Linkers, conjugates and uses thereof

Abstract

The invention relates to the field of bio-pharmaceuticals, in particular to a linker for preparing a targeting molecule-drug conjugate, and a corresponding conjugate, a preparation method and application thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from the following applications:

[0003] International application number PCT / CN2022 / 106025 filed on July 15, 2022. Technical Field

[0004] The present application relates to the field of biopharmaceuticals, and in particular, to a linker for preparing a targeting molecule-drug conjugate, and a corresponding conjugate, a preparation method and use thereof. Background Art

[0005] It has been found that HER2 is overexpressed in several types of cancers including breast cancer and gastric cancer, and it has been shown to be a promising target for cancer therapy. There are multiple treatments targeting HER2 that have been approved, including HER2 tyrosine kinase inhibitors (lapatinib (Lapatinib), tucatinib (Tucatinib)), therapeutic HER2 antibodies (Herceptin, Perjeta) and ADCs targeting HER2 (Kadcyla, Enhertu). These therapeutic agents have significantly improved the survival rate of HER2-positive breast cancer and gastric cancer patients. In particular, Enhertu not only shows a strong effect in HER2 high-expression patients, but also shows signs of efficacy in HER2 medium / low expression patients, which can potentially benefit more cancer patients expressing HER2. Although Enhertu is powerful, it causes more than 10% of interstitial lung disease, which limits its use in a part of patients.

[0006] Enhertu and other commercially available ADCs and most ADCs in clinical trials are prepared by chemical conjugation, using a sulfosuccinimide structure (sulfosuccinimide bond) to conjugate small molecule drugs to targeted antibodies or proteins. The sulfosuccinimide structure is formed by the reaction of a thiol group with a maleimide. However, the sulfosuccinimide bond is unstable. In organisms, reverse Michael addition or exchange with other thiol groups causes cytotoxins to fall off from the ADC and produce off-target toxicity, which will reduce safety and limit clinical applications.

[0007] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. It is an intracellular calcium signal transduction protein and is overexpressed in a variety of tumors. IMMU-132 (also known as SG) and DS-1062 are well-known anti-trop2 ADCs in the clinic. Although SG has shown good anti-tumor efficacy in clinical trials, it has the same side effects as toxic payloads (SN-38), including bone marrow suppression and gastrointestinal toxicity. As for DS1062, in a phase I clinical trial, 48% of patients experienced adverse events higher than grade 3, and 8% of patients suffered from interstitial lung disease. Recently published phase III clinical data on DS-1062 for the treatment of advanced non-small cell lung cancer showed that DS-1062 did not show better safety. In this clinical trial, some grade 5 adverse events were observed. Since the linkers of DS1062 and IMMU-132 both use maleimide groups, and the maleimide group is unstable in the blood, its potential toxin shedding may be one of the reasons for the poor safety mentioned above.

[0008] The protein encoded by the FGFR3 gene is a member of the fibroblast growth factor receptor (FGFR) family and can bind to both acidic and basic fibroblast growth factors (FGFs), and plays an important role in bone development and maintenance. It can be an important target for ADCs.

[0009] The inventors of this case have developed a novel branched linker that does not contain a maleimide group and is conjugated to a specific site of the antibody through the amino acid at the tail, thereby avoiding potential instability in the blood. At the same time, the branched linker has good assembly ability and can be easily and environmentally friendly adapted to a variety of different loads and commercialized linker-load fragments. The formed linker-load structure is easy to separate and purify, which is conducive to the later CMC development. In addition, by conjugating different types of linkers to different antibodies, the stability, compatibility of the linker and the excellent biological activity of the ADC formed by the linker are verified.

[0010] Some conjugates with better molecular stability and better anti-tumor efficacy than the benchmark (e.g., Enhertu) are also provided. In addition, the conjugates may have good physicochemical properties, good pharmacokinetic properties, and good safety. In addition, the highly modular design makes it easy to assemble a variety of drugs. Summary of the invention

[0011] In a first aspect, a compound of formula (I) is provided:

[0012]

[0013] in,

[0014] W is hydrogen, LKb or -C2H4-(PEG) t -(CO)NH2;

[0015] Y is LKa-LKb;

[0016] Each LKa is independently selected from

[0017] Each LKb is independently L 2 ―L 1 ―B;

[0018] Each B is independently a terminal group R 10 , or a combination of the following: 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ;

[0019] R 10 is hydrogen, or a group that can leave when reacting with a group in the carrier;

[0020] Each L 1 independently a cleavable sequence 1 comprising an amino acid sequence capable of being cleaved by an enzyme, and the cleavable sequence 1 comprises 1 to 10 amino acids;

[0021] Each L 2 are independently chemical bonds; or C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、 C 4-10 Cycloalkylene, C 4-10 wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ;

[0022] Ld2 and each Ld1 are independently a chemical bond; or selected from -NH-C1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-, or a natural amino acid, or an oligomeric natural amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or oligomeric natural amino acid is independently unsubstituted or its side chain is replaced by -(CO)-(PEG) j -OR 11 replace;

[0023] -(PEG) t -、-(PEG) i -and-(PEG) j - each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units and having an optional additional C 1-10 Alkylene;

[0024] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 Cycloalkylene; or

[0025] R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group; or

[0026] R 3 and R 4 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group;

[0027] R 11 It is C 1-10 alkyl;

[0028] m is any integer from 1 to 5;

[0029] n is any integer from 2 to 20;

[0030] d is any integer from 1 to 6, in particular 1, 2, 3;

[0031] Each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12, more preferably an integer from 0 to 8, especially 4;

[0032] Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12;

[0033] Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12.

[0034] In a second aspect, a compound having the structure of formula (II) is provided:

[0035]

[0036] in

[0037] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0038] M is LKa-LKb―P;

[0039] P is a load, which is connected to the B part or L of the compound of formula (I) 1 part;

[0040] n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I).

[0041] In a third aspect, a conjugate having a structure of formula (III) is provided:

[0042]

[0043] in,

[0044] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0045] n, d, t, Ld1 and Ld2 are as defined in formula (I);

[0046] M is LKa-LKb―P;

[0047] P is a load, which is connected to the B part or L of the compound of formula (I) 1 part;

[0048] A is the targeting molecule;

[0049] z is an integer of 1-20.

[0050] In a fourth aspect, there is provided an intermediate compound for preparing formula (I), wherein the intermediate compound has a structure of formula (IV):

[0051]

[0052] in,

[0053] Rpg is selected from hydrogen, or a protecting group, preferably selected from acetyl, trifluoroacetyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc);

[0054] W, Ld1, Ld2, n and d are as defined in formula (I).

[0055] In a fifth aspect, provided is a use of the conjugate of the present application or a pharmaceutical composition thereof in the manufacture of a medicament for treating a disease; wherein the disease is a tumor or an autoimmune disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The efficacy of the ADC was shown against SK-BR-3 and MDA-MB-468 co-cultured cell lines.

[0057] Figure 2 The in vivo efficacy of conjugate ADC-2 and conjugate ADC-1 in the JIMT-1CDX model was shown.

[0058] Figure 3 The in vivo efficacy of ADC-2, ADC-3, and Enhertu in the JIMT-1CDX model was shown.

[0059] Figure 4 The in vivo efficacy of conjugate ADC-2 and conjugate ADC-1 in the NCI-N87 CDX model was shown.

[0060] Figure 5 The efficacy of the conjugates ADC-5, ADC-6 and ADC-7 against the proliferation of TROP2-positive tumor cells BxPC-3 was shown.

[0061] Figure 6 The results of internalization assays of conjugates ADC-5 and ADC-6 are shown.

[0062] Figure 7 The efficacy of conjugates ADC-5 and ADC-6 against tumor growth in the TROP2-positive NCI-N87 CDX mouse model was shown.

[0063] Figure 8 The efficacy of conjugates ADC-5 and ADC-6 against tumor growth in the TROP2-positive FaDu CDX mouse model was shown.

[0064] Fig. 9The efficacy of the conjugates ADC-8, ADC-9 and ADC-10 against tumor growth in the FGFR3-positive RT112 / 84CDX mouse model was shown.

[0065] Fig.10 The efficacy of the conjugates ADC-8, ADC-9 and ADC-10 against tumor growth in the FGFR3-positive RT4CDX mouse model was shown. DETAILED DESCRIPTION

[0066] Specific embodiments are provided below to illustrate the technical contents of the present application. Those skilled in the art can easily understand other advantages and effects of the present application through the contents disclosed in this specification. The present application can also be implemented or applied through other different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of the present application.

[0067] definition

[0068] Unless otherwise defined below, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. The technology used herein refers to the technology commonly understood in the art, including variations and equivalent substitutions that are obvious to those skilled in the art. Although it is believed that the following terms can be easily understood by those skilled in the art, elaborating the following definitions will better illustrate the application. When there is a trade name herein, the trade name refers to the corresponding commodity or its active ingredient. All patents cited herein, published patent applications and publications are incorporated herein by reference.

[0069] When a certain amount, concentration or other value or parameter is set forth in the form of a range, a preferred range, or a preferred upper limit or a preferred lower limit, it should be understood that it is equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, regardless of whether the range is explicitly stated. Unless otherwise indicated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the statement "i is an integer from 0 to 20" means that i is any integer from 0 to 20, for example, i can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Other similar statements, such as d, m, n, j and k, should also be understood in a similar manner.

[0070] Unless the context clearly dictates otherwise, singular forms such as "a", "an" and "the" include plural forms. The expression "one (kind) or more (kind)" or "at least one (kind)" may refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 (kinds) or more (kinds).

[0071] The terms "about" and "approximately" when used in conjunction with a numerical variable generally mean that the value of the variable and all values ​​of the variable are within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% of the specified value, or within a wider range.

[0072] The terms "optional" or "optionally" mean that the subsequently described event may but need not occur, and that the description includes instances where said event or circumstance occurs or does not occur.

[0073] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any unspecified elements, steps, or ingredients. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, as well as elements, steps, or ingredients that are optionally present and do not substantially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of.

[0074] The term "targeting molecule" refers to a molecule that has affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, a tumor-specific antigen, etc.). Targeting molecules can deliver a payload to a specific site in the body by targeted delivery. Targeting molecules can recognize one or more targets. The specific target site is defined by the target recognized by the targeting molecule. For example, a targeting molecule that targets a receptor can deliver a cytotoxin to a site containing a large number of receptors. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins for a given antigen, antibody mimetics, scaffold proteins with affinity for a given target, ligands, etc.

[0075] As used herein, the term "antibody" is used in a broad sense, and specifically includes complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they have the desired biological activity. The antibody may belong to any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass, and may be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. The antibody may also be a fully human antibody, a humanized antibody, or a chimeric antibody prepared by a recombinant method. In some embodiments, the antibody may be engineered, for example, by introducing a ligase-specific recognition sequence into the end of the antibody.

[0076] Monoclonal antibody is used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" refers to the characteristic of an antibody being derived from a population of substantially homogeneous antibodies and should not be construed as requiring some specific method to prepare the antibody.

[0077] A complete antibody or full-length antibody essentially comprises one or more antigen-binding variable regions, and one or more light chain constant regions (CL) and one or more heavy chain constant regions (CH), which may include CH1, CH2, CH3 and CH4 depending on the subtype of the antibody. Antigen-binding variable regions (also referred to as fragment variable regions, Fv fragments) typically comprise light chain variable regions (VL) and heavy chain variable regions (VH). The constant region may be a constant region with a native sequence (such as a constant region with a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with a target antigen. The constant region may be recognized by the immune system and interacts with it.

[0078] Antibody fragments may include a portion of a complete antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementary determining regions (CDRs). Fab fragments are antibody fragments obtained by digesting full-length immunoglobulins with papain, or fragments having the same structure produced, for example, by recombinant expression. Fab fragments contain a light chain (comprising VL and CL) and another chain, wherein the other chain contains the variable domain (VH) of the heavy chain and the constant region domain (CH1) of the heavy chain. F(ab')2 fragments are antibody fragments obtained by digesting immunoglobulins with pepsin at pH 4.0-4.5, or fragments having the same structure produced, for example, by recombinant expression. F(ab')2 fragments essentially contain two Fab fragments, wherein each heavy chain portion contains several additional amino acids, including cysteine, which forms a disulfide bond connecting the two fragments. A Fab' fragment is a fragment that contains half of a F(ab')2 fragment (one heavy chain and one light chain). Antibody fragments may contain multiple chains joined together, for example, via disulfide bonds and / or via peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that can produce an antibody that immunospecifically binds to an antigen when inserted into an antibody framework (e.g., by replacing the corresponding region).

[0079] Antibodies according to the present application can be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthetic techniques or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimics) can be expressed by a suitable culture system (e.g., E. coli or mammalian cells). Engineering can refer, for example, to the introduction of a ligase-specific recognition sequence at the end.

[0080] HER2 refers to human epidermal growth factor receptor-2, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0081] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. It is an intracellular calcium signal transduction protein and is overexpressed in a variety of tumors.

[0082] The protein encoded by the FGFR3 gene is a member of the fibroblast growth factor receptor (FGFR) family and can bind to both acidic and basic fibroblast growth factors (FGFs), and plays an important role in bone development and maintenance.

[0083] As used herein, the term "targeting molecule-drug conjugate" is referred to as a "conjugate." Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0084] Small molecule compounds refer to molecules of a size comparable to that of commonly used organic molecules in medicines. This term does not encompass biological macromolecules (e.g., proteins, nucleic acids, etc.), but encompasses low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, the molecular weight of a small molecule compound can be, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, about 200 to about 500 Da.

[0085] Cytotoxin refers to a substance that inhibits or prevents the expression activity of a cell, a cell function, and / or causes cell destruction. The toxicity of the cytotoxin currently used in ADC is higher than that of chemotherapeutic drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, apoptosis regulation. Drugs targeting the microtubule cytoskeleton can be, for example, microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastines, colchicines, and dolastatins. Drugs targeting DNA can be, for example, drugs that directly destroy DNA structure or topoisomerase inhibitors. Examples of drugs that directly destroy DNA structure include, but are not limited to, DNA double-strand breakers, DNA alkylating agents, and DNA intercalators. The DNA double-strand break agent can be, for example, an enediyne antibiotic, including but not limited to dynemicin, esperamicin, neocarzinostatin, uncialamycin, etc. The DNA alkylating agent can be, for example, a DNA dialkylating agent (i.e., a DNA cross-linking agent) or a DNA monoalkylating agent. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, dihydroindole benzodiazepine (IGN) dimer, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (such as DX8951f, DXd-(1) and DXd-(2), the structures of which are depicted below), camptothecins and anthracyclines. Drugs targeting RNA can be, for example, drugs that inhibit splicing, and examples thereof include, but are not limited to, pladienolide. Drugs targeting kinesin-mediated protein transport can be, for example, mitotic kinesin inhibitors, including, but not limited to, kinesin spindle (KSP) inhibitors.

[0086] The spacer is a structure that is located between different structural modules and can separate each structural module in space. The definition of the spacer is not limited by whether it has a certain function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, amino acid derivatives or analogs. "Spacer sequence" refers to an amino acid sequence that acts as a spacer, and examples thereof include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, such as a sequence containing two amino acids such as GA, etc.; or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. A self-removal spacer is a covalent component that is tailored to cause the cleavage association of two chemical bonds after activation of the protective portion in the precursor: after stimulation, the protective portion (such as a cleavable sequence) is removed, thereby generating a cascade of decomposition reactions, so that smaller molecules are released in sequence. Examples of self-removal spacers include, but are not limited to, PABC (p-phenylmethyloxycarbonylaniline), acetals, heteroacetals, and combinations thereof.

[0087] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is attached to the rest of the molecule by a single bond. Alkyl groups can contain from 1 to 20 carbon atoms and are referred to as C1-C 20 Alkyl, such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or their isomers. A divalent group refers to a group obtained by removing a hydrogen atom from a carbon atom having one or more free valence electrons from a corresponding monovalent group. A divalent group has two attachment sites to the rest of the molecule. For example, "alkylene" or "alkylidene" refers to a straight or branched chain saturated divalent hydrocarbon radical. Examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexamethylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, etc.

[0088] As used herein, when one group is combined with another group, the connection of the groups may be linear or branched, as long as a chemically stable structure is formed. The structure formed by such a combination may be connected to the rest of the molecule through any suitable atom in the structure, preferably through a designated chemical bond. For example, when a group selected from -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 When two or more divalent groups of a heterocyclylene group and -(CO)- are combined together to form a combination, the two or more divalent groups may form a linear connection with each other, such as -CR 1 R 2 -C 1-10 Alkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene -(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-C 1-10 Alkylene-(CO)-, -CR 1 R 2 -CR 1 'R 2 '-(CO)-、-CR 1 R 2 -CR 1 'R 2 '-CR 1" R 2" -(CO)-, etc. The resulting bivalent structure can be further linked to other parts of the molecule.

[0089] The term "protecting group" or "Pg" refers to a substituent that is typically used to block or protect a particular functional group while allowing other functional groups on the compound to react. For example, an "amino protecting group" is a substituent that is attached to an amino group to block or protect the amino functional group in the compound. Suitable amino protecting groups include, but are not limited to, acetyl, trifluoroacetyl, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a general description of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0090] As used herein, the expressions "antibody-conjugated drug" and "antibody-drug conjugate" have the same meaning.

[0091] Compound of formula (I)

[0092] In one aspect, a compound of formula (I) is provided:

[0093]

[0094] in,

[0095] W is hydrogen, LKb or -C2H4-(PEG) t -(CO)NH2;

[0096] Y is LKa-LKb;

[0097] Each LKa is independently selected from

[0098] Each LKb is independently L 2 ―L 1 ―B;

[0099] Each B is independently a terminal group R 10 , or a combination of the following: 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ;

[0100] R 10 is hydrogen, or a group that can leave when reacting with a group in the carrier;

[0101] Each L 1 independently a cleavable sequence 1 comprising an amino acid sequence capable of being cleaved by an enzyme, and the cleavable sequence 1 comprises 1 to 10 amino acids;

[0102] Each L 2 are independently chemical bonds; or C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、 C 4-10 Cycloalkylene, C 4-10wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ;

[0103] Ld2 and each Ld1 are independently a chemical bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-, or a natural amino acid, or an oligomeric natural amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or oligomeric natural amino acid is independently unsubstituted or its side chain is replaced by -(CO)-(PEG) j -OR 11 replace;

[0104] -(PEG) t -、-(PEG) i -and-(PEG) j - each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units and having an optional additional C 1-10 Alkylene;

[0105] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 Cycloalkylene; or

[0106] R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group; or

[0107] R 3 and R 4 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group;

[0108] R 11 It is C 1-10 alkyl;

[0109] m is any integer from 1 to 5;

[0110] n is any integer from 2 to 20;

[0111] d is any integer from 1 to 6, especially 1, 2, 3; each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12, more preferably an integer from 0 to 8, especially 4;

[0112] Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12;

[0113] Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12.

[0114] In one embodiment, LKa is selected from Here, m is an integer from 1 to 5.

[0115] In one embodiment, LKa is selected from Here, m is an integer from 1 to 3.

[0116] In one embodiment, LKa is selected from Where m is 1 or 2.

[0117] In one embodiment, L 2 Selected from: -(C2H4-O) p -(CH2)2(CO)-, p is an integer from 0 to 5; more preferably, p is 0, 2 or 4; most preferably, p is 2.

[0118] In one embodiment, each L above 2 The carbonyl group in the structure is connected to L 1 , and the other attachment site is attached to the amide.

[0119] In one embodiment, Ld2 and each Ld1 are independently a chemical bond or

[0120]

[0121] Each i is independently an integer from 0 to 100;

[0122] Each j and k is independently an integer from 1 to 100.

[0123] In one embodiment, Ld1 is wherein i is an integer from 0 to 5; preferably, i is from 0 to 4, more preferably 0, 2 or 4, most preferably 4.

[0124] In one embodiment, Ld2 is a chemical bond.

[0125] In one embodiment, Ld2 is a natural amino acid, or an oligomeric natural amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or oligomeric natural amino acid is independently unsubstituted or its side chain is replaced by -(CO)-(PEG) j -OR 11 replace;

[0126] -(PEG) j - is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units and having an optional additional C at one end. 1-10 Alkylene.

[0127] In one embodiment, each i is independently an integer from 0 to 20, and each j and k is independently an integer from 1 to 20. In one embodiment, each i is independently 0 to 12, and each j and k is independently an integer from 1 to 12.

[0128] In one embodiment, each i is independently an integer from 0 to 8; particularly 4.

[0129] In one embodiment, each j is independently an integer from 8 to 12, specifically 8 or 12.

[0130] In one embodiment, each k is independently an integer from 1 to 7; in particular 1 or 3 or 5.

[0131] In one embodiment, Ld2 and each Ld1 are independently a chemical bond; or a C having an amino group and a carbonyl group at both ends, respectively. 1-20 Alkylene, or a PEG fragment of a certain length with an amino group and a carbonyl group at each end (denoted as -(PEG) i- ), or one or more natural amino acids, the natural amino acids are independently unsubstituted or their side chains are substituted with a PEG fragment of a certain length (denoted as -(PEG) j -)replace.

[0132] In one embodiment, -(PEG) i -Contains -(O-C2H4) i -or-(C2H4-O) i -, with an optional extra C at one end 1-10 Alkylene; -(PEG) j -Contains -(O-C2H4) j -or-(C2H4-O)j -, with an optional extra C at one end 1-10 In very specific cases, -(PEG) i -Contains -C2H4-(O-C2H4) i -or-(C2H4-O) i -C2H4-; in very specific cases, -(PEG) j -Contains -C2H4-(O-C2H4) j -or-(C2H4-O) j -C2H4-.

[0133] In one embodiment, Ld2 is a side chain of -(CO)-(PEG) j -OR 11 Substituted lysine.

[0134] In one embodiment, Ld2 is wherein j is an integer from 1 to 100, preferably an integer from 1 to 20, more preferably an integer from 1 to 12, most preferably an integer from 8 to 12, in particular 8 or 12.

[0135] It should be understood that when there are two or more Ld1, B, L 2 or L 1 When the structure is Ld1, B, L 2 or L 1 The structures of are independently selected. When there are two or more R x (x is 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.), each R x In some embodiments, the "x" in the molecule is indicated with or without an additional prime (') or multiple primes (such as ", '", "", etc.), for example, R, R 1 '、R 1" , R 1" '、R 2 '、R 2" , R 2" ', etc., with or without an extra apostrophe or multiple apostrophes for each R x are independently selected. Other R x , such as R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and "Ld1", "B", "L 2 ” and “L 1 ” should be understood in a similar manner.

[0136] In one embodiment, cleavable sequence 1 is selected from Gly-Gly-Phe-Gly, Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Ala-Ala-Al and combinations thereof; preferably, cleavable sequence 1 is Gly-Gly-Phe-Gly or Val-Cit.

[0137] In one embodiment, R 10 is hydrogen, hydroxyl or

[0138] In one embodiment, R 11 It is C 1-6 Alkyl group, preferably methyl group.

[0139] In one embodiment, W is hydrogen.

[0140] In one embodiment, W is -C2H4-(PEG) t- (CO)NH2, wherein t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12.

[0141] In one specific embodiment, t is 12.

[0142] In one embodiment, n is an integer from 2 to 5, in particular 3.

[0143] In one embodiment, d is any integer from 1 to 4, preferably 1.

[0144] In another aspect, an intermediate compound for preparing formula (I) is provided, which has a structure of formula (IV):

[0145]

[0146] in,

[0147] Rpg is selected from hydrogen, or a protecting group, preferably selected from acetyl, trifluoroacetyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc);

[0148] W, Ld1, Ld2, n and d are as defined in formula (I).

[0149] In one embodiment, Pg is 9-fluorenylmethyleneoxycarbonyl (Fmoc).

[0150] In one embodiment, Ld2 is wherein j is an integer from 1 to 100, preferably an integer from 1 to 20, more preferably an integer from 1 to 12, most preferably an integer from 8 to 12, especially 8 or 12. In one embodiment, R 11 It's methyl.

[0151] In one embodiment, Ld1 is wherein i is an integer from 0 to 5; preferably, i is from 0 to 4, more preferably 0, 2 or 4, most preferably 4.

[0152] In one embodiment, W is hydrogen.

[0153] In one embodiment, n is 3.

[0154] In one embodiment, formula (IV) has the following structure:

[0155]

[0156] The portion containing the recognition sequence for the ligase acceptor or donor substrate

[0157] In one embodiment, the Gn portion of the compound of formula (I) is a recognition sequence of a ligase receptor substrate, which facilitates the enzymatic coupling of the compound of formula (I) with the targeting molecule under ligase catalysis. The targeting molecule is optionally modified and comprises a corresponding recognition sequence of a ligase receptor substrate.

[0158] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of: natural transpeptidase, non-natural transpeptidase, variants thereof and combinations thereof. Non-natural transpeptidase can be but not limited to those obtained by natural transpeptidase engineering. In a preferred embodiment, the ligase is selected from the group consisting of: natural sortase, non-natural sortase and combinations thereof. The types of natural sortase include sortase A, sortase B, sortase C, sortase D, plant lactobacillus (L.plantarum) sortase, etc. (detailed description can be found in US20110321183A1, which is incorporated herein by reference). The type of ligase corresponds to the ligase recognition sequence and is thus used to achieve specific binding between different molecules or structural fragments.

[0159] In some embodiments, the ligase is a sortase selected from the group consisting of sortase A, sortase B, sortase C, sortase D, and Lactobacillus plantarum sortase. In these embodiments, the recognition sequence of the ligase acceptor substrate is selected from the group consisting of oligoglycine, oligoalanine, and a mixture of oligoglycine / alanine with a degree of polymerization of 3 to 10. In a specific embodiment, the recognition sequence of the ligase acceptor substrate is Gn, wherein G is glycine (Gly), and n is an integer from 2 to 10.

[0160] In another specific embodiment, the ligase is sortase A from Staphylococcus aureus. Thus, the ligase recognition sequence can be a typical enzyme recognition sequence, such as LPXTG. In yet another specific embodiment, the recognition sequence of the ligase donor substrate is LPXTGJ, and the recognition sequence of the ligase acceptor substrate is Gn, wherein X can be any single natural or unnatural amino acid; J is absent or is an amino acid fragment comprising 1 to 10 amino acids, optionally labeled. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment comprising 1 to 10 amino acids, wherein each amino acid is independently any natural or unnatural amino acid. In another embodiment, J is G m , wherein m is an integer from 1 to 10. In yet another specific embodiment, the recognition sequence of the ligase donor substrate is LPETG. In another specific embodiment, the recognition sequence of the ligase donor substrate is LPETGG.

[0161] In one embodiment, the ligase is sortase B from Staphylococcus aureus and the corresponding donor substrate recognition sequence may be NPQTN. In another embodiment, the ligase is sortase B from Bacillus anthracis and the corresponding donor substrate recognition sequence may be NPKTG.

[0162] In yet another embodiment, the ligase is sortase A from Streptococcus pyogenes and the corresponding donor substrate recognition sequence can be LPXTGJ, wherein J is as defined above. In another embodiment, the ligase is sortase subfamily 5 from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence can be LAXTG.

[0163] In another embodiment, the ligase is sortase A from Lactobacillus plantarum and the corresponding donor substrate recognition sequence can be LPQTSEQ.

[0164] The ligase recognition sequence may also be other new transpeptidase recognition sequences optimized by artificial screening.

[0165] Moiety containing reactive groups

[0166] Reactive groups for attachment to loads

[0167] In one embodiment, B is a terminal group R 10 , and L 1In the cleavable sequence 1 is connected to the load. In this case, B is not present in the molecule obtained by connecting the cleavable sequence 1 to the load. In one embodiment, B is used to connect to the load. In order to connect to the load, the compound of formula (I) comprises a reactive group. In one embodiment, B in the compound of formula (I) is connected to the load by an amide bond, an ester bond or an ether bond. In one embodiment, the reactive group in B in formula (I) is independently a reactive group (such as a reactive C=O part, a reactive C=CC=O part, an amino group, an amine group, a hydroxyl group, a thiol group) for condensation reaction, nucleophilic addition or electrophilic addition or a reactive group (such as a leaving group connected to O, C, N or S atoms) for substitution reaction. In one embodiment, the reactive group in B is independently selected from carboxyl, active ester, aldehyde group, amino group, amine group, hydroxyl group and thiol group. In a specific embodiment, the reactive group in B for connecting to the load is independently selected from amino group, amine group, hydroxyl group, thiol group, carboxyl and active ester.

[0168] In one embodiment, the reactive groups in B are independently amino, amine or hydroxyl groups, which react with corresponding groups in the support (such as carboxyl, sulfonic acid, phosphoryl with free-OH end, active ester, acyl chloride or isocyanate). In another embodiment, the reactive groups in B are independently carboxyl or active ester, which react with corresponding groups in the support (such as amino, amine or hydroxyl groups).

[0169] In one embodiment, the reactive groups in B are independently amino, hydroxyl or thiol groups, which react with corresponding groups in the support (such as halogen, hydroxyl, thiol, aldehyde). In another embodiment, the reactive groups in B are independently hydroxyl groups, which react with corresponding groups in the support (such as halogen or hydroxyl).

[0170] In one embodiment, each B is independently a terminal group R 10 , or a combination of the following: 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ;

[0171] In one embodiment, Sp1 is selected from the group consisting of PABC, acetal, heteroacetal, and combinations thereof.

[0172] In one embodiment, Sp1 is acetal, heteroacetal or PABC. In one embodiment, heteroacetal is selected from N, O-heteroacetal. In one embodiment, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is connected to cleavable sequence 1, wherein U is absent or U is O, S or N, preferably O or S.

[0173] In one embodiment, Sp1 is PABC.

[0174] In one embodiment, B is R 10 、-NH-CH2-UR 10 Or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , wherein U is absent or U is O, S or N, and g is 1; preferably, U is O or S.

[0175] In one embodiment, B is R 10 .

[0176] In one embodiment, B is -NH-CH2-OR 10 .

[0177] In one embodiment, B is -NH-CH2-O-(CR 1 R 2 ) g -(CO)-R 10 , g is an integer from 1 to 10, preferably 1.

[0178] In one embodiment, B is -NH-CH2-R 10 .

[0179] In one embodiment, R 1 It's hydrogen.

[0180] In one embodiment, R 2 It's hydrogen.

[0181] In one embodiment, R 10 is hydrogen, hydroxyl or In one embodiment, R 10 is hydrogen. In one embodiment, R 10 Is hydroxyl or

[0182] In one embodiment, R 10 It represents the structural part that will not appear in the product molecule produced by the reaction of B and the load.

[0183] Specific examples of compounds of formula (I)

[0184] In one embodiment, each LKa is In one embodiment, formula (I) has the structure of formula (I-1)

[0185]

[0186] In one embodiment, Ld2 is d is 1, W is hydrogen. In one embodiment, the compound of formula (I-1) is as follows:

[0187]

[0188] In one embodiment, Ld2 is a chemical bond, d is 1, and W is -C2H4-(PEG) t -(CO)NH2. In one embodiment, the compound of formula (I-1) is as follows:

[0189]

[0190] In one embodiment, Ld1 is i is 4, n is 3, m is 1, j is 12, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R 11 In one embodiment, the connector 1a has the following structure:

[0191]

[0192] In one embodiment, Ld1 is i is 4, n is 3, m is 2, j is 12, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R11 In one embodiment, the connector 1a has the following structure:

[0193]

[0194]

[0195] In one embodiment, Ld1 is i is 4, n is 3, m is 1, j is 12, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R 11 In one embodiment, the connector 1a has the following structure:

[0196]

[0197]

[0198] In one embodiment, Ld1 is i is 4, n is 3, m is 2, j is 12, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R 11 In one embodiment, the connector 1a has the following structure:

[0199]

[0200]

[0201] In one embodiment, Ld1 is i is 4, n is 3, m is 1, t is 12, L 2Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, the linker 1b has the following structure:

[0202]

[0203]

[0204] In one embodiment, Ld1 is i is 4, n is 3, m is 2, t is 12, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, the linker 1b has the following structure:

[0205]

[0206]

[0207] In one embodiment, Ld1 is i is 4, n is 3, m is 1, t is 12, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, the linker 1b has the following structure:

[0208]

[0209] In one embodiment, Ld1 is i is 4, n is 3, m is 2, t is 12, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, the linker 1b has the following structure:

[0210]

[0211] Compound of formula (I) with a load

[0212] The reactive group contained in B is covalently conjugated with a loading substance containing another reactive group to obtain a compound of formula (I) with a loading substance.

[0213] In yet another aspect, a compound having the structure of formula (II) is provided.

[0214]

[0215] in

[0216] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0217] M is LKa-LKb―P;

[0218] P is a load, which is connected to the B part or L of the compound of formula (I) 1 part;

[0219] n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I).

[0220] Load

[0221] In the present application, the load can be selected from the group consisting of: small molecule compounds, nucleic acids and analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptidomimetics and proteins. In one embodiment, the load is selected from the group consisting of: small molecule compounds, nucleic acid molecules and tracer molecules. In a preferred embodiment, the load is selected from small molecule compounds. In a more preferred embodiment, the load is selected from the group consisting of cytotoxins and fragments thereof.

[0222] In one embodiment, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is selected from the group consisting of: taxanes; maytansines; auristatins; epothilones; combretastatin A-4 phosphate, combretastatin A-4 and its derivatives; indole-sulfonamides; vinca alkaloids, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin 10 and analogs, halichondrin B and eribulin, indole-3-oxoacetamide, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, such as camptothecins and their derivatives, mitoxantrone, and mitoguazone. In a preferred embodiment, the cytotoxin is selected from the group consisting of nitrogen mustards, such as chlorambucil, chlornaphazine, cholestyramide, estramustine, ifosfamide, mechlorethamine, oxychloride mustard, melphalan, novembichin, phenamet, phenesterine, prednimustine, trofosfamide, and uracil mustard. In another preferred embodiment, the cytotoxin is selected from the group consisting of: nitrosoureas, such as carmustine, flubenzuron, formoterol, lomustine, nimustine, ramustine. In one embodiment, the cytotoxin is selected from the group consisting of aziridines.In a preferred embodiment, the cytotoxin is selected from the group consisting of: benzodopa, carboquone, meturedepa and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of: danemycin, esperamicin, neostatin and aclacinomycin. In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, antramycin, bleomycins, actinomycin C, carabicin, carminomycin and cardinophyllin, carminomycin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, Idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferroxorubicin, rodorubicin, rufocromomycin, streptozocin, zinostatin, zorubicin. In another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecene. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, mucin A, bacillocporin A and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs.In a preferred embodiment, the cytotoxin is selected from the group consisting of dimethylfolate, methotrexate, pteropterin, trimetrexate and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, tiamiprine, thioguanine. In yet another embodiment, the cytotoxin is selected from pyrimidine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine. In one embodiment, the cytotoxin is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone. In another embodiment, the cytotoxin is selected from the group consisting of antiadrenal agents. In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of anti-androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprorelin acetate and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors.In another embodiment, the cytotoxin is selected from the group consisting of: vinca alkaloids, colchicines, taxanes, auristatins, maytansines, calicheamicin, doxorubicin, duocarmucin, SN-38, scutellaria cyclotide analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, and pyrrolobenzodiazepine (PBD). In a specific embodiment, the cytotoxin is selected from the group consisting of: vinca alkaloids, colchicines, taxanes, auristatins, and maytansines.

[0223] In a specific embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f.

[0224] In another specific embodiment, the cytotoxin is a maytansinoid, such as DM1 or the like.

[0225] In a specific embodiment, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), etc. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0226] The support contains reactive groups that can react with reactive groups in the compound of formula (I) and thereby covalently conjugate the support with the compound of formula (I). Compounds without reactive groups need to be appropriately derivatized to obtain the support.

[0227] In one embodiment, the cytotoxin is a compound of formula (I)

[0228]

[0229] in,

[0230] a* is 0 or 1;

[0231] The carbon atoms labeled p1* and p2* are each an asymmetric center, and the asymmetric center is in the S configuration, the R configuration, or racemic;

[0232] L 1* Selected from C 1-6 Alkylene, which is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2;

[0233] M* is -CH2-, -NH- or -O-;

[0234] L2* It is C 1-3 Alkylene;

[0235] R 1* and R 2* are each independently selected from hydrogen, C 1-6 Alkyl, halogen and C 1-6 Alkoxy.

[0236] In a specific embodiment, the cytotoxin is a compound of formula (I')

[0237]

[0238] Where g* is any integer from 1 to 6;

[0239] R 1 ' and R 2 ' are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 Cycloalkylene; or

[0240] R 1 ' and R 2 ' together with the carbon atom to which it is attached form a 3- to 6-membered cycloalkyl group.

[0241] In one embodiment, g* is any integer from 1 to 3, preferably 1.

[0242] In one embodiment, R 1 'It's hydrogen.

[0243] In one embodiment, R 2 'It's hydrogen.

[0244] In one embodiment, L 1* Selected from C 1-6 Straight chain alkylene, C 1-6 Branched chain alkylene, C 3-6 Cyclic alkylene and C 3-4 Cycloalkyl-C 1-2 Straight chain alkylene, each of which is independently unsubstituted or substituted with a substituent selected from halogen, -OH and -NH2. 1* Selected from C 1-4 Alkylene, which is unsubstituted or substituted by a substituent selected from halogen, -OH and -NH2. In a preferred embodiment, L 1* Selected from -CH2-, -C2H4-, They are each independently unsubstituted or substituted by at least one substituent selected from halogen, -OH and -NH2. 1* Selected from -CH2-, Wherein "#" marks the position of the carbonyl group. In a more preferred embodiment, L 1* Selected from -CH2-, wherein "#" marks the position of attachment to the carbonyl group. In a specific embodiment, L 1* Selected from -CH2-, wherein "#" marks the position of attachment to the carbonyl group. In a preferred embodiment, the halogen is selected from F, Cl and Br, in particular F.

[0245] In one embodiment, a* is 1, M* is -CH2-, -NH-, or -O-; and L 2* In another embodiment, a* is 1, M* is -CH2-, and L 2* In one embodiment, a* is 0.

[0246] In one embodiment, the carbon atom labeled p1* is in S configuration or racemic, preferably S configuration. In another embodiment, the carbon atom labeled p2* is in S configuration or racemic, preferably S configuration.

[0247] In one embodiment, R 1* and R 2* are each independently selected from hydrogen, C 1-3 Alkyl, halogen and C 1-3 In a preferred embodiment, R 1* and R 2* Each is independently selected from CH3-, F, Cl, Br and CH3O-. In one embodiment, R 1* In another embodiment, R 2* It's F.

[0248] In one embodiment, a* is 0, L 1* Selected from -CH2-, Wherein "#" marks the position of attachment to the carbonyl group. In one embodiment, a* is 1, L 1* yes M* is O, and L 2* It is -C2H4-.

[0249] In one embodiment, a* is 0, R 1* It is Cl, R 2* is F, and L 1* Selected from -CH2-, In one embodiment, a* is 0, R 1* is CH3-, R2* is F, and L 1* Selected from Wherein "#" marks the position of attachment to the carbonyl group.

[0250] In one embodiment, a* is 1, R 1* is CH3-, R 2* It is F, L 1* yes M is O, and L 2* It is -C2H4-.

[0251] In one embodiment, the cytotoxin is selected from:

[0252]

[0253]

[0254]

[0255] In one embodiment, the cytotoxin is selected from:

[0256]

[0257]

[0258] In a preferred embodiment, the cytotoxin is selected from:

[0259]

[0260]

[0261] In a preferred embodiment, the cytotoxin is selected from:

[0262]

[0263] In a more preferred embodiment, the cytotoxin is selected from:

[0264]

[0265] In a specific embodiment, the cytotoxin is selected from:

[0266]

[0267] In one embodiment, the cytotoxin is selected from the following compounds; wherein the wavy bond shows the attachment site to the compound of formula (I).

[0268]

[0269] In some embodiments, the loading material is selected from DX8951f (Compound 9), DXd-(1) (Compound 10), DXd-(2) (Compound 14), Preferred DX8951f, DXd-(1), More preferably, DXd-(1), More preferred

[0270] Preparation of the compound of formula (I) with a load

[0271] In one embodiment, the linker unit and the loading substance are linked via the reactive groups as defined above using any reaction known in the art, including but not limited to condensation reaction, nucleophilic addition, electrophilic addition, and the like.

[0272] In one embodiment, the cargo is a cytotoxin. In one embodiment, the linker-cargo intermediate (numbered LBx) is as shown in the following table.

[0273]

[0274]

[0275] Conjugates and their preparation

[0276] In addition, the compound of formula (I) with a load having a portion comprising a ligase recognition sequence can be conjugated to other molecules comprising a ligase recognition sequence, and can thus be used, for example, to prepare a targeting molecule-drug conjugate, such as an antibody-drug conjugate. Therefore, in yet another aspect, a conjugate is provided, comprising a compound of formula (I), a targeting molecule and a load.

[0277] In yet another aspect, a conjugate having the structure of formula (III) is provided:

[0278]

[0279] in,

[0280] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0281] n, d, t, Ld1 and Ld2 are as defined in formula (I);

[0282] M is LKa-LKb―P;

[0283] P is a load, which is connected to the B part or L of the compound of formula (I) 1 part;

[0284] A is the targeting molecule;

[0285] z is an integer of 1-20.

[0286] In one embodiment, A is an anti-human monoclonal antibody, which is linked to the rest of the conjugate via a modified heavy chain and / or light chain C-terminus, wherein the modified heavy chain and / or light chain C-terminus is modified to contain Leu-Pro-Xaa-Thr, wherein Xaa is any natural or non-natural single amino acid. In one embodiment, z is 2. In a preferred embodiment, A is an anti-human monoclonal antibody, which is linked to the rest of the conjugate via a modified heavy chain and / or light chain C-terminus, wherein the modified heavy chain and / or light chain C-terminus is modified to contain Leu-Pro-Xaa-Thr, wherein Xaa is any natural or non-natural single amino acid, and z is 2.

[0287] Targeting molecules

[0288] In one embodiment, the targeting molecule is an antibody or an antigen-binding fragment thereof.

[0289] In one embodiment, the targeting molecule is an anti-human HER2 antibody or its antigen binding fragment. The example of anti-human HER2 antibody includes but is not limited to Pertuzumab and Trastuzumab. Pertuzumab is bound to the second extracellular domain (ECD2) of HER2 and is approved for the treatment of HER2 positive breast cancer. Trastuzumab is bound to the fourth extracellular domain (ECD4) of HER2 and is approved for the treatment of Her2 positive breast cancer and gastric cancer.

[0290] In a preferred embodiment, the anti-human HER2 antibody is one or more selected from trastuzumab-based engineered anti-HER2 antibodies.

[0291] In one embodiment, the targeting molecule is an anti-human TROP2 antibody or an antigen-binding fragment thereof. Examples of anti-human TROP2 antibodies include, but are not limited to, Trodelvy's antibody (hRS7) and DS1062's antibody (Datopotamab).

[0292] In one embodiment, the targeting molecule is an anti-FGFR3 antibody or an antigen-binding fragment thereof.

[0293] In a preferred embodiment, the anti-human HER2 antibody is a recombinant antibody selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment and an antibody mimetic. In a preferred embodiment, the anti-human Trop2 antibody is a recombinant antibody selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment and an antibody mimetic. In a preferred embodiment, the anti-FGFR3 antibody is a recombinant antibody selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment and an antibody mimetic. In order to be conjugated with the compound of formula (I), the targeting molecule of the present application may include a modified portion connected to the Gn in the compound of formula (I). In one embodiment, the antibody mimetic is selected from scFv, a mini antibody, a bifunctional antibody, and a nano antibody. The introduction position of such modified portions is not limited, for example, when the targeting molecule is an antibody, its introduction position may be but is not limited to being located at the C-terminus or N-terminus of the heavy chain or light chain of the antibody.

[0294] In an alternative embodiment, the modified moiety conjugated to Gn in the compound of formula (I) can be introduced into a non-terminal position of the antibody heavy chain or light chain using, for example, chemical modification methods.

[0295] In one embodiment, the targeting molecule of the present application is an antibody or an antigen-binding fragment thereof, which may include terminal modifications. Terminal modifications refer to modifications at the C-terminus or N-terminus of an antibody heavy chain or light chain, which modification, for example, includes a ligase recognition sequence. In another embodiment, the terminal modifications may also include a spacer Sp2 containing 2-100 amino acids, wherein the antibody, Sp2 and ligase recognition sequence are sequentially connected. In a preferred embodiment, Sp2 is a spacer sequence containing 2-20 amino acids. In a specific embodiment, Sp2 is a spacer sequence selected from the following: GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS, particularly GA.

[0296] In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof includes three types: wild type (LC); C-terminally modified light chain (LCCT), which is modified by directly introducing the ligase recognition sequence LPXTG; and C-terminally modified light chain (LCCT L ), which is modified by introducing a short peptide spacer plus a ligase donor substrate recognition sequence LPXTG. The heavy chain of an antibody or its antigen-binding fragment includes three types: wild type (HC); C-terminally modified heavy chain (HCCT), which is modified by directly introducing a ligase recognition sequence LPXTG; and C-terminally modified heavy chain (HCCT L), which is modified by introducing a short peptide spacer plus a ligase donor substrate recognition sequence LPXTG. X can be any natural or non-natural single amino acid. When z in the compound of formula (III) is 1 or 2, the combination of the above heavy chain and light chain can form 8 preferred antibody molecules, see the amino acid sequence table.

[0297] The conjugate of the present application may further comprise a load. The load is as described above.

[0298] Anti-HER2, TROP2, FGFR3 antibodies are listed as references, but are not limited thereto, and all of the antibodies are used to connect to the linker-load. For example, an anti-human monoclonal antibody can be connected to the rest of the conjugate via a modified heavy chain and / or light chain C-terminus, wherein the modified heavy chain and / or light chain C-terminus is modified to include Leu-Pro-Xaa-Thr, wherein Xaa is any natural or non-natural single amino acid, and z is 2.

[0299] Specific examples of conjugates

[0300] In one embodiment, each LKa is In one embodiment, formula (III) has the structure of formula (III-1):

[0301]

[0302] In one embodiment, Ld2 is Ld1 is d is 1, Q is hydrogen. In one embodiment, the compound of formula (III-1) is as follows:

[0303]

[0304] In one embodiment, Ld2 is a chemical bond, and Ld1 is d is 1, Q is -C2H4-(PEG) t -(CO)NH2. In one embodiment, the compound of formula (III-1) is as follows:

[0305]

[0306] In one embodiment, z is 1 to 4. In one embodiment, z is 2 or 4.

[0307] In one embodiment, z is 2.

[0308] In one embodiment, z is 4.

[0309] In one embodiment, in conjugates III-a, III-b, z is 2 or 4.

[0310] In one embodiment, in conjugates III-a, III-b, z is 2.

[0311] In one embodiment, in conjugates III-a, III-b, z is 4.

[0312] In one embodiment, B in the compound of formula (I) is a terminal group R 10 , and L 1 The cleavable sequence 1 in is linked to a cargo substance to form a compound of formula (II), wherein B is not present in the molecule obtained by linking the cleavable sequence 1 to the cargo substance.

[0313] In one embodiment, i is 4, n is 3, m is 1, j is 12, and L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R 11 In one embodiment, conjugate III-a has the following structure:

[0314]

[0315] In one embodiment, conjugate III-a has the following structure:

[0316]

[0317] In one embodiment, i is 4, n is 3, m is 2, j is 12, and L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R 11 In one embodiment, conjugate III-a has the following structure:

[0318]

[0319] In one embodiment, conjugate III-a has the following structure:

[0320]

[0321] In one embodiment, i is 4, n is 3, m is 1, j is 12, and L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R 11 In one embodiment, conjugate III-a has the following structure:

[0322]

[0323] In one embodiment, conjugate III-a has the following structure:

[0324]

[0325] In one embodiment, i is 4, n is 3, m is 2, j is 12, and L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1, R 11 In one embodiment, conjugate III-a has the following structure:

[0326]

[0327] In one embodiment, conjugate III-a has the following structure:

[0328]

[0329] In one embodiment, i is 4, n is 3, m is 1, t is 12, and L2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, conjugate III-b has the following structure:

[0330]

[0331] In one embodiment, conjugate III-b has the following structure:

[0332]

[0333] In one embodiment, i is 4, n is 3, m is 2, t is 12, and L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, conjugate III-b has the following structure:

[0334]

[0335] In one embodiment, conjugate III-b has the following structure:

[0336]

[0337] In one embodiment, i is 4, n is 3, m is 1, t is 12, and L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 )g -(CO)-R 10 , g is 1. In one embodiment, conjugate III-b has the following structure:

[0338]

[0339] In one embodiment, conjugate III-b has the following structure:

[0340]

[0341] In one embodiment, i is 4, n is 3, m is 2, t is 12, and L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-UR 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , g is 1. In one embodiment, conjugate III-b has the following structure:

[0342]

[0343] In one embodiment, conjugate III-b has the following structure:

[0344]

[0345] Preparation of conjugates

[0346] The conjugate of the present application can be prepared by any method known in the art. In some embodiments, the conjugate is prepared by site-specific conjugation of a targeting molecule catalyzed by a ligase with a compound of formula (I) with a load, wherein the targeting molecule is modified by a ligase recognition sequence. The method comprises steps A and B.

[0347] Step A. Preparation of Linker-Loader Intermediate

[0348] In a preferred embodiment, B in the compound of formula (I) is covalently linked to a carrier having another reactive group through a reactive group.

[0349] The linker-load intermediate prepared using the compound of formula (I) of the present application has a defined structure, a defined composition and high purity, so that when the conjugation reaction is carried out with the antibody, less impurities are introduced or no other impurities are introduced. When such an intermediate is used for ligase-catalyzed site-specific conjugation with a modified antibody containing a ligase recognition sequence, a homologous ADC with highly controllable quality is obtained.

[0350] Step B: Connecting the targeting molecule to the compound of formula (I) with a load

[0351] The targeting molecule of the present application can be conjugated with the compound of formula (I) (ie, the compound of formula (II)) carrying a load by any method known in the art.

[0352] The targeting molecule and the compound of formula (I) with a load are connected to each other through a ligase-specific substrate recognition sequence. The recognition sequence depends on the specific ligase used. In one embodiment, the targeting molecule is an antibody with a terminal modification based on a recognition sequence introduced at the C-terminus of a light chain and / or a heavy chain, and under the catalysis of a wild-type or optimized engineered ligase or any combination thereof and under suitable catalytic reaction conditions, the targeting molecule is conjugated to the compound of formula (II).

[0353] In a specific embodiment, the ligase is sortase A and the conjugation reaction can be represented by the following scheme:

[0354]

[0355] The triangle represents a part of the antibody; while the pentagon represents a part of the compound of formula (II). n, X and J are defined above, respectively. When conjugated with Gn, the corresponding recognition sequence as a receptor substrate, the peptide bond upstream of the glycine in the LPXTGJ sequence is cleaved by sortase A, and the intermediate thus obtained is linked to the free N-terminus of Gn to generate a new peptide bond. The resulting amino acid sequence is LPXTGn. The sequences Gn and LPXTGJ are defined above.

[0356] Metabolism of the conjugate in a physiological environment

[0357] When a part or the whole linker is cleaved in tumor cells, the antitumor compound is partially released to show the antitumor effect of the antitumor compound. Since the linker is cleaved at the connection position with the drug, the antitumor compound is released with its inherent structure to show its inherent antitumor effect.

[0358] In one embodiment, the cleavable sequence 1 (eg, Gly-Gly-Phe-Gly) is cleavable by a lysosomal enzyme (eg, cathepsin B and / or cathepsin L).

[0359] In one embodiment, Sp1 comprises a self-immolative spacer. In one embodiment, Sp1 comprises PABC, acetal or heteroacetal. In one embodiment, L 1 In one embodiment, the linker comprises -Gly-Gly-Phe-Gly-NH-CH2-O-. In one embodiment, -Gly-Gly-Phe-Gly-NH-CH2-O- represents a combination of a restriction enzyme site and a self-excisional spacer, which will cleave in the cell and release the target molecule (such as a drug).

[0360] Specific conjugate table

[0361] In one embodiment, the cargo is a cytotoxin or a fragment thereof. In one embodiment, the antibody is a modified trastuzumab, preferably Ab0001-LCCT L -HC (light chain SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) or Ab0001-LCCT L -HCCT L (Light chain: SEQ ID NO: 3, heavy chain: SEQ ID NO: 4). Ab0001-LCCT L -HC and Ab0001-LCCT L -HCCT L The respective sequences are based on the amino acid sequence of Ab0001 (trastuzumab), with GALPETGG introduced at the C-terminus of the light chain (Ab0001-LCCT L -HC) or introduced into the C-termini of the light and heavy chains (Ab0001-LCCT L -HCCT L ), wherein LPETGG is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence. L -HCCT L In the embodiment, the lysine at the C-terminus of the Ab0001 heavy chain can be maintained as in SEQ ID NO: 4 or removed before the introduction of GALPETGG (the resulting sequence is not shown in the sequence listing). In one embodiment, the antibody-drug conjugate is shown in the following table.

[0362] In one embodiment, the antibody is Ab2 (light chain: SEQ ID NO: 5, heavy chain: SEQ ID NO: 6).

[0363] In one embodiment, the antibody is Ab3 (light chain: SEQ ID NO: 7, heavy chain: SEQ ID NO: 8).

[0364] Nomenclature of ADC: The number in brackets indicates the number of cargo (drug) molecules intended to be attached to the antibody.

[0365]

[0366] Pharmaceutical compositions and pharmaceutical preparations

[0367] Another object of the present application is to provide a pharmaceutical composition comprising a preventively or therapeutically effective amount of the conjugate of the present application and at least one pharmaceutically acceptable carrier.

[0368] The pharmaceutical composition of the present application can be administered in any manner as long as it prevents, alleviates, prevents or cures symptoms of humans or animals. For example, various suitable dosage forms can be prepared according to the route of administration, particularly injection, such as lyophilized powder for injection, injection solution or sterile powder for injection.

[0369] The term "pharmaceutically acceptable" means that when in contact with the patient's tissues within the scope of normal medical judgment, it should not cause excessive toxicity, irritation or allergic response, etc., has a reasonable advantage-disadvantage ratio and is effective for the intended use.

[0370] The term pharmaceutically acceptable carrier refers to a carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline, etc. Pharmaceutically acceptable carriers also include carrier materials that make the composition close to physiological conditions, such as pH regulators, buffers, toxicity regulators, etc., as well as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0371] In one embodiment, the pharmaceutical composition of the present application has an integer or non-integer drug-to-antibody ratio (DAR) of about 1 to about 20, such as about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 3, about 1 to about 2.5, about 1 to about 2. In a specific embodiment, the DAR of the conjugate of the present application is about 2, about 4, about 6 or about 8.

[0372] Treatments and uses

[0373] The conjugates of the present application can be used to treat tumors and / or autoimmune diseases. Tumors treated by the conjugates include tumors characterized by specific tumor-associated antigens or cell surface receptors, and those tumor-associated antigens or cell surface receptors will be recognized by the targeting molecules in the conjugates and can be killed by the cargo / cytotoxins in the conjugates.

[0374] Therefore, in yet another aspect, also provided is use of the conjugate of the present application or the pharmaceutical composition of the present application in the manufacture of a medicament for treating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0375] In another aspect, the conjugate or pharmaceutical composition of the present application for treating tumors or autoimmune diseases is provided.

[0376] In another aspect, a method for treating a tumor or an autoimmune disease is provided, the method comprising administering an effective amount of the conjugate or the pharmaceutical composition of the present application to an individual in need thereof.

[0377] In a preferred embodiment, the conjugate of the present application formed by conjugating an anti-human HER2 antibody to a small molecule cytotoxin can specifically bind to HER2 on the surface of tumor cells and selectively kill tumor cells expressing HER2. In another preferred embodiment, the use of the conjugate of the present application or the pharmaceutical composition of the present application in the manufacture of a medicament for treating a disease, disorder or condition selected from HER2-positive tumors is provided. In a more preferred embodiment, the disease, disorder or condition is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, etc.

[0378] In a preferred embodiment, the conjugate of the present application formed by conjugating an anti-human TROP2 antibody to a small molecule cytotoxin can specifically bind to TROP2 on the surface of tumor cells and selectively kill tumor cells expressing TROP2. In another preferred embodiment, the use of the conjugate (or antibody) of the present application or the pharmaceutical composition of the present application in the manufacture of a medicament for treating a disease, disorder or condition selected from a TROP2-positive tumor is provided. In a more preferred embodiment, the disease, disorder or condition is a TROP2-positive tumor. In one embodiment, the TROP2-positive tumor is selected from the group consisting of: breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, etc.

[0379] In a preferred embodiment, the conjugate of the present application formed by conjugating an anti-FGFR3 antibody to a small molecule cytotoxin can specifically bind to FGFR3 on the surface of tumor cells and selectively kill tumor cells expressing FGFR3. In another preferred embodiment, there is provided the use of the conjugate of the present application or the pharmaceutical composition of the present application in the manufacture of a medicament for treating a disease mediated by FGFR3. Specifically, FGFR3 positive tumors, more especially brain cancer, bladder cancer, urothelial carcinoma, cervical cancer or intrahepatic bile duct carcinoma. In some embodiments, the disease includes a tumor that overexpresses FGFR3 or a tumor with a FGFR3 gene mutation. In some embodiments, the disease is selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial cell sarcoma, synovial tumor, mesothelioma, Ewing's tumor (Ewing's In some embodiments, the disease is selected from the group consisting of: brain cancer, bladder cancer, urothelial carcinoma, cervical cancer, or intrahepatic bile duct cancer. In some embodiments, the disease is glioblastoma.

[0380] The dosage of the conjugate administered to a subject can be adjusted over a wide range. The dosage can vary depending on the particular route of administration and the needs of the subject, and can be determined by a health care professional.

[0381] Beneficial Effects

[0382] The branched linker of the present application does not contain a maleimide group and is conjugated to the antibody at a specific site through the amino acid of the tail, thereby avoiding potential instability in the blood. At the same time, the branched linker has good assembly ability and can be conveniently and environmentally friendly to a variety of different loads and commercial linker-load fragments. The formed linker-load structure is easy to separate and purify, which is conducive to the later development of CMC. In addition, by conjugating different types of linkers with different antibodies, the stability, compatibility of the linker and the excellent biological activity of the ADC formed by the linker are verified.

[0383] The antibody-drug conjugate of the present application uses a specially designed linker-load and is more stable and can achieve great efficacy at a lower DAR, thereby reducing side effects and increasing the therapeutic index.

[0384] The present application utilizes a linking unit with a unique structure and uses a ligase to catalyze the conjugation of a targeting molecule with a load. The conjugate of the present application has good homogeneity, high activity and high selectivity. In addition, the toxicity of the linking unit-load intermediate is much lower than that of the free load, and therefore, the manufacturing process of the drug has no obvious disadvantages, which will be conducive to industrial production.

[0385] The conjugate of the present application achieves at least one of the following technical effects:

[0386] (1) High inhibitory activity against target cells or strong killing effect on target cells.

[0387] (2) Good physicochemical properties (e.g. solubility, physical and / or chemical stability).

[0388] (3) Good pharmacokinetic properties (e.g. good stability in plasma, appropriate half-life and duration of action).

[0389] (4) Good safety (low toxicity to non-target normal cells or tissues, and / or fewer side effects, wider therapeutic range), etc.

[0390] (5) Highly modular design and simple assembly of multiple drugs.

[0391] (6) Low load consumption and low production cost.

[0392] (7) There are almost no isomers and CMC is easy to achieve.

[0393] Examples

[0394] Preparation Example

[0395] In order to more clearly illustrate the purpose and technical solutions, the present application will be further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the present application. Specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

[0396] Instruments, materials, and reagents

[0397] Unless otherwise stated, the instruments and reagents used in the examples are commercially available. The reagents were used directly without further purification. The intermediates used were commercially available or synthesized.

[0398] MS: Thermo Fisher Q Exactive Plus, Water2795-Quattro micro triple quadrupole mass spectrometer

[0399] HPLC: Waters 2695, Agilent 1100, Agilent 1200

[0400] Semi-preparative HPLC: Lisure HP plus 50D

[0401] Flow cytometer: CytoFLEX S

[0402] HIC-HPLC: Butyl-HIC; mobile phase A: 25mM PB, 2M (NH4)2SO4, pH 7.0; mobile phase B: 25mM PB, pH 7.0; flow rate: 0.8ml / min; acquisition time: 25 minutes; injection volume: 20μg; column temperature: 25℃; detection wavelength: 280nm; sample chamber temperature: 8℃.

[0403] SEC-HPLC: column: TSK-gel G3000 SWXL, TOSOH 7.8mm ID×300mm, 5μm; mobile phase: 0.2M KH2PO4, 0.25M KCl, pH 6.2; flow rate: 0.5ml / min; acquisition time: 30 minutes; injection volume: 50μl; column temperature: 25℃; detection wavelength: 280nm; sample plate temperature: 8℃.

[0404] CHO was obtained from Thermo Fisher Scientific; Rink-amide-MBHA-resin was obtained from Nankai Synthesis; SK-BR-3 was obtained from ATCC catalog number HTB-30; NCI-N87 cells were obtained from ATCC catalog number CRL-5822; MDA-MB-468 was obtained from ATCC catalog number HTB-132; JIMT-1 was obtained from Wuxi Apptech; the antibody trastuzumab was prepared based on a known sequence; the optimized recombinant enzyme sortase A from Staphylococcus aureus was prepared in Escherichia coli.

[0405] Example 1: Construction of antibody expression vector, antibody expression, purification and identification

[0406] 1.1 Modified anti-human HER2 antibody Ab0001-LCCT L -HC production

[0407] Antibody Ab0001-LCCT was constructed as follows L-HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) expression plasmid. Ab0001-LCCT L -HC sequence: Based on the amino acid sequence of trastuzumab, GALPETGG is introduced at the C-terminus of the light chain, wherein LPETGG is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence. The plasmid is transfected into CHO cells and a cell population is established, and a high-expressing cell population is screened, and the cell population is cultured in a 5-10 L reactor according to the culture method of trastuzumab, and the supernatant is collected.

[0408] 1.2 Antibody Ab0001-LCCT L Purification of -HC

[0409] Ab0001-LCCT was purified using a combination of MabSelect affinity chromatography and Sepharose S cation exchange chromatography using standard procedures. L -HC, the purified product was dissolved in original trastuzumab drug buffer (5 mM histidine-HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0) and frozen in small aliquots.

[0410] 1.3 Antibody Ab0001-LCCT L -HC quality control

[0411] The purified antibody Ab0001-LCCT was confirmed by SDS-PAGE. L -HC purity is 98.5%; determined by SEC-HPLC, the content of high molecular weight polymers in the sample is less than 0.4%; endotoxin content is less than 0.098EU / mg.

[0412] 1.4 Preparation of other modified anti-human antibodies

[0413] According to a similar method, terminal modifications based on the ligase recognition sequence were introduced into the C-termini of the light chain and / or heavy chain of trastuzumab hRS7 (Ab2) and Ab3, respectively, to obtain modified antibodies.

[0414] The modified anti-human antibodies are listed in Table 1. LPETGG in the terminal modification sequence is a recognition sequence for a ligase donor substrate, and GA is a spacer sequence.

[0415] Table 1. Modified anti-human antibodies

[0416] sequence Sequence introduced at the end <![CDATA[Ab0001-LCCT L -HC light chain]]> SEQ ID NO:1 GALPETGG <![CDATA[Ab0001-LCCT L -HC heavy chain]]> SEQ ID NO:2 -* <![CDATA[Ab0001-LCCT L -HCCT L Light Chain]]> SEQ ID NO:3 GALPETGG <![CDATA[Ab0001-LCCT L -HCCT L Heavy Chain]]> SEQ ID NO:4 GALPETGG Ab2 light chain SEQ ID NO:5 GALPETGG Ab2 heavy chain SEQ ID NO:6 -* Ab3 light chain SEQ ID NO:7 GALPETGG Ab3 heavy chain SEQ ID NO:8 -*

[0417] *: “-” indicates no terminal modification

[0418] Example 2: Preparation of linker-load intermediates

[0419] 2.1 Preparation of intermediate Mc-GGFG-Dxd

[0420] The intermediate Mc-GGFG-Dxd is either commercially available or prepared according to the procedure described in EP 2907824. This compound was used to prepare linker-load intermediates and also for direct attachment to (optionally modified) antibodies to prepare reference ADCs.

[0421] 2.2 Preparation of linker-load 1

[0422]

[0423] Step 1: Preparation of intermediate compound b

[0424] Step 1.1: Preparation of NH2-Asp(OtBu)-Rink amide resin

[0425] Weigh 400 g of Rink amide resin and fully swell it with 2400 mL of DCM. Add 2400 mL of deprotection reagent to completely remove Fmoc, then wash several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin shows blue.

[0426] Weigh 88.87g Fmoc-Asp(OtBu)-OH and 29.19g HOBT and dissolve them in 2000mL DMF and 80mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hours, slowly add it to the reactor together with the resin and stir the reaction under nitrogen at room temperature for 2 to 5 hours, then filter. The resin is washed with DMF and DCM in turn, and in the subsequent ninhydrin test, the resin shows colorless or light yellow.

[0427] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0428] Step 1.2: Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin

[0429] 131.64 g Fmoc-PEG4-OH and 48.64 g HOBT were weighed and dissolved in 2000 mL DMF and 80.0 mL DIC solution. After being placed in an ice bath at -10 ° C for 0.5 hours, it was slowly added to the reactor together with the resin, and stirred at room temperature under nitrogen for 2 to 4 hours, and then filtered. The resin was washed with DMF and DCM in turn, and in the subsequent ninhydrin test, the resin showed colorless or light yellow.

[0430] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0431] Step 1.3: Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0432] Weigh 222.18g Fmoc-Asp(OtBu)-OH and 72.96g HOBT and dissolve them in 2000mL DMF and 80mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hours, slowly add it to the reactor together with the resin and stir the reaction under nitrogen at room temperature for 2 to 4 hours, then filter. The resin is washed with DMF and DCM in turn, and in the subsequent ninhydrin test, the resin shows colorless or light yellow.

[0433] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0434] Step 1.4: Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0435] Weigh 191.75g ​​Dde-Lys(Fmoc)-OH and 48.64g HOBT and dissolve them in 2000mL DMF and 80.0mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hour, slowly add it to the reactor together with the resin and stir the reaction under nitrogen at room temperature for 2 to 4 hours, then filter. The resin is washed with DMF and DCM in turn, and in the subsequent ninhydrin test, the resin shows colorless or light yellow.

[0436] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0437] Step 1.5: Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0438] 170.84 g m-PEG12-CH2CH2COOH and 48.64 g HOBT were weighed and dissolved in 2000 mL DMF and 80.0 mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hours, it was slowly added to the reactor together with the resin and stirred at room temperature under nitrogen for 2 to 4 hours, then filtered. The resin was washed with DMF and DCM in turn, and in the subsequent ninhydrin test, the resin showed colorless or light yellow.

[0439] Step 1.6: Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0440] 2400 mL of the de-Dde reagent was added, and the reaction was stirred at room temperature under nitrogen for 0.5 hours, and then filtered. After repeating the operation 3 times, the resin was washed with DMF and DCM in sequence, and in the subsequent ninhydrin test, the resin showed a blue color.

[0441] Step 1.7: Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0442] Weigh 111.08g Fmoc-Gly-Gly-Gly-OH and 48.64g HOBT and dissolve them in 2000mL DMF and 80.0mL DIC solution. After placing in an ice bath at -10°C for 0.5 hours, slowly add it to the reactor together with the resin and stir the reaction under nitrogen at room temperature for 2 to 4 hours, then filter. The resin was washed with DMF and DCM in turn, and in the subsequent ninhydrin test, the resin showed colorless or light yellow. The resin peptide was washed three times with anhydrous ethanol, filtered and waited for cleavage.

[0443] Step 1.8: Preparation of intermediate compound b

[0444] Add 10000 mL of cleavage reagent (TFA:TIS:H2O=95:2.5:2.5) to a 10 L reactor and cool it to -10±2°C. Add dried and weighed resin. Warm the reactants to room temperature and stir under nitrogen for 2 to 3 hours. Subsequently, filter the resin and wash once with 100 mL of TFA. Combine the filtrate and wash solution.

[0445] 40 L of pre-cooled (below -10°C) cold ether was added to the product solution. The mixture was stirred for 10 minutes and the precipitate was then centrifuged. After centrifugation, the supernatant was discarded, the precipitate was collected and washed with cold ether, and the precipitate was centrifuged again (the speed of each centrifugation was set to 3600 rpm, the centrifugation time was 5 minutes, and the temperature of the centrifuge cavity was -5°C).

[0446] The precipitate was collected as crude compound b. The crude product was purified by preparative HPLC and lyophilized to give pure compound b.

[0447] Step 2: Preparation of intermediate compound a

[0448]

[0449] Step 2.1: Preparation of compound 2

[0450] Compound 1 (1 e.q.) and DMF (5 v / v) were added to the reaction flask, and the mixture was stirred and dissolved under nitrogen protection. After cooling the ice bath to 0-5°C, DIEA (3 e.q.) was added dropwise, and after the dropwise addition, the mixture was stirred at 5°C for 10 minutes. Then, benzyl bromide (1.3 eq) was added dropwise, and after the dropwise addition was completed, it was naturally warmed to room temperature of about 20°C and stirred for 16 hours.

[0451] The reaction solution was slowly poured into ice water, MTBE was added and stirred, and the solution was allowed to stand for separation. The aqueous phase was extracted 4 times with MTBE, the combined organic phase was washed with saturated brine, and then the organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a yellow crude oil, which was applied to the column by a wet method. Elution by PE / EA=6:1 gave a light yellow oil, and the yield was 100%.

[0452] Step 2.2: Preparation of compound 4

[0453] Under nitrogen protection, intermediate 2 (2.0eq), compound 3 (1e.q.) and THF (10v / v) are added to a reaction flask and stirred to dissolve, TsOH (0.1eq) is weighed and added to the reactant, and the reactant is kept at 20 to 22°C for 4 hours. The reaction solution is slowly poured into ice water, extracted 3 times with EA, the combined organic phase is washed with saturated sodium bicarbonate aqueous solution, water and saturated brine successively, the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to obtain a crude product. By mixing the silica gel sample, passing it through the column, eluting with PE / EA=1:1 to collect the product, and concentrating to obtain a white solid with a yield of 40%.

[0454] Step 2.3: Preparation of compound 7

[0455] Under nitrogen protection, compound 4 and DMAc (10v / v) were added to the reaction flask and stirred to dissolve. The reactant was cooled to 14°C to 18°C, DBU (0.5eq) was added dropwise, and the reaction was stirred for 1.5 hours at this temperature, and the reaction of the raw materials was completed by TLC monitoring. The reactant was cooled to 0-5°C, PPTS (0.5eq), EDCI (1e.q.), HOBT (1e.q.) and compound 6 (0.85eq) were added and reacted at 0-10°C for 3 to 4 hours, and the reaction was monitored by LCMS.

[0456] The reaction solution was added to ice water, 2-methyltetrahydrofuran was added and extracted once, and the aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined, washed with 0.5M hydrochloric acid, washed with saturated NaHCO3 aqueous solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, evaporated to dryness, mixed with silica gel, and purified by column. The product was collected by DCM / MeOH elution and concentrated under vacuum to obtain a white solid in 78% yield.

[0457] Step 2.4: Preparation of compound 10

[0458] Under nitrogen protection, intermediate 7 and DMAc (10v / v) were added to the reaction flask and stirred to dissolve. The reactant was cooled to 14°C to 18°C, DBU (0.5eq) was added dropwise, and the reaction was stirred for 1.5 hours at this temperature, and the completion of the reaction was monitored by TLC. The reactant was cooled to 0-5°C, PPTS (0.5eq), EDCI (1e.q.), HOBT (1e.q.) and compound 9 (0.85eq) were added and reacted at 0-10°C for 3 to 4 hours, and the reaction was monitored by LCMS.

[0459] The reaction solution was added to ice water, 2-methyltetrahydrofuran was added and extracted once, and the aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined, washed with 0.5M hydrochloric acid, saturated NaHCO3 aqueous solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, evaporated to dryness, mixed with silica gel, and purified by column. The product was collected by DCM / MeOH elution and concentrated under vacuum to obtain a white solid in 50% yield.

[0460] Step 2.5: Preparation of compound a

[0461] Under nitrogen protection, intermediate 10 was dissolved in DCM (15 v / v), DBU (0.5 eq) was added dropwise at 20 ° C, and the reaction was stirred for 5 hours at 18-22 ° C. The completion of the reaction was monitored by LCMS. The reaction solution was diluted with DCM and wet method was used, purified by column, and the product was collected by DCM: MeOH elution to obtain a white solid with a yield of 82%.

[0462] Step 3: Preparation of compound c

[0463]

[0464] Compound b (400 mg, 0.245 mmol) and compound a (377 mg, 0.539 mmol) were dissolved in DMF (6 ml), and then DIPEA (159 mg, 1.23 mmol) and HATU (233 mg, 0.613 mmol) were added to the reaction solution, and the reaction was stirred at room temperature for 2 hours. After compound b disappeared, it was purified by preparative HPLC, and the prepared solution was lyophilized to obtain 380 mg of product with a yield of 52%. 142 H 207 O 49 N 21 [(M+3H) / 3] + Calculated value: 997.8, found value: 875.9 (fragmentation mass).

[0465] Step 4: Preparation of intermediate compound d

[0466]

[0467] Compound c (380 mg, 0.245 mmol) was dissolved in purified water (80 ml), and palladium hydroxide (38 mg) was added. The system was replaced with hydrogen three times and the reaction was stirred at room temperature. The reaction progress was monitored by HPLC, and the reaction was stopped immediately after the raw material disappeared to prevent the increase of the de-Fmoc product. The reaction solution was filtered and purified by preparative HPLC to obtain 270 mg of product with a yield of 76%. 128 H 195 O 49 N 21 [(M+3H) / 3] + Calculated value: 937.8, found value: 875.9 (fragmentation mass).

[0468] Step 5: Preparation of intermediate compound e

[0469]

[0470] Compound d (270 mg, 0.096 mmol) and 12-1 (120 mg, 0.211 mmol) were dissolved in DMF (5 ml), and then DIPEA (62 mg, 0.48 mmol) and HATU (92 mg, 0.24 mmol) were added to the reaction solution and stirred at room temperature for 2 to 16 hours. After the reaction was completed by HPLC monitoring, the reaction mixture was directly purified by preparative HPLC, and the collected eluent was lyophilized to obtain 235 mg of product with a yield of 66%. 174 H 229 O 55 Cl2F2N 27 [(M+3H) / 3] + Calculated value: 1229.2, experimental value: 1229.3.

[0471] The preparation of 12-1 is shown in 2.3.1 and 2.3.2.

[0472] Step 6: Preparation of Linker-Load 1

[0473]

[0474] Compound e (210 mg, 0.057 mmol) was dissolved in DMF (5 ml), then diethylamine (0.5 ml) was added and the reactants were allowed to react at room temperature for 15 minutes, and the reaction endpoint was monitored by HPLC. After the reaction was completed, it was adjusted to neutrality with 10% TFA aqueous solution under ice bath, and the reactants were purified by preparative HPLC, and 145 mg of product was obtained after freeze drying, with a yield of 73%. 159 H 219 O 53 Cl2F2N 27 [(M+3H) / 3] + Calculated value: 1155.2, experimental value: 1155.3.

[0475] 2.3: Preparation of linker-load 2

[0476] 2.3.1: Preparation of small molecules as cargo

[0477] Intermediate 11

[0478]

[0479] Step A: N-(2-bromo-5-fluorophenyl)acetamide: To a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL) was added concentrated H2SO4 (3 mL), followed by portionwise addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) at room temperature. The mixture was stirred for 3 hours and then poured into 2000 mL of ice water. A precipitate was formed, which was collected by filtration and dried in vacuo at room temperature to give N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ7.68(dd,J=8.9,6.0Hz,1H),7.61(ddd,J=10.7,5.3,3.1Hz,1H),7.02(ddd,J=8.9,8.0,3.1Hz,1H),2.11(s,3H).MS m / z232.0(M+H).

[0480] Step B: N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: To a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (1000 mL) was added dropwise n-BuLi (594 mL, 1.6 M, in n-hexane, 950.22 mmol) at -78 °C over 1 hour. After completion, the mixture was stirred under N2 for 0.5 hour. Then at -78 °C, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise over 0.5 hour, and the mixture was stirred at -78 °C to room temperature for 6 hours. The mixture was poured into 500 mL of saturated NH4Cl aqueous solution at 0 °C. It was extracted with ethyl acetate (500 mL×3), washed with brine (250 mL×2), dried over Na2SO4 and concentrated. The mixture was triturated with (PE / EA=1:1, 100 mL) for 10 min, filtered and the filter cake was collected and dried in vacuo to give N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g) as a yellow solid. MS m / z 206.1 (M-18+H), 246.1 (M+Na).

[0481] Step C: N-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirred mixture of N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) in CH2Cl2 (170 mL) and water (170 mL) were added silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol), and the mixture was stirred at 30°C for 6 hours. The mixture was filtered on celite and washed with CH2Cl2 (100 mL), the filtrate was concentrated and purified by FCC (EA / PE=0-40%) to give N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g) as a light yellow solid. MS m / z 222.1 (M+H).

[0482] Step D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirred mixture of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) in THF (500 mL) at 0°C was added 1-butyl nitrite (8.48 g, 63.28 mmol), followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0°C for 2 hours. After completion, the mixture was acidified with HCl (2N) to adjust pH = 3. The mixture was extracted with ethyl acetate (200 mL×3), washed with brine (100 mL×2), dried over Na2SO4 and concentrated under reduced pressure. The crude mixture was triturated with tert-butyl methyl ether (200 mL) for 10 minutes, filtered and the filter cake was collected and dried in vacuo to give N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12 g) as a yellow solid. MS m / z 251.1 (M+H).

[0483] Step E: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide: To a solution of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (12 g, 47.96 mmol) in acetic anhydride (90 mL) and THF (90 mL) was added 10% Pd / C (1 g), and the mixture was stirred at 25°C for 16 hours under H2 atmosphere. After cooling to 0°C, Et3N (20 mL) was added dropwise, and the mixture was stirred at 0°C for 1 hour. Filtered on celite, the filtrate was poured into ice water (500 mL). Extracted with ethyl acetate (500 mL×3), washed with brine (250 mL×2), dried over Na2SO4 and concentrated. The residue was triturated with tert-butyl methyl ether (120 mL) for 10 minutes, filtered and the filter cake collected and dried in vacuo to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (7.9 g) as a yellow solid. MS m / z 279.1 (M+H).

[0484] Step F: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (7.9 g, 28.39 mmol) in MeOH (150 mL), aqueous HCl (2N, 150 mL) was added, and the mixture was stirred at 50 ° C for 7 hours. After cooling to 0 ° C, saturated aqueous NaHCO3 was added dropwise to adjust pH = 8. It was extracted with ethyl acetate (200 mL×3), washed with brine (200 mL×2), dried over Na2SO4 and concentrated under reduced pressure to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (6.0 g) as a yellow solid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 6.57 (s, 3H), 6.18 (td, J = 11.1, 2.4 Hz, 2H), 4.52 (dt, J = 13.3, 5.0 Hz, 1H), 3.13 (ddd, J = 17.5, 13.0, 4.6 Hz, 1H), 3.00-2.81 (m, 1H), 2.69 (dtd, J = 9.4, 4.6, 2.5 Hz, 1H), 2.09 (s, 3H), 1.79 (qd, J = 13.0, 4.3 Hz, 1H). MS m / z 237.1 (M+H).

[0485] Step G: N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide: NCS (2.26 g, 16.93 mmol) was added portionwise to a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (4.0 g, 16.93 mmol) in DMF (80 mL) at 0 ° C. The mixture was stirred at room temperature for 16 hours. The mixture was poured into 200 mL of ice water. A precipitate was formed, collected by filtration and dried in vacuo at room temperature to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide (4.0 g) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ8.11(d,J=8.0Hz,1H),7.71(s,2H),6.62(d,J=11.9Hz,1H),4.53(ddd,J=13.0,8.0, 4.7Hz,1H),3.18-3.04(m,1H),2.91(ddd,J=17.5,12.4,4.8Hz,1H),2.21-2.08(m,1H),1.99-1.83(m,4H).MS m / z 271.0(M+H).

[0486] Step H: N-(9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide: To N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g, To a mixture of (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol) and o-cresol (10 mL) were added, and the mixture was heated to reflux under N2 for 24 h. The solvent was removed under reduced pressure and the mixture was purified by FCC (THF / CH2Cl2=0-60%) to give N-(9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4.1 g) as a brown solid. MS m / z 498.1 (M+H).

[0487] Step I: A mixture of (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione: A mixture of N-(9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of concentrated aqueous HCl was stirred at 70 °C under N2 for 36 h. The mixture was concentrated under reduced pressure to give crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (2 g) as a brown solid. MS (ESI) m / z 456.1 (M+H).

[0488] 2.3.2: Preparation of intermediate 12 (12-1, 12-2)

[0489]

[0490] 12-1 and 12-2 were prepared from (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (Intermediate 11) as TFA salts by preparative HPLC.

[0491] Table 2

[0492]

[0493] The conditions of the above HPLC are: equipment: Agilent 1200; column: Waters XBridge C18 4.6*50 mm, 3.5 μm; flow rate: 2.0 mL / min; gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min-1.50 min-2.50 min-2.52 min-3.00 min; temperature: 40°C; phase: A: acetonitrile, B: H2O (0.05% TFA); wavelength: 214 nm / 254 nm.

[0494] 2.3.3: Preparation of linker-load 2

[0495]

[0496] opSu is and mixture.

[0497] 2.3.3.1: Preparation of Compound 13 (Step A)

[0498] Weigh 4.33g Fmoc-Gly-Gly-OH and 6.84g Pb(OAc)4 and add to a 500ml single-necked round-bottom flask. Add anhydrous THF / toluene (120 / 40ml) under a nitrogen atmosphere and stir to dissolve. Then add 1.16mL pyridine to the reaction system. Heat the reaction system to 80°C and reflux for 5 hours under a nitrogen atmosphere. Take a sample and monitor the reaction by HPLC.

[0499] The reaction system was cooled to room temperature, filtered, and the filter cake was washed with EA three times. The filtrates were combined and concentrated to dryness. Column chromatography (PE:EA=100:0-50:100) was performed to obtain about 2000 mg of the target product as a white solid with a yield of 44%.

[0500] 2.3.3.2: Preparation of Compound 15 (Step B)

[0501] 200 mg of compound 13 was weighed and added to a 100 ml single-neck round-bottom flask. Then 15 ml of THF was added and stirred to dissolve. Compound 14 (312 mg, 3.0 eq) and TsOH·H2O (15 mg, 0.15 eq) were then added to the reaction system. The reaction system was allowed to react overnight at room temperature. Samples were taken and detected by TLC (PE / EA=1:1) to monitor the reaction. The raw material essentially disappeared, and new spots were detected.

[0502] Saturated sodium bicarbonate solution was added to quench the reaction. Extracted with EA three times. The organic phases were combined and washed with saline, dried over anhydrous magnesium sulfate and concentrated. The crude product was purified by column chromatography (PE:EA=5:1-1:1) to obtain about 80 mg of the target product as a colorless oil with a yield of 29%. MS: [M+H] + =501.1.

[0503] 2.3.3.3: Preparation of Compound 16 (Step C)

[0504] 200mg compound 15 was weighed and added to a 100ml single-necked round-bottom flask. Then 10ml EtOH and 5ml EA were added and completely dissolved. Then, under a nitrogen atmosphere, 40mg palladium carbon was added to the reaction system, and the reaction system was purged with hydrogen three times. The reaction system was kept in a hydrogen environment and stirred at room temperature for 0.5 hours. Samples were taken and detected by TLC (DCM / MeOH=10:1) to monitor the reaction. The raw material essentially disappeared, and new spots were detected.

[0505] The reaction system was filtered and the filter cake was washed 3 times with EA. The filtrates were combined and concentrated to dryness to obtain 200 mg of a white solid product with a yield of 100%. The product can be used directly in the next reaction without purification. MS: [MH] - =409.4.

[0506] 2.3.3.4: Preparation of Compound 21 (Step D)

[0507] Step D-1

[0508] Weigh 2.0g of dichlororesin and put it into a peptide synthesis tube. Add DCM (10ml) and swell at room temperature for 30 minutes. Remove the solvent by vacuum suction. The resin is washed twice with a volume of 7mL of DCM, and the duration of each wash is 1 minute. Remove the solvent by vacuum suction. Then weigh compound 16 (200mg) and add it to a 50ml centrifuge tube. Add DCM (about 10ml). Dissolve the solid by shaking. Add to the above resin. Stir to immerse all the resin in the solution (if there is resin attached to the tube wall, use a small amount of DCM to wash the tube wall). Stir for 4-5 hours. After the reaction is complete, add an appropriate amount of methanol. Stir for 30 minutes. Remove the solvent by vacuum suction. The resin is washed once with DMF, once with methanol, once with DMF, once with methanol and twice with DMF in sequence, with a volume of 10mL and a duration of 1 minute for each wash. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin detection. The resin was colorless and transparent, while the solution was light yellow, indicating that it was qualified for the next coupling step.

[0509] Step D-2

[0510] Deprotection was performed twice by adding 10 mL of a ready-made 20% piperidine / DMF solution, each reaction lasting 10 minutes. After the reaction was complete, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, with a volume of 10 mL and a duration of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dried resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0511] Add 563 mg of Fmoc-Phe-OH and 197 mg of HOBt to a 50 mL centrifuge tube. Then add about 7 mL of DMF. Shake to dissolve the solid. Then add 0.24 mL of DIC. Activate for 10 to 30 minutes to obtain an activated reaction solution.

[0512] Add 3 molar equivalents of activated reaction solution to the resin. Stir so that all resins are fully immersed in the solution (if there is resin attached to the tube wall, a small amount of DCM is used to wash the tube wall). Stirring is carried out for 2-3 hours. After the reaction is completed, the solvent is removed by vacuum suction. The resin is washed twice with DMF in sequence, washed once with methanol, washed once with DMF, washed once with methanol and washed twice with DMF, and for each washing, the volume is 10mL and the duration is 1 minute. Remove the solvent by vacuum suction. Take a small amount of dried resin for ninhydrin detection. The resin is colorless and transparent, and the solution is light yellow, showing that it is qualified for the next coupling step.

[0513] Step D-3

[0514] Deprotection was performed twice by adding 10 mL of a ready-made 20% piperidine / DMF solution, each reaction lasting 10 minutes. After the reaction was complete, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, with a volume of 10 mL and a duration of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dried resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0515] Add 531 mg of Fmoc-GG-OH and 197 mg of HOBt to a 50 mL centrifuge tube. Then add about 10 mL of DMF. Shake to dissolve the solid. Then add 0.24 mL of DIC. Activate for 10 to 30 minutes to obtain an activated reaction solution.

[0516] Add 3 molar equivalents of activated reaction solution to the resin. Stir so that all resins are fully immersed in the solution (if there is resin attached to the tube wall, a small amount of DCM is used to wash the tube wall). Stirring is carried out for 2-3 hours. After the reaction is completed, the reaction solution is removed by vacuum suction. The resin is washed twice with DMF in sequence, washed once with methanol, washed once with DMF, washed once with methanol and washed twice with DMF, and for each washing, the volume is 10mL and the duration is 1 minute. Remove the solvent by vacuum suction. Take a small amount of dried resin for ninhydrin detection. The resin is colorless and transparent, and the solution is light yellow, showing that it is qualified for the next coupling step.

[0517] Step D-4

[0518] Deprotection was performed twice by adding 10 mL of ready-made 20% piperidine / DMF solution, and each reaction lasted 10 minutes. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF in sequence, with a volume of 10 mL and a duration of 1 minute for each washing. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue. Then 462 mg MC-OSu was placed in a 50 mL centrifuge tube and about 10 mL DMF was added. The solid was dissolved by shaking. Then, 0.24 mL DIEA was added to the resin. Stirring was performed to completely immerse all resins in the solution (if there was resin attached to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was performed for 2-3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF in sequence, with a volume of 10 mL and a duration of 1 minute for each washing. The solvent was removed by vacuum suction. A small amount of dried resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was light yellow, indicating that it was qualified for the next coupling step.

[0519] Step D-5

[0520] The resin was washed twice with 10 mL of methanol. The solvent was then fully removed by vacuum suction. The resin was poured out and weighed. A lysis buffer was prepared in a 250 mL conical flask, wherein: the ratio of TFE / DCM was 80% / 20%, and the volume was 7-8 times the weight of the peptide resin. The lysis buffer was added to the peptide resin and shaken thoroughly. The resin was completely immersed in the lysis buffer and cleaved at room temperature for 2-3 hours. The lysis buffer was then filtered out using a simple filter made of a syringe, and the resin was washed with 1-2 ml of DCM and discarded. Then 150 mL of pre-cooled anhydrous ether was added to the lysis buffer, shaken thoroughly, and then left to stand for 20 to 30 minutes. Using a 50 mL centrifuge tube, the above system was centrifuged in a centrifuge at 3500 rpm for 3 minutes, and the supernatant was poured out and discarded. The solid was shaken with pre-cooled anhydrous ether, washed once under ultrasound, centrifuged at 3500 rpm for 3 minutes, and the supernatant was poured out and discarded. The solid was placed in a centrifuge tube and air-dried overnight, and then subjected to preparative purification to obtain 125 mg of a white solid product with a yield of 40%. MS: [MH] - =641.5.

[0521] 2.3.3.5: Preparation of Compound 22 (Step E)

[0522] 150 mg of raw material compound 21 and 55 mg of TSTU were weighed and added to a 10 mL single-necked round-bottom flask, anhydrous DMF (3 mL) was added and stirred for 20 minutes under a nitrogen atmosphere. Then 18 mg of compound 12-1 and 20 μl of DIEA were added to the reaction system in sequence. At room temperature, stirred for 2 hours under a nitrogen atmosphere. Samples were taken and detected by HPLC to monitor the reaction. The raw material peak disappeared completely, and a new peak was detected.

[0523] The reaction system was subjected to preparative purification, and the target product was collected and lyophilized to obtain about 22 mg of a yellow solid product. MS: [M+H] + =1081.0.

[0524] 2.3.3.6: Preparation of linker-load 2 (Step F)

[0525] Compound 22 (30 mg) was weighed and added to a 10 ml single-necked round-bottom flask, and purified water (2 ml) was added. Stirring was performed to dissolve. A DMF solution (2 ml) containing a joint-load intermediate 1 (19.5 mg) was added to the reaction system and stirred. After the reaction was overnight, HPLC was used to monitor the reaction until all raw materials were converted into intermediates. An appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued for another 0.2-20 hours at 0-40 ° C. The reaction was monitored by HPLC until all intermediates were consumed, and then quenched with acetic acid solution.

[0526] The reaction system was subjected to preparative purification, and the target product was collected and lyophilized to obtain about 25 mg of the linker-load 2 as a yellow solid. MS: [(M+3H) / 3] + =1194.4.

[0527] 2.4: Preparation of linker-load 3

[0528]

[0529] opSu is and mixture of

[0530] Step 2.4.1: Preparation of linker-load intermediate 1

[0531]

[0532] The linker-load intermediate 1 was synthesized by conventional solid phase peptide synthesis using Rink-amide-MBHA-resin. The amino acids in the linker unit were protected using Fmoc. The conjugation reagent was selected from HOBt, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the resin was cleaved using trifluoroacetic acid. The product was purified by HPLC, lyophilized and stored for future use. Theoretical mass: 1383.70, measured value: [MH] - =1382.6.

[0533] Step 2.4.2: Preparation of linker-load 3

[0534] The linker-load intermediate 1 and the intermediate MC-GGFG-Dxd (commercially available) were weighed and dissolved in water and DMF, respectively, in a molar ratio of 1:2, and then mixed thoroughly to obtain a mixture, which was reacted at 0-40°C for 0.5-30 hours. Once the reaction was complete, an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued at 0-40°C for another 0.2-20 hours. After the reaction was completed, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain the linker-load 3. Theoretical mass: 3486.52, measured value: [(M+3H) / 3] + =1163.3.

[0535] 2.5: Preparation of linker-load 4

[0536]

[0537] Step A: Synthesis of intermediate (a)

[0538] Fmoc-PEG4-VC-OH (1 equivalent) and p-hydroxybenzyl alcohol (5.0 equivalents) were added to the reaction flask and dissolved in a solvent of DCM: MeOH = 2: 1. Under a nitrogen atmosphere, EEDQ (5.0 equivalents) was added and stirred evenly, and the reaction solution was reacted at room temperature in the dark. The reaction was monitored by HPLC until the reaction was completed (about 3 hours). The reaction system was prepared directly by preparative HPLC, and compound intermediate (a) (white solid, yield 41%) was obtained by freeze-drying the prepared solution. MS (ESI) C 43 H 60 O 11 N6[M+H] + Calculated value: 849.4, experimental value: 849.8.

[0539] Step B: Synthesis of intermediate (b)

[0540] Compound intermediate (a) (1.0 equivalent) was dissolved in DMF and the mixture was cooled to 0°C. p-(Dinitrobenzene) carbonate (4.0 equivalent) and DIPEA (6.0 equivalent) were added to the reactants. The reaction system was stirred at 0°C for 0.5 hours and then recovered at room temperature for 2 hours. The reaction was monitored by HPLC until the reaction was complete. The reaction liquid system was concentrated and purified by column chromatography (eluted with 0-10% MeOH / DCM) to give compound intermediate (b) (white solid, yield 75%). MS (ESI) C 51 H 64 O 15 N7[M+H] + Calculated value: 1015.4, experimental value: 1015.0.

[0541] Step C: Synthesis of intermediate (c)

[0542] Weigh compound intermediate (b) (1.2 equivalents) and HOBT (2.0 equivalents) and dissolve them in DMF, then add MMAE (1.0 equivalents) and DIPEA (10.0 equivalents) and stir the reactants evenly. Allow the mixture to react overnight at room temperature. Monitor the reaction by HPLC until the reaction is complete (about 16 hours) and use the resulting mixture directly in the next reaction.

[0543] Step D: Synthesis of intermediate (d)

[0544] Diethylamine (10% v / v) was added to the reaction solution of the previous step C, and the reaction solution was stirred at room temperature. The reaction was monitored by HPLC until the reaction was completed (about 1.5 hours). The reaction system was directly prepared by preparative HPLC, and compound H0152 (yellow solid, yield 62%) was obtained by freeze drying the prepared solution. MS (ESI) C 69 H 116 O 17 N 11 [M+H]+ calcd: 1370.8, found: 1370.6.

[0545] Step E: Synthesis of intermediate (e)

[0546] Compound intermediate (d) (2.4 equivalents) and compound b (1.0 equivalents) were weighed and dissolved in DMF, and then DIPEA (4.0 equivalents) was added and the reactants were uniformly stirred at 0°C. HATU (2.5 equivalents) was added and the reaction system was stirred at 0°C. The reaction was monitored by HPLC until the reaction was completed (about 2 hours). The reaction system was directly prepared by preparative HPLC, and compound intermediate (e) (yellow solid, yield 50%) was obtained by freeze-drying the preparative solution. MS (ESI) C 212 H 319 O 63 N 31 [M+4H+] 4+ / 4Calculated value: 1084.4, experimental value: 1085.0.

[0547] Step F: Synthesis of intermediate (f)

[0548] The compound intermediate (e) was weighed and dissolved in DMF, and then diethylamine (10% v / w) was added, and the reaction solution was stirred at room temperature. The reaction was monitored by HPLC until the reaction was completed (about 0.5 hours). The reaction system was directly prepared by preparative HPLC, and the compound intermediate (f) (yellow solid, yield 90%) was obtained by freeze drying the prepared solution. MS (ESI) C 197 H 309 O 61 N 31 [M+4H+] 4+ / 4Calculated value: 1028.9, experimental value: 1029.4.

[0549] Example 3 Preparation of Targeting Molecule-Drug Conjugates

[0550] 3.1: The linker-load intermediate is conjugated to the antibody in a site-specific manner by a ligase to form an ADC. The method of the conjugation reaction can be found in WO2015165413A1. ADC-4 was prepared using the same method as Example 3 in WO2015165413A1. The resulting ADCs are listed in the following table:

[0551] ADC Name Connector-Load Antibody DAR value ADC-1 Connector-Load 1 <![CDATA[Ab0001-LCCT L -HC]]> 3.7 ADC-2 Connector-Load 2 <![CDATA[Ab0001-LCCT L -HC]]> 3.6 ADC-3 Connector-Load 3 <![CDATA[Ab0001-LCCT L -HC]]> 3.6 ADC-4 - <![CDATA[Ab0001-LCCT L -HC]]> 1.8 Enhertu - Ab0001 7.5 ADC-5 Connector-Load 1 Ab2 3.5 ADC-6 Connector-Load 2 Ab2 3.6 ADC-7 Connector-Load 3 Ab2 3.6 ADC-8 Connector-Load 1 Ab3 3.6 ADC-9 Connector-Load 2 Ab3 3.7 ADC-10 Connector-Load 3 Ab3 3.6

[0552] 3.2: Preparation of the reference ADC Enhertu by direct attachment of the intermediate MC-GGFG-Dxd to the (optionally modified) antibody (Cys conjugation, ie conjugation via a connection formed by a maleimide structure and the thiol group of Cys). Methods for conjugation reactions are known in the art.

[0553] Effect Example 1: Bystander Killing Effect of the Conjugate in SK-BR-3 and MDA-MB-468 Co-culture

[0554] SK-BR-3 tumor cells (ATCC, HTB-30) were cultured in McCoy'5A medium supplemented with 10% fetal bovine serum at 37°C, 5% CO2. MDA-MB-468 tumor cells (ATCC, HTB-132) were cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 37°C, 0% CO2. The MDA-MB-468-Luc-GFP cell line was constructed by lentiviral infection and sorted by FACS. Co-culture of SK-BR-3 and MDA-MB-468-Luc-GFP was performed at a 4:1 ratio (1×10 4 cells / well) were seeded In 96-well black / transparent bottom polystyrene microplates, keep for 24 hours. Test substances (ADC-1, ADC-2, ADC-3, Enhertu and ADC-4) were applied according to the experimental design and cultivated for 120 hours. MDA-MB-468-Luc-GFP cell counting was performed on BioTek Cytation3 (BioTek, LAB14002). Then, luciferase activity was detected on BioTek SynergyHTX (BioTek, MAB16038) using firefly luciferase reporter gene assay kit (Beyotime, RG006).

[0555] In SK-BR-3 and MDA-MB-468-Luc-GFP co-cultures ( Figure 1 ), ADC-1 and ADC-2 showed similar efficacy. And both ADC-1 and ADC-2 showed efficacy comparable to or even better than Enhertu.

[0556] Effect Example 2: In vivo effects of ADC-2 and ADC-1 in the JIMT-1CDX model

[0557] JIMT-1 tumor cells (DSMZ-ACC 589) were maintained in vitro as monolayer cultures in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% CO2. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment.

[0558] SCID Beige female mice, 6 to 8 weeks old, weighing approximately 18 to 20 g. A total of 27 mice (18 plus 50% spare) were required for the study, all purchased from Shanghai Lingchang Biotechnology Co., Ltd. or other certified suppliers.

[0559] Each mouse was inoculated subcutaneously on the right flank with JIMT-1 tumor cells (5 × 10 6 ) to generate tumors. Animals were randomly divided into groups and when the average tumor volume reached approximately 100 to 200 mm 3 Administration of the test substances (ADC-2, ADC-1) and the number of animals in each group were based on the experimental design.

[0560] After inoculation, animals were checked daily for morbidity and mortality. During routine monitoring, animals were checked for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption, weight gain / loss (weight was measured twice a week), eye / hair matting, and any other abnormal effects. The primary endpoint was to observe whether tumor growth could be delayed or whether mice could be cured.

[0561] Statistical analysis: For comparisons between two groups, an independent sample t-test was used. For comparisons between three or more groups, a one-way ANOVA was performed. If a significant F-statistic (ratio of treatment variance to error variance) was obtained, a multiple comparison procedure was applied after ANOVA. All data were analyzed using SPSS 17.0. p < 0.05 was considered statistically significant.

[0562] According to the results ( Figure 2 ), ADC-1 exhibited comparable or even better efficacy than ADC-2.

[0563] Effect Example 3: In vivo effects of ADC-2, ADC-3 and Enhertu in the JIMT-1CDX model

[0564] Each mouse was inoculated subcutaneously on the right flank with JIMT-1 tumor cells (5×10 6 ) to generate tumors. On the 10th day after tumor inoculation, when the average tumor volume reached 200 mm 3 On the 35th day after drug administration, the average tumor volume of the vehicle control group reached 1,474 mm 3 The T / C and TGI values ​​were calculated using the tumor volume. The calculation formula is as follows: T / C% = TRTV / C RTV ×100%(T RTV : RTV of treatment group; C RTV : RTV of the vehicle control group). Relative tumor volume (RTV) was calculated based on tumor measurement results, and the calculation formula is: RTV = V t / V0, where V0 is the mean tumor volume measured at the time of grouping (i.e., D0), V t is the average tumor volume at one measurement, and T RTV and C RTV The data of the same day were used. Calculation of TGI (%): TGI (%) = [1-(average tumor volume of the treatment group at the end of drug administration-average tumor volume of the treatment group at the beginning of drug administration) / (average tumor volume of the vehicle control group at the end of drug administration-average tumor volume of the vehicle control group at the beginning of drug administration)] × 100%. Treatment with 5 mg / kg ADC-2 (T / C = 6.23%, TGI = 108.47%, p = 0.011) and Enhertu (T / C = 33.51%, TGI = 76.90%, p = 0.041) produced significant antitumor activity, and the average tumor volume was 92 mm 3 and 494mm 3 Treatment with 5 mg / kg ADC-3 (T / C = 43.16%, TGI = 63.73%, p = 0.089) also produced some antitumor activity, with the average tumor volume on day 35 being 636 mm 3 .

[0565] According to the results ( Figure 3 ) showed that there was no statistically significant difference in the efficacy of ADC-3 and Enhertu. And these data also showed that ADC-2 showed better anti-tumor efficacy than Enhertu in the JIMT-1 model.

[0566] According to effect example 2 ( Figure 2 ) and Effect Example 3( Figure 3 ), the conjugate ADC-1 exhibited efficacy comparable to or even better than ADC-2, and further better than Enhertu.

[0567] Effect Example 4: In vivo effect of the conjugate in the NCI-N87 CDX model

[0568] NCI-N87 tumor cells (ATCC, Manassas, VA, catalog number CRL-5822) were maintained in vitro as monolayer cultures in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% CO2. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment.

[0569] BALB / c female nude mice, 6 to 8 weeks old, weighing about 18 to 20 g. A total of 25 mice (18 plus 39% spare) were required for the study, all purchased from Shanghai Lingchang Biotechnology Co., Ltd. or other certified suppliers.

[0570] Each mouse was inoculated subcutaneously on the right flank with NCI-N87 tumor cells (10 × 10 6 ) to generate tumors. Animals were randomly divided into groups and when the average tumor volume reached approximately 150 to 200 mm 3 Administration of the test substances (ADC-2, ADC-1) and the number of animals in each group were based on the experimental design.

[0571] After inoculation, animals were checked daily for morbidity and mortality. During routine monitoring, animals were checked for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption, weight gain / loss (weight was measured twice a week), eye / hair matting, and any other abnormal effects. The primary endpoint was to observe whether tumor growth could be delayed or whether mice could be cured.

[0572] According to the results ( Figure 4 ), conjugate ADC-1 exhibited efficacy comparable to that of ADC-2.

[0573] Effect Example 5: Effect of ADC-5 on the proliferation of TROP2-positive tumor cells BxPC-3

[0574] 1) TROP2-positive human pancreatic cancer cells BxPC-3 were seeded into 96-well cell plates at 100 μl per well (containing 1,000 to 5,000 cells) and cultured overnight in a cell incubator at 37° C., 5% CO 2 , 95% air and 100% humidity.

[0575] 2) Add different concentrations (200, 40, 8, 1.6, 0.32, 0.064, 0.013, 0.0026, 0.00051 and 0.00010 nM) of ADC to BxPC-3 cells cultured overnight. Add 5 μM final concentration of puromycin to the control group. Continue to incubate at 37°C for 120 hours.

[0576] 3) Remove the cell plate from the 37°C cell incubator and equilibrate to room temperature for about 30 minutes. Add 100 μl of CellTiterGlo reagent to each well. After shaking for 2 minutes, place the shaker in the dark at room temperature for 10 minutes and measure the relative light units (RLU) by the Cytation3 microplate analyzer.

[0577] 4) The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 3 and Figure 5 ADCs have a significant inhibitory effect on TROP2-positive cells, among which ADC-5 and ADC-6 have basically the same efficacy, and are significantly better than ADC-7.

[0578] Table 3 Inhibitory effects of different drugs on tumor cell proliferation (IC 50 , nM)

[0579] ADC-5 ADC-6 ADC-7 <![CDATA[IC 50 ,nM]]> 0.2491 0.2543 0.6019

[0580] Effect Example 6: Detection of ADC-5 endocytosis by TROP2-positive tumor cells

[0581] 1) TROP2-positive human gastric cancer cells NCI-N87 and human breast cancer cells MDA-MB-468 were seeded into 96-well cell plates at 100 μl per well (containing 10,000 to 50,000 cells) and incubated with 50 μg / mL fluorescently labeled ADC at 4°C in the dark for 30 minutes.

[0582] 2) After incubation, unbound ADC was washed with PBS, and the cells were incubated in a cell incubator at 37°C, 5% CO2, 95% air and 100% humidity for 10 minutes, 30 minutes, 1 hour, 1.5 hours, 2.5 hours and 3.5 hours, respectively, and then the cells were removed and pre-cooled PBS was added to terminate endocytosis. The cells were ice-bathed for 3 minutes using an acidic buffer with a pH value of 2.5, and the cells were pelleted by centrifugation at 2000 rpm for 3 minutes, and then resuspended using 100 μl FACS buffer per well to prepare for flow cytometry detection.

[0583] 3) The results of endocytosis of the tested ADCs by TROP2-positive tumor cells are shown in Tables 4 and Figure 6 The endocytosis of ADCADC-5 was substantially the same as that of ADC-6 and monoclonal antibody Ab2. Under the same conditions, the negative control human IgG1 did not induce endocytosis of cells, indicating that the preparation of ADC by conjugating the monoclonal antibody to the load does not affect the intracellular endocytosis induced by the monoclonal antibody, and the endocytosis of ADC is target-dependent.

[0584] Table 4 Endocytosis of ADC by tumor cells

[0585]

[0586] Effect Example 7: Effect of ADC-5 on Tumor Growth in the TROP2-Positive NCI-N87 CDX Mouse Model

[0587] 1) Cell culture: NCI-N87 tumor cells (ATCC, catalog number CRL-5822) were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% CO2. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in the exponential growth phase were collected and counted for tumor inoculation.

[0588] 2) Animals: BALB / c female nude mice, 7 to 9 weeks old, weighing about 18 to 22 g. A total of 24 mice were required for the study, and these mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0589] 3) Tumor inoculation and drug administration: Each mouse was subcutaneously inoculated with 0.2 mL (10×10 6 ) NCI-N87 tumor cells (PBS to Matrigel ratio was 1:1). When the average tumor volume reached approximately 150 to 200 mm 3 Animals were randomly divided into groups of 6 mice per group according to tumor size and treated with 3 mg / kg intravenously. The entire process of animal breeding and experiments complied with the requirements of animal welfare.

[0590] 4) Effect evaluation: After drug administration, tumor volume and animal body weight were measured weekly and the results were analyzed according to the formula V = 0.5 × a × b 2 The tumor volume was calculated, where a and b are the long diameter and short diameter of the tumor, respectively. Then, the tumor volume was used to calculate the TGI (%) and the relative tumor proliferation rate T / C (%). The TGI calculation formula for each group is as follows: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i is the average tumor volume of the treatment group on a certain day, T0 is the average tumor volume of the treatment group on the first day of treatment, V i The vehicle control group was compared with T i The average tumor volume on the same day, and V0 is the average tumor volume of the vehicle control group on the first day of treatment. The calculation formula of T / C (%) value is as follows: T / C% = T RTV / C RTV ×100%(TRTV : Relative mean tumor volume (RTV) of the treatment group; C RTV :The vehicle control group was compared with T RTV The relative mean tumor volume (RTV) of each group was calculated as follows: RTV = V t / V0, where V0 is the tumor volume on the first day of treatment, and V t is the tumor volume on a certain day.

[0591] 5) Statistical analysis: For comparison between two groups, an independent sample T test was used. For comparison between three or more groups, a one-way ANOVA was performed. If a significant F-statistic (ratio of treatment variance to error variance) was obtained, a multiple comparison procedure was applied after the ANOVA. All data were analyzed using SPSS 17.0. P < 0.05 was considered to be statistically significant.

[0592] 6) The changes in tumor volume and animal body weight are detailed in Tables 5 and Figure 7 ADC-5 and ADC-6 had comparable inhibitory effects on NCI-N87 tumor volume, and all of the above groups were extremely significant compared to the control group. No significant weight loss was observed in any of the groups administered with ADC, indicating that mice had good tolerance to ADC-5 and ADC-6.

[0593] Table 5 Statistical analysis of experimental results

[0594]

[0595] Notes:

[0596] 1. Mean ± SEM;

[0597] 2. The calculation formula of T / C (%) is as follows: T / C% = T RTV / C RTV × 100%. The relative tumor volume (RTV) of each group was calculated as follows: RTV = V t / V0: V0 is the average tumor volume on the first day of treatment, and V t is the average tumor volume on a particular day;

[0598] 3. The TGI calculation formula for each group is as follows: TGI (%) = [1-(T 42 -T0) / (V 42 -V0)]×100;

[0599] 4. P value is calculated based on tumor volume.

[0600] Effect Example 8: Effect of ADC-5 on Tumor Growth in TROP2-Positive FaDu CDX Mouse Model

[0601] The experiment was carried out according to the method described in Example 7, wherein the administration concentration was set to 2 mg / kg. The changes in tumor volume and animal body weight are shown in Tables 6 and Figure 8 The inhibitory effect of the ADC-5 test group (CR: 6 mice) on FaDu tumor volume was slightly better than that of ADC-6 (CR: 2 mice), and both groups had significant differences compared with the control group. No significant weight loss was observed in the mice in the groups administered with ADC, indicating that the mice had good tolerance to ADC-5 and ADC-6.

[0602] Table 6 Statistical analysis of experimental results

[0603]

[0604] Notes:

[0605] 1. Mean ± SEM;

[0606] 2. The calculation formula of T / C (%) is as follows: T / C% = T RTV / C RTV × 100%. The relative tumor volume (RTV) of each group was calculated as follows: RTV = V t / V0: V0 is the average tumor volume on the first day of treatment, and V t is the average tumor volume on a particular day;

[0607] 3. The TGI calculation formula for each group is as follows: TGI (%) = [1-(T 31 -T0) / (V 31 -V0)]×100;

[0608] 4. P value is calculated based on tumor volume.

[0609] Effect Example 9: Effect of ADC-8 on Tumor Growth in FGFR3-Positive RT112 / 84CDX Mouse Model

[0610] The experiment was carried out according to the method described in Example 7, wherein the administration concentration was set to 5 mg / kg. The changes in tumor volume and animal body weight are shown in Tables 7 and Fig. 9 The inhibitory effects of ADC-8 and ADC-9 on RT112 / 84 tumor volume were comparable and significantly superior to those of ADC-10, and all of the above groups were extremely significant compared to the control group. There was no difference in body weight between each group administered with ADC and the control group, indicating that mice had good tolerance to ADC-8, ADC-9 and ADC-10.

[0611] Table 7 Statistical analysis of experimental results

[0612]

[0613] Notes:

[0614] 1. Mean ± SEM;

[0615] 2. The calculation formula of T / C (%) is as follows: T / C% = T RTV / C RTV × 100%. The relative tumor volume (RTV) of each group was calculated as follows: RTV = V t / V0: V0 is the average tumor volume on the first day of treatment, and V t is the average tumor volume on a particular day;

[0616] 3. The TGI calculation formula for each group is as follows: TGI (%) = [1-(T 27 -T0) / (V 27 -V0)]×100;

[0617] 4. P value is calculated based on tumor volume.

[0618] Effect Example 10: Effect of ADC-8 on Tumor Growth in FGFR3-Positive RT4 CDX Mouse Model

[0619] The experiment was carried out according to the method described in Example 7, wherein the administration concentration was set to 5 mg / kg. The changes in tumor volume and animal body weight are shown in Tables 8 and Fig.10 ADC-8, ADC-9 and ADC-10 all had a certain inhibitory effect on RT4 tumor volume, and ADC-8 and ADC-10 were slightly superior to ADC-9. There was no difference in body weight between each group administered with ADC and the control group, indicating that mice had good tolerance to ADC-8, ADC-9 and ADC-10.

[0620] Table 8 Statistical analysis of experimental results

[0621]

[0622] Notes:

[0623] 1. Mean ± SEM;

[0624] 2. The calculation formula of T / C (%) is as follows: T / C% = T RTV / C RTV × 100%. The relative tumor volume (RTV) of each group was calculated as follows: RTV = V t / V0: V0 is the average tumor volume on the first day of treatment, and V tis the average tumor volume on a particular day;

[0625] 3. The TGI calculation formula for each group is as follows: TGI (%) = [1-(T 40 -T0) / (V 40 -V0)]×100;

[0626] 4. P value is calculated based on tumor volume. Sequence Listing

[0627] SEQ ID No: 1: Ab0001-LCCT L -HC light chain:

[0628] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG

[0629] SEQ ID No:2:Ab0001-LCCT L -HC Heavy Chain:

[0630] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0631] SEQ ID No:3:Ab0001-LCCT L -HCCT L Light chain:

[0632] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG

[0633] SEQ ID No:4:Ab0001-LCCT L -HCCT L Heavy chain:

[0634] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG

[0635] SEQ ID No:5: Ab2 light chain:

[0636] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG

[0637] SEQ ID No:6: Ab2 heavy chain:

[0638] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0639] SEQ ID No:7: Ab3 light chain:

[0640] QSVLTQPPSLSVAPGKTATFTCGGNNIGDKSVHWYRQKPGQAPVLVMYLDTERPSGIPERMSGSNFGNTATLTITRVEAEDEADYYCQVWDSGSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECSGALPETGG

[0641] SEQ ID No:8: Ab3 heavy chain:

[0642] EVQLVQSGAEVKKPGASVKVSCKASGYMFTSYGISWVRQAPGQGLEWMGWVSTYNGDTNYAQKFQGRVTVTTDTSTSTAYMELRSLRSEDTAVYYCARVLGYYDSIDGYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A compound of formula (I): in, W is hydrogen, LKb or -C2H4-(PEG) t -(CO)NH2; Y is LKa-LKb; Each LKa is independently Each LKb is independently L 2 ―L 1 ―B; Each B is independently a terminal group R 10 , or a combination of the following: 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 is hydrogen, or a group that can leave when reacting with a group in the supported substance; Each L 1 independently a cleavable sequence 1 comprising an amino acid sequence capable of being cleaved by an enzyme, and the cleavable sequence 1 comprises 1 to 10 amino acids; Each L 2 are independently chemical bonds; or C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、 C 4-10 Cycloalkylene, C 4-10 wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ; Ld2 and each Ld1 are independently a chemical bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-, or a natural amino acid, or an oligomeric natural amino acid having a degree of polymerization of 2 to 10, said natural amino acid or oligomeric natural amino acid being independently unsubstituted or having a side chain substituted with -(CO)-(PEG) j -OR 11 replace; -(PEG) t -、-(PEG) i -and-(PEG) j - each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units and having an optional additional C 1-10 Alkylene; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 Cycloalkylene; or R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group; or R 3 and R 4 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group; R 11 It is C 1-10 alkyl; m is any integer from 1 to 5; n is any integer from 2 to 20; d is any integer from 0 to 6, in particular 1, 2, 3; Each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12, more preferably an integer from 0 to 8, especially 4; Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12; Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12.

2. The compound according to claim 1, which is selected from 3. A compound according to any one of claims 1 to 2, selected from in, m is any integer from 1 to 5; Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12; Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12.

4. The compound according to any one of claims 1 to 2, wherein Ld2 and each Ld1 are independently a chemical bond or Each k is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each k is independently an integer from 1 to 12, more preferably an integer from 1 to 7, especially 1 or 3 or 5; Preferably, Ld1 is Ld2 is independently selected from a chemical bond or Each i is independently an integer from 0 to 5, preferably an integer from 0 to 4, in particular 0, 2 or 4; Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12.

5. The compound according to any one of claims 1 to 4, wherein Cleavable sequence 1 is selected from Gly-Gly-Phe-Gly, Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Ala-Ala-Ala and combinations thereof; preferably, cleavable sequence 1 is Gly-Gly-Phe-Gly or Val-Cit; and / or Sp1 is selected from PABC, acetal, heteroacetal and a combination thereof; preferably, Sp1 is acetal, heteroacetal or PABC; preferably, the heteroacetal is selected from N,O-heteroacetal; preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-; wherein the -O- or the -NH- is connected to the cleavable sequence 1, and U is not present, or U is O, S or NH, preferably O or S; more preferably, Sp1 is PABC.

6. A compound according to any one of claims 1 to 5, wherein W is hydrogen; and / or R 10 is hydrogen, hydroxyl or R 11 It is C 1-6 Alkyl, preferably methyl; and / or m is an integer from 1 to 3, preferably 1 or 2; n is an integer from 2 to 5, in particular 3; and / or d is 0, or any integer from 1 to 4; preferably 1, 2 or 3; more preferably 1.

7. A compound according to any one of claims 1 to 6, selected from Where R 1 , R 2 and R 10 As defined in claim 1, and g is an integer from 1 to 10, preferably 1.

8. A compound having the structure of formula (II) in Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P; M is LKa-LKb―P; P is a load, which is connected to the B part or L in the compound of formula (I) according to claim 1 1 part; n, d, Ld1, Ld2, t, LKa and LKb are as defined in claim 1; Preferably, M is LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or is a combination of 1) and 2): 1) a self-removing spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 Heterocyclylene and -(CO)-; Preferably, Sp1 is selected from PABC, acetal, heteroacetal and a combination thereof; more preferably, Sp1 is acetal, heteroacetal or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-; wherein the -O- or the -NH- is connected to the cleavable sequence 1, and U is not present, or U is O, S or NH, preferably O or S.

9. A conjugate having the structure of formula (III): in, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P; n, d, t, Ld1 and Ld2 are as defined in claim 1; M is LKa-LKb―P; Preferably, M is LKa-L 2 ―L 1 ―B―P; in Each B is independently absent, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; preferably, B is -NH-CH2-U-, or is absent, or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, g is an integer from 1 to 10, preferably 1; wherein R 1 and R 2 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 Cycloalkylene; or R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group; P is a load, which is connected to the B part or L in the compound of formula (I) according to claim 1 1 part; A is the targeting molecule; z is an integer of 1-20.

10. The conjugate according to claim 9, wherein The conjugate has the structure of the following formula (III-1):

11. The conjugate according to claim 9 or 10, wherein The conjugate has the following structure: Preferably, z is 1 to 4; preferably 2; n is an integer from 2 to 5, preferably, n is 3, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2 to 4, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-U-, or is absent, or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, U is absent, or U is O, g is 1; wherein R 1 and R 2 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 Cycloalkylene; or R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group; Each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12, more preferably an integer from 0 to 8, especially 4; Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12; Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, especially 8 or 12; m is any integer from 1 to 3; in particular 1 or 2.

12. The conjugate according to any one of claims 9 to 11, wherein The targeting molecule is an antibody or an antigen-binding fragment thereof; the antibody or antigen-binding fragment is preferably modified to bind to G in the compound of formula (I). n Partial connection; Preferably, A is an anti-human monoclonal antibody, which is linked to the rest of the conjugate via a modified heavy chain and / or light chain C-terminus, wherein the modified heavy chain and / or light chain C-terminus is modified to contain Leu-Pro-Xaa-Thr, wherein Xaa is any natural or unnatural single amino acid, and z is 2; Preferably, the antibody is an anti-human HER2 antibody, an anti-human TROP2 antibody or an anti-FGFR3 antibody.

13. The conjugate according to any one of claims 9 to 12, wherein The cargo is a cytotoxin or a fragment thereof, which is optionally derivatized so as to be attached to the B part or L part of the compound of formula (I). 1 part; Preferably, the cytotoxin is selected from the group consisting of: a taxane; Maytansines; Auristatins; Epothilones; Compretin A-4 phosphate, Compretin A-4 and its derivatives; Indole-sulfonamides; Vinca alkaloids, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vindesine, anhydrous vinblastine; Dolastatin 10 and analogs; Halichondrin B; Eribulin; Indole-3-oxoacetamide; Podophyllotoxins; 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC); Dictyostelium; Lelymycin; Camptothecin and its derivatives; Mitoxantrone; Mitoguanidine; Nitrogen mustard; Nitrosoureas; Aziridine; Benzodopa; Carboquinone; Metutepa; Uredepa; Danetomycin; Esperamycin; Neostatin; Aclarubicin; Actinomycin; Anthramycin; Bleomycin; Actinomycin C; Carrubicin; Carminomycin; Anticancer Mycin; Carminomycin; Actinomycin D; Daunorubicin; Detorubicin; Adriamycin; Epirubicin; Esorubicin; Idarubicin; Mexiromycin; Mitomycin; Noramycin;Olivomycin; Peplomycin; Porfiromycin; Puromycin; Ferroadriamycin; Rhodorubicin; Streptomelanocin; streptozotocin; linastatin; levorubicin; trichothecenes; T-2 toxin; mucin A; baculosporin A; serpentine; ubenimex; azaserine; 6-diazo-5-oxo-L-norleucine; dimethylfolate; methotrexate; pteropterin; trimetrexate; edatrexate; fludarabine; 6-mercaptopurine; thiopurine; thioguanine; ancitabine; gemcitabine; enocitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine; floxuridine; dimethyltestosterone; drostanolone propionate; cyclothiosteroid; melastane; testolactone; aminoglutethimide; mitotane; trilostane; flutamide; nilutamide; bicalutamide; leuprolide acetate; protein kinase inhibitors; and proteasome inhibitors; and / or Selected from vinca alkaloids, colchicine, taxanes, auristatins, maytansines, calicheamicin, doxorubicin, duocarmycin, SN-38, Nostoc cyclopeptide analogs, derutecan, duocarbamazine, calicheamicin, cintamycin, dolatamcin, pyrrolobenzodiazepine, exatecan and derivatives thereof; and / or Selected from auristatins, in particular MMAE, MMAF or MMAD; and / or Selected from exatecan and its derivatives, such as DX8951f; and / or Selected from DXd-(1) and DXd-(2); preferably DXd-(1).

14. The conjugate according to any one of claims 9 to 13, wherein the loading substance is selected from Specially selected 15. The conjugate according to any one of claims 9 to 14, wherein formula (III) has the following structure: Each z is independently an integer from 1 to 20.

16. A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of the conjugate according to any one of claims 9 to 15 and at least one pharmaceutically acceptable carrier.

17. Use of the conjugate according to any one of claims 9 to 15 or the pharmaceutical composition according to claim 16 in the manufacture of a medicament for treating a disease, wherein the disease is a tumor or an autoimmune disease; preferably a HER2-positive tumor, a TROP2-positive tumor or a FGFR3-positive tumor; Preferably, The HER2-positive tumor is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer and urothelial cancer; The TROP2-positive tumor is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer and urothelial cancer; The FGFR3-positive tumor is selected from brain cancer, bladder cancer, urothelial carcinoma, cervical cancer, multiple myeloma or intrahepatic bile duct carcinoma.

18. A compound of formula (IV), in, Rpg is selected from hydrogen, or a protecting group, preferably selected from acetyl, trifluoroacetyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc); W, Ld1, Ld2, n and d are as defined in claim 1.

19. The compound according to claim 18, wherein the formula (IV) has the following structure:

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