Exatecan derivatives, payload linkers and conjugates thereof

Abstract

FIELD: biopharmaceuticals.SUBSTANCE: derivatives of exatecan, linkers with a payload and their conjugates, as well as antibody-drug conjugates.EFFECT: antitumor activity against HER2-positive tumor cells.88 cl, 10 dwg, 11 tbl, 5 ex

Description

Field of technology to which the present invention relates

[0001] The present invention relates to the field of biopharmaceuticals, in particular to exatecan derivatives, payload linkers and conjugates thereof, as well as antibody-drug conjugates, and to corresponding methods for producing and using them.Background of the invention

[0002] In traditional tumor treatment, chemotherapy is a key treatment strategy. However, off-target toxicity due to nonspecific accumulation in normal tissues, a narrow therapeutic window, and poor tolerability limit the use of chemotherapeutic drugs. In recent decades, targeted therapy using monoclonal antibodies and polypeptides that bind to specific markers on the surface of tumor cells has shown less toxicity than chemotherapy. However, both methods lack efficacy in killing tumor cells.

[0003] The strategy of using toxin-loaded antibodies to selectively kill target cells was first proposed in 1970. Tumor-targeting drug conjugates are mainly antibody-drug conjugates (ADCs), in which a drug is usually linked via a linker to an antibody that specifically recognizes tumor cell surface antigens, and the conjugate contains chemical toxins that effectively kill tumor tissues.

[0004] As of 2021, more than 120 ADCs were in development and clinical trials.

[0005] Most developed cytotoxic agents belong to two main families: tubulin inhibitors (maytansinoids or auristatins) and DNA damaging agents (primarily calicheamicins). All of these are extremely potent cytotoxic drugs, characterized by IC50 values ​​(inhibitory concentrations that inhibit 50% of cells) in the nanomolar and picomolar ranges, as well as an unfavorable toxicity profile when administered systemically. ADC conjugates allow the cytotoxic drug to be concealed in the bloodstream, allowing it to be delivered directly to tumor cells, significantly reducing the toxicity of these potent agents. However, drugs with a more favorable therapeutic index are considered unsuitable for use as cytotoxic agents in conjugates, as their IC50 values ​​are too low to be effective when released in the amounts delivered to tumor cells.

[0006] Studies of ADCs with increased DAR (drug-to-antibody ratio) or bystander effect offer a promising new approach to using less cytotoxic drugs as the cytotoxic agent in conjugates.

[0007] To date, the vast majority of cytotoxic agents in developed conjugates are anti-microtubule agents such as auristatins (e.g., monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)). Brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin are approved ADCs carrying MMAE as the cytotoxic payload.

[0008] Topoisomerase I (TOP1) inhibitors are used as a cytotoxic agent in conjugates in several ADCs. Sacituzumab govitecan carries SN-38, the active metabolite of irinotecan, as a cytotoxic agent. Enhertu (famtrastuzumab deruxtecan (DS8201a), also known as T-Dxd) carries the exatecan derivative deruxtecan (Dxd) as a cytotoxic agent (see publication WO 2014057687 A), and it has recently been approved for the treatment of breast cancer. Based on this, the field requires new small molecule topoisomerase I inhibitors, used alone or as a component of ADCs, with greater activity, stability, and physicochemical properties.

[0009] However, enhertu, as well as other commercially available ADCs and the majority of ADCs in clinical trials, are produced by chemical conjugation using a thiosuccinimide structure (thiosuccinimide linkage) to conjugate a small molecule drug to a targeting antibody or protein. The thiosuccinimide structure is formed by the reaction of a thiol group and a maleimide. The thiosuccinimide linkage is unstable. In vivo, reverse Michael addition or substitution with other thiol groups leads to the release of the cytotoxin from the ADC and off-target toxicity, which reduces safety and limits clinical use. For example, despite its high efficacy, enhertu caused more than 10% of cases of interstitial lung disease, limiting its use in some patients.On the other hand, the chemical coupling reaction is not site-specific, and the resulting ADCs exhibit high heterogeneity. Therefore, new ADCs with higher efficacy and lower toxicity remain urgently needed. Summary of the Invention

[0010] In the first aspect, a compound of formula (I) is provided: where opSu represents R 0 represents C 1-10 alkyl;n is any integer from 2 to 20;k1 and k2 independently represent an integer from 1 to 7;i is an integer from 1 to 100;j is an integer from 1 to 100;P1 and P2 independently represent a payload having the structure of a compound of formula (i'): where a is 0 or 1; each of the carbon atoms labeled p1* and p2* represents an asymmetric center, and the asymmetric center has the S-configuration, the R-configuration, or is racemic;L 1 selected from C 1-6 alkylene which is unsubstituted or is substituted by one substituent selected from halogen, -OH and -NH2; M is -CH2-, -NH- or -O-; L 2 represents C 1-3 alkylene;R 1 and R 2 independently selected from hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0011] In the second aspect, a conjugate having a structure of formula (II) is provided: where A is an antibody or antigen-binding fragment thereof; wherein the antibody or antigen-binding fragment is preferably modified to bind to a (Gly) moiety nin the compound of formula (I);z is an integer from 1 to 20; preferably from 1 to 4; especially 2;P1, P2, R 0 , opSu, n, k1, k2, i and j have the meanings defined above in formula (I).

[0012] In a third aspect, a pharmaceutical composition is provided comprising a conjugate of the invention and at least one pharmaceutically acceptable carrier.

[0013] In a fourth aspect, there is provided the use of the conjugate of the invention or the pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a disease; wherein the disease is a tumor or an autoimmune disease; wherein the tumor preferably comprises HER2-positive tumor cells.

[0014] In the fifth aspect, there is provided a compound having a structure of formula (i):

[0018] where

[0019] k represents an integer from 1 to 7; preferably 1, or 3, or 5, especially 5

[0020] P represents a payload having a structure of a compound of formula (i'), wherein the structure of formula (i') is as defined in formula (I).Brief Description of the Figures

[0021] Figure 1 shows the efficacy of the conjugates in the HER2-high SK-BR-3 cell line.

[0022] Figure 2 shows the efficacy of the conjugates in the HER2-high NCI-N87 cell line.

[0023] Figure 3 shows the efficacy of the conjugates in the HER2-negative cell line MDA-MB-468.

[0024] Figures 4a and 4b show the efficiency of the conjugates in the co-culture system of SK-BR-3 and MDA-MB-468. Figure 4a shows the result obtained by GFP fluorescence. Figure 4b shows the results obtained by luciferase substrate chemiluminescence. Figures 4a and 4b (from left to right) show CH-2-589, CH-2-593, CH-2-518, LC1184(8), and LC302-2-4(4), respectively.

[0025] Figure 5 shows the efficiency of conjugates in the co-culture system of SK-BR-3 and MDA-MB-468 obtained by GFP fluorescence.

[0026] Figure 6 shows the mean change in tumor volume over time in JIMT1 CDX model SCID Beige mice administered 5 mg / kg of the conjugates.

[0027] Figure 7 shows the mean change in tumor volume over time in BALB / c nude mice with Capan-1 CDX model that were administered 5 mg / kg of the conjugates.

[0028] Figures 8a and 8b show the mean change in tumor volume and change in body weight over time in BALB / c nude mice with CDX Capan-1 model that were administered 5 mg / kg of the conjugates.

[0029] Figure 9a and Figure 9b show the mean change in tumor volume and change in body weight over time in BALB / c nude mice with NCI-N87 CDX model that were administered 5 mg / kg of the conjugates.

[0030] Figure 10 shows the stability of the conjugates in serum ex vivo after 0, 24, 48 and 96 hours of incubation in pooled human serum. Detailed disclosure of the present invention

[0031] Specific embodiments of the present invention are presented below to illustrate the technical solutions of the present invention. Those skilled in the art can easily understand other advantages and effects achieved by the present invention through the contents disclosed in the description below. The present invention can also be implemented or applied through other various specific embodiments. Those skilled in the art can make various modifications and variations without departing from the essence of the present invention. Definitions

[0032] Unless otherwise defined below, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art. The techniques used herein are those commonly known to those skilled in the art, including variations and equivalent substitutions that are obvious to those skilled in the art. Although the following terms are believed to be understandable to those skilled in the art, the following definitions are set forth to better illustrate the present invention. When a trade name appears herein, it refers to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0033] When a specific amount, concentration, or other value or parameter is presented as a range of values, a preferred range of values, a preferred upper limit of values, or a preferred lower limit of values, it should be understood that this is equivalent to a specific disclosure of any range formed by combining any upper limit of values ​​or preferred value with any lower limit of values ​​or preferred value, regardless of whether the stated range is explicitly stated. Unless otherwise specified, the numerical ranges given herein include the endpoints of the range and all integer and fractional (decimal) values ​​within the stated range. For example, the expression "i is an integer between 1 and 20" means that i is any integer between 1 and 20, such as i could be 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 j, k and g should also be understood in a similar way.

[0034] Unless the context clearly requires otherwise, singular forms also include plural forms. The expression "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0035] The terms "approximately" and "approximately," when used in connection with a numerical value, generally mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval for the mean) or within ±10% of the specified value or within a wider range.

[0036] The term "optional" or "optional" means that the event described after it may, but need not, occur, and the description includes instances in which the stated event or circumstance occurs or does not occur.

[0037] The terms "comprises," "includes," "comprising," and "having" are open-ended and do not exclude additional, unlisted elements, steps, or ingredients. The term "consisting of" excludes any unlisted element, step, or ingredient. The term "consisting essentially of" means that the scope is limited to the stated elements, steps, or ingredients, as well as elements, steps, or ingredients that are optionally present and that do not materially affect the essential and novel characteristics of the claimed subject matter. It should be understood that the term "comprising" encompasses the terms "consisting essentially of" and "consisting of."

[0038] As used herein, the term "antibody" is used broadly and specifically includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired biological activity. The antibody may belong to any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and may be obtained from any suitable species. In some embodiments, the antibody is of human or murine origin. The antibody may also be a fully human antibody, a humanized antibody, or a chimeric antibody produced by recombinant methods.

[0039] The term "monoclonal antibodies" is used here to refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for a small number of possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. The word "monoclonal" indicates that the antibody's characteristics are obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any specific methods for obtaining the antibody.

[0040] An intact antibody or full-length antibody essentially comprises an antigen-binding variable region(s), as well as a light chain constant region(s) (CL) and a heavy chain constant region(s) (CH), which may include CH1, CH2, CH3, and CH4, depending on the antibody subtype. The antigen-binding variable region (also known as a fragment variable region, Fv fragment) typically comprises a light chain variable region (V l ) and the variable region of the heavy chain (V h ). The constant region may be a constant region with a native sequence (e.g., a constant region with a native human sequence) or a variant of its amino acid sequence. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interact with the immune system.

[0041] An antibody fragment may comprise a portion of an intact antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and an Fd fragment consisting of V domains. h and CH1, an Fv fragment, a fragment of a single-domain antibody (dAb), and an isolated complementarity-determining region (CDR). The Fab fragment is an antibody fragment obtained by cleavage of a full-length immunoglobulin with papain, or a fragment with the same structure, obtained, for example, by recombinant expression. The Fab fragment contains the light chain (containing V l and CL) and another chain, wherein said other chain comprises a heavy chain variable domain (V h) and a heavy chain constant region domain (CH1). The F(ab')2 fragment is an antibody fragment obtained by digestion of immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure obtained, for example, by recombinant expression. The F(ab')2 fragment essentially comprises two Fab fragments, where each portion of the heavy chain contains several additional amino acids, including cysteines, which form disulfide bonds linking the two fragments. The 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 multiple chains linked together, for example, by a disulfide bond and / or a peptide linker. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabodies, 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 can include any antibody fragment that, when inserted into the antibody framework (e.g., by substituting a corresponding region), can result in the formation of an antibody that immunospecifically binds to an antigen.

[0042] The antibodies of the present invention can be produced using techniques well known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, a genetically engineered recombinant antibody (or antibody mimetic) can be expressed using a suitable culture system (e.g., E. coli or mammalian cells). Genetic engineering methods can include, for example, the introduction of a ligase-specific recognition sequence at its ends.

[0043] HER2 refers to human epidermal growth factor receptor 2, which belongs to the epidermal growth factor receptor (EGFR) family of tyrosine kinases. In this application, the terms ErbB2 and HER2 have the same meaning and may be used interchangeably.

[0044] The term "antibody-drug conjugate" as used herein may be used interchangeably with the term "conjugate".

[0045] A small molecular weight compound is a molecule whose size is comparable to that of an organic molecule commonly used in medicine. This term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.), but includes small molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, the molecular weight of the small molecular weight compound may be, for example, from about 100 to about 2000 Da, from about 200 to about 1000 Da, from about 200 to about 900 Da, from about 200 to about 800 Da, from about 200 to about 700 Da, from about 200 to about 600 Da, from about 200 to about 500 Da.

[0046] A spacer is a structure that is located between different structural modules and can spatially separate these modules. The definition of a spacer is not limited to whether it performs a specific function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, where non-amino acid structures can be, but are not limited to, derivatives or analogs of amino acids. "Spacer sequence" refers to an amino acid sequence serving as a spacer, and examples thereof include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, etc., or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc.Self-cleaving spacers are covalent assemblies designed to correlate the cleavage of two chemical bonds following activation of a protective moiety in a precursor: upon stimulation, the protective moiety (e.g., the cleavable sequence) is removed, triggering a cascade of disassembly reactions leading to the sequential release of smaller molecules. Examples of self-cleaving spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetal, heteroacetal, and combinations thereof.

[0047] The term "alkyl" refers to a saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms that is joined to the rest of the molecule by a single bond. Alkyl groups include linear (unbranched) alkyl, branched alkyl, or cyclic alkyl (cycloalkyl), or partially cyclic alkyl (e.g., cyclic alkyl-linear alkyl and cyclic alkyl-branched alkyl). An alkyl group can contain from 1 to 10 carbon atoms, and it refers to C 1-10 an alkyl group, which may be, for example, C 1-6 alkyl group, C 1-4 alkyl group, C 1-3 alkyl group, C 1-2 alkyl, C3alkyl, C4alkyl, C 3-6alkyl. Non-limiting examples of linear alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc. Non-limiting examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.

[0048] The terms "cyclic alkyl" and "cycloalkyl" have the same meaning and are used interchangeably herein. Cyclic alkyl groups may include mono- or polycyclic groups (e.g., having 2 or more fused rings). In a multicyclic cycloalkyl, two or more rings may be joined together, bridged, or spiro-linked. The ring-forming carbon atoms of the cyclic alkyl group may be optionally substituted with oxo (i.e., C(O)). Cyclic alkyl groups may have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbon atoms (C 3-10 ). Examples of cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, and bicyclo[2.1.1]hexyl. In some embodiments, the cyclic alkyl is C 3-6 monocyclic or bicyclic cyclic alkyl, preferably C 3-6 monocyclic cyclic alkyl, especially cyclopropyl.

[0049] The term "partially cyclic" refers to the fact that the group contains one or more cyclic moieties and one or more acyclic (i.e., linear or branched) moieties. Partially cyclic alkyl groups can include cyclic alkyl-linear alkyl groups and cyclic alkyl-branched alkyl groups. Partially cyclic alkyl groups can have 4, 5, 6, 7, 8, 9, or 10 carbon atoms (C 3-10 ), including ring-forming carbon atoms and non-ring-forming carbon atoms. Examples of partially cyclic alkyl groups include, but are not limited to, C 3-9 cyclic alkyl-C1alkyl groups, C 3-8 cyclic alkyl-C2 alkyl groups, C 3-7 cyclic alkyl-C3 linear alkyl groups, C 3-6 cyclic alkyl-C4 linear alkyl groups, C 3-5 cyclic alkyl-C5 linear alkyl groups, C 3-4cyclic alkyl-C6 linear alkyl groups, C3 cyclic alkyl-C7 linear alkyl groups, C 3-7 cyclic alkyl-C3 branched alkyl groups, C 3-6 cyclic alkyl-C4 branched alkyl groups, C 3-5 cyclic alkyl-C5 branched alkyl groups, C 3-4 cyclic alkyl-C6 branched alkyl groups and C3 cyclic alkyl-C7 branched alkyl groups. In some embodiments, the partially cyclic alkyl is C 3-9 cyclic alkyl-C1alkyl group, preferably C 3-6 cyclic alkyl-C 1-2 alkyl group, C 3-6 cyclic alkyl-C1 alkyl group, more preferably C 3-4 cyclic alkyl-C 1-2 alkyl group, especially C 3-4 cyclic alkyl-C1alkyl group, and especially cyclopropylmethyl.

[0050] A divalent radical refers to a group derived from a corresponding monovalent radical by removing one hydrogen atom from a carbon atom with a free valence electron(s). A divalent radical has two connecting sites that are bonded to the rest of the molecule, and the two connecting sites may be on the same atom or on two different atoms of the divalent radical.

[0051] “Alkylene” or “alkylidene” refers to a saturated divalent hydrocarbon group. Alkylene groups include linear, branched, cyclic, or partially cyclic groups. Examples of linear alkylene groups include, but are not limited to, methylene (-CH2-), -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, etc. Examples of branched alkylene groups include, but are not limited to, -CH(CH3)-, -CH(C2H5)-, -CH(CH3)-CH2-, -CH(C3H7)-, -CH(C2H5)-CH2-, -C(CH3)2-CH2-, -(CH(CH3))2-, -CH(CH3)-(CH2)2-, -CH2-CH(CH3)-CH2-, -CH(C4H9)-, -C(CH3)(C3H7)-, -C(C2H5)2-, -CH(C3H7)-CH2-, -CH(C2H5)-CH(CH3)-, -CH(C2H5)-(CH2)2-, -CH2-CH(C2H5)-CH2-, -C(CH3)2-(CH2)2-, -CH2-C(CH3)2-CH2-, -CH(CH3)-(CH2)3-, -CH2-CH(CH3)-(CH2)2-, -CH(C5H 11)-, -C(C2H5)(C3H7)-, -C(CH3)(C4H9)-, -CH(C4H9)-CH2-, -C(C2H5)2-CH2-, -C(CH3)(C3H7)-CH2-, -CH(C2H5)-CH(C2H5)-, -CH(CH3)-CH(C3H7)-, -C(CH3)2-C(CH3)2-, -CH(C3H7)-(CH2)2-, -CH2-CH(C3H7)-CH2-, -CH(C2H5)-C(CH3)2-, -C(CH3)2-CH(CH3)-CH2-, -CH(CH3)-C(CH3)2-CH(CH2-, -CH(CH3)-C(CH3)2-CH(CH2-, -CH(CH3)-C(CH3)2-CH(CH2-, -CH(CH3)-CH(C2H5)-CH2-, -CH(CH3)-CH2-CH(C2H5)-, -CH(CH3)-C(CH3)2-CH2-, -(CH(CH3))3-, -C(CH3)2-(CH2)3-, -CH(CH2H5)-(CH2)3-, -CH2-CH(C2H5)-(CH2)2-, -CH2-CH(CH3)-CH(CH3)-CH2-, -(CH(CH3))2-(CH2)2-, -CH(CH3)-(CH2)2-CH(CH3)-, -(CH2)2-CH(CH3)-(CH2)2-, -CH2-CH(CH3)-(CH2)3-, -CH(CH3)-(CH2)4-, etc.

[0052] The terms "cyclic alkylene" and "cycloalkylene" have the same meaning and are used interchangeably herein. Examples of cyclic alkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, as well as divalent multicyclic alkyl groups containing fused, spiro, or bridged rings. In some embodiments, the cyclic alkylene is C 3-6 cyclic alkylene group, in particular C 3-4 a cyclic alkylene group, especially cyclopropyl.

[0053] Partially cyclic alkylene groups may include divalent radicals in which the two connecting sites that are connected to the rest of the molecule may be on one or more linear or branched alkyl moieties, on one or more cyclic alkyl moieties, or, respectively, on one or more cyclic alkyl moieties and linear or branched alkyl moieties.Examples of partially cyclic alkylene groups include, but are not limited to: (1) cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, as well as divalent multicyclic alkyl groups containing fused, spiro, or bridged rings, each of which is independently substituted with one or more linear or branched alkyl groups; (2) linear or branched alkylene groups, each of which is independently substituted with one or more cyclic alkyl groups; and (3) a group formed by the combination of one or more cyclic alkylene groups and one or more linear or branched alkylene groups, so long as a chemically stable structure is formed. In some embodiments, the partially cyclic alkylene is C. 3-9 cyclic alkyl-C1alkylene group, preferably C 3-6 cyclic alkyl-C 1-2alkylene group, C 3-6 cyclic alkyl-C1alkylene group, more preferably C 3-4 cyclic alkyl-C 1-2 alkylene group, especially C 3-4 cyclic alkyl-C1alkylene group, especially cyclopropylmethylene.

[0054] The compounds of the invention may be present in isotopically labeled or isotopically enriched form containing one or more atoms having an atomic mass or mass number different from the atomic mass or mass number of atoms most commonly found in nature. Isotopes may be radioactive or non-radioactive. Isotopes of atoms such as hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine include, but are not limited to, 2 H, 3 H, 13 WITH, 14 WITH, 15 N, 18 Oh, 32 R, 35 S, 18 F, 36 Cl and 125I. In one embodiment of the invention, the payload (e.g., a compound of formula (i)) may exist in an isotopically labeled or isotopically enriched form, particularly in a deuterated form.

[0055] The expressions “antibody-conjugated drug” and “antibody-drug conjugate” as used herein have the same meaning. The compound of formula (i)

[0056] In one embodiment of the invention, there is provided a compound or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopic compound, metabolite or prodrug thereof, wherein the compound has a structure of formula (i):

[0057] where

[0058] a is 0 or 1;

[0059] each of the carbon atoms labeled p1* and p2* is an asymmetric center, and the asymmetric center has the S configuration, the R configuration, or it is racemic;

[0060] L1 selected from C 1-6 alkylene which is unsubstituted or is substituted by one substituent selected from halogen, -OH and -NH2;

[0061] M represents -CH2-, -NH-, or -O-;

[0062] L 2 represents C 1-3 alkylene;

[0063] R 1 and R 2 independently selected from hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0064] In one embodiment of the invention L 1 selected from C 1-6 linear alkylene, C 1-6 branched alkylene, C 3-6 cyclic alkylene and C 3-4 cyclic alkyl-C 1-2 linear alkylene group, each of which is independently unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2. In one embodiment, L 1 selected from C 1-4alkylene which is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2. In a preferred embodiment, L 1 selected from -СН2-, -С2Н4-, each of which is independently unsubstituted or substituted with at least one substituent selected from halogen, -OH and -NH2. In a preferred embodiment, L 1 selected from -СН2-, where "#" denotes the position of attachment to the carbonyl. In a more preferred embodiment, L 1 selected from -СН2-, where "#" denotes the position of attachment to the carbonyl. In a particular embodiment of the invention, L 1 selected from -СН2- And where "#" denotes the position of attachment to the carbonyl. In a preferred embodiment, the halogen is selected from F, Cl, and Br, especially F.

[0065] In one embodiment, a is 1, M is -CH2-, -NH-, or -O-; and L 2is -C2H4-. In another embodiment, a is 1, M is -CH2-, and L 2 is -СН2-.

[0066] In one embodiment, the carbon atom labeled as p1* has the S-configuration or the racemic configuration, preferably the S-configuration. In another embodiment, the carbon atom labeled as p2* has the S-configuration or the racemic configuration, preferably the S-configuration.

[0067] In one embodiment, each of R 1 and R 2 independently selected from hydrogen, C 1-3 alkyl, halogen and C 1-3 alkoxy. In a preferred embodiment, each of R 1 and R 2 independently selected from CH3-, F, Cl, Br and CH3O-. In one embodiment, R 1 selected from CH3- and Cl. In another embodiment, R 2 represents F.

[0068] In one embodiment of the invention, a is 0, L 1selected from -СН2-, where "#" denotes the position of attachment to the carbonyl. In an embodiment, a is 1, L 1 represents M represents O, and L 2 is -C2H4-.

[0069] In one embodiment of the invention, a is 0, R 1 represents Cl, R 2 represents F, and L 1 selected from -СН2- And In one embodiment, a is 0, R 1 is CH3-, R 2 represents F, and L 1 selected from where "#" denotes the position of attachment to the carbonyl.

[0070] In one embodiment, a is 1, R 1 is CH3-, R 2 represents F, L 1 represents M represents O, and L 2 is -C2H4-.

[0071] In one embodiment, the compound is selected from the following compounds:

[0072] In one embodiment, the compound is selected from the following compounds:

[0073] In a preferred embodiment, the compound is selected from the following compounds:

[0074] In a preferred embodiment, the compound is selected from the following compounds:

[0075] In a more preferred embodiment, the compound is selected from the following compounds:

[0076] In a particular embodiment of the invention, the compound is selected from the following compounds:

[0077] In one aspect, a compound is provided, selected from the following compounds: Compound of formula (I)

[0078] In one aspect, a compound having a structure of formula (I) is provided:

[0079] where

[0080] opSu is

[0081] R 0 represents C 1-10 alkyl;

[0083] k1 and k2 independently represent an integer from 1 to 7;

[0084] i represents an integer between 1 and 100;

[0085] j represents an integer between 1 and 100;

[0086] P1 and P2 independently represent a payload having a compound structure of formula (i'):

[0087] where

[0088] a, carbon atoms labeled as p1* and p2*, L 1 , M, L 2 , R 1 and R 2 , have the values ​​defined in formula (i).

[0089] In one embodiment, the payload is selected from the following compounds:

[0090] In one embodiment, the payload is selected from the following compounds:

[0091] In a preferred embodiment, the payload is selected from the following compounds:

[0092] In a preferred embodiment, the payload is selected from the following compounds:

[0093] In a more preferred embodiment, the payload is selected from the following compounds:

[0094] In a particular embodiment, the payload is selected from the following compounds:

[0095] In one embodiment of R 0 represents C 1-6 alkyl. In a preferred embodiment, R 0 represents C 1-3 alkyl. In a particular embodiment, R 0 is methyl.

[0096] In one embodiment of the invention, n is an integer from 2 to 5. In a particular embodiment of the invention, n is 3.

[0097] In one embodiment of the invention, k1 and k2 are independently equal to 1, or 3, or 5. In a particular embodiment, k1 and k2 are independently equal to 5.

[0098] In one embodiment of the invention, i independently represents an integer from 1 to 20, preferably from 1 to 12, more preferably from 2 to 8. In a particular embodiment, i is 4.

[0099] In one embodiment of the invention, j is independently an integer from 1 to 20, preferably from 1 to 12, more preferably from 8 to 12, especially 8 or 12. In a particular embodiment of the invention, j is 12.

[0100] In one embodiment of the invention, the compound of formula (I) has a structure of formula (I-1):

[0101] where

[0102] PI, P2, opSu, n, i hj have the meanings defined in formula (I).

[0103] In one embodiment of the invention, the compound of formula (I) is selected from the group consisting of the following compounds: Specific embodiments of the compound of formula (I)

[0104] In one embodiment of the invention, the structure of the compound of formula (I) is one of the structures shown in the following table. Obtaining a compound of formula (I)

[0105] In one embodiment of the invention, a compound of formula (I) can be synthesized by coupling a linker to a payload or by coupling a series of suitable building blocks. Such building blocks can be readily generated using retrosynthetic analysis, and any reaction known in the art can be used.

[0106] In one aspect, a compound having a structure of formula (1) is provided:

[0107] where

[0108] k represents an integer from 1 to 7;

[0109] P is a payload having a structure of a compound of formula (i'), wherein the structure of formula (i') is defined above.

[0110] In a preferred embodiment, k is 1, or 3, or 5. In a specific embodiment, k is 5.

[0111] The compound of formula (1) can be synthesized using the compound of formula (i) and other necessary building blocks using a method similar to the synthetic method disclosed in EP 2907824 A (e.g., using the synthetic method for the compound of formula (2) or formula (2b) of EP 2907824 A). Suitable building blocks include, but are not limited to, the linker block HX20113 and the linker block HX20111.

[0112] The maleimide group contained within it can then be reacted with the thiol group of another building block to form a compound of formula (I). The resulting thiosuccinimide is unstable under physiological conditions and can reverse the Michael addition, leading to cleavage at the junction. Moreover, when another thiol compound is present in the system, thiosuccinimide can also undergo thiol exchange with another thiol compound. Both of these reactions cause a drop in payload and lead to toxic side effects. Thiosuccinimide then undergoes a ring-opening reaction to obtain the compound of formula (I).

[0113] The method for the ring-opening reaction can be found in WO 2015 / 165413 A1. The compound containing the ring-opened succinimide moiety can be purified by semi-preparative / preparative HPLC (HPLC) or other suitable separation methods to obtain a compound of high purity and a certain composition, regardless of the efficiency of the succinimide ring-opening reaction.

[0114] In the present invention, by using a payload linker (linker is a low-molecular-weight intermediate), the ring-opened succinimide structure no longer undergoes reverse Michael addition or thiol exchange, and thus the product becomes more stable. A fragment containing a recognition sequence of a ligase acceptor or a donor substrate

[0115] In one embodiment of the invention, the (Gly)n moiety of the compound of formula (I) is a ligase acceptor or donor substrate recognition sequence that facilitates enzyme-catalyzed binding of the compound of formula (I) to an antibody or antigen-binding fragment thereof under catalysis by a ligase. The antibody or antigen-binding fragment thereof may be optionally modified and contain an appropriate ligase acceptor or donor substrate recognition sequence.

[0116] In one embodiment of the invention, the ligase is a transpeptidase. In one embodiment of the invention, the ligase is selected from the group consisting of a naturally occurring transpeptidase, a non-naturally occurring transpeptidase, variants thereof, and a combination thereof. Non-naturally occurring transpeptidase enzymes may be, but are not limited to, enzymes obtained by engineering the natural transpeptidases. In a preferred embodiment of the invention, the ligase is selected from the group consisting of a naturally occurring sortase, a non-naturally occurring sortase, and a combination thereof. Types of naturally occurring sortase include sortase A, sortase B, sortase C, sortase D, L. plantarum sortase, etc. (see US 2011 / 0321183 A1). The type of ligase corresponds to the recognition sequence of the ligase, and therefore it is used to achieve specific conjugation between different molecules or structural fragments.

[0117] In one embodiment of the invention, the fragment (Gly) nthe compound of formula (I) is a ligase acceptor substrate recognition sequence; and the antibody or antigen-binding fragment thereof is optionally modified and comprises a corresponding ligase donor substrate recognition sequence.

[0118] In some embodiments of the invention, the ligase is a sortase selected from sortase A, sortase B, sortase C, sortase D, and L. plantarum sortase.

[0119] In a specific embodiment of the invention, the ligase is sortase A from Staphylococcus aureus. Accordingly, the ligase recognition sequence of the ligase donor substrate may be a typical LPXTG enzyme recognition sequence. In another specific embodiment of the invention, the ligase donor substrate recognition sequence is LXTGJ, where X may be any single amino acid that is natural or non-natural; J is absent or is an amino acid fragment containing 1-10 amino acids, optionally labeled. In one embodiment of the invention, J is absent. In another embodiment of the invention, J is an amino acid fragment containing 1-10 amino acids, where each amino acid independently is any natural or non-natural amino acid. In another embodiment of the invention, J is (Gly) m, where m is an integer from 1 to 10. In another specific embodiment of the invention, the ligase donor substrate recognition sequence is LPETG. In another specific embodiment of the invention, the ligase donor substrate recognition sequence is LPETGG.

[0120] In one embodiment of the invention, the ligase is sortase B from Staphylococcus aureus, and the corresponding substrate-donor recognition sequence may be NPQTN. In another embodiment of the invention, the ligase is sortase B from Bacillus anthracis, and the corresponding substrate-donor recognition sequence may be NPKTG.

[0121] In another embodiment, the ligase is sortase A from Streptococcus pyogenes, and the corresponding substrate-donor recognition sequence may be LPXTGJ, where J has the meanings defined above. In another embodiment, the ligase is a member of subfamily 5 sortases from Streptomyces coelicolor, and the corresponding substrate-donor recognition sequence may be LAXTG.

[0122] In another embodiment of the invention, the ligase is sortase A from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence may be LPQTSEQ.

[0123] The ligase recognition sequence may also be another, completely new transpeptidase recognition sequence optimized by manual screening. Conjugates and their preparation

[0124] Furthermore, a payload-bearing compound (a compound of formula (I)) that has a moiety comprising a ligase recognition sequence can be conjugated to other molecules comprising a ligase recognition sequence and can thus be used, for example, to produce an antibody-small molecule conjugate, such as an antibody-drug conjugate. Accordingly, in another aspect of the invention, there is provided a conjugate that comprises a compound of formula (I) and an antibody or antigen-binding fragment.

[0125] In another aspect of the invention, there is provided a conjugate having a structure of formula (II): where A is an antibody or its antigen-binding fragment; z is an integer from 1 to 20; P1, P2, P 0 , opSu, n, kl, k2, i and j have the meanings defined in formula (I).

[0126] In a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof is modified to bind to a (Gly) fragment n in the compound of formula (I).

[0127] In a preferred embodiment of the invention, z has a value from 1 to 4. In a specific embodiment of the invention, z is 2. The antibody or antigen-binding fragment thereof

[0128] In one embodiment, the antibody is an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 / TNFRSF8 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD44v6 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-CD74 antibody, an anti-CD79b antibody, an anti-CD117 / KITk antibody, an anti-CD123 antibody, an anti-CD138 antibody, an anti-CD142 antibody, an anti-CD174 antibody, an anti-CD227 / MUC1 antibody, an anti-CD352 antibody, an anti-CLDN18.2 antibody, an anti-DLL3 antibody, an anti-ErbB2 / HER2 antibody, an anti-CN33 antibody, an anti-GPNMB antibody, an anti-ENPP3 antibody, an anti- nectin-4, anti-EGFRvIII, anti-SLC44A4 / AGS-5, anti-CEACAM5, anti-PSMA, anti-TIM1, anti-LY6E, anti-LIV1, anti-nectin4, anti-SLITRK6, anti-HGFR / cMet, anti-SLAMF7 / CS1,anti-EGFR, anti-BCMA, anti-AXL, anti-NaPi2B, anti-GCC, anti-STEAP1, anti-MUC16, anti-mesothelin, anti-ETBR, anti-EphA2, anti-5T4, anti-FOLR1, anti-LAMP1, anti-cadherin 6, anti-FGFR2, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-integrin aV antibody, anti-TDGF1 antibody, anti-ephrin A4 antibody, anti-TROP2 antibody, anti-PTK7 antibody, anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, anti-tissue factor antibody or anti-ROR1 / 2 antibody; preferably, the antibody is an anti-HER2 antibody, an anti-FGFR3 antibody, an anti-Trop2 antibody, an anti-HER3 antibody, or an anti-FRα antibody.

[0129] In a preferred embodiment of the invention, the antibody is an anti-HER2 antibody. In one embodiment, the antibody is an anti-human HER2 antibody. 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 HER2-positive gastric cancer.

[0130] In one embodiment of the invention, the antibody is an anti-HER2 antibody or antigen-binding fragment thereof comprising a light chain variable region (V l ) and the variable region of the heavy chain (V h ), where V lcontains LCDR1 containing the amino acid sequence of SEQ ID NO: 1, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and wherein V h comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, HCDR3 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment of the invention, the CDR regions are defined by the KABAT nomenclature. In one embodiment of the invention, the antibody is an anti-HER2 antibody or antigen-binding fragment thereof comprising a light chain variable region (V l ) and the variable region of the heavy chain (V h ), wherein VL has an amino acid sequence that is at least about 85%, at least about 90%, or at least about 95% identical to the sequence of SEQ ID NO: 7, and Vh has an amino acid sequence that is at least about 85%, at least about 90%, or at least about 95% identical to the sequence of SEQ ID NO: 8. In a preferred embodiment of the invention, the HER2 antibody or antigen-binding fragment thereof comprises a light chain variable region (V l ) and the variable region of the heavy chain (V h ), where V lhas the amino acid sequence of SEQ ID NO: 7, and VH has the amino acid sequence of SEQ ID NO: 8. In a specific embodiment, the anti-HER2 antibody comprises a light chain and a heavy chain, wherein the light chain has an amino acid sequence that is at least about 85%, at least about 90%, or at least about 95% identical to a sequence having the amino acid sequence of amino acids 1 to 214 in SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is at least about 85%, at least about 90%, or at least about 95% identical to the sequence of SEQ ID NO: 10. In a preferred embodiment, the anti-human HER2 antibody is one or more antibodies selected from trastuzumab-based engineered anti-HER2 antibodies.In one embodiment of the invention, the antibody is a modified trastuzumab, preferably Ab0001-LCCTL-HC (having the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10). The sequence is Ab0001-LCCT. L -HC is based on the amino acid sequence of trastuzumab, in which GALPETGG is introduced at the C-terminus of the light chain, where LPETGG is the ligase donor substrate recognition sequence and GA is the spacer sequence.

[0131] In a preferred embodiment of the invention, the anti-human HER2 antibody is a recombinant antibody selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment, and an antibody mimetic. In one embodiment, the antibody mimetic is selected from an scFv, a mini-antibody, a diabody, or a nanobody. For conjugation with a compound of formula (I), the antibody or antigen-binding fragment thereof according to the invention may contain a modified moiety for conjugation with a (Gly) moiety. n in the compound of formula (I). The position for the introduction of such a modified fragment is not limited: for example, the insertion position on the antibody or antigen-binding fragment thereof may be located, inter alia, but not limited to, at the C-terminus or at the N-terminus of the heavy chain or light chain of the antibody.

[0132] In an alternative embodiment of the invention, a modified moiety for conjugation with a (Gly) moiety n in the compound of formula (I) can be introduced into a non-terminal position of the heavy chain or light chain of the antibody using, for example, chemical modification methods.

[0133] In one embodiment of the invention, an antibody or an antigen-binding fragment thereof may comprise a terminal modification. A terminal modification refers to a modification of the C-terminus or N-terminus of the heavy chain or light chain of an antibody, which, for example, contains a ligase recognition sequence. In another embodiment of the invention, the terminal modification may further comprise a spacer Sp comprising 2-100 amino acids, wherein the antibody, Sp and the ligase recognition sequence are linked sequentially. In a preferred embodiment of the invention, Sp is a spacer sequence comprising 2-20 amino acids. In a specific embodiment of the invention, Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS, especially GA.

[0134] In a preferred embodiment of the invention, the light chain of the antibody or the antigen-binding fragment thereof comprises 3 types: wild type (LC); C-terminally modified light chain (LCCT), which is modified by directly introducing the LPXTG ligase recognition sequence, and C-terminally modified light chain (LCCT l ), which is modified by introducing short peptide spacers, plus the LPXTG sequence for recognizing the ligase donor substrate. The heavy chain of an antibody or its antigen-binding fragment includes three types: wild type (WT); modified heavy chain with a C-terminus (MCCT), which is modified by directly introducing the LPXTG sequence for ligase recognition; and modified heavy chain (HCCT) l), which is modified by the introduction of short peptide spacers, plus the LPXTG sequence for recognition of the ligase donor substrate. X can be any natural or non-natural single amino acid. When z is 1 or 2 in the compound of formula (II), the combination of the above heavy and light chains can form 8 preferred antibody molecules.

[0135] In one embodiment of the invention, the compound of formula (II) is selected from the group consisting of the following compounds: Specific variants of conjugates

[0136] In one embodiment of the invention, the payload is a cytotoxin or a fragment thereof. In one embodiment of the invention, the antibody-drug conjugate is shown in the following table.

[0137] ADC Nomenclature: The number in parentheses indicates the number of payload molecules (drug substance) that are intended to be combined with the antibody. Obtaining conjugates

[0138] Conjugates (i.e., a compound of formula (II)) of the invention can be prepared by any method known in the art. In some embodiments, the conjugate is prepared by ligase-catalyzed site-specific conjugation of an antibody or antigen-binding fragment and a compound of formula (I), wherein the antibody or antigen-binding fragment thereof is modified with a ligase recognition sequence.

[0139] An antibody or antigen-binding fragment thereof and a compound of formula (I) are linked to each other via ligase-specific substrate recognition sequences. The recognition sequence depends on the particular ligase used. In one embodiment of the invention, the antibody or antigen-binding fragment thereof is an antibody with terminal modifications based on the recognition sequence introduced at the C-terminus of the light chain and / or heavy chain, and the antibody or antigen-binding fragment thereof is conjugated to the compound of formula (I) under catalysis by a wild-type ligase or an optimized engineered ligase, or any combination thereof, under suitable catalytic reaction conditions.

[0140] In a specific embodiment of the invention, the ligase is sortase A, and the conjugation reaction can be represented by the following scheme:

[0141] The triangle represents the antibody part; the pentagon represents the part of the compound of formula (II); and G n is a fragment (Gly) n . The meanings and definitions of n, X, and J are respectively defined above. When conjugated with G n , which is the corresponding acceptor substrate recognition sequence, the glycine peptide bond located upstream of the LPXTGJ sequence is cleaved by sortase A, and the resulting intermediate binds to the free N-terminus of G n with the formation of a new peptide bond. The resulting amino acid sequence is LXTG n . G sequences n and LXTGJ are respectively defined above.

[0142] Therefore, in one embodiment of the invention, the fragment G nin the compound of formula (I) is a ligase acceptor substrate recognition sequence of the ligase; and the antibody is modified to contain a ligase donor substrate recognition sequence for coupling to the G fragment n in a compound of formula (I), wherein it is preferred that (a) the ligase is a sortase selected from sortase A, sortase B, sortase C, sortase D and sortase from L. plantarum; preferably sortase A from Staphylococcus aureus; and / or (b) the recognition sequence of the substrate donor of the ligase is LXTGJ, J is absent or is G m , where G is glycine, m is an integer from 1 to 10, X is any single amino acid that is natural or non-natural; preferably, the ligase donor substrate recognition sequence is LPETGG or LPETGG; and / or the junction of LXTGJ with G n leads to the formation of LXTG n .

[0143] The compound of formula (I) according to the invention has a defined structure, a defined composition, and high purity, so that fewer or no impurities are introduced during the conjugation reaction with the antibody. When such an intermediate is used for site-specific conjugation catalyzed by ligase with a modified antibody containing the ligase recognition sequence, a homogeneous ADC with well-controlled quality is obtained. Metabolism of the conjugate in a physiological environment

[0144] When part or all of the linker is cleaved in tumor cells, the payload is released. Because the linker is cleaved at the point where it connects to the antitumor compound, the antitumor compound is released within its internal structure, exerting its own antitumor effect.

[0145] In one embodiment of the invention, the GGFG (Gly-Gly-Phe-Gly) moiety contained in the compound of formula (I) can be cleaved by lysosomal enzymes (such as cathepsin B and / or cathepsin L).

[0146] In one embodiment of the invention, the compound of formula (I) contains a self-destructive spacer. In one embodiment, the self-destructive spacer is an acetal or a heteroacetal. In one embodiment, the -GGFG-NH-CH2-O- moiety contained in the compound of formula (I) is a combination of a restriction enzyme site and a self-destructive spacer that will cleave in the cell and release a molecule of a target substance (such as an antitumor compound). Pharmaceutical composition and pharmaceutical preparation

[0147] Another object of the invention is to provide a pharmaceutical composition comprising a conjugate according to the invention and at least one pharmaceutically acceptable carrier.

[0148] The pharmaceutical composition of the invention can be administered by any route, provided it provides the effect of preventing, alleviating, preventing, or treating symptoms in humans or animals. For example, various suitable dosage forms can be prepared according to the intended route of administration, especially injectable forms, such as lyophilized powder for injection, injection preparation, or sterile powder for injection.

[0149] The term "pharmaceutically acceptable" means that when the substance comes into contact with the tissues of a patient in the course of ordinary medical practice, no excessive toxicity, irritation or allergic reaction, etc., should occur, and that there is a reasonable balance of advantages and disadvantages, and the substance is effective for the intended use.

[0150] The term "pharmaceutically acceptable carrier" refers to carrier substances that are pharmaceutically acceptable and do not affect the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline, etc. A pharmaceutically acceptable carrier also includes carriers that approximate the composition to physiological conditions, such as pH adjusting agents, buffering agents, toxicity adjusting agents, etc., as well as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0151] In one embodiment, the pharmaceutical composition of the invention has a drug-to-antibody ratio (DAR) of an integer or non-integer from about 1 to about 20, such as from about 1 to about 10, from about 1 to about 8, from about 1 to about 6, from about 1 to about 4, from about 2 to about 5, from about 2 to about 4, or from about 3 to about 4. In a particular embodiment, the conjugate of the invention has a DAR of about 4. In a particular embodiment, the conjugate of the invention has a DAR of about 3.3 to about 3.75, especially about 3.4 to about 3.65. Methods of Treatment and Uses

[0152] The conjugates of the invention are useful for the treatment of tumors and / or autoimmune diseases. Tumors sensitive to treatment with the conjugate include tumors characterized by specific tumor-associated antigens or tumor-associated cell surface receptors, and such tumors are recognized by the targeting molecule (antibody) in the conjugate and can be destroyed by the payload / cytotoxin in the conjugate.

[0153] Accordingly, in another aspect of the present invention, there is also provided the use of the conjugate of the invention or the pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0154] ​​In another aspect, the present invention provides a conjugate of the invention or a pharmaceutical composition of the invention for use in the treatment of a tumor or an autoimmune disease.

[0155] In a further aspect, the present invention provides a method for treating a tumor or an autoimmune disease, the method comprising administering to an individual in need thereof an effective amount of a conjugate of the invention or a pharmaceutical composition of the invention.

[0156] In a preferred embodiment of the invention, a conjugate of the invention formed by conjugating an anti-HER2 antibody and a low molecular weight cytotoxin can specifically bind to HER2 on the surface of a tumor cell and selectively destroy HER 2-expressing tumor cells. In another preferred embodiment of the invention, the anti-HER2 antibody is an anti-human HER2 antibody. In another preferred embodiment of the invention, there is provided the use of a conjugate of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a disease, disorder, or condition selected from HER2-positive tumors. In a more preferred embodiment of the invention, the disease, disorder, or condition is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, etc.

[0157] The dosage of the conjugate administered to a subject can be largely adjusted. The dosage may vary depending on the specific route of administration and the subject's needs, and may be determined at the discretion of the healthcare professional.

[0158] As defined above, the embodiments of the present invention can also be described as follows [1]-

[20] :

[0001] Compound of formula (I): where opSu represents R 0 represents C 1-10 alkyl;n is any integer from 2 to 20;k1 and k2 independently represent an integer from 1 to 7;i is an integer from 1 to 100;j is an integer from 1 to 100;P1 and P2 independently represent a payload having the structure of a compound of formula (i'): where a is 0 or 1; each of the carbon atoms labeled p1* and p2* represents an asymmetric center, and the asymmetric center has the S-configuration, the R-configuration, or is racemic;L 1 selected from C 1-6alkylene which is unsubstituted or is substituted by one substituent selected from halogen, -OH and -NH2; M is -CH2-, -NH- or -O-; L 2 represents C 1-3 alkylene;R 1 and R 2 independently selected from hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0002] Compound [1] having the structure of formula (I-1) where P1, P2, R 0

[0001] .

[0003] The compound according to [1] or [2], where in formula (i'):L 1 selected from C 1-6 linear alkylene, C 1-6 branched alkylene, C 3-6 cyclic alkylene and C 3-4 cyclic alkyl-C 1-2 in a linear alkylene group, each of which is independently unsubstituted or is substituted by one substituent selected from halogen, -OH and -NH2; it is preferred that L 1 selected from C 1-4 linear alkylene, C 1-4branched alkylene, C 3-4 cyclic alkylene and cyclopropylmethylene, each of which is independently unsubstituted or substituted by one substituent selected from halogen, -OH and -NH2;L 1 preferably selected from -CH2-, -C2H4-, , each of which is independently unsubstituted or is substituted by at least one substituent selected from halogen, -OH and -NH2; more preferably, L 1 selected from -СH2-, where "#" denotes the position of attachment to the carbonyl; more preferably, when L 1 selected from -СH2-, where "#" denotes the position of attachment to the carbonyl; in particular, L 1 selected from -СH2-, , where "#" denotes the position of attachment to the carbonyl; and / or the halogen is selected from F, Cl and Br.

[0004] The compound according to any one of items [1] to [3], wherein in formula (i'): M is -CH2-, -NH- or -O-; and L 2represents -C2H4-; or M represents -CH2-, and L 2 is -СH2-.

[0005] The compound according to any one of items [1] to [4], wherein in the formula (i'): the carbon atom marked as p1* has the S-configuration or the racemic configuration, preferably the S-configuration; and / or the carbon atom marked as p2* has the S-configuration or the racemic configuration, preferably the S-configuration.

[0006] A compound according to any one of items [1] to [5], where in formula (i'):R 1 and R 2 each independently selected from hydrogen, C 1-3 alkyl, halogen and C 1-3 alkoxy; preferably each of R 1 and R 2 independently selected from CH3-, F, O, Br and CH3O-; more preferably, R 1 selected from CH3- and Cl; and / or R 2 represents F; further preferred, and is equal to 0, R 1 represents Cl, R 2 represents F, and L 1 selected from -СH2-, or is equal to 0, R1 is CH3-, R 2 represents F, and L 1 selected from , where "#" denotes the position of attachment to the carbonyl; or represents 1, R 1 is CH3-, R 2 represents F, L 1 represents , M represents O, and L 2 is -C2H4-.

[0007] A connection according to any one of items [1] to [6], where the payload is selected from the following connections: preferably from: more preferably from: and especially from:

[0008] Connection according to any of items [1]-[7], where R 0 represents C 1-6 alkyl, preferably C 1-3alkyl; especially methyl; and / or n is an integer from 2 to 5, especially 3; and / or k1 and k2 are independently 1 or 3 or 5, especially 5; i is independently an integer from 1 to 20, preferably from 1 to 12, more preferably from 2 to 8, especially 4; and / or j is independently an integer from 1 to 20, preferably from 1 to 12, more preferably from 8 to 12, especially 8 or 12, especially 12.

[0009] Connection according to any one of items [1] to [8], selected from:

[0010] Conjugate having the structure of formula (II): where A is an antibody or antigen-binding fragment thereof; wherein the antibody or antigen-binding fragment is preferably modified to bind to a (Gly) moiety nin the compound of formula (I);z is an integer from 1 to 20; preferably from 1 to 4; especially 2;

[0001] -[8].

[0011] Conjugate according to

[10] , selected from the following compounds:

[0012] The conjugate according to any one of items

[10] to

[11] , wherein the antibody is an anti-human HER2 antibody, preferably trastuzumab.

[0013] A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a conjugate according to any one of items

[10] -

[12] and at least one pharmaceutically acceptable carrier.

[0014] The pharmaceutical composition according to item

[13] , wherein the conjugate has a drug-to-antibody ratio (DAR) value equal to an integer or non-integer number from 1 to 8, in particular from 3 to 4.

[0015] Use of the conjugate according to any one of items

[10] to

[12] or the pharmaceutical composition according to item

[13] or

[14] in the manufacture of a medicament for treating a disease; wherein the disease is a tumor or an autoimmune disease; preferably a tumor containing HER2-positive tumor cells.

[0016] The use according to item

[15] , wherein the tumor containing HER2-positive tumor cells further includes tumor cells with low HER2 expression; wherein the tumor cells with low HER2 expression express lower levels of HER2 than the HER2-positive tumor cells.

[0017] A compound or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopic compound, metabolite, or prodrug thereof, wherein the compound has a structure of formula (i): where a, the carbon atoms labeled as p1* and p2*, L 1 , M, L 2 , R 1 and R 2

[0001] -[7].

[0018] The connection according to item

[17] , selected from the following connections: preferably selected from: more preferably selected from: especially selected from:

[0019] Compound of formula (1): where k is an integer from 1 to 7; preferably 1, 3, or 5, especially 5

[0001] -[7].

[0020] Connection according to

[19] , selected from the following connections: Beneficial effects

[0159] The novel small molecule topoisomerase I inhibitors provided in the present invention, used either alone or as a component of an ADC, can exhibit greater activity, stability and physicochemical properties compared to the prior art agents.

[0160] The antibody-drug conjugate of the invention utilizes a specially designed payload linker, achieving high efficacy and bystander elimination. At the same time, the conjugate has a lower DAR value, thereby reducing side effects and increasing the therapeutic index, which is particularly important for bystander elimination. The antibody-drug conjugate of the invention is structurally more stable, featuring an open-ring succinimide structure.

[0161] The present invention utilizes a uniquely structured linker and ligase to catalyze the conjugation of a target molecule (antibody) and a payload. The conjugate of the present invention exhibits good homogeneity and high activity. Furthermore, the linker-payload intermediate exhibits significantly lower toxicity than the free payload, resulting in a less hazardous drug production process, which is advantageous for industrial production.

[0162] The conjugate of the present invention achieves at least one of the following technical effects: (1) High inhibitory activity on target cells and good effect on bystanders. (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability). (3) Good pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life and duration of action). (4) Good safety (low toxicity to non-target normal cells or tissues and / or fewer side effects, wider treatment window), etc. (5) Modular design of conjugates, easy assembly for a variety of different drug compounds. Examples

[0163] Examples of receiving

[0164] To more clearly illustrate the objectives and technical solutions, the present invention is further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the invention. Specific experimental methods that are not mentioned in the examples below were carried out in accordance with conventional experimental practice. Equipment, materials and reagents

[0165] Unless otherwise stated, the equipment and reagents used in the examples are commercially available. The reagents can be used directly without further purification.MS (Mass Spectrometry): Thermo Fisher Q Exactive Plus, Water2795-Quattro micro triple quadrupole mass spectrometer.HPLC (HPLC): Waters 2695, Agilent 1100, Agilent 1200.Semi-preparative HPLC: Lisure HP plus 50D.Flow Cytometry: CytoFLEX SHIC-HPLC (HPLC with hydrophobic interaction chromatography): Butyl-HIC; Mobile phase A: 25 mM PB, 2 M (NH4)2SO4, pH 7.0; Mobile phase B: 25 mM PB, pH 7.0; Flow rate: 0.8 mL / min; Exposure time: 25 min; Injection amount: 20 μg; Column temperature: 25°C; Detection wavelength: 280 nm; Sample chamber temperature: 8°C.SEC-HPLC (size-exclusion HPLC): column: TSK-gel G3000 SWXL, TOSOH, internal diameter 7.8 mm × 300 mm, 5 μm; mobile phase: 0.2 M KH2PO4, 0.25 M KCl, pH 6.2; flow rate: 0.5 mL / min; exposure time: 30 min; injection volume: 50 μL; column temperature: 25°C; detection wavelength: 280 nm; sample chamber temperature: 8°C. CHO cells were obtained from Thermo Fisher Scientific; pcDNA 3.3 was obtained from Life Technology; HEK293F cells were obtained from Prejin; PEIMAX transfection reagent was obtained from Polyscience; MabSelect Sure ProA was obtained from GE; Capto S ImpAct was obtained from GE; resins: Rink MBHA-amide resin and dichlororesin were from Nankai Synthesis; HCC1954 was obtained from ATCC CAT# CRL-2338; SK-BR-3 was obtained from ATCC CAT# HTB-30; BT-474 was obtained from ATCC CAT# HTB-20; NCI-N87 cells were obtained from ATCC CAT# CRL-5822; MCF7 was obtained from ATCC CAT# HTB-22; MDA-MB-231 was obtained from ATCC CAT# HTB-26; MDA-MB-468 was obtained from ATCC CAT# HTB-132; CFPAC-1 was obtained from ATCC CAT# CRL-1918; NCI-H2110 was obtained from ATCC CAT# CRL-5924; LMT-1 was obtained from Wuxi Apptech; Sarap-1 was obtained from ATCC CAT# CRL-1573; the antibody trastuzumab was obtained from a known sequence; optimized recombinant enzyme sortase A from Staphylococcus aureus was produced in E. coli.

[0166] In some cases, the order of the reaction schemes described above could be modified to facilitate the reaction or avoid unwanted reaction products. The following examples are provided to provide a more complete understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the present invention in any way. Example 1. Preparation of low-molecular-weight payload compounds. Intermediate 11 Step A: N-(2-bromo-5-fluorophenyl)acetamide: To a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL) was added conc. H2SO4 (3 mL), followed by the addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) portionwise at room temperature. The mixture was stirred for 3 h, then poured into 2000 mL of ice water. The resulting precipitate was collected by filtration and dried under vacuum at room temperature to afford N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 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). LCMS m / z 232.0 (M+H). Step B: N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: To a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (tetrahydrofuran, THF) (1000 mL) was added n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) dropwise over 1 h at -78 °C. Upon completion, the mixture was stirred for 0.5 h under N2. Then 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 at -78 °C to room temperature over 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 saturated brine (250 mL × 2), dried over Na2SO4, and concentrated.The mixture was triturated (PE / EA=1:1, 100 ml) for 10 min, filtered, the precipitate was collected and dried in vacuo to give N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g) as a yellow solid. LCMS m / z 206.1 (N-18+H), 246.1 (M+Na). Step C: N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirred mixture of N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) in CH2Cl2 (170 mL) and water (170 mL) were added silver nitrate (AgNO2) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol), and the mixture was stirred at 30°C for 6 h. The mixture was filtered through celite and washed with CH2Cl2 (100 mL), the filtrate was concentrated and purified by FCC (EA / PE = 0-40%) to give N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g) as a light yellow solid. LCMS m / z 222.1 (M+H).Step D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirred mixture of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) in THF (500 mL) at 0°C was added 1-butyl nitrite (8.48 g, 63.28 mmol) followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0°C for 2 h. After completion, the mixture was acidified with HCl (2 N) to adjust pH to 3. The mixture was extracted with ethyl acetate (200 mL × 3), washed with saturated 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, and the precipitate was collected and dried in vacuo to give N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12 g) as a yellow solid. LCMS m / z 251.1 (M+H).Step E: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-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 under H2 for 16 h. After cooling to 0 °C, Et2N (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). Extracted with ethyl acetate (500 mL × 3), washed with saturated 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 precipitate collected 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. LCMS m / z 279.1 (M+H).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 (2 N, 150 mL), and the mixture was stirred at 50°C for 7 h. After cooling to 0°C, sat. aqueous NaHCO3 solution to adjust pH to 8. Extracted with ethyl acetate (200 mL × 3), washed with saturated brine (200 mL × 2), dried over Na2SO4, and concentrated under reduced pressure to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (6.0 g) as a yellow solid. 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 (cd, J=13.0, 4.3 Hz, 1H). LCMS m / z 237.1 (M+H). Step G: N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-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 DMFA (80 mL) was added NCS (2.26 g, 16.93 mmol) portionwise at 0°C, and the mixture was stirred at room temperature for 16 h. The mixture was poured into 200 mL of ice water. The formed precipitate was collected by filtration and dried in vacuo at room temperature to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g) as a yellow solid. 1H 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). LCMS m / z 271.0 (M+H).Stage H:N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4',6,7]indolizino[1,2-b]quinolin-1-yl)acetamide: To the mixture 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) was added (8)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-pindolysine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol) and o-cresol (10 mL) were added, and the mixture was refluxed under N2 for 24 h.The solvent was removed under reduced pressure and the mixture was purified by FCC (THF / CH2Cl2 = 0-60%) to give N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4.1 g) as a brown solid. LCMS m / z 498.1 (M+H).Stage I:(9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione: mixture N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-and]quinolin-1-yl)acetamide (2.0 g, 4.02 mmol) in 20 ml conc. HCl (aq) was stirred at 70°C under N2 for 36 h. The mixture was concentrated under reduced pressure to give crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (2 g) as a brown solid.LCMS (ESI) m / z 456.1 [M+H]. + Intermediate connection 12 Intermediate 12: (9S)-1-amino-4-bromo-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrobromide was synthesized from 2-bromo-5-fluoroaniline using a procedure similar to that described above for intermediate 11. LCMS (ESI) m / z 500.0 [M+H] + Intermediate connection 13 Intermediate 13: (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride was synthesized from 2-bromo-5-fluoro-4-methoxyaniline using a procedure similar to that described above for intermediate 11. LCMS (ESI) m / z 452.1 [M+H] + Procedure for C730 and C731 C730 and C731 were prepared by preparative HPLC from (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (intermediate 11) as the trifluoroacetic acid (TFA) salt. HPLC conditions are as specified above: Equipment: Agilent 1200; Chromatography column: Waters XBridge C18 4.6 × 50 mm, 3.5 μm; Flow: 2.0 mL / min; Gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min- 0.50 min-2.50 min-2.52 min-3.00 min; Temperature: 40°C; Mobile phase: A: acetonitrile, B: H2O (0.05% TFA); Wavelengths: 214 nm / 254 nm. Procedure for C518 Stage J:N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide: To solution of crude hydrochloride (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (50 mg, 0.10 mmol) in 2 ml dimethylformamide (DMF) was added 2-hydroxyacetic acid (11.6 mg, 0.15 mmol), HATU (57.9 mg, 0.15 mmol), and then Et3N (20.5 mg, 0.20 mmol) was added dropwise at 0°C, and the mixture was stirred at 0°C to room temperature for 2 h. The mixture was poured into 10 mL of ice water, extracted with ethyl acetate (20 mL × 3), washed with saturated brine (20 mL × 2), dried over Na2SO4, and concentrated.The residue was purified by preparative HPLC to give N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (6 mg) as yellow solid and K-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4,:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (5 mg) as a yellow solid. The resulting compounds, shown in Table II below, were reacted and further processed in the same manner as in the example for C518. The HPLC conditions are as specified above: Equipment: Agilent 1200; Chromatography column: Waters XBridge C18 4.6 × 50 mm, 3.5 μm; Flow: 2.0 mL / min; Gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min-1.50 min-2.50 min-2.52 min-3.00 min; Temperature: 40°C; Mobile phase: A: Acetonitrile, B: H2O (0.05% TFA); Wavelengths: 214 nm / 254 nm. The resulting compounds listed in Table III below were synthesized from intermediate 12 or intermediate 13 using a procedure similar to that described above for C518. HPLC conditions are as specified above: Equipment: Agilent 1200; Chromatography column: Waters XBridge C18 4.6 × 50 mm, 3.5 μm; Flow: 2.0 mL / min; Gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min-1.50 mi-2.50 min-2.52 min-3.00 min; Temperature: 40°C; Mobile phase: A: acetonitrile, B: H2O (0.05% TFA); Wavelengths: 214 nm / 254 nm. Procedure for C589 Stadnya K:(S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide: To a solution of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dionemesylate (50 mg, 0.10 mmol, (S)-3-hydroxybutanoic acid (15.6 mg, 0.15 mmol), HATU (57.9 mg, 0.15 mmol) were added in 2 mL of DMF, then Et3N (20.5 mg, 0.20 mmol) was added dropwise at 0 °C, and the mixture was stirred from 0 °C to room temperature for 2 h. The mixture was poured into 10 mL of ice water, extracted with ethyl acetate (20 mL × 3), washed with saturated brine (20 mL × 2), dried over Na2SO4, and concentrated.The residue was purified by preparative HPLC to give (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4|:6,7]indolizino[1,2-o]quinoline-1-yl)-3-hydroxybutanamide (9.4 mg) as a yellow solid. The resulting compounds shown in Table IVI below were reacted and worked up in the same manner as in Example C 589. The HPLC parameters listed above are: Equipment: Agilent 1200; Chromatographic column: Waters XBridge CIS 4.6 × 50 mm, 3.5 μm; Flow: 2.0 mL / min; Gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min-1.50 min-2.50 min-2.52 min-3.00 min; Temperature 40°C; Mobile phase A: acetonitrile, B: H2O (D, 0.5% TFA); Wavelengths: 214 nm / 254 nm. Example 2. Preparation of linkers

[0167] 2.1 Obtaining the HX20113 linker

[0168] HX20113 was synthesized by conventional solid-phase polypeptide synthesis using Rink MBHA-amide resin. Fmoc was used to protect the amino acid in the linker fragment. 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, lyophilized, and stored for further use. The theoretical mass was 1207.59, measured: [MH] value = 1206.7.

[0169] Obtaining the HX20111 linker

[0170] HX20111 was synthesized by conventional solid-phase polypeptide synthesis using Rink's MBHA-amide resin. Fmoc was used to protect the amino acid in the linker fragment. 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, lyophilized, and stored for further use. Theoretical mass: 1383.70, measured: [MN] - =1332.6. Example 3. Obtaining intermediate linker products with a payload

[0171] 3.1. Preparation of the intermediate product Mc-GGFG-Dxd

[0172] The intermediate Mc-GGFG-Dxd is commercially available or can be prepared according to the procedures described in EP 2907824. This compound was used to prepare the intermediate of the payload linker (the compound of formula (I)), and was also used to directly couple to the antibody (optionally modified) to obtain the reference ADCs LCI 184(8) and LC1184(4).

[0173] 3.2. Receipt of H0019

[0174] Synthesis of НХ21008-а

[0175] 4.33 g of Fmoc-Gly-Gly-OH and 6.84 g of Pb(OAc)4 were weighed and added to a 500 mL single-neck round-bottomed flask. Anhydrous THF / toluene (120 / 40 mL) was added under a nitrogen atmosphere and stirred to dissolve. Then 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 collected and analyzed by HPLC to monitor the reaction.

[0176] The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (EA) three times. The filtrates were combined and concentrated to dryness. Column chromatography (PE:EA=100:0 ~ 50:100) was performed to obtain approximately 2000 mg of the target product as a white solid with a yield of 44%.

[0177] Synthesis of H0005-a

[0178] 200 mg of HX21008-a was weighed and added to a 100 mL single-neck round-bottomed flask. Then 15 mL of THF was added and stirred until dissolved. Then H0005 (316 mg, 3.0 equiv.) and TsOH⋅H2O (15 mg, 0.15 equiv.) were added to the reaction system. The reaction in the reaction system was carried out overnight at room temperature. Samples were collected and analyzed by thin-layer chromatography (TLC) (PE / EA=1:1) to monitor the reaction. The starting materials almost disappeared, and a new point was detected on the chromatogram.

[0179] Saturated sodium bicarbonate solution was added to quench the reaction. Extraction was performed with EA three times. The organic phases were combined and washed with brine, dried over anhydrous magnesium sulfate, and concentrated. The crude product was purified by column chromatography (PE:EA=5:1~1:1) to obtain approximately 165 mg of the target product as a colorless oil in 60% yield. MS: [M+H] + =503.4.

[0180] Synthesis of H0005-b

[0181] 200 mg of H0005-a was weighed and added to a 100 mL single-neck round-bottomed flask. Then 10 mL of EtOH and 5 mL of EA were added until completely dissolved. Then 40 mg of palladium-on-carbon was added to the reaction system under a nitrogen atmosphere, and the reaction system was purged with hydrogen gas three times. The reaction system was maintained under a hydrogen atmosphere and stirred for 0.5 hour at room temperature. Samples were collected and analyzed by thin-layer chromatography (TLC) (DCM / MeOH=10:1) to monitor the reaction. The starting materials almost disappeared, and a new point was detected on the chromatogram.

[0182] The reaction system was filtered, and the filter cake was washed with EA three times. The filtrates were combined and concentrated to dryness to obtain 200 mg of the product as a white solid in 100% yield. The product was directly used in the subsequent reaction without purification. [M+H] + =413.3.

[0183] Synthesis H0005

[0184] 2.0 g of dichlororesin was weighed and placed in a polypeptide synthesis tube. Dichloromethane DCM (10 mL) was added and allowed to swell at room temperature for 30 minutes. The solvent was removed by vacuum suction. The resin was washed twice with 7 mL of DCM, each washing time being 1 minute. The solvent was removed by vacuum suction. Then H0005-b (200 mg) was weighed and added to a 50 mL centrifuge tube. DCM (approximately 10 mL) was added, and the solid was dissolved by shaking. Then the resulting mixture was added to the above resin. Stirring was continued until all the solution was absorbed into the resin (if the resin stuck to the walls of the tube, a small amount of DCM was used to wash the walls of the tube). Stirring was continued for 4-5 hours. After the reaction was complete, the appropriate amount of methanol was added. Stirring was continued for 30 minutes. The solvent was removed by vacuum suction.The resin was washed sequentially once with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each washing for 1 minute. The solvent was removed by vacuum aspiration. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, while the solution had a yellowish tint, indicating its suitability for the next coupling step.

[0185] Deprotection was performed twice by adding 10 mL of a prepared 20% piperidine / DMF solution and reacting for 10 minutes each time. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially with 10 mL of DMF twice, methanol once, DMF once, methanol once, and DMF twice, each washing for 1 minute. The solvent was removed by vacuum aspiration. A small amount of dry resin was collected for ninhydrin detection. Both the resin and solution were dark blue.

[0186] 563 mg Fmoc-Phe-OH and 197 mg HOBt were added to a 50 mL centrifuge tube. Then approximately 7 mL DMF was added. The solid was dissolved by shaking. Then 0.24 mL DIC was added. The mixture was activated for 10-30 minutes to obtain an activated reaction solution.

[0187] Three molar equivalents of the activated reaction solution were added to the resin. Stirring was carried out until the solution was completely absorbed by the resin (if the resin adhered to the walls of the tube, a small amount of DCM was used to rinse the walls of the tube). Stirring was carried out for 2-3 hours. After completion of the reaction, the solvent was removed by vacuum suction. The resin was washed sequentially twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, in a volume of 10 mL, with each wash duration of 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 was yellowish, indicating suitability for the next coupling step.

[0188] Deprotection was performed twice by adding 10 mL of a prepared 20% piperidine / DMF solution and reacting for 10 minutes each time. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each washing for 1 minute. The solvent was removed by vacuum aspiration. A small amount of dry resin was collected for ninhydrin detection. Both the resin and the solution were dark blue.

[0189] 531 mg Fmoc-GG-OH and 197 mg HOBt were added to a 50 mL centrifuge tube. Then approximately 10 mL DMF was added. The solid was dissolved by shaking. Then 0.24 mL DIC was added. Activation was carried out for 10-30 minutes to obtain an activated reaction solution.

[0190] Three molar equivalents of the activated reaction solution were added to the resin. Stirring was performed to completely absorb the solution by the resin (if the resin adhered to the walls of the tube, a small amount of DCM was used to wash the walls of the tube). Stirring was continued for 2-3 hours. After completion of the reaction, the reaction solution was removed by vacuum suction. The resin was successively washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, in a volume of 10 mL, with each wash duration of 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 was yellowish, indicating suitability for the next coupling step.

[0191] Deprotection was performed twice by adding 10 mL of a prepared 20% piperidine / DMF solution and reacting for 10 minutes each time. After completion of the reaction, the solution was removed by vacuum suction. The resin was successively washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each washing for 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. Then, 462 mg of MC-OSu was placed in a 50 mL centrifuge tube, and approximately 10 mL of DMF was added. The solid was dissolved by shaking. Then, 0.24 mL of DIEA was added to the resin. Stirring was continued until the solution was completely absorbed by the resin (if the resin stuck to the walls of the test tube, a small amount of DCM was used to rinse the walls). Stirring was continued for 2-3 hours.After the reaction was complete, the reaction solution was removed by vacuum aspiration. The resin was washed sequentially: twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each washing for 1 minute. The solvent was removed by vacuum aspiration. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, while the solution was yellowish, indicating its suitability for the next coupling step.

[0192] The resin was washed twice with 10 ml of methanol. The solvent was then thoroughly removed by vacuum aspiration. The resin was poured out and weighed. Lysis buffer was prepared in a 250 ml conical flask with a TFE / DCM ratio of 80% / 20% and a volume 7-8 times the weight of the peptide resin. The lysis buffer was added to the peptide resin and shaken well. The resin was completely soaked in lysis buffer, and lysis was carried out at room temperature for 2-3 hours. The lysis buffer was then filtered using a simple syringe filter, the resin was washed with 1-2 ml of DCM, and discarded. Then, 150 ml of pre-chilled anhydrous ether was added to the lysis buffer, the mixture was shaken well, and incubated for 20-30 minutes. Using a 50 ml centrifuge tube, the above system was centrifuged in a centrifuge at 3500 rpm for 3 minutes, and the supernatant was decanted and discarded.The solid was shaken with pre-chilled anhydrous ether, washed once with ultrasound, centrifuged at 3500 rpm for 3 min, and the supernatant decanted and discarded. The solid was placed in a centrifuge tube and allowed to air dry overnight before being subjected to preparative purification to afford 125 mg of product as a white solid in 40% yield. [M+H] + = 645.4.

[0193] Synthesis H0013

[0194] 150 mg of the starting material H0005 and 55 mg of TSTU were weighed and added to a 10 mL single-neck round-bottomed flask. Anhydrous DMF (3 mL) was added under a nitrogen atmosphere and stirred for 20 min. Then, 18 mg of TSN00643 and 20 μL of DIEA were successively added to the reaction system. Stirring was carried out at room temperature for 2 hours under a nitrogen atmosphere. Samples were collected and detected by HPLC to monitor the reaction. The peak of the starting materials completely disappeared, and new peaks were detected.

[0195] The reaction system was subjected to preparative purification, the target product was collected and lyophilized to obtain approximately 22 mg of the product as a yellowish solid. [M+H] + = 1062.7.

[0196] Synthesis of H0019

[0197] H0013 (30 mg) was weighed and added to a 10 mL single-neck round-bottomed flask, followed by purified water (2 mL). Stirring was continued until dissolved. A DMF solution (2 mL) containing HX20111 (19.5 mg) was added to the reaction mixture and stirred. After an overnight reaction, HPLC was used to monitor the reaction until all the starting material had been converted to intermediate compounds. Then, the reaction system was cooled to 0 ~ 10 °C, and 0.5% LiOH solution was added to the reaction system at a controlled temperature until the pH of the reaction system became approximately 12. The reaction was stirred for 20 minutes, and the reaction progress was monitored using HPLC until all the intermediate compounds were consumed, and then 30% acetic acid (1.5 mL) was added to the reaction system to quench the reaction.

[0198] The reaction system was subjected to preparative purification, the target product was collected and lyophilized to obtain approximately 25 mg of the product as a yellowish solid. [(M+3H) / 3] + = 1182.3.

[0199] 3.3. Receipt of H0020, H0021, H0034 and H0035

[0200] The payload linker intermediates H0020, H0021, H0034, and H0035 can be prepared using similar synthetic methods and reagents. The intermediates H0014, H0015, H0026, and H0032 were synthesized using a method similar to that for H0013, and then used to synthesize H0020, H0021, H0034, and H0035, respectively.

[0201] LC-Ms data are shown in the following table:

[0202] 3.4. Obtaining LB302-2-4

[0203] HX20111 and the intermediate MC-GGFG-Dxd (molar ratio ~1:2) were weighed and dissolved in water and DMF, respectively, and then thoroughly mixed to obtain a mixture, which was reacted at 0-40°C for 0.5-30 hours. After completion of the reaction, 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 another 0.2-20 hours. After completion of the reaction, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain the payload linker intermediate LB302-2-4. Theoretical mass: 3486.52, measured: [(M+3H) / 3] + = 1163.3.

[0204] 3.5. Obtaining LB302-2-1

[0205] LB3 02-2-1 can be prepared using similar synthesis methods and reagents. Example 4. Construction of antibody expression vector, antibody expression, purification and identification

[0206] 4.1. Preparation of modified anti-human HER2 antibody Ab0001-LCCTL-HC

[0207] Plasmids for antibody expression Ab0001-LCCT L -HC (light chain SEQ ID NO: 9, heavy chain: SEQ ID NO: 10) was constructed as follows. Antibody sequence Ab0001-LCCT L -NS: based on the amino acid sequence of trastuzumab, and GALPETGG was introduced at the C-terminus of the light chain, where LPETGG is the ligase donor substrate recognition sequence and GA is the spacer sequence. The plasmids were transfected into CHO cells, the cell population was grown, and it was screened for high-expressing cells. The cells were cultured according to the trastuzumab culture process in a 5-10 liter reactor, and the supernatant was collected.

[0208] 4.2. Purification of Ab0001-LCCT antibody L -HC

[0209] Cleaning Ab0001-LCCT L -NS was performed in a standard manner using a combination of MabSelect affinity chromatography and Sepharose S cation exchange chromatography, and purified products were dissolved in Trastuzumab original drug buffer (5 mM histidine-HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0), and aliquots were frozen in small quantities.

[0210] 4.3. Quality control of Ab0001-LCCT antibodies L -HC

[0211] The purity of the above purified antibody Ab0001-LCCT L -HC was 98.5% according to SDS-PAGE; the high molecular weight polymer content in the sample was less than 0.4% according to SEC-HPLC; the endotoxin content was less than 0.098 EU / mg.

[0212] 4.4. Production of other modified anti-human antibodies

[0213] According to a similar method, a terminal modification based on the ligase recognition sequence was introduced into the C-terminus of the light and / or heavy chain of trastuzumab, respectively, to obtain a modified antibody.

[0214] Modified anti-human HER2 antibodies based on Ab0001 (trastuzumab) are listed in the following table. LPETGG in the terminal modification sequence is the ligase donor substrate recognition sequence, and GA is the spacer sequence.Modified anti-human HER2 antibodies Example 5. Preparation of antibody-lower molecular weight compound conjugates

[0215] 5.1. The payload linker intermediates were respectively conjugated to the antibody using ligase in a site-specific manner to form ADCs. The conjugation reaction is disclosed in WO 2015 / 165413A1. The resulting ADCs are listed in the following table:

[0216] 5.2. Reference ADCs LC1184(8) and LC1184(4) were prepared, respectively, by direct coupling of the intermediate Mc-GGFG-Dxd with Ab0001-LCCT L -НС (Cys conjugation, i.e., conjugation through bonds formed by the maleimide structure(s) with the thiol group(s) of Cys). The method for carrying out the conjugation reaction is known in the art. LC1184(8) contains eight Mc-GGFG-Dxd fragments introduced into reduced interchain cysteines. LC1184(4) contains four Mc-GGFG-Dxd fragments introduced into reduced interchain cysteines. Example of the effect of action 1. The effect of low-molecular compounds on cell proliferation

[0217] Aim of the study: In this experiment, cell viability was recorded using CellTiter-Glo to evaluate the effects of small molecules on the proliferation of the HER2-high tumor cell line SK-BR-3 and to analyze the effects of small molecules on tumor cell proliferation.

[0218] Materials and equipment

[0219] PBS: Gibco, Cat# 10010-023); Trypsin: Gibco, Cat# 25200056); FBS: Gibco, Cat# 10270-106); CellTiter-Glo® Luminescent Cell Viability Assay: Promega, Cat# G7573); McCoy'5A: Gibco, Cat# 16600-082); L15: Hyclone, Cat# SH30525.01); DMEM: Gibco, Cat# 11995-065). 96-well plate: Corning / 3603; 96-well deepwell plate: Thermo / 278743; 15ml EP tubes: Thermo / 339650; 1.5ml EP tubes: Beaver.

[0220] Cell covering -1 day

[0221] (1) Microscopic examination of cells

[0222] (2) Cell digestion: fermentation with 2ml 0.25% trypsin for 3 minutes;

[0223] (3) Centrifugation: 1000 rpm for 5 minutes;

[0224] (4) Cell Counting

[0225] A. Cell dilution:

[0226] B. Cell seeding: 100 µl / well.

[0227] C. Incubation: 37°C, 5% CO2, overnight.

[0228] Cellular tests

[0229] (1) Microscopic examination

[0230] (2) Obtaining medicinal compound:

[0231] A. Preparation of sample preparation buffer: The desired amount of sample preparation buffer was prepared using the culture medium (10% FBS) for the test cells;

[0232] B. Sample preparation: The sample was diluted from the first concentration to the desired concentration in a 96-well deep-well plate.

[0233] Test compounds: Ten final concentrations were prepared by diluting with cell medium to the following concentrations: 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, and 0.000512 nM.

[0234] Positive control puromycin: A 2.5 mg / mL stock solution diluted with cell medium was used. A 250-fold dilution was prepared beforehand, and then a 2-fold dilution was performed upon compound application until the test puromycin concentration reached 5 μg / mL.

[0235] (3) Compound application: For each concentration of the diluted compound series, 100 μL / well were applied in triplicate.

[0236] (4) After adding the drug, the cells were incubated in an incubator for 120 hours.

[0237] Cell viability test

[0238] (1) Preparation of detection reagents: CellTiter-Glo luminescent cell viability assay reagent was allowed to warm up to room temperature while protected from light.

[0239] (2) Cell preparation: The test cells were removed from the incubator and equilibrated at room temperature (25°C) for 30 minutes.

[0240] (3) ATP detection: The culture medium was removed and 100 μL of DMEM per well, 50 μL of CTG per well were added to the 96-well plate, protected from light with aluminum foil and shaken with a shaking and mixing device at 200 rpm at room temperature for 10 minutes.

[0241] (4) Detection procedure: After setting the procedure, the black-walled, transparent-bottomed plate was placed into the instrument without a lid.

[0242] (5) Data collection.

[0243] 5. Data processing and analysis

[0244] GraphPad software was used for data processing. The cell proliferation inhibition rate was plotted against the concentration of the control sample or test sample using a four-parameter curve-fitting model, and R values ​​were obtained accordingly. 2 (rounded to 4 significant digits) and IC 50 (rounded to 4 significant digits) or EU 50 for the control sample and the test sample. If necessary, the relative biological activity A for the sample, i.e., the activity coefficient, was calculated. Dxd was used as the control sample for calculating the activity coefficient.

[0245] Cell proliferation inhibition rate (%) = (luminescence value of drug-treated group - luminescence value of puro group) / (luminescence value of blank control group - luminescence value of puro group) × 100; where puro group refers to the puromycin-treated group

[0246] Sample activity ratio (%) = (IC value 50 control sample / IC value 50 test sample) × 100

[0247] Activity coefficient of control sample (%) = (IC value 50 control sample / IC value 50 previous control sample) × 100

[0248] 6. Results taken into account

[0249] R 2 ≥0.950; CV% between parallel samples ≤ 30%.

[0250] Results obtained:

[0251] The results are shown in the following table. Effect of Action Example 2: Effect of HER2-Targeted Conjugates on Cell Proliferation

[0252] Aim of the study: In this experiment, cell viability was recorded using the CellTiter-Glo assay to evaluate the effect of ADC on the proliferation of two types of HER2-high cells, such as SK-BR-3, NCI-N87 tumor cell lines, and HER2-negative MDA-MB-468 tumor cell line, and the effect of ADC on tumor cell proliferation was also evaluated.

[0253] Materials and equipment

[0254] PBS: Gibco, Cat# 10010-023); Trypsin: Gibco, Cat# 25200056); FBS: Gibco, Cat# 10270-106); CellTiter-Glo® Luminescent Cell Viability Assay: Promega, Cat# G7573); McCoy'SA: Gibco, Cat# 16600-082); L15: Hyclone, Cat# SH30525.01); DMEM: Gibco, Cat# 11995-065). 96-well plate: Corning / 3603; 96-well deepwell plate: Thermo / 278743; 15ml EP tubes: Thermo / 339650; 1.5ml EP tubes: Beaver.

[0255] Cell covering -1 day

[0256] (1) Microscopic examination of cells

[0257] (2) Cell digestion: fermentation with 2ml 0.25% trypsin for 3 minutes;

[0258] (3) Centrifugation: 1000 rpm for 5 minutes;

[0259] (4) Cell Counting

[0260] A. Cell dilution:

[0261] B. Cell seeding: 100 µl / well.

[0262] C. Incubation: 37°C, 5% CO2, overnight.

[0263] Cellular tests

[0264] (1) Microscopic examination

[0265] (2) Obtaining medicinal compound:

[0266] A. Preparation of sample preparation buffer: The desired amount of sample preparation buffer was prepared using the culture medium (10% FBS) for the test cells;

[0267] B. Sample preparation: The sample was diluted from the first concentration to the desired concentration in a 96-well deep-well plate.

[0268] Test samples: Ten final concentrations were prepared by diluting with cell medium to the following concentrations: 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0.000512, and 0.0001024 nM.

[0269] Positive control puromycin: A 10 mg / mL stock solution diluted with cell medium was used. A 100-fold dilution was prepared beforehand, and then a 10-fold dilution was performed upon compound application to achieve a test concentration of 5 μg / mL puromycin.

[0270] (4) Compound application: For each concentration of the diluted series, the compound was applied in triplicate at 100 μL / well.

[0271] (5) After adding the drug, the cells were incubated in an incubator for 120 hours.

[0272] Determination of cell viability

[0273] (1) Preparation of detection reagents: CellTiter-Glo Luminescent Cell Viability Assay Reagent was allowed to warm up to room temperature while protected from light.

[0274] (2) Cell preparation: The test cells were removed from the incubator and equilibrated at room temperature (25°C) for 30 minutes.

[0275] (3) ATP detection: The culture medium was removed, DMEM 100 μL / well and CTG 50 μL / well were added to a 96-well plate protected from light with aluminum foil and shaken with a shaking and mixing device at 200 rpm at room temperature for 10 minutes.

[0276] (4) Detection procedure: After setting the procedure, the black-walled, transparent-bottomed plate was placed into the instrument without a lid.

[0277] (5) Data collection.

[0278] 5. Data processing and analysis

[0279] GraphPad software was used for data processing. The cell proliferation inhibition rate was plotted against the concentration of the control sample or test sample using a four-parameter curve-fitting model, and R values ​​were obtained accordingly. 2 (rounded to 4 significant digits) and IC 50 (rounded to 4 significant digits) or EU 50 for the control sample and the test sample.

[0280] Cell proliferation inhibition rate (%) = (luminescence value of drug-treated group - luminescence value for puro group) / (luminescence value of blank control group - luminescence value for puro group) × 100; where puro group refers to the puromycin-treated group.

[0281] 6. Results taken into account

[0282] R 2 ≥0.950; CV% between parallel samples ≤ 30%.

[0283] Results obtained:

[0284] The results are shown in the following table and Figures 1, 2 and 3.

[0285] In HER2-highly expressing SK-BR-3 and NCI-N87 cells, the conjugates (CH-2-589, CH-2-593, CH-2-518, LC302-2-1(4), and LC302-2-4(4)) demonstrated very high efficacy. The IC50 values ​​for the conjugates were lower than those for the small molecule Dxd payload. In HER2-negative cells, the conjugates showed minimal efficacy. Cytotoxicity was not observed in HER2-negative cells. MDA-MB-468 demonstrated conjugate stability with reduced off-target release of the payload, and therefore good safety. This indicates that the conjugates exhibit HER2-dependent cytotoxic activity in a dose-dependent manner, demonstrating good targeting ability, very high efficacy, and good safety.Experimental example 3: Evaluation of the bystander killing effect

[0286] Objective of the experiment. The bystander killing activity of ADCs (CH-2-589, CH-2-593, CH-2-518, and LC302-2-4(4)) on MDA-MB-468 cells was tested in a coculture system of HE112-positive SK-BR-3 tumor cell line and HE112-negative MDA-MB-468 tumor cell line, and the bystander killing effect was compared with that of a reference ADC (LC1184(8)) with a structure similar to DS8201a.

[0287] Materials and equipment

[0288] L15: Hyclone, SH30525.01; McCoy's 5A: Gibco, 16600-082; FBS: Gibco, 10099-141; Firefly Luciferase Reporter Gene Assay Kit: Beyotime, RG006; 50ml Centrifugal Tube: Corning, 430829; 15ml Centrifugal Tube: Corning, 430791; 96-Well Plate: Corning, 3599; CO2 Cell Incubator: Thermo; Centrifuge: Thermos, L550; Microscope: Nikon, ISZ; Cell Counter: Countstar, IC1000; Cytation3 Plate Reader: BioTek, Cytation3; Multi-mode microplate reader: BioTek, Synergy HTX; SK-BR-3: ATCC, HTB-30; MDA-MB-468-Luc-GFP: produced in-house using lentiviral infection.

[0289] Experimental method

[0290] 1) Cell collection and counting.

[0291] 2) The cell density was adjusted to 1×10 5 cells / ml.

[0292] 3) The cell mixture was prepared with the ratio of SKBR3:MDA-MB-468-Luc-GFP = 4:1.5 ml.

[0293] Cell Coverage: 1×10 4cells per well, 100 µl per well

[0294] Test samples: Three final concentrations were prepared by diluting with cell medium to the following concentrations: 50 nM, 10 nM, and 1 nM.

[0295] 4) The drug was administered the next day at a rate of 100 μl per well.

[0296] 5) Incubated in an incubator with 5% CO2 at 37°C for 120 hours.

[0297] Washed with PBS to remove dead cells and detected using a Cytation3 plate reader;

[0298] Biyuntian Luciferase Reporter Gene Assay Kit: Lysis was performed in an incubator at 37°C for 10 minutes using 100 µl of lysis buffer. The mixture was centrifuged at 2000 rpm for 5 minutes. A 100 µl supernatant was collected. 100 µl of substrate was added. Chemiluminescence was recorded using a microplate reader (amplification: 200).

[0299] Results

[0300] Two methods (GFP fluorescence counting and luciferase substrate luminescence assay) were used to determine the effects of bystander death. See Figures 4a, 4b, and 5).

[0301] All conjugates (CH-2-593, CH-2-589, CH-2-518, and 1X302-2-4(4)) showed very high bystander killing effects when analyzed using both methods. CH-2-593 showed a bystander killing effect comparable to or greater than that of LC1184(8). Example of effect size 4. In vivo evaluation of conjugates

[0001]

[0303] 5×10 6 LMT-1 human breast cancer cells (HER2 medium) were inoculated subcutaneously into the right flank of SCID Beige mice. After 7 days, when the tumor volume reached an average of 142 mm 3, tumor-bearing mice were intravenously injected with LC 1184(8) and five other different conjugates at a dose of 5 mg / kg. Tumor volume was measured twice a week with a caliper. LC1184(8) showed better efficacy than LC1184(4), suggesting that with the same payload, a higher DAR will lead to better efficacy. LC302-2-1(4) and LC302-2-4(4) showed better efficacy than LC1184(8) (Figure 6). Thus, the conjugate of the invention achieves high efficacy with a low DAR value.

[0304] 4.2. Capan-1 Human Pancreatic Cancer Xenograft Model (1)

[0305] To establish a xenograft model, BALB / c nude mice were subcutaneously inoculated into the right flank with 5×10 6Capan-1 human pancreatic cancer cells (with a low HER2 level). After 8 days, when the tumor volume reached an average of 178 mm3, the tumor-bearing mice were intravenously injected with LC 1184(8) and five other different conjugates at a dose of 5 mg / kg. Tumor volume was measured twice a week with a caliper. LC1184(8) showed better efficacy than LC1184(4). LC302-2-l(4) and LC302-2-4(4) showed comparable efficacy to LC1184(8) (Figure 7).

[0306] 4.3. Capan-1 Human Pancreatic Cancer Xenograft Model (2)

[0307] Purpose of the study: The aim of the study is to evaluate the in vivo antitumor efficacy of compounds CH-2-589, CH-2-593, CH-2-518 and LC302-2-4(4) in the treatment of human pancreatic cancer subcutaneous xenograft model Capan-1 in female nude BALB / c mice.

[0308] Study outline:

[0309] Cell culture: Capan-1 tumor cells (ATCC-NTB-79) were maintained in vitro as a monolayer culture in IMDM medium supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic solution at 37°C in an atmosphere of 5% CO2. Tumor cells were regularly subcultured twice a week by treatment with trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0310] Animals: BALB / c nude mice, female, 6-8 weeks old, body weight approximately 18-20 g. A total of 48 animals (30 plus 60%) were used in the study, purchased from Shanghai Lingchang Laboratory Animal Co., LTD or other certified suppliers.

[0311] Tumor inoculation: Each mouse was subcutaneously inoculated in the right flank with Capan-1 tumor cells (5×10 6) in 0.2 ml PBS with Matrigel (1:1) for tumor development. For the efficacy study, animals were randomized, and treatment was started when the average tumor volume reached approximately 150-200 mm3. The administration of the test drug and the number of animals in each group are shown in the following experimental design table. Groups and procedures Animal housing: An acclimatization period of approximately one week was allowed between animal collection and tumor inoculation to acclimate the animals to the laboratory environment. Mice were housed in a special pathogen-free environment and in individual ventilated cages (3 mice per cage). All objects, bedding, and water were sterilized before use. When working in the room with mice, personnel were provided with textile or vinyl gloves. The electronic tag was clearly labeled with symbols and symbols indicating the number of animals, sex, strain, collection date, treatment administered, study number, group number, and treatment start date. Food and water containers were changed twice a week. The target conditions for the minimal environmental and lighting regime were as follows:

[0313] Temperature 20 ~ 26°C

[0314] Humidity 40~70%

[0315] Light cycle: 12 hours light and 12 hours dark.

[0316] Diet: All animals had free access to a standard, certified commercial laboratory diet. Maximum permissible concentrations of contaminants in the diet were monitored and regularly analyzed by the manufacturer. Animals had free access to autoclaved municipal tap water suitable for human consumption. The diet was deemed free of known contaminants that could influence tumor growth.

[0317] Group Allocation: Before treatment, all animals were weighed and their tumor volumes were measured. Because tumor volume can influence the effectiveness of any treatment, mice were assigned to groups using Excel-based randomization software that performs stratified randomization based on tumor volumes. This ensures that all groups are comparable at baseline.

[0318] Observations: The protocol and any amendments or procedures related to the care and use of animals in this study were reviewed and approved by the WuXi AppTec Institutional Animal Care and Use Committee (IACUC) prior to experimentation. During the study, animal care and use were conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). After inoculation, animals were examined daily for morbidity and mortality. During regular monitoring, animals were examined for any effects of tumor growth and treatment on normal behavior, such as mobility, food and water consumption, weight gain / loss (body weight was measured twice a week), eye clouding, coat color changes, and any other abnormal effects. Mice mortality and observed clinical signs were recorded in each subgroup of animals.

[0319] Endpoints: The primary endpoint was to determine whether tumor growth could be delayed or whether mice could be cured. Tumor size was measured twice a week in two dimensions using a caliper, and the volume was expressed as mm 3 according to the formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Tumor size was then used to calculate TGI (%) values ​​and the relative tumor proliferation rate T / C (%). TGI was calculated for each group of animals using the formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100; where Ti is the mean tumor volume in the treatment group on a given day, T0 is the mean tumor volume in the treatment group on the first day of treatment, Vi is the mean tumor volume in the vehicle-treated control group on the same day as Ti, and V0 is the mean tumor volume in the vehicle-treated group on the first day of treatment.

[0320] The T / C value (in %) was calculated for each group using the formula: T / C% = T RTV / C RTV × 100% (where T RTV - relative mean tumor volume (RTV) of the treatment group; C RTV - relative mean tumor volume (RTV) of the group receiving the vehicle on the same day as T RTV ). The relative tumor volume (RTV) was calculated for each group using the formula: RTV = V t / V0; where V0 is the tumor volume on the first day of treatment, V t - tumor volume on a given day.

[0321] Research Completion:

[0322] 1) Body weight loss: Any animal that lost 20% of body weight on any day was humanely euthanized or turned over to a veterinarian.

[0323] 2) Tumor Burden: Tumor burden should not exceed 3000 mm3. Individual animals were euthanized immediately when tumor burden reached 3000 mm3. Ulceration: If tumor ulceration occurred, the following procedures were used:• Animals with ulcerated tumors were observed at least 3 times per week and increased in frequency up to daily depending on clinical signs.• Ulcerated tumors that were not sloughed were cleaned with an appropriate wound cleansing solution (e.g., Novalsan). Antibiotic cream was applied to the ulcer / lesion only as directed by a veterinarian. Euthanasia criteria included the presence of a lesion (meeting one or more of the following):• Does not heal or form an eschar within 1 week.• Lesion diameter greater than 5 mm.• Lesion becomes cavitated.If signs of infection (e.g. presence of pus) or bleeding developed, or if the animal showed signs of discomfort (e.g. excessive licking and biting at a specific location) or systemic signs of disease (lethargy, decreased activity, decreased food intake, weight loss), then a veterinarian's opinion was used to exclude the animal from the experiment.

[0324] 3) Clinical Signs: Animals were euthanized if they were considered moribund (unless the IACUC granted special permission based on adequate justification included in the protocol, and if the animals were given enhanced supportive care, such as warm subcutaneous fluids (SQ), gel food, placing a food bowl near the animal to allow it to reach the food, placing the cage on a heating pad for additional warmth, etc. Note: a moribund state indicates that the animal is unlikely to survive). Questions regarding these endpoints were discussed with a veterinarian.

[0325] Clinical examples of illnesses and diseases included the following:• Hunched over.• Persistent recumbent position and unresponsive to handling or other stimuli.• Signs of severe organ or systemic failure.• Inanition.• Hypothermia.• CNS: seizures.• Respiratory: rapid breathing, labored breathing, cough, wheezing.• Gastrointestinal: diarrhea lasting >2 days, jaundice.

[0326] Any animal exhibiting the above clinical problems is humanely euthanized by CO2 exposure.

[0327] In case of unexpected death, no autopsy was performed.

[0328] Statistical analysis: An independent sample t-test was used to compare two groups. One-way analysis of variance was used to compare three or more groups. If a significant F-statistic (the ratio of the variance in the treatment group to the error variance) was obtained, multiple comparison procedures were used after ANOVA analysis. All data were analyzed using SPSS 17.0. A p value of <0.05 was considered statistically significant.

[0329] Results obtained:

[0330] All conjugates showed significant tumor inhibition effects. (Figure 8a and Figure 8b).

[0331] 4.4. NCI-N87 human gastric cancer xenograft model.

[0332] Aim of the study: The aim of the study is to evaluate the in vivo antitumor efficacy of CH-2-589, CH-2-593, CH-2-518 and LC302-2-4(4) in the treatment of NCI-N87 human gastric cancer subcutaneous xenograft model in female BALB / c nude mice.

[0333] Study outline:

[0334] Cell culture: NCI-N87 tumor cells (ATCC, Manassas, VA, Cat# CRL-5822) were maintained in vitro as a monolayer culture in RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in a 5% CO2 atmosphere. Tumor cells were regularly subcultured twice a week by treatment with trypsin-EDTA. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0335] Animals: BALB / c mice, female, 6-8 weeks old, body weight approximately 18-22g. A total of 36 mice (30 plus 20%) were used in the study, which were purchased from Shanghai Lingchang Laboratory Animal Co., LTD or other certified suppliers.

[0336] Tumor inoculation: Each mouse was subcutaneously inoculated in the right flank with NCI-N87 tumor cells (10×10 6) in 0.2 ml PBS with Matrigel (1:1) for tumor development. For efficacy studies, animals were randomized, and treatment was started when the average tumor volume reached approximately 150-200 mm 3 The administration of the test drug and the number of animals in each group are shown in the following experimental design table.Groups and procedures

[0337] Results obtained:

[0338] All conjugates showed significant tumor inhibition effects. (Figure 9a and Figure 9b). Example of the effect of action 5. Stability of conjugates in serum ex vivo.

[0339] To study the ex-vivo serum stability, LC302-2-1(4), LC302-2-4(4), and LC1184(8) conjugates were inoculated into pooled human serum at 37°C, respectively. After 0, 24, 48, and 96 hours, the conjugates were captured with antigen, deglycosylated with glycosidase, and dissociated with acid. The supernatant was collected and centrifuged, and then detected by high-resolution LCMS to determine the DAR value.

[0340] LC302-2-1(4) and LC302-2-4(4) were found to be quite stable after incubation in serum for 96 hours. No significant decrease in DAR was observed for LC302-2-1(4) and LC302-2-4(4) after incubation for 96 hours. However, the DAR for LC1184(8) decreased from 7.8 to 2.6 after incubation for 96 hours (Figure 10). 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EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>< / insdseq> < / sequencedata> < / st26sequencelisting> <---

Claims

1. Compound of formula (I) where opSu represents R 0 represents C 1-3 alkyl; n is any integer from 2 to 5; k1 and k2 independently represent an integer from 1 to 7; i is an integer between 1 and 20; j is an integer between 1 and 20; P1 and P2 independently represent a payload having the structure of formula (i') a is equal to 0; each of the carbon atoms labeled p1* and p2* represents an asymmetric center and the asymmetric center has the S-configuration, the R-configuration, or is racemic; L 1 selected from -CH2-, where "#" denotes the position of attachment to the carbonyl; R 1 selected from Cl and Br; R 2represents F.

2. The compound according to claim 1, having a structure of formula (I-1) where P1, P2, R 0 , opSu, n, i and j have the meanings defined in paragraph 1.

3. The compound according to claim 1, where in formula (i') L 1 selected from -CH2-, where "#" denotes the position of attachment to the carbonyl.

4. The compound according to claim 3, where in formula (i') L 1 selected from -CH2- where "#" denotes the position of attachment to the carbonyl.

5. A compound according to any one of claims 1 to 4, wherein in formula (i') the carbon atom designated as p1* has the S-configuration or the racemic configuration.

6. The compound according to claim 5, wherein in formula (i') the carbon atom designated as p1* has the S-configuration.

7. A compound according to any one of claims 1 to 4, wherein in formula (i') the carbon atom designated as p2* has the S-configuration or the racemic configuration.

8. The compound according to claim 7, wherein in formula (i') the carbon atom designated as p2* has the S-configuration.

9. A compound according to any one of paragraphs 1-4, where in formula (i') R 1 represents Cl.

10. The compound according to any one of paragraphs 1-3, where a is equal to 0, R 1 represents Cl, R 2 represents F and L 1 selected from -CH2- 11. The compound according to any one of paragraphs 1-4, where a is equal to 0, R 1 represents Cl, R 2 represents F and L 1 represents 12. A connection according to any one of paragraphs 1-4, where the payload is selected from:

13. A connection according to any one of paragraphs 1-4, where the payload is selected from:

14. The connection according to any one of paragraphs 1-4, where R 0 is methyl.

15. A compound according to any one of paragraphs 1-4, where n is 3.

16. A compound according to any one of claims 1-4, where k1 and k2 are independently equal to 1, or 3, or 5.

17. The compound according to claim 16, where k1 and k2 are independently equal to 5.

18. A compound according to any one of claims 1 to 4, wherein i and j independently represent a number from 1 to 12.

19. The compound according to claim 18, where i is a number from 2 to 8.

20. The compound according to item 19, where i is equal to 4.

21. The compound according to claim 18, where j is a number from 8 to 12.

22. The compound according to item 21, where j is equal to 12.

23. A conjugate having a structure of formula (II) where A is an antibody or an antigen-binding fragment thereof; z is an integer between 1 and 4; opSu is R 0 represents C 1-3 alkyl; n is any integer from 2 to 5; k1 and k2 independently represent an integer from 1 to 7; i is an integer between 1 and 20; j is an integer between 1 and 20; P1 and P2 independently represent a payload having the structure of formula (i') where a is equal to 0; each of the carbon atoms labeled p1* and p2* represents an asymmetric center and the asymmetric center has the S-configuration, the R-configuration, or is racemic; L 1 selected from -CH2-, where "#" denotes the position of attachment to the carbonyl; R 1 selected from Cl and Br; R 2 represents F.

24. The conjugate according to claim 23, where in formula (i') L 1 selected from -СН2-, where "#" denotes the position of attachment to the carbonyl.

25. The conjugate according to claim 24, where in formula (i') L 1selected from -CH2-, where "#" denotes the position of attachment to the carbonyl.

26. The conjugate of any one of claims 23-25, wherein in formula (i') the carbon atom designated as p1* has the S-configuration or the racemic configuration.

27. The conjugate of claim 26, wherein in formula (i') the carbon atom designated as p1* has the S-configuration.

28. The conjugate according to any one of claims 23-25, wherein in formula (i') the carbon atom designated as p2* has the S-configuration or the racemic configuration.

29. The conjugate of claim 28, wherein in formula (i') the carbon atom designated as p2* has the S-configuration.

30. The conjugate according to any one of paragraphs 23-25, where in formula (i') R 1 represents Cl.

31. The conjugate according to claim 23 or 24, where a is 0, R 1 represents Cl, R 2 represents F and L 1 selected from -CH2-, 32. The conjugate according to any one of paragraphs 23-25, where a is 0, R 1 represents Cl, R2 represents F and L 1 represents 33. The conjugate of any one of paragraphs 23-25, wherein the payload is selected from:

34. The conjugate according to any one of paragraphs 23-25, where R 0 is methyl.

35. The conjugate according to any one of claims 23-25, where n is 3.

36. The conjugate of any one of claims 23-25, wherein k1 and k2 are independently equal to 1, or 3, or 5.

37. The conjugate according to claim 36, where k1 and k2 are independently equal to 5.

38. The conjugate of any one of claims 23-25, wherein i and j independently represent a number from 1 to 12.

39. The conjugate of claim 38, where i is a number from 2 to 8.

40. The conjugate according to claim 39, where i is equal to 4.

41. The conjugate according to claim 38, j is a number from 8 to 12.

42. The conjugate according to claim 41, where j is 12.

43. The conjugate of claim 23, wherein the antibody or antigen-binding fragment is modified to bind to a fragment (Gly) n in a compound of formula (I) as defined in any one of claims 1 to 22.

44. The conjugate according to any one of claims 23-43, where z is 2.

45. The conjugate of any one of claims 23-44, wherein the antibody is an anti-HER2 antibody.

46. ​​A pharmaceutical composition having antitumor activity against HER2-positive tumor cells, comprising a prophylactically or therapeutically effective amount of a conjugate according to any one of claims 23-45 and at least one pharmaceutically acceptable carrier.

47. The pharmaceutical composition of claim 46, wherein the conjugate has a drug to antibody ratio (DAR) equal to an integer or non-integer number from 1 to 8.

48. The pharmaceutical composition of claim 47, wherein the conjugate has a drug to antibody ratio (DAR) equal to an integer or non-integer number from 3 to 4.

49. Use of a conjugate according to any one of claims 23-45 or a pharmaceutical composition according to any one of claims 46-48 in the manufacture of a medicinal product for the treatment of a disease; wherein the disease is a tumor containing HER2-positive tumor cells.

50. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (i) where a is equal to 0; each of the carbon atoms labeled p1* and p2* represents an asymmetric center and the asymmetric center has the S-configuration, the R-configuration, or is racemic; L 1 selected from -CH2-, where "#" denotes the position of attachment to the carbonyl; R 1 selected from Cl and Br; R 2 represents F.

51. The compound according to claim 50, where in formula (i) L 1 selected from -СН2- where "#" denotes the position of attachment to the carbonyl.

52. A compound according to claim 50 or 51, wherein in formula (i) the carbon atom designated as p1* has the S-configuration or the racemic configuration.

53. A compound according to claim 52, wherein in formula (i) the carbon atom designated as p1* has the S-configuration.

54. A compound according to claim 50 or 51, wherein in formula (i) the carbon atom designated as p2* has the S-configuration or the racemic configuration.

55. A compound according to claim 54, wherein in formula (i) the carbon atom designated as p2* has the S-configuration.

56. The compound according to claim 50 or 51, where in formula (i) R 1 represents Cl.

57. The compound according to claim 50, where a is 0, R 1 represents Cl, R 2 represents F and L 1 selected from -CH2- 58. The compound according to claim 50 or 51, where a is 0, R 1 represents Cl, R 2 represents F and L 1represents 59. Compound of formula (1) where k is an integer from 1 to 7; P is a payload having the structure of formula (i') where a is equal to 0; each of the carbon atoms labeled p1* and p2* represents an asymmetric center and the asymmetric center has the S-configuration, the R-configuration, or is racemic; L 1 selected from -CH2-, where "#" denotes the position of attachment to the carbonyl; R 1 selected from Cl and Br; R 2 represents F.

60. The compound according to claim 59, where in formula (i') L 1 selected from -CH2- where "#" denotes the position of attachment to the carbonyl.

61. A compound according to claim 59 or 60, wherein in formula (i') the carbon atom designated as p1* has the S-configuration or the racemic configuration.

62. The compound according to claim 61, wherein in formula (i') the carbon atom designated as p1* has the S-configuration.

63. A compound according to claim 59 or 60, wherein in formula (i') the carbon atom designated as p2* has the S-configuration or the racemic configuration.

64. The compound according to claim 63, wherein in formula (i') the carbon atom designated as p2* has the S-configuration.

65. The compound according to claim 59 or 60, where in formula (i') R 1 represents Cl.

66. The compound according to claim 59, where a is 0, R 1 represents Cl, R 2 represents F and L 1 selected from -CH2- 67. The compound according to claim 59 or 60, where a is 0, R 1 represents Cl, R 2 represents F and L 1 represents 68. The connection according to claim 59 or 60, wherein the payload is selected from:

69. The compound according to claim 59, where k is 1, or 3, or 5.

70. The compound according to claim 69, where k is 5.

71. Compound of formula (I) where opSu represents R 0 represents C 1-3 alkyl; n is any integer from 2 to 5; k1 and k2 are independently equal to 1, 3, or 5; i is an integer between 1 and 20; j is an integer between 1 and 20; P1 and P2 independently represent a payload with the following structure:

72. The compound according to claim 71, having a structure of formula (I-1) where P1, P2, R 0 , opSu, n, i and j have the meanings defined in paragraph 71.

73. The compound according to item 71 or 72, where R 0 is methyl; and / or n is 3; and / or i is 4; and / or j is 8 or 12.

74. The compound according to item 71 or 72, where R 0 is methyl; n is 3; i is 4; j is 12.

75. The compound according to item 71 or 72, selected from:

76. A conjugate having a structure of formula (II) where A is an antibody against HER2; the antibody is modified to bind to the (Gly) moiety n in a compound of formula (I) as defined in any one of paragraphs 71 to 75; z is an integer between 1 and 4; opSu is R 0 represents C1-3 alkyl; n is any integer from 2 to 5; k1 and k2 independently represent 1, 3, or 5; i is an integer between 1 and 20; j is an integer between 1 and 20; P1 and P2 independently represent a payload having the structure of formula (i') 77. The conjugate according to claim 76, where R 0 is methyl; and / or n is 3; and / or i is 4; and / or j is 8 or 12.

78. The conjugate according to claim 76, where R 0 represents methyl; n represents 3; i represents 4; j represents 12.

79. The conjugate of claim 76, selected from:

80. The conjugate of any one of claims 76-79, wherein the antibody is trastuzumab.

81. A pharmaceutical composition having antitumor activity against HER2-positive tumor cells, comprising a prophylactically or therapeutically effective amount of a conjugate according to any one of claims 76-80 and at least one pharmaceutically acceptable carrier.

82. The pharmaceutical composition of claim 81, wherein the conjugate has a drug to antibody ratio (DAR) equal to an integer or non-integer number from 1 to 8.

83. The pharmaceutical composition of claim 81, wherein the conjugate has a drug to antibody ratio (DAR) equal to an integer or non-integer number from 3 to 4.

84. Use of a conjugate according to any one of claims 76-80 or a pharmaceutical composition according to any one of claims 81-83 in the manufacture of a medicinal product for the treatment of a disease; wherein the disease is a tumor containing HER2-positive tumor cells.

85. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has a structure selected from the following formulas:

86. Compound of formula (1) where k is an integer from 1 to 7; R has the following structure:

87. The compound according to claim 86, where k is 1, or 3, or 5.

88. The compound according to item 86, selected from:

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