Conjugate and preparation method and application thereof

Abstract

The invention provides a conjugate as well as a preparation method and application thereof. Conjugates of Formula 1: M-[(L1) a-(L2) b-(D) c] 1 wherein M is a biomacromolecule having a nucleophilic function, M is linked to L1 by means of the nucleophilic function of M, D is a functional molecule, L2 is a linker, and L1 is a compound of Formula I. I

Description

Background Technology

[0001] Protein modification can endow proteins with new properties and functions. A common protein modification strategy involves modifying amino acid residues on proteins. Because lysine and cysteine ​​have high affinity reactivity, protein modifications are often targeted at these two amino acids.

[0002] In 2019, three antibody-drug conjugates (ADCs) received FDA approval: Fam-trastuzumab Deruxtecan (for the treatment of unresectable or metastatic HER2-positive breast cancer), Polotuzumab Vedotin (for the treatment of relapsed / refractory diffuse large B-cell lymphoma), and Enfortumab Vedotin (for the treatment of locally advanced or metastatic urothelial carcinoma). These ADCs are synthesized by modifying the reduced interchain disulfide bonds on the antibody, with maleimide as the linker. It is well known that maleimide-thiol adducts are unstable and readily undergo thiol exchange reactions under physiological conditions. Antibody-drug conjugates (ADCs) synthesized via the addition reaction between maleimide and thiol groups can lead to premature drug release, which may severely affect the stability and efficacy of the ADC.

[0003] Despite the progress made by chemists in improving the stability of maleimides and finding new stable linkers, problems remain unsolved. Therefore, there is an urgent need to develop a linker that meets the requirements for conjugate preparation, especially one that balances activity and stability. Summary of the Invention

[0004] This application provides a potential linker that holds promise for the preparation of conjugates (e.g., ADCs). This linker is capable of reacting with nucleophilic functional groups (e.g., -SH of cysteine) of biomolecules with relatively high efficiency and / or chemoselectivity. In this application, the linker and / or conjugates containing and / or prepared via the linker remain stable in both in vitro and in vivo environments. For example, the conjugates (e.g., ADCs) described in this application can kill target cells more effectively and / or more efficiently due to their better stability and / or safety (especially at relatively high concentrations). In this application, the conjugates address the balance between activity and stability.

[0005] On the one hand, this application provides a conjugate of Formula 1, M-[(L1)] a -(L2) b -(D) c ]1, where L1 is a compound of formula I, R is -F or -OH, where M is a biomacromolecule, and M is linked to L1 via its nucleophilic functional group; L2 is a linker, and L2 is linked to R1, R3, or R2; D is a functional molecule; a is an integer from 1 to 10; b and c are each independently integers from 0 to 10, provided that b and c are not simultaneously 0; R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group; and R... 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0006] In some embodiments, the nucleophilic functional group of M is selected from: -SH, -NH2, -SeH, -OH, and

[0007] On one hand, this application provides a conjugate of Formula 2, MS-[(L1)a-(L2)b-(D)c]2, wherein MS is a biomacromolecule containing cysteine, and MS is linked to L1 using the cysteine, where L1 is a compound of Formula I. R is -F or -OH, L2 is a linker, L2 is connected to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independent integers from 0 to 10, provided that b and c are not simultaneously 0, where R1 is H, an optionally substituted alkyl group or an optionally substituted aryl group, where R 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0008] In some embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, M is selected from the group consisting of proteins, DNA, RNA, and viruses.

[0009] In some embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, M is a biomacromolecule expressed on the cell surface.

[0010] In some embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, M is an antigen-binding protein or a fragment thereof.

[0011] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, M is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-engineered antibody, a human antibody, a single-chain antibody scFv, or an antibody fragment.

[0012] In some embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0013] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, L2 is selected from the group consisting of: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG. NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidone-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0014] In some embodiments, D has a biological function in the conjugate of Formula 1 and / or the conjugate of Formula 2.

[0015] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, D and / or its derivatives are capable of inhibiting the growth of tumor cells.

[0016] In some embodiments, D is a drug in the conjugate of Formula 1 and / or the conjugate of Formula 2.

[0017] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, D is selected from the group consisting of: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0018] In some embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; new interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0019] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R 1' It is -H.

[0020] In some embodiments, R1 is -H in the conjugate of Formula 1 and / or the conjugate of Formula 2.

[0021] In some embodiments, R3 is -H in the conjugate of Formula 1 and / or the conjugate of Formula 2.

[0022] In some embodiments, R2 is an optionally substituted phenyl group in the conjugate of Formula 1 and / or the conjugate of Formula 2.

[0023] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0024] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... R4 is selected from the following groups: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0025] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... Where R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 is selected from the following group: And H.

[0026] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, where R7 is selected from the following group:

[0027] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 is selected from the following group:

[0028] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and R9 is selected from the following group: H and

[0029] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and

[0030] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0031] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is... Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Select from the following groups: -CF3, -CN, and -OCH3.

[0032] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, R2 is an optionally substituted alkyl group -CH=CH-R. 12 , where R 12 for Where R 13 It is -CH2N3.

[0033] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, the ring is an optionally substituted cyclic olefin or an optionally substituted aryl-cyclic olefin.

[0034] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, the ring is selected from the group consisting of:

[0035] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, L1 is selected from the group consisting of:

[0036]

[0037]

[0038]

[0039] In some embodiments, in the conjugates of Formula 1 and / or Formula 2, the conjugates are selected from:

[0040]

[0041]

[0042] On the other hand, this application provides a conjugate of formula 3, (L1) a -(L2) b -(D) c 3, where L1 is a compound of formula III. L2 is a linker, and L2 is connected to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not simultaneously 0, wherein R1 is H, an optional alkyl or an optional aryl, wherein R2 is H, an optional alkyl or an optional aryl, wherein R3 is H, an optional alkyl or an optional aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0043] In some embodiments, in the conjugate of Formula 3, L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0044] In some embodiments, in the conjugate of Formula 3, L2 is selected from: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidone-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0045] In some embodiments, D has a biological function in the conjugate of Formula 3.

[0046] In some embodiments, in the conjugate of Formula 3, D and / or its derivatives are able to inhibit the growth of tumor cells.

[0047] In some embodiments, D is a drug in the conjugate of Formula 3.

[0048] In some embodiments, in the conjugate of Formula 3, D is selected from the group consisting of: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0049] In some embodiments, in the conjugate of Formula 3, D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; new interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0050] In some embodiments, R1 is -H in the conjugate of Formula 3.

[0051] In some embodiments, R3 is -H in the conjugate of Formula 3.

[0052] In some embodiments, in the conjugate of Formula 3, R2 is an optionally substituted phenyl group.

[0053] In some embodiments, in the conjugate of Formula 3, R2 is R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0054] In some embodiments, in the conjugate of Formula 3, R2 is R4 is selected from the following groups: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0055] In some embodiments, in the conjugate of Formula 3, R2 is Where R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 is selected from the following group: And H.

[0056] In some embodiments, in the conjugate of Formula 3, R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, where R7 is selected from the following group:

[0057] In some embodiments, in the conjugate of Formula 3, R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 is selected from the following group:

[0058] In some embodiments, in the conjugate of Formula 3, R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and R9 is selected from the following group: H and

[0059] In some embodiments, in the conjugate of Formula 3, R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and

[0060] In some embodiments, in the conjugate of Formula 3, R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from: -COOH, -NH2 and Where R 11 Selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0061] In some embodiments, in the conjugate of Formula 3, R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Select from the following groups: -CF3, -CN, and -OCH3.

[0062] In some embodiments, in the conjugate of Formula 3, R2 is an optionally substituted alkyl group -CH=CH-R. 12 , where R 12 for Where R 13 It is -CH2N3.

[0063] In some embodiments, in the conjugate of Formula 3, the ring is an optionally substituted cycloalkene or an optionally substituted aryl-cycloalkene.

[0064] In some embodiments, in the conjugate of Formula 3, the ring is selected from:

[0065] In some embodiments, in the conjugate of Formula 3, L1 is selected from the group consisting of:

[0066]

[0067]

[0068]

[0069] In some embodiments, in the conjugate of Formula 3, the conjugate is selected from:

[0070]

[0071]

[0072] On the other hand, this application provides a method for preparing a conjugate, the method comprising the following steps: by making the conjugate of Formula 3: Combined with M, the conjugate of formula 1 is obtained: Where M is a biomacromolecule, and M is linked to L1 via its nucleophilic functional group; L2 is a linker, and L2 is linked to R1, R3, or R2 in Formula 1; D is a functional molecule; a is an integer from 1 to 10; b and c are each independently an integer from 0 to 10, provided that b and c are not simultaneously 0; R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group; and R... 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0073] In some embodiments, in the method, the nucleophilic functional group of M is selected from the group consisting of -SH, -NH2, -SeH, -OH, and

[0074] A method for preparing a conjugate, the method comprising the following steps: by making the conjugate of formula 3: Combined with M, we obtain the conjugate of formula 2: R is -OH or -F, where MS is a biomolecule containing cysteine, MS is linked to L1 using the cysteine, L2 is a linker and is linked to R1, R3 or R2 in Formula 3, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently integers from 0 to 10, provided that b and c are not simultaneously 0, where R1 is H, an optionally substituted alkyl group or an optionally substituted aryl group, and R... 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0075] In some embodiments, M is selected from proteins, DNA, RNA, and viruses in the method.

[0076] In some embodiments, M is a biomacromolecule expressed on the cell surface in the method.

[0077] In some embodiments, M is an antigen-binding protein or a fragment thereof in the method.

[0078] In some embodiments, in the method, M is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody scFv, or antibody fragment.

[0079] In some embodiments, M in the method comprises a functional group for nucleophilic addition reactions.

[0080] In some embodiments, in the method, L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, pre-charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0081] In some embodiments, in the method, L2 is selected from the group consisting of: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), and maleimide PEG. NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidone-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0082] In some embodiments, D has a biological function in the method.

[0083] In some embodiments, D and / or its derivatives are able to inhibit the growth of tumor cells in the method.

[0084] In some embodiments, D is a drug in the method.

[0085] In some embodiments, in the method, D is selected from the group consisting of: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives and DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0086] In some embodiments, in the method, D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; new interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0087] In some implementations, R1 is -H in the method.

[0088] In some implementations, R3 is -H in the method.

[0089] In some embodiments, R2 is an optionally substituted phenyl group in the method.

[0090] In some embodiments, R2 is... R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0091] In some embodiments, R2 is... R4 is selected from the following groups: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0092] In some embodiments, R2 is... Where R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 is selected from the following group: And H.

[0093] In some embodiments, R2 is... Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, where R7 is selected from the following group:

[0094] In some embodiments, R2 is... Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 is selected from the following group:

[0095] In some embodiments, R2 is... Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and R9 is selected from the following group: H and

[0096] In some embodiments, R2 is... Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and

[0097] In some embodiments, R2 is... Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0098] In some embodiments, R2 is... Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Select from the following groups: -CF3, -CN, and -OCH3.

[0099] In some embodiments, R2 is an optionally substituted alkyl group -CH=CH-R in the method. 12 , where R 12 for Where R 13 It is -CH2N3.

[0100] In some embodiments, the ring in the method is an optionally substituted cycloalkene or an optionally substituted aryl-cycloalkene.

[0101] In some embodiments, in the method, the ring is selected from:

[0102] In some embodiments, the method is carried out at a temperature ranging from about 16°C to about 37°C.

[0103] In some embodiments, the method is carried out at a pH in the range of about 7.4 to about 8.

[0104] In some embodiments, the method is carried out using a catalyst.

[0105] In some embodiments, the method further includes the step of purifying the conjugate of Formula 3.

[0106] On the other hand, this application provides a compound of formula III, or a pharmaceutically acceptable salt thereof:

[0107] III, wherein R1 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0108] In some embodiments, R1 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0109] In some embodiments, the compound is selected from:

[0110] On the other hand, this application provides a compound of formula IV, or a pharmaceutically acceptable salt thereof: R1 is selected from the following groups: NH-(CH2)n1-CO-R2, -OH, NH-(CH2) n2 -R3、NH-(CH2CH2-O) n3 -(CH2)n4-NH-CO-O-R4, or Where n1, n2, n3, or n4 are independent integers from 1 to 10, and R2 is selected from the following group: R3 is selected from the following group: Where R4 is

[0111] In some embodiments, R1 is selected from the group consisting of: NH-(CH2)2-CO-R2, -OH, NH-(CH2)-R3, NH-(CH2CH2-O)3-(CH2)2-NH-CO-O-R4.

[0112] In some embodiments, the compound is selected from the group consisting of:

[0113] On the other hand, this application provides a compound of formula V, or a pharmaceutically acceptable salt thereof: R1, R2, and R4 are optional substituents, R3 is selected from the group consisting of H, optional substituted alkyl, -F3, optional substituted alkyl, -N, or O-optionally substituted alkyl, and R5 is selected from the group consisting of -COOH, -NH2, and

[0114] In some implementations, R1 is H.

[0115] In some implementations, R2 is H.

[0116] In some implementations, R4 is H.

[0117] In some implementations, R3 is selected from the group consisting of H, CF3, CN, and OCH3.

[0118] In some embodiments, the compound is selected from:

[0119] On the other hand, this application provides a compound of formula VI, or a pharmaceutically acceptable salt thereof:

[0120]

[0121] On the other hand, this application provides a pharmaceutical composition comprising the conjugate of this application and a pharmaceutically acceptable carrier.

[0122] On the other hand, this application provides a method for modulating the tumor microenvironment of a subject, the method comprising administering to the subject a conjugate of this application, or a pharmaceutical composition of this application.

[0123] On the other hand, this application provides a method for modulating the immune response of a subject, the method comprising administering to the subject a conjugate of this application, or a pharmaceutical composition of this application.

[0124] On the other hand, this application provides a method for preventing and / or treating a disease in a subject in need, the method comprising administering to the subject a conjugate of this application, or a pharmaceutical composition of this application.

[0125] In some implementations, the disease includes tumors and / or autoimmune diseases.

[0126] On the other hand, this application provides a diagnostic reagent comprising the conjugate of this application.

[0127] In some embodiments, the diagnostic reagent is labeled.

[0128] In some embodiments, the marker is selected from: radioactive markers, fluorophores, chromophores, imaging agents, and metal ions.

[0129] Other aspects and advantages of this application will become apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of this application are shown and described. It will be appreciated that this application can have other different embodiments, and certain details may be modified in various obvious ways without departing from this disclosure. Therefore, the drawings and description are to be regarded as exemplary rather than restrictive in nature.

[0130] By incorporating via reference

[0131] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if the individual publications, patents or patent applications were specifically and separately indicated to be incorporated by reference. Attached Figure Description

[0132] The novel features of the invention are set forth in detail in the appended claims. The features and advantages of the invention will be better understood by referring to the following detailed description of exemplary embodiments using the principles of the invention, along with the accompanying drawings (also referred to herein as “figure” and “FIG.”):

[0133] Figure 1 The reaction process for screening candidate Michael acceptors as linkers is shown.

[0134] Figure 2 The chemical structure of the Michael acceptor candidate is shown.

[0135] Figure 3 The chemical structure of the modified Michael acceptor candidate is shown.

[0136] Figure 4 The reaction process for screening modified candidate Michael acceptors as linkers is shown.

[0137] Figure 5 The chemical structure of the Michael acceptor candidate is shown.

[0138] Figure 6 The reaction process for screening modified candidate Michael acceptors as linkers is shown.

[0139] Figure 7 The chemical structure of the previously reported linker is shown.

[0140] Figure 8 The reaction process between sfGFP E124C and the previously reported linker is shown.

[0141] Figure 9 The reaction results between sfGFP E124C and the previously reported linker are shown.

[0142] Figure 10 The experimental results comparing the linker of this application with those of previously reported linkers are shown.

[0143] Figure 11 a to Figure 11 c illustrates the stability of the connector in this application.

[0144] Figure 12 a to Figure 12 c shows the generation of conjugates using the linkers of this application and verifies the stability of the conjugates.

[0145] Figure 13 a to Figure 13 c shows the mass spectrometry results of the conjugates obtained by the linkers of this application, demonstrating their stability in serum.

[0146] Figures 14a to 14c The steps for generating conjugates using the linkers of this application are shown.

[0147] Figures 15a to 15c The mass spectrometry results of the conjugate with a DAR of 3.2 obtained by linker of this application are shown.

[0148] Figure 16 a to Figure 16 b shows the mass spectrometry results of the conjugate with a DAR of 3.8 obtained by the linker of this application.

[0149] Figure 17 a to Figure 17 h shows the results of a tumor cell killing assay of the conjugates obtained through the linkers of this application.

[0150] Figure 18 The LC-MS chromatogram and mass spectrum of SSF-PEG4-vc-PAB-MMAE 1 are shown.

[0151] Figure 19 The LC-MS chromatogram of SSF-PEG4-GGFG-Dxd3 is shown.

[0152] Figure 20 The process of studying the stability of the linker is shown.

[0153] Figure 21 The hydrolytic stability results for MA 5 and MA 2 are shown.

[0154] Figure 22 A to Figure 22 B shows a comparison of MA 2 with previously reported stable Cys-specific labeling reagents.

[0155] Figure 23 The MS / MS spectrum of the GFP fragment modified with MA 2 is shown.

[0156] Figure 24 A to Figure 24 G shows Cys-specific modifications performed on different proteins using SSF.

[0157] Figure 25 A to Figure 25 D shows the results of the antitumor activity of the conjugates obtained through the linkers of this application.

[0158] Figure 26 A to Figure 26 B shows a comparison of the stability of MA 2 and maleimide in aqueous buffer.

[0159] Figure 27 A to Figure 27H illustrates the preparation of SSF-ssDNA conjugates obtained through the linkers of this application and their application in single-cell sequencing.

[0160] Figure 28 The deconvolutional intact protein MS of the conjugate obtained by the linker of this application is shown.

[0161] Figure 29 The deconvolutional intact protein MS of the conjugate obtained through the linker of this application is shown.

[0162] Figure 30 The results of cell viability assays for cell line (N87) are shown. Detailed Implementation

[0163] While various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and alternatives will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0164] As used herein, the term "conjugate" generally refers to any substance formed by individual parts linked together. In a conjugate, the individual parts may bind to each other at one or more active sites. Furthermore, the individual parts may be covalently or non-covalently related or linked and exhibit different stoichiometric molar ratios. Conjugates can include peptides, polypeptides, proteins, prodrugs metabolized in vivo to active agents, polymers, nucleic acid molecules, small molecules, binding agents, mimics, synthetic drugs, inorganic molecules, organic molecules, and radioisotopes. For example, conjugates can contain drugs and antigen-binding proteins, and can be antibody-drug conjugates (ADCs).

[0165] As used herein, the term "ADC" generally refers to the linking of an antigen-binding protein to a drug. This linking can be a covalent bond or a non-covalent interaction, such as through electrostatic forces. Various linkers can be used to form immunoconjugates. Additionally, immunoconjugates can be provided as fusion proteins, which can be expressed from polynucleotides encoding the immunoconjugate. As used herein, a "fusion protein" refers to a protein produced by the conjugation of two or more genes or gene segments that originally encode individual proteins (including peptides and polypeptides).

[0166] As used in this article, the term "biological macromolecule" generally refers to biological molecules such as nucleic acids, proteins, antibodies, carbohydrates, polysaccharides, and lipids.

[0167] As used herein, the term "linker" generally refers to a chemical part or bond that attaches two or more molecules. A linker can be any molecular component capable of joining or connecting two or more scaffolds. A linker can be a molecule whose function is to act as a flexible connector between modules in a scaffold, or it can be a molecule with additional functions. In this disclosure, linkers can be used to attach fucose or fucose derivatives to an active portion. Linkers of different lengths allow for attachment of fucose or fucose derivatives at different distances from the active portion.

[0168] As used herein, the term "functional molecule" generally refers to any molecule that is a component of the conjugate of this application and can play a role in the function of the conjugate.

[0169] As used herein, the term "biological function" generally refers to any activity or process performed in biology by the functional molecules of this application. For example, a biological function may include any activity or process performed in vitro and / or in vivo by functional molecules, and a biological function may include any activity or process performed in vitro and / or in vivo by conjugates containing functional molecules.

[0170] As used herein, the term "functional group" generally refers to a biomolecular group capable of participating in addition reactions (e.g., nucleophilic addition reactions). In this application, the nucleophilic addition reaction can be a chemical addition reaction in which a nucleophile forms a sigma bond with an electron-deficient substance. Nucleophilic addition reactions can convert carbonyl groups into a variety of functional groups. For example, the nucleophilic functional group of a biomolecular group can be -SH, -NH2, -SeH, -OH, or...

[0171] As used herein, the term "addition reaction" generally refers to an organic reaction in which two or more molecules combine to form a larger molecule (adduct). This addition reaction can include both electrophilic and nucleophilic additions. The addition reaction can be limited to compounds with multiple bonds, such as molecules with carbon-carbon double bonds (olefins) or triple bonds (alkynes), and compounds with rings, which are also considered to be unsaturated rings. For example, molecules containing carbon-heterodouble bonds (such as carbonyl (C=O) groups or imine (C=N) groups) can undergo addition reactions.

[0172] As used herein, the term "antigen-binding protein" generally refers to a polypeptide molecule that specifically binds to an antigenic determinant. For example, an antigen-binding protein may target a specific site, such as an entity (e.g., an effector moiety or a second antigen-binding moiety) that can be attached to the tumor matrix by a specific type of tumor cell or antigenic determinant. Further, as defined herein, an antigen-binding protein may comprise an antibody and fragments thereof. For example, an antigen-binding protein may comprise an antibody antigen-binding domain comprising a variable region of the antibody heavy chain and a variable region of the antibody light chain. For example, an antigen-binding protein may comprise an antibody constant region as further defined herein and known in the art. A valid heavy chain constant region may comprise five isotypes: α, δ, ε, γ, or μ. A valid light chain constant region may comprise two isotypes: κ and λ. A valid light chain constant region may comprise one of two isotypes: κ and λ.

[0173] As used herein, the term "antibody" generally refers to a polypeptide or protein complex that specifically binds to an antigenic epitope or a mimic of it. Antibodies include intact antibodies or binding fragments thereof that compete with the intact antibody for specific binding, and include chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies. In some embodiments, antibodies are referred to as immunoglobulins and include various classes and subtypes such as IgA (IgA1 and IgA2), IgD, IgE, IgM, and IgG (IgG1, IgG3, and IgG4). In some embodiments, as used herein, the term "antibody" refers to polyclonal or monoclonal antibodies and organofunctional fragments. Antibodies include modified or derivatized antibody variants that retain the ability to specifically bind to epitopes. Antibodies are capable of selectively binding to target antigens or epitopes. Antibodies can include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized and other chimeric antibodies, single-chain antibodies (scFvs), Fab fragments, F(ab')2 fragments, and disulfide-linked Fvs (sdFv) fragments. In some embodiments, the antibody is derived from any source, such as mice or humans, including their chimeric antibodies. In some embodiments, the antibody is humanized.

[0174] As used herein, the term "derivative" generally refers to a compound that is expected to exhibit similar (e.g., physical, and / or chemical, and / or biological) activities to the host (parent) compound. For example, a derivative can be a precursor, metabolite, salt, and / or ester of the host compound.

[0175] As used herein, the term "drug" generally refers to any agent that is harmful to cell growth and proliferation and can be used to reduce, inhibit, or destroy cells or malignant tumors. For example, a drug may contain toxins. For example, a drug may contain chemotherapeutic agents.

[0176] As used herein, the term "cytokine" generally refers to molecules that mediate and / or regulate biological or cellular functions or processes (e.g., immunity, inflammation, and hematopoiesis). In this application, cytokines may also include "lymphokines," "chemokines," "monokines," and "interleukins." Examples of cytokines may include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. For example, cytokines may include IL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. For example, cytokines may be human cytokines. As used in this article, the term "cytokine" can also refer to Sauve et al., *Proceedings of the National Academy of Sciences of the United States of America* 88, 4636-40 (1991); Hu et al., *Blood* 101, 4853-4861 (2003); and U.S. Patent Application Publication No. 2003 / 0124678; Shanafelt et al., *Nature Biotechnol* 18, 1197-1202 (2000); and Heaton et al., *Cancer Research*. Res) 53,2597-602 (1993) and U.S. Patent No. 5,229,109; wild-type cytokines, such as the IL-2 mutant described in U.S. Patent Application Publication No. 2007 / 0036752; International Publication No. 2008 / 0034473; International Publication No. 2009 / 061853; PCT Patent Application PCT / EP2012 / 051991. This includes cytokine variants containing one or more amino acid mutations in the corresponding amino acid sequence. Additionally, cytokine variants, such as IL-15 variants, are also described herein. For example, cytokines can be mutated to eliminate glycosylation.

[0177] As used herein, the term "aryl" generally refers to a hydrocarbon ring system having carbon atoms with a hydrocarbon ring radical (i.e., a monocyclic hydrocarbon ring) or two to four fused rings, wherein the cyclic hydrocarbon ring may be aromatic with five or six carbon atoms, and each ring forming the hydrocarbon ring system may be aromatic and independently have five or six carbon atoms. Examples of aryl groups may include phenyl, naphthyl (i.e., naphthalene), and anthracene. For example, aryl may preferably include phenyl.

[0178] As used herein, the term "alkyl" generally refers to at least one carbon atom (e.g., 1 to 20 carbon atoms. "1 to 20 carbon atoms" can refer to a straight-chain and / or branched group of an alkyl group having up to 1, 2, 3, etc., including up to 20 carbon atoms), and saturated aliphatic (i.e., non-aromatic) acyclic hydrocarbons (i.e., groups consisting of carbon and hydrogen atoms) include adjacent carbon-carbon double or triple bonds. For example, an alkyl group can contain 1 to 10 carbon atoms. For example, an alkyl group can contain 1 to 6 carbon atoms.

[0179] As used herein, the term "drug" generally refers to any substance that alters the physiological function of a subject. In this application, a drug may comprise any compound having the desired biological activity and reactive functionality, and which can be used to prepare the conjugate of this application. The desired biological activity may include activities that can be used to diagnose, cure, reduce, treat, or prevent diseases in humans or other animals. Therefore, as long as they possess the necessary reactive functional groups, these compounds may be associated with the term "drug" as can be found in the official Chinese Pharmacopoeia (e.g., in the official Homeopathic Pharmacopoeia, or in the official National Formulary, or any of its revised versions). Exemplary drugs may be described in the Physician's Desk Reference (PDR) and the Orange Book maintained by the U.S. Food and Drug Administration (FDA). New drugs may continue to be discovered and developed, and this application also incorporates these new drugs into the term "drug" of the drug conjugate of this application.

[0180] As used herein, the term "Toll-like receptor agonist" generally refers to any agonist of Toll-like receptors. In this application, Toll-like receptors can be recognized by TLRs, which can activate immune cell responses. TLRs may include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. For example, Toll-like receptor agonists may include vaccine adjuvants used in antitumor therapies, as these adjuvants can activate immune cells and induce inflammation.

[0181] As used herein, the term "Sting agonist" generally refers to an agent capable of binding to and activating STING. For example, activation of STING activity may include stimulation of inflammatory cytokines, including interferons such as type 1 interferons (including IFN-α, IFN-β) and type 3 interferons (e.g., CXCL9, CCL4, CXCL11, CCL5, CCL3, or CCL8). STING agonist activity may also include stimulation of TANK-binding kinase (TBK) 1 phosphorylation, interferon regulatory factor (IRF) activation (e.g., IRF3 activation), interferon-γ-induced protein (IP-10), or the secretion of other inflammatory proteins and cytokines. The activity of a STING agonist can be determined, for example, by the compound's ability to stimulate STING pathway activation, using interferon stimulation assays, reporter gene assays (e.g., hSTING wt assays or THP-1 dual assays), TBK1 activation assays, IP-10 assays, or other assays known to those skilled in the art. The activity of STING agonists can also be determined by the compound's ability to increase the transcriptional level of genes encoding proteins that are activated by STING or the STING pathway. For example, such activity can be detected using RNA-seq assays.

[0182] As used herein, the term "pharmaceuticalally acceptable carrier" generally refers to a non-API (API stands for active pharmaceutical ingredient) used to form a pharmaceutical product, such as a disintegrant, binder, filler, or lubricant. Pharmaceutically acceptable carriers conform to established government standards (including those issued by the U.S. Food and Drug Administration and the European Food and Drug Administration) and are generally safe for human use. For example, pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, and / or sterile injectable solutions or dispersions only prior to use.

[0183] The term "tumor" generally refers to a malignant tumor characterized by disordered or uncontrolled cell growth. For example, a tumor can include primary malignancies (e.g., those whose cells have not migrated to sites other than the original tumor site in the subject's body) and secondary malignancies (e.g., those caused by metastasis, where tumor cells have migrated to a secondary site different from the original tumor site). Tumors can include solid tumors and / or non-solid tumors.

[0184] The term "tumor microenvironment" generally refers to the complex microenvironment surrounding tumor cells. For example, the tumor microenvironment can include surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and / or extracellular matrix (ECM). For instance, the tumor microenvironment may harbor cancer stem cells and other molecules that contribute to tumor development and progression. Therefore, during treatment, targeting and manipulating cells and factors within the tumor microenvironment can help control malignant tumors and achieve positive health outcomes.

[0185] The term "immune response" generally refers to a subject's defense against foreign substances and / or pathogens. An immune response may result in an immune reaction, for example, through the recognition and binding of antigens by its specific antibodies or by previously sensitized lymphocytes.

[0186] The term "autoimmune disease" generally refers to any disease and / or condition caused by an immune-mediated attack on a subject's own organs. Examples of autoimmune diseases can include rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and / or vasculitis.

[0187] As used herein, the term "treatment" generally refers to improving a disease or symptom (i.e., slowing or stopping or reducing the progression of a disease (e.g., a tumor) or at least one of its clinical symptoms). For example, treatment may include reducing or improving at least one physical parameter, including those that the patient may not be able to recognize.

[0188] As used in this article, the term "prevention" generally refers to preventive treatment of a disease or symptom; or delaying the onset or progression of a disease or symptom.

[0189] As used herein, unless otherwise specified herein or obviously contradicted by the context, the terms “a,” “an,” and “the,” and similar terms used in the context of this application (especially in the context of the claims) shall be construed as covering both the singular and the plural.

[0190] Conjugate

[0191] On the one hand, this application provides a conjugate of Formula 1, M-[(L1)] a -(L2) b -(D) c ]1, where L1 is a compound of formula I, R is -F or -OH, where M is a biomacromolecule, and M is linked to L1 via its nucleophilic functional group; L2 is a linker, and L2 is linked to R1, R3, or R2; D is a functional molecule; a is an integer from 1 to 10; b and c are each independently integers from 0 to 10, provided that b and c are not simultaneously 0; R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group; and R... 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0192] For example, the nucleophilic functional group of M can be selected from: -SH, -NH2, -SeH, -OH, and

[0193] On the other hand, this application provides a conjugate of Formula 2, MS-[(L1)a-(L2)b-(D)c]2, wherein MS is a biomacromolecule containing cysteine, and MS is linked to L1 using the cysteine, where L1 is a compound of Formula I. R is -F or -OH, L2 is a linker, L2 is connected to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independent integers from 0 to 10, provided that b and c are not simultaneously 0, where R1 is H, an optionally substituted alkyl group or an optionally substituted aryl group, where R 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0194] For example, M can be selected from: protein, DNA, RNA, and virus.

[0195] For example, M can be a biological macromolecule expressed on the cell surface.

[0196] For example, M can be an antigen-binding protein or a fragment thereof.

[0197] For example, M can be a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody scFv, or antibody fragment.

[0198] For example, M can contain functional groups for nucleophilic addition reactions.

[0199] For example, L2 can be selected from the following group: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0200] For example, L2 can be selected from the following group: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidone-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0201] For example, D can have biological functions.

[0202] For example, D and / or its derivatives may be able to inhibit the growth of tumor cells.

[0203] For example, D can be a drug.

[0204] For example, D can be selected from the following group: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, scabra, maytansine, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0205] For example, D can be MMAE or its derivatives; melphalan or its derivatives; lenalidomide or its derivatives; IL-2 or its derivatives; new interleukin-2 / 15 or its derivatives; T785 or its derivatives; or MSA-2 or its derivatives.

[0206] For example, R1' can be -H. For example, R1 is -H. . For example, R3 is -H.

[0207] For example, R2 can be an optionally substituted phenyl group.

[0208] For example, R2 can be R4 can be selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0209] For example, R2 can be R4 can be selected from the following groups: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0210] For example, R2 can be Where R4 can be -O-(CH2)n1-COO-R5, n1 can be an integer from 1 to 10, and R5 can be selected from the following group: And H.

[0211] For example, R2 can be Where R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, and R6 can be -(CH2)n3-CO-R7, n3 can be an integer from 1 to 10, and R7 can be selected from the following group:

[0212] For example, R2 is Where R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, and R6 can be -(CH2)n4-R8, n4 can be an integer from 1 to 10, and R8 can be selected from the following group:

[0213] For example, R2 can be Where R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, R6 can be -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 can be an integer from 1 to 10, n6 can be an integer from 1 to 10, and R9 can be selected from the following group: H and

[0214] For example, R2 can be R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 It can be selected from the following groups: -COOH, -NH2 and

[0215] For example, R2 can be R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 It can be selected from the following groups: -COOH, -NH2 and Where R 11 It may be selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0216] For example, R2 can be R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 It can be selected from the following groups: -COOH, -NH2 and Where R 11 You can choose from the following groups: -CF3, -CN, and -OCH3.

[0217] For example, R2 can be an substituted alkyl group -CH=CH-R. 12 , where R 12 It can be Where R 13 It can be -CH2N3.

[0218] For example, the ring can be an optionally substituted cyclic olefin or an optionally substituted aryl-cyclic olefin.

[0219] For example, the ring can be selected from the following group:

[0220] For example, L1 can be selected from the following group:

[0221]

[0222]

[0223] For example, the conjugate can be selected from the following group:

[0224]

[0225]

[0226] For example, the conjugates of Formula 1 or Formula 2 can be as follows:

[0227]

[0228] and / or Where R is -F or -OH.

[0229] On the other hand, this application provides a conjugate of formula 3, (L1) a -(L2) b -(D) c 3, where L1 is a compound of formula III. L2 is a linker, and L2 is connected to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not simultaneously 0, wherein R1 is H, an optional alkyl or an optional aryl, wherein R2 is H, an optional alkyl or an optional aryl, wherein R3 is H, an optional alkyl or an optional aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0230] In some embodiments, in the conjugate of Formula 3, L2 is selected from: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0231] For example, L2 can be selected from: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidone-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0232] For example, D can have biological functions. For example, D and / or its derivatives can be able to inhibit the growth of tumor cells. For example, D can be a drug.

[0233] For example, D can be selected from the following group: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists. For example, D can be MMAE or its derivatives; melphalan or its derivatives; lenalidomide or its derivatives; IL-2 or its derivatives; new interleukin-2 / 15 or its derivatives; T785 or its derivatives; or MSA-2 or its derivatives.

[0234] For example, R1 can be -H.

[0235] For example, R3 can be -H.

[0236] For example, R2 can be an optionally substituted phenyl group.

[0237] For example, R2 can be R4 can be selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0238] For example, R2 can be R4 can be selected from the following groups: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0239] For example, R2 can be Where R4 can be -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 can be selected from the following group: And H.

[0240] For example, R2 can be Where R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, and R6 can be -(CH2)n3-CO-R7, n3 can be an integer from 1 to 10, and R7 can be selected from the following group:

[0241] For example, R2 is Where R4 can be -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 can be -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 can be selected from the following group:

[0242] For example, R2 can be Where R4 can be -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, R6 can be -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 can be an integer from 1 to 10, n6 can be an integer from 1 to 10, and R9 can be selected from the following group: H and

[0243] For example, R2 can be R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 It can be selected from the following groups: -COOH, -NH2 and

[0244] For example, R2 can be R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 It can be selected from the following groups: -COOH, -NH2 and Where R 11 It may be selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0245] For example, R2 can be R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 It can be selected from the following groups: -COOH, -NH2 and Where R 11 You can choose from the following groups: -CF3, -CN, and -OCH3.

[0246] For example, R2 can be an substituted alkyl group -CH=CH-R. 12 , where R 12 It can be Where R 13 It can be -CH2N3.

[0247] For example, the ring can be an optionally substituted cyclic olefin or an optionally substituted aryl-cyclic olefin.

[0248] For example, the ring can be selected from the following group:

[0249] For example, L1 can be selected from the following group:

[0250]

[0251]

[0252] For example, the conjugate may be selected from the group consisting of:

[0253]

[0254]

[0255] method

[0256] On the other hand, this application provides a method for preparing a conjugate, the method comprising the following steps: by making the conjugate of Formula 3: Combined with M, the conjugate of formula 1 is obtained: M is a biological macromolecule, and M is linked to L1 via its nucleophilic functional group. L2 is a linker, and L2 is linked to R1, R3, or R2 in Formula 1. D is a functional molecule, a is an integer from 1 to 10, b and c are each independently integers from 0 to 10, provided that b and c are not simultaneously 0. R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group. 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0257] For example, the nucleophilic functional group of M can be selected from the following group: -SH, -NH2, -SeH, -OH and

[0258] On the other hand, this application provides a method for preparing a conjugate, the method comprising the following steps: by making the conjugate of Formula 3: Combined with M, we obtain the conjugate of formula 2: R is -OH or -F, where MS is a biomolecule containing cysteine, MS is linked to L1 using the cysteine, L2 is a linker and is linked to R1, R3 or R2 in Formula 3, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently integers from 0 to 10, provided that b and c are not simultaneously 0, where R1 is H, an optionally substituted alkyl group or an optionally substituted aryl group, and R... 1' R1 is H or an isotope thereof, wherein R2 is H, an optionally substituted alkyl or an optionally substituted aryl, wherein R3 is H, an optionally substituted alkyl or an optionally substituted aryl, and optionally, the C connecting R1 and the C connecting R2 form a ring.

[0259] In this application, the method can be carried out at a temperature ranging from about 16°C to about 37°C. For example, the method can be carried out at a temperature of at least about 16°C, at least about 17°C, at least about 18°C, at least about 19°C, at least about 20°C, at least about 21°C, at least about 22°C, at least about 23°C, at least about 24°C, at least about 25°C, at least about 26°C, at least about 27°C, at least about 33°C, at least about 34°C, at least about 35°C, at least about 36°C, or at least about 37°C.

[0260] In this application, the method can be carried out at a pH in the range of about 7.4 to about 8. For example, the method can be carried out at a pH of at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, or at least about 8.0.

[0261] In this application, the method can be carried out using a catalyst. For example, the catalyst may contain Pd(OAc)2.

[0262] In this application, the method may further include the step of purifying the conjugate of Formula 3.

[0263] In this application, the conjugation in this method may include addition reactions (e.g., nucleophilic addition reactions), which may fall under the category of "click chemistry". For example, the -SH group of M may participate in the addition reaction in this method.

[0264] compound

[0265] On the other hand, this application provides a compound of formula III, or a pharmaceutically acceptable salt thereof: R1 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0266] For example, R1 can be selected from the following group: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0267] In some cases, the compound of formula III may be one of the compounds in Table 1.

[0268] Table 1

[0269]

[0270]

[0271] On the other hand, this application provides a compound of formula IV, or a pharmaceutically acceptable salt thereof: R1 is selected from the following groups: NH-(CH2)n1-CO-R2, -OH, NH-(CH2) n2 -R3、NH-(CH2CH2-O) n3-(CH2)n4-NH-CO-O-R4, or Where n1, n2, n3, or n4 are independent integers from 1 to 10, and R2 is selected from the following group: R3 is selected from the following group: Where R4 is

[0272] For example, R1 can be selected from the following group: NH-(CH2)2-CO-R2, -OH, NH-(CH2)-R3, NH-(CH2CH2-O)3-(CH2)2-NH-CO-O-R4.

[0273] In some cases, the compound of formula IV can be one of the compounds in Table 2.

[0274] Table 2

[0275]

[0276] On the other hand, this application provides a compound of formula V, or a pharmaceutically acceptable salt thereof: R1, R2, and R4 are optional substituents, R3 is selected from the group consisting of H, optional substituted alkyl, -F3, optional substituted alkyl, -N, or O-optionally substituted alkyl, and R5 is selected from the group consisting of -COOH, -NH2, and

[0277] For example, R1 can be H. For example, R2 can be H. For example, R4 can be H.

[0278] For example, R3 can be selected from: H, CF3, CN and OCH3.

[0279] In some cases, the compound of formula V can be one of the compounds in Table 3.

[0280] Table 3

[0281]

[0282] On the other hand, this application provides a compound of formula VI, or a pharmaceutically acceptable salt thereof:

[0283]

[0284] As used herein, the term "Formula III" (or Formula IV, V, VI) can also be defined as all forms of compounds comprising "Formula III" (or Formula IV, V, VI), including hydrates, solvates, isomers, crystalline and amorphous forms, isomorphs, polymorphs, and their metabolites. For example, compounds of Formula VI or their pharmaceutically acceptable salts may exist in both non-solventized and solvated forms. When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content will depend on humidity and drying conditions. In such cases, non-stoichiometry will serve as the standard.

[0285] Compounds of Formula III (or Formula IV, V, VI) may have asymmetric carbon atoms. For example, the carbon-carbon bonds in compounds of Formula VI may be depicted herein using solid lines, solid wedges, or dashed wedges. Using solid lines to depict bonds with asymmetric carbon atoms is intended to indicate that all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) are included at that carbon atom. Using solid or dashed wedges to depict bonds with asymmetric carbon atoms is intended to indicate that only the stereoisomers shown are included. Compounds of this application may contain more than one asymmetric carbon atom. In these compounds, using solid lines to depict bonds with asymmetric carbon atoms is intended to indicate that all possible stereoisomers are included. For example, unless otherwise stated, it is intended that compounds of Formula III (or Formula IV, V, VI) may exist as enantiomers and diastereomers, or as racemic mixtures and mixtures thereof. Solid lines are used to depict bonds with one or more asymmetric carbon atoms in compounds of Formula III (or Formula IV, V, VI), and solid or dashed wedges are used to depict bonds with other asymmetric carbon atoms in the same compound, intended to indicate the presence of a mixture of diastereomers.

[0286] The compounds of this application (e.g., compounds of “Formula III” (or Formula IV, V, VI)) may exist as cage compounds or other complexes. The scope of this invention includes complexes such as cage compounds, drug-host inclusion compounds, etc., wherein the drug and host are present in stoichiometric or non-stoichiometric amounts compared to the aforementioned solvates. It also includes complexes of “Formula III” (or Formula IV, V, VI) containing two or more organic and / or inorganic components, which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of such complexes, see Haleblian, *J. Pharm. Sci.*, 64(8), 1269-1288 (August 1975).

[0287] Stereoisomers of Formula III (or Formula V, Formula V, Formula VI) include cis and trans isomers of compounds of Formula III (or Formula V, Formula V, Formula VI), optical isomers such as R and S enantiomers, diastereomers, geometric isomers, rotational isomers, conformational isomers, and tautomers, including compounds exhibiting more than one isomer type; and mixtures thereof (such as racemic and diastereomer pairs). Also included are acid addition salts or base addition salts in which the counterion is optically active (e.g., D-lactate or L-lysine) or racemic (e.g., DL-tartrate or DL-arginine).

[0288] Pharmaceutical Compositions and Uses

[0289] On the other hand, this application provides a pharmaceutical composition comprising the conjugate of this application and a pharmaceutically acceptable carrier.

[0290] In this application, the pharmaceutical composition may comprise the conjugate of this application presented by a pharmaceutically acceptable carrier. The carrier may be a solid product, a liquid, or both, and may be formulated with the compound into a unit-dose composition (e.g., a tablet) containing 0.05% to 95% of the active compound by weight. Other pharmacologically active substances may also be present.

[0291] In this application, the conjugates and / or pharmaceutical compositions of the present invention may be administered via any suitable route.

[0292] On the other hand, this application provides a method for modulating the tumor microenvironment of a subject, the method comprising administering to the subject a conjugate of this application, or a pharmaceutical composition of this application.

[0293] On the other hand, this application provides a method for modulating the immune response of a subject, the method comprising administering to the subject a conjugate of this application, or a pharmaceutical composition of this application.

[0294] On the other hand, this application provides a method for preventing and / or treating a disease in a subject in need, the method comprising administering to the subject a conjugate of this application, or a pharmaceutical composition of this application.

[0295] This method can be in vitro, ex vivo, or in vivo. For example, the conjugate of this application can be applied to one or more cells in vitro. Alternatively, the conjugate of this application can be applied to a subject in need.

[0296] On the other hand, this application provides that the conjugate of this application is used for the prevention and / or treatment of diseases in subjects in need.

[0297] On the other hand, this application provides a method for preparing a drug for treating diseases.

[0298] For example, the disease could be a tumor. For example, the tumor could be a solid tumor. For example, the tumor could be a non-solid tumor. For example, the solid tumor could include sarcomas and carcinomas. A sarcoma could refer to a tumor in a blood vessel, bone, adipose tissue, ligament, lymphatic vessel, muscle, or tendon. A carcinoma could refer to a tumor that forms in epithelial cells. Imagine the solid tumor as a non-lymphoma solid tumor. For example, the solid tumor could be named according to the cell type that forms it.

[0299] For example, the disease can include tumors and / or autoimmune diseases.

[0300] For example, autoimmune diseases can include glomerulonephritis, pulmonary hemorrhage nephritis syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjögren's syndrome, Crohn's disease, Wright's syndrome, ankylosing spondylitis, Lyme arthritis, Guillain-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

[0301] Generally, the conjugates of this application can be administered in an effective amount to treat the diseases described herein. The conjugates of this application can be in the form of pharmaceutical compositions suitable for such routes and administered via any suitable route at a dose effective for the intended treatment. The therapeutically effective dose of the conjugate required to treat the progression of a medical condition can be readily determined by those skilled in the art using preclinical and clinical methods familiar in the medical field. As used herein, the term "therapeuticly effective dose" generally refers to the amount of the conjugate being administered that will, to some extent, alleviate one or more symptoms of the treated disease.

[0302] Dosing regimens for conjugates and / or compositions containing such conjugates can be based on a variety of factors, including patient type, age, weight, sex, and medical condition; severity of condition; route of administration; and the activity of the specific compound used. Therefore, dosing regimens can vary considerably.

[0303] Suitable subjects according to the present invention include mammalian subjects. For example, the subject can be a mammal, such as a human.

[0304] On the other hand, this application provides a diagnostic reagent comprising the conjugate of this application.

[0305] For example, the diagnostic reagent can be labeled.

[0306] For example, the label can be selected from: radioactive labels, fluorophores, chromophores, imaging agents, and metal ions.

[0307] On the other hand, this application provides the following implementation methods:

[0308] 1. A conjugate of Formula 1, M-[(L1)] a -(L2) b -(D) c ]1,

[0309] Where L1 is a compound of formula I, R is -F or -OH.

[0310] M is a biological macromolecule, and M is linked to L1 using its nucleophilic functional group.

[0311] L2 is a connector, and L2 is connected to R1, R3, or R2.

[0312] D is a functional molecule.

[0313] a is an integer from 1 to 10.

[0314] b and c are each independent integers from 0 to 10, provided that b and c are not both 0 at the same time.

[0315] Wherein R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0316] Where R 1' It is H or its isotopes.

[0317] Wherein R2 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0318] Wherein R3 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0319] Optionally, the C connecting R1 and the C connecting R2 form a loop.

[0320] 2. The conjugate according to claim 1, wherein the nucleophilic functional group of M is selected from the group consisting of -SH, -NH2, -SeH, -OH, and

[0321] 3. A conjugate of formula 2,

[0322] MS-[(L1)a-(L2)b-(D)c]2,

[0323] MS is a biomolecule containing cysteine, and MS is linked to L1 using the cysteine, where L1 is a compound of formula I. R is -F or -OH.

[0324] L2 is a connector, and L2 is connected to R1, R3, or R2.

[0325] D is a functional molecule.

[0326] a is an integer from 1 to 10.

[0327] b and c are each independent integers from 0 to 10, provided that b and c are not both 0 at the same time.

[0328] Wherein R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0329] Where R 1' It is H or its isotopes.

[0330] Wherein R2 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0331] Wherein R3 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0332] Optionally, the C connecting R1 and the C connecting R2 form a loop.

[0333] 4. The conjugate according to any one of claims 1 to 3, wherein M is selected from the group consisting of proteins, DNA, RNA, and viruses.

[0334] 5. The conjugate according to any one of claims 1 to 4, wherein M is a biomacromolecule expressed on the cell surface.

[0335] 6. The conjugate according to any one of claims 1 to 5, wherein M is an antigen-binding protein or a fragment thereof.

[0336] 7. The conjugate according to any one of claims 1 to 6, wherein M is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody scFv, or antibody fragment.

[0337] 8. The conjugate according to any one of claims 1 to 7, wherein L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0338] 9. The conjugate according to any one of claims 1 to 8, wherein L2 is selected from the group consisting of: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG. NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC or 2,5-dioxopyrrolidine-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0339] 10. The conjugate according to any one of claims 1 to 9, wherein D has a biological function.

[0340] 11. The conjugate according to any one of claims 1 to 10, wherein D and / or its derivatives are capable of inhibiting the growth of tumor cells.

[0341] 12. The conjugate according to any one of claims 1 to 11, wherein D is a drug.

[0342] 13. The conjugate according to any one of claims 1 to 12, wherein D is selected from: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0343] 14. The conjugate according to any one of claims 1 to 13, wherein D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; new interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0344] 15. The conjugate according to any one of claims 1 to 14, wherein R 1' It is -H.

[0345] 16. The conjugate according to any one of claims 1 to 15, wherein R1 is -H.

[0346] 17. The conjugate according to any one of claims 1 to 16, wherein R3 is -H.

[0347] 18. The conjugate according to any one of claims 1 to 17, wherein R2 is an optionally substituted phenyl group.

[0348] 19. The conjugate according to any one of claims 1 to 18, wherein R2 is R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0349] 20. The conjugate according to any one of claims 1 to 19, wherein R2 is R4 is selected from: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0350] 21. The conjugate according to any one of claims 1 to 18, wherein R2 is Where R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 is selected from the following group: And H.

[0351] 22. The conjugate according to any one of claims 1 to 18, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, where R7 is selected from:

[0352] 23. The conjugate according to any one of claims 1 to 18, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 is selected from the following group:

[0353] 24. The conjugate according to any one of claims 1 to 18, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and R9 is selected from the following group: H and

[0354] 25. The conjugate according to any one of claims 1 to 18, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and

[0355] 26. The conjugate according to any one of claims 1 to 18, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0356] 27. The conjugate according to any one of claims 1 to 18, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Selected from: -CF3, -CN, and -OCH3.

[0357] 28. The conjugate according to any one of claims 1 to 18, wherein R2 is an optionally substituted alkyl group -CH=CH-R 12 , where R 12 for Where R 13 It is -CH2N3.

[0358] 29. The conjugate according to any one of claims 1 to 28, wherein the ring is an optionally substituted cycloalkene or an optionally substituted aryl-cycloalkene.

[0359] 30. The conjugate according to any one of claims 1 to 29, wherein the ring is selected from the group consisting of:

[0360] 31. The conjugate according to any one of claims 1 to 30, wherein L1 is selected from the group consisting of:

[0361]

[0362]

[0363]

[0364] 32. The conjugate according to any one of claims 1 to 31, wherein the conjugate is selected from the group consisting of:

[0365]

[0366]

[0367]

[0368] 33. A conjugate of formula 3,

[0369] (L1) a -(L2) b -(D) c 3,

[0370] Where L1 is a compound of formula III,

[0371]

[0372] L2 is a connector, and L2 is connected to R1, R3, or R2.

[0373] D is a functional molecule.

[0374] a is an integer from 1 to 10.

[0375] b and c are each independent integers from 0 to 10, provided that b and c are not both 0 at the same time.

[0376] Wherein R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0377] Wherein R2 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0378] Wherein R3 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0379] Optionally, the C connecting R1 and the C connecting R2 form a loop.

[0380] 34. The conjugate according to claim 33, wherein L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0381] 35. The conjugate according to any one of claims 33 to 34, wherein L2 is selected from the group consisting of: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG. NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidone-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0382] 36. The conjugate according to any one of claims 33 to 35, wherein D has a biological function.

[0383] 37. The conjugate according to any one of claims 33 to 36, wherein D and / or its derivatives are capable of inhibiting the growth of tumor cells.

[0384] 38. The conjugate according to any one of claims 33 to 37, wherein D is a drug.

[0385] 39. The conjugate according to any one of claims 33 to 38, wherein D is selected from the group consisting of: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0386] 40. The conjugate according to any one of claims 33 to 39, wherein D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; new interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0387] 41. The conjugate according to any one of claims 33 to 40, wherein R1 is -H.

[0388] 42. The conjugate according to any one of claims 33 to 41, wherein R3 is -H.

[0389] 43. The conjugate according to any one of claims 33 to 42, wherein R2 is an optionally substituted phenyl group.

[0390] 44. The conjugate according to any one of claims 33 to 43, wherein R2 is R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0391] 45. The conjugate according to any one of claims 33 to 43, wherein R2 is R4 is selected from: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0392] 46. ​​The conjugate according to any one of claims 33 to 43, wherein R2 is Where R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 is selected from the following group: and -H.

[0393] 47. The conjugate according to any one of claims 33 to 43, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, where R7 is selected from:

[0394] 48. The conjugate according to any one of claims 33 to 43, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 is selected from the following group:

[0395] 49. The conjugate according to any one of claims 33 to 43, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and R9 is selected from the following group: H and

[0396] 50. The conjugate according to any one of claims 33 to 43, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and

[0397] 51. The conjugate according to any one of claims 33 to 43, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0398] 52. The conjugate according to any one of claims 33 to 43, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Select from the following groups: -CF3, -CN, and -OCH3.

[0399] 53. The conjugate according to any one of claims 33 to 43, wherein R2 is an optionally substituted alkyl group -CH=CH-R. 12 , where R 12 for Where R 13 It is -CH2N3.

[0400] 54. The conjugate according to any one of claims 33 to 53, wherein the ring is an optionally substituted cycloalkene or an optionally substituted aryl-cycloalkene.

[0401] 55. The conjugate according to any one of claims 33 to 54, wherein the ring is selected from:

[0402] 56. The conjugate according to any one of claims 33 to 55, wherein the L1 is selected from the group consisting of:

[0403]

[0404]

[0405]

[0406] 57. The conjugate according to any one of claims 33 to 56, wherein the conjugate is selected from the group consisting of:

[0407]

[0408]

[0409] 58. A method for preparing a conjugate, the method comprising the following steps:

[0410] By making the conjugate of formula 3: Combined with M, the conjugate of formula 1 is obtained:

[0411] M is a biological macromolecule, and M is linked to L1 using its nucleophilic functional group. L2 is a linker, and L2 is linked to R1, R3, or R2 in Formula 1.

[0412] D is a functional molecule.

[0413] a is an integer from 1 to 10.

[0414] b and c are each independent integers from 0 to 10, provided that b and c are not both 0 at the same time.

[0415] Wherein R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0416] Where R 1' It is H or its isotopes.

[0417] Wherein R2 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0418] Wherein R3 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0419] Optionally, the C connecting R1 and the C connecting R2 form a loop.

[0420] 59. The method of claim 58, wherein the nucleophilic functional group of M is selected from the group consisting of -SH, -NH2, -SeH, -OH, and

[0421] 60. A method for preparing a conjugate, the method comprising the following steps:

[0422] By making the conjugate of formula 3: Combined with M, we obtain the conjugate of formula 2: R is -OH or -F.

[0423] MS is a biomolecule containing cysteine, and MS utilizes the cysteine ​​to link with L1.

[0424] L2 is a connector, and L2 is connected to R1, R3, or R2 in Equation 3.

[0425] D is a functional molecule.

[0426] a is an integer from 1 to 10.

[0427] b and c are each independent integers from 0 to 10, provided that b and c are not both 0 at the same time.

[0428] Wherein R1 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0429] Where R 1' It is H or its isotopes.

[0430] Wherein R2 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0431] Wherein R3 is H, an optionally substituted alkyl group, or an optionally substituted aryl group.

[0432] Optionally, the C connecting R1 and the C connecting R2 form a loop.

[0433] 61. The method according to any one of claims 58 to 60, wherein M is selected from the group consisting of proteins, DNA, RNA, and viruses.

[0434] 62. The method according to any one of claims 58 to 61, wherein M is a biomolecule expressed on the cell surface.

[0435] 63. The method according to any one of claims 58 to 62, wherein M is an antigen-binding protein or a fragment thereof.

[0436] 64. The method according to any one of claims 58 to 63, wherein M is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody scFv, or antibody fragment.

[0437] 65. The method according to any one of claims 58 to 64, wherein M comprises a functional group for nucleophilic addition reactions.

[0438] 66. The method according to any one of claims 58 to 65, wherein L2 is selected from: cuttable linkers, non-cuttable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0439] 67. The method according to any one of claims 58 to 66, wherein L2 is selected from: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG. NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidone-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

[0440] 68. The method according to any one of claims 58 to 67, wherein D has a biological function.

[0441] 69. The method according to any one of claims 58 to 68, wherein D and / or its derivatives are capable of inhibiting the growth of tumor cells.

[0442] 70. The method according to any one of claims 58 to 69, wherein D is a drug.

[0443] 71. The method according to any one of claims 58 to 70, wherein D is selected from: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives and DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists and STING agonists.

[0444] 72. The method according to any one of claims 58 to 71, wherein D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; new interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0445] 73. The method according to any one of claims 58 to 72, wherein R1 is -H.

[0446] 74. The method according to any one of claims 58 to 73, wherein R3 is -H.

[0447] 75. The method according to any one of claims 58 to 74, wherein R2 is an optionally substituted phenyl group.

[0448] 76. The method according to any one of claims 58 to 75, wherein R2 is R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0449] 77. The method according to any one of claims 58 to 75, wherein R2 is R4 is selected from the following groups: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0450] 78. The method according to any one of claims 58 to 75, wherein R2 is Where R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 is selected from the following group: and -H.

[0451] 79. The method according to any one of claims 58 to 75, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, where R7 is selected from the following group:

[0452] 80. The method according to any one of claims 58 to 75, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 is selected from the following group:

[0453] 81. The method according to any one of claims 58 to 75, wherein R2 is Where R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and R9 is selected from the following group: H and

[0454] 82. The method according to any one of claims 58 to 75, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and

[0455] 83. The method according to any one of claims 58 to 75, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Selected from the following group: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0456] 84. The method according to any one of claims 58 to 75, wherein R2 is Where R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Selected from: -CF3, -CN, and -OCH3.

[0457] 85. The method according to any one of claims 58 to 75, wherein R2 is an optionally substituted alkyl group -CH=CH-R. 12 , where R 12 for Where R 13 It is -CH2N3.

[0458] 86. The method according to any one of claims 58 to 85, wherein the ring is an optionally substituted cycloalkene or an optionally substituted aryl-cycloalkene.

[0459] 87. The method according to any one of claims 58 to 86, wherein the ring is selected from:

[0460] 88. The method according to any one of claims 58 to 87, wherein the method is performed at a temperature in the range of about 16°C to about 37°C.

[0461] 89. The method according to any one of claims 58 to 88, wherein the method is carried out at a pH in the range of about 7.4 to about 8.

[0462] 90. The method according to any one of claims 58 to 89, wherein the method is carried out with a catalyst.

[0463] 91. The method according to any one of claims 58 to 90, wherein the method further comprises the step of purifying the conjugate of formula 3.

[0464] 92. A compound of formula III, or a pharmaceutically acceptable salt thereof: R1 is selected from the following group: -OH, -PO3H2, -SeH, -SH, optional substituted alkyl-OH, optional substituted alkyl-halogen, optional substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optional substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0465] 93. The compound according to claim 92, wherein R1 is selected from the group consisting of -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0466] 94. The method according to any one of claims 92 to 93, wherein the compound is selected from the group consisting of:

[0467]

[0468]

[0469] 95. A compound of formula IV, or a pharmaceutically acceptable salt thereof:

[0470]

[0471] R1 is selected from the following groups: NH-(CH2)n1-CO-R2, -OH, NH-(CH2) n2 -R3、NH-(CH2CH2-O) n3 -(CH2)n4-NH-CO-O-R4, or

[0472] Where n1, n2, n3, or n4 are independent integers from 1 to 10.

[0473] R2 is selected from the following group:

[0474] R3 is selected from the following group:

[0475] Where R4 is

[0476] 96. The compound according to claim 95, wherein R1 is selected from the group consisting of NH-(CH2). 2 -CO-R2, -OH, NH-(CH2)-R3, NH-(CH2CH2-O)3-(CH2)2-NH-CO-O-R4.

[0477] 97. The method according to any one of claims 95 to 96, wherein the compound is selected from the group consisting of: 97

[0478]

[0479] 98. A compound of formula V, or a pharmaceutically acceptable salt thereof:

[0480] R1, R2, and R4 are arbitrary substituents.

[0481] R3 is selected from the following group: H, optionally substituted alkyl-F3, optionally substituted alkyl-N or O-optionally substituted alkyl.

[0482] R5 is selected from the following groups: -COOH, -NH2, and

[0483] 99. The compound according to claim 98, wherein R1 is H.

[0484] 100. The compound according to any one of claims 98 to 99, wherein R2 is H.

[0485] 101. The compound according to any one of claims 98 to 100, wherein R4 is H.

[0486] 102. The compound according to any one of claims 98 to 101, wherein R3 is selected from the group consisting of H, CF3, CN and OCH3.

[0487] 103. The compound according to any one of claims 98 to 102, wherein the compound is selected from:

[0488] 104. A compound of formula VI, or a pharmaceutically acceptable salt thereof:

[0489] 105. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 57 and a pharmaceutically acceptable carrier.

[0490] 106. A method for modulating the tumor microenvironment of a subject, the method comprising administering to the subject the conjugate of any one of claims 1 to 57, or the pharmaceutical composition of claim 105.

[0491] 107. A method for modulating an immune response in a subject, the method comprising administering to the subject the conjugate of any one of claims 1 to 57, or the pharmaceutical composition of claim 105.

[0492] 108. A method for preventing and / or treating a disease in a subject in need, the method comprising administering to the subject the conjugate of any one of claims 1 to 57, or the pharmaceutical composition of claim 105.

[0493] 109. The method of claim 108, wherein the disease includes tumors and / or autoimmune diseases.

[0494] 110. The method of claim 109, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

[0495] 111. A diagnostic reagent comprising the conjugate according to any one of claims 1 to 57.

[0496] 112. The diagnostic reagent of claim 111, wherein the diagnostic reagent is labeled.

[0497] 113. The diagnostic reagent according to claim 112, wherein the label is selected from: radioactive labels, fluorophores, chromophores, imaging agents, and metal ions.

[0498] Example

[0499] The following embodiments are provided to provide a complete disclosure and description of how to make and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but certain experimental errors and biases should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used, such as bp: one or more base pairs; kb: one thousand or several thousand bases; pl: one or several picoli; s or sec: one or several seconds; min: one or several minutes; h or hr: one or several hours; aa: one or more amino acids; nt: one or more nucleotides; im: intramuscular; ip: intraperitoneal; sc: subcutaneous; etc.

[0500] Silica gel column chromatography

[0501] Silica gel column chromatography was performed using silica gel 60 (200-300 mesh). Analytical thin-layer chromatography (TLC) was performed using silica gel (silica gel 60F254). TLC used short-wave ultraviolet light as a visualization agent, KMnO4 and heat energy as developing agents, and was performed on pre-coated silica gel plates.

[0502] SSF-DNA Synthesis

[0503] To an Eppendorf tube containing 5'-NH2-20 / 59nt ssDNA (50 μM final concentration, 1 equivalent) in PBS (50 mM, pH 8.0), add SSF-NHS (100 equivalent, 100 mM in DMF) and DMF. Vortex the reaction mixture and shake at 30°C. After incubation overnight, analyze the reaction mixture by LC-MS.

[0504] Mal-DNA Synthesis

[0505] Add SSF-NHS (100 equivalents, 100 mM in DMF) and DMF to an Eppendorf tube containing 5'-NH2-20 (50 μM final concentration, 1 equivalent) in PBS (50 mM, pH 8.0). Vortex the reaction mixture and shake at 30 °C. After incubation overnight, analyze the reaction mixture by LC-MS.

[0506] Hydrolytic stability of SSF-DNA and Mal-DNA

[0507] SSF-20nt ssDNA / Mal-20nt ssDNA (final concentration 100 μM) was incubated in PBS (50 mM, pH 7.4) with shaking at 37 °C for 48 h. 20 μL of the reaction mixture was analyzed by LC-MS.

[0508] Synthesis of protein-DNA conjugates

[0509] SSF / Mal-DNA (56 μL, 450 mM in H2O) and PBS (10 μL, pH 8.0, 50 mM) were added to a solution of Nb-PD-L1 or other proteins (50 μL, 100 μM, HEPES buffer). The reaction mixture was incubated at 37 °C for 12 h to produce homogeneous protein-DNA conjugates.

[0510] LC-MS

[0511] LC-MS analysis of protein conjugation and protein-DNA (GFP-20ntssDNA, neo2-20ntssDNA): LC-MS was performed on an Acquity UPLC protein BEH C4 column (1.7 mm, 2.1 × 50 mm) on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system. The mobile phases were solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) at a flow rate of 0.5 mL / min. The gradient used was: no gradient 95% H2O for 2 min, then 95% to 10% H2O over 4 min, then 10% H2O over 1 min, then 10% to 95% H2O over 1 min, and then 95% H2O for 2 min. The electrospray ionization source was operated in positive ion mode with a capillary voltage of 2.0 kV and an orifice voltage of 40 V. Nitrogen was used as the desolventizing gas at a total flow rate of 850 L / h. Following the manufacturer's instructions, the total mass spectrum was reconstructed from the ion series using the MaxEnt algorithm (version 4.1, from Waters) pre-installed on MassLynx software. To obtain the ion series, the dominant peak in the chromatogram was selected for integration and further analysis.

[0512] LC-MS analysis of SSF-DNA, Mal-DNA, and Nb-PD-L1-ssDNA: LC*MS was performed on an Acquity UPLC protein BEH C8 column (1.7 mm, 2.1 × 50 mm) on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system. Solvent A (10 mM ammonium formate aqueous solution) and solvent B (100% methanol) were used as the mobile phases at a flow rate of 0.5 mL / min. The gradient used was: no gradient 95% H2O for 2 min, then 95% to 5% H2O over 4 min, then 5% H2O over 1 min, then 5% to 95% H2O over 1 min, and then 95% H2O for 2.5 min. The electrospray ionization source was operated in negative mode with a capillary voltage of 2.0 kV and an orifice voltage of 80 V. Nitrogen was used as the desolventizing gas at a total flow rate of 850 L / h. According to the manufacturer's instructions, the total mass spectrum was reconstructed from the ion series using the MaxEnt algorithm (version 4.1, from Waters) pre-installed on MassLynx software. To obtain the ion series, the dominant peak of the chromatogram was selected for integration and further analysis.

[0513] LC-MS analysis of peptide-MA conjugation: LC*MS was performed on a Xevo SQDetector 2 mass spectrometer coupled with an Acquity UPLC BEH300 C18 column (1.7 mm, 2.1 × 50 mm). Solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile) were used as the mobile phases at a flow rate of 0.4 mL / min. Method A: Gradient used: No gradient, 90% H2O for 2 min, then 90% to 10% H2O over 5 min, then 10% H2O for 1 min, then 10% to 90% H2O over 1 min, then 95% H2O for 1 min. Method B: Gradient used: No gradient, 90% H2O for 2 min, then 90% to 70% H2O over 15 min, then 70% to 10% H2O for 20 min, then 10% to 90% H2O over 1 min, then 95% H2O for 2 min. The electrospray ionization source was operated in positive ion mode with a capillary voltage of 2.0 kV and an orifice voltage of 40 V. Nitrogen was used as the desolventizing gas at a total flow rate of 850 L / h. The total mass spectrum was reconstructed from the ion series using the MaxEnt algorithm (version 4.1, from Waters) pre-installed on MassLynx software, according to the manufacturer's instructions. To obtain the ion series, the dominant peak of the chromatogram was selected for integration and further analysis.

[0514] LC-MS / MS analysis of protein conjugation: For in-gel digestion, the labeled GFP was first broken down by SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. The GFP band was removed, the gel was cut into small particles, and transferred to pre-cleaned microcentrifuge tubes. The resulting gel particles were desalted twice with 50% ACN in 25 mM ammonium bicarbonate (ABC), and then dehydrated in ACN. The gel particles were rehydrated with 20 mM DTT in 25 mM ABC and incubated at 55 °C for 45 min. The gel particles were washed with 25 mM ABC and dehydrated again with acetonitrile, and then incubated in 55 mM iodoacetamide in 25 mM ABC at room temperature in the dark for 30 min. The treated gel particles were washed with 25 mM ABC and dehydrated again with acetonitrile. Then, the gel particles were rehydrated with trypsin solution (20 ng / μL) and incubated at 37 °C for 16 h. To extract trypsin peptides, gel particles were immersed in an ACN / water / FA solution (v:v:v = 50:45:5) and vortexed for 30 min. The solution was carefully removed, and the extraction was repeated once. The extracts were combined and dried in a vacuum centrifuge. LC-MS / MS was performed on an Orbitrap Fusion Lumosmass spectrometer (Thermo Fisher Scientific) coupled with an Easy-nLC 1200LC system. The peptide sample was loaded onto an analytical column (1.9 μm). C18 (250 mm * 75 μm inner diameter) and eluted with a gradient for 65 min. The mass spectrometer was run in data-dependent mode. Full-scan spectra were acquired in the 350–1500 m / z range using an Orbitrap mass analyzer. MS / MS fragmentation was performed using HCD mode. The normalized collision energy was 30 V. Raw data were analyzed using Pfind3 and searched for bovine proteomes in the UniProt database. Cysteine ​​carbamoyl methylation was set as a fixed modification. Methionine oxidation and cysteine ​​residue modification were set as variable modifications. Peptides modified with 2b-1a or 2b+1a were considered correctly identified when the score (PSM score, peptide match score) was higher than 26, and the modified sites were manually verified.

[0515] NMR

[0516] NMR measurements were performed on a Bruker AVANCE III-400 or 500 spectrometer in deuterium chloroform (CDCl3). 1 H NMR and 13 C NMR spectra were recorded on 400 MHz or 500 MHz and 100 MHz or 125 MHz spectrometers, respectively. 19FNMR spectra were recorded on a 376 MHz or 470 MHz spectrometer. Chemical shifts were reported relative to internal TMS (δ 0.00 for 1H NMR), chloroform ( 1 H NMR (δ 7.26) and chloroform ( 13 The δ value for C NMR is δ77.00. The following abbreviations are used for multiplicity: s: singlet, d: doublet, dd: doublet doublet, t: triplet, q: quartet, m: multiplet, br: broad signal in the proton spectrum. Mass spectrometry was performed using ESI-MS (LCQ Fleet, Thermo Fisher Scientific).

[0517] Cell barcoding and scRNA-seq

[0518] A mixture of Jurkat, A549, JIMT-1, and MDA-MB-231 cells was stained with Nb-PD-L1-59ntssDNA at 4°C for 30 min. After washing and assessing cell number and viability, the cells were pooled and loaded onto Singleton cells. The scRNA-seq library was prepared according to the manufacturer's instructions (Syngene (Nanjing) Biotechnology Co., Ltd., Nanjing, China) using a microarray targeting 20,000 cells. After amplification, cDNA and the Nb-PD-L1-59ntssDNA tag were separated using SPRI size selection (SPRI 0.6× and 1.4×). The Nb-PD-L1-59ntssDNA tag library was quantified (Qubit, Invitrogen) and amplified using primers SGR-bead-1 / SGR-tag-1, and indexed by additional PCR using primers SGR-bead-2 / SGR-tag-2. Finally, the Nb-PD-L1-59ntssDNA tag library and transcriptome library were analyzed using a BioAnalyzer High Sensitivity DNA Kit (Agilent) and sequenced on an Illumina NovaSeq6000.

[0519] Primers for preparing Nb-PD-L1 labeled libraries

[0520]

[0521] protein sequence

[0522] Engineered Neo2 sequence: Engineered Neo2 consists of 124 amino acids and 1 free cysteine. MLVNRICGKGIDGGSPKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILE EIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFSAVDHH HHHH (SEQ ID NO.5)

[0523] Engineered Nb-Pd-L1 sequence: Engineered Nb-Pd-L1 consists of 143 amino acids and 1 free cysteine. MDQVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTS GSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGA FQYWGQGTQVTVSSLPETGGCHHHHHH (SEQ ID NO. 6)

[0524] GFP sequence: Engineered GFP consists of 243 amino acids and 3 free cysteine ​​residues. MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRICLKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGGGGLEHHHHHHH (SEQ ID NO.7)

[0525] Trastuzumab light chain: DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.8);

[0526] Trastuzumab heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESLTQPEGKNNTTKPPKVSNKALPAPIEKTISKSRKQPREPQVYTLPPSREEMTKNQVSLTCLVKSLKSPK(SEQ ID NO.9)

[0527] The amino acid sequences of the light and heavy chains of KN026 can be found in US2018 / 0291103, and the amino acid sequences of the light and heavy chains of KN046 can be found in US20210095031A1.

[0528] DNA sequence

[0529] 20ntssDNA: 5'-NH2-C6-AGC AGC ACA GAG GTC AGA TG (SEQ ID NO.10)

[0530] 59ntssDNA: 5'-NH2-C6-TGT CAA GAT GCT ACC GTT CAG AGC GCA AGA CAC TCCACA AAA AAA AAA AAA AAA AAA*A*A*Thiomodification (SEQ ID NO.11)

[0531] Data Analysis

[0532] Raw sequence readings were processed using the CeleScope pipeline (version 1.3.1) with default parameters (https: / / github.com / singleron-RD / CeleScope). The Nb-PD-L1-59ntssDNA tag library was processed using CeleScope's new barcode processing plugin ("teg"), which is inspired by previous scRNA-seq multiplexing algorithms. Gene expression matrix was then analyzed using R.

[0533] GFP modified with MA

[0534] MA (1 μL, 20 mM in DMSO) and PBS (15 μL, pH 7.4 / 9.0, 50 mM) were added to GFP solution (4 μL, 250 μM, HEPES buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain GFP-MA.

[0535] Linker stability study

[0536] GSH (0.8 μL, 100 mM) and PBS (20 μL, pH 8.0, 50 mM) were added to the GFP-linker solution (20 μL, 40 μM, PBS solution) at 37 °C and kept for 48 h. Figure 20 )

[0537] Example 1: Screening Michael acceptors as connectors

[0538] Michael acceptors are commonly used reagents for addition reactions of cysteine ​​with antibodies. A novel Michael acceptor has been investigated to demonstrate its efficient and chemoselective reaction with cysteine ​​on antibodies. Furthermore, modification of the Michael acceptor with antibodies did not result in serum degradation. Based on this, candidate Michael acceptors were used as reaction reagents, with green fluorescent protein (GFP) serving as a template protein for screening. Considering reactivity, chemoselectivity, and stability, Michael acceptors capable of highly efficient reactions with cysteine ​​were screened.

[0539] The reaction process is shown in Figure 1 Furthermore, the chemical structures of the following Michael acceptor candidates are in... Figure 2 It is displayed in the middle.

[0540] Table 4. Screening of Michael's acceptors

[0541]

[0542] 1Reaction conditions: sfGFP E124C (320 μM, 5 μL), Michael acceptor (6.4 μL, 5 mM), PBS buffer (40 μL, pH=8), 37℃, 2 h. 2 The bonus number has no affiliation.

[0543] The Michael acceptor MA 2 was named Reagent 1 or PhESF.

[0544] Example 2: Modification of Connectors

[0545] Reagent 1 in Modification Example 1 (the chemical structure of the modified Michael acceptor candidate is as follows) Figure 3 (as shown in the image).

[0546] The effect of different substituents on the addition reaction (e.g., addition activity) was investigated when the benzene ring of reagent 1 was modified with different substituents (Michael acceptors 1-1 to 1-4) (Table 5). Michael acceptors 1-1 to 1-4 reacted with sfGFP-TEV. When the para-thiofluorine group was attached to the strongly electron-withdrawing trifluoromethoxy group, the addition activity was significantly reduced (item 4); when the electron-donating methoxy and hydroxymethyl groups were attached to the para-thiofluorine group and the electron-withdrawing trifluoromethyl group was attached to the meta-position, the addition activity did not change significantly (items 2-3, item 5). When the sulfonyl fluoride group was attached to allyl 1-5, the addition activity was significantly reduced (item 6).

[0547] The reaction process is visible Figure 4 .

[0548] Table 5: Modification of sfGFP-TEV by the derivative of reagent 1

[0549]

[0550] Example 3: Modification of Connectors

[0551] Using different configurations of alkenyl sulfonyl fluoride (the chemical structure of the modified Michael acceptor candidate is in...) Figure 5 The effects of different configurations of olefin sulfonyl fluoride on addition reactions were investigated (Table 6). When 5 equivalents of the Michael acceptor were used to react with sfGFP E124C, Michael acceptors 1, 1a, 1b, and 1a-1 all exhibited good chemoselectivity; however, Michael acceptor 1b-1 reacted with both cysteine ​​and lysine. When 20 equivalents of the Michael acceptor were used to react with sfGFP E124C, only Michael acceptor 1 showed good chemoselectivity.

[0552] The reaction process is visible Figure 6 .

[0553] Table 6

[0554]

[0555] Example 4: Characteristics of the selected connector

[0556] 4.1 Selectivity

[0557] To further verify the reactivity, chemoselectivity, and stability of Michael acceptor 1, linkers reported in previous literature (e.g., linkers used in ADCs) were selected as comparative examples to demonstrate that Michael acceptor 1 has good reactivity selectivity.

[0558] The previously reported chemical structure of the linker is in Figure 7 The reaction process between sfGFP E124C and the previously reported linker is shown in the figure. Figure 8 The reaction results are in Figure 9 The reaction conditions are as follows: sfGFP E124C (320 μM, 5 μL), linker (5 mM, 6.4 μL), PBS buffer (40 μL, pH=8), 37℃, 2 h.

[0559] 4.2 Competitiveness

[0560] To verify their differences in reactivity, comparative examples were conducted in a competitive test. Figure 10 The results showed that Michael acceptor 1 was moderately reactive: this means that it was more reactive than alkenylsulfonamide and alkynyl phosphate, but less reactive than maleimide (5) and carbonylacrylamide (4) (Table 7).

[0561] Table 7

[0562] Item 1 reagents Ratio (sfGFP E124C-1:sfGFP E124C-x) 1 1,2 sfGFP E124C-1:sfGFP E124C-2>99:1 2 1,3 sfGFP E124C-1:sfGFP E124C-3=6:1 3 1,4 sfGFP E124C-1:sfGFP E124C-4=1:5 4 1,5 sfGFP E124C-1:sfGFP E124C-5<1:99

[0563] 1 Reaction conditions: sfGFP E124C (320 μM, 5 μL), 1 (5 mM, 3.2 μL), x (5 mM, 3.2 μL), PBS buffer (40 μL, pH=8), 37℃, 2 h.

[0564] 4.3 Stability

[0565] Linkers 5 and 4, which exhibited higher reactivity, were used as controls to verify the stability of sfGFP-modified PhESF as a linker. After modification, sfGFP was added to a buffer containing GSH and incubated at 37°C for different times, followed by mass spectrometry analysis. The mass spectrometry results showed that linkers 4 and 5, containing modified sfGFP, underwent significant thiol exchange. However, under the same conditions, Michael acceptor 1 did not undergo thiol exchange. Figure 11 a to Figure 11c).

[0566] Example 5: Generation of conjugates containing linkers and antibodies

[0567] Using Michael acceptor 1 modified antibodies (e.g., Herceptin) Figure 12 a to Figure 12 b), and verify that modification of the antibody does not affect its binding ability to the antigen. Figure 12 c).

[0568] Example 6: Further modification of the connector

[0569] Michael acceptor 1 has no other modifiable groups. Therefore, Michael acceptor 1 was modified to have an azide group, and then the modified Michael acceptor 1 antibody was used, and the stability of the conjugated antibody was tested in serum.

[0570] Mass spectrometry analysis revealed that the modified antibody did not undergo elimination in serum after 7 days. Figure 13 a to Figure 13 C). In Figure 13 middle, Figure 13 a shows Herceptin chemoselectively modified with N3-PhESF; Figure 13 b shows the mass spectrometry results of the N3-PhESF modified antibody; Figure 13 c shows the results of Herceptin-PhESF-N3 in the serum stability test.

[0571] Example 7: Generation of conjugates containing linkers and drugs

[0572] Michael acceptor 1 was used as a linker to synthesize the ADC. To improve the solubility of Michael acceptor 1, polyethylene glycol was modified onto it. Figure 14a Then, the toxin MMAE is conjugated to a cleavable VC-PAB ( Figure 14b ), and finally undergo a condensation reaction to obtain the ADC: PhESF-PEG4-MMAE( Figure 14c ).

[0573] Example 8: Generating an ADC

[0574] 8.1DAR = 3.2

[0575] The PhESF-PEG4-MMAE obtained in Example 7 was reacted with the reduced antibody to synthesize an ADC (Herceptin-PhESF-PEG4-MMAE) with a DAR of approximately 3.2. As a comparative example, an ADC (Herceptin-Mal-MMAE) with a DAR of approximately 3.2 was also synthesized.

[0576] First, the stability of the two ADCs in serum was studied, and it was found that the DAR value of the ADC using Michael acceptor 1 as a linker did not decrease significantly after 7 days, while the DAR value of the ADC using maleimide decreased by 70%. Figures 15a to 15c ).

[0577] Figures 15a to 15c The results of the ADC stability test are shown. Figure 15a The results of ADC stability testing in human serum are shown; Figure 15b The mass spectrometry results of Herceptin-PhESF-PEG4-MMAE in serum at different time points are shown; Figure 15c The mass spectrometry results of Herceptin-PhESF-PEG4-MMAE in serum at different time points are shown.

[0578] 8.2 DAR = 3.8

[0579] The DAR value of ADCs using MMAE as the drug is generally no more than 4. To enhance the killing effect, an ADC with a DAR value of approximately 3.8 was synthesized and verified using mass spectrometry. Figure 16 a to Figure 16 b). As a comparative example, an ADC (Herceptin-Mar-MMAE) with a DAR of approximately 3.8 was also synthesized.

[0580] Figure 16 a shows the structure and mass spectrometry results of Herceptin-PhESF-PEG4-MMAE; Figure 16 b shows the structure and mass spectrometry results of Herceptin-Mal-MMAE.

[0581] Example 9: Tumor Cell Killing Assay

[0582] Tumor cell killing assays were performed using two ADCs (Herceptin-PhESF-PEG4-MMAE and Herceptin-Mal-MMAE prepared in Example 8). The results showed that both ADCs were effective against HER2. + The killing efficiency of cells SKBR3, NCI-N87, and MDA-MB-435 was similar. Figure 17 a to Figure 17 c).

[0583] Herceptin-PhESF-PEG4-MMAE on IC50 of 3 cell types 50 The values ​​were 18.3 ng / mL, 7.76 ng / mL, and 14.66 ng / mL, respectively, and the IC50 values ​​of Herceptin-Mal-MMAE were 24.48 ng / mL, 10.94 ng / mL, and 12.03 ng / mL, respectively.

[0584] For HER -Cellular MDA-MB-231 and HER - Cellular MDA-MB-435, neither of the two ADCs showed a significant killing effect. Figure 17 d to Figure 17 e).

[0585] Herceptin-Mal-MMAE was found to significantly kill HER2 at high concentrations. - Cells, while Herceptin-PhESF-PEG4-MMAE did not show significant killing effect at high concentrations ( Figure 17 f).

[0586] Then, bystander lethality tests were conducted on the ADC, and it was found that stable Michael acceptor 1 did not affect its bystander lethality. Figure 17 g).

[0587] Figure 17 a to Figure 17 h shows the results of the tumor cell killing assay. Figure 17 a shows the killing effect on SKBR3 cells. Figure 17 b shows the killing effect on NCI-N87 cells; Figure 17 c shows the HER + The killing effect of MDA-MB-435 cells; Figure 17 d shows the HER - The killing effect of MDA-MB-435 cells; Figure 17 e shows the killing effect on MDA-MB-231 cells; Figure 17 f shows the killing effect of relatively high concentrations of ADC on MDA-MB-231 cells; Figure 17 g shows the results of the ADC bystander kill experiment, and Figure 17 h shows the killing effect of ADC at a concentration of 10 μg / mL on MDA-MB-231 cells.

[0588] Example 10: Chemical Synthesis and Analysis

[0589] Example 10.1: Synthesis of SSF-PEG4-PAB-MMAE 1

[0590]

[0591] Compound S2 was synthesized based on a previously disclosed procedure. 1H NMR (400MHz, CDCl3): δ7.52(2H,d,J=8.8Hz), 6.68(2H,d,J=8.8Hz), 4.07(2H,t, J=4.6Hz),4.00(2H,s),3.83(2H,t,J=4.6Hz),3.70-3.65(12H,m),1.46(9H,s); 13 C NMR (100MHz, CDCl3): δ169.68,158.69,138.17,117.06,82.92,81.58,70.83,70.71,70.60,69.60,69.03,67.53,28.12. HRMSm / z(ESI):C 20 H 31 IO7[M+H] + Calculated value: 511.1193, measured value: 511.1192.

[0592] tert-Butyl(E)-14-(4-(2-(fluorosulfonyl)vinyl)phenoxy)-3,6,9,12-tetraoxatetradecanoate (compound S4):

[0593] A 20 mL reaction tube, dried in an oven, containing AgTFA (2.4 mmol, 1.2 equivalents), Pd(OAc)₂ (22 mg, 5 mol%), acetone (5 mL), S₂ (1.02 g, 2 mmol), and ethylene S₃ (440 mg, 4.0 mmol, 2 equivalents) was added. The resulting mixture was refluxed at 60 °C for 12 h. The crude product was purified by silica gel column chromatography to give S₄ (403 mg, 82%). 1 H NMR (400MHz, CDCl3): δ7.71(1H,d,J=15.2Hz), 7.48(2H,d,J=8.8Hz), 6.97(2H,d,J=8.8Hz), 6.70(1H,dd,J= 15.2Hz,2.4Hz),4.17(2H,t,J=4.6Hz),4.00(2H,s),3.86(2H,t,J=4.6Hz),3.72-3.64(12H,m),1.46(9H,s); 13 C NMR (100MHz, CDCl3): 169.65,162.50,148.60,131.08,123.68,115.43,114.82,114.54,81.59,70.87,70.70,70.58,69.45,69.00,67.75,28.09; 19 F(376MHz, CDCl3): δ+63.01; HRMSm / z(ESI): C22 H 33 FO9S[M+H]+ Calculated value: 493.1908, measured value: 493.1904

[0594] (E)-14-(4-(2-(fluorosulfonyl)vinyl)phenoxy)-3,6,9,12-tetraoxatetradecanoic acid (compound S5)

[0595]

[0596] A 20 mL reaction tube, dried in an oven, containing S4 (286 mg, 0.5 mmol), TFA (2 mL), and CH2Cl2 (2 mL) was added. The resulting mixture was incubated at room temperature for 4 h. The crude product was purified by silica gel column chromatography to give S5 (192 mg, 90%). 1 H NMR (400MHz, CDCl3): δ7.73(1H,d,J=15.2Hz), 7.49(2H,d,J=8.8Hz), 6.98(2H,d,J=8.8Hz), 6.70(1H,dd,J=15 .2Hz,2.4Hz),4.19(2H,t,J=4.6Hz),4.11(2H,s),3.89(2H,t,J=4.6Hz),3.75-3.71(4H,m),3.68-3.65(8H,m); 13 C NMR (100MHz, CDCl3): 171.77,162.38,148.61,131.10,123.77,115.46,114. 87,114.59,71.13,70.90,70.58,70.35,70.23,70.15,69.44,69.17,67.53; 19 F(376MHz, CDCl3): δ+63.00; HRMSm / z(ESI): C 18 H 25 FO9S[MH] - Calculated value: 435.1125, measured value: 435.1138.

[0597] SSF-PEG4-vc-PAB-MMAE 1

[0598]

[0599] Add the contents of an oven-dried reaction tube (20 mL) containing EDC (0.1 mmol, 2 equivalents), DIPEA (0.15 mmol, 3 equivalents), dry DMF (2 mL), S5 (22 mg, 0.05 mmol, 1 equivalent), and S6 (67 mg, 0.06 mmol, 1.2 equivalents). Incubate the resulting mixture at room temperature for 12 h. The crude product was purified by preparative HPLC to give S6 (26 mg, 34%). HRMSm / z (ESI): C 76 H 117 FN 10 O 20 S[M+H] + Calculated value: 1541.8229, measured value: 1541.8230. Product 1 was analyzed by LC-MS.

[0600] LC-MS chromatogram and mass spectrum of 1

[0601] The result is Figure 18 As shown in the image.

[0602] Example 10.2: Synthesis of SSF-PEG4-GGFG-Dxd 3

[0603]

[0604] (E)-2-(4-(((S)-10-benzyl-1-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15,18-hexaoxa-3,20,23,26,29-pentaoxa-5,8,11,14,17-pentazatrione-31-yl)oxy)phenyl)ethylene-1-sulfonyl fluoride (compound SSF-PEG4-GGFG-Dxd 3)

[0605]

[0606] Add oven-dried reaction tubes (20 mL) containing EDC (0.04 mmol, 2 equivalents), DIPEA (0.06 mmol, 3 equivalents), dry DMF (2 mL), S7 (16.8 mg, 0.02 mmol, 1 equivalent), and S5 (10.5 mg, 0.024 mmol, 1.2 equivalents). Incubate the resulting mixture at room temperature for 12 h. The crude product was purified by preparative HPLC to give 3 (8.0 mg, 32%). HRMSm / z (ESI): C 76 H 117 FN10 O 20 S[M+Na] + Calculated value: 1281.4238, measured value: 1281.4196. Product 3 was analyzed by LC-MS.

[0607] The result is Figure 19 It is displayed in the middle.

[0608] Example 10.3: Synthesis of MA 16-Biotin

[0609]

[0610] (E)-2-(4-(azidomethyl)phenyl)ethylene-1-sulfonyl fluoride (compound MA 16):

[0611] Under an inert atmosphere, diphenylphosphoazide (DPPA) (2 mmol, 1 equivalent) was added dropwise to an anhydrous DMF (2 mL) solution of S9 (432 mg, 2 mmol, 1 equivalent). The mixture was cooled to 0 °C, and diazabicycloundecene (DBU) (2 mmol, 1 equivalent) was added dropwise. The resulting mixture was stirred at room temperature for 12 h and quenched with water. The solution was extracted with DCM. The organic layers were combined with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give MA 16 (120 mg, 25%). 1 H NMR (400MHz, CDCl3): δ7.81 (1H, d, J = 15.4Hz), 7.58 (2H, d, J = 6.8Hz), 7.43 (2H, d, J = 6.8Hz), 6.88 (1H, dd, J = 15.4Hz, 2.8Hz), 4.43 (2H, s); 13 C NMR (100MHz, CDCl3): 148.01, 140.31, 129.48, 128.93, 118.61, 118.33, 54.16; 19 F(376MHz, CDCl3): δ+62.32; HRMSm / z(ESI): C9H8FN3O2S[M+H] + Calculated value: 242.0400, measured value: 242.0402.

[0612] (E)-2-(4-((4-(15-oxo-19-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-2,5,8,11-tetraoxa-14-azanonadecanyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)ethylene-1-sulfonyl fluoride (compound MA 16-biotin)

[0613]

[0614] A 20 mL oven-dried reaction tube containing CuSO4·5H2O (0.005 mmol, 0.1 equivalent), BTTP (0.01 mmol, 0.2 equivalent), ASC (0.02 mmol, 0.4 equivalent), DMSO (1.5 mL), H2O (0.5 mL), S10 (0.055 mmol, 1.1 equivalent), and MA 16 (0.05 mol, 1 equivalent) was added. The resulting mixture was reacted at 37 °C for 12 h. The crude product was purified by silica gel column chromatography to give MA 16-biotin (31 mg, 89%). 1 H NMR (400MHz, CDCl3): δ7.80 (1H, s), 7.58 (1H, d, J = 15.2Hz), 7.45 (1H, d, J = 8.0Hz), 7.19-7 .13(3H,m),5.40(2H,s),4.40(2H,s),4.22-4.19(1H,m),4.03-4.00(1H,m),3.38-3.33(4H ,m),3.31-3.28(6H,m),3.23-3.20(2H,m),3.05-3.00(3H,m),2.92-2.87(1H,m),2.64-2. 60(1H,m),2.42-2.39(1H,m),1.91(2H,t,J=7.4Hz),1.41-1.28(4H,m),1.16-1.10(2H,m); 13 C NMR (100MHz, CDCl3): 174.83,164.69,147.83,139.64,131.68,129.65,128.57,119.23,118.95,70.12,70.0 1,70.07,69.82,69.52,69.12,63.43,62.10,60.39,55.57,53.15,39.61,38.98,35.28,28.33,28.06,25.43; 19 F(376MHz, CDCl3): δ+59.81; HRMSm / z(ESI): C 30 H 43 FN6O8S2[M+H] + Calculated value: 699.2946, measured value: 699.2942.

[0615] Example 10.4: Synthesis of SSF-NHS

[0616]

[0617] Compound S10 was synthesized based on a previously disclosed procedure. ¹H NMR (400 MHz, CDCl₃): δ 7.54 (2H, d, J = 8.9 Hz), 6.66 (2H, d, J = 8.9 Hz), 3.92 (2H, t, J = 6.4 Hz), 2.40 (2H, t, J = 7.4 Hz), 1.83–1.74 (2H, m), 1.73–1.67 (2H, m), 1.56–1.78 (2H, m); HRMS m / z (ESI): C₁₂H₁₅IO₃[MH] - Calculated value: 332.9988, measured value: 332.9985.

[0618] tert-butyl 6-(4-iodophenoxy)hexanoate (compound S12):

[0619] A 20 mL reaction tube, dried in an oven, was added to contain S11 (501 mg, 1.5 mmol, 1 equivalent), tBuOH (5 equivalents), and anhydrous DCM (2 mL). The mixture was cooled to 0 °C, and DMAP (0.1 equivalent) was added dropwise. Then DCC (1.1 equivalent) was added. The resulting mixture was stirred overnight at room temperature. The crude product was purified by silica gel column chromatography to give S12 (444.6 mg, 76%). 1 H NMR (400MHz, CDCl3): δ7.53(2H,d,J=8.9Hz), 6.66(2H,d,J=8.9Hz), 3.91(2H,t,J=6.4Hz), 2.24(2H, t,J=7.4Hz),1.81-1.74(2H,m),1.68-1.60(2H,m),1.51-1.46(2H,m),1.44(9H,s); HRMSm / z(ESI): C 16 H 23 IO3[M+H] + Calculated value: 391.0770, measured value: 391.0773.

[0620] tert-Butyl(E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoate (compound S13):

[0621] A 20 mL oven-dried reaction tube containing AgTFA (0.6 mmol, 1.2 equivalents), Pd(OAc)₂ (5.5 mg, 5 mol%), acetone (2 mL), S₁₂ (1.02 g, 0.5 mmol), and ethylene S₃ (220 mg, 1 mmol, 2 equivalents) was added. The resulting mixture was refluxed at 60 °C for 12 h. The crude product was purified by silica gel column chromatography to give S₁₃ (163.7 mg, 88%). 1 H NMR (400MHz, CDCl3): δ7.74(1H,d,J=15.4Hz), 7.49(2H,d,J=8.8Hz) 6.93(2H,d,J=8.8Hz), 6.68(1H,dd,J=2.8Hz), 4.02(2H,t,J=6.4Hz),2.25(2H,t,J=7.4Hz),1.86-1.79(2H,m),1.70-1.63(2H,m),1.54-1.51(2H,m),1.44(9H,s); 19 F(376MHz, CDCl3): δ+63.07; HRMSm / z(ESI): C 18 H 25 FO5S[M+H] + Calculated value: 373.1485, measured value: 373.1486.

[0622] (E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoic acid (compound S14):

[0623] Add an oven-dried reaction tube (20 mL) containing S12 (93 mg, 0.25 mmol), TFA (2 mL), and DCM (2 mL). Stir the resulting mixture at room temperature for 4 h. The crude product was purified by silica gel column chromatography to obtain S13 (72 mg, 92%). ¹H NMR (400 MHz, CDCl₃): δ 7.74 (¹H, d, J = 15.4 Hz), 7.49 (2H, d, J = 8.8 Hz), 6.94 (2H, d, J = 8.8 Hz), 6.69 (¹H, dd, J = 2.6 Hz), 4.02 (2H, t, J = 6.4 Hz), 2.41 (2H, t, J = 7.4 Hz), 1.91–1.81 (2H, m), 1.77–1.69 (2H, m), 1.58–1.52 (2H, m); 19 F(376MHz, CDCl3): δ+63.05; HRMSm / z(ESI): C 14 H 17 FO5S[MH] -Calculated value: 315.0702, measured value: 315.0703.

[0624] 2,5-Dioxopyrrolidone-1-yl(E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoate (compound SSF-NHS)

[0625] A 20 mL oven-dried reaction tube containing S14 (63 mg, 0.2 mmol), N-hydroxysuccinimide (1.2 equivalents), EDC (1.2 equivalents), and CH2Cl2 (4 mL) was added. The resulting mixture was stirred at room temperature for 12 h. After completion, the reaction was quenched with 20 mL of water and extracted three times with 20 mL of DCM. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated to give SSF-NHS (67 mg, 81%). 1 H NMR (400MHz, CDCl3): δ7.74(1H,d,J=15.4Hz), 7.49(2H,d,J=8.8Hz) 6.94(2H,d,J=8.8Hz), 6.69(1H,dd,J=15.4Hz 2.6Hz),4.03(2H,t,J=6.4Hz),2.84(4H,s),2.66(2H,t,J=7.4Hz),1.88-1.82(4H,m),1.64-1.59(2H,m); 13 C NMR (100MHz, CDCl3): 169.17,168.46,162.70,148.68,131.12,123.48,115.31,114.35,67.87,33.96,30.84,28.54,25.60,24.29; 19 F(376MHz, CDCl3): δ+63.06; HRMSm / z(ESI): C 18 H 20 FNO7S[M+H] + Calculated value: 436.0842, measured value: 436.0827.

[0626] Example 10.5: Synthesis of SSF-OSO2F

[0627]

[0628] A 20 mL reaction tube, dried in an oven, containing SN38 (98.3 mg, 0.25 mmol), Et3N (3 equivalents), and DCM (2 mL) was added. The mixture was stirred at room temperature for 24 h under an SO2F2 balloon. The reaction was quenched by adding brine (30 mL) and extracted with DCM (30 mL). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give SN38-OSO2F (100.5 mg, 85%) in the form of a yellow foam. 1H NMR (400MHz, DMSO): δ8.37(1H,d,J=2.6Hz), 8.33(1H,d,J=9.4Hz) 7.66(1H,s)5.60(1H,d,J=16.4Hz), 5.41-5.37(3H,m),3.29-3.27(2H,m),1.99-1.94(2H,m),1.43(3H,t,J=7.6Hz),1.01(3H,t,J=7.4Hz).

[0629] In vivo cytotoxicity studies

[0630] Cells were seeded at 5,000 cells per well in the presence of 5% CO2 at 37°C and incubated for 24 h. Serial dilutions of SN38 and SN38-OSO2F were added to the cells in complete growth medium, and the cells were incubated at 37°C for 96 h in the presence of 5% CO2. Cell viability was evaluated using a Cell Counting-Lite 2.0 luminescent cell viability assay (Vazyme, DD1101-01). Cell viability was plotted as the percentage of untreated cells. Each measurement was performed in triplicate.

[0631] Figure 30 The results of cell viability assays for SN38 and SN38-OSO2F using cell line (N87) are shown.

[0632] Step 10.7: Synthesis of SSF-PEG4-T785

[0633]

[0634] Add the contents of a 20 mL oven-dried reaction tube containing EDC (0.1 mmol, 2 equivalents), DIPEA (0.15 mmol, 3 equivalents), dry DMF (2 mL), T785 (15.6 mg, 0.05 mmol, 1 equivalent), and S5 (26 mg, 0.06 mmol, 1.2 equivalents). Incubate the resulting mixture at room temperature for 12 h. The crude product was purified by preparative HPLC to give SSF-PEG4-T785 (15.6 mg, 24%). HRMSm / z (ESI): C36 H 48 FN5O8S[M+H] + Calculated value: 730.3286, measured value: 730.3253.

[0635] Example 11: Synthesis of Antibody-Biotin Conjugate

[0636] Example 11.1: Synthesis of trastuzumab-MA 16-Cy5.5 conjugate

[0637] Cysteine-selective protein modification using MA 16

[0638] MA 16 (1 μL, 20 mM in DMSO) and PBS (15 μL, pH 7.5, 50 mM) were added to the protein solution (4 μL, 250 μM in PBS buffer). After incubation at 37 °C for 2 h, the solution was desalted, and the product was obtained before LC-MS analysis.

[0639] Antibody reduction

[0640] TCEP·HCl (2.6 μL, 25 mM in PBS) was added to an antibody solution (KN026, KN046, trastuzumab) (40 μL, 160 μM in PBS), and the resulting solution was incubated at 37 °C for 2 h. Before LC-MS analysis, the solution was desalted to obtain the reduced antibody.

[0641] MA 16-biotin (1.6 μL, 5 mM in DMSO) and PBS (34.5 μL, pH 7.4, 50 mM) were added to the antibody solution (4 μL, 100 μM in PBS buffer). After incubation at 37 °C for 2 h, the solution was desalted, and the antibody-biotin conjugate was obtained before LC-MS analysis.

[0642] Synthesis of trastuzumab-MA 16-Cy5.5 conjugate

[0643] MA 16 (1.6 μL, 5 mM in DMSO) and PBS (34.5 μL, pH 7.4, 50 mM) were added to trastuzumab solution (4 μL, 100 μM in PBS buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain trastuzumab-MA 16.

[0644] DBCO-Cy5.5 (0.8 μL, 5 mM in DMSO) and PBS (15 μL, pH 7.4, 50 mM) were added to trastuzumab-MA 16 solution (4 μL, 50 μM in PBS buffer). After incubation at 37 °C for 12 h, the solution was desalted, and trastuzumab-MA 16-Cy5.5 was obtained before LC-MS analysis.

[0645] SDS-PAGE analysis of antibody conjugates

[0646] Mix 2 μL of trastuzumab, trastuzumab-MA 16-Cy5.5, and trastuzumab-MA 16-Biotin with 10 μL of ultrapure water and 4 μL of SDS-PAGE loading buffer containing 2-mercaptoethanol. Heat the sample to 95°C or for 10 min and load it completely onto the SDS-PAGE gel.

[0647] Example 11.2: Synthesis of Trastuzumab-1 (Herceptin-PhESF-MMAE)

[0648] 1 (i.e., SSF-PEG4-PAB-MMAE 2.6 μL, 25 mM in DMSO): Add DMSO (17.4 μL) and PBS (280 μL, pH = 7.4, 50 mM) to trastuzumab solution (100 μL, 130 μM, PBS buffer). After incubation at 25 °C for 2 h, desalt the solution to obtain trastuzumab-1.

[0649] Example 11.3: Synthesis of Trastuzumab-2 (Herceptin-Mal-MMAE)

[0650] 2 (i.e., Mal-VC-PAB-MMAE 1.8 μL, 25 mM in DMSO): Add 18.2 μL of DMSO and 280 μL of PBS (pH = 7.4, 50 mM) to trastuzumab solution (100 μL, 130 μM, PBS buffer). After incubation at 25°C for 2 h, the solution was desalted to obtain trastuzumab-2. Mal-VC-PAB-MMAE was purchased from Shandong Meitai Pharmaceutical Co., Ltd.

[0651] Example 11.4: Synthesis of Trastuzumab-3 (Herceptin-PhESF-Dxd)

[0652] 3 (i.e., SSF-PEG4-GGFG-Dxd 1.5 μL, 25 mM in DMSO): Add 0.5 μL of DMSO and 10 μL of PBS (pH = 9.0, 50 mM) to a trastuzumab solution (10 μL, 130 μM, PBS buffer). After incubation at 37°C for 12 h, the solution is desalted to obtain trastuzumab-3.

[0653] Figure 28 MS of the deconvolutiond intact protein of trastuzumab-3 is shown.

[0654] Step 11.5: Synthesis of Trastuzumab-T785

[0655] SSF-PEG4-T785 (13 μL, 10 mM in DMSO): DMSO (27 μL) and PBS (560 μL, pH 9.0, 50 mM) were added to trastuzumab solution (200 μL, 130 μM, PBS buffer). After incubation at 25 °C for 2 h, the solution was desalted to obtain trastuzumab-T785.

[0656] Figure 29 MS of the deconvolutiond intact protein of trastuzumab-T785 is shown.

[0657] Example 12: Analysis of MA 16-related conjugates

[0658] SDS-PAGE analysis of antibody conjugates

[0659] Mix 2 μL of trastuzumab, trastuzumab-MA 16-Cy5.5, and trastuzumab-MA 16-Biotin with 10 μL of ultrapure water and 4 μL of SDS-PAGE loading buffer containing 2-mercaptoethanol. Heat the sample to 95°C or for 10 min and load it completely onto the SDS-PAGE gel.

[0660] The results of modifying trastuzumab using a detection probe derived from MA 16 are as follows: Figure 24 As shown.

[0661] Figure 24 A shows the synthetic regimen for attaching MA16-Biotin and MA16-Cy5.5 to trastuzumab. The reaction conditions for trastuzumab-MA16-Biotin were: 20 μM trastuzumab, 400 μM MA16-Biotin, PBS, 37°C, 2 h. The reaction conditions for trastuzumab-MA16 were: 20 μM trastuzumab, 400 μM MA16, PBS, 37°C, 2 h. The reaction conditions for trastuzumab-MA16-Cy5.5 were: 10 μM trastuzumab-MA16, 200 μM DBCO-Cy5.5, PBS, 37°C, 12 h. Figure 24 B and Figure 24 C shows the SDS-PAGE gel, Western blot, and LC-MS analyses of trastuzumab before and after reaction with MA16-biotin.

[0662] Flow cytometry assay

[0663] Cells were suspended in flow cytometry buffer (PBS containing 2% FBS) containing trastuzumab-MA 16-biotin or control trastuzumab and incubated at 4°C for 45 min. After washing twice with flow cytometry buffer, cells were further incubated with APC-streptavidin (BioLegend, 405207) at 4°C for 30 min, resuspended, and washed twice with flow cytometry buffer. Analysis was performed using an Agilent flow cytometer (Angilent NovoCyte Quanteon). Isotype control antibody staining was used to determine the threshold for positive and negative cells. Agilent Novoexpress was used for all flow data analysis.

[0664] Figure 24 D shows flow cytometry analysis of cancer cells stained with trastuzumab-MA16-biotin. NCI-N87 (HER2+) and MDA-MB-468 (HER2-) cells were incubated with trastuzumab-MA16-biotin, while the control group was treated with trastuzumab. After staining, cells were further stained with SA-APC to detect biotin.

[0665] Figure 24 E and Figure 24 F shows the analysis of trastuzumab before and after Cy5.5 modification by fluorescence imaging (right), Coomassie staining (left), and LC-MS.

[0666] Fluorescence imaging

[0667] MDA-MB-231 and NCI-N87 cells were grown overnight at 37°C on sterile glass coverslips or slides. After brief washing with DPBS, sections were incubated with 20 μg / mL trastuzumab-MA 16-Cy5.5 at 37°C for 1 h. MDA-MB-231 cells were transfected with a plasmid encoding GFP. The co-cultured cells were then imaged under a microscope at 40X magnification.

[0668] Figure 24 G shows fluorescence imaging (scale bar 50 μm) of specific cell surface HER2 detection enabled by trastuzumab-MA16-Cy5.5.

[0669] Example 13: Analysis of trastuzumab-related conjugates

[0670] Example 13.1: Human Stability Study

[0671] In the Eppendorf assay, for each sample, 90 μL of human serum was mixed with 10 μL of either trastuzumab-1 (20 mg / mL) or trastuzumab-2 to obtain a final solution of 0.2 mg / mL ADC in human serum. Samples were incubated in humans and mice at 37°C for 3 and 7 days, respectively. Samples were incubated in mouse serum at 37°C for 3 days. Samples from day 0 were directly processed further.

[0672] Example 13.2: In vitro cytotoxicity study

[0673] Cells were seeded at 5,000 cells per well in the presence of 5% CO2 at 37°C and incubated for 24 h. Serial dilutions of trastuzumab-1, trastuzumab-2, and trastuzumab were added to the cells in complete growth medium, and the cells were incubated at 37°C for 96 h in the presence of 5% CO2. Cell viability was evaluated using a Cell Counting-Lite 2.0 luminescent cell viability assay (Vazyme, DD1101-01). Cell viability was plotted as the percentage of untreated cells. Each measurement was performed in triplicate.

[0674] Example 13.3: Bystander lethality test

[0675] A mixture of SKBR-3 and MDA-MB-231 cells was seeded at a 1:1 ratio in 96-well plates, and MDA-MB-231 cells were seeded separately in 96-well plates at the same density and incubated at 37°C and 5% CO2 for 24 h. Then, 2 μg / mL of trastuzumab, trastuzumab-1, and trastuzumab-2 were added, and the cells were incubated at 37°C and 5% CO2 for 96 h. MDA-MB-231 is a stable cell line overexpressing luciferase. Cell viability was evaluated using a dual-luciferase reporter assay kit (Vazyme, DL101-01).

[0676] Example 13.4: Xenotransplantation experiment in nude mice

[0677] All in vivo studies were conducted in accordance with the local guidelines of the Laboratory Animal Management and Use Committee (Approval No.: IACUC-2101001). NCI-N87 cells (2 million) were subcutaneously inoculated into specific pathogen-free female nude mice. Tumor-bearing mice were randomly assigned to treatment or control groups. Tumors were treated when the average tumor volume reached approximately 100 mm². 3 Up to 200mm 3 Dosing was initiated on day 0. Each substance, 1 mg / kg trastuzumab-1 or trastuzumab-2, along with a mediator (PBS), was administered intravenously to mice on days 0 and 14. Tumor volume was defined as 1 / 2 * length * width. 2 The tumor size was recorded every three days.

[0678] The result is Figure 25 It is displayed in the middle.

[0679] Figure 25 A shows the antitumor activity of ADC (5 mg / kg) in the BALB / c nude mouse NCI-N87 tumor xenograft model. Figure 25 B shows the antitumor activity of the ADC (1 mg / kg) in the BALB / c nude mouse NCI-N87 tumor xenograft model. Tumor volumes of seven mice in each group are shown individually. Figure 25 C shows Figure 25 The Kaplan-Mayer survival analysis of the study shown in B. Figure 25 D showed that neutropenia was observed in rats after a 20 mg / kg ADC dose. Four animals were administered trastuzumab-1, trastuzumab-2, or a solvent, and samples were taken to obtain hematological markers.

[0680] Example 13.5: Safety Study

[0681] All in vivo studies were conducted in accordance with the local guidelines of the Laboratory Animal Management and Use Committee (Approval No.: ZJCLA-IACUC-20040026). In 12- to 14-week-old female rats, ADC or PBS was administered intravenously at 20 mg / kg (four rats per dose group, randomly assigned). Four serum samples were collected before and several days after administration for hematological analysis.

[0682] Example 14: Analysis of MA 2-related conjugates

[0683] Hydrolytic stability of MA 2 and MA 5

[0684] At 37℃, MA 2 (i.e. 5mM final concentration) or MA 5 (i.e. The final concentration of 5 mM was determined by shaking in 100 μL of 50 mM PBS (pH 9.0) for 48 h. Then, 1 μL of the reaction mixture and 15 μL of PBS (pH 7.4, 50 mM) were added separately to GFP solution (4 μL, 250 μM, HEPES buffer). After incubation at 37 °C for 2 h, the solution was desalted, and the product was obtained before LC-MS analysis.

[0685] Hydrolysis stability results in Figure 21 It is displayed in the middle.

[0686] The stability results of SSF(MA 2) and maleimide in aqueous buffer were as follows: Figure 26 As shown in the image.

[0687] Figure 26A shows the reaction mixture of 5'-maleimide-ssDNA with shaking at 37 °C for 48 h. The reaction mixture was then analyzed by LC-MS. The 5'-maleimide-ssDNA was completely hydrolyzed to 5'-maleic acid-ssDNA. Figure 26 B demonstrates the hydrolytic stability of SSF: 5'-maleimide-ssDNA was shaken at 37 °C for 48 h. The reaction mixture was then analyzed by LC-MS, and only the starting material was obtained.

[0688] Modification of GFP-TEV by MA 2 analogues

[0689] MA 2 or MA 2 analogues (i.e., compounds 1a, 1a-1, 1b, 1b-1) (1 μL, 20 mM in DMSO) and PBS (15 μL, pH 7.4, 50 mM) were added to GFP-TEV solution (4 μL, 250 μM in HEPES buffer). After incubation at 37 °C for 2 h, the solution was desalted, and the product was obtained before LC-MS analysis.

[0690] Dynamics Experiment

[0691] MA 2 or a previously stable linker (i.e., compounds 2, 3, 6, 7) (1 μL, 5 mM in DMSO) or PBS (15 μL, pH 7.4, 50 mM) was added to a GFP solution (4 μL, 250 μM, HEPES buffer). After incubation at 37 °C for different times (5 min, 10 min, 30 min, 60 min, 90 min, 120 min, and 240 min), the solution was desalted to obtain the product prior to LC-MS analysis.

[0692] The result is Figure 22 It is displayed in the middle. Figure 22 MA 2 was compared with previously reported stable Cys-specific labeling reagents.

[0693] Figure 22 A shows the chemical structure of the reported stable Cys-specific labeling reagent. The arrow points to the cysteine ​​reaction site. Figure 22 B shows the reaction kinetics of GFP (50 μM) with 5 equivalents of labeled reagent.

[0694] Competitive Experiment

[0695] MA 2 (0.5 μL, 20 mM in DMSO), 2-5 (0.5 μL, 20 mM in DMSO), and PBS (15 μL, pH 7.4, 50 mM) were added to GFP solution (4 μL, 250 μM in HEPES buffer). After incubation at 37 °C for 2 h, the solution was desalted, and the product was obtained before LC-MS analysis.

[0696] MS / MS spectra of the MA2-modified GFP fragment were obtained in... Figure 23 It is displayed in the middle.

[0697] Figure 24 The study demonstrates Cys-specific modifications to different proteins using SSF.

[0698] Figure 24 A shows the reaction protocols of MA2 with different proteins. Figure 24 B shows the deconvolutioned intact protein MS of the protein-MA6 conjugate, including neo2, Nb-PD-L1, GFP, KN046, trastuzumab, and KN026.

[0699] Determination of binding affinity of trastuzumab-MA2 conjugate

[0700] SKBR3 cells were conjugated with different concentrations of trastuzumab-MA6 or trastuzumab on ice for 30 min in 200 μL flow cytometry buffer (PBS containing 2% FBS). After conjugation, the cells were washed twice with PBS and then incubated with trastuzumab-Cy5.5 on ice for 30 min. The cells were resuspended and washed twice more with flow cytometry buffer, and analyzed using an Agilent flow cytometer (Angilent NovoCyte Quanteon).

[0701] Example 15: Conjugation integrity study of Nb-PD-L1-20ntssDNA based on LC-MS / SDS-PAGE in the presence of 10% human serum

[0702] A mixture of Nb-PD-L1-20nt ssDNA (2 μL, 50 μM), human serum (2 μL), and PBS (16 μL, pH 8.0) was incubated at 37°C in the dark for 24 h, 48 h, and 72 h. The reaction mixture was analyzed by LC-MS and SDS-PAGE. SDS-PAGE: 10 μL of Nb-PD-L1-20nt ssDNA was mixed with 5 μL of SDS-PAGE loading buffer containing 2-mercaptoethanol. The sample was heated to 95°C, held for 10 min, and then completely loaded onto the SDS-PAGE gel.

[0703] Figure 27 This study demonstrates the site-specific DNA-protein conjugates constructed from SSF-ssDNA and the application of Nb-PD-L1 ssDNA in single-cell RNA sequencing.

[0704] Figure 27 A shows a protocol for protein modification using an SSF-ssDNA probe. Figure 27 B shows the deconvolution mass spectra of DNA-protein conjugates constructed using 20nt SSF-ssDNA or 59nt SSF-ssDNA probes. Figure 27 C shows the conjugate integrity study of Nb-PD-L1-20ntssDNA based on LC-MS in the presence of 10% human serum, as well as deconvolution mass spectra of samples collected at specific time points. Figure 27 D shows a flowchart of Nb-PD-L1-ssDNA used for CITE-seq, which is used to detect target cells at the single-cell level using transcriptomics. Figure 27 E shows single-cell expression profile clustering based on transcriptome. Cyan: Jurkat; Red: A549; Green: JIMT-1; Purple: MDA-MB-231. Figure 27 G shows the superposition of Figure 27 F shows the relative intensity of Nb-PD-L1-ssDNA targeting on the UMAP projection. Figure 27 G shows a violin plot depicting the mRNA expression levels of PD-L1 (CD274) in four cell lines. Figure 27 H shows a violin plot of scale (z-score) normalized UMI counts describing the 59nt-ssDNA barcode (Nb-PD-L1 binding strength) in four cell lines.

[0705] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The invention is not limited to the specific embodiments provided in the specification. Although the invention has been described with reference to the foregoing description, the description and illustration of embodiments herein are not restrictive. Many variations, modifications, and alternatives will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, as these depend on various conditions and variables. It should be understood that the invention can be practiced in various alternative ways to the embodiments described herein. Therefore, it is contemplated that the invention should equally cover any such alternatives, modifications, variations, or equivalents. The appended claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A conjugate of Formula 1, M-[(L1)] a -(L2) b -(D) c ]1, where L1 is a compound of formula I, I, R are -F or -OH, where M is a protein, DNA, RNA, or virus, M is linked to L1 using its nucleophilic functional group, L2 is a linker and is linked to R2, D is a drug, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, where R1 is H, and R... 1' It is H or its isotopes, where R2 is R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, alkyl-OH, alkyl-halogen, alkyl-N3, -B(OH)2, -halogen, -OTf, alkyl-NH2, -O-alkyl-C≡CH, -CO-NH-C≡CH-alkyl, wherein the alkyl group is an alkyl group with 1 to 10 carbon atoms, or R4 is -O-(CH2)n1-COO-R5, where n1 is an integer from 1 to 10, and R5 is selected from the following group: And H, or R4 is -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, where n3 is an integer from 1 to 10, and R7 is selected from: Alternatively, R4 can be -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 can be -(CH2)n4-R8, where n4 is an integer from 1 to 10, and R8 is selected from the following group: Alternatively, R4 can be -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 can be -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, where n5 and n6 are both integers from 1 to 10, and R9 is selected from the following group: H and Or R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Select from the following groups: -CF3, -CN, and -OCH3, where R3 is H.

2. The conjugate according to claim 1, wherein the nucleophilic functional group of M is selected from the group consisting of -SH, -NH2, -SeH, -OH, and 3. The conjugate according to any one of claims 1 to 2, wherein M is an antigen-binding protein or a fragment thereof.

4. The conjugate according to any one of claims 1 to 3, wherein L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

5. The conjugate according to any one of claims 1 to 4, wherein L2 is selected from the group consisting of: VC-PAB, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), maleimide PEG. NHS, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimide-4-(maleimidemethyl)cyclohexanecarboxylate (sulfo-SMCC or 2,5-dioxopyrrolidine-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-ester (CX1-1).

6. The conjugate according to any one of claims 1 to 5, wherein D and / or its derivatives are capable of inhibiting the growth of tumor cells.

7. The conjugate according to any one of claims 1 to 6, wherein D is selected from: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, saccharidin, maytansin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA small groove adhesives, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucoside, etoposide phosphate; nitrogen mustard, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

8. The conjugate according to any one of claims 1 to 7, wherein D is MMAE, melphalan, lenalidomide, IL-2, new interleukin-2 / 15, T785, or MSA-2.

9. The conjugate according to any one of claims 1 to 8, wherein R2 is R4 is selected from: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

10. The conjugate according to any one of claims 1 to 9, wherein L1 is selected from the group consisting of:

11. The conjugate according to any one of claims 1 to 10, wherein the conjugate is selected from the group consisting of:

12. A conjugate of formula 3, (L1)a-(L2)b-(D)c 3, wherein L1 is a compound of formula III. III. L2 is a connector, and L2 is connected to R2. D is the drug, a is an integer from 1 to 10, b and c are each an independent integer from 0 to 10, provided that b and c are not both 0. R1 is H, and R2 is... R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, alkyl-OH, alkyl-halogen, alkyl-N3, -B(OH)2, -halogen, -OTf, alkyl-NH2, -O-alkyl-C≡CH, -CO-NH-C≡CH-alkyl, wherein the alkyl group is an alkyl group with 1 to 10 carbon atoms, or R4 is -O-(CH2)n1-COO-R5, where n1 is an integer from 1 to 10, and R5 is selected from the following group: And H, or R4 is -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, where n3 is an integer from 1 to 10, and R7 is selected from: Alternatively, R4 can be -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 can be -(CH2)n4-R8, where n4 is an integer from 1 to 10, and R8 is selected from the following group: Alternatively, R4 can be -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 can be -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, where n5 and n6 are both integers from 1 to 10, and R9 is selected from the following group: H and Or R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Select from the following groups: -CF3, -CN, and -OCH3, where R3 is H.

13. The conjugate according to claim 12, wherein L1 is selected from the group consisting of:

14. A method for preparing a conjugate, the method comprising the following steps: By making the conjugate of formula 3: 3 is combined with M to obtain the conjugate of formula 1: M is a protein, DNA, RNA, or virus, and M is linked to L1 using its nucleophilic functional group. L2 is a linker and is linked to R2 in Equation 1. D is a drug, a is an integer from 1 to 10, b and c are each independent integers from 0 to 10, provided that b and c are not simultaneously 0, where R1 is H, and R... 1' It is H or its isotopes, where R2 is R4 is selected from the following group: -OH, -PO3H2, -SeH, -SH, alkyl-OH, alkyl-halogen, alkyl-N3, -B(OH)2, -halogen, -OTf, alkyl-NH2, -O-alkyl-C≡CH, -CO-NH-C≡CH-alkyl, wherein the alkyl group is an alkyl group with 1 to 10 carbon atoms, or R4 is -O-(CH2)n1-COO-R5, where n1 is an integer from 1 to 10, and R5 is selected from the following group: And H, or R4 is -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, where n3 is an integer from 1 to 10, and R7 is selected from: Alternatively, R4 can be -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 can be -(CH2)n4-R8, where n4 is an integer from 1 to 10, and R8 is selected from the following group: Alternatively, R4 can be -O-(CH2)n2-CO-NH-R6, where n2 is an integer from 1 to 10, and R6 can be -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, where n5 and n6 are both integers from 1 to 10, and R9 is selected from the following group: H and Or R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, where R 10 Selected from the following groups: -COOH, -NH2 and Where R 11 Select from the following groups: -CF3, -CN, and -OCH3, where R3 is H.

15. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

16. A method for preventing and / or treating a disease in a subject in need, the method comprising administering to the subject the conjugate of any one of claims 1 to 11, or the pharmaceutical composition of claim 15, in the preparation of a medicament for treating a disease, said disease including tumors and / or autoimmune diseases.

17. A diagnostic reagent comprising the conjugate according to any one of claims 1 to 11.

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