Glutamyl-containing linkers, macromolecular conjugate drugs, and medical uses thereof

Abstract

The present disclosure relates to glutamyl-containing linker-drugs and macromolecule conjugate drugs, to pharmaceutical compositions comprising the macromolecule conjugate drugs, and to uses of the macromolecule conjugate drugs for treating a disease or condition or lessening the severity of the disease or condition.

Description

Technical Field

[0001] This disclosure pertains to the field of medicinal chemistry, and specifically relates to glutamine-containing linker-drugs and macromolecular conjugates, pharmaceutical compositions comprising such macromolecular conjugates, and the use of such macromolecular conjugates for treating diseases or conditions or reducing the severity of said diseases or conditions. Background Technology

[0002] Antibody-drug conjugates (ADCs) are composed of antibodies and effector molecules covalently linked by linkers. They combine the advantages of strong targeting of antibodies and high activity of effector molecules, and are hailed as "biological missiles" for precise delivery of effector molecules (Signal Transduction and Targeted Therapy, 2022, 7(1): 93).

[0003] Traditional antibody-drug conjugates (ADCs) primarily enter cells via antibody-mediated endocytosis, where they are cleaved by enzymes in lysosomes to release active molecules, thereby achieving targeted killing. However, this mechanism is highly dependent on high antigen expression and efficient endocytosis, and the distribution of antibodies deep within solid tumors is limited due to their large molecular size and limited tissue penetration. Furthermore, tumor cells may develop resistance through decreased endocytic efficiency, downregulation of antigen expression, or lysosomal dysfunction, further limiting the efficacy of these ADCs. Traditional ADCs are particularly ineffective against certain tumor targets that do not rely on endocytosis or have low antigen expression levels. Therefore, developing ADCs that can respond to specific enzymes or chemical conditions in the tumor extracellular microenvironment, achieving linker cleavage and drug release, has become an important direction for expanding the application scope and improving the efficacy of ADCs. Summary of the Invention

[0004] Gamma-glutamyl transpeptidase (GGT) and its isoenzymes are enzymes that recognize and catalyze the transfer of glutamyl groups from catalytic substrates (such as glutathione) to receptors. They participate in many important physiological processes and are overexpressed in various cancers, including ovarian and cervical cancer, where they are closely associated with tumor cell invasion and drug resistance. The inventors have constructed a novel ADC by incorporating the glutamyl group chemical fragment recognized by GGT into a linker. This novel ADC relies on the release of active drugs from GGT enzymes expressed on the cell membrane or from isoenzymes present in the cytoplasm, expanding the applicability of cancer targets. Compared to traditional ADCs, its small-molecule cytotoxic release outside the cell allows for deeper penetration into the tumor, maximizing the bystander effect.

[0005] This disclosure provides a linker comprising a glutamyl unit, a linker-drug conjugate comprising the linker, and a macromolecular conjugated drug comprising the linker. The macromolecular conjugated drug comprises a macromolecular portion, a linker, and a drug portion. The linker is configured such that when the macromolecular conjugated drug is localized to a tumor microenvironment, the glutamyl unit is specifically recognized and cleaved by gamma-glutamyl transpeptidase (GGT), thereby releasing the drug portion. In some embodiments, the glutamyl unit is linked to the drug portion or to other segments in the linker via its γ-carboxyl group. In some embodiments, the γ-carboxyl group of the glutamyl unit forms an amide bond or an ester bond with the drug portion or to other segments in the linker. In some embodiments, the linker further comprises a self-eliminating spacer. One end of the self-eliminating spacer is linked to the glutamyl unit, and the other end is linked to the drug portion. When the glutamyl unit is cleaved by GGT, the self-eliminating spacer can spontaneously degrade, releasing the drug portion.

[0006] Specifically, this disclosure covers the following aspects.

[0007] 1. Linker-drug conjugates

[0008] The first aspect of this disclosure relates to a compound of formula I, its salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label, wherein

[0009] L is a self-eliminating spacer; R stands for a connector used to link macromolecules; D is either 0 or 1; C represents an active molecular group, selected from drug groups, cytotoxic groups, detection reagent groups, diagnostic reagent groups, and targeting carrier groups.

[0010] In some implementations, in Formula I As a whole or In this configuration, L1 is attached to the carbonyl group of a glutamyl group, and the other end is attached to C; Ar is a 5-6 aryl or a 5-6 heteroaryl group; each R1 is independently hydrogen or C. 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro, or cyano; r is 0, 1, 2, or 3; L1 is -NH- or -N(R) a (CH2) n N(R a )-C(O)-O-, where R a Each independently is H or C 1-6Alkyl group, n is 1, 2, 3 or 4; R is -X-L2-L3-R2, where X is -C(O)-, -O-, -NH-, -C(O)NH-, -NHC(O)-, -NCH3-, -CH2O-, -CH(R b )O-、-C(R b )2O-、-CH2NH-、-CH(R b )-、-C(R b )2NH-, -S-, -S(O)- or -S(O)2-, where R b Each independently is C 1-6 Alkyl group, or X is absent; L2 is -(CH2CH2O). k -(CH2) l -、-CH[-(CH2) l -NHC(O)-(CH2CH2O) k -CH3]-、-(CH2) m -or In this context, each k is an independent integer between 1 and 12, each l is an independent integer between 1 and 12, m is an integer between 1 and 30, or L2 does not exist; L3 is... , , , , , , , , , , , , , , , , , , , , , , , , , or Where each i is an independent integer between 0 and 6, each j is an independent integer between 0 and 8, or L3 does not exist; R2 is , , , , , , , , , , , , , , , , , , , , , Or H, where e is an integer between 0 and 12, and v is an integer between 0 and 48.

[0011] In some embodiments, C is an antitumor drug, an antiviral drug, an antibiotic, an antibacterial drug (such as rifampin and its analogues (e.g., Rifalogue, KRM-1657)), or an immunomodulatory drug, and is attached to an adjacent carbonyl group or L via an N atom or an O atom in the C molecule, or to an adjacent L via an N atom in the C molecule in the form of a quaternary ammonium group.

[0012] In some embodiments, Ar is phenyl, pyridinyl, pyrimidinyl, furanyl, imidazolyl, pyrroleyl, thiazolyl, pyrazolyl, or thiophenyl.

[0013] In some implementations, R a Each independently is H or C 1-4 Alkyl group. In some embodiments, R a Each is independently H, methyl, ethyl, n-propyl, or n-butyl. In some embodiments, R a It can be H or -CH3.

[0014] In some implementations, n is 1, 2, or 3.

[0015] In some implementations, L1 is -NH- or -N(R) a (CH2)2N(R) a )-C(O)-O-, where R a The definition is as described in any embodiment of this disclosure.

[0016] In some implementations, X is -C(O)-, -O-, -NH-, -C(O)NH-, or -NHC(O)-, or X is not present.

[0017] In some implementations, X is -C(O)-, -O-, -NH-, or does not exist.

[0018] In some implementations, L2 is -(CH2CH2O). k-(CH2) l -,-CH[-(CH2) l -NHC(O)-(CH2CH2O) k -CH3]- or -(CH2) m - where k, l, and m are defined as described in any embodiment of this disclosure.

[0019] In some embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, k is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, k is 2, 3, 4, 5, 6, 7, or 8.

[0020] In some embodiments, l is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, l is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, l is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, l is 1, 2, 3, 4, 5, or 6. In some embodiments, l is 1, 2, 3, or 4. In some embodiments, l is 1. In some embodiments, l is 2. In some embodiments, l is 3. In some embodiments, l is 4. In some embodiments, l is 5. In some embodiments, l is 6.

[0021] In some implementations, m is an integer between 1 and 20. In some implementations, m is an integer between 1 and 15. In some implementations, m is an integer between 1 and 10. In some implementations, m is an integer between 1 and 8. In some implementations, m is an integer between 1 and 6. In some implementations, m is an integer between 1 and 4. In some implementations, m is 1, 2, 3, or 4.

[0022] In some implementations, L2 is -(CH2). m - or does not exist, m is defined as described in any embodiment of this disclosure.

[0023] In some implementations, L3 is , or Alternatively, L3 may not exist, where i and j are defined as described in any embodiment of this disclosure.

[0024] In some implementations, R2 is , , , , , , , where e is defined as described in any embodiment of this disclosure.

[0025] In some implementations, R2 is , or , where e is defined as described in any embodiment of this disclosure.

[0026] In some embodiments, e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, e is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, e is 1, 2, 3, 4, 5, or 6. In some embodiments, e is 1, 2, 3, or 4. e is 1, 2, or 3.

[0027] In some implementations, R2 is , , , , , , , , or .

[0028] In some implementations, each R1 is independently hydrogen, C 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro, or cyano. In some embodiments, R1 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. In some embodiments, R1 is fluorine, chlorine, bromine, iodine, hydroxyl, nitro, or cyano.

[0029] In some implementations, r is 0 or 1.

[0030] In some embodiments, C is a cytotoxic agent, a microtubule inhibitor (e.g., MMAE), a DNA alkylating agent, a DNA chimera, an RNA polymerase inhibitor, a kinase inhibitor, a topoisomerase inhibitor, a spindle kinase inhibitor, an antimetabolite, a Toll-like receptor agonist (e.g., a TLR7 agonist), or a STING agonist.

[0031] In some embodiments, Ar is phenyl, furanyl, imidazolyl, or thiophene.

[0032] In some implementations, L1 is -NH- or -N(R) a (CH2)2N(R) a )-C(O)-O-, where R a It is H or -CH3; In some implementations, X is -C(O)NH- or -NHC(O)-, or X is not present.

[0033] In some implementations, L2 is not present.

[0034] In some implementations, L3 is not present.

[0035] In some implementations, R2 is , , or .

[0036] In some implementations, L3 is Or it may not exist, where i is defined as described in any embodiment of this disclosure.

[0037] In some embodiments, i is 1, 2, 3, 4, 5, or 6. In some embodiments, i is 1. In some embodiments, i is 2. In some embodiments, i is 3. In some embodiments, i is 4. In some embodiments, i is 5. In some embodiments, i is 6.

[0038] In some embodiments, j is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, j is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, j is 1. In some embodiments, j is 2. In some embodiments, j is 3. In some embodiments, j is 4. In some embodiments, j is 5. In some embodiments, j is 6.

[0039] In some implementations, R2 is L3 is By introducing a cyclohexyl group at the proximal end of maleimide, the stability of maleimide-derived thioethers can be enhanced (Eur. J. Biochem. 101, 395-399 (1979), reducing off-target effects caused by anti-Michael addition).

[0040] In some implementations, R2 is L3 is By introducing oxygen atoms into the cyclohexyl group, the stability of maleimide-derived thioethers can be further increased, and the water solubility of the linker can also be increased (Sci Rep. 2016 Aug 9:6:30835.).

[0041] In some implementations, R2 is L3 is Wherein i is defined as described in any embodiment of this disclosure, by introducing an amino group at the proximal end of maleimide, the maleimide is induced to spontaneously hydrolyze and open the ring, forming a stable ring-opening coupling form (Bioconjugate Chem. 2015, 26, 1, 145–152).

[0042] In some implementations, each R1 is independently hydrogen or nitro.

[0043] In some implementations, r is 1.

[0044] In some implementations, D is 1.

[0045] In some embodiments, C is auristatin, monomethylauricatine E (MMAE), monomethylauricatine F (MMAF) and its derivatives, maytansine and its derivatives (e.g., maytansine-like substances, DM1, DM3, DM4, paclitaxel, calcitrinin, pyroxine, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxic substances and their derivatives, SN-38, exatecan, doxorubicin, TLR7 agonist or lapatinib with CAS 1821304-87-3).

[0046] In some implementations, Ar is phenyl.

[0047] In some implementations, L3 is -NH-.

[0048] In some implementations, X does not exist.

[0049] In some implementations, R2 is .

[0050] In some implementations, R1 is hydrogen.

[0051] In some embodiments, the compound represented by Formula I is selected from: .

[0052] 2. Applications of Linker-Drug Conjugates

[0053] The second aspect of this disclosure relates to the use of compounds of formula I as described in the first aspect of this disclosure, their salts, solvates, hydrates, enantiomers, diastereomers, racemates, tautomers, polymorphs, or isotopic markers in the preparation of macromolecular conjugates.

[0054] 3. Macromolecular conjugates

[0055] The third aspect of this disclosure relates to a macromolecule conjugated with a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label thereof, formed by conjugation of a macromolecule and a compound of Formula I as described in the first aspect of this disclosure, its salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label thereof, wherein the macromolecule is a protein, antibody, polypeptide, enzyme, protein ligand, or protein scaffold.

[0056] In some embodiments, the macromolecular conjugate drug is as shown in Formula II, wherein

[0057] C and D are defined as described in any embodiment of this disclosure; As a whole or Where Ar, L1, R1, and r are defined as described in any embodiment of this disclosure; R' is -X-L2-L3-R2'-, where X, L2, and L3 are defined as described in any embodiment of this disclosure; R2' is , , , , , , , , , , , , , , , , Or H, where v is an integer between 0 and 48, and k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; A is a macromolecule selected from albumin, antibody, polypeptide, enzyme, protein ligand and protein scaffold; E is an integer or decimal between 1 and 20.

[0058] In some implementations, A is albumin or an antibody or its antigen-binding fragment.

[0059] In some embodiments, A is coupled to the site of the R2' group via an S or N atom in the macromolecule. Alternatively, A may couple to the site of the R2' group by forming an oxime bond through the reaction of the carbonyl group in the macromolecule with the hydroxylamine group in the R2' group. superior.

[0060] In some implementations, R2' is , , , , , ,or , where k1 is defined as described in any embodiment of this disclosure.

[0061] In some embodiments, E is an integer or decimal between 1 and 15. In some embodiments, E is an integer or decimal between 1 and 12. In some embodiments, E is an integer or decimal between 1 and 10. In some embodiments, in the compounds shown in Formula II, E is an integer or decimal between 1 and 8, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8.

[0062] In some implementations, A is albumin, monoclonal antibody (MAB), bispecific antibody, multispecific antibody, or an antigen-binding fragment thereof.

[0063] In some embodiments, A is a monoclonal antibody, bispecific antibody, or multispecific antibody with a thiol or amino group as the coupling site, or a monoclonal antibody, bispecific antibody, or multispecific antibody with a site-directed mutation or modification using a thiol or amino group as the coupling site, or a monoclonal antibody, bispecific antibody, or multispecific antibody with a site-directed mutation using a carbonyl group in acetylphenylalanine (pAF) as the coupling site.

[0064] In some implementations, R2' is , , ,or , where k1 is defined as described in any embodiment of this disclosure.

[0065] In some implementations, A is selected from: anti-HER2 humanized monoclonal antibody mil40, trastuzumab (Herceptin), pertuzumab (PERJETA), zotuximab (Zolbetuximab, IMAB362), magrolimab, cetuximab (ERBITUX), panitumumab (VECTIBIX), rituximab (RITUXAN), alemtuzumab (CAMPATH), teimozumab (ZEVALIN), tosimomumab (BEXXAR), oflamuzumab (ARZERRA), bevacizumab (AVASTIN), ipilimumab (YERVOY), denosumab (XGEVA), pembrolizumab (KEYTRUDA), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi ), sacituzumab, rovalpituzumab and their antigen-binding fragments.

[0066] In some embodiments, in the compound represented by Formula II, A is trastuzumab.

[0067] In some embodiments, A in the compound represented by Formula II is zotuximab.

[0068] In some embodiments, in the compound shown in Formula II, A is molotovicillin.

[0069] In some embodiments, A is albumin. In some embodiments, A is human serum albumin.

[0070] In some embodiments, A is an antibody or its antigen-binding fragment, comprising: (a) The following three heavy chain variable regions are complement-determining regions: (i) VH CDR1, which has the CDR1 sequence contained in the heavy chain as shown in SEQ ID NO: 1, or has a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR1 sequence contained in the heavy chain. (ii) VH CDR2, having the CDR2 sequence contained in the heavy chain as shown in SEQ ID NO: 1, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR2 sequence contained in the heavy chain; and (iii) VH CDR3, having the CDR3 sequence contained in the heavy chain as shown in SEQ ID NO: 1, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of one or two amino acids) compared to the CDR3 sequence contained in the heavy chain. and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the CDR1 sequence contained in the light chain as shown in SEQ ID NO: 2, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR1 sequence contained in the light chain. (v) VL CDR2, having the CDR2 sequence contained in the light chain as shown in SEQ ID NO: 2, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the light chain; and (vi) VL CDR3, having the CDR3 sequence contained in the light chain as shown in SEQ ID NO: 2, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the light chain.

[0071] In some embodiments, A is an antibody or its antigen-binding fragment, comprising: (a) The following three heavy chain variable regions are complement-determining regions: (i) VH CDR1, having the CDR1 sequence contained in the heavy chain as shown in SEQ ID NO: 3, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR1 sequence contained in the heavy chain. (ii) VH CDR2, having the CDR2 sequence contained in the heavy chain as shown in SEQ ID NO: 3, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR2 sequence contained in the heavy chain; and (iii) VH CDR3, having the CDR3 sequence contained in the heavy chain as shown in SEQ ID NO: 3, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the heavy chain. and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the CDR1 sequence contained in the light chain as shown in SEQ ID NO: 4, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR1 sequence contained in the light chain. (v) VL CDR2, having the CDR2 sequence contained in the light chain as shown in SEQ ID NO: 4, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the light chain; and (vi) VL CDR3, having the CDR3 sequence contained in the light chain as shown in SEQ ID NO: 4, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the light chain.

[0072] In some embodiments, A is an antibody or its antigen-binding fragment, comprising: (a) The following three heavy chain variable regions are complement-determining regions: (i) VH CDR1, having the CDR1 sequence contained in the heavy chain as shown in SEQ ID NO: 5, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR1 sequence contained in the heavy chain. (ii) VH CDR2, having the CDR2 sequence contained in the heavy chain as shown in SEQ ID NO: 5, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the heavy chain; and (iii) VH CDR3, having the CDR3 sequence contained in the heavy chain as shown in SEQ ID NO: 5, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the heavy chain. and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the CDR1 sequence contained in the light chain as shown in SEQ ID NO: 6, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR1 sequence contained in the light chain. (v) VL CDR2, having the CDR2 sequence contained in the light chain as shown in SEQ ID NO: 6, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the light chain; and (vi) VL CDR3, having the CDR3 sequence contained in the light chain as shown in SEQ ID NO: 6, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the light chain.

[0073] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0074] In some implementations, the CDR1, CDR2, and CDR3 contained in the heavy chain variable region, and / or the CDR1, CDR2, and CDR3 contained in the light chain variable region, are defined by the Kabat, Chothia, AbM, or IMGT numbering system.

[0075] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:2. In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:4. In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:6.

[0076] In some embodiments, the macromolecular conjugate is selected from:

[0077] in A represents A, and A and E are defined as described in any embodiment of this disclosure, and k1 is defined as described in any embodiment of this disclosure.

[0078] In some embodiments, the macromolecular conjugate is selected from:

[0079] in, The antibody, E, is defined as described in any embodiment of this disclosure. The antibody or albumin is preferred, preferably human serum albumin. In some embodiments, the antibody or albumin is linked to the # site via an N atom in the molecule. In some embodiments, E is about 4 or about 8. In some embodiments, E is about 4.

[0080] 4. Pharmaceutical Composition

[0081] The fourth aspect of this disclosure relates to a pharmaceutical composition comprising a macromolecular conjugate drug as described in any embodiment of the third aspect of this disclosure, a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph or isotopic label, and one or more pharmaceutically acceptable carriers and / or excipients.

[0082] In some embodiments, the macromolecular conjugate drug, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label is present in an effective amount in the pharmaceutical composition.

[0083] The pharmaceutical compositions disclosed herein can be prepared into various dosage forms, including but not limited to tablets, capsules, solutions, suspensions, granules, or injections, using conventional methods in the art, and administered via routes such as oral or non-enteric, such as intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

[0084] 5. Pharmaceutical uses of macromolecular conjugates

[0085] The fifth aspect of this disclosure relates to the use of any embodiment of the macromolecular conjugate drug described in the third aspect of this disclosure, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemic mixture, tautomer, polymorph, or isotopic label in the preparation of a medicament for treating a disease or condition or reducing the severity of said disease or condition, wherein said disease or condition is selected from tumors, infectious diseases, hematological diseases, metabolic diseases, and inflammation.

[0086] In some implementations, the disease or condition is selected from cancer, lymphoma, lymphoid tumor, blastoma, sarcoma, and leukemia.

[0087] In some implementations, the cancer is selected from: breast cancer, squamous cell carcinoma, lung cancer, peritoneal cancer, gastric cancer, endometrial adenocarcinoma, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, urethral cancer, hepatocellular carcinoma, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, gallbladder cancer, esophageal cancer, bile duct cancer, head and neck cancer, and metastatic tumors.

[0088] In some embodiments, the cancer is selected from epithelial squamous cell carcinoma, HER2-positive breast cancer, colon cancer, rectal cancer, gastrointestinal cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma.

[0089] 6. Preparation method

[0090] In some embodiments, the macromolecular conjugate drugs described in this disclosure can be synthesized via the following routes.

[0091]

[0092] Among them: C, E, As defined in any embodiment of this disclosure, M represents -L2-L3-, and L2, L3, and k1 are defined as described in any embodiment of this disclosure.

[0093] Compound 3 was prepared by amide condensation of compound 1 (N-fluorenylmethoxycarbonyl-L-glutamic acid 5-(9-fluorenylmethyl) ester) and compound 2, and then activated by di(p-nitrophenyl) carbonate to obtain activated intermediate 4. Intermediate 4 was linked with active molecule C (including microtubule inhibitor MMAE, TLR7 agonist and topoisomerase inhibitor Dxd) to prepare compound 5, and then deprotected with piperidine to obtain load 6. The antibody coupling reaction was carried out in two steps. In the first step, an azide group 7 was modified on the naked amino group of the antibody to obtain compound 8. In the second step, load 6 was coupled to compound 8 by Click reaction, and finally the final macromolecular conjugated drug 9 was prepared.

[0094] In some embodiments, the macromolecular conjugate drugs described in this disclosure can be synthesized via the following routes.

[0095]

[0096] Among them: C, E, As defined in any embodiment of this disclosure, M represents -L2-L3-, and L2, L3, and k1 are defined as described in any embodiment of this disclosure.

[0097] Intermediate 11 was prepared by amide condensation of N-fluorenylmethoxycarbonyl-L-glutamic acid 5-(9-fluorenylmethyl) ester and 2-(methylamino)ethylcarbamate tert-butyl ester (10), and intermediate 12 was prepared by deprotection of the Boc protecting group with hydrochloric acid; intermediate 14 was prepared by reaction of compound 2 with p-nitrophenyl chloroformate (13), and intermediate 15 was prepared by reaction with intermediate 12; activated intermediate 16 was prepared by activation with di(p-nitrophenyl) carbonate, intermediate 17 was prepared by reaction with active molecule C, and loaded intermediate 18 was prepared by deprotection of the Fmoc protecting group with piperidine; loaded intermediate 18 was coupled to compound 8 by Click reaction to finally prepare the final macromolecular conjugated drug 19.

[0098] In some embodiments, the macromolecular conjugate drugs described in this disclosure can be synthesized via the following routes.

[0099]

[0100] Among them: C, E, As defined in any embodiment of this disclosure, M represents -L2-L3-, and L2, L3, and k1 are defined as described in any embodiment of this disclosure.

[0101] Intermediate 21 was prepared by condensation of N-fluorenylmethoxycarbonyl-L-glutamic acid 5-(9-fluorenylmethyl) ester with (4-amino-3-nitrophenyl)methanol (20), and intermediate 22 was prepared by reduction with iron powder under acidic conditions. Intermediate 22 was prepared by amide condensation to obtain intermediate 23, and activated intermediate 24 was prepared by di(p-nitrophenyl) carbonate. Intermediate 25 was prepared by reaction with active molecule C, and loaded 26 was prepared by removing the Fmoc protecting group with piperidine. Loaded 26 was coupled to compound 8 by Click reaction to finally prepare the final macromolecular conjugated drug 27.

[0102] definition

[0103] In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0104] As used herein, the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0105] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both singular and plural forms in the context of describing this application (especially in the context of the following claims).

[0106] As used herein, the term “and / or” should be considered as a specific disclosure of each of two or more specified features or elements, and any combination of two or more features or elements. Therefore, the term “and / or” as used in phrases, such as “A and / or B” herein, is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).

[0107] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residue in the variable region of an antibody that is responsible for antigen binding. Nanobodies contain three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0108] As used herein, the term “about” is understood to mean within + / -20%, + / -18%, + / -15%, + / -12%, + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1%, + / -0.5%, + / -0.4%, + / -0.3%, + / -0.2%, and + / -0.1% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term “about”.

[0109] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the essential properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, conservative substitution generally refers to replacing a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0110] As used herein, the term "pharmaceutically acceptable" means that the substance described is not only physiologically acceptable to a subject, but also has pharmaceutical value. For example, the description of "pharmaceutically acceptable salt" indicates that the salt is not only physiologically acceptable to a subject, but also refers to a synthetic substance with pharmaceutical value, such as a salt formed as an intermediate during chiral resolution. Although such intermediate salt cannot be directly administered to a subject, it can play a role in obtaining the final product of this application.

[0111] In this disclosure, compounds (including compounds of Formula I or macromolecular conjugates of Formula II) may be used either in their own form or in the form of their salts (e.g., pharmaceutically acceptable salts). The term "pharmaceutically acceptable salt" refers to a salt of the compound of this application that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Pharmaceutically acceptable salts of compounds of Formula I or macromolecular conjugates of Formula II include salts formed with pharmaceutically acceptable inorganic or organic acids, or with pharmaceutically acceptable inorganic or organic bases. Examples of suitable acid addition salts include salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pyric acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, or tannic acid. Other acids, such as oxalic acid, while not pharmaceutically acceptable on their own, can be used to prepare salts as intermediates to obtain the compounds of this application and their pharmaceutically acceptable salts. Examples of suitable base addition salts include salts formed with sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, or procaine. Methods for preparing pharmaceutically acceptable salts of the compounds of this application are known to those skilled in the art. When referring to compounds of this disclosure, this includes compounds of Formula I or macromolecular conjugates of Formula II and their pharmaceutically acceptable salts.

[0112] As used herein, the term "pharmaceuticalally acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including but not limited to binders, diluents, fillers, disintegrants, wetting agents, lubricants, colorants, flavoring agents, solubilizers, osmotic regulators, or other conventional additives. Typical pharmaceutically acceptable carriers and / or excipients include, for example, microcrystalline cellulose, starch, crospovidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium dodecyl sulfate, or magnesium stearate.

[0113] As used herein, the term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group, preferably having 1-6, 1-4, or 1-3 carbon atoms. For example, "C 1-6"Alkyl" refers to a saturated, straight-chain or branched monovalent hydrocarbon group having 1-6 carbon atoms. 1-4 "Alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group having 1 to 4 carbon atoms. Typical examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, etc.

[0114] As used herein, the term "5-6 aryl" refers to an unsaturated aromatic carbocyclic group with 5-6 carbon atoms, consisting of a monocyclic or two- or more fused rings having a conjugated π-electron system. Typical examples of "5-6 aryl" include, but are not limited to, phenyl.

[0115] As used herein, the term "5-6-membered heteroaryl" refers to an aryl group as defined herein, in which at least one ring member is a heteroatom selected from nitrogen, oxygen, or sulfur. Typical examples of "5-6-membered heteroaryl" include, but are not limited to, pyridinyl, pyrimidinyl, furanyl, imidazolyl, pyrroleyl, thiazolyl, pyrazolyl, thiophenyl, etc.

[0116] As used in this article, the term "macromolecule drug conjugates (MDCs)" refers to complex macromolecular drugs formed by covalently binding macromolecular carriers (such as albumin, antibodies, peptides, enzymes, protein ligands, and protein scaffolds) with therapeutically active molecules (such as cytotoxins, small molecule drugs, and radionuclides) through chemical linkers. Typical examples of macromolecule drug conjugates include antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), and albumin-drug conjugates.

[0117] As used herein, the drug macromolecule conjugation ratio refers to the number of therapeutically active molecules (payloads) attached to each macromolecule (such as an antibody or its antigen-binding fragment or albumin), denoted, for example, by the letter E in Formula II of this disclosure.

[0118] As used herein, the term "solvate" refers to a substance formed by the association of a compound of Formula I or a macromolecular conjugate of Formula II, or a pharmaceutically acceptable salt thereof, with an organic solvent molecule, including but not limited to methanol, ethanol, propanol, acetonitrile, etc. The compound of Formula I or the macromolecular conjugate of Formula II, or a pharmaceutically acceptable salt thereof, may also form hydrates with water.

[0119] Unless otherwise specified, the compounds disclosed herein (including compounds of Formula I or macromolecular conjugates of Formula II) may exist in stereoisomer form. The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. Stereoisomers are isomers resulting from different spatial arrangements of atoms in a molecule, including meso compounds, racemic compounds, enantiomers, diastereomers, cis-trans isomers, conformational isomers, or mixtures thereof. Similarly, the compounds of this application may exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0120] The compounds disclosed herein (including compounds of Formula I or macromolecular conjugates of Formula II) may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0121] This disclosure covers all possible crystalline forms or polymorphs of the compounds of this disclosure (including the compounds shown in Formula I or the macromolecular conjugates shown in Formula II), which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0122] As used herein, the term "isotope label" refers to the substitution of one or more atoms in a compound with the same number of atoms but with a different atomic mass or mass number than the dominant atomic mass or mass number found in nature. Examples of isotopes suitable for inclusion in compounds of this disclosure (including compounds of Formula I or macromolecular drug conjugates of Formula II) include, but are not limited to, hydrogen isotopes such as 2 H, 3 H; carbon isotopes, for example 11 C, 13 C and 14 C; Chlorine isotopes, for example 36 Cl; fluorine isotopes, for example 18 F; Iodine isotopes, for example 123 I and 125 I; nitrogen isotopes, for example 13 N and 15 N; oxygen isotopes, for example 15 O, 17 O and 18 O; and sulfur isotopes such as 35 S.

[0123] As used herein, the term "treatment" refers to a method performed to achieve a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, relieving symptoms, narrowing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also mean prolonging survival compared to expected survival (if no treatment were received).

[0124] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for treating a disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0125] It should also be noted that the dosage and method of administration of the macromolecular conjugates disclosed herein depend on numerous factors, including the patient's age, weight, sex, natural health condition, nutritional status, the activity intensity of the compound, the timing of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage ranges from 0.01 to 1000 mg / kg body weight / day. In this disclosure, the dosing regimen can be adjusted to achieve optimal therapeutic or preventative effects. For example, it can be administered as a single dose, multiple times over a period of time, or the dosage can be proportionally reduced or increased depending on the urgency of the treatment situation.

[0126] Abbreviations / Tags

[0127] ADC (antibody-drug conjugate): Antibody-drug conjugate; EEDQ (Ethyl 1,2-dihydro-2-ethoxy-1-quinolinecarboxylate): 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; DCM (Dichloromethane): Dichloromethane; THF (Tetrahydrofuran): Tetrahydrofuran; HOBT (1-Hydroxybenzotriazole): 1-Hydroxybenzotriazole; DIPEA ( N,N-Diisopropylethylamine: Diisopropylethylamine; DMF ( N,N -Dimethylformamide): N,N -Dimethylformamide; THPTA (Tris(3-hydroxypropyltriazolylmethyl)amine): Tris(3-hydroxypropyltriazolylmethyl)amine; Fmoc (9-Fluorenylmethyloxycarbonyl): 9-fluorenylmethoxycarbonyl; MMAE (Monomethyl auristatin E): Monomethyl auristatin E; EA (Ethyl acetate): Ethyl acetate; Boc ( t -Butyloxycarbonyl); tert-Butyloxycarbonyl; EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride): 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; AcOH (Acetic acid): Glacial acetic acid; DMA ( N,N -Dimethylacetamide): N,N -Dimethylacetamide; DAR (Drug Antibody Ratio): Drug-antibody conjugate ratio.

[0128] Beneficial effects

[0129] The linker-drug conjugates provided in this disclosure contain a glutamyl fragment, exhibiting excellent enzymatic cleavage performance. The macromolecular drug conjugates containing the linker provided in this disclosure exhibit good structural stability, ideal enzymatic decomposition performance, and satisfactory in vitro and in vivo efficacy and safety.

[0130] Sequence information

[0131] Descriptions of the sequences involved in this disclosure are provided in the table below.

[0132] Table 1: Sequence Information Attached Figure Description

[0133] Figure 1The results of the drug macromolecular conjugation ratio test for macromolecular conjugated drug II-1 are shown.

[0134] Figure 2 The results showed that the macromolecular conjugate II-1 inhibited the proliferation of tumor cells SKBR3, BT474, and NCI-N87.

[0135] Figure 3 The results showed that the macromolecular conjugate II-2 inhibited the proliferation of tumor cells NUGC4.

[0136] Figure 4 The results of a pharmacodynamic study of the macromolecular conjugate II-1 in NCI-N87 tumor-bearing mice are presented.

[0137] Figure 5 The results of a pharmacodynamic study of the macromolecular conjugate II-2 in NUGC4 tumor-bearing mice are presented. Detailed Implementation

[0138] The following specific embodiments further illustrate the substantive content of this disclosure. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this disclosure. In the following embodiments, unless specific conditions are specified, conventional conditions or manufacturer recommendations are followed. Raw materials whose manufacturers are not specified are all commercially available conventional products.

[0139] While many of the materials and operating methods used in the following embodiments are well known in the art, this disclosure is still intended to describe them in as much detail as possible. It will be apparent to those skilled in the art that, unless otherwise stated, the materials and operating methods used in the following embodiments are well known in the art.

[0140] Example 1: Preparation of macromolecular conjugate II-1

[0141] 1) (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-hydroxybut-3-yn-1-yl)phenyl)- L -Glutamate ((9) H -fluoren-9-yl)methyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)- N 5 -(4-(1-hydroxybut-3-yn-1-yl)phenyl)-L Preparation of glutaminate

[0142] (S)-5-((9) H -fluorene-9-yl)methoxy)-4-((((9) H -fluorene-9-yl)methoxy)carbonyl)amino)-5-oxopentanoic acid ((S)-5-((9) H -fluoren-9-yl)methoxy)-4-((((9 H 1-(4-aminophenyl)but-3-yn-1-ol (0.106 g, 0.66 mmol) and EEDQ (0.163 g, 0.66 mmol) were added to a 50 mL round-bottom flask, followed by 10 mL of anhydrous DCM. The mixture was stirred at room temperature for 24 h. After the reaction was complete, a large amount of white solid precipitated out. 1N HCl aqueous solution was added to the reaction solution, and the mixture was filtered. The filter cake was dried under vacuum to obtain a white solid (0.306 g, 67%). 1 H-NMR (600 MHz, DMSO- d6 ): δ 9.891(s, 1H), 7.945 (d, J = 7.8 Hz, 1H), 7.898-7.862 (m, 4H), 7.745-7.740 (m, 2H), 7.696 (d, J = 7.2 Hz, 2H), 7.538 (d, J = 8.4 Hz, 2H),7.416-7.370 (m, 4H), 7.325-7.259 (m, 6H), 5.435 (d, J = 4.2 Hz, 1H), 4.626(dd, J = 10.8 Hz, 6.6 Hz, 1H), 4.374-4.358 (m, 2H), 4.307 (t, J = 7.2 Hz, 2H), 4.244 (q, J = 7.2 Hz, 2H), 4.204-4.175 (m, 1H), 2.703 (t, J = 3.0 Hz, 1H), 2.468 (dd, J= 2.4 Hz, 6.6 Hz, 1H), 2.447-2.410 (m, 2H), 2.078 (td, J =13.2 Hz, 7.8 Hz, 1H), 2.078 (td, J = 13.2 Hz, 7.8 Hz, 1H), 1.861 (td, J =14.4 Hz, 8.4 Hz, 1H). MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 44 H 39 N2O6691.27, found 691.29.

[0143] 2) (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((4-nitrophenoxy)carbonyl)oxy)but-3-yne-1-yl)phenyl)- L -Glutamate (9 H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((4-nitrophenoxy)carbonyl)oxy)but-3-yn-1-yl)phenyl)- L Preparation of glutaminate

[0144] (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-hydroxybut-3-yn-1-yl)phenyl)- L⁻ glutamic acid (0.1 g, 0.145 mmol) and p-nitrophenyl chloroformate (0.058 g, 0.29 mmol) were added to a 50 mL round-bottom flask, followed by 6 mL of anhydrous THF. Pyridine (0.057 g, 0.725 mmol) was diluted in 1 mL of anhydrous THF and slowly added dropwise to the reaction mixture. During the slow addition of pyridine, the reaction mixture gradually became clear, and the reaction was carried out at room temperature for 5 h. The reaction mixture was then added to deionized water, extracted twice with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate overnight. The concentrated crude product was purified by column chromatography to give a white solid (0.088 g, 71% yield). 1 H-NMR (600 MHz, DMSO- d6 ): δ 10.41 (s, 1H), 8.304 (d, J = 9.0 Hz, 1H), 7.948 (d, J =7.8 Hz, 1H), 7.899-7.863 (m, 4H), 7.745-7.729 (m, 2H), 7.696 (d, J = 7.2 Hz, 2H), 7.640 (d, J = 8.4 Hz, 2H), 7.538 (d, J = 9.0 Hz, 2H), 7.442-7.259 (m,10H), 5.770 (t, J = 6.0 Hz, 1H), 4.380-4.362 (m, 2H), 4.311 (t, J = 7.2 Hz,2H), 4.264-4.182 (m, 3H), 2.987-2.885 (m, 3H), 2.450 (br, 2H), 2.101-2.070(m, 1H), 1.913-1.838 (m, 1H), 1.365-1.232 (m, 4H). MS (ESI) m / z: ESI-MS m / z(M+Na) + Calculated for C 51 H 41 N3O 10 Na 878.28, found 878.28.

[0145] 3) (9) H -fluorene-9-yl)methyl N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 5 -(4-(1-(((( S )-1-(((S)-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenyl)- L -Glutamate (9 H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((( S )-1-((( S )-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4 -yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenyl)- L Preparation of glutaminate

[0146] (9)H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((4-nitrophenoxy)carbonyl)oxy)but-3-yne-1-yl)phenyl)- L β-Glutamate (0.088 g, 0.10 mmol) was added to a 25 mL round-bottom flask, followed by 3 mL of anhydrous DMF. After the compound was completely dissolved, MMAE (0.06 g, 0.083 mmol), HOBT (0.011 g, 0.083 mmol), and DIPEA (0.011 g, 0.083 mmol) were added sequentially, and the mixture was stirred at room temperature. After reacting for 9 h, the solvent was removed by pressure concentration, and the sample was mixed. The crude product was purified by column chromatography to obtain a white solid (0.048 g, 40% yield). 1 H-NMR (600 MHz, DMSO- d6 ): δ 7.809-7.668 (m, 4H), 7.577-7.523 (m, 5H), 7.398-7.276 (m, 11H), 7.251-7.211 (m, 1H), 6.583 (br, 1H), 5.848-5.678 (m, 2H), 5.007-4.394 (m, 8H), 4.305-3.996 (m, 6H), 3.518-3.253 (m, 7H), 3.138-2.824 (m, 5H), 2.757-2.700 (m, 2H), 2.435-1.606 (m, 15H), 1.427-1.237 (m,18H), 1.079-0.743 (m, 20H). MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 84 H 104 N7O 14 1434.756, found 1434.824.

[0147] 4) N 5 -(4-(1-(((( S )-1-(((S)-1-(((3 R 4 S 5 S )-1-(( S )-2-((1R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamoyl)oxy)but-3-yne-1-yl)phenyl)- L -Glutamine (I-1, N 5 -(4-(1-(((( S )-1-((( S )-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4 -yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenyl)- L Preparation of glutamine)

[0148] (9) H -fluorene-9-yl)methyl N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((( S )-1-(((S)-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1S ,2 R )-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenyl)- L -Glutamate (0.04 g, 0.028 mmol) was added to a 10 mL round-bottom flask. 100 μL of piperidine was diluted in 1 mL of anhydrous DMF and slowly added dropwise to the round-bottom flask. The solution immediately turned yellow. The mixture was stirred at room temperature for 2 h. The solvent was removed by concentration under reduced pressure, and the sample was mixed. The crude product was purified by column chromatography to obtain a white solid (0.027 g, 93% yield). 1 H-NMR (600 MHz, DMSO- d6 ): δ 10.455-10.348 (m, 1H), 8.030-7.926 (m, 1H), 7.722-7.644(m, 1H), 7.579-7.468 (m, 2H), 7.407-7.374 (m, 1H), 7.316-7.254 (m, 4H),7.188-7.101 (m, 2H), 5.672-5.537 (m, 1H), 5.447 (br, 1H), 4.765-4.330 (m,3H), 4.257-3.780 (m, 3H), 3.600-3.090 (m, 13H), 3.004-2.890 (m, 4H), 2.849-2.716 (m, 4H), 2.442-2.416 (m, 1H), 2.278 (br, 1H), 2.135-1.893 (m, 4H), 1.816-1.724 (m, 3H), 1.623-1.483 (m, 4H), 1.322 (br, 1H), 1.054-0.645 (m,22H). MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 55 H 84 N7O 12 1034.61, found 1034.62.

[0149] 5) Preparation of antibodies modified with azide groups

[0150] Herceptin (26.25 mg, 1.75 × 10⁻⁶) -4 mmol (purchased from Roche / Genentech) was added to a 5 mL EP tube, and compound 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (0.407 mg, 1.05 × 10⁻⁶ mmol, purchased from Roche / Genentech) was added dropwise to the tube. -3 A DMA solution of 1 mmol was prepared. After reacting overnight at room temperature with stirring, the conjugation product was transferred to PBS to prepare azide-modified Herceptin antibody 1, where E represents the drug macromolecule conjugation ratio, which is approximately 4. Herceptin is a marketed antibody that targets the HER2 antigen on tumor cells.

[0151] 6) Preparation of macromolecular conjugate II-1

[0152] Herceptin (2.5 mg, 1.67 × 10⁻⁶) modified with an azide group (2.5 mg, 1.67 × 10⁻⁶) -5 Add 0.26 mg (2.5 × 10 mmol) to a 2 mL EP tube, and then add compound I-1 (0.26 mg, 2.5 × 10 mmol) dropwise. -4 A DMA solution of 0.04 mg (mmol) was then added sequentially. -4 mmol), THPTA (0.22 mg, 5.01×10 -4 mmol) and sodium ascorbate (1.49 mg, 7.51 × 10 ...). -3 A mmol) aqueous solution was added to the reaction system, and the reaction was carried out overnight at room temperature with stirring. The conjugated product was then transferred to PBS to prepare the target macromolecular conjugated drug II-1. Here, E represents the macromolecular conjugation ratio, which is 3-4. Herceptin is a marketed antibody that targets the HER2 antigen on tumor cells.

[0153]

[0154] Example 2: Preparation of compounds I-2 and I-3

[0155] Following the method for preparing compound I-1 in step 4) of Example 1, compounds I-2 and I-3 were prepared by replacing MMAE with a TLR7 agonist with CAS registration number 1821304-87-3 and eczetcan, respectively.

[0156]

[0157] Compound I-2: 1 H-NMR (600 MHz, DMSO- d6 ): δ 10.396 (s, 1H), 9.598 (s,1H), 7.702-7.683 (m, 2H), 7.579 (d, J = 7.8 Hz, 2H), 7.509 (d, J = 7.8 Hz,1H), 7.393 (d, J = 2.4 Hz), 7.276 (d, J = 8.4 Hz, 2H), 7.182-7.133 (m, 1H),7.003 (s, 1H), 6.796 (d, J = 7.8 Hz, 1H), 6.682 (d, J = 7.8 Hz, 1H), 6.134(d, J = 2.4 Hz, 1H), 5.609 (t, J = 6.0 Hz, 1H), 5.216 (s, 2H), 3.858 (s, 3H),3.522 (dd, J = 12.6 Hz, 2H), 2.972 (t, J = 5.4 Hz, 6H), 2.811 (t, J = 2.4 Hz,1H), 2.704-2.695 (m, 2H), 2.304 (br, 3H), 2.043-1.955 (m, 2H), 1.671-1.620(m, 8H), 1.548-1.540 (m, 3H), 1.351-1.296 (m, 4H), 0.862 (t, J = 7.2 Hz, 3H).MS (ESI) m / z: ESI-MS m / z (M+H) + calculated for C 40 H 52 N9O6 754.396, found754.542.

[0158] Compound I-3: 1 H-NMR (600 MHz, DMSO- d6): δ 10.449-10.359 (m, 1H), 8.185-8.130 (m, 1H), 7.741 (d, J = 10.2 Hz, 1H), 7.582 (br, 1H), 7.380-7.283 (m,3H), 6.521 (s, 1H), 5.728 (dt, J = 19.8 Hz, 5.6 Hz, 1H), 5.442 (br, 1H), 5.284-5.191 (m, 3H), 3.169-2.767 (m, 7H), 2.348 (d, J = 11.4 Hz, 4H), 2.155-1.844 (m, 7H), 1.627 (s, 1H), 0.871 (t, J = 7.2 Hz, 3H). MS (ESI) m / z: ESI-MSm / z (M+H) + Calculated for C 40 H 39 FN5O9 752.265, found 725.279.

[0159] Example 3: Preparation of macromolecular conjugates II-2, II-3 and II-4

[0160] Following the preparation method of macromolecular conjugate II-1 in Example 1, Herceptin was replaced with Zolbetuximab (purchased from Astellas), albumin (purchased from Zhejiang Hisun Pharmaceutical Co., Ltd.), and Magrolimab (purchased from Beijing Sinopharm Co., Ltd.), respectively, to prepare macromolecular conjugates II-2, II-3, and II-5, where E represents the macromolecular conjugation ratio. In II-2, E is 7-8, and in II-3 and II-5, E is 3-4.

[0161]

[0162] Example 4: Preparation of macromolecular conjugates II-5 and II-6

[0163] 1) Preparation of antibodies modified with azide groups

[0164] Referring to the method for preparing azide-modified antibodies in step 5) of Example 1, 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate was used to replace 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate to prepare azide-modified Herceptin antibody 2, where E represents the drug macromolecule conjugation ratio, which is approximately 4. Herceptin is a marketed antibody that targets the HER2 antigen on tumor cells.

[0165]

[0166] 2) Preparation of macromolecular conjugates II-5 and II-6

[0167] Referring to the preparation method of macromolecular conjugate II-1 in Example 1, macromolecular conjugates II-5 and II-6 were prepared using compounds I-2 or I-3, and Herceptin antibody 2 modified with an azide group, respectively, as raw materials. Here, E represents the macromolecular conjugation ratio, which is 3-4. Herceptin is a marketed antibody that targets the HER2 antigen on tumor cells.

[0168]

[0169] Example 5: Preparation of the macromolecular conjugate drug II-7

[0170] 1) (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(2-((tert-Butoxycarbonyl)(methyl)amino)ethyl)- N 5 -methyl- L -Glutamate (9 H -fluoren-9-yl)methyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)- N 5-(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)- N 5 -methyl- L Preparation of glutaminate

[0171] (S)-5-((9) H -fluorene-9-yl)methoxy)-4-((((9) H fluorene-9-yl)methoxy)carbonyl)amino)-5-oxopentanoic acid (0.25 g, 0.46 mmol), tert-butyl(2-(methylamino)ethyl)(methyl)carbamate (0.087 g, 0.46 mmol) and EEDQ (0.114 g, 0.46 mmol) were added to a 50 mL round-bottom flask, followed by 10 mL of anhydrous DCM. The mixture was stirred at room temperature for 24 h. The solvent was removed by pressure concentration, and the crude product was purified by column chromatography to give a white solid (0.254 g, 77%). 1 H-NMR (600 MHz, DMSO- d6 ): δ 7.902-7.872 (m, 5H), 7.727-7.687 (m, 5H), 7.417-7.386 (m, 4H), 7.319-7.274 (m, 4H), 4.377-4.364 (m, 2H), 4.294 (t, J =7.8 Hz, 2H), 4.235 (t, J = 6.6 Hz, 2H), 4.157 (br, 1H), 3.396 (br, 1H), 3.346(br, 1H), 3.262 (br, 2H), 2.887 (s, 2H), 2.823 (s, 1H), 2.751 (br, 3H),2.340-2.256 (m, 2H), 1.987-1.910 (m, 1H), 1.819-1.795 (m, 1H). MS (ESI) m / z:ESI-MS m / z (M+H) + Calculated for C 43 H 48 N3O7718.34, found 718.35.

[0172] 2) (9) H -fluorene-9-yl)methyl N 2 -(((9H -fluorene-9-yl)methoxy)carbonyl)- N 5 -methyl- N 5 -(2-(methylamino)ethyl)- L -Glutamate (9 H -fluoren-9-yl)methyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)- N 5 -methyl- N 5 -(2-(methylamino)ethyl)- L Preparation of glutaminate

[0173] (9) H -fluorene-9-yl)methylN 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(2-((tert-Butoxycarbonyl)(methyl)amino)ethyl)- N 5 -methyl- L 1-Glutamate (0.2 g, 0.279 mmol) was added to a 25 mL round-bottom flask, and 2 mL of ethyl acetate solution of 2N HCl was added. The mixture was stirred overnight at room temperature, and a large amount of white precipitate was precipitated. The solvent was removed by pressure concentration, and the solid was dried under vacuum to obtain a white solid (170 mg, 99% yield). 1 H-NMR (600 MHz, DMSO- d6 ): δ7.934-7.878 (m, 5H), 7.742-7.691 (m, 4H), 7.424-7.393 (m, 4H), 7.327-7.278 (m,4H), 4.424-4.361 (m, 2H), 4.343-4.276 (m, 2H), 4.243 (t, J = 6.6 Hz, 2H),4.213-4.168 (m, 1H), 3.637-3.517 (m, 2H), 3.073-2.997 (m, 2H), 2.920 (s, 2H),2.538 (t, J= 10.8 Hz, 3H), 2.460-2.290 (m, 2H), 1.989-1.932 (td, J = 13.8Hz, 7.2Hz, 1H), 1.834-1.773 (m, 1H), 1.232 (s, 1H). MS (ESI) m / z: ESI-MS m / z(M+H) + Calculated for C 38 H 40 N3O5 618.29, found 618.30.

[0174] 3) Preparation of 4-(1-hydroxybut-3-yn-1-yl)phenyl(4-nitrophenyl) carbonate

[0175] 4-(1-hydroxybut-3-yn-1-yl)phenol (0.2 g, 1.23 mmol) was added to a 25 mL round-bottom flask, followed by 5 mL of anhydrous THF. The mixture was then cooled to 0°C in an ice bath. p-Nitrophenyl chloroformate (0.248 g, 1.23 mmol) was dissolved in 3 mL of anhydrous THF and slowly added dropwise to the reaction mixture. The solution color changed from pink to pale yellow. Next, DIPEA was added dropwise to the reaction mixture; the solution color remained unchanged. The reaction mixture was then allowed to stand at room temperature overnight. The solvent was removed by pressure concentration, and the crude product was purified by column chromatography to give a white solid (0.295 g, 73% yield). 1 H-NMR (600 MHz, DMSO- d6 ): δ 8.364(d, J = 9.0 Hz, 2H), 7.708 (d, J = 9.0 Hz, 2H), 7.479 (d, J = 9.0 Hz, 2H), 7.362 (d, J = 8.4 Hz, 2H), 5.606 (d, J = 4.2 Hz, 1H), 4.726 (dd, J = 10.8 Hz, 6.0 Hz, 1H), 2.744 (t, J= 3.0 Hz, 1H), 2.536 (ddd, J = 9.0 Hz, 6.6 Hz, 3.0Hz, 2H). MS (ESI) m / z: ESI-MS m / z (M+Na) + Calculated for C 17 H 13 NO6Na 350.07, found 350.06.

[0176] 4) (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(2-(((4-(1-hydroxybut-3-ynyl)phenoxy)carboxyl)(methyl)amino)ethyl)- N 5 -methyl- L -Glutamate (9 H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(2-(((4-(1-hydroxybut-3-yn-1-yl)phenoxy)carbonyl)(methyl)amino)ethyl)- N 5 -methyl- L Preparation of glutaminate

[0177] 4-(1-hydroxybut-3-yn-1-yl)phenyl(4-nitrophenyl) carbonate (0.73 g, 1.18 mmol), (9 H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -methyl- N 5 -(2-(methylamino)ethyl)- LGlutamate (0.464 g, 1.416 mmol) and HOBT (0.159 g, 1.18 mmol) were added to a 100 mL round-bottom flask, followed by 25 mL of anhydrous DMF. After the compounds were completely dissolved, DIPEA was added dropwise, and the solution changed from colorless to yellow. The mixture was stirred overnight at room temperature. The solvent was removed by pressure concentration, and the solution was reconstituted with DCM, washed once with water, once with saturated brine, and dried overnight with anhydrous sodium sulfate. The concentrated crude product was purified by column chromatography to give a white solid (0.882 g, 93%). 1 H-NMR (600MHz, DMSO- d6 ): δ 7.955-7.871 (m, 5H), 7.736-7.676 (m, 4H), 7.418-7.275 (m,10H), 7.067-7.008 (m, 2H), 5.529 (d, J = 3.0 Hz, 1H), 4.663 (br, 1H), 4.396-4.182 (m, 7H), 3.521-3.401 (m, 4H), 3.024-3.014 (m, 1H), 2.945-2.864 (m, 7H),2.733-2.709 (m, 3H), 2.429-2.301 (m, 2H), 1.993-1.812 (m, 2H). MS (ESI) m / z:ESI-MS m / z (M+H) + Calculated for C 49 H 48 N3O8 805.34, found 805.35.

[0178] 5)(9 H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -methyl- N 5 -(2-(methyl((4-(1-((((4-nitrophenoxy)carbonyl)oxy)but-3-yn-1-yl)phenoxy)carbonyl)amino)ethyl)- L -Glutamate (9 H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N5 -methyl- N 5 -(2-(methyl((4-(1-(((4-nitrophenoxy)carbonyl)oxy)but-3-yn-1-yl)phenoxy)carbonyl)amino)ethyl)- L Preparation of glutaminate

[0179] (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(2-(((4-(1-hydroxybut-3-ynyl)phenoxy)carboxyl)(methyl)amino)ethyl)- N 5 -methyl- L Using glutamate as a raw material, (9) was synthesized according to step 2) in Example 1. H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((4-nitrophenoxy)carbonyl)oxy)but-3-yne-1-yl)phenyl)- L The method using β-glutamate esters yielded a white solid M24 (70% yield). This was used directly in the next reaction.

[0180] 6)(9 H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(2-(((4-(1-(((( S )-1-((( S )-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamoyl)oxy)but-3-yne-1-yl)phenoxy)carbonyl)(methyl)amino)ethyl)- N 5 -methyl- L -Glutamate (9 H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(2-(((4-(1-(((( S )-1-((( S )-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)ami no)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenoxy)carbonyl)(methyl)amino)ethyl)- N 5 -methyl- L Preparation of glutaminate

[0181] (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)-N 5 -methyl- N 5 -(2-(methyl((4-(1-((((4-nitrophenoxy)carbonyl)oxy)but-3-yn-1-yl)phenoxy)carbonyl)amino)ethyl)- L Using glutamate as a raw material, (9) was synthesized according to step 3) in Example 1. H -fluorene-9-yl)methyl N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((( S )-1-(((S)-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutyl)amino)-3-methyl-1-oxobutyl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenyl)- L The method of preparing glutamate yields a white solid (79% yield). 1 H-NMR (600 MHz, DMSO- d6 ): δ 7.892-7.870 (m, 5H), 7.728-7.615 (m, 4H), 7.539-7.431(dd, J = 13.8 Hz, 7.2 Hz, 4H), 7.313-6.981 (m, 10H), 5.691-5.592 (m, 1H), 5.422-5.336 (m, 1H), 4.524-4.189 (m, 9H), 4.048-3.936 (m, 2H), 3.592-3.518(m, 4H), 3.401 (br, 1H), 3.250-2.702 (m, 21H), 2.441-2.253 (m, 4H), 2.181 (t, J = 7.8 Hz, 1H), 2.129 (d, J= 3.0 Hz, 1H), 1.994-1.981 (m, 1H), 1.912 (s,1H), 1.561-1.466 (m, 3H), 1.234 (s, 12H), 1.015-0.652 (m, 28H). MS (ESI) m / z:ESI-MS m / z (M+H) + calculated for C 89 H 112 N8O 16 1549.820, found 1549.835.

[0182] 7) N 5 -(2-(((4-(1-(((( S )-1-((( S )-1-(((3 R ,4 S ,5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenoxy)carbonyl)(methyl)amino)ethyl)- N 5 -methyl- L -glutamine (I-4) Preparation

[0183] Using the pyrrolidine prepared in step 6) H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)-N 5 -(2-(((4-(1-(((( S )-1-((( S )-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)ami no)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)but-3-yn-1-yl)phenoxy)carbonyl)(methyl)amino)ethyl)- N 5 -methyl- L Using glutaminate as a raw material, a white solid I-4 (88% yield) was prepared by referring to the method for preparing compound I-1 in step 4) of Example 1. 1 H-NMR (600 MHz, DMSO- d6 ): δ 8.020-6.969 (m, 12H), 5.726-5.602(m, 1H), 5.451 (br, 1H), 4.762-4.360 (m, 3H), 4.005-3.946 (m, 2H), 3.601-2.746 (m, 32H), 2.439-2.415 (m, 2H), 2.276 (br, 1H), 2.134-1.227 (m, 13H), 1.054-0.642 (m, 23H). MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 60 H 93 N8O 14 1149.673, found 1149.7005.

[0184] 8) Preparation of the macromolecular conjugate II-7

[0185] Following the preparation method of macromolecular conjugate II-1 in Example 1, macromolecular conjugate II-7 was prepared using compound I-4 and the azide-modified Herceptin antibody 2 prepared in Example 4 as raw materials, where E represents the macromolecular conjugation ratio, which is 3-4. Herceptin is a marketed antibody that targets the HER2 antigen on tumor cells.

[0186]

[0187] Example 6: Preparation of macromolecular conjugate drug III-1

[0188] 1) (9) H -fluoren-9-yl)methyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)- N 5 -(4-(hydroxymethyl)-2-nitrophenyl)- L Preparation of glutaminate

[0189] (S)-5-((9) H -fluorene-9-yl)methoxy)-4-((((9) H (-fluorene-9-yl)methoxy)carbonyl)amino)-5-oxovaleric acid (0.4 g, 0.73 mmol), (4-amino-3-nitrophenyl)methanol (0.111 g, 0.66 mmol), and EEDQ (0.163 g, 0.66 mmol) were added to a 50 mL round-bottom flask, followed by 10 mL of anhydrous DCM. The mixture was stirred at room temperature for 24 h. After the reaction was complete, a large amount of white solid precipitated out. 1N HCl aqueous solution was added to the reaction solution, and the mixture was filtered. The filter cake was dried under vacuum to obtain a white solid (0.373 g, 81%). MS (ESI) m / z: ESI-MS m / z (M+H) + calculated for C 41 H 35 N3O8 697.24, found 697.26.

[0190] 2) (9) H -fluoren-9-yl)methylN 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(2-amino-4-(hydroxymethyl)phenyl)- L Preparation of glutaminate

[0191] (9) H -fluoren-9-yl)methyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)- N 5 -(4-(hydroxymethyl)-2-nitrophenyl)- L Glutamate (0.3 g) was added to a 25 mL round-bottom flask, followed by 2 mL of glacial acetic acid and 0.9 g of reduced iron powder. The mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction solution was filtered through diatomaceous earth and washed with ethyl acetate. Extraction was performed using ethyl acetate / water. The ethyl acetate layer was washed with saturated brine and dried overnight with anhydrous sodium sulfate. The solvent was removed by pressure concentration, and the crude product was purified by column chromatography to give a white solid (0.243 g, 85% yield). MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 41 H 37 N3O6 667.27, found 667.23.

[0192] 3) (9) H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(2-(hept-6-ynamido)-4-(hydroxymethyl)phenyl)- L Preparation of glutaminate

[0193] (9) H -fluoren-9-yl)methylN 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(2-amino-4-(hydroxymethyl)phenyl)- L Glutaminate (0.20 g, 0.30 mmol), hept-6-ynoic acid (0.035 g, 0.28 mmol), and EEDQ (0.089 g, 0.36 mmol) were added to a 50 mL round-bottom flask, followed by 10 mL of anhydrous DCM. The mixture was stirred at room temperature for 24 h. The solvent was removed by pressure concentration, and the crude product was purified by column chromatography to obtain a white solid (0.154 g, 71%). MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 48 H 45 N3O7775.33, found 775.35.

[0194] 4) (9) H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(2-(hept-6-ynamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)- L Preparation of glutaminate

[0195] Referring to step 2 of Example 1 (9) H -fluorene-9-yl)methyl N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- N 5 -(4-(1-(((4-nitrophenoxy)carbonyl)oxy)but-3-yne-1-yl)phenyl)- L The method for preparing glutamate yielded a white solid (154 mg, 85%). MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 55 H48 N4O 11 940.33, found 940.38.

[0196] 5)(9 H -fluoren-9-yl)methyl N 2 -(((9 H -fluoren-9-yl)methoxy)carbonyl)- N 5 -(2-(hept-6-ynamido)-4-((((( S )-1-((( S )-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohepta n-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)methyl)phenyl)- L Preparation of glutaminate

[0197] Following the method described in step 3) of Example 1, a white solid (154 mg, 85%) was prepared. MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 88 H 110 N8O 15 1518.81, found 1518.87.

[0198] 6) N 5 -(2-(hept-6-ynamido)-4-(((((S )-1-((( S )-1-(((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohepta n-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)methyl)phenyl)- L Preparation of glutamine (I-5)

[0199] Following the preparation method of compound I-1 in step 4) of Example 1, white solid I-5 (99 mg, 89%) was prepared. MS (ESI) m / z: ESI-MS m / z (M+H) + Calculated for C 59 H 90 N8O 13 1118.66, found 1118.68.

[0200] 7) Preparation of macromolecular conjugate III-1

[0201] Following the method for preparing macromolecular conjugate drug II-1 in step 6) of Example 1, compound I-1 was replaced with compound I-5 to prepare macromolecular conjugate drug III-1. Here, E represents the macromolecular conjugation ratio, which is 3-4, and refers to Herceptin, a marketed antibody targeting the HER2 antigen of tumor cells.

[0202]

[0203] Example 7: Quality Control Study

[0204] This embodiment evaluated the DAR (drug-dependent irradiation) of the macromolecular conjugate (e.g., II-1) prepared according to the embodiments of this disclosure. Figure 1The results showed that the molecular weight of the macromolecular conjugate II-1 increased by approximately 4243 Da compared to that of Herceptin. Since modifying Herceptin with one I-1 increases the molecular weight by approximately 1308 Da, each antibody molecule in the macromolecular conjugate II-1 conjugates approximately 3.2 I-1 small molecule drugs.

[0205] Upon testing, the E values ​​of other macromolecular conjugates prepared in the embodiments of this disclosure (such as II-3 to II-7 and III-1) were all between 3 and 4, and the E value of II-2 was between 7 and 8.

[0206] Example 8: Cytotoxicity Study

[0207] This embodiment evaluated the in vitro cytotoxicity of the test drugs, including the macromolecular conjugate and the toxin MMAE, prepared according to the embodiments of this disclosure. The cell lines tested included the HER2-positive human breast adenocarcinoma cell line SKBR3, the HER2-positive Herceptin-resistant BT474 cell line (BT474-HDR), and the HER2-positive gastric cancer cell line NCI-N87 (all cell lines were purchased from ATCC).

[0208] The reagents, instruments, and consumables used in the experiment are listed in the table below:

[0209] The experimental procedure is as follows: 1) Cell thawing Thaw the vial containing the target cells by gently stirring it in a 37 °C water bath. After the contents are thawed, the vials are removed from the water bath and decontaminated by immersion or spraying with 70% ethanol. Transfer the contents of the vial to a centrifuge tube containing 9 mL of complete DMEM culture medium and centrifuge (200 g; 5 min). The cells were resuspended in culture medium, precipitated, and dispensed into 75 cm² basins. 2 In the culture flask; Incubate the culture at 37 °C, 5% CO2 in Galaxy ® The cells were cultured in a CO2 incubator (48R, #CO48312044) with an oxygen concentration of 20% and a carbon dioxide concentration of 5%.

[0210] 2) Unfold the cells

[0211] Cells were passaged three times a week in a medium containing 10% FBS (heat-inactivated) and 1% penicillin-streptomycin solution at a ratio of 1:4. For passaged cells, adherent cells were first rinsed with trypsin / EDTA solution (3 mL). Then, trypsin / EDTA (3 mL, T75 flask) was added and the cells were evenly coated by swirl. The culture was incubated at 37 °C until cell separation. Cell separation was verified under a microscope. An equal volume of cell culture medium was added to inactivate trypsin. The separated cells were collected, centrifuged at 200 g for 5 minutes, and then resuspended in fresh culture medium.

[0212] 3) Cell inoculation

[0213] Collect cells and count the number of cells; Add 100 μL of cell suspension with adjusted density to the specified 96-well cell culture plate. The final cell density is approximately 5,000-10,000 cells / well.

[0214] Cover the container and incubate it in a 37°C, 5% CO2 incubator for 24 hours. The oxygen concentration in the incubator is 20%.

[0215] 4) Prepare the test drug

[0216] The test drug solution was serially diluted at a ratio of 1:3. The initial maximum concentration of the test drug macromolecular conjugate II-1 and MMAE was approximately 100 nM. 5) Co-incubation of drugs and cells Remove the culture medium from the 96-well plates inoculated with cells, add 150 μL of fresh culture medium to each well, and then add 50 μL of the prepared test drug to each well. Incubate the cells at 37 °C in a 5% CO2, 20% O2 incubator for 96 h.

[0217] 6) Reading the board

[0218] Remove the plates from the incubator and allow them to equilibrate at room temperature for 15 minutes; CellTiter Glo reagent was incubated at 37 °C before the experiment; Dilute CellTiter-Glo reagent with PBS at a volume ratio of 3:7 to prepare a detection solution, and add the diluted detection solution to the wells to be tested; The board was then placed at room temperature and vibrated for 8 minutes, then left to stand for 2 minutes, and finally read on an EnSpire reader for signal detection.

[0219] 7) Data Analysis

[0220] The survival percentage is expressed as follows: %Viability = 100 × (Sample / HC) HC cells are those that have not been treated with the test drug.

[0221] The cytotoxicity results of the macromolecular conjugate II-1 showed ( Figure 2 All cell lines showed high sensitivity to MMAE, with an IC50 value of [missing information]. 50 The concentrations ranged from 0.20 nM to 0.67 nM. The macromolecular conjugate II-1 exhibited potent inhibitory activity against cell proliferation in SKBR3, BT-474, and NCI-N87 cell lines, with an IC50 concentration of 0.20 nM to 0.67 nM. 50 The values ​​were 0.13 nM, 1.16 nM, and 0.59 nM, respectively. Figure 2 The above results confirm that the macromolecular conjugate II-1 has proliferation-inhibiting activity consistent with the toxin MMAE in HER2-positive tumor cell lines.

[0222] Testing revealed that other antibodies prepared in this embodiment of the present disclosure, which are macromolecular conjugates of Herceptin (e.g., II-6 and III-1), also exhibited the same inhibitory activity against proliferation as the small molecule drugs they were conjugated to on HER2-positive tumor cell lines.

[0223] Example 9: Drug Release Mechanism Study

[0224] To investigate the drug release mechanism of the macromolecular conjugates prepared in this embodiment, this embodiment tested the in vitro cytotoxicity of Zolbetuximab, Zolbetuximab-VC-MMAE, and macromolecular conjugate II-2 on claudin 18.2 antigen-positive human gastric cancer cell line NUGC4 (purchased from ATCC) expressing γ-glutamyl transpeptidase (GGT). The cell line culture procedure was as described in Example 8.

[0225] Cytotoxicity results showed ( Figure 3 NUGC4 cells are highly sensitive to MMAE, and the activity of MMAE in the NUGC4 cell line is IC50. 50 The concentration was 0.13 nM. The macromolecular conjugate II-2 exhibited potent inhibitory activity against cell proliferation in the NUGC4 cell line, with an IC50 concentration of 0.13 nM. 50 The value is 1.37 nM, while Zolbetuximab-VC-MMAE is only 27.25 nM. Zolbetuximab's IC... 50 Value greater than 100 nM ( Figure 3 This result indicates that the macromolecular conjugate drug (e.g., II-2) prepared in the embodiments of this disclosure has cell proliferation inhibition activity comparable to MMAE. Since the human gastric cancer cell line NUGC4 expresses GGT on the cell membrane, this preliminarily confirms that the macromolecular conjugate drug prepared in the embodiments of this disclosure is a drug that releases GGT enzyme expressed on the cell membrane.

[0226] Example 10: Pharmacodynamic study in NCI-N87 tumor-bearing mice

[0227] This embodiment evaluates the antitumor effect of macromolecular conjugates (e.g., II-1) prepared according to the embodiments of this disclosure on NCI-N87 tumor-bearing mice.

[0228] When the tumor volume grows to ~100 mm 3 NCI-N87 tumor-bearing mice were randomly divided into four groups: PBS group, Herceptin administration group, II-1 administration group, and Herceptin-VC-MMAE administration group (prepared according to the method disclosed in Angew Chem Int Ed Engl. 2024, Apr.15;63(16):e202310318). The mice were administered the medication via tail vein injection once a week for a total of two administrations. Tumor size was measured every two days using calipers. The tumor volume was calculated using the formula 1 / 2 × length × width × width (mm). When the tumor volume of any mouse exceeded 2000 mm... 3 At that time, the mice were euthanized.

[0229] The results show that ( Figure 4 In the NCI-N87 tumor xenograft model, compared with the PBS group and the Herceptin-treated group, both different doses of II-1 and Herceptin-VC-MMAE significantly inhibited tumor growth. Compared with the Herceptin-VC-MMAE-treated group, the macromolecular conjugate II-1 showed superior antitumor efficacy at the same dose of 3 mg / kg. These results indicate that the macromolecular conjugate (e.g., II-1) prepared in this embodiment has excellent antitumor potential.

[0230] Example 11: Pharmacodynamic study in NUGC4 tumor-bearing mice

[0231] This embodiment evaluates the antitumor effect of macromolecular conjugates (e.g., II-2) prepared according to the present disclosure in NUGC4 tumor-bearing mice that are positive for claudin18.2 antigen.

[0232] When the tumor grows to ~50 mm 3Mice were randomly assigned to seven groups: a PBS group, a Zolbetuximab (IMMAB362) administration group (10 mg / kg), a high-dose Zolbetuximab-VC-MMAE administration group (prepared according to the method disclosed in Angew Chem Int Ed Engl. 2024, Apr. 15;63(16):e202310318) administration group (10 mg / kg), a low-dose Zolbetuximab-VC-MMAE administration group (5 mg / kg), a high-dose II-2 administration group (10 mg / kg), a low-dose II-2 administration group (5 mg / kg), and a II-2 group (2 mg / kg). The drugs were administered via tail vein injection once a week for a total of three administrations. Tumor size was measured twice weekly using calipers. Mice were sacrificed on day 28 after the start of administration.

[0233] The results show that ( Figure 5 In the NUGC4 xenograft model, at the same dosage of 10 mg / kg and 5 mg / kg, the macromolecular conjugate II-2 showed a significant antitumor effect compared to the IMAB362-VC-MMMAE group. These results indicate that the GGT enzyme-dependent macromolecular conjugate prepared in the embodiments of this disclosure (e.g., II-2) has superior antitumor potential compared to the cathepsin-dependent compound IMAB362-VC-MMAE.

[0234] Although specific embodiments of this disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are all within the scope of this disclosure. The full scope of this disclosure is given by the appended claims and any equivalents thereof.

Claims

1. The compound represented by Formula I, its salts, solvates, hydrates, enantiomers, diastereomers, racemates, tautomers, polymorphs, or isotopic labels, wherein... L is a self-eliminating spacer; R stands for connector used to link macromolecules; D is either 0 or 1; C represents an active molecular group, selected from drug groups, cytotoxic groups, detection reagent groups, diagnostic reagent groups, and targeting carrier groups.

2. The compound of claim 1, its salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label, wherein... As a whole or In this configuration, L1 is attached to the carbonyl group of a glutamyl group, and the other end is attached to C; Ar is a 5-6 aryl or a 5-6 heteroaryl group; each R1 is independently hydrogen or C. 1-6 Alkyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro, or cyano; r is 0, 1, 2, or 3; L1 is -NH- or -N(R) a (CH2) n N(R a )-C(O)-O-, where R a Each independently is H or C 1-6 Alkyl group, n is 1, 2, 3 or 4; R is -X-L2-L3-R2, where X is -C(O)-, -O-, -NH-, -C(O)NH-, -NHC(O)-, -NCH3-, -CH2O-, -CH(R b )O-、-C(R b )2O-、-CH2NH-、-CH(R b )-、-C(R b )2NH-, -S-, -S(O)- or -S(O)2-, where R b Each independently is C 1-6 Alkyl group, or X is absent; L2 is -(CH2CH2O). k -(CH2) l -、-CH[-(CH2) l -NHC(O)-(CH2CH2O) k -CH3]-、-(CH2) m -or In this context, each k is an independent integer between 1 and 12, each l is an independent integer between 1 and 12, m is an integer between 1 and 30, or L2 does not exist; L3 is... , , , , , , , , , , , , , , , , , , , , , , , , , or Where each i is an independent integer between 0 and 6, each j is an independent integer between 0 and 8, or L3 does not exist; R2 is , , , , , , , , , , , , , , , , , , , , , Or H, where e is an integer between 0 and 12, and v is an integer between 0 and 48.

3. The compound, its salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label of claim 2, characterized in that the following features One or more: - C is an antitumor drug, antiviral drug, antibiotic, antibacterial drug or immunomodulatory drug, and is attached to the adjacent carbonyl group or L through the N atom or O atom in the C molecule, or is attached to the adjacent L through the N atom in the C molecule in the form of a quaternary ammonium group; - Ar is phenyl, pyridinyl, pyrimidinyl, furanyl, imidazolyl, pyrroloyl, thiazolyl, pyrazolyl, or thiophenyl; - L1 is -NH- or -N(R) a (CH2)2N(R) a )-C(O)-O-, where R a It is H or -CH3; - X is -C(O)-, -O-, -NH-, -C(O)NH- or -NHC(O)-, or X does not exist; - L2 is -(CH2) m -or does not exist; - L3 is , or Or L3 does not exist; - R2 is , , , , , , , , or ; - Each R1 is independently composed of hydrogen and C. 1-4 Alkyl, fluorine, chlorine, bromine, iodine, hydroxyl, nitro or cyano; - r is 0 or 1.

4. The compound, its salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label of claim 3, characterized by one or more of the following features: - C represents cytotoxins, tubulin inhibitors, DNA alkylating agents, DNA chimeras, RNA polymerase inhibitors, kinase inhibitors, topoisomerase inhibitors, spindle kinase inhibitors, antimetabolites, Toll-like receptor agonists, or STING agonists. - Ar is phenyl, furanyl, imidazolyl or thiophene; - L1 is -NH- or -N(R) a (CH2)2N(R) a )-C(O)-O-, where R a It is H or -CH3; - X is -C(O)NH- or -NHC(O)-, or X does not exist; - L2 does not exist; - L3 does not exist; - R2 is , , or ; - Each R1 is independently either hydrogen or nitro; - r is 1; - D is 1.

5. The compound, its salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label of claim 4, characterized by one or more of the following features: - C represents orlistatine, monomethyl orlistatine E, monomethyl orlistatine F, maytansine, maytansine-like substances, DM1, DM3, DM4, paclitaxel, calcitrol, pyroxine, doxorubicin, camptothecin, pyrrolobenzodiazepines, SN-38, ixotecan, doxorubicin, TLR7 agonist or lapatinib with CAS number 1821304-87-3; - Ar represents phenyl; - L3 is -NH-; - X does not exist; - R2 is ; - R1 is hydrogen.

6. The compound of claim 5, its salt, solvate, hydrate, enantiomer, diastereomer, racemic mixture, tautomer, polymorph, or isotopic label, wherein the compound is selected from: 。 7. Use of the compound, salt, solvate, hydrate, enantiomer, diastereomer, racemic mixture, tautomer, polymorph or isotopic label of any one of claims 1 to 6 in the preparation of macromolecular conjugated drugs.

8. A macromolecule-conjugated drug, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label, formed by conjugation of a macromolecule and a compound according to any one of claims 1 to 6, its salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label, wherein the macromolecule is a protein, antibody, polypeptide, enzyme, protein ligand, or protein scaffold.

9. The macromolecular conjugate of claim 8, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label, wherein the macromolecular conjugate is as shown in Formula II, wherein... The definitions of C and D are as described in any one of claims 1 to 6; As a whole or Wherein Ar, L1, R1, and r are defined as described in any one of claims 2 to 6; R' is -X-L2-L3-R2'-, where X, L2, and L3 are defined as described in any one of claims 2 to 6; R2' is , , , , , , , , , , , , , , , , Or H, where v is an integer between 0 and 48, and k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; A is a macromolecule selected from albumin, antibody, polypeptide, enzyme, protein ligand and protein scaffold; E is an integer or decimal between 1 and 20.

10. The macromolecular conjugated drug of claim 9, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemic mixture, tautomer, polymorph, or isotopic label, characterized by the following features One or more: - A is albumin or antibody or its antigen-binding fragment; - A is coupled to the R2' group through the S or N atom in the macromolecule. Alternatively, A may couple to the site of the R2' group by forming an oxime bond through the reaction of the carbonyl group in the macromolecule with the hydroxylamine group in the R2' group. superior; - R2' is , , , , , ,or , where k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; - E is an integer or decimal between 1 and 12.

11. The macromolecular conjugated drug of claim 10, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemic mixture, tautomer, polymorph, or isotopic label, characterized in that the following features One or more: - A represents albumin, monoclonal antibody, bispecific antibody, multispecific antibody, or its antigen-binding fragment; - R2' is , , ,or , where k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

12. The macromolecular conjugated drug of claim 10, and its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph, or isotopic label, wherein... A is selected from: anti-HER2 humanized monoclonal antibody mil40, trastuzumab, pertuzumab, zotuximab, molotovicillin, cetuximab, panitumumab, rituximab, alemtuzumab, tiimomab, tosimob, oflambumab, bevacizumab, ipilimumab, denosumab, pembrolizumab, nivolumab, avelumab, atezolizumab, durvalumab, sacituzumab, rovalpituzumab and their antigen-binding fragments; or A represents albumin; or A represents an antibody or its antigen-binding fragment, which includes: (a) The following three heavy chain variable regions are complement-determining regions: (i) VH CDR1, which has the CDR1 sequence contained in the heavy chain as shown in SEQ ID NO: 1, or has a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR1 sequence contained in the heavy chain. (ii) VH CDR2, having the CDR2 sequence contained in the heavy chain as shown in SEQ ID NO: 1, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR2 sequence contained in the heavy chain; and (iii) VH CDR3, having the CDR3 sequence contained in the heavy chain as shown in SEQ ID NO: 1, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of one or two amino acids) compared to the CDR3 sequence contained in the heavy chain. and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the CDR1 sequence contained in the light chain as shown in SEQ ID NO: 2, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR1 sequence contained in the light chain. (v) VL CDR2, having the CDR2 sequence contained in the light chain as shown in SEQ ID NO: 2, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the light chain; and (vi) VL CDR3, having the CDR3 sequence contained in the light chain as shown in SEQ ID NO: 2, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the light chain; or A represents an antibody or its antigen-binding fragment, which includes: (a) The following three heavy chain variable regions are complement-determining regions: (i) VH CDR1, having the CDR1 sequence contained in the heavy chain as shown in SEQ ID NO: 3, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR1 sequence contained in the heavy chain. (ii) VH CDR2, having the CDR2 sequence contained in the heavy chain as shown in SEQ ID NO: 3, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR2 sequence contained in the heavy chain; and (iii) VH CDR3, having the CDR3 sequence contained in the heavy chain as shown in SEQ ID NO: 3, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the heavy chain. and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the CDR1 sequence contained in the light chain as shown in SEQ ID NO: 4, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR1 sequence contained in the light chain. (v) VL CDR2, having the CDR2 sequence contained in the light chain as shown in SEQ ID NO: 4, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the light chain; and (vi) VL CDR3, having the CDR3 sequence contained in the light chain as shown in SEQ ID NO: 4, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the light chain; or A represents an antibody or its antigen-binding fragment, which includes: (a) The following three heavy chain variable regions are complement-determining regions: (i) VH CDR1, having the CDR1 sequence contained in the heavy chain as shown in SEQ ID NO: 5, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR1 sequence contained in the heavy chain. (ii) VH CDR2, having the CDR2 sequence contained in the heavy chain as shown in SEQ ID NO: 5, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the heavy chain; and (iii) VH CDR3, having the CDR3 sequence contained in the heavy chain as shown in SEQ ID NO: 5, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the heavy chain. and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the CDR1 sequence contained in the light chain as shown in SEQ ID NO: 6, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the CDR1 sequence contained in the light chain. (v) VL CDR2, having the CDR2 sequence contained in the light chain as shown in SEQ ID NO: 6, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR2 sequence contained in the light chain; and (vi) VL CDR3, having the CDR3 sequence contained in the light chain as shown in SEQ ID NO: 6, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in the light chain.

13. The macromolecular conjugated drug of claim 12, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemic mixture, tautomer, polymorph, or isotopic label, characterized in that the following features One or more: The permutations described in any of (i)-(vi) are conservative permutations; - The CDR1, CDR2 and CDR3 contained in the heavy chain variable region, and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region are defined by the Kabat, Chothia, AbM or IMGT numbering system. - The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:2; or the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:4; or the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:

6.

14. The macromolecular conjugate of claim 12, its pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemic mixture, tautomer, polymorph, or isotopic label, wherein the macromolecular conjugate is selected from: in A and E are defined as described in any one of claims 10-12, and k1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

15. A pharmaceutical composition comprising any one of the macromolecular conjugated drugs of claims 8-14, a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemate, tautomer, polymorph or isotopic label, and one or more pharmaceutically acceptable carriers and / or excipients.

16. Use of any macromolecular conjugated drug, pharmaceutically acceptable salt, solvate, hydrate, enantiomer, diastereomer, racemic, tautomer, polymorph, or isotopic label thereof, as described in any one of claims 8-14, in the preparation of a medicament for treating a disease or condition or for reducing the severity of said disease or condition, wherein said disease or condition is selected from tumors, infectious diseases, hematological diseases, metabolic diseases, and inflammation.

17. The use of claim 16, wherein the disease or condition is selected from cancer, lymphoma, lymphoid tumor, blastoma, sarcoma, and leukemia.

18. The use according to claim 17, wherein the cancer is selected from: breast cancer, squamous cell carcinoma, lung cancer, peritoneal cancer, gastric cancer, endometrial adenocarcinoma, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, urethral cancer, hepatocellular carcinoma, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, gallbladder cancer, esophageal cancer, bile duct cancer, head and neck cancer, and metastatic tumors.

19. The use according to claim 17, wherein the cancer is selected from epithelial squamous cell carcinoma, HER2-positive breast cancer, colon cancer, rectal cancer, gastrointestinal cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma.

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