Linker, conjugate and uses thereof

Abstract

The present disclosure relates to the field of biopharmaceuticals, and in particular, to linkers for preparing targeted molecule-drug conjugates, corresponding conjugates, and their preparation processes and uses.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the priority of International Application PCT / CN2022 / 106025 filed on July 15, 2022.

[0002] The present disclosure relates to the field of biopharmaceuticals, and in particular, to linkers for preparing targeted molecule - drug conjugates, corresponding conjugates, and their preparation processes and uses.

Background Art

[0003] HER2 has been found to be overexpressed in several types of cancers such as breast cancer and gastric cancer, and has been proven to be a promising target for cancer treatment. Multiple HER2 - targeted therapies have been approved, including HER2 tyrosine kinase inhibitors (lapatinib, tucatinib), therapeutic HER2 antibodies (Herceptin, Pertuzumab), and HER2 - targeted ADCs (Kadcyla, Enhertu). These therapeutic agents have significantly improved the survival rate of patients with HER2 - positive breast cancer and gastric cancer. In particular, Enhertu has shown high efficacy not only in patients with high HER2 but also in patients with medium / low HER2, and there is a prospect that more HER2 - expressing cancer patients will benefit. Despite its high efficacy, Enhertu induces more than 10% of interstitial lung disease, so its use in some patients is restricted.

[0004] Enhertu, like other commercially available ADCs and most ADCs in clinical trials, is prepared by chemical conjugation that conjugates small - molecule drugs to target antibodies or proteins through a thiol - succinimide structure (thiol - succinimide linkage). The thiol - succinimide structure is formed by the reaction of a thiol group and maleimide. However, the thiol - succinimide linkage is not stable. In vivo, the detachment of cytotoxins from ADCs and off - target toxicity occur due to retro - Michael addition or exchange with other thiol groups, resulting in reduced safety and restricted clinical application. ​

[0005] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. It is an intracellular calcium signal transducer and is overexpressed in various tumors. IMMU-132 (also known as SG) and DS-1062 are clinically well-known anti-trop2 ADCs. SG has shown excellent anti-tumor effects in clinical trials, but has the same side effects as the toxic payload (SN-38), such as myelosuppression and gastrointestinal toxicity. For DS1062, in the phase I clinical trial, 48% of patients had adverse events above grade 3, and 8% of patients had interstitial lung disease. The recently published phase III clinical data of DS-1062 in the treatment of advanced non-small cell lung cancer indicate that DS-1062 is not excellent in terms of safety. In this clinical trial, several grade 5 adverse events were observed. Since the linkers of both DS1062 and IMMU-132 use maleimide groups that are unstable in the blood, their potential toxin excretion can be one of the causes of the above low safety.

[0006] The protein encoded by the FGFR3 gene is a member of the fibroblast growth factor receptor (FGFR) family, can bind to acidic and basic fibroblast growth factors (FGF), and plays an important role in bone development and maintenance. It may be an important target for ADCs.

[0007] The inventors have developed a novel branched linker that does not contain a maleimide group and conjugates to a specific site of the antibody via the terminal amino acid to avoid potential instability in the blood. On the other hand, the branched linker has strong binding ability, can adapt to various payloads, is convenient as a linker-payload fragment and environmentally friendly and can be commercialized. The formed linker-payload structure is easy to separate and purify and is useful for late-stage CMC development. In addition, by conjugating different types of linkers to different antibodies, the stability of the linker, compatibility, and excellent biological activity of the ADC formed by the linker are verified.

[0008] Some conjugates are also provided that have higher molecular stability and antitumor efficacy compared to benchmarks, such as Enhertu. In addition, these conjugates can have excellent physicochemical properties, excellent pharmacokinetic properties, and high safety. Furthermore, due to the advanced modular design, they are easy to conjugate with multiple drugs.

Summary of the Invention

[0009] In a first aspect, a compound of formula (I),

Chemical formula

Chemical formula

[0010] Each LKb is independently L 2 ―L 1 ―B, Each B is independently a terminal group R 10 Or 1) a self-destructive spacer Sp1, 2) a bond, or -CR 1 R 2 -, C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 Heterocyclylene and -(CO)-, one or a combination of two or more of the selected divalent groups, and 3) a combination with the terminal group R 10 And, R 10 Is hydrogen or a group that can be detached when reacting with a group in the payload, Each L 1 Is independently a cleavable sequence 1 containing an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 contains 1 to 10 amino acids, Each L 2is independently a bond, or one or more -CH2- structures in the alkylene are optionally -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -,

Chemical formula

[0011] In a second aspect, a compound having the structure of formula (II), wherein

Chemical formula

[0012] In a third aspect, a conjugate having the structure of formula (III),

Chemical formula

[0013] In a fourth aspect, an intermediate compound having the structure of formula (IV) for the preparation of formula (I),

Chemical formula

[0014] In a fifth aspect, use of the conjugate of the present disclosure 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, providing the use.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

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Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0016] In the following, specific embodiments will be shown to explain the technical content of the present disclosure. A person skilled in the art can easily understand other advantages and effects of the present disclosure from the content disclosed in this specification. The present disclosure may be implemented or applied by other different specific embodiments. A person skilled in the art can make various modifications and variations without departing from the spirit of the present disclosure.

[0017] Definition Unless otherwise defined herein, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art. The technologies used in this specification refer to technologies generally understood in the relevant field and include obvious modifications and equivalent substitutions for a person skilled in the art. Although the following terms are considered to be well understood by a person skilled in the art, their definitions are described below to better explain the present disclosure. The product names described in this specification refer to the corresponding products or their active ingredients. All patents, published patent applications, and publications cited in this specification are incorporated herein by reference.

[0018] It should be understood that when a specific quantity, concentration, or other value or parameter is described in the form of a range, a preferred range, or a preferred upper limit or a preferred lower limit, any range combined with any upper limit or preferred value and any lower limit or preferred value is equivalent to being specifically disclosed, regardless of whether it is explicitly stated. Unless otherwise specified, the numerical ranges listed in this specification are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the expression "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 expressions such as d, m, n, j, and k should be understood in the same way.

[0019] Unless the context indicates otherwise, singular forms such as "a" and "the" include plural forms. The expressions "one or more" or "at least one" may represent 1, 2, 3, 4, 5, 6, 7, 8, and 9 or more.

[0020] When the terms "about" and "approximately" are used in connection with a numerical variable, they generally mean that the value of the variable and all values of the variable are within the range of experimental error (e.g., within the 95% confidence interval of the mean value), or within ±10% of the specified value, or within a wider range.

[0021] The term "optional" or "optionally" means that the event described thereafter may occur, but does not necessarily occur, and the expression includes the case where the event or situation occurs or does not occur.

[0022] Expressions such as "comprising", "including", "containing", and "having" are open-ended and do not exclude additional unenumerated elements, steps, or components. The expression "consisting of" excludes any element, step, or component not expressly indicated. The expression "consisting essentially of" means limiting the scope to the specified element, step, or component, and any optionally present elements, steps, or components that do not substantially affect the important and novel features of the claimed subject matter. The expression "comprising" should be understood to include the expressions "consisting essentially of" and "consisting of".

[0023] The term "targeting molecule" means a molecule having an affinity for a specific target (e.g., receptor, cell surface protein, cytokine, tumor-specific antigen, etc.). The targeting molecule can deliver the payload to a specific site in the body by target delivery. The targeting molecule 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 targeting a receptor can deliver a cytotoxic agent to a site containing a large amount of the receptor. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins of a given antigen, antibody mimetics, scaffold proteins having an affinity for a given target, ligands, etc.

[0024] As used herein, the term "antibody" is used in a broad sense and includes intact 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 be of any subtype (such as IgG, IgE, IgM, IgD, and IgA, etc.) or subclass, and may be derived from any suitable species. In some embodiments, the antibody is of human or mouse origin. The antibody may be a fully human antibody, a humanized antibody, or a chimeric antibody prepared by recombinant methods. In some embodiments, the antibody can be engineered, for example, by introduction of a ligase-specific recognition sequence at the terminus.

[0025] Monoclonal antibody is used herein to refer to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for a few possible natural variations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" refers to the fact that the properties of the antibody are derived from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method for generating the antibody.

[0026] An intact antibody or full-length antibody essentially comprises an antigen-binding variable region, a light chain constant region (CL), and a heavy chain constant region (CH), and the heavy chain constant region (CH) may include CH1, CH2, CH3, and CH4 depending on the antibody subtype. The antigen-binding variable region (also known as the fragment variable region, Fv fragment) typically includes a light chain variable region (VL) and a heavy chain variable region (VH). The constant region may be a constant region having a natural sequence (such as a constant region having a human natural sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region may be recognized by and interact with the immune system.

[0027] An antibody fragment may comprise a part of an intact antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fd fragment consisting of VH and CH1 domains, Fv fragment, single-domain antibody (dAb) fragment, and isolated complementarity-determining region (CDR). A Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin or a fragment having the same structure as that generated, for example, by recombinant expression or the like. A Fab fragment contains a light chain (including VL and CL) and another chain, and the other chain contains a variable domain of a heavy chain (VH) and a constant region domain of a heavy chain (CH1). An F(ab’)2 fragment is an antibody fragment obtained by pepsin digestion of an immunoglobulin at pH 4.0 - 4.5 or a fragment having the same structure as that generated, for example, by recombinant expression or the like. An F(ab’)2 fragment essentially contains two Fab fragments, and each heavy chain portion contains some additional amino acids including cysteine that forms a disulfide bond connecting the two fragments. A Fab’ fragment is a fragment containing half of an F(ab’)2 fragment (one heavy chain and one light chain). An antibody fragment may contain a plurality of chains linked via, for example, disulfide bonds and / or peptide linkers. Examples of antibody fragments include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd’ fragments, and other fragments including modified fragments. An antibody fragment typically contains at least or about 50 amino acids and typically at least or about 200 amino acids. An antigen-binding fragment may include any antibody fragment that, when inserted into an antibody framework (e.g., by substitution of a corresponding region), can give rise to an antibody that binds immunospecifically to an antigen.

[0028] The antibodies according to the present disclosure can be prepared using techniques known in the art such as, for example, recombinant techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art. For example, a genetically engineered recombinant antibody (or antibody mimetic) can be expressed in a suitable culture system (e.g., E. coli or mammalian cells). The manipulation may refer to, for example, introducing a ligase-specific recognition sequence at the end.

[0029] HER2 refers to human epidermal growth factor receptor-2 and 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.

[0030] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. It is an intracellular calcium signal transducer and is overexpressed in various tumors.

[0031] The protein encoded by the FGFR3 gene is a member of the fibroblast growth factor receptor (FGFR) family, can bind to acidic and basic fibroblast growth factors (FGF), and plays an important role in bone development and maintenance.

[0032] As used herein, the term "targeted molecule-drug conjugate" is referred to as "conjugate". Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0033] A small molecule compound refers to a molecule of a size comparable to that of organic molecules commonly used in pharmaceuticals. The term does not include biopolymers (e.g., proteins, nucleic acids, etc.), but includes low molecular weight peptides such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. or their derivatives. Typically, the molecular weight of the small molecule compound may 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.

[0034] A cytotoxic agent refers to a substance that inhibits or blocks the expression activity and cell functions of cells and / or causes cell destruction. The cytotoxic agents commonly used in current ADCs are more toxic than chemotherapeutic drugs. Examples of cytotoxic agents include, but are not limited to, drugs that target target sites such as the microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and apoptosis regulation. Drugs that target the microtubule cytoskeleton can be, for example, microtubule stabilizers or microtubule protein polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of microtubule protein polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastines, colchicines, and dolastatin. DNA-targeting drugs can be, for example, drugs that directly disrupt the DNA structure or topoisomerase inhibitors. Examples of drugs that directly disrupt the DNA structure include, but are not limited to, DNA double-strand breakers, DNA alkylating agents, and DNA intercalators. DNA double-strand breakers can be, for example, enediyne antibiotics, including, but not limited to, dynemicin, esperamicin, neocarzinostatin, uncialamycin, etc. DNA alkylating agents can be, for example, DNA bis-alkylating agents (i.e., DNA cross-linking agents) or DNA mono-alkylating agents. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, 1-(chloromethyl)-2,3-dihydrogen-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, dihydroindolobenzodiazepine (IGN) dimers, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1), and DXd-(2), the structures of which are shown below), camptothecins, and anthracyclines. Drugs that target RNA can be, for example, drugs that inhibit splicing, examples of which 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 protein (KSP) inhibitors.

[0035] A spacer is a structure that is located between different structural modules and can spatially separate the structural modules. The definition of a spacer is not limited by whether it has a specific function or can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures. Among them, the non-amino acid structures can be, but are not limited to, amino acid derivatives or analogs. The "spacer sequence" refers to an amino acid sequence that acts as a spacer, and examples thereof include a single amino acid, a sequence containing a plurality of amino acids, such as a sequence containing two amino acids such as GA, or sequences containing, but not limited to, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. A self-destructing spacer is a covalent assembly that is adjusted to cleave two chemical bonds correlatively after activation of the protected part of the precursor. Upon stimulation, the protected part (e.g., a cleavable sequence) is removed, triggering a cascade of degradation reactions, and as a result, smaller molecules are sequentially released. Examples of self-destructing spacers include, but are not limited to, PABC (p-benzyloxycarbonylaniline), acetals, heteroacetals, and combinations thereof.

[0036] The term "alkyl group" means a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, and the saturated aliphatic hydrocarbon group is linked to the rest of the molecule by a single bond. The alkyl group may contain 1 to 20 carbon atoms, i.e., C1-C 20It can be an alkyl group, for example, a C1-C4 alkyl group, a C1-C3 alkyl group, a C1-C2 alkyl group, a C3 alkyl group, a C4 alkyl group, or a C3-C6 alkyl group. Non-limiting examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-methylbutyl group, a 1-ethylpropyl group, a 1,2-dimethylpropyl group, a neopentyl group, a 1,1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 2,3-dimethylbutyl group, a 1,3-dimethylbutyl group, or a 1,2-dimethylbutyl group, or isomers thereof, but are not limited thereto. A divalent radical means a group obtained by removing one hydrogen atom from a carbon atom having a free valence electron of the corresponding monovalent radical. A divalent radical has two linking sites linked to the rest of the molecule. For example, an "alkylene group" or an "alkylidene group" refers to a straight-chain or branched-chain saturated divalent hydrocarbon group. Examples of the alkylene group include methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene group, ethylpropylene group, etc., but are not limited thereto.

[0037] As used herein, when one group is combined with another group, if a chemically stable structure is formed, the linkage of the groups may be linear or branched. The structure formed by such a combination can be linked to other parts of the molecule by any suitable atom in the structure, preferably by a specified chemical bond. For example, -CR 1 R 2 -, C 1-10 alkylene, C4-10 Cycloalkylene, C 4-10 When two or more divalent groups selected from cycloalkylene, C, heterocyclylene, and -(CO)- are combined to form a combination, the two or more divalent groups are, for example, -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., can form a linear linkage with each other. The resulting divalent structure can be further linked to other parts of the molecule.

[0038] The term "protecting group" or "Pg" refers to a substituent that can generally be employed to block or protect a specific functionality while not reacting with other functional groups on a compound. For example, an "amino protecting group" is a substituent attached to an amino group that blocks or protects the amino functionality in a compound. Suitable amino protecting groups include, but are not limited to, acetyl, trifluoroacetyl, t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and 9-fluorenylmethyloxycarbonyl (Fmoc). For a review of protecting groups and their use, see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0039] As used herein, the expressions "antibody-conjugated drug" and "antibody-drug conjugate" have the same meaning.

[0040] Compound of formula (I) In one aspect, a compound of formula (I),

Chem.

Chem.

[0041] each LKb is independently L 2 ―L 1 ―B, each B is independently a terminal group R 10 or a combination of 1) a self-destructive spacer Sp1, 2) one or more combinations of a divalent group selected from a bond, -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-, and 3) a terminal group R 10 and, R 10 is hydrogen or a group that can be cleaved when reacting with a group in the payload, each L 1 is independently a cleavable sequence 1 containing an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 contains 1 to 10 amino acids, each L 2 is independently a bond or one or more -CH2- structures in alkylene are optionally -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -,

Chem.

[0042] In one embodiment, LKa is

Chemical formula

[0043] In one embodiment, LKa is

Chemical formula

[0044] In one embodiment, LKa is

Chemical formula

[0045] In one embodiment, L 2 is -(C2H4-O) p-Selected from -(CH2)2(CO)-, p is an integer from 0 to 5, more preferably p is 0, 2 or 4, and most preferably p is 2.

[0046] In one embodiment, L 2 In each of the above structures of, the carbonyl group is linked to L 1 and the other linking sites are linked to the amide.

[0047] In one embodiment, Ld2 and each Ld1 are independently a bond or

Chemical formula

[0048] In one embodiment, Ld1 is

Chemical formula

[0049] In one embodiment, Ld2 is a bond.

[0050] In one embodiment, Ld2 is independently unsubstituted or a natural amino acid or natural amino acid oligomer having a degree of polymerization of 2 to 10 substituted with -(CO)-(PEG) j -OR 11 where -(PEG) -(PEG) j - is a PEG fragment having an optional additional C 1-10 alkylene at one end and containing a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units.

[0051] In one embodiment, each i is independently an integer from 0 to 20, and each j and k are independently integers from 1 to 20. In one embodiment, each i is independently from 0 to 12, and each j and k are independently integers from 1 to 12.

[0052] In one embodiment, each i is independently an integer from 0 to 8, particularly 4.

[0053] In one embodiment, each j is independently an integer from 8 to 12, particularly 8 or 12.

[0054] In one embodiment, each k is independently an integer from 1 to 7, particularly 1, or 3 or 5.

[0055] In one embodiment, Ld2 and each Ld1 are independently a bond, or a C alkylene having an amino and a carbonyl at each end, respectively, or a PEG fragment of a specific length having an amino and a carbonyl at each end (-(PEG)-), or one or more natural amino acids that are independently unsubstituted or substituted on the side chain with a PEG fragment of a specific length (-(PEG)-). 1-20 alkylene, or a PEG fragment of a specific length having an amino and a carbonyl at each end (-(PEG)-), or one or more natural amino acids that are independently unsubstituted or substituted on the side chain with a PEG fragment of a specific length (-(PEG)-). i -), or one or more natural amino acids that are independently unsubstituted or substituted on the side chain with a PEG fragment of a specific length (-(PEG)-). j -).

[0056] In one embodiment, -(PEG)- i - is -(O-C2H4)- i - or -(C2H4-O)- i - and includes optional additional C alkylene at one end, and -(PEG)- 1-10 - is -(O-C2H4)- j - or -(C2H4-O)- j - and includes optional additional C alkylene at one end. A very specific -(PEG)- j - includes -C2H4-(O-C2H4)- 1-10 - or -(C2H4-O)-C2H4-, and a very specific -(PEG)- i - includes -C2H4-(O-C2H4)- i - or -(C2H4-O)- i -C2H4-, and a very specific -(PEG)- j - includes -C2H4-(O-C2H4)- j - or -(C2H4-O)-j -C2H4- is included.

[0057] In one embodiment, Ld2 is a lysine substituted with -(CO)-(PEG) j -OR 11 on the side chain.

[0058] In certain embodiments, Ld2 is

Chemical formula

[0059] When there are two or more Ld1, B, L 2 or L 1 structures in the molecule, it is understood that the respective structures of Ld1, B, L 2 or L 1 are independently selected. When there are two or more R x (x is 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) in the molecule, each R x is independently selected. In some embodiments, "x" in the molecule has additional apostrophes (') or multiple apostrophes ('', ''', '''', etc.), for example, R, R 1’ , R 1’’ , R 1’’’ , R 2’ , R 2’’ , R 2’’’ , etc., or is shown without an apostrophe, where each R x is independently selected regardless of the presence or absence of additional apostrophes or multiple apostrophes. Other Rs such as R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , etc., and "Ld1", "B", "L x " and "L 2 " and "L 1 " should be understood similarly.

[0060] In one embodiment, the cleavable array 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, and preferably, the cleavable array 1 is Gly-Gly-Phe-Gly or Val-Cit.

[0061] In one embodiment, R 10 is hydrogen, hydroxy, or

Chemical formula

[0062] In one embodiment, R 11 is C 1-6 alkyl, preferably methyl.

[0063] In one embodiment, W is hydrogen.

[0064] In one embodiment, W is -C2H4-(PEG) t -(CO)NH2, where t is independently an integer from 1 to 100, preferably from 1 to 20, and preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12.

[0065] In a particular embodiment, t is 12.

[0066] In one embodiment, n is an integer from 2 to 5, particularly 3.

[0067] In one embodiment, d is any integer from 1 to 4, preferably 1.

[0068] In another aspect, an intermediate compound having the structure of formula (IV) for the preparation of formula (I),

Chemical formula

[0069] In one embodiment, Pg is 9-fluorenylmethyloxycarbonyl (Fmoc).

[0070] In one embodiment, Ld2 is

Chemical formula

[0071] In one embodiment, Ld1 is

Chemical formula

[0072] In one embodiment, W is hydrogen.

[0073] In one embodiment, n is 3.

[0074] In one embodiment, formula (IV) has the following structure.

Chemical formula

[0075] Portion containing a recognition sequence of a ligase acceptor or donor substrate In one embodiment, G of the compound of formula (I) n moiety is a recognition sequence of a ligase acceptor substrate and promotes the enzyme-catalyzed coupling of the compound of formula (I) and a targeting molecule under the catalysis of a ligase. The targeting molecule is optionally modified and contains the corresponding recognition sequence of the ligase acceptor substrate.

[0076] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of a natural transpeptidase, a non-natural transpeptidase, variants thereof, and combinations thereof. Non-natural transpeptidase enzymes can be obtained by manipulating natural transpeptidases, but are not limited thereto. In a preferred embodiment, the ligase is selected from the group consisting of a natural sortase, a non-natural sortase, and combinations thereof. Species of natural sortase include sortase A, sortase B, sortase C, sortase D, sortase L. plantarum, etc. (for a detailed description, see US20110321183A1, which is incorporated herein by reference). The type of ligase corresponds to the ligase recognition sequence and is used to achieve specific conjugation between different molecules or structural fragments.

[0077] In some embodiments, the ligase is a sortase selected from sortase A, sortase B, sortase C, sortase D, and sortase L. plantarum. In these embodiments, the recognition sequence of the ligase acceptor substrate is selected from the group consisting of oligomeric glycine, oligomeric alanine, and oligomeric glycine / alanine mixtures having a degree of polymerization of 3 to 10. In a particular embodiment, the recognition sequence of the ligase acceptor substrate is Gn, where G is glycine (Gly) and n is an integer from 2 to 10.

[0078] In another specific embodiment, the ligase is sortase A derived from Staphylococcus aureus. Accordingly, the ligase recognition sequence can be the typical recognition sequence LPXTG of the enzyme. 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, where X can be any natural or non-natural single amino acid, and J is either absent or an amino acid fragment containing 1 to 10 amino acids, optionally labeled. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment containing 1 to 10 amino acids, where each amino acid is independently any natural or non-natural amino acid. In another embodiment, J is G m where 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.

[0079] In one embodiment, the ligase is sortase B derived from Staphylococcus aureus, and the corresponding donor substrate recognition sequence can be NPQTN. In another embodiment, the ligase is sortase B derived from Bacillus anthracis, and the corresponding donor substrate recognition sequence can be NPKTG.

[0080] In yet another embodiment, the ligase is sortase A derived from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence can be LPXTGJ, where J is as defined above. In another embodiment, the ligase is sortase subfamily 5 derived from Streptomyces cellulare, and the corresponding donor substrate recognition sequence can be LAXTG.

[0081] In yet another embodiment, the ligase is sortase A derived from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence can be LPQTSEQ.

[0082] The ligase recognition sequence may be any completely new transpeptidase recognition sequence optimized by manual screening.

[0083] Portion containing a reactive group Reactive groups for linking to the payload In one embodiment, B is the terminal group R 10 and the cleavable sequence 1 in L 1 is linked to the payload. In this case, B is not present in the molecule obtained by linking the cleavable sequence 1 and the payload. In one embodiment, B is used for linking to the payload. For linking to the payload, the compound of formula (I) contains a reactive group. In one embodiment, B in the compound of formula (I) is linked to the payload via an amide bond or an ester bond or an ether bond. In one embodiment, the reactive groups in B in formula (I) are independently reactive groups for condensation reactions, nucleophilic additions or electrophilic additions (e.g., reactive C=O moieties, reactive C=C-C=O moieties, amino groups, amine groups, hydroxy groups or thiol groups), or reactive groups for substitution reactions (e.g., leaving groups attached to O, C, N or S atoms). In one embodiment, the reactive groups in B are independently selected from carboxyl groups, active esters, aldehyde groups, amino groups, amine groups, hydroxy groups and thiol groups. In certain embodiments, the reactive groups in B for linking to the payload are independently selected from amino groups, amine groups, hydroxy groups, thiol groups, carboxyl groups and active esters.

[0084] In one embodiment, the reactive groups in B are independently amino groups, amine groups or hydroxy groups and react with the corresponding groups in the payload (e.g., carboxyl groups, sulfonic acid groups, phosphoryl groups having a free - OH terminus, active esters, acid chlorides or isocyanate groups). In another embodiment, the reactive groups in B are independently carboxyl groups or active esters and react with the corresponding groups in the payload (e.g., amino groups, amine groups or hydroxy groups).

[0085] In one embodiment, the reactive group in B is independently an amino group, a hydroxy group or a thiol group, and reacts with the corresponding group (e.g., halogen, hydroxy group, aldehyde group) in the payload. In another embodiment, the reactive group in B is independently a hydroxy group and reacts with the corresponding group (e.g., halogen or hydroxy group) in the payload.

[0086] In one embodiment, each B is independently a terminal group R 10 or is a combination of 1) a self-immolative spacer Sp1, 2) one or more combinations of divalent groups selected from a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-, and 3) a terminal group R 10 and.

[0087] In one embodiment, Sp1 is selected from PABC, acetal, heteroacetal and combinations thereof.

[0088] In one embodiment, Sp1 is an acetal, a heteroacetal or PABC. In one embodiment, the heteroacetal is selected from N,O-heteroacetals. In one embodiment, Sp1 is -O-CH2-U- or -NH-CH2-U-, where -O- or -NH- is linked to a cleavable sequence 1 and U is absent or U is O, S or N, preferably O or S.

[0089] In one embodiment, Sp1 is PABC.

[0090] In one embodiment, B is R 10 , -NH-CH2-U-R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10and here U is absent or U is O, S or N, g is 1, and preferably U is O or S.

[0091] In one embodiment, B is R 10 is.

[0092] In one embodiment, B is -NH-CH2-O-R 10 is.

[0093] In one embodiment, B is -NH-CH2-O-(CR 1 R 2 ) g -(CO)-R 10 and g is an integer from 1 to 10, preferably 1.

[0094] In one embodiment, B is -NH-CH2-R 10 is.

[0095] In one embodiment, R 1 is hydrogen.

[0096] In one embodiment, R 2 is hydrogen.

[0097] In one embodiment, R 10 is hydrogen, hydroxy or

Chemical formula

Chemical formula

[0098] In one embodiment, R 10 represents a structural moiety that does not appear in the product molecule resulting from the reaction of B and the payload.

[0099] Specific embodiments of the compound of formula (I) In one embodiment, each LKa is

Chemical formula

Chemical formula

[0100] In one embodiment, Ld2 is

Chemical formula

Chemical formula

[0101] In one embodiment, Ld2 is a bond, d is 1, and W is -C2H4-(PEG) t -(CO)NH2. In one embodiment, the compound of formula (I-1) is as follows.

Chemical formula

[0102] In one embodiment, Ld1 is

Chemical formula

[0103]

Chemical formula

Chemical formula

Chemical formula

[0104] In one embodiment, Ld1 is

Chemical formula

[0105]

Chemical formula

Chemical formula

Chemical formula

[0106] In one embodiment, Ld1 is [Chemical formula] where i is 4, n is 3, m is 1, j is 12, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-U-R 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 where U is O, g is 1, R 11 is methyl. In one embodiment, linker I-a has the following structure.

[0107] [Chemical formula] [Chemical formula] [Chemical formula]

[0108] In one embodiment, Ld1 is [Chemical formula] where i is 4, n is 3, m is 2, j is 12, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-U-R 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10where U is O, g is 1, and R 11 is methyl. In one embodiment, linker I-a has the following structure.

[0109]

Chemical formula

Chemical formula

Chemical formula

[0110] In one embodiment, Ld1 is

Chemical formula

[0111]

Chemical formula

Chemical formula

Chemical formula

[0112] In one embodiment, Ld1 is

Chemical formula

[0113]

Chemical formula

Chemical formula

Chemical formula

[0114] In one embodiment, Ld1 is

Chemical formula

[0115] [Chemical] [Chemical] [Chemical]

[0116] In one embodiment, Ld1 is [Chemical] where i is 4, n is 3, m is 2, t is 12, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-U-R 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 where U is O and g is 1. In one embodiment, linker I-b has the following structure.

[0117] [Chemical] [Chemical] [Chemical]

[0118] Compound of formula (I) having a payload The reactive group contained in B provides a compound of formula (I) having a payload by covalently conjugating with a payload containing another reactive group.

[0119] In yet another aspect, a compound having the structure of formula (II), [Chemical formula] In the formula, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P, M is LKa-LKb-P, P is the payload linked to the B part or L 1 part of the compound of formula (I), n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I), to provide a compound.

[0120] Payload In the present disclosure, the payload can be selected from the group consisting of small molecule compounds, nucleic acids and their analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptidomimetics, and proteins. In one embodiment, the payload is selected from the group consisting of small molecule compounds, nucleic acid molecules, and tracer molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from the group consisting of cytotoxins and their fragments.

[0121] In one embodiment, the cytotoxin is selected from the group consisting of drugs targeting the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrous vinblastine, 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 camptothecins and their derivatives, DNA topoisomerase inhibitors such as mitoxantrone, mitoguazone. In a preferred embodiment, the cytotoxin is selected from the group consisting of nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, phenamet, phenesterine, prednimustine, trofosfamide, uracil mustard. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of nitrosoureas such as carmustine, flubenzuron, formoterol, lomustine, nimustine, ranimustine. 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, carbocon, 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 dynemicin, esperamicin, neocarzinostatin, and aclacinomycin.In another preferred embodiment, the cytotoxic agent is selected from the group consisting of actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, calminomycin, and cardinophilin, calminomycin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, iron adriamycin, rhodrubicin, lufocromomycin, streptozocin, dinostatin, zorubicin. In yet another preferred embodiment, the cytotoxic agent is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxic agent is selected from the group consisting of T-2 toxin, verrucarin A, bacrocrypolin A, and anguidine. In one embodiment, the cytotoxic agent is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxic agent is selected from the group consisting of folic acid analogs. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of dimethyl folic acid, methotrexate, pteropterin, trimetrexate, and edatrexate. In one embodiment, the cytotoxic agent is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of fludarabine, 6-mercaptopurine, thiampurine, thioguanine. In yet another embodiment, the cytotoxic agent is selected from pyrimidine analogs. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, floxuridine. In one embodiment, the cytotoxic agent is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone. In another embodiment, the cytotoxic agent is selected from the group consisting of antiadrenal drugs.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 antiandrogens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide 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 vinblastines, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocarmycin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dorastatin, and pyrrolobenzodiazepine (PBD). In a particular embodiment, the cytotoxin is selected from the group consisting of vinblastines, colchicines, taxanes, auristatins, and maytansinoids.

[0122] In a particular embodiment, the cytotoxin is exatecan or a derivative thereof such as DX8951f.

[0123] In another particular embodiment, the cytotoxin is a maytansinoid such as DM1.

[0124] In a particular 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.

[0125] The payload contains a reactive group that can react with the reactive group in the compound of formula (I) to covalently conjugate the payload to the compound of formula (I). Compounds that do not contain a reactive group require appropriate derivatization to obtain the payload.

[0126] In one embodiment, the cytotoxin is a compound of the following formula (i), [Chemical formula] wherein, a* is 0 or 1, each of the carbon atoms marked with p1* and p2* respectively is an asymmetric center, and the asymmetric center is in the S configuration, R configuration or racemate, L 1* is selected from unsubstituted or C alkylene substituted with one substituent selected from halogen, -OH and -NH2, 1-6 selected from alkylene, M* is -CH2-, -NH- or -O-, L 2* is C 1-3 alkylene, R 1* and R 2* are each independently selected from hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0127] In a specific embodiment, the cytotoxin is a compound of the following formula (i'), [Chemical formula] wherein, g* is any integer from 1 to 6, 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 they are attached form a 3- to 6-membered cycloalkyl group.

[0128] In one embodiment, g* is any integer from 1 to 3, preferably 1.

[0129] In one embodiment, R 1’is hydrogen.

[0130] In one embodiment, R 2’ is hydrogen.

[0131] In one embodiment, L 1* is C 1-6 linear alkylene, C 1-6 branched alkylene, C 3-6 cyclic alkylene and C 3-4 cyclic alkyl-C 1-2 selected from linear alkylene groups, each of which is independently unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2. In one embodiment, L 1* is selected from unsubstituted or C substituted with one substituent selected from halogen, -OH and -NH2 1-4 alkylene. In a preferred embodiment, L 1* is -CH2-, -C2H4-,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0132] In one embodiment, a* is 1, M* is -CH2-, -NH-, or -O-, and L 2* is -C2H4-. In another embodiment, a* is 1, M* is -CH2-, and L 2* is -CH2-. In one embodiment, a* is 0.

[0133] In one embodiment, the carbon atom marked with p1* has an S configuration or is a racemate, preferably an S configuration. In another embodiment, the carbon atom marked with p2* has an S configuration or is a racemate, preferably an S configuration.

[0134] In one embodiment, R 1* and R 2* are each independently selected from hydrogen, C 1~3 alkyl, halogen, and C 1~3 alkoxy. In a preferred embodiment, R 1* and R 2* are each independently selected from CH3-, F, Cl, Br, and CH3O-. In one embodiment, R 1* is selected from CH3- and Cl. In another embodiment, R 2* is F.

[0135] In one embodiment, a* is 0 and L 1* is -CH2-,

Chemical formula

Chemical formula

[0136] In one embodiment, a* is 0, and R 1* is Cl, and R 2* is F, and L 1* is -CH2-,

Chemical formula

Chemical formula

[0137] In one embodiment, a* is 1, and R 1* is CH3-, and R 2* is F, and L 1* is

Chemical formula

[0138] In one embodiment, the cytotoxin is selected from the following.

[0139]

Chemical formula

Chemical formula

Chemical formula

[0140] In one embodiment, the cytotoxin is selected from the following.

Chemical formula

Chem.

[0141] In a preferred embodiment, the cytotoxin is selected from the following.

Chem.

Chem.

[0142] In a preferred embodiment, the cytotoxin is selected from the following.

Chem.

[0143] In a more preferred embodiment, the cytotoxin is selected from the following.

Chem.

[0144] In a particular embodiment, the cytotoxin is selected from the following.

Chem.

[0145] In one embodiment, the cytotoxin is selected from the following compounds, where the wavy bond indicates the linking site for linking to the compound of formula (I).

Chem.

[0146] In some embodiments, the payload is DX8951f (Compound 9), DXd-(1) (Compound 10), DXd-(2) (Compound 14),

Chem.

Chemical formula

Chemical formula

Chemical formula

[0147] Preparation of the compound of formula (I) having a payload In one embodiment, the linking unit and the payload are linked via the reactive groups defined above by any reaction known in the art including, but not limited to, condensation reactions, nucleophilic addition, electrophilic addition, etc.

[0148] In one embodiment, the payload is a cytotoxic agent. In one embodiment, the linking unit-payload intermediate (numbered LBx) is as shown in the following table.

[0149]

Table 1-1

Table 1-2

[0150] Conjugate and its preparation Furthermore, the compound of formula (I) having a payload and having a portion containing a ligase recognition sequence can be conjugated with other molecules containing the ligase recognition sequence, thereby, for example, can be used in the preparation of targeted molecule-drug conjugates such as antibody-drug conjugates. Thus, in yet another aspect, a conjugate comprising a compound of formula (I), a targeting molecule, and a payload is provided.

[0151] In yet another aspect, a conjugate having the structure of formula (III), wherein:

Chemical formula

[0152] In one embodiment, A is an anti-human monoclonal antibody 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 include Leu-Pro-Xaa-Thr, and 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 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 include Leu-Pro-Xaa-Thr, and Xaa is any natural or non-natural single amino acid, and z is 2.

[0153] Targeting molecule In one embodiment, the targeting molecule is an antibody or an antigen-binding fragment thereof.

[0154] In one embodiment, the targeting molecule is an anti-human HER2 antibody or an antigen-binding fragment thereof. Examples of anti-human HER2 antibodies include, but are not limited to, pertuzumab and trastuzumab. Pertuzumab binds to the second extracellular domain (ECD2) of HER2 and is approved for the treatment of HER2-positive breast cancer. Trastuzumab binds to the fourth extracellular domain (ECD4) of HER2 and is approved for the treatment of Her2-positive breast cancer and gastric cancer.

[0155] In a preferred embodiment, the anti-human HER2 antibody is one or more selected from anti-HER2 antibodies engineered based on trastuzumab.

[0156] 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, the trodelvy antibody (hRS7) and the DS1062 antibody (datopotamab).

[0157] In one embodiment, the targeting molecule is an anti-FGFR3 antibody or an antigen-binding fragment thereof.

[0158] In a preferred embodiment, the anti-human HER2 antibody is a recombinant antibody selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetics. In a preferred embodiment, the anti-human Trop2 antibody is a recombinant antibody selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetics. In a preferred embodiment, the anti-FGFR3 antibody is a recombinant antibody selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetics. For conjugation with the compound of formula (I), the targeting molecule of the present disclosure may include a modifying moiety for linking to Gn in the compound of formula (I). In one embodiment, the antibody mimetic is selected from scFv, minibody, diabody, nanobody. The introduction position of such a modifying moiety is not limited. For example, when the targeting molecule is an antibody, the introduction position may be, but is not limited to, the C-terminus or N-terminus of the heavy chain or light chain of the antibody.

[0159] In an alternative embodiment, the modifying moiety for conjugation with Gn in the compound of formula (I) may be introduced at a non-terminal position of the heavy or light chain of the antibody, for example, using chemical modification methods.

[0160] In one embodiment, the targeting molecule of the present disclosure is an antibody or an antigen-binding fragment thereof that may include a terminal modification. The terminal modification refers to a modification at the C-terminus or N-terminus of the heavy or light chain of the antibody and includes, for example, a ligase recognition sequence. In another embodiment, the terminal modification may further include a spacer Sp2 containing 2 to 100 amino acids, where the antibody, Sp2, and the ligase recognition sequence are linked in sequence. In a preferred embodiment, Sp2 is a spacer sequence containing 2 to 20 amino acids. In a particular embodiment, Sp2 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, and particularly GA.

[0161] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment includes three types: wild type (LC), C-terminal modified light chain (LCCT) modified by direct introduction of the ligase recognition sequence LPXTG, and C-terminal modified light chain (LCCT L ) modified by introduction of a short peptide spacer + ligase donor substrate recognition sequence LPXTG. The heavy chain of the antibody or its antigen-binding fragment includes three types: wild type (HC), C-terminal modified heavy chain (HCCT) modified by direct introduction of the ligase recognition sequence LPXTG, and C-terminal modified heavy chain (HCCT L ). X may be any natural or non-natural single amino acid. When z in the compound of formula (III) is 1 or 2, the above combinations of heavy and light chains can form eight preferred antibody molecules as shown in the amino acid sequence listing.

[0162] The conjugate of the present disclosure may further include a payload. The payload is as described above.

[0163] Anti-HER2, TROP2, and FGFR3 antibodies are listed without limitation for reference of all antibodies and are used to conjugate to the linker-payload. For example, an anti-human monoclonal antibody can be conjugated to the remaining portion of the conjugate (linker-payload portion) via a modified heavy chain and / or light chain C-terminus, where the modified heavy chain and / or light chain C-terminus is modified to include Leu-Pro-Xaa-Thr, Xaa is any natural or non-natural single amino acid, and z is 2.

[0164] Specific embodiments of the conjugate In one embodiment, each LKa is

Chemical formula

Chemical formula

[0165] In one embodiment, Ld2 is

Chemical formula

Chemical formula

Chemical formula

[0166] In one embodiment, Ld2 is a bond, and Ld1 is

Chemical formula

[0167] In one embodiment, z is from 1 to 4. In one embodiment, z is 2 or 4.

[0168] In one embodiment, z is 2.

[0169] In one embodiment, z is 4.

[0170] In one embodiment, in conjugates III-a and III-b, z is 2 or 4.

[0171] In one embodiment, in conjugates III-a and III-b, z is 2.

[0172] In one embodiment, in conjugates III-a and III-b, z is 4.

[0173] In one embodiment, B in the compound of formula (I) is the terminal group R 10 and the cleavable sequence 1 in L 1 is linked to the payload to form the compound of formula (II), where B does not exist in the molecule obtained by linking the cleavable sequence 1 and the payload.

[0174] In one embodiment, i is 4, n is 3, m is 1, j is 12, and L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, and L 1 is Gly-Gly-Phe-Gly, and B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R10 where U is O, g is 1, and R 11 is methyl. In one embodiment, conjugate III-a has the following structure. [Chemical formula]

[0175] In one embodiment, conjugate III-a has the following structure. [Chemical formula]

[0176] In one embodiment, i is 4, n is 3, m is 2, j is 12, and L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, and L 1 is Gly-Gly-Phe-Gly, and B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 where U is O, g is 1, and R 11 is methyl. In one embodiment, conjugate III-a has the following structure. [Chemical formula]

[0177] In one embodiment, conjugate III-a has the following structure. [Chemical formula]

[0178] In one embodiment, i is 4, n is 3, m is 1, j is 12, and L 2 is -(C2H4-O) p-(CH2)2(CO)-, p is 2, and L 1 is Val-Cit, and B is PABC-NH-CH2-U-R 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 where U is O, g is 1, and R 11 is methyl. In one embodiment, conjugate III-a has the following structure.

Chemical formula

[0179] In one embodiment, conjugate III-a has the following structure.

Chemical formula

[0180] In one embodiment, i is 4, n is 3, m is 2, j is 12, and L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, and L 1 is Val-Cit, and B is PABC-NH-CH2-U-R 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 where U is O, g is 1, and R 11 is methyl. In one embodiment, conjugate III-a has the following structure.

Chemical formula

[0181] In one embodiment, conjugate III-a has the following structure. [Chemical formula]

[0182] In one embodiment, i is 4, n is 3, m is 1, t is 12, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 and U is O, g is 1. In one embodiment, conjugate III-b has the following structure. [Chemical formula]

[0183] In one embodiment, conjugate III-b has the following structure. [Chemical formula]

[0184] In one embodiment, i is 4, n is 3, m is 2, t is 12, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Gly-Gly-Phe-Gly, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 and U is O, g is 1. In one embodiment, conjugate III-b has the following structure. [Chemical formula]

[0185] In one embodiment, conjugate III-b has the following structure.

Chemical formula

[0186] In one embodiment, i is 4, n is 3, m is 1, t is 12, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-U-R 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 and g is 1. In one embodiment, conjugate III-b has the following structure.

Chemical formula

[0187] In one embodiment, conjugate III-b has the following structure.

Chemical formula

[0188] In one embodiment, i is 4, n is 3, m is 2, t is 12, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is Val-Cit, B is PABC-NH-CH2-U-R 10 or PABC-R 10 or PABC-NH-CH2-U-(CR 1 R 2) g -(CO)-R 10 wherein g is 1. In one embodiment, conjugate III-b has the following structure.

Chemical formula

[0189] In one embodiment, conjugate III-b has the following structure.

Chemical formula

[0190] Preparation of the conjugate The conjugates of the present disclosure 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 and a payload under a ligase catalyst of a compound of formula (I), wherein the targeting molecule is modified by a ligase recognition sequence. The method includes step A and step B.

[0191] Step A. Preparation of the linker-payload intermediate In a preferred embodiment, B in the compound of formula (I) is covalently linked to a payload containing another reactive group via a reactive group.

[0192] The linker-payload intermediate prepared from the compound of formula (I) of the present disclosure has a clear structure, a clear composition, and high purity. Therefore, when conjugated with an antibody, few impurities are introduced, or no other impurities are introduced. When such an intermediate is used for site-specific conjugation with a modified antibody containing a ligase recognition sequence under a ligase catalyst, a homogeneous ADC with a highly controllable quality can be obtained.

[0193] Step B. Linking the targeting molecule to the compound of formula (I) having a payload The targeting molecule of the present disclosure can be conjugated to a compound of formula (I) (i.e., a compound of formula (II)) having a payload by any method known in the art.

[0194] The targeting molecule and the compound of formula (I) having a payload are linked to each other via a ligase-specific recognition sequence of the substrate. The recognition sequence depends on the particular ligase used. In one embodiment, the targeting molecule is an antibody with a terminal modification based on the recognition sequence introduced at the C-terminus of the light chain and / or heavy chain, and the targeting molecule conjugates with the compound of formula (II) under the catalytic action of a wild-type or optimized engineered ligase or any combination thereof, and suitable catalytic reaction conditions.

[0195] In a particular embodiment, the ligase is sortase A, and the conjugation reaction can be represented by the following scheme.

[0196]

Chemical formula

[0197] The triangle represents a part of the antibody, and the pentagon represents a part of the compound of formula (II). n, X, and J are as defined above. When conjugating with the corresponding recognition sequence Gn of the acceptor substrate, the peptide bond upstream of glycine in the LPXTGJ sequence is cleaved by sortase A, and the resulting intermediate is linked to the free N-terminus of Gn to form a new peptide bond. The resulting amino acid sequence is LPXTGn. The sequences Gn and LPXTGJ are as defined above.

[0198] Metabolism of the conjugate in a physiological environment When part or all of the linker is cleaved in tumor cells, the anti-tumor compound moiety is released to exhibit the anti-tumor effect of the anti-tumor compound. Since the linker is cleaved at the drug-linking position, the anti-tumor compound is released in its own unique structure to exhibit its own unique anti-tumor effect.

[0199] In one embodiment, the cleavable sequence 1 (such as Gly-Gly-Phe-Gly) can be cleaved by lysosomal enzymes (such as cathepsin B and / or cathepsin L).

[0200] In one embodiment, Sp1 includes a self-destructive spacer. In one embodiment, Sp1 includes PABC, acetal, or heteroacetal. In one embodiment, L 1 is Gly-Gly-Phe-Gly. In one embodiment, the linker includes -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-destructive spacer, and is cleaved intracellularly to release the target molecule (such as a drug).

[0201] Table of specific conjugates In one embodiment, the payload 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). The sequences of Ab0001-LCCT L -HC and Ab0001-LCCT L -HCCT L are each based on the amino acid sequence of Ab0001 (trastuzumab), and GALPETGG is introduced at the C-terminus of the light chain (Ab0001-LCCT L ) or at the C-termini of the light chain and heavy chain (Ab0001-LCCT L -HCCT L ), where LPETGG is the recognition sequence of the ligase donor substrate and GA is the spacer sequence. Ab0001-LCCT L -HCCT LIn this case, the lysine at the C-terminus of the heavy chain of Ab0001 may be maintained as in SEQ ID NO: 4, or may be removed before the introduction of GALPETGG (the resulting sequence is not shown in the sequence listing). In one embodiment, the antibody-drug conjugate is as shown in the following table.

[0202] In one embodiment, the antibody is Ab2 (light chain: SEQ ID NO: 5, heavy chain: SEQ ID NO: 6).

[0203] In one embodiment, the antibody is Ab3 (light chain: SEQ ID NO: 7, heavy chain: SEQ ID NO: 8).

[0204] ADC nomenclature: The numbers in parentheses represent the number of payload (drug) molecules to be conjugated to the antibody.

[0205]

Table 2

[0206] Pharmaceutical composition and pharmaceutical formulation Another object of the present disclosure is to provide a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of the conjugate of the present disclosure and at least one pharmaceutically acceptable carrier.

[0207] The pharmaceutical composition of the present disclosure may be administered by any method as long as the effect of preventing, alleviating, preventing, or curing human or animal symptoms is achieved. For example, various suitable dosage forms, particularly injectable freeze-dried powders, injections, or injections such as sterile powders for injection, can be prepared according to the administration route.

[0208] The term "pharmaceutically acceptable" means that it does not cause undue toxicity, irritation, allergic reaction, etc. when contacting the patient's tissue within the scope of normal medical judgment, has a reasonable benefit / defect ratio, and is effective for the intended use.

[0209] 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 and the like. Pharmaceutically acceptable carriers include carrier materials that bring the composition closer to physiological conditions, such as pH adjusters, buffers, toxicity adjusters, etc., and also include sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0210] In one embodiment, the pharmaceutical composition of the present disclosure has a drug-antibody ratio (DAR) that is an integer or non-integer from 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 certain embodiments, the conjugate of the present disclosure has a DAR of about 2, about 4, about 6, or about 8.

[0211] Therapeutic method and use The conjugate of the present disclosure is useful for the treatment of tumors and / or autoimmune diseases. Tumor conjugate therapy includes tumors characterized by specific tumor-related antigens or cell surface receptors, and tumors that can be recognized by the targeting molecule in the conjugate and killed by the payload / cytotoxin in the conjugate.

[0212] Accordingly, in yet another aspect, there is further provided the use of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, disorder, or condition selected from tumors or autoimmune diseases.

[0213] In another aspect, there is provided the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure for use in the treatment of tumors or autoimmune diseases.

[0214] In a further aspect, there is provided a method of treating a tumor or autoimmune disease, the method comprising administering to a subject in need thereof an effective amount of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure.

[0215] In a preferred embodiment, the conjugate of the present disclosure formed by the conjugation of an anti-human HER2 antibody and a small molecule cytotoxin can specifically bind to HER2 on the surface of tumor cells and can selectively kill HER2-expressing tumor cells. In another preferred embodiment, there is provided the use of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, disorder or symptom selected from HER2-positive tumors. In a more preferred embodiment, the disease, disorder or symptom is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, etc.

[0216] In a preferred embodiment, the conjugate of the present disclosure formed by the conjugation of an anti-human TROP2 antibody and a small molecule cytotoxin can specifically bind to TROP2 on the surface of tumor cells and can selectively kill TROP2-expressing tumor cells. In another preferred embodiment, there is provided the use of the conjugate (or antibody) of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, disorder or symptom selected from TROP2-positive tumors. In a more preferred embodiment, the disease, disorder or symptom is a TROP2-positive tumor. In one embodiment, the TROP2-positive tumors are selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial cancer, etc.

[0217] In a preferred embodiment, the conjugate of the present disclosure formed by the conjugation of an anti-FGFR3 antibody and a small molecule cytotoxin can specifically bind to FGFR3 on the surface of tumor cells and can selectively kill FGFR3-expressing tumor cells. In another preferred embodiment, provided is the use of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating FGFR3-mediated diseases. The disease is specifically an FGFR3-positive tumor, more specifically brain cancer, bladder cancer, urothelial cancer, cervical cancer, or intrahepatic cholangiocarcinoma. In some embodiments, the disease includes tumors that overexpress FGFR3 or tumors with FGFR3 gene mutations. In some embodiments, the disease is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma. In some embodiments, the disease is selected from brain cancer, bladder cancer, urothelial cancer, cervical cancer, or intrahepatic cholangiocarcinoma. In some embodiments, the disease is glioblastoma.

[0218] The dosage of the conjugate administered to a subject can be adjusted within a fairly wide range. The dosage may vary depending on the specific route of administration and the needs of the subject, and may also follow the judgment of a medical professional.

[0219] Beneficial effects This branched linker does not contain a maleimide group and conjugates to a specific site of an antibody via a terminal amino acid, thus avoiding potential instability in the blood. On the other hand, the branched linker has strong binding ability, can adapt to various payloads, is convenient as a linker-payload fragment and environmentally friendly and can be commercialized. The formed linker-payload structure is easy to separate and purify and is useful for late-stage CMC development. Furthermore, by conjugating different types of linkers to different antibodies, the stability of the linker, its compatibility, and the excellent biological activity of the ADC formed by the linker are verified.

[0220] The antibody-drug conjugate of the present disclosure uses a specially designed linker-payload, is more stable, can achieve high efficacy with a relatively low DAR, thereby reducing side effects and improving the therapeutic index.

[0221] The present disclosure utilizes a linking unit with a unique structure and catalyzes the conjugation of a targeting molecule and a payload with a ligase. The conjugate of the present disclosure has high homogeneity, high activity and high selectivity. Furthermore, the toxicity of the linking unit-payload intermediate is much lower than that of the free payload, so there is less adverse impact on the drug manufacturing process and it is advantageous for industrial production.

[0222] The conjugate of the present disclosure achieves at least one of the following technical effects. (1) High inhibitory activity against target cells, or a strong killing effect on target cells. (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability). (3) Good pharmacokinetic properties (e.g., high stability in plasma, appropriate half-life and duration of action). (4) High safety (low toxicity to normal cells or tissues outside the target, and / or few side effects, wide therapeutic window), etc. (5) Advanced modular design, can be easily conjugated with multiple drugs. (6) Low payload consumption, low production cost. (7) Few isomers, and CMC can be easily developed.

[0223] Examples Preparation Example To more clearly explain the object and technical solution of the invention, the present disclosure will be further described by specific examples below. It should be understood that these examples are not intended to limit the scope of the present disclosure. Specific experimental methods not described in the following examples are carried out according to conventional experimental methods.

[0224] Apparatus, Materials and Reagents Unless otherwise specified, the apparatus and reagents used in the examples are commercially available. The reagents can be used directly without further purification. The intermediates used are either commercially available products or synthetic products. MS: Thermo Fisher Q Exactive Plus, Waters2795 - Quattro micro triple quadrupole mass spectrometer HPLC: Waters2695, Agilent1100, Agilent1200 Semi - preparative HPLC: Lisure HP plus 50D Flow cytometry: CytoFLEX S HIC - HPLC: Butyl - HIC, Mobile phase A: 25 mM PB, 2M (NH4)2SO4, pH 7.0, Mobile phase B: 25 mM PB, pH 7.0, Flow rate: 0.8 ml / min, Collection time: 25 min, Sample injection volume: 20 μg, Column temperature: 25 °C, Detection wavelength: 280 nm, Sample chamber temperature: 8 °C. SEC - HPLC: Column: TSK - gel G3000 SWXL, TOSOH 7.8 mm ID × 300 mm, 5 μm, Mobile phase: 0.2M KH2PO4, 0.25M KCl, pH 6.2, Flow rate: 0.5 ml / min, Collection time: 30 min, Sample injection volume: 50 μl, Column temperature: 25 °C, Detection wavelength: 280 nm, Sample tray temperature: 8 °C. CHO was obtained from Thermo Fisher Scientific. Rink-amide-MBHA-resin was obtained from Nankai synthesis. SK-BR-3 was obtained from ATCC CAT# HTB-30. NCI-N87 cells were obtained from ATCC CAT# CRL-5822. MDA-MB-468 was obtained from ATCC CAT# HTB-132. JIMT-1 was obtained from Wuxi Apptech. The antibody trastuzumab was prepared according to a known sequence. The optimized recombinant enzyme sortase A derived from Staphylococcus aureus was prepared in Escherichia coli.

[0225] Example 1 Construction, expression, purification and identification of antibody expression vectors 1.1 Modified anti-human HER2 antibody Ab0001-LCCT L -HC production Antibody Ab0001-LCCT L -HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) expression plasmid was constructed as follows. Antibody Ab0001-LCCT L The sequence of -HC was based on the amino acid sequence of trastuzumab, with GALPETGG introduced at the C-terminus of the light chain, where LPETGG is the recognition sequence of the ligase donor substrate and GA is the spacer sequence. The plasmid was transfected into CHO cells, a cell population was established, a high-expression cell population was screened, and cultured in a 5-10 L reactor with reference to the culture process of trastuzumab, and the supernatant was collected.

[0226] 1.2 Antibody Ab0001-LCCT L -HC purification Ab0001-LCCT L Purification of -HC was carried out using a standard process combining MabSelect affinity chromatography and Sepharose S cation exchange chromatography. The purified product was dissolved in the original trastuzumab drug buffer (5 mM histidine HCl, 2% trehalose, 0.009% polysorbate 20, PH 6.0) and frozen in small aliquots.

[0227] 1.3 Antibody Ab0001-LCCT L -Quality control of HC The purified antibody Ab0001-LCCT L -The purity of HC is 98.5% by SDS-PAGE, the high molecular weight polymer content of the sample is less than 0.4% by SEC-HPLC, and the endotoxin content is less than 0.098 EU / mg.

[0228] 1.4 Preparation of other modified anti-human antibodies According to the same method, terminal modifications based on the ligase recognition sequence were introduced into the C-terminus of the light chain and / or heavy chain of trastuzumab, hRS7 (Ab2), and Ab3 respectively to obtain modified antibodies.

[0229] The modified anti-human antibodies are shown in Table 1. LPETGG in the terminal modification sequence is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence.

[0230]

Table 3

[0231] Example 2 Preparation of linker-payload intermediate 2.1 Preparation of intermediate Mc-GGFG-Dxd The intermediate Mc-GGFG-Dxd is a commercial product or is prepared according to the procedure described in EP2907824. This compound is for preparing a linker-payload intermediate and is also for directly linking to an (optionally modified) antibody to prepare a reference ADC.

[0232] 2.2 Preparation of linker-payload 1

Chemical formula

[0233] Step 1: Preparation of intermediate compound b Step 1.1 Preparation of NH2-Asp(OtBu)-Rink amide resin 400 g of Rink amide resin was weighed and completely swollen with 2400 mL of DCM. 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.

[0234] 88.87 g of Fmoc-Asp(OtBu)-OH and 29.19 g of HOBT were weighed and dissolved in a solution of 2000 mL of DMF and 80 mL of DIC. After being placed in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, stirred and reacted at room temperature with nitrogen for 2 - 5 h, and then filtered. The resin was washed successively with DMF and DCM, and showed colorless or light yellow in the subsequent ninhydrin test.

[0235] 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.

[0236] Step 1.2 Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin 131.64 g of Fmoc-PEG4-OH and 48.64 g of HOBT were weighed and dissolved in a solution of 2000 mL of DMF and 80.0 mL of DIC. After being placed in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, stirred and reacted at room temperature with nitrogen for 2 - 5 h, and then filtered. The resin was washed successively with DMF and DCM, and showed colorless or light yellow in the subsequent ninhydrin test.

[0237] 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.

[0238] Step 1.3 Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Weighed 222.18 g of Fmoc-Asp(OtBu)-OH and 72.96 g of HOBT, and dissolved them in 2000 mL of DMF and 80 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, stirred and reacted at room temperature for 2 - 5 h with nitrogen, and then filtered. The resin was washed successively with DMF and DCM, and showed colorless or light yellow in the subsequent ninhydrin test.

[0239] Added 2400 mL of deprotection reagent to completely remove Fmoc, and then washed it several times at room temperature with DMF and DCM. In the subsequent ninhydrin test, the resin showed blue.

[0240] Step 1.4 Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Weighed 191.75 g of Dde-Lys(Fmoc)-OH and 48.64 g of HOBT, and dissolved them in 2000 mL of DMF and 80.0 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, stirred and reacted at room temperature for 2 - 5 h with nitrogen, and then filtered. The resin was washed successively with DMF and DCM, and showed colorless or light yellow in the subsequent ninhydrin test.

[0241] Added 2400 mL of deprotection reagent to completely remove Fmoc, and then washed it several times at room temperature with DMF and DCM. In the subsequent ninhydrin test, the resin showed blue.

[0242] Step 1.5 Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Weighed 170.84 g of m-PEG12-CH2CH2COOH and 48.64 g of HOBT, and dissolved them in 2000 mL of DMF and 80.0 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, stirred and reacted at room temperature for 2 - 5 h with nitrogen, and then filtered. The resin was washed successively with DMF and DCM, and showed colorless or light yellow in the subsequent ninhydrin test.

[0243] Step 1.6 Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 2400 mL of Dde removal reagent was added, and the reaction was stirred at room temperature for 0.5 h under nitrogen, and then filtered. After repeating the operation three times, the resin was washed with DMF and DCM in sequence, and the resin showed blue in the subsequent ninhydrin test.

[0244] Step 1.7 Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 111.08 g of Fmoc-Gly-Gly-Gly-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After being placed in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, and the reaction was stirred at room temperature for 2 - 5 h with nitrogen, and then filtered. The resin was washed successively with DMF and DCM, and showed colorless or light yellow in the subsequent ninhydrin test. The resin peptide was washed three times with absolute ethanol, filtered, and prepared for cleavage.

[0245] Step 1.8 Preparation of intermediate compound b 10000 mL of cleavage reagent (TFA:TIS:H2O = 95:2.5:2.5) was added to a 10 L reactor and cooled to -10 ± 2 °C. The dried and weighed resin was added. The temperature was raised to room temperature and the reaction was stirred for 2 - 3 h under nitrogen. Then, the resin was filtered and washed once with 100 mL of TFA. The filtrate and the washing solution were combined.

[0246] 40 L of pre-cooled (-less than 10 °C) cold ether was added to the product solution. The mixture was stirred for 10 minutes, and then the precipitate was centrifuged. After centrifugation, the supernatant was discarded, the precipitate was collected, washed with cold ether, and the precipitate was centrifuged again (the centrifugation speed for each time was 3600 rpm, the centrifugation time was 5 minutes, and the centrifugation cavity temperature was -5 °C).

[0247] The precipitate was recovered as crude compound b. The crude product was purified by preparative HPLC and lyophilized to obtain pure compound b.

[0248] Step 2: Preparation of intermediate compound a

Chemical formula

[0249] Step 2.1 Preparation of compound 2 Compound 1 (1 equivalent) and DMF (5 v / v) were added to a reaction flask, and the mixture was stirred and dissolved under nitrogen protection. After cooling the ice bath to 0 - 5 °C, DIEA (3 equivalents) was added dropwise. After the addition, the mixture was stirred at 5 °C for 10 min. Subsequently, benzyl bromide (1.3 equivalents) was added dropwise. After the addition was complete, the temperature was allowed to rise naturally to room temperature of about 20 °C and stirred for 16 h.

[0250] 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, then the organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude yellow oil, which was poured onto a column by the wet method. Elution with PE / EA = 6:1 gave a light yellow oil with a yield of 100%.

[0251] Step 2.2 Preparation of compound 4 Under nitrogen protection, intermediate 2 (2.0 equivalents), compound 3 (1 equivalent) and THF (10 v / v) were added to a reaction flask, stirred and dissolved, TsOH (0.1 equivalent) was weighed and added to the reaction, and the reaction was maintained at 20 - 22 °C for 4 h. The reaction solution was slowly poured into ice water, extracted 3 times with EA, the combined organic phase was washed successively with saturated aqueous sodium bicarbonate solution, water and saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain a crude product. The product was recovered by elution with PE / EA = 1:1 by passing through a column and mixing silica gel samples, concentrated to obtain a white solid with a yield of 40%.

[0252] Step 2.3 Preparation of compound 7 Under nitrogen protection, compound 4 and DMAc (10 v / v) were added to a reaction flask and stirred to dissolve. The reaction was cooled to 14 - 18 °C, and DBU (0.5 equivalent) was added dropwise. The mixture was stirred at this temperature for 1.5 h to react, and the completion of the reaction of the raw materials was monitored by TLC. The reaction was cooled to 0 - 5 °C, and PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent) and compound 6 (0.85 equivalent) were added, and the reaction was carried out at 0 - 10 °C for 3 - 4 h, and the reaction was monitored by LCMS.

[0253] The reaction solution was added to ice water, 2 - methyltetrahydrofuran was added for extraction once, and the aqueous phase was extracted twice with 2 - methyltetrahydrofuran. The organic phases were combined, washed with 0.5 M hydrochloric acid, saturated aqueous NaHCO3 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 recovered by elution with DCM / MeOH and concentrated under vacuum to obtain a white solid in a yield of 78%.

[0254] Step 2.4 Preparation of compound 10 Under nitrogen protection, intermediate 7 and DMAc (10 v / v) were added to a reaction flask and stirred to dissolve. The reaction was cooled to 14 - 18 °C, and DBU (0.5 equivalent) was added dropwise. The mixture was stirred at this temperature for 1.5 h to react, and the completion of the reaction was monitored by TLC. The reaction was cooled to 0 - 5 °C, and PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent) and compound 9 (0.85 equivalent) were added, and the reaction was carried out at 0 - 10 °C for 3 - 4 h, and the reaction was monitored by LCMS.

[0255] The reaction solution was added to ice water, 2 - methyltetrahydrofuran was added for extraction once, and the aqueous phase was extracted twice with 2 - methyltetrahydrofuran. The organic phases were combined, washed with 0.5 M hydrochloric acid, saturated aqueous NaHCO3 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 recovered by elution with DCM / MeOH and concentrated under vacuum to obtain a white solid in a yield of 50%.

[0256] Step 2.5 Preparation of Compound a Under nitrogen protection, Intermediate 10 was dissolved in DCM (15 v / v), DBU (0.5 equivalent) was added dropwise at 20 °C, and the mixture was stirred at 18 - 22 °C for 5 h to react. The completion of the reaction was monitored by LCMS. The reaction solution was diluted with DCM and purified by column using the wet method, and the product was recovered by elution with DCM:MeOH to obtain a white solid in a yield of 82%.

[0257] Step 3: Preparation of Compound c

Chemical formula

[0258] Compound b (400 mg, 0.245 mmol) and Compound a (377 mg, 0.539 mmol) were dissolved in DMF (6 ml), then DIPEA (159 mg, 1.23 mmol) and HATU (233 mg, 0.613 mmol) were added to the reaction solution, and the mixture was stirred at room temperature for 2 h to react. After the disappearance of Compound b, it was purified by preparative HPLC, and the preparation solution was lyophilized to obtain 380 mg of the product in a yield of 52%. C 142 H 207 O 49 N 21 [(M + 3H) / 3] + Calculated value: 997.8, Measured value: 875.9 (fragmentation mass).

[0259] Step 4: Preparation of Intermediate Compound d

Chemical formula

[0260] Compound c (380 mg, 0.245 mmol) was dissolved in purified water (80 ml), and palladium hydroxide (38 mg) was added. The reaction system was replaced with hydrogen three times and stirred at room temperature to react. The progress of the reaction was monitored by HPLC, and the reaction was stopped immediately when 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 the product in a yield of 76%. C 128H 195 O 49 N 21 [(M + 3H) / 3] + Calculated value: 937.8, measured value: 875.9 (fragmentation mass).

[0261] Step 5: Preparation of Intermediate Compound e [Chemical formula]

[0262] 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, followed by stirring at room temperature for 2 - 16 h. After monitoring the completion of the reaction by HPLC, the reaction mixture was directly purified by preparative HPLC, and the recovered eluate was lyophilized to obtain 235 mg of the product in a 66% yield. C 174 H 229 O 55 Cl2F2N 27 [(M + 3H) / 3] + Calculated value: 1229.2, measured value: 1229.3.

[0263] The preparation of 12-1 is shown in 2.3.1 and 2.3.2.

[0264] Step 6: Preparation of Linker-Payload 1 [Chemical formula]

[0265] Compound e (210 mg, 0.057 mmol) was dissolved in DMF (5 ml), and then diethylamine (0.5 ml) was added and reacted at room temperature for 15 min. The end point of the reaction was monitored by HPLC. After the reaction was completed, it was adjusted to neutral with 10% aqueous TFA solution under an ice bath, and the reaction product was purified by preparative HPLC. After lyophilization, 145 mg of the product was obtained in a 73% yield. C 159 H 219 O 53Cl2F2N 27 [(M + 3H) / 3] + Calculated value: 1155.2, measured value: 1155.3.

[0266] 2.3 Preparation of Linker - Payload 2 2.3.1 Preparation of Payload Small Molecule Intermediate 11 [Chemical Structure]

[0267] Step A: N-(2-Bromo-5-fluorophenyl)acetamide: Concentrated H2SO4 (3 mL) was added dropwise to a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL), followed by 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) at room temperature. The mixture was stirred for 3 h and then poured into 2000 mL of ice water. A precipitate was formed, 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 1H NMR (400 MHz, DMSO-d6) δ 7.68 (dd, J = 8.9, 6.0 Hz, 1H), 7.61 (ddd, J = 10.7, 5.3, 3.1 Hz, 1H), 7.02 (ddd, J = 8.9, 8.0, 3.1 Hz, 1H), 2.11 (s, 3H). MS m / z 232.0 (M + H).

[0268] Step B: N-(5-Fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) was added dropwise to a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (1000 mL) at -78 °C over 1 h. After completion, the mixture was stirred under N2 for 0.5 h. Subsequently, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise at -78 °C over 0.5 h, and the mixture was stirred from -78 °C to room temperature for 6 h. The mixture was poured into 500 mL of saturated aqueous NH4Cl solution at 0 °C. 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 cake was recovered and dried in vacuo to obtain 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).

[0269] Step C: N-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: Silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol) were added 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), and the mixture was stirred at 30 °C for 6 h. The mixture was filtered through Celite, washed with CH2Cl2 (100 mL), and the filtrate was concentrated and purified by FCC (EA / PE = 0~40%) to obtain 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).

[0270] 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 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 h. After completion, the mixture was acidified with HCl (2N) to adjust the pH to 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 min, filtered, the cake was recovered, 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).

[0271] Step E: N,N’-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: To a solution of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-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 h under a H2 atmosphere. After cooling to 0 °C, Et3N (20 mL) was added dropwise, and the mixture was stirred at 0 °C for 1 h. It was filtered through celite, and the filtrate was poured into ice water (500 mL). It was 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 min, filtered, the cake was recovered, and dried in vacuo to give N,N’-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g) as a yellow solid. MS m / z 279.1 (M+H).

[0272] Step F: N,N'-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g, 28.39 mmol) in MeOH (150 mL) was added aqueous HCl solution (2N, 150 mL), and the mixture was stirred at 50 °C for 7 h. After cooling to 0 °C, saturated aqueous NaHCO3 was added dropwise to adjust the pH to 8. The mixture was extracted with ethyl acetate (200 mL × 3), washed with brine (200 mL × 2), dried over Na2SO4 and concentrated under reduced pressure to obtain N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (6.0 g) as a yellow solid. 1 1H 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).

[0273] Step G: N-(8-Amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (4.0 g, 16.93 mmol) in DMF (80 mL) was added NCS (2.26 g, 16.93 mmol) at 0 °C little by little, and the mixture was stirred at room temperature for 16 h. The mixture was poured into 200 mL of ice water. A precipitate was formed and collected by filtration and dried in vacuo at room temperature to obtain N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide (4.0 g) as a yellow solid. 11H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 8.0 Hz, 1H), 7.71 (s, 2H), 6.62 (d, J = 11.9 Hz, 1H), 4.53 (ddd, J = 13.0, 8.0, 4.7 Hz, 1H), 3.18 - 3.04 (m, 1H), 2.91 (ddd, J = 17.5, 12.4, 4.8 Hz, 1H), 2.21 - 2.08 (m, 1H), 1.99 - 1.83 (m, 4H). MS m / z 271.0 (M+H).

[0274] 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-[d]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide: To a mixture of N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g, 14.78 mmol) in toluene (400 mL) were added (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), and the mixture was heated to reflux for 24 h under N2. 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-[d]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).

[0275] Step I: (9S)-1-Amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]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[d]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[d]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).

[0276] 2.3.2 Preparation of Intermediate 12 (12-1, 12-2)

Chemical formula

[0277] (9S)-1-Amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (Intermediate 11) was prepared as the TFA salt of 12-1 and 12-2 by preparative HPLC.

[0278]

Table 4

[0279] The above HPLC conditions: Equipment: Agilent 1200, Chromatography column: Waters XBridge C18 4.6*50mm, 3.5um, Flow rate: 2.0 mL / min, Gradient elution: 5.0% - 95.0% - 95.0% - 5.0% - 5.0%, 0.00min - 1.50min - 2.50min - 2.52min - 3.00min, Temperature: 40°C, Mobile phase: A: Acetonitrile, B: H2O (0.05% TFA), Wavelength: 214nm / 254nm.

[0280] 2.3.3 Preparation of Linker - Payload 2 [Chemical formula]

[0281] opSu is [Chemical formula] a mixture of.

[0282] 2.3.3.1 Preparation of Compound 13 (Step A) Weighed 4.33 g of Fmoc - Gly - Gly - OH and 6.84 g of Pb(OAc)4 and added them to a 500 ml one - neck round - bottom flask. Added anhydrous THF / toluene (120 / 40 ml) under a nitrogen atmosphere and stirred to dissolve. Subsequently, 1.16 mL of pyridine was added to the reaction system. The reaction system was heated to 80°C and refluxed for 5 hours under a nitrogen atmosphere. Samples were taken and detected by HPLC to monitor the reaction.

[0283] The reaction system was cooled to room temperature, filtered, and the filter cake was washed 3 times with EA. The filtrates were combined and concentrated until dry. Column chromatography was performed (PE:EA = 100:0~50:100) to obtain the target product as about 2000 mg of a white solid with a yield of 44%.

[0284] 2.3.3.2 Preparation of Compound 15 (Step B) 200 mg of Compound 13 was weighed and added to a 100 mL one-neck round-bottom flask. Subsequently, 15 mL of THF was added and stirred to dissolve. Subsequently, Compound 14 (312 mg, 3.0 equiv) and TsOH·H2O (15 mg, 0.15 equiv) were added to the reaction system. The reaction system was reacted overnight at room temperature. Samples were taken and detected by TLC (PE / EA = 1:1) to monitor the reaction. The raw material almost disappeared and new spots were detected.

[0285] Saturated sodium hydrogen carbonate solution was added to quench the reaction. Extraction was performed 3 times with EA. The organic phases were combined, washed with physiological saline, dried over anhydrous magnesium sulfate, and concentrated. The crude product was purified by column chromatography (PE:EA = 5:1~1:1) to obtain the target product as a colorless oil in a yield of 29% at about 80 mg. MS: [M+H] + = 501.1.

[0286] 2.3.3.3 Preparation of Compound 16 (Step C) 200 mg of Compound 15 was weighed and added to a 100 mL one-neck round-bottom flask. Subsequently, 10 mL of EtOH and 5 mL of EA, which were completely dissolved, were added. Subsequently, 40 mg of palladium carbon was added to the reaction system under a nitrogen atmosphere, and the reaction system was purged 3 times with hydrogen gas. The reaction system was maintained under a hydrogen atmosphere and stirred at room temperature for 0.5 h. Samples were taken and detected by TLC (DCM / MeOH = 10:1) to monitor the reaction. The raw material almost disappeared and new spots were detected.

[0287] The reaction system was filtered, and the filter cake was washed 3 times with EA. The filtrates were combined and concentrated until dry to obtain the product as a white solid in a yield of 100% at 200 mg. The product can be directly used in the next reaction without purification. MS: [M-H] - = 409.4.

[0288] 2.3.3.4 Preparation of Compound 21 (Step D) Step D-1 2.0 g of dichloro resin was weighed and placed into a polypeptide synthesis tube. DCM (10 ml) was added and it was swollen at room temperature for 30 minutes. The solvent was removed by vacuum suction. The resin was washed twice with DCM, with a volume of 7 mL and a time of 1 minute for each wash. The solvent was removed by vacuum suction. Subsequently, 200 mg of Compound 16 was weighed and added to a 50 ml centrifuge tube. DCM (about 10 ml) was added. The solid was dissolved by shaking. It was added to the above resin. It was stirred to immerse all the resin in the solution (if resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). It was stirred for 4 - 5 hours. After the reaction was completed, an appropriate amount of methanol was added. It was stirred for 30 min. The solvent was removed by vacuum suction. The resin was washed in sequence once with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, with a volume of 7 mL and a time of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was suitable for the next coupling step.

[0289] Step D-2 It was deprotected twice by adding 10 mL of a ready-made 20% piperidine / DMF solution, with a reaction time of 10 minutes each time. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed in sequence twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, with a volume of 7 mL and a time of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. Both the resin and the solution were dark blue.

[0290] 563 mg of Fmoc-Phe-OH and 197 mg of HOBt were added to a 50 ml centrifuge tube. Subsequently, about 7 mL of DMF was added. The solid was dissolved by shaking. Subsequently, 0.24 mL of DIC was added. It was activated for 10 - 30 minutes to obtain an activated reaction solution.

[0291] 3 molar equivalents of the activation reaction solution were added to the resin. The mixture was stirred to completely immerse the resin in the solution (if resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the solvent was removed by vacuum suction. The resin was washed successively twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, with a volume of 7 mL and a time of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution had a yellowish color, indicating that it was suitable for the next coupling step.

[0292] Step D-3 Deprotection was carried out twice by adding 10 mL of a ready-made 20% piperidine / DMF solution, with a reaction time of 10 minutes each time. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed successively twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, with a volume of 7 mL and a time of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. Both the resin and the solution were dark blue.

[0293] 531 mg of Fmoc-GG-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Subsequently, approximately 10 mL of DMF was added. The solid was dissolved by shaking. Subsequently, 0.24 mL of DIC was added. Activation was carried out for 10 - 30 minutes to obtain the activation reaction solution.

[0294] 3 molar equivalents of the activation reaction solution were added to the resin. The mixture was stirred to completely immerse the resin in the solution (if resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. The resin was washed successively twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, with a volume of 7 mL and a time of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution had a yellowish color, indicating that it was suitable for the next coupling step.

[0295] Step D-4 Deprotection was carried out twice by adding 10 mL of a commercially available 20% piperidine / DMF solution, with a reaction time of 10 minutes each time. 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. For each washing, the volume was 7 mL and the time was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. Both the resin and the solution were dark blue. Subsequently, 462 mg of MC-OSu was placed in a 50 mL centrifuge tube, and about 10 mL of DMF was added. The solid was dissolved by shaking. Subsequently, 0.24 mL of DIEA was added to the resin. The resin was stirred to be completely immersed in the solution (if the resin adhered to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out 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. For each washing, the volume was 7 mL and the time was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution had a yellowish color, indicating that it was suitable for the next coupling step.

[0296] Step D-5 The resin was washed twice with 10 mL of methanol. Subsequently, the solvent was completely removed by vacuum suction. The resin was poured out and weighed. The dissolution buffer was prepared in a 250 mL Erlenmeyer flask, where the ratio of TFE / DCM was 80% / 20%, and the volume was 7 - 8 times the weight of the peptide resin. The dissolution buffer was added to the peptide resin and shaken well. The resin was completely immersed in the dissolution buffer and dissolved at room temperature for 2 - 3 hours. Then, the dissolution buffer was filtered through a simple filter made with a syringe, and the resin was washed with 1 - 2 mL of DCM and discarded. Subsequently, 150 mL of pre-cooled anhydrous ether was added to the dissolution buffer, shaken well, and then left to stand for 20 - 30 minutes. The above system was centrifuged in a centrifuge at 3500 rpm for 3 minutes using a 50 mL centrifuge tube, and the supernatant was poured out and discarded. The solid was shaken with pre-cooled anhydrous ether, washed once under ultrasonic waves, 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 a product as a 125 mg white solid with a yield of 40%. MS: [M - H] - = 641.5.

[0297] 2.3.3.5 Preparation of Compound 22 (Step E) 150 mg of starting compound 21 and 55 mg of TSTU were weighed and added into a 10 mL one-neck round-bottom flask. Anhydrous DMF (3 mL) was added under a nitrogen atmosphere and stirred for 20 min. Subsequently, 18 mg of compound 12 - 1 and 20 μL of DIEA were sequentially added to the reaction system. The mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction was monitored by sampling and detecting the sample by HPLC. The starting material peak completely disappeared, and a new peak was detected.

[0298] The reaction system was subjected to preparative purification to recover the target product and freeze-dried to obtain a product as a ~22 mg yellowish solid. MS: [M + H] + = 1081.0.

[0299] 2.3.3.6 Preparation of Linker-Payload 2 (Step F) Compound 22 (30 mg) was weighed and placed into a 10 mL single-neck round-bottom flask, and purified water (2 mL) was added. It was stirred until dissolved. A DMF solution (2 mL) containing linker-payload intermediate 1 (19.5 mg) was added to the reaction system and stirred. After reacting overnight, the reaction was monitored by HPLC until all the raw materials were converted into the intermediate. 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 carried out at 0 - 40 °C for an additional 0.2 - 20 h. The reaction was monitored by HPLC until all the intermediates were consumed, and then it was quenched with acetic acid solution.

[0300] The reaction system was subjected to preparative separation and purification, and the target product was recovered and freeze-dried to obtain about 25 mg of a yellowish solid, linker-payload 2. MS: [(M + 3H) / 3] + = 1194.4.

[0301] 2.4 Preparation of Linker-Payload 3

Chem.

[0302] opSu is

Chem.

[0303] Step 2.4.1: Preparation of Linker-Payload Intermediate 1

Chem.

[0304] The linker-payload intermediate 1 is synthesized by conventional solid-phase polypeptide synthesis using Rink-amide-MBHA resin. Fmoc was used to protect the amino acids in the linking unit. The conjugation reagent was selected from HOBT, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the resin was cleaved with trifluoroacetic acid. The product was purified by HPLC and lyophilized for storage for use. Theoretical mass: 1383.70, measured value: [M-H] - = 1382.6.

[0305] Step 2.4.2: Preparation of linker-payload 3 The linker-payload intermediate 1 and intermediate MC-GGFG-Dxd (purchased as a commercial product) with a molar ratio of ~1:2 were weighed and dissolved in water and DMF respectively, and then mixed well to obtain a mixture, which was reacted at 0 - 40 °C for 0.5 - 30 h. When the reaction was completed, an appropriate amount of Tris Base solution or other solution to promote the ring-opening reaction was directly added to the reaction mixture, and the reaction was carried out at 0 - 40 °C for another 0.2 - 20 h. After the reaction was completed, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain linker-payload 3. Theoretical mass: 3486.52, measured value: [(M + 3H) / 3] + = 1163.3.

[0306] 2.5 Preparation of linker-payload 4

Chemical formula

[0307] Step A: Synthesis of intermediate (a) Fmoc-PEG4-VC-OH (1 equiv) and p-hydroxybenzyl alcohol (5.0 equiv) were added to the reaction flask and dissolved in a solvent of DCM:MeOH = 2:1. Under a nitrogen atmosphere, EEDQ (5.0 equiv) was added and stirred uniformly, and then the reaction solution was reacted in the dark at room temperature. The reaction was monitored by HPLC until completion (about 3 h). The reaction system was directly prepared by preparative HPLC, and the prepared solution was lyophilized to obtain compound intermediate (a) (white solid, yield 41%). C 43 H 60 O 11 N6[M+H] + The calculated value of MS (ESI) for [M+H] is 849.4, and the measured value is 849.8.

[0308] Step B: Synthesis of intermediate (b) Compound intermediate (a) (1.0 equiv) was dissolved in DMF, and the mixture was cooled to 0 °C. P-(dinitrobenzene) carbonate (4.0 equiv) and DIPEA (6.0 equiv) were added to the reactants. The reaction system was stirred at 0 °C for 0.5 h, and then stirred again at room temperature for 2 h. The reaction was monitored by HPLC until completion. The reaction solution system was concentrated and purified by column chromatography (eluted with 0 - 10% MeOH in DCM) to obtain compound intermediate (b) (white solid, yield 75%). C 51 H 64 O 15 N7[M+H] + The calculated value of MS (ESI) for [M+H] is 1015.4, and the measured value is 1015.0.

[0309] Step C: Synthesis of intermediate (c) Compound intermediate (b) (1.2 equiv) and HOBT (2.0 equiv) were weighed and dissolved in DMF, and then MMAE (1.0 equiv) and DIPEA (10.0 equiv) were added and stirred uniformly to react. The mixture was reacted at room temperature overnight. The reaction was monitored by HPLC until completion (about 16 h), and the obtained mixture was directly used in the next reaction.

[0310] Step D: Synthesis of intermediate (d) 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 completion (about 1.5 h). The reaction system was directly prepared by preparative HPLC, and the prepared solution was lyophilized to obtain Compound H0152 (yellow solid, yield 62%). C 69 H 116 O 17 N 11 Calculated MS (ESI) value of [M+H]+: 1370.8, measured value: 1370.6.

[0311] Step E: Synthesis of Intermediate (e) Compound Intermediate (d) (2.4 equivalents) and Compound b (1.0 equivalent) were weighed and dissolved in DMF, and then DIPEA (4.0 equivalents) was added and the reaction was stirred uniformly 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 completion (about 2 h). The reaction system was directly prepared by preparative HPLC, and the prepared solution was lyophilized to obtain Compound Intermediate (e) (yellow solid, yield 50%). C 212 H 319 O 63 N 31 [M + 4H+] 4+ Calculated MS (ESI) value of / 4: 1084.4, measured value: 1085.0.

[0312] Step F: Synthesis of Intermediate (f) 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 completion (about 0.5 h). The reaction system was directly prepared by preparative HPLC, and the prepared solution was lyophilized to obtain Compound Intermediate (f) (yellow solid, yield 90%). C 197 H 309 O 61 N 31 [M + 4H+] 4+ Calculated MS (ESI) value of / 4: 1028.9, measured value: 1029.4.

[0313] Example 3 Preparation of Targeted Molecule-Drug Conjugate 3.1 The linker-payload intermediates were each site-specifically conjugated to an antibody by ligase to form an ADC. For the method of the conjugation reaction, reference may be made to WO2015165413A1. ADC-4 was prepared by the same method as Example 3 in WO2015165413A1. The obtained ADCs are as shown in the following table.

[0314] [Table 5]

[0315] 3.2 The reference ADC enherts were prepared by directly linking the intermediate MC-GGFG-Dxd to an (optionally modified) antibody (Cys conjugation, i.e., conjugation via a linkage formed by the maleimide structure of Cys and a thiol group). The method of the conjugation reaction is known in the art.

[0316] Effect Example 1 Bystander killing effect of the conjugate in the SK-BR-3 and MDA-MB-468 co-culture SK-BR-3 tumor cells (ATCC, HTB-30) were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. 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 in a 0% CO2 atmosphere. The MDA-MB-468-Luc-GFP cell line was constructed by lentiviral infection method and sorted by FACS. The co-culture of SK-BR-3 and MDA-MB-468-Luc-GFP was at a ratio of 4:1 (1×10 4Cells / well) were seeded in Corning® 96-well black / clear bottom polystyrene microplates for 24 h. Test substances (ADC-1, ADC-2, ADC-3, Enhertu, and ADC-4) were administered according to the experimental design and incubated for 120 h. The MDA-MB-468-Luc-GFP cell count was performed by BioTek Cytation3 (BioTek, LAB14002). And the luciferase activity was detected by Firefly Luciferase Reporter Gene Assay Kit (Beyotime, RG006) in BioTek Synergy HTX (BioTek, MAB16038).

[0317] The SK-BR-3 and MDA-MB-468-Luc-GFP co-culture (Figure 1), ADC-1 and ADC-2 showed similar efficacy. Both ADC-1 and ADC-2 showed efficacy equal to or greater than that of Enhertu.

[0318] Effect Example 2: In vivo effects of ADC-2 and ADC-1 in the JIMT-1 CDX model JIMT-1 tumor cells (DSMZ-ACC589) were maintained in vitro as monolayer cultures in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics-antifungals at 37 °C in a 5% CO2 atmosphere in air. The tumor cells were passaged regularly twice a week by trypsin-EDTA treatment.

[0319] SCID beige, female, 6 - 8 weeks old, body weight about 18 - 20 g. A total of 27 animals (18 + 50% reserve) were required for the study and were obtained from Shanghai Lingchang biotechnology co. LTD. or other approved vendors.

[0320] To the right flank of each mouse, (5×10 6 ) JIMT-1 tumor cells in 0.2 mL of PBS containing Matrigel (1:1) were subcutaneously inoculated for tumor formation. In the efficacy study, the animals were randomized and the average tumor volume was about 100 - 200 mm 3Treatment was initiated when it reached. The administration of the test substances (ADC-2, ADC-1) and the number of animals in each group followed the experimental design.

[0321] After inoculation, the morbidity and mortality rates of the animals were examined daily. During regular monitoring, the animals were examined for any effects of tumor growth and treatment on normal behaviors such as motor ability, food and water intake, weight gain or loss (weight was measured twice a week), and thinning of the eyes / hair, as well as any other abnormal effects. The main evaluation item was to confirm whether tumor growth could be delayed or the mice could be cured.

[0322] Statistical analysis: For comparisons between two groups, an independent samples t-test was used. For comparisons between three or more groups, one-way ANOVA was performed. When a significant F statistic (the 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.

[0323] According to the results (Figure 2), ADC-1 showed the same or higher efficacy than ADC-2.

[0324] Effect Example 3: In vivo effects of ADC-2, ADC-3, and Enhertu in the JIMT-1 CDX model In the right flank of each mouse, JIMT-1 tumor cells (5×10 6 ) in a 0.2 mL mixture of PBS and Matrigel (PBS:Matrigel = 1:1) were subcutaneously inoculated for tumor formation. Treatment was initiated on the 10th day after tumor inoculation when the average tumor volume reached 200 mm 3 . The average tumor volume of the vehicle control group reached 1,474 mm 3 on the 35th day after administration. T / C and TGI values were calculated based on the tumor volume. The calculation formula is T / C% = T RTV / C RTV ×100% (T RTV : RTV of the treatment group, C RTV : RTV of the vehicle control group). The relative tumor volume (RTV) was calculated based on the tumor measurement results, and the calculation formula is RTV = V t / V0, where V0 is the average tumor volume measured at the time of grouping (i.e., D0), and V t is the average tumor volume in a single measurement, and T RTV and C RTV collected data on the same day. Calculation of TGI (%): TGI (%) = [1 - (average tumor volume at the end of treatment in the treatment group - average tumor volume at the start of treatment in the treatment group) / (average tumor volume at the end of treatment in the vehicle control group - average tumor volume at the start of treatment in the vehicle control group)] × 100%. Treatment with ADC-2 (T / C = 6.23%, TGI = 108.47%, p = 0.011) and Enhertu (T / C = 33.51%, TGI = 76.90%, p = 0.041) at 5 mg / kg showed significant antitumor activity, with average tumor volumes of 92 mm 3 and 494 mm 3 respectively. Treatment with ADC-3 (T / C = 43.16%, TGI = 63.73%, p = 0.089) at 5 mg / kg also showed certain antitumor activity, with an average tumor volume of 636 mm 3 on day 35.

[0325] According to the results (Figure 3), there was no statistically significant difference in the efficacy between ADC-3 and Enhertu. These data also suggested that ADC-2 showed higher antitumor efficacy than Enhertu in the JIMT-1 model.

[0326] According to the results of Effect Example 2 (Figure 2) and Effect Example 3 (Figure 3), the conjugate of ADC-1 showed efficacy equal to or higher than that of ADC-2 and even higher efficacy than Enhertu.

[0327] Effect Example 4: In vivo effect of the conjugate in the NCI-N87 CDX model 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-antifungal at 37°C in a 5% CO2 atmosphere in air. The tumor cells were passaged regularly twice a week by trypsin-EDTA treatment.

[0328] BALB / c nude, female, 6 - 8 weeks old, body weight approximately 18 - 20 g. A total of 25 animals (18 + 39% reserve) were required for the test and were obtained from Shanghai Lingchang biotechnology co., LTD. or other approved vendors.

[0329] To the right flank of each mouse, 0.2 mL of NCI - N87 tumor cells (10×10 6 ) in PBS and Matrigel (1:1) was subcutaneously inoculated for tumor growth. In the efficacy test, the animals were randomized and treatment was initiated when the average tumor volume reached approximately 150 - 200 mm 3 . The administration of the test articles (ADC - 2, ADC - 1) and the number of animals in each group were in accordance with the experimental design.

[0330] After inoculation, the morbidity and mortality of the animals were examined daily. At regular monitoring, the animals were examined for any effects of tumor growth and treatment on normal behaviors such as locomotor ability, food and water intake, weight gain or loss (weight was measured twice a week), thinning of fur / coat color of eyes, etc., and any other abnormal effects. The main evaluation item was to confirm whether the tumor growth could be delayed or the mice could be cured.

[0331] According to the results (Figure 4), the conjugate of ADC - 1 showed the same efficacy as ADC - 2.

[0332] Effect Example 5: Effect of ADC - 5 on the proliferation of TROP2 - positive tumor cells BxPC - 3 1) TROP2 - positive human pancreatic cancer cells BxPC - 3 were inoculated into 96 - well cell plates at 100 μl / well (containing 1000 - 5000 cells) and cultured overnight in a cell incubator at 37°C, 5% CO2, 95% air and 100% humidity.

[0333] 2) ADCs at different concentrations (200, 40, 8, 1.6, 0.32, 0.064, 0.013, 0.0026, 0.00051, and 0.00010 nM) were added to BxPC-3 cells cultured overnight. Puromycin at a final concentration of 5 μM was added to the control group. Incubation was continued at 37 °C for 120 h.

[0334] 3) The cell plates were taken out from the cell incubator at 37 °C and equilibrated for about 30 min until they reached room temperature. 100 μl of CellTiterGlo reagent was added to each well. After shaking for 2 min, the oscillator was left in the dark at room temperature for 10 min, and the relative light units (RLU) were measured using a Cytation3 microplate analyzer.

[0335] 4) The results of the inhibitory effects of different drugs on the growth of tumor cells are shown in Table 3 and Figure 5. The ADCs have an obvious inhibitory effect on TROP2-positive cells. Among them, ADC-5 and ADC-6 are essentially the same and are significantly superior to ADC-7.

[0336]

Table 6

[0337] Example of Effect 6: Detection of Endocytosis of ADC-5 by TROP2-Positive Tumor Cells 1) TROP2-positive human gastric cancer cells NCI-N87 and human breast cancer cells MDA-MB-468 were incubated in a 96-well cell plate at 100 μl (10,000 - 50,000 cells) / well and incubated with 50 μg / mL of fluorescently labeled ADC at 4 °C in the dark for 30 min.

[0338] 2) After incubation, unbound ADC was cleaned with PBS, and the cells were incubated in a cell incubator at 37 °C, 5% CO2, 95% air, and 100% humidity for 10 min, 30 min, 1 h, 1.5 h, 2.5 h, and 3.5 h, respectively. Then the cells were taken out, and pre-cooled PBS was added to terminate endocytosis. An acid buffer solution with a pH value of 2.5 was used in an ice bath for 3 minutes, and the cells were precipitated by centrifugation at 2000 rpm for 3 minutes. Then the cells were resuspended in 100 μl / well of FACS buffer and prepared for flow cytometry detection.

[0339] 3) The results of endocytosis of TROP2-positive tumor cells against the detected ADC are shown in Table 4 and Figure 6. The endocytosis of ADC-5 among the ADCs is essentially the same as that of ADC-6 and monoclonal antibody Ab2. Under the same conditions, the negative control of human IgG1 did not induce endocytosis of cells, indicating that the preparation of ADC by conjugating a monoclonal antibody with a payload does not affect the intracellular endocytosis induced by the monoclonal antibody, and the endocytosis of ADC is target-dependent.

[0340]

Table 7

[0341] Example of Effect 7: Effect of ADC-5 on Tumor Growth in a TROP2-Positive NCI-N87 CDX Mouse Model 1) Cell culture: NCI-N87 tumor cells (ATCC, catalog number CRL-5822) were maintained in vitro as a monolayer culture in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal at 37 °C in a 5% CO2 atmosphere in air. The tumor cells were passaged regularly twice a week by trypsin-EDTA treatment. For tumor inoculation, cells growing in the exponential growth phase were collected and counted.

[0342] 2) Animals: BALB / c female nude mice, 7 - 9 weeks old, with a body weight of approximately 18 - 22 g. A total of 24 mice were required for the experiment and were obtained from Shanghai Lingchang Biotechnology Co., Ltd.

[0343] 3) Tumor inoculation and administration: 0.2 mL (10×10 6 ) of NCI - N87 tumor cells (the ratio of PBS to Matrigel was 1:1) were subcutaneously inoculated into the right flank of each mouse. When the average tumor volume reached approximately 150 - 200 mm 3 , the animals were randomly divided into groups of 6 according to the tumor size and treated by intravenous injection at a dose of 3 mg / kg. The entire process of animal breeding and experiment met the requirements of animal welfare.

[0344] 4) Effect evaluation: After administration, the tumor volume and animal body weight were measured weekly. The tumor volume was calculated using the formula V = 0.5×a×b 2 , where a and b are the major and minor diameters of the tumor, respectively. Subsequently, the tumor growth inhibition rate (TGI, %) and the tumor relative growth rate (T / C, %) were calculated based on the tumor volume. The calculation formula for TGI in each group is TGI(%) = [1 - (T i - T0) / (V i - V0)]×100, where T i is the average tumor volume of the treatment group on a specific day, T0 is the average tumor volume of the treatment group on the first day of treatment, V i is the average tumor volume of the vehicle control group on the same day as T i , and V0 is the average tumor volume of the vehicle control group on the first day of treatment. The calculation formula for the T / C(%) value is T / C% = T RTV / C RTV ×100% (T RTV : relative average tumor volume (RTV) of the treatment group, C RTV : relative average tumor volume (RTV) of the vehicle control group on the same day as T RTV ). The relative tumor volume (RTV) of each group was calculated as 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 specific day.

[0345] 5) Statistical analysis: Independent-sample t-tests were used for comparisons between two groups. One-way ANOVA was performed for comparisons among three or more groups. When a significant F statistic (the ratio of treatment variance to error variance) was obtained, multiple comparison procedures were applied after the ANOVA. All data were analyzed using SPSS 17.0. P < 0.05 was considered a statistically significant difference.

[0346] 6) Changes in tumor volume and body weight of animals are shown in detail in Table 5 and Figure 7. The inhibitory effects of ADC-5 and ADC-6 on NCI-N87 tumor volume were the same, and all of the above groups were highly significant compared to the control group. No significant weight loss was observed in each ADC administration group, indicating that mice have good tolerance to ADC-5 and ADC-6.

[0347]

Table 8

[0348] Effect Example 8: Effect of ADC-5 on tumor growth in a TROP2-positive FaDu CDX mouse model This experiment was carried out with reference to the method described in Effect Example 7, but the administration concentration was set at 2 mg / kg here. Changes in tumor volume and body weight of animals are shown in Table 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 of the above groups showed a significant difference compared to the control group. No significant weight loss was observed in each ADC administration group, indicating that mice have good tolerance to ADC-5 and ADC-6.

[0349]

Table 9

[0350] Effect Example 9: Effect of ADC-8 on tumor growth in an FGFR3-positive RT112 / 84 CDX mouse model This experiment was carried out with reference to the method described in Effect Example 7, but the administration concentration was set at 5 mg / kg here. The changes in tumor volume and animal body weight are shown in Table 7 and Figure 9. The inhibitory effects of ADC-8 and ADC-9 on the RT112 / 84 tumor volume are the same, significantly exceeding ADC-10, and all of the above groups are extremely significant compared to the control group. There is no difference in body weight between each ADC administration group and the control group, indicating that mice have good tolerance to ADC-8, ADC-9, and ADC-10.

[0351]

Table 10

[0352] Effect Example 10: Effect of ADC-8 on tumor growth in FGFR3-positive RT4 CDX mouse model This experiment was carried out with reference to the method described in Effect Example 7, but the administration concentration was set at 5 mg / kg here. The changes in tumor volume and animal body weight are shown in Table 8 and Figure 10. ADC-8, ADC-9, and ADC-10 all have a certain inhibitory effect on the RT4 tumor volume, and ADC-8 and ADC-10 are slightly superior to ADC-9. There is no difference in body weight between each ADC administration group and the control group, indicating that mice have good tolerance to ADC-8, ADC-9, and ADC-10.

[0353]

Table 11

[0354] Sequence listing SEQ ID NO: 1: Ab0001-LCCT L -HC light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG Sequence number 2: Ab0001-LCCT L -HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 3: Ab0001-LCCT L -HCCT L Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG Sequence number 4: Ab0001-LCCT L -HCCT L Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG Sequence number 5: Ab2 light chain: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG Sequence number 6: Ab heavy chain: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 7: Ab light chain: QSVLTQPPSLSVAPGKTATFTCGGNNIGDKSVHWYRQKPGQAPVLVMYLDTERPSGIPERMSGSNFGNTATLTITRVEAEDEADYYCQVWDSGSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECSGALPETGG Accession number 8: Ab heavy chain: EVQLVQSGAEVKKPGASVKVSCKASGYMFTSYGISWVRQAPGQGLEWMGWVSTYNGDTNYAQKFQGRVTVTTDTSTSTAYMELRSLRSEDTAVYYCARVLGYYDSIDGYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A compound of formula (I), wherein 【Chemical 1】 in the formula,[[]]END]] W is hydrogen, LKb or -C ２ H ４ -(PEG) ｔ -(CO)NH ２ and is Y is LKa - LKb, each LKa is independently 【Chemical 2】 and Each LKb is independently L ２ - L １ - B, and Each B is independently a terminal group R １０ or is 1) a self-destructing spacer Sp1 and 2) one or a combination of two or more selected from the group consisting of a bond, or -CR １ R ２ -, C １－１０ alkylene, C ４－１０ cycloalkylene, C ４－１０ heterocyclylene, and -(CO)-, and 3) a combination with the terminal group R １０ and, R １０ is a group that can be removed when reacting with hydrogen or a group in the payload, Each L １ is independently a cleavable sequence 1 containing an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 contains 1 to 10 amino acids, Each L ２ is independently a bond or one or more —CH ２ — structures in alkylene are optionally —CR ３ R ４ —, —O—, —(CO)—, —S(=O) ２ —, —NR ５ —, 【Chemical Formula 2-1】 C ４－１０ cycloalkylene, C ４－１０ heterocyclylene, phenylene-replaced C ２－２０ alkylene, wherein said cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted with 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-O-R ９ and is substituted with at least one substituent selected therefrom, Ld2 and each Ld1 are independently a bond or -NH-C １－２０ alkylene-(CO)-, -NH-(PEG) ｉ -(CO)-, or are independently unsubstituted or substituted on the side chain with -(CO)-(PEG) ｊ -OR １１ and are natural amino acids or natural amino acid oligomers having a degree of polymerization of 2 to 10 substituted with -(PEG) ｔ - and -(PEG) ｉ - and -(PEG) ｊ - each is a PEG fragment having an optional additional C ２ H ４ at one end, containing a specified number of consecutive -(O-C ２ H ４ structural units or consecutive -(C １－１０ alkylene at one end, which is a PEG fragment having an optional additional C R １ 、R ２ 、R ３ 、R ４ 、R ５ 、R ６ 、R ７ 、R ８ 、R ９ are each independently hydrogen, halogen, -C １－１０ alkyl, -C １－１０ haloalkyl, C ４－１０ cycloalkylene, or R １ and R ２ together with the carbon atoms to which they are attached form a 3- to 6-membered cycloalkyl group, or R ３ and R ４ together with the carbon atoms to which they are attached form a 3- to 6-membered cycloalkyl group, R １１ is C １－１０ alkyl, and m is any integer from 1 to 5, n is any integer from 2 to 20, d is any integer from 0 to 6, particularly 1, 2, 3, each i is independently an integer from 0 to 100, preferably from 0 to 20, preferably each i is independently an integer from 0 to 12, more preferably from 0 to 8, particularly 4, each j is independently an integer from 1 to 100, preferably from 1 to 20, preferably each j is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, each t is independently an integer from 1 to 100, preferably from 1 to 20, preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, a compound.[[]]END]]

2. 【Fig. 3】 The compound according to claim 1, selected from

3. 【Fig. 4-1】 【Chemical Formula 4-2】 selected from in the formula,[[]]END]] m is any integer from 1 to 5, each t is independently an integer from 1 to 100, preferably from 1 to 20, preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, each j is independently an integer from 1 to 100, preferably from 1 to 20, preferably each j is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, the compound according to any one of claims 1 to 2.[[]]END]]

4. Ld2 and each Ld1 are independently a bond or 【Chemical Formula 5】 and each k is independently an integer from 1 to 100, preferably from 1 to 20, preferably each k is independently an integer from 1 to 12, more preferably from 1 to 7, particularly 1, or 3 or 5, preferably, Ld1 is 【Chemical Formula 6】 and Ld2 is independently a bond or 【Chemical Formula 7】 selected from each i is independently an integer from 0 to 5, preferably from 0 to 4, particularly 0, 2 or 4, each j is independently an integer from 1 to 100, preferably from 1 to 20, preferably each j is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, the compound according to any one of claims 1 to 2.[[]]END]]

5. The 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, the cleavable sequence 1 is Gly - Gly - Phe - Gly or Val - Cit, and / or Sp1 is selected from PABC, acetal, heteroacetal and combinations thereof, preferably Sp1 is acetal, heteroacetal or PABC, preferably said heteroacetal is selected from N,O-heteroacetals, preferably Sp1 is -O-CH ２ -U- or -NH-CH ２ -U-, wherein said -O- or -NH- is linked to cleavable sequence 1, U is absent or U is O, S or NH, preferably O or S, more preferably Sp1 is PABC, the compound according to any one of claims 1 to 4.

6. W is hydrogen, and / or R １０ is hydrogen, hydroxy, or [Chemical Formula 8] and R １１ 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, particularly 3, and / or d is 0 or any integer from 1 to 4, preferably 1, 2 or 3, more preferably 1, the compound according to any one of claims 1 to 5.

7. 【Fig. 9-1】 【Chemical Formula 9-2】 【Chemical Formula 9-3】 【Chemical Formula 9-4】 【Chemical Formula 9-5】 【Chemical Formula 9-6】 selected from In the formula, R １ , R ２ and R １０ are as defined in claim 1, g is an integer from 1 to 10, preferably 1, and the compound according to any one of claims 1 to 6.

8. A compound having the structure of formula (II), 【Chemical Formula 10】 wherein Q is hydrogen, -C ２ H ４ -(PEG) ｔ -(CO)NH ２ or LKb-P, M is LKa-LKb-P, P is a payload linked to the B moiety or the L moiety of the compound of formula (I) as defined in claim 1 １ and is n, d, Ld1, Ld2, t, LKa and LKb are as defined in claim 1, Preferably, M is LKa-L ２ -L １ -B-P, each B is independently non-existent, or 1) a self-destructive spacer Sp1, and 2) a combination with one or more combinations of divalent groups selected from bonding, or -CR １ R ２ -, C １－１０ alkylene, C ４－１０ cycloalkylene, C ４－１０ heterocyclylene and -(CO)-, or a combination of one or more combinations thereof Preferably, Sp1 is selected from PABC, acetal, heteroacetal and combinations thereof, more preferably, Sp1 is acetal, heteroacetal or PABC, still more preferably, said heteroacetal is selected from N,O-heteroacetal, more preferably, Sp1 is -O-CH ２ -U- or -NH-CH ２ -U-, wherein said -O- or -NH- is linked to the cleavable sequence 1, and U is absent or U is O, S or NH, preferably O or S, a compound.

9. A conjugate having the structure of formula (III), 【Chemical 11】 wherein Q is hydrogen, -C ２ H ４ -(PEG) ｔ -(CO)NH ２ 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, and Each B does not exist independently, or is a combination of 1) a self-destructive spacer Sp1 and 2) one or more combinations of divalent groups selected from bonding, or -CR １ R ２ -, C １－１０ alkylene, C ４－１０ cycloalkylene, C ４－１０ heterocyclylene and -(CO)-, and preferably, B is -NH-CH ２ -U-, or does not exist, or -NH-CH ２ -U-(CR １ R ２ ) ｇ -(CO)-, g is an integer from 1 to 10, preferably 1, R １ and R ２ are each independently hydrogen, halogen, -C １－１０ alkyl, -C １－１０ haloalkyl, C ４－１０ cycloalkylene, or R １ and R ２ together with the carbon atoms to which they are attached form a 3- to 6-membered cycloalkyl group, P is a payload linked to the B moiety or the L moiety of the compound of formula (I) as defined in claim 1, １ and A is a targeting molecule, z is an integer from 1 to 20, the conjugate.

10. The conjugate according to claim 9, having the structure of the following formula (III-1). 【Chemical 12】

11. Having the following structure, 【Chemical Formula 13-1】 【Chemical Formula 13-2】 preferably, z is from 1 to 4, preferably 2, n is an integer from 2 to 5, preferably n is 3, and L ２ is -(C ２ H ４ -O)- ｐ -(CH ２ ), ２ and p is from 2 to 4, and L １ is Gly-Gly-Phe-Gly, and B is -NH-CH ２ -U- or does not exist or -NH-CH ２ -U-(CR １ R ２ ), ｇ and -(CO)-, and U does not exist or U is O, and g is 1, and 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 atoms to which they are attached form a 3- to 6-membered cycloalkyl group, each i is independently an integer from 0 to 100, preferably from 0 to 20, preferably each i is independently an integer from 0 to 12, more preferably from 0 to 8, particularly 4, each j is independently an integer from 1 to 100, preferably from 1 to 20, preferably each j is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, each t is independently an integer from 1 to 100, preferably from 1 to 20, preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, m is any integer from 1 to 3, particularly 1 or 2, the conjugate according to claim 9 or 10.

12. The targeted molecule is an antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment is preferably modified to be linked to the G ｎ moiety in the compound of formula (I), Preferably, A is an anti-human monoclonal antibody linked to the remainder of the conjugate via a modified heavy chain and / or light chain C-terminus, provided that the modified heavy chain and / or light chain C-terminus is modified to include Leu-Pro-Xaa-Thr, Xaa is any natural or non-natural single amino acid, z is 2, preferably, the antibody is an anti-human HER2 antibody, an anti-human TROP2 antibody or an anti-FGFR3 antibody, the conjugate according to any one of claims 9 to 11.

13. The payload is a cytotoxin or a fragment thereof, and is optionally derivatized for attachment to the B moiety or the L １ moiety in the compound of formula (I), Preferably, the cytotoxin is a taxane, a maytansinoid, an auristatin, an epothilone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, an indole sulfonamide, a vinca alkaloid such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrous vinblastine, dolastatin 10 and its analogs, halicondrin B, eribulin, indole-3-oxoacetamide, a podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, a camptothecin and its derivatives, mitoxantrone, mitoguazone, a nitrogen mustard, a nitrosourea, an aziridine, benzodopa, carbocone, meturedepa, uredepa, dynemicin, esperamicin, neocarzinostatin, aclacinomycin, actinomycin, anthramycin, a bleomycin, actinomycin C, calicheamicin, calimomycin, cardinophilin, calimomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, a mitomycin, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferric doxorubicin (ferricselected from the group consisting of adriamycin, rhodomycin, leucochromomycin, streptozocin, dinostatin, zorubicin, trichothecene, T-2 toxin, verucarin A, basiliporin A, anguidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethyl folic acid, methotrexate, pteropterin, trimethoprim, edatrexate, fludarabine, 6-mercaptopurine, thiampurine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, floxuridine, calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitor and proteasome inhibitor, and / or Selected from vinblastines, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocarmycin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, drastansine, pyrrolobenzodiazepine, exatecan and its derivatives, and / or, Selected from auristatins, particularly MMAE, MMAF or MMAD, and / or, Selected from exatecan and its derivatives such as DX8951f, and / or, The conjugate according to any one of claims 9 to 12, selected from DXd-(1) and DXd-(2), preferably DXd-(1).

14. The payload is 【Chemical Formula 14】 selected from In particular, 【Chemical Formula 15】 The conjugate according to any one of claims 9 to 13, selected from

15. Formula (III) has the following structure, 【Chemical Formula 16-1】 【Chemical Formula 16-2】 【Chemical 16-3】 【Chemical Formula 16-4】 【Chemical 16-5】 each z is independently an integer from 1 to 20, the conjugate according to any one of claims 9 to 14.

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 an 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 cancer, cervical cancer, multiple myeloma or intrahepatic cholangiocarcinoma.

18. A compound of formula (IV), 【Chemical 17】 wherein Rpg is selected from hydrogen or a protecting group, preferably selected from acetyl, trifluoroacetyl, t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc), W, Ld1, Ld2, n and d are as defined in claim 1, the compound.

19. The compound according to claim 18, wherein the formula (IV) has the following structure. 【Chemical Formula 18】

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