP buffer" herein refers to Bis-tris propane buffer.

[0349] The term "Heppso buffer" herein refers to N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid buffer.

[0350] The term "POPSO buffer" herein refers to piperazine-N,N'-bis(2-hydroxy-propanesulfonic acid) buffer.

[0351] The term "EPPS buffer" herein refers to 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer.

[0352] The term "Tris buffer" herein refers to tris(hydroxymethyl)aminomethane buffer.

[0353] 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, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer or MES buffer.

[0354] In some embodiments, the first buffer system and the second buffer system are MES buffers.

[0355] In some embodiments, the concentration of the first buffer system and the second buffer system is 10-100 mM (mmol / L).

[0356] In some embodiments, the pH value of the first buffer system and the second buffer system is 5.5 to 8.0. In some embodiments, the pH value of the buffer system is 5.8 to 8.0. In some embodiments, the pH value of the first buffer system and the second buffer system is 6.0 to 7.4. In some embodiments, the pH value of the first buffer system and the second buffer system is 6.7 to 7.4. In some embodiments, the pH value of the first buffer system and the second buffer system is 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2 or 7.4.

[0357] In some embodiments, the first buffer system and the second buffer system are MES buffer, and the pH value of the MES buffer is 5.8 to 6.7.

[0358] The term "transition metal ion" refers to elements of Groups 4 to 12, determined by their typical chemical properties, i.e., coordinated ions in various oxidation states, colored complexes, and catalytic properties as elements or ions (or both). Sc and Y in Group 3 are also generally considered transition metals.

[0359] In some embodiments, the transition metal ion is selected from the group consisting of: Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof.

[0360] In some embodiments, the transition metal ion is Zn 2+ .

[0361] In some embodiments, there is no specific limitation on the salt of the transition metal ion, as long as the transition metal ion is soluble in the reaction solution so that free transition metal ions can be released in the reaction solution. In some embodiments, the salt of the transition metal ion is chloride, nitrate, sulfate, acetate, iodide, bromide, formates or tetrafluoroborate.

[0362] In some embodiments, Zn 2+ The salt is ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate or zinc tetrafluoroborate. In some embodiments of the present invention, Zn 2+ The salt is ZnCl2.

[0363] Those skilled in the art will appreciate that the incubation temperature and incubation time in step (a) depend on the specific antibody to be coupled. In some embodiments, in step (a), the incubation temperature is 0°C to 37°C, 0°C to 25°C or 0°C to 15°C, in step (a), the incubation time is 0.2h to 24h, optionally, in step (a), the incubation temperature is 0°C to 10°C, and in step (a), the incubation time is 2h to 16h.

[0364] In some embodiments, in step (a), the incubation temperature is 0° C. to 15° C., 0° C. to 10° C., 0° C. to 8° C., 0° C. to 6° C. In some embodiments, in step (a), the incubation temperature is 4° C., 8° C., 12° C., 15° C., 18° C., 24° C., 30° C., 35° C., or 37° C.

[0365] In some embodiments, in step (a), the incubation time is 0.5h to 24h, 0.5h to 20h, 0.5h to 16h, 0.5h to 12h, 0.5h to 8h or 0.5h to 6h. In some embodiments, the incubation time is 0.25h, 0.3h, 0.5h, 0.7h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 7h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h.

[0366] In some embodiments, in step (a), the molar ratio of the first reducing agent to the antibody is 2: 1 to 3: 1, and the incubation time is 0.5h to 9h. In some embodiments, in step (a), the molar ratio of the first reducing agent to the antibody is 2.8: 1 to 3: 1, and the incubation time is 1h to 9h. In some embodiments, in step (a), 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 is 0.5, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h or 9.5h.

[0367] In some embodiments, in step (a), the incubation temperature is 0°C to 25°C, and in step (a), the incubation time is 0.5h to 24h. In some embodiments, in step (a), the incubation temperature is 0°C to 15°C, and in step (a), the incubation time is 0.5h to 24h. In some embodiments, in step (a), the incubation temperature is 0°C to 10°C, and in step (a), the incubation time is 2h to 16h.

[0368] In some embodiments, in step (c), there is no specific limitation on the second reducing agent, as long as the second reducing agent can reduce the interchain disulfide bonds 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.

[0369] In some embodiments, in step (c), in the absence of transition metal ions, there is no specific limit to the concentration of the second reducing agent, as long as the second reducing agent can completely reduce the interchain disulfide bonds in the antibody, but is not limited thereto. In some embodiments of the present invention, the molar ratio of the second reducing agent to the 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, 6:1 to 8:1. In some embodiments, the molar ratio of the second reducing agent to the antibody is 20:3.

[0370] In some embodiments, in step (c), the incubation time of the second reducing agent is 0.5h to 24h, or 5h to 20h. In some embodiments, in step (c), the incubation time of the second reducing agent is 6h to 18h, 8h to 18h, 8h to 15h, or 8h to 12h. In some embodiments, in step (c), the incubation time of the second reducing agent is 8h or 12h.

[0371] In some embodiments, in step (c), the transition metal ion is introduced to selectively reduce two interchain disulfide bonds in the interchain disulfide bonds. 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 antibody to the second reducing agent is 2.5:1 to 20:1, and / or the incubation time is 1h to 24h. 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 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 or 24 h. 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 antibody to the second reducing agent is 3: 1 to 15: 1, and the incubation time is 1 h to 12 h. 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 antibody to the second reducing agent is 2.5:1 to 15:1, and the incubation time is 12h to 24h.

[0372] In some embodiments, in step (c), the transition metal ion is introduced to selectively reduce one of the interchain disulfide bonds. 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 antibody to the second reducing agent is 0.8:1 to 2.5:1, and / or the incubation time is 0.5h to 24h. 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, 1:100. In some embodiments, in step (c), the molar ratio of the second reducing agent to the 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 0.5h,

[0373] 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22 or 24h. 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 antibody to the second reducing agent is 0.8:1 to 2:1, and the incubation time is 0.5h to 24h. 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 antibody to the second reducing agent is 2:1 to 2.5:1, and the incubation time is 1h to 9h.

[0374] In some embodiments, in step (c), the incubation temperature of the second reducing agent is 0° C. to 37° C., or 5° C. to 30° C. In some embodiments, in step (c), the incubation temperature of the second reducing agent 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, in step (c), the incubation temperature of the second reducing agent is 25° C.

[0375] In some embodiments, in step (b) and step (d), the reaction temperature with the reduced thiol group 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, in step (b) and step (d), the reaction temperature with the reduced thiol group is 24° C.

[0376] In some embodiments, in step (b) and step (d), the reaction time with the reduced thiol group is 0.5h to 6h, 0.5h to 5h, 0.5h to 4h, 0.5h to 3h, 0.5h to 3h, 0.5h to 2h, 0.5h to 1h. In some embodiments, in step (b) and step (d), the reaction time with the reduced thiol group is 0.5h, 1h, 2h or 3h.

[0377] In some embodiments, in step (b) and step (d), the reaction temperature and reaction time with the reduced thiol group are independent.

[0378] In some embodiments, in step (b) and step (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 step (b) and step (d), the reaction temperature with the reactive group is 25° C.

[0379] In some embodiments, in step (b) and step (d), the reaction time with the reactive group is 2h to 12h, 2h to 10h, 4h to 10h, 6h to 10h or 8h to 10h. In some embodiments, in step (b) and step (d), the reaction time with the reactive group is 8h.

[0380] In some embodiments, in step (b) and step (d), the reaction temperature and reaction time with the reactive group are independent.

[0381] In certain embodiments, in step (b), the metal chelator can capture the excessive transition metal ions. In certain embodiments, there is no specific limitation on the metal chelator, as long as the metal chelator can capture the excessive transition metal ions and will not affect the reduction of the disulfide bonds in the antibody. In certain 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).

[0382] 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.

[0383] In some embodiments, in step (b), the molar ratio of the metal chelator to the 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, 40:1 to 55:1.

[0384] In some embodiments, in step (d), the molar ratio of the metal chelator to the 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, 2:1 to 6:1.

[0385] In some embodiments, dialysis, ultrafiltration or gel filtration is used to filter out excess metal chelator and the complex of the metal chelator and the transition metal ion.

[0386] In some embodiments, in step (b), the modification reagent 1 is in excess based on the amount of the antibody.

[0387] In some embodiments, in step (b), the molar ratio of the first sulphur bridging agent to the antibody is 5: 1 to 1: 1, 2: 1 to 1: 1, 1.5: 1 to 1: 1, 1.2: 1 to 1: 1, or 1.1: 1 to 1: 1. In some embodiments, in step (b), the molar ratio of the first sulphur bridging agent to the antibody is 1.05: 1.

[0388] In some embodiments, in step (b), when the first linker-payload reacts with the reduced thiol group, the molar ratio of the first linker-payload to the antibody is 5: 1 to 1: 1, 2: 1 to 10: 1, 3: 1 to 10: 1, 4: 1 to 9: 1, or 5: 1 to 7: 1. In some embodiments, in step (b), when the first linker-payload reacts with the reduced thiol group, the molar ratio of the first linker-payload to the antibody is 5: 1.

[0389] In some embodiments, in step (b), when the first linker-payload reacts with the reactive group in the first sulfide 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.

[0390] In some embodiments, in step (d), the modification reagent 2 is in excess based on the amount of the antibody.

[0391] In some embodiments, in step (d), the molar ratio of the second sulphur bridging agent to the 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 the second sulphur bridging agent to the antibody is 5: 1, 4.5: 1, 4: 1, 3.8: 1, 3.5: 1 or 3.2: 1.

[0392] In some embodiments, in step (d), when the second linker-payload reacts with the reduced thiol group, the molar ratio of the second linker-payload to the 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, 12: 1 to 10: 1. In some embodiments, in step (d), when the first linker-payload reacts with the reduced thiol group, the molar ratio of the second linker-payload to the antibody is 35:3.

[0393] In some embodiments, in step (d), when the second linker-payload reacts with the reactive group in the second sulfide bridging reagent, the molar ratio of the second linker-payload to the 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.

[0394] In some embodiments, the method further comprises the following steps:

[0395] Optionally, introducing a compound containing at least one thiol group to consume excess of the first linker-payload in step (b) and / or excess of the second linker-payload in step (d);

[0396] The antibody with site-specific modification of thiol group obtained in step (b) and / or step (d) is purified and recovered.

[0397] In some embodiments, there is no specific limitation on the compound that consumes the excess of the first linker-payload and / or the second linker-payload, as long as the compound contains at least one thiol group. In some embodiments, the compound is cysteine.

[0398] By purifying and recovering the antibody with site-specific modification of thiol group in step (b) and / or step (d), the content of antibody with site-specific modification of thiol group can be made higher. In some embodiments, the antibody with site-specific modification of thiol group is purified by desalting column, size exclusion chromatography, ultrafiltration, dialysis and / or similar methods. In some embodiments, the antibody with site-specific modification of thiol group is purified by desalting column purification. If necessary, further enrichment (e.g., D2) can be used in some cases using hydrophobic interaction chromatography (HIC).

[0399] In certain embodiments, there is no specific limitation on the antibody. According to the antigen associated with the disease, a person skilled in the art can select a suitable antibody that can be used for the bioconjugation process of the present invention. In certain embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody or a multispecific antibody.

[0400] As used herein, the term "antibody" refers to any immunoglobulin that binds to a specific antigen. A natural complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and the first, second and third constant regions, while each light chain consists of a variable region and a constant region. The heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG and IgM.

[0401] In some embodiments, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof.

[0402] The term "human antibody" herein refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or an amino acid sequence of an antibody derived from a non-human source (utilizing a human antibody repository) or other human antibody coding sequences. The human antibody definition specifically excludes humanized antibodies comprising non-human antigen binding residues.

[0403] The term "humanized antibody" herein refers to a chimeric antibody comprising amino acid residues from a non-human heavy chain variable region (HVR) and amino acid residues from human FR. In certain embodiments, a humanized antibody will comprise substantially all or at least one (usually two) variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to HVRs of non-human antibodies, and all or substantially all FRs correspond to FRs of human antibodies. Alternatively, a humanized antibody may 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 been humanized.

[0404] The term "chimeric antibody" herein refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the rest of the heavy chain and / or light chain is derived from a different source or species.

[0405] The term "hinge region" herein refers to the portion of an antibody comprising a heavy chain molecule connecting the CH1 domain and the CH2 domain. The hinge region comprises about 25 amino acid residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently.

[0406] The term "Fab fragment" herein refers to the region of the antibody structure that is capable of binding to an antigen. It consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and constant region fragments), and the light chain and the heavy chain are connected by a disulfide bond. Fab fragments can be obtained by protease digestion of full-length antibodies. Under the action of papain, human immunoglobulin G can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into F(ab')2 fragments and pFc' fragments. The F(ab')2 fragment can be further reduced to form two Fab' fragments.

[0407] The term "Fc region" herein refers to a monomeric protein, a dimeric protein or a heterodimeric protein having at least an immunoglobulin CH2 and CH3 domains. The CH2 and CH3 domains may form at least a portion of a dimer region of a protein / molecule (eg, an antibody).

[0408] In some embodiments, the antibody refers to an immunoglobulin and is a molecule containing an antigen binding site that immunospecifically binds to an antigen. In some embodiments, the antibody is classified as IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the antibody is classified as IgG.

[0409] In some embodiments, the class of the antibody is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is IgG1 or IgG4.

[0410] In some embodiments, the antibody is wild-type. The term "wild-type" herein refers to naturally occurring and free of mutations.

[0411] In some embodiments, the antibody is an engineered antibody having two amino acid substitutions of two interchain cysteines that form an interchain disulfide bond in the hinge region.

[0412] In some embodiments, the amino acid substitution is selected from the following substitutions: cysteine ​​to alanine, leucine, arginine, lysine, asparagine, methionine, aspartic acid, phenylalanine, proline, glutamine, serine, glutamic acid, threonine, glycine, tryptophan, histidine, tyrosine, isoleucine or valine, respectively.

[0413] In some embodiments, the amino acid substitution is selected from the following substitutions: cysteine ​​to asparagine, glutamine, serine, threonine or tyrosine, respectively.

[0414] In some embodiments, the amino acid substitution is selected from the following: cysteine ​​to serine.

[0415] In some embodiments, the antibody comprises at least one mutation in the Fc region. In some embodiments, the at least one mutation modulates effector function, or reduces or eliminates Fc-g receptor binding.

[0416] In some embodiments, the one or more mutations are to stabilize the antibody and / or increase half-life. In some instances, the one or more mutations are to regulate Fc receptor interactions to reduce or eliminate Fc effector functions, such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In other instances, the one or more mutations are to regulate glycosylation.

[0417] In some embodiments, the one or more mutations are located in the Fc region. In some instances, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some instances, the mutation comprises L234 and L235. In some instances, the mutation comprises L234A and L235A. In some cases, the residue positions are referenced to IgG1.

[0418] In some embodiments, the Fc region comprises a mutation at residue position L234, L235, D265, N21, K46, L52, or P53, or a combination thereof. In some instances, the mutation comprises a combination of L234 and L235 with a mutation at residue position K46, L52, or P53. In some cases, the residue positions are referenced to IgG1.

[0419] In some embodiments, the Fc region comprises mutations at L234, L235 and K46. In some cases, the Fc region comprises mutations at L234, L235 and L52. In some cases, the Fc region comprises mutations at L234, L235 and P53. In some cases, the Fc region comprises mutations at D265 and N21. In some cases, the residue positions are with reference to IgG1.

[0420] In some instances, the Fc region comprises L234A, L235A, D265A, N21G, K46G, L52R or P53G, or a combination thereof. In some instances, the Fc region comprises a combination of L234A and L235A and K46G, L52R or P53G. In some cases, the Fc region comprises L234A, L235A and K46G. In some cases, the Fc region comprises L234A, L235A and L52R. In some cases, the Fc region comprises L234A, L235A and P53G. In some cases, the Fc region comprises D265A and N21G. In some cases, the residue position is with reference to IgG1.

[0421] In some embodiments, the Fc region comprises a mutation at residue position L233, L234, D264, N20, K45, L51 or P52. In some instances, the Fc region comprises a combination of a mutation at L233 and L234 and a mutation at residue position K45, L51 or P52. In some cases, the Fc region comprises a mutation at L233, L234 and K45. In some cases, the Fc region comprises a mutation at L233, L234 and L51. In some cases, the Fc region comprises a mutation at L233, L234 and K45. In some cases, the Fc region comprises a mutation at L233, L234 and P52. In some instances, the Fc region comprises a mutation at D264 and N20. In some cases, positions equivalent to residues L233, L234, D264, N20, K45, L51, or P52 in an IgG1, IgG2, IgG3, or IgG4 framework are contemplated.

[0422] In some embodiments, the Fc region comprises L233A, L234A, D264A, N20G, K45G, L51R or P52G. In some instances, the Fc region comprises L233A and L234A. In some instances, the Fc region comprises a combination of L233A and L234A with K45G, L51R or P52G. In some cases, the Fc region comprises L233A, L234A and K45G. In some cases, the Fc region comprises L233A, L234A and L51R. In some cases, the Fc region comprises L233A, L234A and K45G. In some cases, the Fc region comprises L233A, L234A and L51R. In some cases, the Fc region comprises L233A, L234A and K45G. In some cases, the Fc region comprises L233A, L234A and P52G. In some instances, the Fc region comprises D264A and N20G. In some cases, the residue position is with reference to IgG1.

[0423] In some embodiments, the human IgG constant region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), for example, using amino acid modifications as described in 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, 13(10):3863-72; Res, 67(18):8882-8890; Stavenhagen et al., 2008 Adv. Enzyme Regul., 48:152-164; Alegre et al., 1992 J Immunol, 148:3461-3468; Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

[0424] In some embodiments, the antibody of IgG1, IgG2, IgG3 or IgG4 is a human antibody or a humanized antibody. Information of IgG1, IgG2, IgG3 or IgG4 can be obtained at NCBI or UniProt (https: / / www.uniprot.org / ).

[0425] In some embodiments, the antibody is a bispecific antibody. In some embodiments of the invention, the antibody is an IgG1-like bispecific antibody.

[0426] In some embodiments, a person skilled in the art may select a suitable method to prepare a bispecific antibody. In some embodiments of the present invention, the bispecific antibody may be prepared by a knob-in-holes technique (Ridgway JBB, Presta LG, Paul C. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization ("knobs-into-holes" engineering of heavy chain heterodimerization antibody CH3 domain) [J]. Protein Engineering (7): 617 (2023-08-11)., format chain exchange (FORCE) technology, universal 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 (a new method for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1) [J]. Journal of Biological Chemistry (Journal of Biological Chemistry) 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]. Antibodies (mAbs), 2022, 14 (1), e2018960), CrossMAb technology (Klein C, Schaefer W, Regula JT. The use of CrossMAb technology for the generation of bi-and multispecific antibodies [J]. Antibodies (mAbs), 2016, 8 (6), P1010-P1020.) or a combination thereof.

[0427] The term "knob-to-hole structure" herein is used in its broadest sense and covers various situations, such as a CH1 domain of one heavy chain having a knob mutation and a CH1 domain of another heavy chain having a hole mutation, a CH2 domain of one heavy chain having a knob mutation and a CH2 domain of another heavy chain having a hole mutation, and / or a CH3 domain of one heavy chain having a knob mutation and a CH3 domain of another heavy chain having a hole mutation. For example, in general, a "knob-in-hole structure" can refer to a modification within the interface in the CH3 domains between two antibody heavy chains: i) in the CH3 domain of one heavy chain (the first CH3 domain), one amino acid residue is substituted by another amino acid residue carrying a large side chain, thereby generating a protrusion ("knob") in the interface of the first CH3 domain; ii) in the CH3 domain of the other heavy chain (the second CH3 domain), one amino acid residue is substituted by another amino acid residue carrying a smaller side chain, thereby generating a cavity ("hole") within the interface of the second CH3 domain, into which the protrusion ("knob") in the first CH3 domain can be placed.

[0428] In some embodiments, the antibody is selected from any one of a cytotoxic antibody, a cell proliferation inhibitor, a cell activation and interaction regulator, a human immune system regulator, an antigen neutralizer, an antibody immunospecific for a viral antigen, or an antibody immunospecific for a microbial antigen.

[0429] In some embodiments, the antibody can be a target-specific antibody. In some embodiments, the antibody can be, but is not limited to, anti-HER2 antibody, anti-FAP antibody, anti-OX-40 antibody, anti-41BB antibody, anti-angiopoietin 2 antibody, anti-ant-IL-4Rα antibody, anti-BCMA antibody, anti-Blys antibody, anti-BTNO2 antibody, anti-C5 antibody, anti-CD122 antibody, anti-CD13 antibody, anti-CD133 antibody, anti-CD137 antibody, anti-CD138 antibody, anti-CD16a antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody Antibodies, anti-CD27 antibodies, anti-CD28 antibodies, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD38 antibodies, anti-CD40 antibodies, anti-CD47 antibodies, anti-CD-8 antibodies, anti-CD79 antibodies, anti-CEA antibodies, anti-CGPR / CGRPR antibodies, anti-CSPGs antibodies, anti-CTLA4 antibodies, anti-CTLA-4 domain antibodies, anti-DLL-4 antibodies, anti-EGFR antibodies, anti-EpCAM antibodies, anti-factor IXa antibodies, anti-factor X antibodies, anti-GITR antibodies , 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 antibody, anti-SARS-CoV-2 antibody, anti-Te38 antibody, anti-TIM-3 antibody, anti-TNF antibody, anti-TNFα antibody, anti-TROP-2 antibody, anti-TWEAK antibody, anti-VEGF antibody, or anti-VEGFR antibody.

[0430] In some embodiments, the antibody has targeting specificity, and it targets HER2 (human epidermal growth factor receptor 2), TROP2 (TACSTD2, tumor-associated calcium signal transducer 2), BCMA (TNFRSF17, TNF receptor superfamily member 17).

[0431] In some embodiments, the antibody can be trastuzumab, sacituzumab, belantamab, risankizumab, eptinezumab, teprotumumab, polatuzumab, tafasitamab, rovelizumab, romosozumab, dostarlimab, enfortumab or ublituximab. In some embodiments, the antibody is trastuzumab, sacituzumab or belantamab.

[0432] In some embodiments, the antibodies can be purchased commercially or produced by any method known to those skilled in the art.

[0433] In some embodiments, the first sulfide bridging reagent and the second sulfide bridging reagent independently contain at least two substituents that allow re-bridging of the thiol group.

[0434] In some embodiments, the first sulphur bridging agent and the second sulphur bridging agent are independently selected from, but not limited to, the group consisting of:

[0435]

[0436] In some embodiments, the reactive groups independently contain azide and / or dibenzocyclooctyne (DBCO).

[0437] In some embodiments, the sulfide-bridging reagent is linked to the reactive group through an alkyl group or polyethylene glycol (PEG).

[0438] In some embodiments, the first sulphur bridging agent carrying a reactive group and the second sulphur bridging agent carrying a reactive group are independently selected from, but not limited to, the group consisting of:

[0439]

[0440] 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.

[0441] In some embodiments, the first sulphur bridging agent carrying a reactive group and the second sulphur bridging agent carrying a reactive group are dibromomaleimide-PEG4-N3 having the formula:

[0442]

[0443] In some embodiments, the linkers in the first linker-payload and the second linker-payload are selected from any one of the linkers having the following characteristics: one end is capable of linking to the reduced thiol group of the antibody or the reactive group of the sulfide bridging reagent, and the other end is capable of linking to the payload.

[0444] The term "linker" herein refers to a substituted molecule containing at least two substituents, one of which can covalently bind to a drug molecule and the other of which can covalently couple to a reactive group of an antibody or a sulfide-bridging agent.

[0445] In some embodiments, when the first linker-payload and the second linker-payload react with the reduced thiol group, the linkers of the first linker-payload and the second linker-payload independently include a cleavable linker or a non-cleavable linker. The cleavable linker can be a chemically unstable linker and an enzyme-cleavable linker. Due to its high plasma stability and good intracellular cleavage selectivity and effectiveness, the enzyme-cleavable linker is widely selected as a candidate for cleavable linkers in ADC. In some embodiments, the enzyme-cleavable linker includes the following structure: -maleimidocaproyl-(-MC-), -maleimidocaproyl-peptide portion-(-MC-peptide portion-), -p-aminobenzyl alcohol-(-PAB-), or -peptide portion-. In some embodiments, the peptide portion is a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.

[0446] 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).

[0447] 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).

[0448] In some embodiments, the tetrapeptide can be, but is not limited to, glycine-glycine-phenylalanine-glycine (GGFG).

[0449] In some embodiments, when the first linker-payload and / or the second linker-payload reacts with a reduced thiol group, the linkers of the first linker-payload and the second linker-payload can independently be, but are not limited to, 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. In some embodiments, when the first linker-payload and / or the second linker-payload reacts with a reduced thiol group, the linkers of the first linker-payload and the second linker-payload can independently be, but are not limited to, MC-AAD-PAB, MC-GVC-PAB, MC-GGG-PAB, MC-FFK-PAB, MC-EVC-PAB or MC-GFK-PAB.

[0450] In some embodiments, the linker comprises a drug-carrying maleimide, a drug-carrying organic chloride, a drug-carrying organic bromide, a drug-carrying organic iodide, and / or a drug-carrying vinyl pyrimidine.

[0451] In some embodiments, when the first linker-payload and / or the second linker-payload reacts with the reactive group in the sulfide bridging reagent, the linker of the first linker-payload and / or the second linker-payload further comprises an azide group and / or dibenzocyclooctyne (DBCO). In some embodiments, when the linker of the first linker-payload and / or the second linker-payload contains an azide group, the reactive group of the sulfide bridging group contains DBCO. In some embodiments, when the linker of the first linker-payload and / or the second linker-payload contains DBCO, the reactive group of the sulfide bridging group contains an azide group.

[0452] In some embodiments, when the first linker-payload and / or the second linker-payload react with the reactive group in the sulfide bridging reagent, the linkers of the first linker-payload and the second linker-payload are independently selected from, but not limited to, any one of the group consisting of:

[0453]

[0454] wherein n is 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3, and m 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, and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0455] As used herein, the term "capping agent" refers to a compound that does not carry a drug but contains at least one substituent that can be covalently coupled to an antibody.

[0456] In some embodiments, the capping agent is a cleavable linker or a non-cleavable linker. In some embodiments, the capping agent is (2-aminoethyl)maleimide.

[0457] In some embodiments, there is no particular limitation on the payload, as long as the payload contains at least one substituent that allows attachment from the payload to the linker.

[0458] The term "payload" herein refers to any cytotoxic molecule having at least one substituent or partial structure that allows attachment 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 condition.

[0459] In some embodiments, the payload is a cytotoxic drug, a cytokine, a nucleic acid, a radionuclide, a kinase, or a 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 can be, but is not limited to, an anticancer agent, an antiviral agent, or an antimicrobial agent.

[0460] In some embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of the cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric tumor (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver malignancies, anal cancer, penile cancer, and head and neck cancer.

[0461] Exemplary payloads are monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), monomethyl auristatin EF (MMAF), calicheamicin (CLM), maytansine (DM1), maytansine alkaloid, duocarmycin, anthracycline, pyrrolobenzodiazepine dimer, amatoxin, quinoline alkaloid, Dxd, doxorubicin hydrochloride, methotrexate, erlotinib, bortezomib, fulvestrant, sunitinib mesylate imatinib, letrozole, finasunate, platinums (such as oxaliplatin, carboplatin and cisplatin), finasunate, fluorouracil, rapamycin, folinic acid, lapatinib, lonafarnib, sorafenib, gefitinib, capmtothecin, topotecan, moss statin, adezelesin, anthracycline, kazelesin, bisoprolol, dolastatin, auristatin, duocarmycin, eleutherobin, taxanes (such as paclitaxel or docetaxel), cyclophosphamide, doxorubicin, vincristine, prednisone or prednisolone, other alkylating agents (such as nitrogen mustard, chlorambucil and ifosfamide), antimetabolites (such as azathioprine or mercaptopurine), vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, etoposide phosphate, epipodophyllotoxin, actinomycin, daunorubicin, valrubicin, idarubicin, edrecolomab, epirubicin, bleomycin, plicamycin or mitomycin, and their salts.

[0462] In some embodiments, the payload is deludecanoic acid (DXd), cyanine 3 (Cy3), MMAE, MMAD or MMAF. In some embodiments of the invention, the payload is MMAE, DXd or Cy3.

[0463] The linker-payload is a chemical moiety that is synthesized by connecting the linker to the payload. Depending on the desired payload and the selected linker, those skilled in the art can select a suitable method to couple them together. For example, some conventional coupling methods (such as amine coupling methods) can be used to form the desired linker-payload, which still contains a reactive group for coupling to the antibody by a covalent bond. In the present invention, a drug-maleimide complex (i.e., maleimide-connected drug) is used as an example of a payload carrying a reactive group. In ADC preparation, the most common reactive group that can be combined with a thiol group is maleimide. In addition, organic chlorides, bromides, and iodides are also often used.

[0464] The linker-payload can be any physically active compound, or any compound used to diagnose, prevent or treat a disease. In some embodiments, when the first linker-payload and / or the second linker-payload reacts with the reduced thiol group, the first linker-payload and / or the second linker-payload are independently MC-VC-PAB-MMAE, MC-VC-PAB-MMAD and MC-VC-PAB-MMAF.

[0465] In some embodiments, the first sulfide bridging reagent carrying the first linker-payload and the second sulfide bridging reagent carrying the second linker-payload independently have the following formula:

[0466]

[0467] Wherein Q is selected from the group consisting of:

[0468]

[0469] S is selected from, but not limited to, a cleavable linker or a non-cleavable linker, S is selected from the group consisting of:

[0470]

[0471]

[0472] wherein n is 0-20, m is 0-20, optionally, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0473] T is the effective load.

[0474] In some embodiments, the first sulfide bridging reagent carrying the first linker-payload and the second sulfide bridging reagent carrying the second linker-payload are independently selected from, but not limited to, the group consisting of:

[0475]

[0476] In some embodiments, the payload of the first sulphur bridging agent carrying the first linker-payload and the payload of the second sulphur bridging agent carrying the second linker-payload are different or the same. In some embodiments, the linker of the first sulphur bridging agent carrying the first linker-payload and the linker of the second sulphur bridging agent carrying the second linker-payload may be different or the same. In some embodiments, the first sulphur bridging agent carrying the first linker-payload and the second sulphur bridging agent carrying the second linker-payload may be different or the same.

[0477] In some embodiments, the preparation method of the D2-type ADC comprises the following steps:

[0478] (a1) incubating the compound according to the present invention used as the first reducing agent and the antibody in the first buffer system in the presence of an effective amount of the transition metal ion to selectively reduce the interchain disulfide bonds in the antibody; or

[0479] Incubating the composition according to the present invention and the antibody in the first buffer system to selectively reduce the interchain disulfide bonds in the antibody, wherein the compound according to the present invention is used as the first reducing agent;

[0480] (b1) introducing an excess of the metal chelator and an excess of the first linker-payload to react with the reduced thiol group from step (a1).

[0481] In some embodiments, in step (b1), the reaction temperature is 15°C to 25°C, and in step (b1), the reaction time is 0.5h to 2h.

[0482] In some embodiments, the D2-type ADC has a uniformity of up to 53%, 55%, 60%, 65%, 70%, 75%, 80%, 84%, 87%, 89%, 90%, 91%, 92% or 94%.

[0483] In some embodiments, the content of D0 and D4 contained in the ADC prepared by the preparation method of the D2-type ADC is 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, the content of D0 and D4 contained in the ADC prepared by the preparation method of the D2-type ADC is less than 20% of the total weight of D0, D2, D4, D6 and D8.

[0484] In some embodiments, the preparation method of the D2+D6 type ADC comprises the following steps:

[0485] (c2) incubating the reaction product from step (b1) and the second reducing agent in the second buffer system to reduce the interchain disulfide bonds in the reaction product from step (b1);

[0486] (d2) introducing the incubation product from step (c2) and an excess of the second linker-payload to react with the reduced thiol group from step (c2).

[0487] In some embodiments, the uniformity of the D2+D6 ADC is as high as 75%, 80%, 85%, or even 90%.

[0488] In some embodiments, the preparation method of the D2+D3 type ADC comprises the following steps:

[0489] (d3) introducing the incubation product from step (c2) and an excess of a second sulfide bridging reagent carrying the second linker-payload to react with the reduced thiol group from step (c2).

[0490] In some embodiments, the preparation method of the D2+D3 type ADC comprises the following steps:

[0491] (d3`) introducing the incubation product from step (c2) and an excess of the second sulfide bridging reagent carrying a reactive group to re-bridge the reduced thiol group from step (c2), and then incubating an excess of the second linker-payload in the second buffer system to react with the reactive group of the sulfide bridging group.

[0492] In some embodiments, the preparation method of the D1-type ADC comprises the following steps:

[0493] (b4) introducing an excess of a metal chelator and an excess of a first sulfide bridging reagent carrying the first linker-payload to react with the reduced thiol group from step (a1).

[0494] In some embodiments, the preparation method of the D1-type ADC comprises the following steps:

[0495] (b4') introducing an excess of a metal chelator and the first sulfide bridging reagent carrying the reactive group to re-bridge the reduced thiol group from step (a1), and then incubating an excess of the first linker-payload in the first buffer system to react with the reactive group of the sulfide bridging group.

[0496] In some embodiments, the uniformity of the D1-type ADC is generally as high as 75%, 80%, 85%, or even 90%.

[0497] In some embodiments, the preparation method of the D1+D6 type ADC comprises the following steps:

[0498] (c5) incubating the reaction product from step (b4) or step (b4′) and the second reducing agent in the second buffer system to reduce the interchain disulfide bonds in the reaction product from step (b4) or (b4′);

[0499] (d5) introducing the incubation product from step (c5) and an excess of the second linker-payload to react with the reduced thiol group from step (c5).

[0500] In some embodiments, the preparation method of the D1+D3 type ADC comprises the following steps:

[0501] (d6) introducing the incubation product from step (c5) and an excess of a second sulfide bridging reagent carrying the second linker-payload to react with the reduced thiol group from step (c5).

[0502] In some embodiments, the preparation method of the D1+D3 type ADC comprises the following steps:

[0503] (d6`) introducing the incubation product from step (c5) and an excess of the second sulfide bridging reagent carrying a reactive group to re-bridge the reduced thiol group from step (c5), and then incubating an excess of the second linker-payload in the second buffer system to react with the reactive group of the sulfide bridging group.

[0504] In some embodiments, the preparation method of the D0+D6 type ADC comprises the following steps:

[0505] (b7) introducing an excess of a metal chelator and an excess of a first sulphur bridging agent to react with the reduced thiol group from step (a1).

[0506] (c7) incubating the reaction product from step (b7) and the second reducing agent in the second buffer system to reduce the interchain disulfide bonds in the reaction product from step (b7);

[0507] (d7) introducing the incubation product from step (c7) and an excess of the second linker-payload to react with the reduced thiol group from step (c7).

[0508] In some embodiments, the uniformity of the D0+D6 ADC is as high as 75%, 80%, 85%, or even 90%.

[0509] In some embodiments, the preparation method of the D0+D3 type ADC comprises the following steps:

[0510] (d8) introducing the incubation product from step (c7) and an excess of a second sulfide bridging reagent carrying the second linker-payload to react with the reduced thiol group from step (c7).

[0511] In some embodiments, the preparation method of the D0+D3 type ADC comprises the following steps:

[0512] (d8`) introducing the incubation product from step (c7) and an excess of the second sulfide bridging reagent carrying a reactive group to re-bridge the reduced thiol group from step (c7), and then incubating an excess of the second linker-payload in the second buffer system to react with the reactive group of the sulfide bridging group.

[0513] In some embodiments, an antibody with site-specific modification (D1+D4 type ADC, D1+D2 type ADC) is prepared by a method comprising steps (a), (b), (c) and (d), wherein the modification reagent 1 is a first sulfur bridge reagent carrying a first linker-payload, and the modification reagent 2 is a second linker-payload. At the same time, a transition metal ion is introduced in step (c).

[0514] In some embodiments, an antibody with site-specific modification (D1+D4 type ADC, D1+D2 type ADC) is prepared by a method comprising steps (a), (b), (c) and (d), wherein the modification reagent 1 is a first sulfur bridge reagent carrying a reactive group that reacts with the first linker-payload, and the modification reagent 2 is a second linker-payload. At the same time, a transition metal ion is introduced in step (c).

[0515] In some embodiments, an antibody with site-specific modification (D2+D4 ADC, D2+D2 ADC) is prepared by a method comprising steps (a), (b), (c) and (d), wherein the modification agent 1 is the first linker-payload and the modification agent 2 is the second linker-payload. At the same time, a transition metal ion is introduced in step (c).

[0516] In some embodiments, an antibody with site-specific modification (D0+D4 type ADC) is prepared by a method comprising steps (a), (b), (c) and (d), wherein the modification reagent 1 is a first sulphur bridge reagent or a capping reagent, and the modification reagent 2 is a second linker-payload. At the same time, a transition metal ion is introduced in step (c).

[0517] Various analytical methods can be used to determine the yield and isomeric mixture of the ADC. In certain embodiments, the analytical method is HIC-HPLC. HIC-HPLC can separate ADCs with different numbers of drugs loaded on antibodies. The drug loading level can be determined based on the ratio of absorbance (e.g., at 250nm and 280nm). For example, if the drug can absorb at 250nm, the antibody absorbs at 280nm. Therefore, the ratio of 250 / 280 will increase with the increase of drug loading.

[0518] Compared with ADCs produced by conventional conjugation processes, the uniformity of the ADCs of the present invention is improved using the bioconjugation process described herein, without the need for protein engineering or ligase, simple operation, and reduced cost.

[0519] The process of generating ADC with uniform D2 by selectively reducing one of the four interchain disulfide bonds on IgG antibodies circumvents any need for protein engineering or enzyme catalysis, but is based on natural interchain disulfide bonds, requiring only novel reducing agents and transition metal ions. Therefore, compared with conventional ADC preparation processes, the process of the present invention is less complicated and significantly improves the uniformity of the resulting antibody drug conjugate.

[0520] In some embodiments, the preparation method of the D1-type ADC comprises the following steps:

[0521] (A1) incubating 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) at 4°C for 4 h;

[0522] (B1) introducing EDTA (0.6 mM) and excess dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol groups from step (A1) at a temperature of 24 °C and a reaction time of 3 h, and then recovering the product using a desalting column;

[0523] (C1) The product from step (B1) was incubated with DBCO-Cy3 (0.02 mM) in MES (20 mM, pH 6.7) at 25°C for 8 h, and the resulting D1-type ADC was recovered using a desalting column.

[0524] In some embodiments, the preparation method of the D0+D6 type ADC comprises the following steps:

[0525] (A2) incubating 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) at 4°C for 4 h;

[0526] (B2) introducing EDTA (0.6 mM) and excess dibromomaleimide (0.013 mM) to react with the reduced thiol groups from step (A2) at 24 °C for 3 h, and then recovering the product using a desalting column;

[0527] (C2) In MES (20 mM, pH 6.7), the product from step (B2) and TCEP (0.08 mM) were introduced at a reaction temperature of 25° C. and a reaction time of 12 h;

[0528] (D2) The second linker-payload (MC-GGFG-DXd, 0.14 mM) was introduced into step (C2), and the reaction mixture was placed at 24°C for 1 h, and then the resulting D0+D6 type ADC was recovered using a desalting column.

[0529] In some embodiments, the preparation method of the D2+D6 type ADC comprises the following steps:

[0530] (A3) incubating 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) at a temperature of 4°C and an incubation time of 4 h;

[0531] (B3) introducing EDTA (0.6 mM) and excess MC-MMAF (0.06 mM) to react with the reduced thiol groups from step (A3) at 24 °C for 1 h, and then recovering the product using a desalting column;

[0532] (C3) In MES (20 mM, pH 6.7), the product from step (B3) and TCEP (0.08 mM) were introduced at a reaction temperature of 25 °C and a reaction time of 8 h;

[0533] (D2) The second linker-payload (MC-GGFG-DXd, 0.14 mM) was introduced into step (C3), and the reaction mixture was placed at 24°C for 1 h, and then the resulting D2+D6 type dual payload ADC was recovered using a desalting column.

[0534] In some embodiments, the preparation method of the D2-type ADC comprises the following steps:

[0535] (A4) Incubate the first reducing agent (0.02 mM) and trastuzumab (0.012 mM) in MES buffer (pH 6.7, 20 mM) in the presence of ZnCl2 (0.24 M). The incubation temperature is 4°C and the incubation time is 4 h;

[0536] (B4) Add EDTA (0.6 mM) to capture Zn 2+ ;

[0537] (C4) introducing MC-VC-PAB-MMAE (0.06 mM) to react with the reduced thiol groups from step (1) at a reaction temperature of 24 °C and a reaction time of 1 h;

[0538] (D4) introduction of cysteine ​​(0.08 mM) to consume excess MC-VC-PAB-MMAE;

[0539] (E4) Purify the obtained ADC using a desalting column.

[0540] Antibodies with site-specific modification of thiol groups

[0541] The present invention provides an antibody with site-specific modification of a thiol group prepared by the method of the present invention.

[0542] In some embodiments, an antibody having a site-specific modification of a thiol group is conjugated to Modifying Agent 1 and / or Modifying Agent 2.

[0543] In some embodiments, the Modifying Agent 1 and / or the Modifying Agent 2 is covalently linked to a reduced thiol group in the hinge region of the antibody.

[0544] In some embodiments, the Modifying Agent 1 and / or the Modifying Agent 2 is covalently linked to a reduced thiol group in the Fab region of the antibody.

[0545] In some embodiments, an antibody with a site-specific modification of a thiol group is coupled with a modification agent 1 to form a D2-type ADC or a D1-type ADC. In some embodiments of the present invention, an antibody with a site-specific modification of a thiol group is coupled with a modification agent 1 and a modification agent 2 to form a D2+D6-type ADC, a D2+D3-type ADC, a D1+D6-type ADC, a D1+D3-type ADC, a D0+D6-type ADC, a D0+D3-type ADC, a D0+D4-type ADC, a D2+D4-type ADC, a D1+D4-type ADC, a D2+D2-type ADC, or a D1+D2-type ADC.

[0546] In some embodiments, the D2-type ADC is trastuzumab-[MC-VC-PAB-MMAE]2, gosartuzumab-[MC-VC-PAB-MMAE]2, or mabelantuzumab-[MC-VC-PAB-MMAE]2.

[0547] In some embodiments, the D1-type ADC is trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1.

[0548] In some embodiments, the D0+D6 type ADC is trastuzumab-[maleimide]1[MC-GGFG-DXd]6.

[0549] In some embodiments, the D2+D6 type ADC is trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6.

[0550] In some embodiments, the ADC comprises 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.

[0551] Use of antibodies with site-specific modification of thiol groups

[0552] The present invention provides use of an antibody having a thiol group site-specific modification according to the present invention in producing a therapeutic agent for preventing, diagnosing or treating a disease.

[0553] The term "treatment" as used herein for any disease refers to alleviating or ameliorating the disease (i.e., slowing or stopping the progression of the disease or at least one of its clinical symptoms); or alleviating or improving at least one physical parameter or biomarker associated with the disease, including physical parameters or biomarkers that may not be discernible by the patient. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above effects. For tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, delaying tumor development, or a combination of these means.

[0554] As used herein, the term "prevention" as applied to any disease refers to preventive treatment of the disease; or delaying the onset or progression of the disease.

[0555] In some embodiments, the disease is a tumor or cancer. In some embodiments, the disease is an autoimmune disease, etc.

[0556] In some embodiments, cancer can include, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphatic malignancies. More specific examples of the cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric tumor (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver malignancies, anal cancer, penile cancer, and head and neck cancer.

[0557] Pharmaceutical composition

[0558] The present invention also provides a pharmaceutical composition, comprising an antibody with site-specific modification of a thiol group prepared by the above method, and at least one pharmaceutically acceptable carrier.

[0559] The pharmaceutical compositions provided herein can be formulated in any manner known in the art, such as, the pharmaceutical compositions provided herein can be formulated in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration.

[0560] A pharmaceutical composition is formulated to be compatible with its intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal administration).

[0561] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0562] Suitable components can include, for example, antioxidants, fillers, adhesives, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers (such as sugar and cyclodextrin). Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated benzyl alcohol and / or propyl gallate. As disclosed herein, in the composition comprising the antibody or its Fab provided herein and conjugate, one or more antioxidants (such as methionine) are included to reduce the oxidation of the antibody or its Fab. This reduction of oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and extending the shelf life to the maximum extent. Therefore, in certain embodiments, a pharmaceutical composition comprising one or more antibodies or its Fab disclosed herein and one or more antioxidants (such as methionine) is provided.

[0563] In some embodiments, the pharmaceutical composition can be a liquid solution, suspension or emulsion. In some embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as, for example, a liquid solution, suspension, emulsion or a solid form suitable for generating a liquid solution, suspension or emulsion. Injectable preparations can include sterile and / or non-pyrogenic solutions for injection, sterile soluble dry products (such as lyophilized powders) for mixing with solvents before use, including subcutaneous injection tablets, sterile suspensions for injection, sterile insoluble dry products for combination with solvents before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0564] In some embodiments, the pharmaceutical composition is combined with other therapeutic agents. There is no specific limitation on other therapeutic agents, as long as the other therapeutic agents can reduce the side effects of the pharmaceutical composition or increase the effectiveness of the pharmaceutical composition. The other therapeutic agents are anticancer agents, anti-autoimmune disease agents, antiemetics, antiallergic agents, etc.

[0565] In some embodiments, the anticancer agent may include, but is not limited to, erlotinib, bortezomib, fulvestrant, sunitinib mesylate imatinib, letrozole, finasunate, platinums (such as oxaliplatin, carboplatin and cisplatin), finasunate, fluorouracil, rapamycin, folinic acid, lapatinib, lonafarnib, sorafenib, gefitinib, capmtothecin, topotecan, bryostatin, adezelesin, anthracyclines, kazelesin, bisoprolol, dolastatin, auristatin, beta-blockers, bisoprolol ... Carcinomycin, eleutherobin, taxanes (such as paclitaxel or docetaxel), cyclophosphamide, doxorubicin, vincristine, prednisone or prednisolone, other alkylating agents (such as nitrogen mustard, chlorambucil and ifosfamide), antimetabolites (such as azathioprine or mercaptopurine), other microtubule inhibitors (vinca alkaloids, like vincristine, vinblastine, vinorelbine and vindesine, and taxanes), podophyllotoxins (etoposide, teniposide, etoposide phosphate and epipodophyllotoxin), topoisomerase inhibitors, other cytotoxins (such as actinomycin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin, etc.).

[0566] In some embodiments, anti-autoimmune disease agents may include, but are not limited to, ibuprofen, loxoprofen, naproxen, diclofenac, indomethacin, meloxicam, lornoxicam, nabumetone, celecoxib, acetaminophen, glucocorticoids, azathioprine, cyclophosphamide, and the like.

[0567] In some embodiments, during and after administration of the ADC of the present invention, the patient may feel nauseous. Therefore, an antiemetic may be administered to prevent nausea (upper abdomen) and vomiting. The antiemetic may include, but is not limited to, aprepitant, ondansetron, granisetron hydrochloride, lorazepam, dexamethasone, prochlorperazine, casopitant, etc.

[0568] In some embodiments, during and after administration of the ADC of the present invention, the patient may experience an allergic reaction. Therefore, antiallergic agents can be used to minimize the risk of allergic reactions. Antiallergic agents include dexamethasone, beclomethasone, hydrocortisone, prednisolone, prednisone, methylprednisolone, hydroxyzine, cyproheptadine, bronchodilators, terbutaline, etc.

[0569] The present invention provides 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 prepared by the above method or a pharmaceutical composition according to the present invention.

[0570] The term "subject" herein refers to mammals, primates (e.g., human male or female), dogs, rabbits, guinea pigs, pigs, rats, and mice. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0571] The term "therapeutically effective amount" herein refers to the amount of an antibody (such as the ADC of the present invention) with a site-specific modification of a thiol group that will elicit a biological or medical response in a subject, such as improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease, etc. The therapeutically effective amount will vary with the type and severity of the condition to be alleviated. It should also be understood that for any particular subject, the specific dosing regimen should be adjusted over time according to individual needs 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 various factors, such as the type of disease, the patient's age, weight, sex, medical condition, severity, route of administration, and the specific antibody used. In some embodiments, the therapeutically effective amount can vary greatly, 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 can include clinical effects and / or laboratory values ​​such as toxic effects.

[0572] It will be apparent to those skilled in the art that other suitable modifications and alterations to the invention described herein are apparent and can be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. The invention has now been described in detail and will be more clearly understood by reference to the following examples, which are intended for illustrative purposes only and are not intended to be limiting. Further, unless otherwise specifically stated, the reagents and solvents described in this specification can be readily obtained from commercial suppliers.

[0573] Example

[0574] The invention is further described in the following examples, which are not intended to limit the scope of the invention described in the claims.

[0575] Reagents and manufacturers

[0576] Trastuzumab is commercially available from Roche.

[0577] Gosartuzumab and mabelantuzumab are commercially available from MedChemExpress.

[0578] TCEP is commercially available from Bidepharm.

[0579] EDTA is commercially available from Aladdin.

[0580] DMA (dimethylacetamide) can be purchased from Aldrich Sigma.

[0581] MC-VC-PAB-MMAE can be purchased from Levena biopharma.

[0582] MC-GGFG-DXd is commercially available from Levena.

[0583] Dibromomaleimide is commercially available from Aladdin.

[0584] Desalting columns (type: 40K, 0.5 mL, reference number: 87766, batch number: SJ251704) can be purchased from ThermoScientific.

[0585] The 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), tert-butyldiphenylsilyl chloride (TBDPSCl), all of which can be purchased from,

[0586] Synthetic Procedure A of Compounds According to the Invention

[0587] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and an amine reagent (0.5 mmol, depending on the structure of the compound) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product.

[0588] Synthesis Procedure A-1 of Compounds According to the Invention

[0589] The product prepared by synthesis procedure A was dissolved in DCM (3 mL) and TFA (0.3 mL) was added. The mixture was stirred for 1 h and LCMS showed that the reaction was complete. The mixture was concentrated and the residue was dissolved in distilled water and washed twice with EtOAc. The water layer was freeze-dried to obtain the corresponding product.

[0590] Synthetic Procedure B for Compounds According to the Invention

[0591] Under N2 atmosphere, HOBt (67.5 mg, 0.5 mmol, 1 eq) and EDCI (95.5 mg, 0.5 mmol, 1 eq) were added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and an amine reagent (0.5 mmol, depending on the structure of the compound) was added. The reactant was stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 chromatographic column to give the corresponding product.

[0592] Synthesis Procedure B-1 of Compounds According to the Invention

[0593] The product prepared by synthesis procedure B was dissolved in DCM (3 mL) and TFA (0.3 mL) was added. The mixture was stirred for 1 h and LCMS showed that the reaction was complete. The mixture was concentrated and the residue was dissolved in distilled water and washed twice with EtOAc. The water layer was freeze-dried to obtain the corresponding product.

[0594] Example 1: Synthesis of TCEP-NO

[0595]

[0596] 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 DMF (degassed) under an inert atmosphere. The solution was added to TCEP (528 μmol, 150 mg, 3 eq.) dissolved in 1.5 mL degassed DMF containing DIPEA (704 μmol, 123 μL, 4 eq.) under an inert atmosphere. The reaction was stirred at room temperature for 60 min. Afterwards, DMF was removed in vacuo and the residue was added to CH3COOH (0.1 N, 5 mL) and EtOAc (2 mL). The resulting mixture was stirred for 5 min and filtered to give compound 2 (61.2 mg, 12.2%) as a white solid. Without further purification, EtOAc (2 mL), 5% TFA and 5% TIPS were added. The reaction continued for another 2 h. H2O (5 mL) and EtOAc (10 mL) were then introduced. The aqueous phase was further washed twice with EtOAc (10 mL) and concentrated to give TCEP-NO (13 mg). For compound 2 (also known as TCEP-19-int1), MS [MH] - =506.2, C 28 H 30 The exact mass of NO6P was calculated to be 507.18. 1 H NMR (400 MHz, DMSO-d6): δ7.40-7.15 (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 exact mass of NO6P was calculated to be 265.07. 1 H-NMR (400 MHz, deuterium oxide): δ 2.93-2.85 (m, 4H), 2.73-2.56 (m, 8H).

[0597] Example 2: Synthesis of TCEP-3NO

[0598]

[0599] Under an inert atmosphere, compound 1 tritylhydroxylamine (NH2-O-Trt, 528 μmol, 145.5 mg, 3 equivalents), EDC (528 μmol, 101 mg, 3 equivalents) and HOBt (880 μmol, 134.5 mg, 5 equivalents) were dissolved in 4 mL DMF (degassed). Under an inert atmosphere, this solution was added to TCEP (176 μmol, 50 mg, 1 equivalent) dissolved in 4 mL of degassed DMF containing DIPEA (704 μmol, 123 μl, 4 equivalents). The reactants were stirred at room temperature for 60 min. Afterwards, DMF was removed in vacuo, and the residue was added to 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 was added EtOAc (2 mL), 5% TFA and 5% TIPS without further purification. The reaction was continued for another 2 h. H2O (5 mL) and EtOAc (10 mL) were then introduced. The aqueous phase was further 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 exact mass calculated for 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).

[0600] Example 3: Synthesis of TCEP-CO

[0601]

[0602] Compound 4 (176 μmol, 43.2 mg, 1 eq., di-tert-butyliminodiacetate, 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 DMF (degassed) 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. Afterwards, the DMF was removed in vacuo and the residue was added to CHCOOH (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. Without further purification, EtOAc (2 mL), 5% TFA and 5% TIPS were added. The reaction was continued for another 2 h. H2O (5 mL) and EtOAc (10 mL) were then introduced. The aqueous phase was further washed twice with EtOAc (10 mL) and concentrated to give TCEP-CO (8 mg). For TCEP-CO, MS [MH] - =363.86, C9H 16 The exact mass of NO6P was calculated to be 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).

[0603] Example 4: Synthesis of TCEP-1

[0604]

[0605] 1.TCEP-1-int2

[0606] Under N2 atmosphere, HOBt (1.64 g, 12.0 mmol, 1.2 equiv.) and EDCI (2.32 g, 12 mmol, 1.2 equiv.) were added to a DMF (30 mL) solution of TCEP-1-int1 (3.0 g, 10.0 mmol, 1.0 equiv., Fmoc-glycine, Bidepharm), followed by DIPEA (4.4 mL, 25.2 mmol, 2.5 equiv.). The mixture was stirred for 30 min, and compound 1 triphenylmethylhydroxylamine (2.78 g, 10 mmol, 1.0 equiv., 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 a crude product TCEP-1-int2 (4.5 g, 80% yield, white solid), which was used directly in the next step without further purification.

[0607] 2.TCEP-1-int3

[0608] DBU (5 mL) was added to a solution of TCEP-1-int2 (4.5 g, 8.1 mmol, 1.0 equivalent) in DMF (25 mL). The resulting mixture was stirred at room temperature for 0.5 h, and LCMS showed that the reaction was complete. The mixture was poured into ice water and extracted with EtOAc, dried in vacuo, and purified with a flash tower (EtOAc / petroleum ether=0-50%) to give the product TCEP-1-int3 (2.0 g, yield 77%, white solid).

[0609] 3.TCEP-1

[0610] TCEP-1 was synthesized in the synthetic procedure B-1, wherein TCEP-1-int3 was an amine reagent, to obtain TCEP-1 (45.1 mg, 28%) as a white solid. MS [MH] - =321.15, C 11 H 19 The exact mass of N2O7P was calculated to be 322.25. 1 H-NMR (400 MHz, deuterium oxide): δ 3.99 (s, 0.64H), 3.87 (s, 1.34H), 2.96–2.81 (m, 6H), 2.63-2.56 (m, 6H).

[0611] Example 5: Synthesis of TCEP-2

[0612]

[0613] TCEP-2 was synthesized in the synthetic procedure B-1, wherein compound 5 (t-butyl glycine, Bidepharm) was used as an amine reagent to obtain TCEP-2 (52.3 mg, 34%) as a white solid. MS [MH] - =306.18, C 11 H 18 The exact mass of NO7P was calculated to be 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).

[0614] Example 6: Synthesis of TCEP-3

[0615]

[0616] TCEP-3 was synthesized in Procedure B, where compound 6 (DL-phenylalanine, Adamas) was the amine reagent to give TCEP-3 (65.0 mg, 33%) as a white solid. MS [MH] - =396.24, C 18 H 24 The exact mass of NO7P was calculated to be 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).

[0617] Example 7: Synthesis of TCEP-4

[0618]

[0619] 1.TCEP-4-int1

[0620] To a solution of ethanolamine (610 mg, 10 mmol, 1.0 eq., Adamas) in DMC (20 mL) was added imidazole (15 mmol, 1.5 eq.), followed by TBDPSCl (10 mmol, 1.0 eq., Adamas) at 0 °C. The mixture was stirred at room temperature for 2 h. TLC showed that the reaction was complete, the reaction mixture was washed with water and brine, the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to give a crude product, which was used directly in the next step without further purification.

[0621] 2.TCEP-4

[0622] TCEP-4 was synthesized in the synthetic procedure B-1, wherein TCEP-4-int1 was an amine reagent, to obtain TCEP-4 (60.0 mg, 40.8%) as a white solid. MS [MH] - =292.25, C 11 H 20 The exact mass of NO6P was calculated to be 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).

[0623] Example 8: Synthesis of TCEP-5

[0624]

[0625] TCEP-5 was synthesized in Procedure B, where (2-phenoxy-ethylamine, Bidepharm) was the amine reagent to give TCEP-5 (105.0 mg, 56.8%) as a white solid. MS [MH] - =368.24, C 17 H 24 The exact mass of NO6P was calculated to be 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).

[0626] Example 9: Synthesis of TCEP-6

[0627]

[0628] 1.TCEP-6-int1

[0629] To a solution of N-methylhydroxylamine hydrochloride (830 mg, 10 mmol, 1.0 eq., Bidepharm) in DMC (20 mL) was added imidazole (15 mmol, 1.5 eq.), followed by TBDPSCl (10 mmol, 1.0 eq., Adamas) at 0 °C. The mixture was stirred at room temperature for 2 h. TLC showed that the reaction was complete, and the reaction mixture was washed with water and brine, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to give a crude product, which was used directly in the next step without further purification.

[0630] 2.TCEP-6

[0631] TCEP-6 was synthesized in Procedure A-1, where TCEP-6-int1 was an amine reagent to obtain TCEP-6 (13.0 mg, 9.3%) as a white solid. MS [M+H] + =280.22, C 10 H 18 The exact mass of NO6P was calculated to be 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). Example 10: Synthesis of TCEP-7

[0632]

[0633] TCEP-7 was synthesized in the synthetic procedure B, wherein (aniline, Adamas) was the amine reagent to obtain TCEP-7 (73.0 mg, yield: 45.0%) as a white solid. MS [MH] - =324.21, C 15 H 20 The exact mass of NO5P was calculated to be 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).

[0634] Example 11: Synthesis of TCEP-8

[0635]

[0636] TCEP-8 was synthesized in Synthesis Procedure B, where (benzylamine, Adamas) was used as the amine reagent to obtain TCEP-8 as a white solid (85.6 mg, yield: 50.5%). MS [MH]- =338.23, C 16 H 22 The exact mass of NO5P was calculated to be 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).

[0637] Example 12: Synthesis of TCEP-9

[0638]

[0639] TCEP-9 was synthesized in Synthesis Procedure A, where (4-aminobenzene-1,2-diol, Bidepharm) was used as an amine reagent to obtain TCEP-9 (63.8 mg, 35.7%) as a white solid. MS [MH] - =356.20, C 15 H 20 The exact mass of NO7P was calculated to be 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).

[0640] Example 13: Synthesis of TCEP-10

[0641]

[0642] TCEP-10 was synthesized in Synthesis Procedure A, where (5-amino-2-hydroxybenzoic acid, Bidepharm) was used as the amine reagent to obtain TCEP-10 (53.7 mg, 27.9%) as a white solid. MS [MH] - =384.20, C 16 H 20 The exact mass of NO8P was calculated to be 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).

[0643] Example 14: Synthesis of TCEP-11

[0644]

[0645] TCEP-11 was synthesized in the synthetic procedure B, wherein (bis(pyridin-2-ylmethyl)amine, Shanghai Jizhi Biochemical Technology Co., Ltd.) was used as an amine reagent to obtain TCEP-11 (10.5 mg, 4.9%) as a brown solid. MS [M+H] + =432.24, C 21 H 26 The exact mass of N3O5P was calculated to be 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).

[0646] Example 15: Synthesis of TCEP-12

[0647]

[0648]

[0649] TCEP-12 was synthesized in Synthesis Procedure A, where (5-amino-8-hydroxyquinoline, Bidepharm) was used as the amine reagent to obtain TCEP-12 (33.2 mg, 16.9%) as a white solid. MS [MH] - =391.24, C 18 H 21 The exact mass calculated for N2O6P is 392.11. 1 H 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).

[0650] Example 16: Synthesis of TCEP-15

[0651]

[0652] TCEP-15 was synthesized in Synthesis Procedure B, where (bis(pyridin-2-yl)methylamine, Bidepharm) was the amine reagent to give TCEP-15 (21.7 mg, 10.4%) as a white solid. MS [M+H] + =418.26, C 20 H 24 The exact mass of N3O5P was calculated to be 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).

[0653] Example 17: Synthesis of TCEP-18

[0654]

[0655] 1.TCEP-18-int1

[0656] Phenylphosphine (110mg, 1.0mmol, Adamas) was dissolved in acetonitrile (5mL, degassed) in a round-bottom flask dried over a straight flame in N2(g). Potassium hydroxide (10N, 10μL) was added to the mixture, and the resulting solution was cooled to 0°C. Tert-butyl acrylate (0.44mL, 3.0mmol, Adamas) was added. After complete addition of tert-butyl acrylate, the reactant was heated to 50°C and stirred for 8h. The reaction mixture was dissolved in EtOAc (10mL), then washed with brine (2×5mL). The organic matter was dried with Na2SO4, filtered and concentrated. The residue was purified by a flash tower (EtOAc / petroleum ether=0-20% (v / v)) to obtain the product TCEP-18-int1 (254mg, 69.4%) as a transparent liquid.

[0657] 2.TCEP-18

[0658] Under N2 atmosphere, a solution of TCEP-18-int1 (254 mg, 0.69 mmol) in HCl / 1,4-dioxane (4M, Adamas) was stirred at room temperature for 2 h. LCMS showed that the reaction was complete, and the mixture was concentrated to remove 1,4-dioxane. The resulting residue was dissolved in water and freeze-dried to give TCEP-18 (152.7 mg, 88.2%) as a white solid. MS [MH] - =253.19, C 12 H 15The exact mass of O4P was calculated to be 254.07. 1 H NMR (400MHz, DMSO-d6): δ7.74 (dd, J=10.9, 7.3Hz, 2H), 7.62-7.49 (m, 3H), 2.46-2.35 (m, 2H), 2.33-2.00 (m, 6H).

[0659] Example 18: Synthesis of TCEP-19

[0660]

[0661] Under N2 atmosphere, HATU (380 mg, 1.0 mmol, 2.5 eq.) was added to a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 eq.) in DMF (3 mL) at 0°C, followed by DIPEA (174 μL, 1.0 mmol, 2.5 eq.). The mixture was stirred for 30 min, and glycine tert-butyl ester (1 mmol, Adamas) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to obtain the claimed 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 showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in 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 exact mass of N3O8P is calculated to be 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).

[0662] Example 19: Synthesis of TCEP-20

[0663]

[0664] Under N2 atmosphere, HATU (380 mg, 1.0 mmol, 2.5 equiv) was added to a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 equiv) in DMF (3 mL) at 0°C, followed by DIPEA (174 μL, 1.0 mmol, 2.5 equiv). The mixture was stirred for 30 min and sodium 3-aminopropane-1-sulfonate (1 mmol, Adamas) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to obtain the claimed 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 showed that the reaction was complete. The mixture was concentrated and the residue was dissolved 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 exact mass of PS2 was calculated to be 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).

[0665] Example 20: Synthesis of TCEP-21

[0666]

[0667] Under N2 atmosphere, HATU (380 mg, 1.0 mmol, 2.5 equiv) was added to a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 equiv) in DMF (3 mL) at 0°C, followed by DIPEA (174 μL, 1.0 mmol, 2.5 equiv). The mixture was stirred for 30 min and diethanolamine (1 mmol, Bidepharm) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to obtain the claimed 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 showed that the reaction was complete. The mixture was concentrated and the residue was dissolved in distilled water (10 mL) and washed with EtOAc (2*5 mL). The aqueous layer was lyophilized to give TCEP-20 (13.8 mg, yield: 7.8) as a white solid. MS[M+H]+ =440.27, C 17 H 34 The exact mass of N3O8P was calculated to be 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).

[0668] Example 21: Synthesis of TCEP-23

[0669]

[0670] 1.TCEP-23-int1

[0671] To a solution of 2-(aminooxy)tetrahydro-2H-pyran (1.17 g, 10 mmol, 2.0 equiv., Bidepharm) in DMF (15 mL) was added DIPEA (3.5 mL, 20 mmol, 4 equiv.) followed by 2-(bromomethyl)pyridine hydrobromide (1.3 g, 5.0 mmol, 1.0 equiv., 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*3). The organic layer was washed with brine (30 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by flash column to give TCEP-23-int1 (800 mg, 80%) as a colorless oil.

[0672] 2.TCEP-23

[0673] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and TCEP-23-int1 (100 mg, 0.5 mmol, 1.0 eq) was added. The reactants were 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 showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved 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 H21 The exact mass calculated for N2O6P is 356.31. 1 H-NMR (400 MHz, deuterium oxide): δ8.73 (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).

[0674] Example 22: Synthesis of TCEP-24

[0675]

[0676] TCEP-24 was synthesized in the synthetic procedure A, wherein (4-aminobenzoic acid, Bidepharm) was used as the amine reagent to obtain TCEP-24 (21.5 mg, yield: 10.4%) as a white solid. MS [MH] - =412.22, C 17 H 20 The exact mass of NO9P was calculated to be 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).

[0677] Example 23: Synthesis of TCEP-25

[0678]

[0679] 1.TCEP-25-int1

[0680] Pd / C (150 mg) and two drops of AcOH were added to a MeOH (25 mL) solution of 2-pyridinecarboxaldehyde (1.0 g, 10 mmol, 1.0 equivalents of Adamas) and glycine tert-butyl ester (1.3 g, 10.0 mmol, 1.0 equivalents). The mixture was degassed 3 times and purged with H2, then stirred at room temperature for 16 h under H2 atmosphere. The reaction mixture was filtered through a Celite pad, and the filtrate was concentrated, then purified with a flash tower to give TCEP-25-int1 (1.6 g, 72.0%) as a yellow oil.

[0681] 2.TCEP-25

[0682] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and TCEP-25-int1 (111 mg, 0.5 mmol, 1.0 eq) was added. The reactants were 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 showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in distilled water and lyophilized to give TCEP-25 (51.3 mg, 17.2%) as a white solid. MS [M+H] + =399.25, C 17 H 23 The exact mass of N2O7P was calculated to be 398.12. 1 H NMR (400 MHz, deuterium oxide): δ8.60 (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).

[0683] Example 24: Synthesis of TCEP-26

[0684]

[0685] 1.TCEP-26-int1

[0686] Under N2 atmosphere, HATU (4.3 mg, 11.0 mmol, 2.2 equiv) was added to a solution of Fmoc-iminodiacetoacetic acid (1.8 g, 5.0 mmol, 1.0 equiv, Bidepharm) in DMF (30 mL), followed by DIPEA (2.0 mmol, 4.0 equiv). The mixture was stirred for 30 min, and tritylhydroxylamine (3.0 mg, 11.0 mmol, 2.2 equiv) was added. The reactant 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.

[0687] 2.TCEP-26-int2

[0688] DBU (2 mL) was added to a DMF (10 mL) solution of TCEP-26-int1 (2.0 g, 2.3 mmol, 1.0 equivalent). The mixture was stirred at room temperature for 1 h, then poured into ice water (100 mL), extracted with EtOAc (50 mL * 3). The organic layers were combined, washed with brine, dried over Na2SO4 and filtered. The filtrate was concentrated and purified by a flash tower (EtOAc / petroleum ether = 0-50% (v / v) to obtain TCEP-26-int2 (1.2 g, 80%).

[0689] 3.TCEP-26

[0690] The compound was synthesized in the synthetic procedure A-1, wherein TCEP-26-int2 was an amine reagent, to obtain TCEP-26 (31.5 mg, yield: 21.3%) as a white solid. MS [M+H] + =396.17, C 13 H 22 The exact mass of N3O9P is calculated to be 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).

[0691] Example 25: Synthesis of TCEP-28

[0692]

[0693] 1.TCEP-28-int1

[0694] To a solution of o-tritylhydroxylamine (1.4 g, 5.0 mmol, 1.0 equiv) in DMF (15 mL) was added DIPEA (1.7 mL, 10 mmol, 2 equiv), followed by tert-butyl bromoacetate (1.0 g, 5.0 mmol, 1.0 equiv, 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*3). The organic layer was washed with brine (30 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by flash tower to give TCEP-28-int1 (1.4 g, 70%) as a white solid.

[0695] 2.TCEP-28-int2

[0696] To a solution of TCEP-28-int1 (1.4 g, 3.6 mmol, 1.0 equiv) 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 to give TCEP-28-int2 (380 mg, 71.8%) as a colorless oil.

[0697] 3.TCEP-28

[0698] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq.) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq.) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq.). The mixture was stirred for 30 min, and TCEP-28-int2 (73.5 mg, 0.5 mmol, 1.0 eq.) was added. The reactants were 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 showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in distilled water and lyophilized to give TCEP-28 (43.6 mg, 27.1%) as a white solid. MS[MH]-=322.16, C 11 H 18 The exact mass of NO8P was calculated to be 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).

[0699] Example 26: Synthesis of TCEPA

[0700]

[0701] TCEPA was synthesized in Synthesis Procedure A-1, where 4-methoxybenzylamine was used as the amine reagent to give TCEPA (13.5 mg, 11%). MS [M+H] + =250.18,C9H 16 The exact mass of NO5P was calculated to be 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).

[0702] Example 27: Synthesis of TCEP-30

[0703]

[0704] TCEP-30 was synthesized in the synthetic procedure A-1, wherein [L-tyrosine tert-butyl ester, Adamas] was used as the amine reagent to obtain TCEP-30 (25.3 mg, 12.25%) as a white solid. MS [M+H] + =414.23, C 18 H 24 The exact mass of NO8P was calculated to be 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).

[0705] Example 28: Synthesis of TCEP-31

[0706]

[0707] TCEP-31 was synthesized in Synthesis Procedure A, where (DL-3-(4-fluorophenyl)alanine, Bidepharm) was used as the amine reagent to obtain TCEP-31 (27.1 mg, 13.10%) as a white solid. MS [M+H] + =416.01, C 18 H 23 The exact mass of NO7P was calculated to be 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).

[0708] Example 29: Synthesis of TCEP-32

[0709]

[0710] TCEP-32 was synthesized in Synthesis Procedure A, where (DL-4-cyanophenylalanine, Bidepharm) was used as the amine reagent to obtain TCEP-32 (18.5 mg, 8.76%) as a white solid. MS [M+H] + =423.24, C 19 H 23The exact mass of N2O7P was calculated to be 422.12. 1 H 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).

[0711] Example 30: Synthesis of TCEP-33

[0712]

[0713] TCEP-33 was synthesized in Synthesis Method A, where (DL-4-nitrophenylalanine, Bidepharm) was used as an amine reagent to obtain TCEP-33 (20.7 mg, 9.37%) as a white solid. MS [M+H] + =443.24, C 19 H 23 The exact mass of N2O9P was calculated to be 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).

[0714] Example 31: Synthesis of TCEP-34

[0715]

[0716] TCEP-34 was synthesized in Synthesis Procedure A, where (N-benzylhydroxylamine hydrochloride, Bidepharm) was used as the amine reagent to obtain TCEP-34 (15.7 mg, 8.85%) as a white solid. MS [M+H] + =356.05, C 16 H 22 The exact mass of NO6P was calculated to be 355.12. 1H 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).

[0717] Example 32: Synthesis of TCEP-35

[0718]

[0719] Synthesis TCEP-35 was synthesized in Synthesis Procedure A, where (N-phenylhydroxylamine, Bidepharm) was used as the amine reagent to obtain TCEP-35 (17.1 mg, 10.0%) as a white solid. MS [M+H] + =341.97, C 15 The exact mass calculated for 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).

[0720] Example 33: Synthesis of TCEP-37

[0721]

[0722] 1.TCEP-37-int1

[0723] To a solution of 2,4-dimethoxybenzaldehyde (1.66 g, 10.0 mmol, 1.0 equiv., Adamas) in MeOH (25 mL) was added o-methylhydroxylamine hydrochloride (1.66 g, 20.0 mmol, 2.0 equiv., Bidepharm). The resulting mixture was stirred at room temperature for 16 h, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in AcOH (20 mL), NaBH3CN was added and stirred at room temperature for 5 h. LCMS showed that 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 organic layers were combined, dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and purified by flash column (EtOAc / petroleum ether=0-50%) to obtain the product TCEP-37-int1 (N-(2,4-dimethoxybenzyl)-O-methylhydroxylamine, 1.5 g, 76.1%, colorless oil).

[0724] 2.TCEP-37

[0725] TCEP-37 was synthesized in the synthesis procedure A-1, wherein TCEP-73-int1 was used as an amine reagent to obtain TCEP-37 (12.8 mg, 9.14%) as a white solid. MS [M+H] + =280.18, C 10 H 18 The exact mass of NO6P was calculated to be 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).

[0726] Examples 34 to 66: Preparation of D2-type ADC

[0727] The ADC was prepared using a one-pot reaction:

[0728] (1) ZnCl2 (0.24 mM) and a reducing agent (0.02 mM) were added to a monoclonal antibody solution (0.012 mM, dissolved in MES buffer, pH 6.7, 20 mM), and the reaction mixture was placed at 4°C for 4 h, 8 h, and 12 h, respectively;

[0729] (2) Add EDTA (0.6 mM) to capture Zn 2+ ;

[0730] (3) MC-VC-PAB-MMAE (0.06 mM) dissolved in DMA was introduced and the reaction was continued at 24 °C for 1 h;

[0731] (4) adding cysteine ​​(0.08 mM) to consume excess MC-VC-PAB-MMAE;

[0732] (5) Purify the reaction mixture using a desalting column.

[0733] The monoclonal antibody and reducing agent used, the molar ratio of the antibody and the reducing agent, and the incubation time in step (1) are as follows: Meanwhile, in Example 40, the buffer system is a MOPS buffer, and the pH value is 7.4.

[0734]

[0735]

[0736] Examples 67 to 81 and Comparative Example 10: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates with different molar ratios of ZnCl2 to reducing agent The preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates is similar to the preparation of D2-type ADC of Example 34, but the dosage of ZnCl2 is adjusted in step (1). The dosage of ZnCl2 and the molar ratio of ZnCl2 to reducing agent are as follows:

[0737] serial number Serial number <![CDATA[ZnCl2(mM)]]> <![CDATA[ZnCl2 / TCEP-NO (molar ratio)]]> E67 632 0.008 0.4 E68 633 0.02 1 E69 599 0.04 2 E70 600 0.08 4 E71 601 0.12 6 E72 602 0.16 8 E73 603 0.2 10 E74 605 0.28 14 E75 606 0.32 16 E76 729 0.48 24 E77 / 0.6 30 E78 733 1.2 60 E79 / 1.4 70 E80 / 2.5 125 E81 / 4 200 C10 / 5 250

[0738] “E” is an abbreviation of Embodiment, and “C” is an abbreviation of Comparative Example in Application.

[0739] Examples 82 to 85: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates at different molar ratios of antibody to reducing agent The preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates is similar to the preparation of D2-type ADC in Example 34, but the dosage of the antibody in step (1) or the incubation time in step (1) is adjusted. The dosage of the antibody and the molar ratio of the antibody to the reducing agent are as follows:

[0740] Example Antibody (mM) Antibody / TCEP-NO (molar ratio) Incubation time in step (1) (h) 82 0.011 1:1.8 4 83 0.01 1:2.0 1 84 0.008 1:2.5 1 85 0.0067 1:3.0 1

[0741] Examples 86 to 101: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate using different buffers The preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate was similar to the preparation of ADC of Example 34, but the following buffer adjustments were made:

[0742] Example Serial number Buffer system pH 86 561 Bis-Tris buffer 6.7 87 562 PIPES Buffer 6.7 88 563 MOPS Buffer 6.7 89 564 BES buffer 6.7 90 566 HEPES buffer 6.7 91 567 ADA Buffer 6.7 92 568 PB 6.7 93 626 DIPSO Buffer 7.4 94 627 MOBS Buffer 7.4 95 628 MOPSO Buffer 7.4 96 629 TES Buffer 7.4 97 630 ACES Buffer 7.4 98 631 TAPSO Buffer 7.4 99 / MES buffer 5.8 100 / MES buffer 6.4 101 / MES buffer 6.7

[0743] All buffers are commercially available from Macklin.

[0744] Examples 102 to 113: In step (1), the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is prepared at different incubation temperatures or incubation times. The preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to the preparation of the ADC of Example 34, but the following incubation temperature or incubation time is not adjusted in (1):

[0745] Example Incubation temperature (℃) Incubation time (h) 102 15 4 103 24 4 104 37 4 105 4 0.25 106 4 0.5 106 4 0.75 108 4 1 109 4 2 110 4 3 111 4 4 112 4 5 113 4 6

[0746] Example 114: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugate by Engineering Antibodies

[0747] The preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate was similar to the preparation of the ADC of Example 34, but an engineered antibody was used.

[0748] The engineered antibody is a mutant of trastuzumab with a cysteine ​​to serine mutation replacing the disulfide bond between the heavy and light chains (ordered from Biointron).

[0749] Example 115: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (D1-type ADC)

[0750] 1. Synthesis of dibromomaleimide-PEG4-N3

[0751]

[0752] 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 minutes. DCM (10 mL) was then added, the organic phase was washed with H2O, dried over MgSO4, and the solvent was removed in vacuo to give the title compound 1 (139 mg, 0.4 mmol, 80%).

[0753] A solution of azido-PEG4-amine (105 mg, 0.4 mmol, 1 eq., Xi'an Kangfuno Biotechnology 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).

[0754] After 30 minutes, 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 MgSO4. After vacuum concentration, column chromatography purification (100% EtOAc as mobile phase) was performed to give dibromomaleimide-PEG4-N3 (title product 2) as a light yellow oil (150 mg, 0.3 mmol, 75%).

[0755] 2. Preparation of Trastuzumab-[Maleimide-PEG4-N3-DBCO-Cy3]1

[0756] (1) Incubating 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 ZnCl2 (0.24 mM) at a temperature of 4 °C and a time of 4 h;

[0757] (2) introducing EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group obtained in step (1), the reaction temperature is 24 ° C and the reaction time is 3 h, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[0758] (3) Trastuzumab-maleimide-PEG4-N3 and DBCO-Cy3 (0.02 mM) were incubated in MES (20 mM, pH 6.7) at 25 °C for 8 h, and then trastuzumab-maleimide-PEG4-N3-DBCO-Cy3 was recovered using a desalting column.

[0759] Example 116: Preparation of trastuzumab-[maleimide]1[MC-GGFG-DXd]6 (D0+D6 type ADC)

[0760] (1) incubating 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 ZnCl2 (0.24 mM) at a temperature of 4°C and a time of 4 h;

[0761] (2) introducing EDTA (0.6 mM) and dibromomaleimide (0.013 mM) to react with the reduced thiol group from step (1), the reaction temperature was 24 ° C and the reaction time was 3 h, and then the product was recovered using a desalting column to obtain trastuzumab-[maleimide] 1;

[0762] (3) In MES (20 mM, pH 6.7), trastuzumab-[maleimide] 1 and TCEP (0.08 mM) were introduced at a reaction temperature of 25 °C and a reaction time of 12 h;

[0763] (4) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (3), and the reaction mixture was placed at 24° C. for 1 h, and then trastuzumab-[maleimide] 1[MC-GGFG-DXd] 6 was recovered using a desalting column.

[0764] Example 117: Preparation of trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 (D2+D6 type ADC)

[0765] (1) incubating the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) of Example 1 in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl2 (0.24 mM) at a temperature of 4°C and a time of 4 h;

[0766] (2) introducing EDTA (0.6 mM) and excess MC-MMAF (0.06 mM) to react with the reduced thiol group from step (1), the reaction temperature is 24 ° C and the reaction time is 1 h, and then using a desalting column to recover the product to obtain trastuzumab [MC-MMAF] 2;

[0767] (3) In MES (20 mM, pH 6.7), trastuzumab-[MC-MMAF]2 and TCEP (0.08 mM) were introduced at a reaction temperature of 25 °C and a reaction time of 8 h;

[0768] (4) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (3), and the reaction mixture was placed at 24°C for 1 h, and then the resulting trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 was recovered using a desalting column.

[0769] Examples 118 to 119: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 (D2+D2 type ADC)

[0770] (1) incubating 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 ZnCl2 (0.12 mM) at a temperature of 4°C and a time of 4 h;

[0771] (2) introducing EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.048 mM) to react with the reduced thiol group from step (1), the reaction temperature was 24 ° C and the reaction time was 1 h, and then the product was recovered using a desalting column to obtain trastuzumab-[MC-VC-PAB-MMAE]2;

[0772] (3) incubating ZnCl2 (1.2 mM), the second reducing agent TCEP-3 (0.0144 mM) / TCEP-6 (0.0216 mM) and the product from step (2) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 4 h;

[0773] (4) Introducing EDTA (3 mM) to capture Zn 2+, and introduce MC-GGFG-DXd (0.1 mM) to react with the reduced thiol groups from step (3), the reaction temperature is 24° C. and the reaction time is 1 h;

[0774] (5) Purify the reaction mixture using a desalting column.

[0775] Example 120: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 (D1+D2 type ADC)

[0776] (1) Incubating 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 ZnCl2 (0.24 mM) at a temperature of 4 °C and a time of 4 h;

[0777] (2) introducing EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group obtained in step (1), the reaction temperature is 25° C. and the reaction time is 1 h, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[0778] (3) Incubate trastuzumab-[maleimide-PEG4-N3]1 from step (2) and DBCO-Cy3 (0.02 mM) in MES (20 mM, pH 6.7) at a reaction temperature of 25°C and a reaction time of 6 h, and then recover trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 using a desalting column.

[0779] (4) incubating ZnCl2 (1.2 mM), a second reducing agent TCEP-3 (0.0144 mM) and the product from step (3) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 4 h;

[0780] (5) Introducing EDTA (3 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 25° C. and the reaction time was 1 h;

[0781] (6) Purify the reaction mixture using a desalting column.

[0782] Examples 121 to 122: Preparation of Trastuzumab-[Maleimide]1[MC-VC-PAB-MMAE]4 (D0+D4 ADC)

[0783] (1) incubating 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 ZnCl2 (0.24 mM) at a temperature of 4°C and a time of 4 h;

[0784] (2) introducing EDTA (0.6 mM) and dibromomaleimide (0.013 mM) to react with the reduced thiol group from step (1), the reaction temperature is 24 ° C and the reaction time is 3 h, and then using a desalting column to recover the product to obtain trastuzumab-maleimide;

[0785] (3) incubating ZnCl2 (0.36 mM), the second reducing agent TCEP-3 (0.036 mM) / TCEP-6 (0.048 mM) and the product from step (2) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 16 h;

[0786] (4) Introducing EDTA (0.6 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 24° C. and the reaction time was 1 h;

[0787] (5) Purify the reaction mixture using a desalting column.

[0788] Examples 123 to 124: Preparation of trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 (D2+D4 type ADC)

[0789] (1) Incubating 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 ZnCl2 (0.24 mM) at a temperature of 4 °C and a time of 4 h;

[0790] (2) introducing EDTA (0.6 mM) and MC-GGFG-DXd (0.072 mM) to react with the reduced thiol group from step (1), the reaction temperature was 24 ° C and the reaction time was 1 h, and then the product was recovered using a desalting column to obtain trastuzumab-[MC-GGFG-DXd]2;

[0791] (3) incubating ZnCl2 (0.36 mM), the second reducing agent TCEP-3 (0.036 mM) / TCEP-6 (0.048 mM) and the product from step (2) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 16 h;

[0792] (4) Introducing EDTA (0.6 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 24° C. and the reaction time was 1 h;

[0793] (5) Purify the reaction mixture using a desalting column.

[0794] Example 125: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 (D1+D4 type ADC)

[0795] (1) Incubating 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 ZnCl2 (0.24 mM) at a temperature of 4 °C and a time of 4 h;

[0796] (2) introducing EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group from step (1), the reaction temperature is 25 ° C and the reaction time is 6 h, and then the product is recovered using a desalting column to obtain trastuzumab-maleimide-PEG4-N3;

[0797] (3) Trastuzumab-maleimide-PEG4-N3 and DBCO-Cy3 (0.02 mM) were incubated in MES (20 mM, pH 6.7) at 25 °C for 6 h, and then trastuzumab-maleimide-PEG4-N3-DBCO-Cy3 was recovered using a desalting column.

[0798] (4) incubating ZnCl2 (0.36 mM), a second reducing agent TCEP-3 (0.0408 mM) and the product from step (3) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 16 h;

[0799] (5) Introducing EDTA (0.6 mM) to capture Zn 2+, and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 24° C. and the reaction time was 1 h;

[0800] (6) Purify the reaction mixture using a desalting column.

[0801] Comparative Examples 1 to 9: Preparation of ADC without using transition metal ions

[0802] The preparation of D2-type ADC is as follows:

[0803] (1) TCEP-NO, TCEP-3NO or TCEP-CO (0.02 mM) was added to a monoclonal antibody solution (0.012 mM, dissolved in MES buffer, pH 6.7, 20 mM), and the reaction mixture was placed at 4°C for 4 h, 8 h or 12 h, respectively;

[0804] (2) MC-VC-PAB-MMAE (0.06 mM) dissolved in DMA was introduced and the reaction was continued at 24 °C for 1 h;

[0805] (3) adding cysteine ​​(0.08 mM) to consume excess MC-VC-PAB-MMAE;

[0806] (4) Purify the reaction mixture using a desalting column.

[0807] The monoclonal antibodies and reducing agents used were as follows:

[0808] Comparison Examples Serial number ADC Antibody reducing agent 1 381 Trastuzumab-MC-VC-PAB-MMAE Trastuzumab TCEP-NO 2 683 Gosartuzumab-MC-VC-PAB-MMAE Gosartumomab TCEP-NO 3 684 Mabelantuzumab-MC-VC-PAB-MMAE Mabelantuzumab TCEP-NO 4 693 Trastuzumab-MC-VC-PAB-MMAE Trastuzumab TCEP-3NO 5 677 Gosartuzumab-MC-VC-PAB-MMAE Gosartumomab TCEP-3NO 6 679 Mabelantuzumab-MC-VC-PAB-MMAE Mabelantuzumab TCEP-3NO 7 378 Trastuzumab-MC-VC-PAB-MMAE Trastuzumab TCEP-CO 8 673 Gosartuzumab-MC-VC-PAB-MMAE Gosartumomab TCEP-CO 9 675 Mabelantuzumab-MC-VC-PAB-MMAE Mabelantuzumab TCEP-CO

[0809] Comparative Example 11: Preparation of ADC using TCEP

[0810] (1) TCEP (0.02 mM) was added to a trastuzumab solution (0.012 mM, in MES buffer, pH 6.7, 20 mM), and the reaction mixture was placed at 4 °C for 4 h;

[0811] (2) MC-VC-PAB-MMAE (0.06 mM) dissolved in DMA was introduced and the reaction was continued at 24 °C for 30 min;

[0812] (3) adding cysteine ​​(0.08 mM) to consume excess MC-VC-PAB-MMAE;

[0813] (4) Purify the reaction mixture using a desalting column.

[0814] Comparative Example 12: Preparation of ADC using TCEP

[0815] (1) ZnCl2 (0.24 mM) and TCEP (0.02 mM) were added to a trastuzumab solution (0.012 mM, dissolved in MES buffer, pH 6.7, 20 mM), and the reaction mixture was placed at 4 °C for 4 h;

[0816] (2) Add EDTA (0.6 mM) to capture Zn 2+ ;

[0817] (3) MC-VC-PAB-MMAE (0.06 mM) dissolved in DMA was introduced and the reaction was continued at 24 °C for 30 min;

[0818] (4) adding cysteine ​​(0.08 mM) to consume excess MC-VC-PAB-MMAE;

[0819] (5) Purify the reaction mixture using a desalting column.

[0820] Homogeneity determination

[0821] Drug / antibody ratio (DAR) and product distribution were analyzed at 30°C using HIC-HPLC (Agilent 1200) with a TSK gel butyl-NPR column (4.6 mm IDX 3.5 cm) (available from Tosoh Biosciences) at a flow rate of 0.5 mL / min. 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 flushing procedure was as follows:

[0822] Time [min] Solvent A[%] Solvent B[%] 0.00 100.0 0.0 2.00 100.0 0.0 15.00 0.0 100.0 17.00 0.0 100.0 18.00 100.0 0.0 20.00 100.0 0.0

[0823] The results of Examples 34 to 36 and Comparative Examples 1 to 3 are shown in Table 1. Figures 1 to 6 Shown in.

[0824] According to the results shown in Table 1, different D2 ratios and D4 ratios were obtained for the ADCs of Examples 34 to 36 and Comparative Examples 1 to 3 prepared by TCEP-NO, indicating that MC-VC-PAB-MMAE was successfully linked to trastuzumab, gosatuzumab or mabelantuzumab. TCEP-NO can be used as a reducing agent in antibody modification and ADC preparation. Compared with C1 to C3, the ADCs of Examples 34 to 36 prepared by TCEP-NO significantly increased the D2 ratio. This indicates that TCEP-NO has a good effect in the presence of Zn 2+ TCEP-NO has reduction selectivity in the case of TCEP-NO and can be used to prepare D2-type ADC.

[0825] Table 1

[0826] Example Attached photos D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) 34 1 4.55 91.09 4.36 0 0 35 2 5.98 84.97 9.06 0 0 36 3 1.77 89.2 9.04 0 0 C1 4 69.85 30.15 0 0 0 C2 5 58.11 37.6 4.29 0 0 C3 6 38.79 48.82 12.39 0 0

[0827] “E” is an abbreviation for “Example.” “C” is an abbreviation for Comparative Example.

[0828] The results of Examples 37 to 39 and Comparative Examples 4 to 6 are shown in Table 2, and the chromatograms are shown in Table 2. Figures 7 to 12 shown.

[0829] According to the results shown in Table 2, different D2 ratios and D4 ratios were obtained for the ADCs of Examples 37 to 39 and C4 to C6 prepared by TCEP-3NO, indicating that MC-VC-PAB-MMAE was successfully linked to trastuzumab, gosatuzumab or mabelantuzumab. TCEP-3NO can be used as a reducing agent in antibody modification and ADC preparation. Compared with C4 to C6, the ADCs of Examples 37 to 39 prepared by TCEP-3NO significantly increased the D2 ratio. This indicates that TCEP-3NO has a good effect in the presence of Zn 2+ TCEP-3NO has reduction selectivity in the case of TCEP-3NO and can be used to prepare D2-type ADC.

[0830] Table 2

[0831] Example Attached photos D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) 37 7 2.87 91.47 5.67 0 0 38 8 2.85 87.86 9.29 0 0 39 9 1.52 87.62 10.86 0 0 C4 10 52.13 42.34 5.53 0 0 C5 11 45.05 45.92 9.04 0 0 C6 12 38.56 49.01 12.43 0 0

[0832] The results of Examples 40 to 42 and Comparative Examples 7 to 9 are shown in Table 3, and the chromatograms are shown in Table 3. Figures 13 to 17 shown.

[0833] According to the results shown in Table 3, different D2 ratios and D4 ratios were obtained for the ADCs of Examples 40 to 42 and C7 to C9 prepared by TCEP-CO, indicating that MC-VC-PAB-MMAE was successfully linked to trastuzumab, gosatuzumab or mabelantuzumab. TCEP-CO can be used as a reducing agent in antibody modification and ADC preparation. Compared with C7 to C9, the ADCs of Examples 40 to 42 prepared by TCEP-CO significantly increased the D2 ratio. This indicates that TCEP-CO has a good effect in the presence of Zn 2+ TCEP-CO has reduction selectivity in the case of TCEP-CO and can be used to prepare D2-type ADC.

[0834] Table 3

[0835] Example Attached photos D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) 40 13 4.87 90.51 4.63 0 0 41 14 9.91 81.26 8.83 0 0 42 15 3.94 85.44 10.62 0 0 C7 - - - - - - C8 16 42.92 48.2 8.88 0 0 C9 17 42.54 50 7.46 0 0

[0836] For the coupling process using the same steps but without adding transition metal ions in step (a) as a negative control (see Tables 1 to 3), the present invention successfully demonstrated that the combination of transition metal ions and a novel reducing agent can increase the D2 level in ADC. In addition, it was demonstrated that the ADC product generated by this new process has a high Fc and / or Fab preference. By using the process of the present invention to produce antibody drug conjugates, the uniformity of the antibody drug conjugates is greatly improved.

[0837] According to Table 4 and Figures 18 to 23 The results of Examples 43 to 66 and Comparative Examples 11 to 12 shown in FIG. 1 and FIG. 2 are similar to those of the example 43 to 66 and the comparative examples 11 to 12 using the Zn-free 2+ Compared with the traditional method of TCEP, the compounds of the present invention can improve the uniformity of D2-type ADC, among which TCEP-6 has the best selective reduction ability, and the D2 content is as high as 94.25%. At the same time, the selective reduction ability of TCEP-NO, TCEP-3NO, TCEP-CO, TCEP-1, TCEP-19, TCEP-20, TECP-21, TCEP-23, TCEP-24, TCEP-26, TCEP-28, TCEP-34, TCEP-35 and TCEP-37 is also very outstanding, and the D2 content is as high as 84%, 87%, or even 90% or 93%.

[0838] Table 4

[0839]

[0840] The results of Examples 67 to 81 and Comparative Example 10 are shown in Table 5, and the chromatograms are shown in Table 5. Figures 24 to 37 According to the results shown in Table 5, the content of D2 was increased by adding transition metal ions. The D2 ratio increased with the increase of Zn 2+ / TCEP-NO molar ratio increases from 0.4 to 6. After that, the D2 ratio reaches a plateau. 2+ When the molar ratio of Zn / TCEP-NO is as high as 200:1 and 250:1, the content of D2 is lower than that of Zn in the range of 2:1 to 125:1. 2+ / TCEP-NO molar ratio. This indicates that transition metal ions, especially Zn 2+ / TCEP-NO ratio plays a key role in determining the D2 ratio and reduction selectivity.

[0841] Table 5

[0842]

[0843]

[0844] The results of Examples 82 to 85 are shown in Table 6, and the chromatograms are shown in Table 6. Fig.38 According to the results shown in Table 6 and Examples 46, 49, 53 and 54, when the antibody / TCEP-NO molar ratio is 1:0.9 to 1:3.0, the D2 ADC content is as high as 55%, 60%, 70%, 75%, and even 80%, 85% or 90%. When the antibody / TCEP-NO molar ratio is 1:2 and 1:2.5, the reduction time is shortened to 1h, and the D2 content is greater than 80%.

[0845] Table 6

[0846]

[0847] The results of Examples 86 to 101 are shown in Table 7, and the chromatograms are shown in Table 7. Figures 39 to 52 As shown in the results in Table 7, different buffers significantly affect the reduction kinetics and selectivity. The buffer systems in Examples 86 to 101 help increase the content of D2-type ADC.

[0848] Table 7

[0849]

[0850] The results of Examples 102 to 113 are shown in Table 8, and the chromatograms are shown in Table 8. Figure 53 to Figure 55 As shown in Table 8, when the reduction temperature is 4°C to 37°C and the reduction time is 0.25h to 6h, the content of D2 type ADC is as high as 80%. The content of D2 increases as the reduction time of step (1) increases from 0.25h to 1h and reaches a plateau after 1h, which indicates that the reaction kinetics is very rapid.

[0851] Table 8

[0852]

[0853] The results of Example 114 are shown in Table 9, and the chromatogram is shown in Fig.56 As shown. According to the structure shown in Table 9, the content of D2 prepared by engineering antibodies is as high as 96%. These results show that this method is also applicable to antibodies with simple mutations and may have better reduction selectivity in some mutant antibodies.

[0854] Table 9

[0855]

[0856] As shown in Table 10 and Fig.57 As shown in Table 11 and Fig.58 As shown in Table 12 and Fig.59As shown, the results confirm that the content of D2+D6 type ADC is generally as high as 81.31%. These results show that the method of the present invention can perform site-specific modification on antibodies and prepare different types of ADCs while improving uniformity.

[0857] Table 10

[0858] Example Attached photos ADC Type D0 (wt%) D1 (wt%) D2 (wt%) 115 57 D1 11.0 83.61 5.39

[0859] Table 11

[0860]

[0861] Table 12

[0862] Example Attached photos ADC Type D2+D4 / D4+D2(%) D2+D6(%) D4+D4 / D0+D8(%) 117 59 D2+D6 7.49 81.31 11.2

[0863] In step (3) of Examples 118 to 119, one of the interchain disulfide bonds in the D2-type ADC is reduced. Fig.60 As shown, the results indicate that the D2+D2 ADC content is typically as high as 68% or 70%, which indicates that the process of this method is beneficial to the site-specific modification of D2+D2 antibodies and can improve uniformity.

[0864] Table 13

[0865]

[0866] As shown in Table 14 and Fig.61 As shown, the results indicate that the D1+D2 ADC content is typically as high as 80% or 83%, which indicates that the process of this method is beneficial to the site-specific modification of D1+D2 antibodies and can improve uniformity.

[0867] Table 14

[0868] serial number Attached photos D1(％) D2(％) D1+D2(%) D4(％) D2+D4(%) E120 61 6.87 3.04 83.21 3.06 3.82

[0869] In step (3) of Examples 121 to 122, two of the interchain disulfide bonds in the D2-type ADC are reduced. Fig.62 As shown, the results indicate that the D0+D4 ADC content is usually as high as 55% or 61%, which indicates that the process of this method is beneficial to the site-specific modification of the D0+D4 antibody and can improve the uniformity.

[0870] Table 15

[0871] serial number Attached photos reducing agent D0(％) / / E121-Step (2) 62A TCEP-NO 100 / / serial number Attached photos reducing agent D0+D2(%) D0+D4(%) D0+D6(%) E121-Step (5) 62B TCEP-3 18.96 61.27 19.77 E122-Step (5) 62C TCEP-6 21.23 61.85 16.92

[0872] As shown in Table 16 and Fig.63As shown, the results indicate that the D2+D4 ADC content is typically as high as 70%, 75%, or even 78% or 80%, which indicates that the process of this method is beneficial to the site-specific modification of D2+D4 antibodies and can improve uniformity.

[0873] Table 16

[0874]

[0875] As shown in Table 17 and Fig.64 As shown, the results indicate that the D1+D4 ADC content is usually as high as 60%, 65%, or even 70%, which indicates that the process of this method is beneficial to the site-specific modification of D1+D4 antibodies and can improve uniformity.

[0876] Table 17

[0877]

[0878]

[0879] Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are presently unforeseeable or possibly unforeseeable may occur to applicants or other persons skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. A compound having the following formula (I): or a salt, solvate or stereoisomer thereof, characterized in that X, Y and Z are each independently covalently linked to the phosphorus atom via a PC bond, wherein the PC bond is PC(sp 3 ) or PC(sp 2 ); X is formula (II): L1 is selected from the group consisting of: -CH(R1)-, -C(CH3)(R1)-, -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, aryl groups each independently selectively substituted by a group containing at least one coordinating atom selected from N, O and S, and heteroaryl groups each independently selectively substituted by a group containing at least one coordinating atom selected from O and S; R1, R2 and R3 are each independently H, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, C1-C5 hydroxylaminealkyl, C1-C5 N-hydroxyamidealkyl, aryl or heteroaryl; or R2 or R3 and L2 form a 5-6 membered optionally substituted ring; A is optionally present and is -C(O)-, or -C(O)J-; J is an organic group comprising an amino group or an imino group and a carbonyl group, wherein the amino group or the imino group forms an amide group with -C(O), and the carboxyl group is optionally covalently linked to L2; L2 is optionally present, L2 serves as a transition metal chelator motif and is -N(R4)(R5) or hydroxyl; R4 and R5 are each independently hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring, and R4 or R5 and R2 or R3 form a 5-6 membered optionally substituted ring; R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4; R4 and R5 are not hydroxyl groups at the same time; Y is the same as X, Z is the same as X, or Y and Z are each independently a 5-6 membered optionally substituted saturated heterocyclic group, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, an aryl group, a C1-C5 carboxyalkyl group, a 5-6 membered optionally substituted cycloalkyl group, or -C(O)Q is an ester group, an imide group or an amide group, X, Y and Z are not -CH2CH2C(O)OH at the same time.

2. The compound according to claim 1, characterized in that L1 is -CH(R1)CH(R2)-, R1 and R2 are each independently H, methyl, isopropyl, hydroxymethyl, hydroxyethyl, carboxymethyl, carboxyethyl, N-hydroxyacetamido, phenyl, 2-pyridyl, 4-pyridyl or 4-imidazolyl, or R2 and L2 form a 5-6 membered optionally substituted ring.

3. The compound according to claim 1 or 2, characterized in that L1 is -CH(R1)CH(R2)-; R1 is H, and R2 and R4 of L2 form a 5-6 membered optionally substituted ring; L2 is -N(R4)(R5), and R5 is hydroxyl.

4. The compound according to claim 1 or 2, characterized in that L1 is an optionally substituted phenyl group linked to A in the ortho, meta or para position, A is -C(O)-; L2 is -N(R4)(R5) or hydroxyl; R4 is hydrogen and R5 is hydroxyl.

5. The compound according to claim 4, characterized in that L1 is a phenyl group substituted by a hydroxyl group or a carboxyl group at the ortho position or the meta position, and the phenyl group is connected to A at the para position.

6. The compound according to claim 1 or 2, characterized in that L1 is a phenyl group which is selectively substituted at the ortho, meta or para position by a hydroxyl group, a halogen group, a carboxyl group, a sulfonyl group, an amino group, a methoxy group or an ethoxy group, A and L2 do not exist.

7. The compound according to claim 1 or 2, characterized in that L1 is optionally substituted 4-pyridyl or optionally substituted 4-quinolyl, and A and L2 are absent.

8. The compound according to claim 7, characterized in that L1 is 9. The compound according to claim 1 or 2, characterized in that L1 is -CH(R1)CH(R2)-, R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamide, R2 is H.

10. The compound according to claim 1 or 2, characterized in that L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl.

11. The compound according to claim 1 or 2, characterized in that L1 is -CH(R1)CH(R2)-; R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamide; R2 is H; A is -C(O)-; L2 is -N(R4)(R5); R4 is hydrogen, and R5 is hydroxyl.

12. The compound according to claim 1 or 2, characterized in that L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-; L2 is -N(R4)(R5); R4 is hydrogen, an optionally substituted 5-6 membered saturated heterocyclic group, R5 is hydroxyl.

13. The compound according to claim 12, characterized in that R4 is 14. The compound according to claim 1 or 2, characterized in that L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-; L2 is -N(R4)(R5); R4 and R5 form a 5-6 membered optionally substituted ring.

15. The compound according to claim 14, characterized in that L2 is 16. The compound according to claim 1, characterized in that L1 is -CH(R1)CH(R2)-, R1 and R2 are each independently H.

17. The compound according to claim 1 or 16, characterized in that L2 is -N(R4)(R5); R4 is hydrogen, C1-C5 alkyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted aryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring; R5 is a hydroxyl group, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

18. The compound according to claim 1 or 17, characterized in that R4 is hydrogen, methyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), a 5-6 membered saturated heterocyclic group containing a heteroatom N or O, a benzyl group, a benzyl group substituted with a hydroxyl group on the phenyl ring, a phenyl group optionally substituted with a hydroxyl group, a halogen group or a carboxyl group, a heteroaralkyl group containing a heteroatom N, or R4 and R5 form a 5-6 membered ring; R5 is a hydroxyl group, R6 is hydrogen, C1-C5 alkyl, C1-C5 hydroxyalkyl, or heteroarylalkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

19. The compound according to claim 18, characterized in that R4 is Hydrogen or <h2 style=";text-align:left;direction:ltr">-(CH2)n<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> (OCH2CH2O)n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> CH(R6)CO(R7), R5 is a hydroxyl group, R6 is hydrogen, methyl, hydroxymethyl or R7 is hydroxyl or -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0.

20. The compound according to claim 1 or 16, characterized in that L2 is -N(R4)(R5); R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or optionally substituted heteroarylalkyl, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

21. The compound according to claim 20, characterized in that R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or a 6-membered heteroarylalkyl group, R6 is hydrogen, R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

22. The compound according to claim 21, characterized in that R4 and R5 are independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or R6 is hydrogen, R7 is hydroxyl or -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0.

23. The compound according to claim 1 or 16, characterized in that L2 is -N(R4)(R5); R4 is hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, optionally substituted C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl; R5 is hydrogen, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

24. The compound according to claim 23, characterized in that R4 is hydrogen, C0-C3 hydroxyalkyl, C1-C3 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), phenyl optionally substituted by carboxyl, hydroxyl, amino, halogen, pyridyl, amino optionally substituted by 2-methylpyridine, benzyl optionally substituted by carboxyl, hydroxyl, amino or halogen, arylalkoxy, pyridyl substituted by carboxyl, bipyridyl, R5 is hydrogen, R6 is hydrogen, amino, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, aryl, aralkyl optionally substituted by hydroxy, halogen, cyano or nitro, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

25. The compound according to claim 24, characterized in that R4 is hydrogen, hydroxy, ethylhydroxy, methoxy, R5 is hydrogen.

26. The compound according to claim 23, characterized in that R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl, carboxyethyl, benzyl, benzyl substituted by -OH, F, -CN or -NO2, N-hydroxyacetamido, R7 is hydroxyl, -NH(CH2CONH)n 3 OH; n 1 and n 3 Each is independently digitized 0, 1, 2, 3, 4, n 2 Is the number 0.

27. The compound according to claim 23, characterized in that R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl, benzyl, carboxyethyl, N-hydroxyacetamido, heteroaryl or heteroaralkyl; R7 is hydroxyl, -NH(CH2CONH)n 3 OH; n 1 is the number 0 or 2, n 2 is a number 0 or 1, n 3 Is the number 0.

28. The compound according to claim 1, characterized in that J is a peptide residue, including a single amino 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(OCH2CH2O)n 4 CH2COOH,n 4 For numbers 2-10, 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. The compound according to claim 28, characterized in that J is the residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine.

30. The compound according to claim 28 or 29, characterized in that A is -C(O)J-, J is a residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxyl.

31. The compound according to claim 1, characterized in that Y and Z are each independently Q is -NHOH, -NHCH2CH2SO3H, -N(CH2CH2OH)2, -NHCH2COOH, -NHCH(CH3)COOH, -NH(CH2CH2O)3CH3.

32. The compound according to claim 1, characterized in that The compound is selected from the group consisting of:

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

34. The composition according to claim 33, characterized in that The transition metal ion is Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof, optionally, the transition metal ion is Zn 2+ .

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 1:250, optionally, 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:

200.

36. 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, characterized in that: Under an inert atmosphere, at least one carboxyl group in the following formula III is connected to the transition metal chelator portion by introducing a condensation reagent. On the heteroatom Among them, X' is L1 is selected from the group consisting of: -CH(R1)-, -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, aryl groups each independently selectively substituted by a group containing at least one coordinating atom selected from N, O and S, and heteroaryl groups each independently selectively substituted by a group containing at least one coordinating atom selected from O and S; R1, R2 and R3 are each independently H, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, C1-C5 hydroxylaminealkyl, C1-C5 N-hydroxyamidealkyl, aryl or heteroaryl; or R2 or R3 and L2 form a 5-6 membered optionally substituted ring; A' is -COOH or -C(O)J-COOH; J is an organic group comprising an amino group or an imino group and a carbonyl group, wherein the amino group or the imino group forms an amide group with -C(O), and the carboxyl group is optionally covalently linked to L2; L2 is optionally present, L2 serves as a transition metal chelator motif and is -N(R4)(R5) or hydroxyl; R4 and R5 are independently hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring, and R4 or R5 and R2 or R3 form a 5-6 membered optionally substituted ring; R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4; R4 and R5 are not hydroxyl groups at the same time; Y' is the same as X'; Z' is the same as X', or Y and Z are each independently a 5-6 membered optionally substituted saturated heterocyclic group, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, an aryl group, a C1-C5 carboxyalkyl group, a 5-6 membered optionally substituted cycloalkyl group, or -C(O)Q is an ester group, an imide group or an amide group.

38. The method according to claim 37, characterized in that The transition metal chelator part can be composed of 2-phenoxy-ethylamine, aniline, benzylamine, 4-aminobenzene-1,2-diol, 5-amino-2-hydroxybenzoic acid, bis(pyridin-2-ylmethyl)amine, 5-amino-8-hydroxyquinoline, bis(pyridin-2-yl)methylamine, 4-aminophthalic acid, L-tyrosine tert-butyl ester, DL-3-(4-fluorophenyl)alanine, DL-4-cyanophenylalanine, DL-4-nitrophenylalanine, N-benzylhydroxylamine hydrochloride, N-phenylhydroxylamine, supply.

39. The method according to claim 37 or 38, characterized in that The structure of formula III is 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. The use according to claim 40, characterized in that The antibody is modified by selectively reducing the interchain SS bonds. Optionally, the antibody is modified by selectively reducing one of the interchain SS bonds.

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

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

44. The method according to claim 43, characterized in that The number of the thiol groups is 1, 2, 3, 4, 5, 6, 7 or 8.

45. The method according to claim 44, characterized in that The interchain disulfide bonds link the two heavy chains above the hinge region or link the heavy chains to the light chains in the Fab region.

46. ​​The method according to claim 44, characterized in that The interchain disulfide bonds connect the two heavy chains in the hinge region and connect the heavy chains to the light chains in the Fab region.

47. The method according to claim 43, characterized in that The method comprises the following steps: (a) incubating the compound according to any one of claims 1 to 32 or its salt, solvate, stereoisomer and the antibody as a first reducing agent in a first buffer system in the presence of the transition metal ion to selectively reduce the interchain disulfide bonds in the antibody; or Incubating the composition according to any one of claims 33 to 36 and the antibody in the first buffer system to selectively reduce the interchain disulfide bonds in the antibody, wherein the compound according to any one of claims 1 to 32 is used as the first reducing agent; (b) introducing a metal chelator and a modifying agent 1 to react with the reduced thiol group from step (a), wherein the modifying agent 1 is a capping agent, a first linker-payload or a first sulfide bridging agent, and optionally, the first sulfide bridging agent carries the first linker-payload or a reactive group.

48. The method according to claim 47, characterized in that The method further comprises the following steps: (c) incubating the reaction product from step (b) with a second reducing agent in a second buffer system to reduce the interchain disulfide bonds in the reaction product, and optionally, introducing the transition metal ion; (d) introducing the incubation product from step (c) and a modification reagent 2 to react with the reduced thiol group from step (c), optionally introducing the metal chelator, wherein the modification reagent 2 is a second linker-payload or a second sulfide bridge reagent, optionally, the second sulfide bridge reagent carries the second linker-payload or reactive group.

49. The method according to claim 47 or 48, characterized in that The first sulphur bridging reagent and the second sulphur bridging reagent each independently contain at least two substituents that allow re-bridging of the thiol group.

50. The method according to claim 47 or 48, characterized in that The first sulphur bridging agent and the second sulphur bridging agent are independently selected from the group consisting of:

51. The method according to claim 47 or 48, characterized in that The reactive groups each independently contain an azide group and / or dibenzocyclooctyne DBCO.

52. The method of claim 47, wherein: The molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:250, optionally, the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:60, more optionally, the molar ratio of the first reducing agent to the transition metal ion is 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. The method according to claim 47, characterized in that The molar ratio of the first reducing agent to the antibody is 3:1 to 0.5:1, optionally, the molar ratio of the first reducing agent to the antibody is 3:1 to 1:1, more optionally, the molar ratio of the first reducing agent to the antibody is 2:1 to 1:

1.

54. The method according to claim 47, characterized in that 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 and Tris buffer.

55. The method according to claim 54, characterized in that 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. Optionally, the first buffer system and the second buffer system are both MES buffer.

56. The method according to claim 54, characterized in that The concentrations of the first buffer system and the second buffer system are 10 mM to 100 mM.

57. The method according to claim 54, characterized in that The pH value of the first buffer system and the second buffer system is 5.5 to 8. Optionally, the pH value of the first buffer system and the second buffer system is 6.0 to 7.

4. More optionally, the pH value of the first buffer system and the second buffer system is 6.7 to 7.

4.

58. The method according to claim 47, characterized in that The transition metal ion is selected from the group consisting of: Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof, optionally, the transition metal ion is Zn 2+ .

59. The method according to claim 47, characterized in that In step (a), the incubation temperature is 0°C to 37°C, 0°C to 25°C or 0°C to 15°C, and in step (a), the incubation time is 0.2h to 24h, optionally, in step (a), the incubation temperature is 0°C to 10°C, and in step (a), the incubation time is 2h to 16h.

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

61. The method according to claim 48, wherein 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 h to 24 h.

62. The method according to 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 0.5h to 24h.

63. The method according to claim 47, characterized in that When the first sulphur bridging reagent carries the reactive group, the step (b) comprises the following steps: The metal chelator and the first sulfide bridging reagent carrying the reactive group are introduced to re-bridge the reduced thiol group from step (a), and then the first linker-payload is incubated in the first buffer system to react with the reactive group of the sulfide bridging group.

64. The method according to claim 48, characterized in that When the second sulphur bridging reagent carries the reactive group, the step (d) comprises the following steps: The incubation product from step (c) and the second thiol bridging reagent carrying the reactive group are introduced to re-bridge the reduced thiol group from step (c), and optionally, the metal chelator is introduced, and then the second linker-payload is incubated in the second buffer system to react with the reactive group of the thiol bridging group.

65. The method of claim 47, wherein: The method comprises the following steps: (a1) incubating the compound according to any one of claims 1 to 32 used as the first reducing agent and the antibody in the first buffer system in the presence of an effective amount of the transition metal ion to selectively reduce the interchain disulfide bonds in the antibody; or Incubating the composition according to any one of claims 33 to 36 and the antibody in the first buffer system to selectively reduce the interchain disulfide bonds in the antibody, wherein the compound according to any one of claims 1 to 32 is used as the first reducing agent; (b1) introducing an excess of the metal chelator and an excess of the first linker-payload to react with the reduced thiol group from step (a1).

66. The method according to claim 65, characterized in that The antibody with site-specific modification of thiol groups is a D2-type ADC.

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

68. The method according to claim 67, characterized in that The antibody with site-specific modification of thiol groups is a D2+D6 type ADC.

69. The method according to claim 47 or 48, characterized in that The method further comprises the following steps: Optionally, introducing a compound containing at least one thiol group to consume excess of the first linker-payload in step (b) and / or excess of the second linker-payload in step (d); The antibody with site-specific modification of thiol group obtained in step (b) and / or step (d) is purified and recovered.

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

71. The method according to claim 70, characterized in that The antibody is an engineered antibody having two amino acid replacements of two interchain cysteines that form an interchain disulfide bond in the hinge region, optionally, the amino acid replacements are selected from the following replacements: cysteine ​​to alanine, to leucine, to arginine, to lysine, to asparagine, to methionine, to aspartic acid, to phenylalanine, to proline, to glutamine, to serine, to glutamic acid, to threonine, to glycine, to tryptophan, to histidine, to tyrosine, to isoleucine or to valine, more optionally, the amino acid replacement is selected from the following replacements: cysteine ​​to serine.

72. The method according to claim 47 or 48, characterized in that The linkers in the first linker-payload and the second linker-payload are selected from any one of the linkers having the following characteristics: one end is capable of being linked to the reduced thiol group of the antibody or the reactive group of the sulfide bridging reagent, and the other end is capable of being linked to the payload.

73. The method according to claim 47 or 48, characterized in that The payload is selected from any one containing at least one substituent that allows the payload to be attached to a linker. Optionally, the payload is a cytotoxic drug, a cytokine, a nucleic acid, a radionuclide, a kinase or a derivative thereof.

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

75. The antibody with site-specific modification of thiol groups according to claim 74, characterized in that: The antibody having the site-specific modification of the thiol group is coupled to the modification reagent 1 and / or the modification reagent 2.

76. The antibody with site-specific modification of thiol groups according to claim 74 or 75, characterized in that: The antibody with site-specific modification of thiol groups is a D2-type ADC, a D1-type ADC, a D2+D6-type ADC, a D2+D3-type ADC, a D1+D6-type ADC, a D1+D3-type ADC, a D0+D6-type ADC, a D0+D3-type ADC, a D2+D2-type ADC, a D2+D4-type ADC, a D1+D2-type ADC, a D1+D4-type ADC or a D0+D4-type ADC.

77. Use of an antibody having site-specific modification of a thiol group according to any one of claims 74 to 76 in the production of a therapeutic agent for preventing, diagnosing or treating a disease.

78. A pharmaceutical composition comprising the antibody having site-specific modification of a thiol group 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 site-specific modification of a thiol group according to any one of claims 74 to 76 or a pharmaceutical composition according to claim 78.

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