Exatecan derivatives and their linker-payloads and conjugates

Abstract

To provide a novel ADC with higher potency and lower toxicity.SOLUTION: The present disclosure relates to the biopharmaceutical field, in particular, exatecan derivatives, linker-payloads and conjugates thereof, and antibody-drug conjugates. An example thereof is a compound of formula (II) below, where A represents an antibody or an antigen binding fragment thereof, and P1 and P2 independently have a payload with an exatecan derivative structure.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals, and more particularly to exatecan derivatives, their linker-payload and conjugates, antibody-drug conjugates, and corresponding methods for manufacturing and using them. [Background technology]

[0002] In conventional cancer treatment, chemotherapy is one of the main treatment strategies. However, off-target toxicity due to nonspecific accumulation in normal tissue, short treatment gaps, and low resistance limit the development of chemotherapy drugs. For decades, targeted therapies using monoclonal antibodies and polypeptides that bind to tumor cell surface-specific markers have shown less toxicity than chemotherapy. However, neither of these is highly effective in killing tumor cells.

[0003] In 1970, the therapeutic strategy of using toxin-carrying antibodies to selectively kill target cells was first discovered. Tumor-targeted drug conjugates primarily consist of ADCs, which couple antibodies that specifically recognize tumor cell surface antigens with a chemotoxin via a linker, allowing the chemotoxin to effectively kill tumor tissue. As of 2021, 120 ADCs were in clinical development.

[0004] Most cytotoxic payloads belong to two families: tubulin inhibitors (maytansinoids or auristatin) and DNA damage agents (mainly calicheamicin). Both are highly effective cytotoxic drugs, and IC 50 Although the inhibitory concentration (50% cell suppression) is within the nanomolar and picomolar range, it has the characteristic of having an unfavorable toxicity spectrum when administered systemically. By conjugating cytotoxic drugs to ADCs, they can be hidden in the bloodstream and delivered directly to tumor cells, thereby significantly reducing the toxicity of these potent drugs. Generally, drugs with excellent therapeutic indices are not suitable for development as warheads, and their IC 50The level is too low, and it is thought that it cannot perform its function when delivered to tumor cells in a certain amount.

[0005] Several ADC studies, by working to increase DAR or exploit bystander effects, offer promising new methods that allow less cytotoxic drugs to be used as warheads.

[0006] To date, most of the warheads developed have been tubulin inhibitors such as auristatins (e.g., monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)). Rentuximab vedotin, polatzumab Vedotin and enfortumab vedotin are both ADCs that carry approved MMAE payloads.

[0007] Topoisomerase I (TOP 1) inhibitors are used as warheads in various ADCs. Sacituzumab govitecan carries the active metabolite SN-38 of irinotecan as its warhead. Enhertu (fam-trastuzumab deruxtecan (DS8201a), also known as T-Dxd) carries the exatecan derivative deruxtecan (Dxd) as its warhead (Patent Document 1), and has recently been approved for use in the treatment of breast cancer. Based on this, there is a strong demand in this field for novel small molecule topoisomerase I inhibitors that have higher activity, stability, and physicochemical properties, whether used alone or as a component of ADCs.

[0008] However, on the one hand, Enhertu and most other commercially available ADCs and ADCs used in clinical trials are manufactured by chemical coupling, using a sulfosuccinimide structure (sulfosuccinimide linkage) to couple small molecule drugs to target antibodies or proteins. The sulfosuccinimide structure is formed by the reaction of a mercapto group with maleimide. Sulfosuccinimide linkage is unstable. In vivo, reverse Michael addition or exchange with other mercapto groups causes cytotoxins to detach from the ADC and induces off-target toxicity, thereby reducing safety and limiting clinical application. For example, Enhertu is highly potent but causes interstitial lung disease in over 10% of patients, which limits its use in some patients. On the other hand, chemical coupling reactions are not site-specific, and the resulting ADCs exhibit a high degree of heterogeneity.

[0009] There is still a strong demand for novel ADCs with higher potency and lower toxicity. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2014057687 [Overview of the project]

[0011] In one embodiment, the present invention provides a compound of formula (I). [ka] Here, opSu is [ka] or [ka] And, R 0 is C 1~10 It is an alkyl group, n is an arbitrary integer from 2 to 20, k1 and k2 are independently integers from 1 to 7, i is an integer from 1 to 100, j is an integer from 1 to 100, P1 and P2 are independently payloads having the structure shown in formula (i’),

Chemical formula

[0012] The second aspect, the present invention provides a conjugate having the structure shown in formula (II).

Chemical formula

[0013] In a third aspect, the present invention provides a pharmaceutical composition comprising the conjugate of the present invention and at least one pharmaceutically acceptable carrier.

[0014] In a fourth aspect, the present invention provides the use of the conjugate or pharmaceutical composition of the present invention in the manufacture of a drug for treating a disease, wherein the disease is a tumor or an autoimmune disease, preferably a tumor comprising HER2-positive tumor cells.

[0015] In a fifth aspect, the present invention provides a compound having the structure shown in formula (i). [ka] Here, a, p1 * A carbon atom with a mark attached, p2 * A carbon atom with L attached. 1 M, L 2 , R 1 and R 2 This is defined in equation (I).

[0016] In a sixth aspect, the present invention provides a compound of formula (1). [ka] Here, k is an integer from 1 to 7, preferably 1, 3, or 5, and particularly 5. P is a payload having the structure shown in equation (i'), which is defined in equation (I). [Brief explanation of the drawing]

[0017] [Figure 1] This figure shows the efficacy of the conjugate in the HER2-high-expressing cell line SK-BR-3. [Figure 2]This figure shows the efficacy of the conjugate in the HER2-high-expression cell line NCI-N87. [Figure 3] This figure shows the efficacy of the conjugate in the HER2-negative cell line MDA-MB-468. [Figure 4] This figure shows the efficacy of the conjugate in the SK-BR-3 and MDA-MB-468 co-culture system. Figure 4a shows the results of GFP fluorescence detection. Figure 4b shows the results of luciferase substrate chemiluminescence detection. In Figures 4a and 4b, from left to right are CH-2-589, CH-2-593, CH-2-518, LC1184(8), and LC302-2-4(4), respectively. [Figure 5] This figure shows the efficacy of the conjugate in the SK-BR-3 and MDA-MB-468 co-culture system, which passes GFP fluorescence detection. [Figure 6] This figure shows the time course of the mean tumor volume in SCID Beige mouse JIS 1 CDX models administered 5 mg / kg of the conjugate. [Figure 7] This figure shows the time course of the mean tumor volume in BALB / c nude mouse Capan-1 CDX models administered 5 mg / kg of the conjugate. [Figure 8] This figure shows the time course of mean tumor volume and body weight in BALB / c nude mouse Capan-1 CDX models administered 5 mg / kg of the conjugate. [Figure 9] This figure shows the time course of mean tumor volume and body weight in BALB / c nude mouse NCI-N 87 CDX models administered 5 mg / kg of the conjugate. [Figure 10] This figure shows the in vitro serum stability of the conjugate after being mixed with human serum and incubated for 0, 24, 48, and 96 hours. [Modes for carrying out the invention]

[0018] The technical details of the present invention will be described below with reference to specific embodiments. Those skilled in the art will readily understand other advantages and effects of the present invention from the contents disclosed in the specification. The present invention can be implemented or applied by further different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of the invention.

[0019] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The term "technical" as used herein refers to the technically understood art, including modifications and equivalent substitutions that are obvious to those skilled in the art. The following terms are considered to be readily understood by those skilled in the art, but the invention may be more... To further clarify, the following definitions are provided. Where a trade name is mentioned herein, it refers to the corresponding product or its active ingredient. All patents, patent applications, and publications cited herein are incorporated herein by reference.

[0020] Where a quantity, concentration, or other value, or parameter is indicated as a range, preferred range, or preferred upper and lower limits, this should be understood to specifically disclose the entire range formed by a pair of upper or preferred values ​​and lower or preferred values, regardless of whether the range is disclosed separately. Where a range of a number is disclosed herein, unless otherwise specified, the range is intended to include all integers and fractions (decimals) within the range, as well as its endpoints. For example, the expression "i is an integer between 1 and 20" means that i is any integer between 1 and 20, for example, i may 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 be understood in the same way.

[0021] Unless the context clearly indicates a different meaning, singular forms such as "one kind" and "the kind" include the plural form. Expressions like "one kind or more kinds" or "at least one kind" can represent 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0022] When used with variable values, the terms "approximately" or "about" usually 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 of the mean) or within ±10% of the specific value, or a wider range.

[0023] The terms “preferably” or “optionally” mean that the events described later may, but may not, occur, and such descriptions include circumstances under which the events or circumstances may or may not occur.

[0024] The expressions “to include,” “to contain,” “to have,” and “to have” are non-restrictive and do not exclude other unlisted elements, steps, or components. The expression “to consist of…” excludes elements, steps, or components that are not listed. The expression “substantially consisting of…” means that the scope is limited to a specific element, step, or component, along with any element, step, or component that does not substantially affect the basic and novel features of the claimed subject matter. It should be understood that the expression “to include” includes the expressions “substantially consisting of…” and “to consist of…”.

[0025] As used herein, the term “antibody” is used in its broadest sense and includes, in particular, complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity. Antibodies may be any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and may originate from any suitable species. In some embodiments, the antibodies are derived from humans or mice. Antibodies may be fully human antibodies, humanized antibodies, or chimeric antibodies produced by recombinant methods.

[0026] As used herein, monoclonal antibodies refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except in the case of possible naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific to a single antigen site. The term "monoclonal" describes the characteristic of an antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method.

[0027] A complete antibody or full-length antibody basically contains an antigen-binding variable region, a light chain constant region (CL), and a heavy chain constant region (CH), and may also contain CH1, CH2, CH3, and CH4 depending on the antibody subtype. The antigen-binding variable region (also called a fragment variable region, Fv fragment) is generally the light chain variable region (V L ) and heavy chain variable region (V H The constant region may be a constant region with a natural sequence (e.g., a constant region with a human natural sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with a target antigen. The constant region can be recognized by and interact with the immune system.

[0028] The antibody fragment may include a portion of a complete antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments are Fab, Fab′, F(ab′)2, V H and CH1 domain The Fab fragment comprises an Fd fragment, an Fv fragment, a single-domain antibody (dAb) fragment, and an isolated complementarity-determining region (CDR). The Fab fragment is an antibody fragment obtained by digesting full-length immunoglobulin with papain, or a fragment having the same structure produced, for example, by recombinant expression. The Fab fragment contains a light chain (V L and CL) and other chains, the other chains being, Variable region of heavy chain (V H ) and one steady region (CH1) of the heavy chain. F(ab′)2 section A fragment is an antibody fragment obtained by digesting immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure produced, for example, by recombinant expression. An F(ab′)2 fragment basically contains two Fab fragments, each heavy chain portion containing several additional amino acids and cysteine ​​that forms a disulfide bond linking the two fragments. A Fab′ fragment is a fragment containing one of the F(ab′)2 fragments (one heavy chain and one light chain). The above antibody fragment may contain multiple chains linked by, for example, disulfide bonds and / or peptide linkers. Examples of antibody fragments further include single-chain Fv(scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, and other fragments including modified fragments. Antibody fragments generally contain at least or about 50 amino acids and generally at least or about 200 amino acids. The antigen-binding fragment may include any antibody fragment that, when inserted into an antibody framework (for example, by replacing a corresponding region), can yield an antibody that binds immunospecifically to the antigen.

[0029] The antibodies of the present invention can be manufactured using techniques well known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthesis techniques, or other techniques known in the art. For example, a recombinant antibody (or antibody analog) can be expressed in a suitable culture system (e.g., Escherichia coli (E. coli) or mammalian cells). The said operation may refer, for example, to introducing a ligase-specific recognition sequence to its terminal.

[0030] HER2 refers to human epidermal growth factor acceptor-2 and belongs to the epidermal growth factor (EGFR) acceptor tyrosine kinase family. In this application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0031] As used herein, the term “antibody-drug conjugate” is referred to as “conjugate.”

[0032] Small molecule compounds generally refer to molecules that are roughly the same size as organic molecules used in drugs. This term does not include biomacromolecules (e.g., proteins, nucleic acids, etc.), but does include low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides. Generally, the molecular weight of small molecule compounds may be, for example, about 100 to 2000 Da, about 200 to 1000 Da, about 200 to 900 Da, about 200 to 800 Da, about 200 to 700 Da, about 200 to 600 Da, or about 200 to 500 Da.

[0033] A "spacer" refers to a structure located between different structural modules that can separate them from space. The definition of a spacer does not limit whether it has a certain function or whether it is cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, and non-amino acid structures may be, but are not limited to, amino acid derivatives or analogs. A "spacer sequence" refers to an amino acid sequence that acts as a spacer, and examples include, but are not limited to, a single amino acid or a sequence containing multiple amino acids, such as a sequence containing two amino acids like GA, or such as GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGGS, etc. Since a self-destructing spacer is a covalent assembly, after the protective portion in the precursor is activated, the two chemical bonds degrade sequentially, and after the protective portion (e.g., a cleavable sequence) is activated and removed, a cascade-type decomposition reaction occurs, releasing smaller molecules according to priority. Examples of self-destructing spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0034] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group composed of carbon and hydrogen atoms, which is bonded to the rest of the molecule by a single bond. Alkyl groups include linear alkyl groups, branched alkyl groups, or cyclic alkyl groups (cycloalkyl groups) or partially cyclic alkyl groups (e.g., cycloalkyl group-linear alkyl group and cycloalkyl group-branched alkyl group). Alkyl groups may have 1 to 10 carbon atoms. 1-10 They are called alkyl groups, for example, C 1~6 Alkyl alkyl group, C 1~4 Alkyl alkyl group, C 1~3 Alkyl alkyl group, C 1~2 Alkyl alkyl group, C3 alkyl group, C4 alkyl group, C 3~6These are alkyl groups. Non-limiting examples of linear alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups. 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 groups.

[0035] The terms "cyclic alkyl group" and "cycloalkyl group" have the same meaning and can be used interchangeably in this specification. Cycloalkyl groups may include monocyclic or polycyclic (e.g., having two or more fused rings) groups. In polycyclic cycloalkyl groups, two or more rings may be fused, crosslinked, or spirocondensed. The carbon atoms forming the ring of a cycloalkyl group may optionally be substituted with oxo (i.e., C(O)). Cycloalkyl groups may have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbon atoms (C 3~10 ) has This may also be done. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, and bicyclo[2.1.1]hexyl groups. In some embodiments, the cycloalkyl group is C 3~6 It is a monocyclic or bicyclic cycloalkyl group, preferably C 3~6 Monocyclic cycloalkyl groups, particularly cyclopropyl groups.

[0036] The term "partially cyclic" refers to a group that contains one or more cyclic parts and one or more acyclic (i.e., linear or branched) parts. Partially cyclic alkyl groups may include cycloalkyl-linear alkyl groups and cycloalkyl-branched alkyl groups. Partially cyclic alkyl groups consist of 4, 5, 6, 7, 8, 9, or 10 carbon atoms (C) including the ring-forming carbon atoms and the acyclic carbon atoms. 3~10 ) may have. An example of a partially cyclic alkyl group is C 3~9 Cycloa Lukyl group - C1 alkyl group, C 3~8 Cycloalkyl group - C2 alkyl group, C 3~7 Cycloalkyl group - C3 linear alkyl group, C 3~6 Cycloalkyl group - C4 linear alkyl group, C 3~5 Cycloalkyl group - C5 linear alkyl group, C 3~4 Cycloalkyl groups - C6 linear alkyl groups, C3 Cycloalkyl group - C7 linear alkyl group, C 3~7 Cycloalkyl-C3 branched-chain alkyl group, C 3~6 Cycloalkyl group - C4 branched alkyl group, C 3~5 Cycloalkyl group - C5 branched alkyl group, C 3~4 This includes, but is not limited to, cycloalkyl-C6 branched alkyl groups and C3-cycloalkyl-C7 branched alkyl groups. In some embodiments, A segmental cyclic alkyl group is C 3~9 It is a cycloalkyl group - C1 alkyl group, preferably, C 3~6 Cycloalkyl group-C 1~2 Alkyl alkyl group, C 3~6 Cycloalkyl group - C1 alkyl group And moreover, C 3~4 Cycloalkyl group-C 1~2 Alkyl groups, especially C 3~4 Cycloalkyl groups - C1 alkyl groups, particularly cyclopropyl group - methyl group.

[0037] A divalent group is a group obtained by removing one hydrogen atom from a carbon atom that has free valence electrons in the corresponding monovalent group. A divalent group has two bonding sites that attach to the rest of the molecule, and these two bonding sites may be on the same atom or two different atoms of the divalent group.

[0038] An alkylene group or alkylidene group refers to a saturated divalent hydrocarbon group. Alkylene groups include linear, branched, cyclic, or partially cyclic groups. Examples of linear alkylene groups include the methylene group (-CH2-), -(CH2)2-, This includes, but is not limited to, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, etc. Examples of branched alkylene groups are -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-CH2-, -CH(C2H5)-CH(CH3)-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(C2 H5)-(CH2)3-, -CH2-CH(C2H5)-(CH2)2-, -CH2-CH(CH3)-CH(CH3)-CH2-, -(CH(CH3))2-(CH2)2-, -CH(CH3) This includes -(CH2)2-CH(CH3)-, -(CH2)2-CH(CH3)-(CH2)2-, -CH2-CH(CH3)-(CH2)3-, -CH(CH3)-(CH2)4-, etc. It is not limited to them.

[0039] The terms "cycloalkylene group" and "cyclic alkylene group" have the same meaning and can be used interchangeably in this specification. Examples of cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene groups, as well as divalent polycyclic alkyl groups including fused rings, spiro rings, or crosslinking rings. In some embodiments, the cycloalkylene group is C 3~6 It is a cycloalkylene group, and especially C 3~4 This refers to cycloalkylene groups, particularly cyclopropyl groups.

[0040] A partially cyclic alkylene group may contain a divalent group, and the two bonding sites attached to the rest of the molecule may both be on one or more linear or branched alkyl groups, or both be on one or more cycloalkyl groups, or each may be on a cycloalkyl group and a linear or branched alkyl group. Examples of partially cyclic alkylene groups include, but are not limited to, (1) a divalent polycyclic alkyl group including a condensed ring, spiro ring, or crosslinking ring, and a cyclopropylene group, cyclobutylene group, cyclopentylene group, cyclohexylene group, cycloheptylene group, and cyclooctylene group, each independently substituted with one or more linear or branched alkyl groups; (2) a linear or branched alkylene group, each independently substituted with one or more cycloalkyl groups; and (3) a group obtained by combining one or more cycloalkylene groups and one or more linear or branched alkylene groups, provided that it forms a chemically stable structure. In some embodiments, the partially cyclic alkylene group is C 3~9 It is a cycloalkyl-C1 alkylene group, preferably C 3~6 Cyclo Alkyl-C 1~2 Alkylene group, C 3~6 It is a cycloalkyl-C1 alkylene group, more, C 3~4 Cycloalkyl group-C 1~2 Alkylene group, especially C 3~4 This refers to a cycloalkyl-C1 alkylene group, particularly a cyclopropyl-methylene group.

[0041] The compounds of the present invention can exist in isotope-labeled or isotope-enriched forms containing one or more atoms having atomic masses and mass numbers different from those most common in nature. The isotopes may be radioactive or non-radioactive. Isotopes of atoms such as hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine are: 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 32 P, 35S, 18 F, 36 Cl and 125 This includes, but is not limited to, I. In one embodiment, the payload (e.g., the compound of formula (i)) may exist in an isotopically labeled or isotopically enriched form, particularly in a deuterated form.

[0042] As used herein, the terms “drug that can be conjugated to an antibody” and “antibody-drug conjugate” have the same meaning.

[0043] Compound of formula (i) In one embodiment, the present invention provides a compound or a pharmaceutically acceptable salt thereof, stereoisomer, solvate, polymorph, tautomer, isotope, metabolite, or prodrug having the structure shown in formula (i). [ka] Here, a is either 0 or 1. p1 * A carbon atom with a p2 attached * Each carbon atom with the symbol attached is a chiral center, and the chiral centers are in an S configuration, an R configuration, or racemized. L 1 C 1~6 Selected from alkylene groups, unsubstituted or with halogens, -OH and It is substituted with one substituent selected from bi-NH2, M is -CH2-, -NH-, or -O-. L 2 is C 1~3 It is an alkylene group, R 1 and R 2 These are, independently, hydrogen and C 1~6 Alkyl alkyl groups, halogens, and C 1~6 Selected from alkoxy groups.

[0044] In one embodiment, L 1 C 1~6 Linear alkylene group, C1~6 A branched alkylene group, C 3~6 A cycloalkylene group and C 3~4 A cycloalkyl group - C 1~2 Selected from a linear alkylene group, and the above groups are independently unsubstituted or substituted by one substituent selected from halogen, -OH, and -NH2. In one embodiment, L is selected from a C alkylene group, and is unsubstituted or substituted by one substituent selected from halogen, -OH, and -NH2. In a preferred embodiment, L 1 is 1~4 an alkylene group selected from, and is unsubstituted or substituted by one substituent selected from halogen, -OH, and -NH2. In a preferred embodiment, L is 1 -CH2-, -C2 H4-,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0045] In one embodiment, a is 1, M is -CH2-, -NH-, or -O-, and L 2 is -C2H4-. In another embodiment, a is 1, M is -CH2-, and L 2 It is -CH2-.

[0046] In one embodiment, p1 * The carbon atoms with the mark are in an S configuration or racemized, which is preferable. In another embodiment, p2 * The carbon atoms with the mark are in an S configuration or racemic configuration. It is a modified version, preferably in an S configuration.

[0047] In one embodiment, R 1 and R 2 These are, independently, hydrogen and C 1~3 Alkyl alkyl groups, halogens, and C 1~3 Selected from alkoxy groups. In one preferred embodiment, R 1 and R 2 Each of these is independently selected from CH3-, F, Cl, Br, and CH3O-. In one embodiment, R 1 This is selected from CH3- and Cl. In another embodiment, R 2 It is F.

[0048] In one embodiment, L 1 -CH2-, [ka] Selected from, where "#" indicates the position where it bonds with the carbonyl group. In one embodiment, a is 1 and L 1 teeth [ka] And M is O, L2 It is -C2H4-.

[0049] In one embodiment, a is 0, and R 1 is Cl, and R 2 is F and L 1 -CH2-, [ka] Selected from. In one embodiment, a is 0 and R 1 It is CH3-, and R 2 is F , L 1 teeth, [ka] Selected from the options, where "#" indicates the position where it bonds to the carbonyl group.

[0050] In one embodiment, a is 1, and R 1 It is CH3-, and R 2 is F and L 1 teeth [ka] And M is O, L 2 It is -C2H4-.

[0051] In one embodiment, the above compound is [ka] [ka] [ka] Selected from.

[0052] In one embodiment, the above compound is [ka] [ka] Selected from.

[0053] In one preferred embodiment, the above compound is [ka] [ka] Selected from.

[0054] In one preferred embodiment, the above compound is [ka] Selected from.

[0055] In a more preferred embodiment, the compound is [ka] Selected from.

[0056] In a special embodiment, the above compound is [ka] Selected from.

[0057] In one aspect, the present invention is [ka] C730 And, [ka] C731 The present invention provides compounds selected from the following.

[0058] Compound of formula (I) In one embodiment, the present invention provides a compound having the structure shown in formula (I). [ka] Here, opSu is [ka] or [ka] And, R 0 is C 1~10 It is an alkyl group, n is any integer between 2 and 20. k1 and k2 are independent integers between 1 and 7. i is an integer between 1 and 100. j is an integer between 1 and 100. P1 and P2 are payloads that independently have the structure shown in equation (i'), [ka] Here, a, p1 * A carbon atom with a mark attached, p2 * A carbon atom with L attached. 1 M, L 2 , R 1 and R 2 This is as defined in equation (i).

[0059] In one embodiment, the payload is: [ka] [ka] [ka] Selected from.

[0060] In one embodiment, the payload is [ka] [ka] Selected from.

[0061] In one preferred embodiment, the payload is [ka] [ka] Selected from.

[0062] In one preferred embodiment, the payload is [ka] Selected from.

[0063] In a more preferred embodiment, the payload is [ka] Selected from.

[0064] In a special embodiment, the payload is [ka] Selected from.

[0065] In one embodiment, R 0 is C 1~6 It is an alkyl group. In a preferred embodiment, R 0 is C1 ~3 It is an alkyl group. In a special embodiment, R 0 This is a methyl group.

[0066] In one embodiment, n is an integer between 2 and 5. In a special embodiment, n is 3.

[0067] In one embodiment, k1 and k2 are independently 1, 3, or 5. In a special embodiment, k1 and k2 are independently 5.

[0068] In one embodiment, i is an independent integer between 1 and 20, preferably between 1 and 12, and more preferably between 2 and 8. In a special embodiment, i is 4.

[0069] In one embodiment, j is an independent integer between 1 and 20, preferably between 1 and 12, more preferably between 8 and 12, and particularly 8 or 12. In a special embodiment, j is 12.

[0070] In one embodiment, the compound of formula (I) has the structure shown in formula (I-1). [ka] Here, P1, P2, R 0 opSu, n, i, and j are defined in formula (I). ru.

[0071] In one embodiment, the compound of formula (I) is [ka] BH-1 and [ka] BH-2 Selected from.

[0072] Specific embodiments of the compound of formula (I) In one embodiment, the structure of the compound of formula (I) is as shown in the table below. [Table 1-1] [Table 1-2]

[0073] Production of the compound of formula (I) In one embodiment, the compound of formula (I) can be produced by binding a linker and a payload or by binding a series of suitable building blocks. Such building blocks can be easily designed by retrosynthetic analysis, and any method known in the art can be used.

[0074] In one aspect, the present invention provides a compound having the structure shown in formula (1). [Chemical formula] Here k is an integer from 1 to 7, P is a payload having the structure shown in formula (i'), and the structure shown in formula (i') is as defined above.

[0075] In a preferred embodiment, k is 1, 3 or 5. In a particular embodiment, k is 5.

[0076] The compound of formula (1) can be synthesized by a method similar to the synthesis method disclosed in European Patent Application Publication No. 2907824 (for example, the synthesis method of formula (2) or (2b) in European Patent Application Publication No. 2907824) using the compound of formula (i) and other necessary building blocks. Suitable building blocks include, but are not limited to, the binding unit HX20113 and the binding unit HX20111. [Chemical formula]

[0077] Next, the maleimide group can react with a mercapto group in another building block of the compound of formula (I). The resulting sulfosuccinimide is unstable under physiological conditions and readily undergoes a reverse Michael addition reaction, leading to decomposition of the binding site. Furthermore, if another sulfhydryl compound is present in the system, thiosuccinimide may undergo thiol exchange with the other sulfhydryl compound. Both of these reactions result in payload loss and toxic side effects. Next, the sulfosuccinimide is subjected to a ring-opening reaction. Then, the compound of formula (I) can be obtained.

[0078] The method for the ring-opening reaction can be found in International Publication No. 2015165413. Compounds containing the ring-opening moiety for succinimide can be purified by half-part / preparative HPLC or other suitable separation method to achieve high purity and accurate composition, regardless of the efficiency of the succinimide ring-opening reaction.

[0079] In the present invention, when applied to a linker-payload (linker-small molecule intermediate), the ring-open succinimide structure does not undergo reverse Michael addition or thiol exchange, resulting in a more stable product.

[0080] A portion containing a ligase acceptor or donor substrate recognition sequence. In one embodiment, the compound of formula (I) (Gly) n The part is a ligase acceptor or The donor substrate recognition sequence promotes ligase-catalyzed enzymatic coupling between the compound of formula (I) and an antibody or antigen-binding fragment. The antibody or its antigen-binding fragment may be modified and contain a ligase acceptor or a corresponding recognition sequence for the donor substrate.

[0081] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from natural transpeptidases, non-natural transpeptidases, their variants, and combinations thereof. Non-natural transpeptidases may, but are not limited to, those obtained by manipulating natural transpeptidases. In a preferred embodiment, the ligase is selected from natural sortase enzymes, non-natural sortase enzymes, and combinations thereof. Examples of natural sortase enzymes include Sortase A, Sortase B, Sortase C, Sortase D, and Sortase L. plantarum (U.S. Patent Application Publication No. 20110321183). The type of ligase corresponds to the ligase recognition sequence, thereby different classifications. It is used to achieve specific conjugates between child or constituent fragments. In one embodiment, the compound of formula (I) (Gly) n The portion is the ligase acceptor substrate. The recognition sequence is, and the antibody or its antigen-binding fragment may be modified and include the corresponding recognition sequence of the ligated donor substrate.

[0082] In some embodiments, the ligase is a sortase enzyme selected from sortase A, sortase B, sortase C, sortase D, and sortase L. plantarum.

[0083] In a particular embodiment, the ligase is Sortase A derived from Staphylococcus aureus. Therefore, the ligase recognition sequence may be the representative recognition sequence of the enzyme, LPXTG. In another particular embodiment, the ligase donor substrate recognition sequence is LPXTGJ, and the ligase acceptor substrate recognition sequence is G m and Here, X is any single natural or non-natural amino acid, and J is either absent or an amino acid fragment containing 1 to 10 amino acids, which may have a tag. In one embodiment, J is absent. In another embodiment, J is an amino acid fragment containing 1 to 10 amino acids, each of which is independently any natural or non-natural amino acid. In yet another embodiment, J is (Gly) m And here, m is an integer from 1 to 10. Another special In one embodiment, the ligated donor substrate recognition sequence is LPETG. In another special embodiment, the ligated donor substrate recognition sequence is LPETGG.

[0084] In one embodiment, the ligase may be Sortase B derived from Staphylococcus aureus, and the corresponding donor substrate recognition sequence may be NPQTN. In another embodiment, the ligase may be Sortase B derived from Bacillus anthracis, and the corresponding donor substrate recognition sequence may be NPKTG.

[0085] In another embodiment, the ligase may be Sortase A derived from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be LPXTGJ, where J is as defined above. In yet another embodiment, the ligase may be Sortase subfamily 5 derived from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence may be LAXTG. In another embodiment, the ligase may be Sortase A derived from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence may be LPQTSEQ. The ligase recognition sequence may also be any other novel transpeptidase recognition sequence that has been manually selected and optimized.

[0086] Conjugates and their preparation In addition, a compound having a payload with a part containing a ligase recognition sequence (compound of formula (I)) can conjugate with other molecules containing a ligase recognition sequence, and thus can be used for the preparation of antibody-small molecule conjugates such as antibody-drug conjugates. Therefore, in another aspect, the present invention provides a conjugate comprising a compound of formula (I) and an antibody or an antigen-binding fragment.

[0087] In another aspect, the present invention provides a conjugate having the structure shown in formula (II). [Chemical formula] Here, A is an antibody or an antigen-binding fragment thereof, z is an integer from 1 to 20, P1, P2, R 0 , opSu, n, k1, k2, i and j are as defined in formula (I). as described.

[0088] In a preferred embodiment, the antibody or an antigen-binding fragment thereof is modified to bind to the (Gly) part in the compound of formula (I). n part.

[0089] In a preferred embodiment, z is from 1 to 4. In a special embodiment, z is 2.

[0090] An antibody or an antigen-binding fragment thereof In one embodiment, the antibodies are anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD25 antibodies, anti-CD30 / TNFRSF8 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD4 4v6 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117 / KITk antibody, anti-CD123 antibody, anti-CD138 antibody, anti- CD142 antibody, anti-CD174 antibody, anti-CD227 / MUC1 antibody, anti-CD352 antibody, anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2 / HER2 antibody, anti-CN33 antibody, anti-GPNMB antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRvIII antibody, anti-SLC44A4 / AGS-5 antibody, anti-CEACAM5 antibody, anti-PSMA antibody, Anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-Nectin4 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody, anti-BCMA antibody, anti-AXL antibody, anti-NaPi 2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-Mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-LAMP1 antibody, anti-Cadherin6 antibody, anti- The antibody is FGFR2 antibody, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-integrinαV antibody, anti-TDGF1 antibody, anti-EphrinA4 antibody, anti-TROP2 antibody, anti-PTK7 antibody, anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, anti-TissueFactor antibody, or anti-ROR1 / 2 antibody, preferably anti-HER2 antibody, anti-FGFR3 antibody, anti-Trop2 antibody, anti-HER3 antibody, or anti-FRα antibody.

[0091] In one preferred embodiment, 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. No. Pertuzumab binds to the second extracellular domain (ECD2) of HER2 and is approved for use in the treatment of HER2-positive breast cancer. Trastuzumab binds to the fourth extracellular domain (ECD4) of HER2 and is approved for use in the treatment of HER2-positive breast cancer and gastric cancer.

[0092] In one embodiment, the antibody is an anti-HER2 antibody or its antigen-binding fragment, and the light chain variable domain (V L ) and heavy chain variable domain (V H ) including, of which V L LCDDR1, LCD It includes R2 and LCDR3, where LCDR1 contains the amino acid sequence of SEQ ID NO:1, LCDR2 contains the amino acid sequence of SEQ ID NO:2, and LCDR3 contains the amino acid sequence of SEQ ID NO:3, and of which, V H HCDR1, HCDR 2 and HCDR3 are included, where HCDR1 contains the amino acid sequence of SEQ ID NO:4, HCDR2 contains the amino acid sequence of SEQ ID NO:5, and HCDR3 contains the amino acid sequence of SEQ ID NO:6. In one embodiment, CDR is defined by KABAT. In one embodiment, the antibody is an anti-HER2 antibody or its antigen-binding fragment, and the light chain variable domain (V L ) and heavy chain variable domain (V H ) including, of which V L is SEQ ID NO: It has an amino acid sequence that shows at least 85%, at least 90%, or at least 95% sequence identity with 7, and V H This is less than SEQ ID NO: 8 It has an amino acid sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity. In one preferred embodiment, the anti-HER2 antibody or its antigen-binding fragment has a light chain variable domain (V L ) and heavy chain variable domain (V H ) including, of which V L is SEQ ID NO: It has an amino acid sequence of 7, and also VH This is amino acid SEQ ID NO: 8 It has an acid sequence. In a particular embodiment, the anti-HER2 antibody comprises a light chain and a heavy chain, of which the light chain has an amino acid sequence showing at least 85%, at least 90%, or at least 95% sequence identity to amino acid sequence 1-214 of SEQ ID NO: 9, and the heavy chain has an amino acid sequence showing at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 10. In a preferred embodiment, the anti-human HER2 antibody is one or more selected from engineered anti-HER2 antibodies based on trastuzumab. In one embodiment, the antibody is modified trastuzumab, preferably Ab0001-LCCT L -HC (Light Chain SEQ ID NO:9) (It has a heavy chain SEQ ID NO:10). Antibody Ab0001-LCCT L -H The C sequence is based on the amino acid sequence of trastuzumab, with GALPETGG introduced at the C-terminus of the light chain. Of these, LPETGG is the ligase donor substrate recognition sequence, and GA is the spacer sequence.

[0093] In one preferred embodiment, the anti-human HER2 antibody is a recombinant antibody and is selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetic compounds. In one embodiment, the antibody mimetic is selected from scFv, mini-antibodies, bi-antibodies, and nano-antibodies. With respect to conjugation with the compound of formula (I), the antibody of the present invention or its antigen-binding fragment is (Gly) in the compound of formula (I). n You can include the modifying part in order to conjugate it to the part. The location where such modified portions are introduced is not limited; for example, the introduction site of the antibody or its antigen-binding fragment may be at the C-terminus or N-terminus of the antibody's heavy or light chain, but is not limited to these locations.

[0094] In one preferred embodiment, (Gly) in the compound of formula (I) n Parts and Conjugate The modification portion for this purpose can be introduced, for example, by chemical modification to a position other than the end of the antibody's heavy or light chain.

[0095] In one embodiment, the antibody or its antigen-binding fragment may include terminal modifications. Terminal modifications refer to modifications at the C-terminus or N-terminus of the heavy or light chain of the antibody, including, for example, a ligase recognition sequence. In another embodiment, a spacer Sp containing 2 to 100 amino acids of terminal modifications is used. It may also contain, in order, an antibody, Sp, and a ligase recognition sequence. In one preferred embodiment, Sp is a spacer sequence containing 2 to 20 amino acids. In a special embodiment, Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGGS, particularly GA.

[0096] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment may be wild-type (LC), a C-terminally modified light chain (LCCT) modified by directly introducing the ligase recognition sequence LPETGG, and a C-terminally modified light chain (LCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition sequence LPXTG. L It includes three types: antibodies or their antigenic bonds. The heavy chain of the composite fragment consists of the wild type (HC), a C-terminal modified light chain (HCCT) modified by directly introducing the ligase recognition sequence LPXTG, and a C-terminal modified light chain (HCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition sequence LPXTG. L ) It includes three types. X may be any natural or unnatural single amino acid. When z in the compound of formula (II) is 1 or 2, the heavy chain and light chain can be combined to form eight preferred antibody molecules.

[0097] In one embodiment, the compound of formula (II) is [ka] and [ka] Selected from. Specific implementations of conjugate In one embodiment, the payload is a cytotoxin or a fragment thereof. In one embodiment, the antibody-drug conjugate is as shown in the table below.

[0098] ADC nomenclature: The number in parentheses indicates the number of payload (drug) molecules intended to bind to the antibody.

[0099] [Table 2]

[0100] Preparation of Conjugates The conjugate of the present invention (i.e., the compound of formula (II)) can be used in any method known in the art. It can be prepared by the following: In some embodiments, the conjugate is prepared by site-specifically conjugating an antibody or antigen-binding fragment with a compound of formula (I) using a ligase catalyst, wherein the antibody or antigen-binding fragment is modified by a ligase recognition sequence.

[0101] An antibody or its antigen-binding fragment and the compound of formula (I) are mutually bound by a substrate ligase-specific recognition sequence. The recognition sequence depends on the specific ligase used. In one embodiment, the antibody or its antigen-binding fragment is an antibody in which terminal modifications based on the recognition sequence have been introduced at the C-terminus of the light chain and / or heavy chain, and the antibody or its antigen-binding fragment is conjugated with the compound of formula (I) under appropriate catalytic reaction conditions using a wild-type or optimized engineered ligase or any combination thereof as a catalyst.

[0102] In a specific embodiment, the ligase is Sortase A, and the conjugation reaction may be represented by the following solution. [ka] The triangle represents a part of the antibody, the pentagon represents a part of the compound of formula (I), and G n is (G ly) n This represents a part. n, X, and J are as defined above. The corresponding recognition sequence G of the receptor substrate n When conjugating with, in the LPXTGJ sequence The upstream peptide bond of glycine is broken down by Sortase A, and the resulting intermediate is G n It binds to the free N-terminus to generate a new peptide bond. The resulting amino The acid sequence is LPXTG n This is the array G. n And LPXTGJ are as defined above.

[0103] Therefore, in one embodiment, the G of the compound of formula (I) n The part is the ligase acceptor. The substrate recognition sequence is modified to include the ligated donor substrate recognition sequence, thereby enabling the G of the compound of formula (I). n Combine into parts, Preferably, (a) The ligase is a sortase enzyme selected from Sortase A, Sortase B, Sortase C, Sortase D and Sortase L. plantarum, preferably Sortase A derived from Staphylococcus aureus, and / or (b) The ligase donor substrate recognition sequence is LPXTGJ, where J is absent or G m Here, G is glycine, m is an integer from 1 to 10, and X is any natural or non-natural compound. The ligase donor substrate recognition sequence is a natural single amino acid, preferably LPXTG or LPETGG, and / or LPXTG is G n Combine with LPXTG n Generates.

[0104] The compound of formula (I) of the present invention has a precise structure, precise composition, and high purity, so when it is used in a conjugation reaction with an antibody, very few impurities are introduced, or no other impurities are introduced at all. When such an intermediate is used in site-specific conjugation with a modified antibody containing a ligase-recognition sequence catalyzed by a ligase, a homogeneous ADC with highly controllable quality can be obtained.

[0105] Metabolism of conjugates in the physiological environment When part or all of the linker is degraded in tumor cells, the payload is released. Because the degradation occurs at the site where the linker is bound to the antitumor compound, the antitumor compound is released in its intrinsic structure and exhibits its intrinsic antitumor effect.

[0106] In one embodiment, the GGFG (Gly-Gly-Phe-Gly) moiety in the compound of formula (I) may be degraded by a lysosomal enzyme (e.g., cathepsin B and / or cathepsin L).

[0107] In one embodiment, the compound of formula (I) contains a self-destructing spacer. In one embodiment, the self-destructing spacer is an acetal or heteroacetal. In one embodiment, the -GGFG-NH-CH2-O- portion of the compound of formula (I) is a restriction enzyme site and a self-destructing spacer It represents a combination of molecules that are broken down in cells and release target molecules (e.g., antitumor compounds).

[0108] Pharmaceutical compositions and pharmaceutical preparations Another object of the present invention is to provide a pharmaceutical composition comprising the conjugate of the present invention and at least one pharmaceutically acceptable carrier.

[0109] The pharmaceutical composition of the present invention can be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing, or treating symptoms in humans or animals. For example, it can be prepared in various appropriate dosage forms depending on the route of administration, in particular as injectable preparations such as lyophilized powder for injection or sterile powder for injection.

[0110] The term "pharmaceutically acceptable" means that, within the bounds of sound medical judgment, it can come into contact with patient tissue without causing excessive toxicity, irritation, or allergic reactions, has a reasonable benefit-risk ratio, and is effective for its intended use.

[0111] The term "pharmaceutically acceptable carrier" refers to a carrier material that is pharmaceutically acceptable and does not impair the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline. Pharmaceutically acceptable carriers further include carrier materials that bring the composition closer to physiological conditions, such as pH adjusters, buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate.

[0112] In one embodiment, the drug-to-antibody ratio (DAR) of the pharmaceutical composition of the present invention is an integer or non-integer between about 1 and about 20, for example, an integer or non-integer between about 1 and about 10, about 1 and about 8, about 1 and about 6, about 1 and about 4, about 2 and about 5, about 2 and about 4, or about 3 and about 4. In a particular embodiment, the conjugate of the present invention has a DAR of about 4. In a particular embodiment, the conjugate of the present invention has a DAR between about 3.3 and about 3.75, particularly about 3.4 and about 3.65.

[0113] Treatment methods and use The conjugates of the present invention can be used to treat tumors and / or autoimmune diseases. Tumors sensitive to treatment with the conjugates include tumors characterized by specific tumor-associated antigens or cell surface acceptors, where these tumor cells are recognized by the conjugate's target molecule (antibody) and further killed by the conjugate's payload / cytotoxin.

[0114] Accordingly, in another aspect, the present invention further provides the use of the conjugate or pharmaceutical composition of the present invention in the manufacture of a drug for treating a disease, symptom or condition selected from tumors or autoimmune diseases.

[0115] In another aspect, the present invention relates to a conjugate for treating tumors or autoimmune diseases. The present invention provides a pharmaceutical composition or a vegetarian.

[0116] In a further embodiment, the present invention provides a method for treating a tumor or autoimmune disease, comprising administering an effective amount of the conjugate or pharmaceutical composition of the present invention to an individual in need.

[0117] In one preferred embodiment, the conjugate formed by the binding of an anti-HER2 antibody and a small molecule cytotoxin according to the present invention can specifically bind to HER2 on the surface of tumor cells and selectively kill tumor cells expressing HER2. In another preferred embodiment, the anti-HER2 antibody is an anti-human HER2 antibody. In yet another preferred embodiment, the present invention provides the use of the conjugate or pharmaceutical composition of the present invention in the manufacture of a drug for treating a disease, disorder, or condition selected from HER2-positive tumors. In a more preferred embodiment, the above disease, disorder, or condition is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma, among others.

[0118] The dose of the conjugate administered to the subject can be adjusted to a considerable extent. The dose will vary depending on the specific route of administration and the subject's needs, and can be determined by a medical professional.

[0119] As defined above, embodiments of the present invention may be described as follows: [1] to

[20] [1] A compound of formula (I), [ka] Here, opSu is [ka] or [ka] And, R 0 is C 1~10 It is an alkyl group, n is any integer between 2 and 20. k1 and k2 are independent integers between 1 and 7. i is an integer between 1 and 100. j is an integer between 1 and 100. P1 and P2 are payloads that independently have the structure shown in equation (i'), [ka] Here, a is either 0 or 1. p1 * A carbon atom with a p2 attached * Each carbon atom with the attached symbol is a chiral center, and the chiral centers are in an S configuration, an R configuration, or racemized configuration. L 1 C 1~6 Selected from alkylene groups, unsubstituted or with halogens, -OH and It is substituted with one substituent selected from bi-NH2, M is -CH2-, -NH-, or -O-. L 2 is C 1~3 It is an alkylene group, R 1 and R 2 These are, independently, hydrogen and C 1~6 Alkyl alkyl groups, halogens, and C 1~6 A compound selected from alkoxy groups.

[0120] [2] Having the structure shown in formula (I-1), [ka] Here, P1, P2, R 0 opSu, n, i and j are as defined in claim 1. The compound according to claim [1].

[0121] [3] In equation (i'), L 1 C 1~6 Linear alkylene group, C 1~6 Branched alkylene group, C 3~6 Cyclo Alkile n group and C 3~4 Cycloalkyl group-C 1~2 Selected from linear alkylene groups, each of which is independently unsubstituted or one selected from halogen, -OH and -NH2 Substituting with substituents, Preferably, L 1 C 1~4 Linear alkylene group, C 1~4 Branched alkylene group, C 3~4 Cyclo Alkile Selected from an NH group and a cyclopropyl methylene group, each of which is independently unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH2. It is done, Preferably, L 1 -CH2-, -C2H4-, [ka] Selected from, each of the groups is independently unsubstituted or substituted with at least one substituent selected from halogen, -OH and -NH2. More preferably, L 1 -CH2-, [ka] Selected from, where "#" indicates the position where it bonds to the carbonyl group. More preferably, L 1 -CH2-, [ka] Selected from, where "#" indicates the position where it bonds to the carbonyl group. In particular, L 1 -CH2-, [ka] and [ka] Selected from, where "#" indicates the position where it bonds to the carbonyl group. and / or, The compound according to claim [1] or [2], wherein the halogen is selected from F, Cl, and Br.

[0122] [4] In equation (i'), M is -CH2-, -NH-, or -O-, L 2 It is -C2H4-, or, M is -CH2-, L 2 is -CH2-, any one of claims [1] to [3] The compounds described above.

[0123] [5] In equation (i'), p1 * The carbon atoms with the attachment are in an S configuration or racemized, preferably in an S configuration. Yes, and / or, p2 * The carbon atoms with the attachment are in an S configuration or racemized, preferably in an S configuration. A compound according to any one of claims [1] to [4].

[0124] [6] In equation (i'), R 1 and R 2 These are, independently, hydrogen and C 1~3 Alkyl alkyl groups, halogens, and C 1~3 Selected from alkoxy groups, Preferably, R 1 and R 2Each of these is independently selected from CH3-, F, Cl, Br, and CH3O-. more, R1 is selected from CH3- and Cl, and / or R 2 F is, More preferably, a is 0, R 1 is Cl, and R 2 is F and L 1 -CH2-, [ka] Selected from, or, a is 0, R 1 It is CH3-, and R 2 is F and L 1 teeth, [ka] Selected from, where "#" indicates the position where it bonds to the carbonyl group, or, a is 1, R 1 It is CH3-, and R 2 is F and L 1 teeth [ka] And M is O, L 2 is -C2H4-, any one of claims [1] to [5] The compounds described above.

[0125] [7] The aforementioned payload is [ka] [ka] Selected from, Preferably, [ka] Selected from, more, [ka] Selected from, especially, [ka] A compound selected from any one of claims [1] to [6].

[0126] [8] R 0 is C 1~6 It is an alkyl group, preferably C 1~3 Alkyl groups, especially methyl groups, and / or, n is an integer between 2 and 5, in particular 3, and / or k1 and k2 are independently 1, 3, or 5, and especially 5. i is an independent integer between 1 and 20, preferably between 1 and 12, more preferably between 2 and 8, particularly 4, and / or The compound according to any one of claims [1] to [7], wherein j is an independent integer between 1 and 20, preferably between 1 and 12, more preferably between 8 and 12, particularly 8 or 12.

[0127] [9] [ka] [ka] A compound according to any one of claims [1] to [8], selected from the following.

[0128]

[10] Having the structure shown in formula (II), [ka] Here, A is an antibody or its antigen-binding fragment, and the antibody or its antigen-binding fragment is preferably modified to form (Gly) in the compound of formula (I). n Combine into parts, z is an integer from 1 to 20, preferably from 1 to 4, and especially 2. P1, P2, R 0 , opSu, n, k1, k2, i and j are the terms of claims [1] to [8] A conjugate as defined in any one of the following clauses.

[0129]

[11] [ka] The conjugate according to claim

[10] , which is selected from the following.

[0130]

[12] The conjugate according to claim

[10] or

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

[0131]

[13] A pharmaceutical composition comprising a conjugate according to any one of claims

[10] to

[12] in an effective amount for prevention or treatment, and at least one pharmaceutically acceptable carrier.

[0132]

[14] The pharmaceutical composition according to claim

[13] , wherein the drug-to-antibody ratio (DAR) of the conjugate is an integer or non-integer between 1 and 8, and particularly an integer or non-integer between 3 and 4.

[0133]

[15] Use of the conjugate according to any one of claims

[10] to

[12] or the pharmaceutical composition according to claim

[13] or

[14] in the manufacture of a drug for treating a disease, wherein the disease is a tumor or an autoimmune disease, preferably a tumor comprising HER2-positive tumor cells.

[0134]

[16] The tumor containing the aforementioned HER2-positive tumor cells further contains HER2-low-expressing tumor cells, The use according to claim

[15] , wherein the HER2 expression level of the HER2-low-expressing tumor cells is lower than that of the HER2-positive tumor cells.

[0135]

[17] Having the structure shown in formula (i), [ka] Here, a, p1 * A carbon atom with a mark attached, p2 * A carbon atom with L attached. 1 M, L 2 , R 1 and R 2 A compound or a pharmaceutically acceptable salt thereof, stereoisomer, solvate, polymorph, tautomer, isotope, metabolite, or prodrug as defined in any one of claims [1] to [7].

[0136]

[18] [ka] [ka] Selected from, Preferably, [ka] Selected from, more, [ka] Selected from, especially, [ka] The compound according to claim

[17] , which is selected from the following.

[0137]

[19] A compound of formula (1), [ka] Here, k is an integer from 1 to 7, preferably 1, 3, or 5, and particularly 5. P is a payload having the structure shown in formula (i'), wherein the structure shown in formula (i') is defined in any one of claims [1] to [7].

[0138]

[20] [ka] The conjugate according to claim

[19] , which is selected from the following.

[0139] The novel small molecule topoisomerase I inhibitor according to the present invention can exhibit higher activity, stability, and physicochemical properties than the prior art when used alone or as a component of an ADC.

[0140] The antibody-drug conjugate of the present invention can achieve high efficacy and bystander killing effect using a specially designed linker-payload. Furthermore, because it has a low DAR, it can reduce side effects and improve the therapeutic index, which is very important for bystander killing. The antibody-drug conjugate of the present invention has a more stable structure, such as an open-ring succinimide structure.

[0141] This invention utilizes a linker with a unique structure and uses a ligase to catalyze the conjugation of a target molecule (antibody) and a payload. The conjugate produced by this invention is highly homogeneous and highly active. Furthermore, the linker unit-payload intermediate has much lower toxicity than the free payload, thus posing a low hazard in the drug manufacturing process and making it useful for industrial production.

[0142] The conjugate of the present invention achieves at least one of the following technical effects. (1) It has high inhibitory activity against target cells and high bystander killing effect.

[0143] (2) It has excellent physicochemical properties (e.g., solubility, physical and / or chemical stability).

[0144] (3) It has excellent pharmacokinetic properties (e.g., high stability in plasma, appropriate half-life and duration of action).

[0145] (4) Safety (low toxicity and / or few adverse effects on non-target normal cells or tissues, wide range of diseases) It excels in areas such as treatment range.

[0146] (5) The highly modular design allows for easy combination of various drugs. [Examples]

[0147] Preparation Examples To more clearly explain the object and technical means of the present invention, the present invention will be further described below with reference to specific examples. Please understand that these examples are for illustrative purposes only and do not limit the scope of the present invention. Any specific experimental methods not mentioned in the following examples will be carried out according to conventional experimental methods. Equipment, materials, and reagents

[0148] Unless otherwise specified, the equipment and reagents used in the examples are all commercially available. The reagents can be used as is without purification.

[0149] MS: Thermo Fisher Q Exactive Plus, Water2795-Quattro micro triple quadrupole mass spectrometer HPLC: Waters 2695, Agilent 1100, Agilent 1200 Half-portion HPLC: Lisure HP plus 50D Flow cytometer: CytoFLEX S HIC-HPLC: Butyl-HIC, mobile phase A: 25mM PB, 2M (NH4 )2SO4, pH 7.0, mobile phase B: 25mM PB, pH 7.0, flow rate: 0.8 mL / min; collection time: 25 min, sample injection volume: 20 μg; column temperature: 25°C; detection wavelength: 210 nm; sample chamber temperature: 8 °C. SEC-HPLC: Chromatography column: TSK-gel G3000 SWXL, TOSOH 7.8mm ID×300mm, 5μm, mobile phase: 0.2M KH2P O4, 0.25M KCl, pH 6.2, flow rate: 0.5 mL / min; collection time: 30 min, sample injection volume: 50 μl, column temperature: 25°C; detection wavelength: 210 nm; sample dish temperature: 8°C. CHO cells were obtained from Thermo Fisher Scientific, pcDNA3.3 from Life Technology, HEK293F from Prejin, PEIMAX transfection reagent from Polyscience, MabSelect Sure ProA from GE, and Capto S ImpAct was obtained from GE, Rink-amide-MBHA resin and dichloro resin were obtained 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 and NCI-N87 (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, and NCI-H2110 was obtained from ATCC CAT# It was obtained from CRL-5924, JIMT-1 was obtained from Wuxi Apptech, Capan-1 was obtained from ATCC CAT# CRL-1573, the antibody trastuzumab was manufactured based on a known sequence, and it was used against Escherichia coli (E. coli) and Staphylococcus aureus (Sta We produce an optimized recombinase Sortase A derived from phylococcus aureus.

[0150] In some cases, the order in which the above reaction scheme is carried out can be changed to accelerate the reaction or avoid unwanted reaction products. To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the invention in any way. [Examples]

[0151] Preparation of small payload molecules Intermediate 11 [ka] Step A: N-(2-bromo-5-fluorophenyl)acetamide: While stirring, concentrated H2SO4 (3 mL) was added to a solution of acetic anhydride (214 g, 2.10 mol) and acetic acid (500 mL), and then 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) was added in small portions at room temperature. After stirring the mixture for 3 hours, it was placed in 2000 mL of ice water. A precipitate formed, which was collected by filtration and vacuum-dried at room temperature to obtain yellow solid N-(2-bromo-5-fluorophenyl)acetamide (105 g). 1 H NMR(400MHz,DMSO-d6)δ7.68(dd,J=8.9,6.0Hz, 1H),7.61(ddd,J=10.7,5.3,3.1Hz,1H),7.02(ddd,J=8.9,8.0,3.1Hz,1H),2.11(s,3H).LCMSm / z232.0[M+H] + .

[0152] Step B: N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide:-78 o While stirring with C, N-(2-bromo-5-fluorophosphate) n-BuLi (594 mL, 1.6 M, in n-hexane, 950.22 mmol) was added dropwise to a solution of phenylacetamide (105 g, 452.48 mmol) and THF (1000 mL). After completion, the mixture was stirred in N2 for 0.5 hours. Then, -78 o At C, within 0.5 hours, add a solution of cyclobutanone (38.06 g, 542.98 mmol) and THF (50 mL) dropwise, and mix the contents at -78 o The mixture was stirred at room temperature for 6 hours from C. o In C, the mixture was placed in a 500 mL saturated NH4Cl aqueous solution, extracted with ethyl acetate (500 mL x 3), and then rinsed with saline solution. The mixture was washed with (250 mL x 2), dried over Na2SO4, and concentrated. The mixture was polished with (PE / EA = 1:1, 100 mL) for 10 min, filtered, and the filtration cake was collected. Vacuum drying was performed to obtain yellow solid N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g). LCMS m / z 206.1 (M-18+H), 24 6.1(M+Na).

[0153] Step C: N-(3-fluoro-8-oxo-5,6,7,8-tetralin-1-yl)acetamide: While stirring, add N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24g, 107.50 mmol) and CH2Cl2( A mixture of 170 mL of water and 170 mL of silver nitrate (AgNO3) (5.48 g) (32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.0 Add 1 mmol) to the mixture and mix for 30 minutes. o The mixture was stirred for 6 hours in C. The mixture was filtered through diatomaceous earth and then C. Wash with H2Cl2 (100 mL), concentrate the filtrate, and obtain FCC (EA / PE = 0-40%). By purifying the solution using LCMS, a pale yellow solid N-(3-fluoro-8-(oxo)-5,6,7,8-tetralin-1-yl))acetamide (14 g)W was obtained. LCMS m / z 222.1 [M+H] + .

[0154] Step D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetralin-1-yl)acetamide: 0 o At C, while stirring, N-(3- A mixture of fluoro-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (14 g, 63.28 mmol) and THF (500 mL) was mixed with 1-butyl nitrite (8.48 g, 63.28 mmol), and then t-BuOK (8.52 g, 75.94 mmol) was added. oThe mixture was stirred in C for 2 hours. After completion, the mixture was treated with HCl(2N). The mixture was acidified with ethyl acetate (200 mL x 3), washed with brine (100 mL x 2), dried over Na2SO4, and concentrated under reduced pressure. The crude product of the mixture was polished with methyl tert-butyl ether (200 mL) for 10 min, filtered, and the filter cake was collected. Vacuum drying was performed to obtain a yellow solid N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetralin-1-yl)acetamide (12 g). LCMS m / z 251.1 [M+H] + .

[0155] Step E: Add 10% Pd / C (1g) to a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetralin-1,7-diyl) diacetamide (12g, 47.96 mmol) with acetic anhydride (90 mL) and THF (90 mL), and mix the contents for 25 minutes. o The mixture was stirred for 16 hours under an H2 atmosphere in C. o Cooling to C After that, add Et3N (20 mL) dropwise and mix 0 o The mixture was stirred in C for 1 hour. Filtered through diatomaceous earth, the filtrate was placed in ice water (500 mL). Extraction was performed with ethyl acetate (500 mL x 3), washed with brine (250 mL x 2), dried over Na2SO4, and concentrated. The residue was polished with methyl tert-butyl ether (120 mL) for 10 minutes, filtered, and the filter cake was collected. Vacuum drying yielded a yellow solid N,N′-(3-fluoro-8-oxo-5,6,7,8-tetralin-1,7-diyl)diacetamide (7.9 g). LCMS m / z 279.1 [M+H] + .

[0156] Step F: Add aqueous HCl (2N, 150 mL) to a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetralin-1,7-diyl)diacetamide (7.9 g, 28.39 mmol) and MeOH (150 mL), and mix the mixture for 50 minutes. o Stirred in C for 7 hours. o After cooling to C, add saturated NaHCO3 aqueous solution dropwise. The pH was adjusted to 8. Extraction was performed with ethyl acetate (200 mL x 3), washed with saline solution (200 mL x 2), dried over Na2SO4, and concentrated under reduced pressure to obtain a yellow solid N,N'-(3-fluoro-8-oxo-5,6,7,8-tetralin-1,7-diyl)diacetamide (6.0 g). 1 H NMR(400MHz,Chloroform-d)δ6.57(s ,3H),6.18(td,J=11.1,2.4Hz,2H),4.52(dt,J=13.3,5.0Hz,1H),3.13(ddd,J=17.5,13.0,4.6H z,1H),3.00-2.81(m,1H),2.69(dtd,J=9.4,4.6,2.5Hz,1H),2.09(s,3H),1.79(qd,J=13.0,4.3Hz,1H).LCMSm / z237.1[M+H] + .

[0157] Step G: N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl)acetamide: 0 o C, N,N′-(3-fluoro- 4.0 g, 16.93 mmol) of 8-oxo-5,6,7,8-tetralin-1,7-diyl)diacetamide was added in fractional amounts to a solution of DMF (80 mL) and NCS (2.26 g, 16.93 mmol). The mixture was stirred at room temperature for 16 hours. The mixture was placed in 200 mL of ice water. A precipitate formed, which was collected by filtration and vacuum-dried at room temperature to obtain a yellow solid N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl)acetamide (4.0 g). 1 1H NMR (400 MHz) z,DMSO-d6)δ8.11(d,J=8.0Hz,1H),7.71(s,2H) ,6.62(d,J=11.9Hz,1H),4.53(ddd,J=13.0,8.0,4.7Hz,1H),3.18-3.04(m,1H),2.91(d dd,J=17.5,12.4,4.8Hz,1H),2.21-2.08(m,1H),1.99-1.83(m,4H).LCMSm / z271.0[M+H] + .

[0158] Step H: N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)acetamide:N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetralin-2-yl)acetamide (4.0g To a mixture of (S)-4-ethyl-4-hydroxyl-7,8-dihydro-1H-pyrano[3,4-f]indridine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol), and orthocresol (10 mL) were added. The mixture was heated under N2 reflux and held for 24 hours. The solvent was removed by reducing the pressure, and the mixture was refrigerated in a FCC. Purification by (THF / CH2Cl2=0-60%) yielded a brown solid N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)acetamide (4.1g). LCMSm / z498.1[M+H] + .

[0159] Step I: (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13-dione:70 o C, N-((9S)-4-chloro-9-ethyl-5-fluoro-9- A mixture of hydroxyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of concentrated HCl aqueous solution was stirred under N2 for 36 hours. The mixture was concentrated under reduced pressure to obtain a brown solid. The crude product (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13-dione hydrochloride (2g) was obtained. LCMS(ESI) m / z 456.1[M+H] + .

[0160] Intermediate 12 [ka] Intermediate 12: (9S)-1-amino-4-bromo-9-ethyl-5-fluoro-9-hydroxyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13-dione hydrobromide was synthesized from 2-bromo-5-fluoroaniline by a method similar to that of intermediate 11. LCMS(ESI) m / z 500.0[M+H] + .

[0161] Intermediate 13 [ka] Intermediate 13: (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxyl-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13-dione hydrochloride was synthesized from 2-bromo-5-fluoro-4-methoxyaniline by a method similar to that of intermediate 11. LCMS(ESI)m / z452.1[M+H] + .

[0162] Methods C730 and C731 [ka] C730 and C731 were prepared in TFA salt form from (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13-dione hydrochloride (intermediate 11) by preparative HPLC.

[0163] Table I [Table 3]

[0164] Method C518 [ka] Step J: N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide:crude product (9S)-1-amino-4-chloro-9-ethyl-5-fluoro -9-hydroxyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13-dione hydrochloride (50 mg, 0.10 mmol) was mixed with 2 mL of DMF, to which 2-hydroxyacetic acid (11.6 mg, 0.15 mmol) and HATU (57.9 mg, 0.15 mmol) were added. o Add Et3N (20.5 mg, 0.20 mmol) dropwise to C and mix the contents 0 o Stir from C to room temperature for 2 hours. The mixture was placed in 10 mL of ice water, extracted with ethyl acetate (20 mL x 3), washed with brine (20 mL x 2), dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC to obtain a yellow solid N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)-2-hydroxyacetate Mid (6 mg) and a yellow solid N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide (5 mg) were obtained.

[0165] The compounds prepared in Table II below were reacted and worked up in the same manner as in Example C518.

[0166] Table II [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8]

[0167] The compounds prepared in Table III below were synthesized from intermediate 12 or intermediate 13 using a method similar to that for C518, as described above.

[0168] Table III [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7]

[0169] Method C589 [ka] Step K: (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxyl-4-methyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)-3-hydroxylbutyroamide:(1S,9S) -1-amino-9-ethyl-5-fluoro-9-hydroxyl-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-10,13-dionemethanesulfonate (50 mg, 0.10 mmol, CAS: 169869-90-3) was mixed with 2 mL of DMF to which (S)-3-hydroxybutyrate (15.6 mg, 0.15 mmol) and HATU (57.9 mg, 0.15 mmol) were added. o Add Et3N (20.5 mg, 0.20 mmol) dropwise to C and mix the contents 0 o The mixture was stirred for 2 hours from C to room temperature. The solution was placed in L ice water, extracted with ethyl acetate (20 mL x 3), washed with saline solution (20 mL x 2), dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC to obtain a yellow solid (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxyl-4-methyl-10,13-di(oxo)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolidino[1,2-b]quinoline-1-yl)-3-hydroxylbutyroamide (9.4 mg).

[0170] The compounds prepared in Table IV below were reacted and worked up in the same manner as in Example C589.

[0171] Table IV [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Examples]

[0172] Preparation of the linker 2.1 Preparation of Linker HX20113 [ka] HX20113 was synthesized using a general solid-phase polypeptide synthesis method for Rink-amide-MBHA-resins. Fmoc protects the amino acids in the binding unit. The conjugate reagent was selected from HOBT, HOAt / DIC, DCC, EDCI, or HATU. After synthesis, the resin was decomposed with trifluoroacetic acid. The product was purified by HPLC and lyophilized for use. Theoretical mass: 1207.59, measured mass: [MH] - = 1206.7.

[0173] Preparation of Linker HX20111 [ka] HX20111 was synthesized using a common solid-phase polypeptide synthesis method for Rink-amide-MBHA-resins. Fmoc protects the amino acids in the binding unit. The conjugate reagent was selected from HOBT, HOAt / DIC, DCC, EDCI, or HATU. After synthesis, the resin was decomposed with trifluoroacetic acid. The product was purified by HPLC and lyophilized for use. Theoretical mass: 1383.70, measured value: [MH] - = 1382.6. [Examples]

[0174] Preparation of linker-payload intermediates 3.1 Preparation of the intermediate Mc-GGFG-Dxd The intermediate Mc-GGFG-Dxd can be purchased commercially or prepared by the method described in European Patent Application Publication No. 2907824. This compound can be used to prepare the linker-payload intermediate (compound of formula (I)) and can also be used to prepare control ADC LC1184(8) and control ADC LC1184(4) by direct conjugation with a modified antibody.

[0175] 3.2 Preparation of H0019 [ka] Synthesis of HX21008-a Weigh 4.33g of Fmoc-Gly-Gly-OH and 6.84g of Pb(OAc)4. The mixture was then placed in a 500 mL round-bottom flask. Anhydrous THF / toluene (120 / 40 ml) was added under a nitrogen atmosphere and dissolved by stirring. Next, 1.16 mL of pyridine was added to the reaction system. Under a nitrogen atmosphere, the reaction system was heated to 80°C and refluxed for 5 hours. The reaction was monitored by sampling and measurement by HPLC.

[0176] The reaction system was cooled to room temperature, filtered, and the filtered cake was washed three times with EA. The filtrates were combined and concentrated to dryness. 2000 mg of the target product was obtained by purification by column chromatography (PE:EA = 100:0 to 50:100), with a yield of 44%.

[0177] Synthesis of H0005-a 200 mg of HX21008-a was weighed and placed in a 100 ml round-bottom flask. Next, 15 ml of THF was added and dissolved by stirring. Then, H0005 (316 mg, 3.0 eq) and TsOH·H2O (15 mg, 0.15 eq) were added. The reaction system was left at room temperature. The mixture was allowed to react overnight. The reaction was monitored by sampling and measuring by TLC (PE / EA=1:1). The starting material virtually disappeared, and new spots appeared.

[0178] The reaction was quenched by adding a saturated sodium bicarbonate solution. Extraction was performed three times with EA. The organic phases were combined and washed with brine. The crude product was dried over anhydrous magnesium sulfate and concentrated. Purification by column chromatography (PE:EA = 5:1 to 1:1) yielded approximately 165 mg of the target product, a colorless oily substance, with a yield of 60%. MS:[M+H] + =503.4. Synthesis of H0005-b 200 mg of H0005-a was weighed and placed in a 100 ml round-bottom flask. Next, 10 ml of EtOH and 5 ml of EA were added and completely dissolved. Then, 40 mg of palladium carbon was added to the reaction system under a nitrogen gas atmosphere, and nitrogen gas was passed through the reaction system three times. The reaction system was kept under a hydrogen gas atmosphere and stirred at room temperature for 0.5 hours. The reaction was monitored by sampling and measuring by TLC (DCM / MeOH = 10:1). The starting materials virtually disappeared, and new spots appeared.

[0179] The reaction system was filtered, and the filter cake was washed three times with EA. The filtrates were combined and concentrated to dryness to obtain 200 mg of a white solid product, with a yield of 100%. The product was used directly in the next reaction without purification. [M+H] + =413.3. Synthesis of H0005 2.0 g of dichlororesin was weighed and placed in a peptide synthesis tube. 10 ml of DCM was added and the mixture was allowed to swell at room temperature for 30 minutes. The solvent was removed by vacuum suction. The resin was washed twice with DCM, each time at a rate of 7 mL for 1 minute. The solvent was removed by vacuum suction. Next, 200 mg of H0005-b was weighed and placed in a 50 ml centrifuge tube. Approximately 10 ml of DCM was added and the solid was dissolved by shaking. This was added to the above resin. The mixture was stirred to immerse all of the resin in the solution (if resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). The mixture was stirred for 4-5 hours. After the reaction was complete, an appropriate amount of methanol was added. The mixture was stirred for 30 minutes. The solvent was removed by vacuum suction. The resin was washed once with DMF, once with methanol, once with DMF, once with tanol, and twice with DMF, each time at a rate of 10 mL for 1 minute. The solvent was removed by vacuum aspiration. Ninhydrin was detected in a small amount of dry resin. The resin was colorless and transparent, and the solution was pale yellow, suggesting that it passed the coupling procedure in the next step.

[0180] Deprotection was performed twice by adding 10 mL of a commercially available 20% piperidine / DMF solution, with each reaction lasting 10 minutes. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially with DMF twice, methanol once, DMF once, tanol once, and DMF twice, each time at a volume of 10 mL for 1 minute. The solvent was removed by vacuum aspiration. Ninhydrin detection was performed on a small amount of dried resin. Both the resin and the solution were deep blue.

[0181] 563 mg of Fmoc-Phe-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Then approximately 7 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIC was added. The mixture was activated for 10 to 30 minutes to obtain the activated reaction solution.

[0182] Three molar equivalents of the activation reaction solution were added to the resin. The mixture was stirred to completely immerse the resin in the solution (if resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). The mixture was stirred for 2-3 hours. After the reaction was complete, the solvent was removed by vacuum suction. The resin was washed sequentially with DMF twice, methanol once, DMF once, tanol once, and DMF twice, each time at a volume of 10 mL for 1 minute. The solvent was removed by vacuum suction. Ninhydrin detection was performed on a small amount of dried resin. The resin was colorless and transparent, and the solution was pale yellow, suggesting that it had passed the coupling step.

[0183] Deprotection was performed twice by adding 10 mL of a commercially available 20% piperidine / DMF solution, with each reaction lasting 10 minutes. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially with DMF twice, methanol once, DMF once, tanol once, and DMF twice, each time at a volume of 10 mL for 1 minute. The solvent was removed by vacuum aspiration. Ninhydrin detection was performed on a small amount of dried resin. Both the resin and the solution were deep blue.

[0184] 531 mg of Fmoc-Phe-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Then approximately 10 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIC was added. The mixture was activated for 10 to 30 minutes to obtain the activated reaction solution.

[0185] Three molar equivalents of the activated reaction solution were added to the resin. The mixture was stirred to completely immerse the resin in the solution (if any resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). The mixture was stirred for 2-3 hours. After the reaction was complete, the reaction solution was removed by vacuum suction. The resin was washed sequentially with DMF twice, methanol once, DMF once, tanol once, and DMF twice, each time at a volume of 10 mL for 1 minute. The solvent was removed by vacuum suction. Ninhydrin detection was performed on a small amount of dried resin. The resin was colorless and transparent, and the solution was pale yellow, suggesting that it had passed the coupling step.

[0186] Deprotection was performed twice by adding 10 mL of a commercially available 20% piperidine / DMF solution, with each reaction lasting 10 minutes. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially with DMF twice, methanol once, DMF once, tanol once, and DMF twice, each time at a volume of 10 mL for 1 minute. The solvent was removed by vacuum aspiration. Ninhydrin detection was performed on a small amount of dried resin. Both the resin and the solution were deep blue.

[0187] 462 mg of MC-OSu and 10 mL of DMF were added to a 50 mL centrifuge tube. The solid was dissolved by shaking. Next, 0.24 mL of DIEA was added to the resin. The mixture was stirred to completely immerse the resin in the solution (if resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). The mixture was stirred for 2-3 hours. After the reaction was complete, the reaction solution was removed by vacuum suction. The resin was washed sequentially with DMF twice, methanol once, DMF once, tanol once, and DMF twice, each time at a volume of 10 mL for 1 minute. The solvent was removed by vacuum suction. Ninhydrin detection was performed on a small amount of dried resin. The resin was colorless and transparent, and the solution was pale yellow, suggesting that it had passed the coupling step.

[0188] The resin was washed twice with 10 mL of methanol. Next, the solvent was completely removed by vacuum aspiration. The resin was removed and weighed. A lysis buffer was prepared in a 250 mL Erlenmeyer flask so that the TFE / DCM ratio was 80% / 20% and the volume was 7-8 times the weight of the peptide resin. The lysis buffer was added to the peptide resin and mixed uniformly. The resin was completely immersed in the lysis buffer and dissolved at room temperature for 2-3 hours. Next, the lysis buffer was filtered through a simple filter made with a syringe, and the resin was washed with 1-2 mL of DCM and discarded. Then, 150 mL of pre-cooled anhydrous ether was added to the lysis buffer and mixed uniformly, and then allowed to stand for 20-30 minutes. The above system was centrifuged in a 50 mL centrifuge tube at 3500 rpm for 3 minutes, and the supernatant was removed and discarded. The solid was shaken in pre-cooled anhydrous ether, ultrasonically washed once, centrifuged at 3500 rpm for 3 minutes, and the supernatant was removed and discarded. The solid was placed in a centrifuge tube and air-dried overnight, then preparatively purified to obtain 125 mg of a white solid product, with a yield of 40%. [M+H] + =645. 4. Synthesis of H0013 150 mg of starting material H0005 and 55 mg of TSTU were weighed and placed in a 10 mL round-bottom flask. Anhydrous DMF (3 mL) was added under a nitrogen atmosphere and the mixture was stirred for 20 minutes. Next, 18 mg of TSN 00643 and 20 μl of DIEA were added sequentially to the reaction system. The mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. The reaction was monitored by sampling and measurement by HPLC. The starting material peak virtually disappeared, and a new peak appeared.

[0189] The reaction system was preparatively purified, the target product was collected, and freeze-dried to obtain a pale yellow solid product of approximately 22 mg. [M+H] + =1062.7. Synthesis of H0019 Weigh out H0013 (30 mg) and place it in a 10 mL round-bottom flask with a neck, then add purified water (2 (ml) was added. It was stirred to dissolve. A DMF solution (2 mL) containing HX20111 (19.5 mg) was added to the reaction system and stirred. After reacting overnight, the reaction was monitored by HPLC until all the starting materials were converted to the intermediate. Next, the reaction system was cooled to 0-10°C, and while controlling the temperature, a 0.5% LiOH solution was added to the reaction system until the pH of the reaction system was approximately 12. The reaction mixture was stirred for 20 minutes, and the reaction was monitored by HPLC until all the intermediate was consumed. Then, the reaction was quenched by adding 30% acetic acid (1.5 mL) to the reaction system.

[0190] The reaction system was preparatively purified, the target product was collected, and freeze-dried to obtain a pale yellow solid product of approximately 25 mg. [(M+3H) / 3] + =1182.3. 3.3 H0020, H0021, H0034 and H0035 Linker-payload intermediates H0020, H0021, H0034, and H0035 can be prepared using similar synthetic routes and reagents. Intermediates H0014, H0015, H0026, and H0032 were synthesized using a method similar to that of H0013, and were subsequently used in the synthesis of H0020, H0021, H0034, and H0035, respectively.

[0191] [ka] [ka] The LC-Ms data is shown in the table below. [Table 7] 3.4 Preparation of LB302-2-4 [ka] HX 20111 and the intermediate MC-GGFG-Dxd (in a molar ratio of approximately 1:2) were weighed, 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 the reaction was complete, an appropriate amount of Tris-Base solution or another solution that promotes ring-opening was directly added to the reaction mixture, and the reaction was further reacted at 0–40°C for a total of 0.2–20 hours. After the reaction was complete, the product was purified by half-portion / preparative HPLC and lyophilized to obtain the linker-payload intermediate LB 302-2-4. Theoretical mass: 3486.52, measured mass: [(M+3H) / 3] + = 1163.3.

[0192] 3.5 Preparation of LB302-2-1 LB302-2-1 can be prepared using a similar synthetic route and reagents.

[0193] [ka] [Examples]

[0194] Construction of antibody expression vectors, antibody expression, purification, and identification. 4.1 Modified anti-human HER2 antibody Ab0001-LCCT L - Preparation of HC Antibody Ab0001-LCCT L -HC (Light chain: SEQ ID NO: 9, Heavy chain: SEQ The expression plasmid for ID NO:10) is constructed as follows: Antibody Ab0001-LCCT L -HC sequence: Based on the amino acid sequence of trastuzumab, GA is added to the C-terminus of the light chain. LPETGG was introduced, with LPETGG being the recognition sequence for the ligated donor substrate and GA being the spacer sequence. The plasmid was transfected into CHO cells to establish a cell population and screen for high-expression cell populations. These cells were then cultured in a 5-10 L reactor following the trastuzumab culture procedure, and the supernatant was collected.

[0195] 4.2 Antibody Ab0001-LCCT L - Purification of HC Ab0001-LCCT L - HC purification is performed using MabSelect affinity chromatography. The process was carried out using a standard procedure combining tography and Sepharose S cation exchange chromatography, and the purified product was returned to the original trastuzumab drug buffer (5 mM). Histidine-HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0) was dissolved and frozen in equal amounts and small portions.

[0196] 4.3 Antibody Ab0001-LCCT L - Mass control of HC The antibody Ab 0001-LCCT was purified by the above SDS-PAGE method. L - Purity of HC The concentration was 98.5%, and SEC-HPLC detected that the high molecular weight polymer content in the sample was less than 0.4%, and the endotoxin content was less than 0.098 EU / mg.

[0197] 4.4 Preparation of other modified anti-human antibodies Based on a similar method, ligase-recognition-based terminal modifications were introduced to the C-terminuses of the light and / or heavy chains of trastuzumab, respectively, to obtain modified antibodies.

[0198] The modified anti-human HER2 antibodies based on Ab0001 (trastuzumab) are shown in the table below. In the terminal modification sequence, LPETGG is the ligated donor substrate recognition sequence, and GA is the spacer sequence.

[0199] Modified anti-human HER2 antibody [Table 8] [Examples]

[0200] Preparation of antibody-small molecule conjugates 5.1 The linker-payload intermediates are each site-specifically conjugated with the antibody by ligase to form ADCs. The conjugation reaction method can be found in International Publication No. 2015165413. The generated ADCs are shown in the table below. [Table 9]

[0201] 5.2 Control ADCs LC 1184(8) and LC 1184(4) use the intermediate Mc-GGFG-Dxd in Ab 0001-LCCT L - Directly binds to HC (Cysco It is prepared by conjugation (i.e., by conjugation by a bond formed between the maleimide structure and the mercapto group of Cys). The method of the conjugation reaction is well known in this field. Eight Mc-GGFG-Dxd are introduced into LC 1184(8) by reduced interchain cysteine. Reduced interchain cysteine ​​is introduced into LC 1184(4) This will enable the installation of four Mc-GGFG-Dxd units.

[0202] Example 1: The effect of small molecules on cell proliferation Research Objective: This experiment aims to evaluate the effects of small molecule compounds on the proliferation of the high-HER2 tumor cell line SK-BR-3 by measuring cell activity using CellTiter-Glo, and to analyze the effects of small molecule compounds on tumor cell proliferation.

[0203] Materials and equipment PBS: Gibco, Cat#10010-023); Trypsin: Gibco, Cat#25200056); FBS: Gibco, Cat#10270-106); CellTiter-Glo(registered trademark) 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 deep-well plate: Thermo / 278743; 15mL microcentrifuge tube: Thermo / 339650; 1.5mL microcentrifuge tube: Beaver.

[0204] Cell inoculation - day 1 (1) Microscopic examination of cells (2) Digestion: Digested with 2 mL of 0.25% trypsin for 3 minutes. (3) Centrifugal separation: 1000 rpm, 5 min; (4) Cell counting A. Cell dilution: B. Cell inoculation: 100 μl / well C. Incubation: 37°C, 5% CO2, overnight. Cell examination

[0205] (1) (Microscopic examination) (2) Preparation of drugs: A. Preparation of sample preparation buffer: The required amount of sample preparation buffer was prepared using the culture medium (10% FBS) of the test cells. B. Sample preparation: The sample was diluted from the first concentration to the desired concentration in a 96-deep-well plate.

[0206] Test compound: Prepared by diluting in cell culture medium to 10 final concentrations: 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, and 0.000512 nM.

[0207] A stock solution of 2.5 mg / ml puromycin was used as a positive control and diluted in cell culture medium. A 250-fold dilution was performed, and then a 2-fold dilution was performed again to achieve a puromycin test concentration of 5 μg / ml when administering the compound.

[0208] (3) Administration of the compound: The compound was administered so that each concentration in the dilution series had three overlapping values. 100 μl / well.

[0209] (4) After administering the drug, the cells were incubated in an incubator for 120 hours.

[0210] Cell activity test (1) Preparation of detection reagent: CellTiter-Glo Luminescent Ce The Viability Assay reagent was heated to room temperature while protected from light.

[0211] (2) Cell preparation: The test cells were removed from the incubator and allowed to equilibrate at room temperature (25°C) for 30 minutes.

[0212] (3) ATP measurement: Discard the culture medium, add 100 μl / well of DMEM, add 50 μl / well of CTG to a 96-well plate, shield from light with aluminum foil, and shake at 200 rpm for 10 minutes at room temperature using a vortex mixer.

[0213] (4) Measurement program: After setting the program, the black wall transparent bottom plate was placed on the equipment without attaching the cover.

[0214] (5) Data collection 5. Data Processing and Analysis Data processing was performed using GraphPad software. A 4-Lameter curve model was used to plot the cell proliferation inhibition rate against the concentration of the control or test sample, and data fitting was performed to obtain the R-values ​​for the control and test samples, respectively. 2 (Round to four significant figures) (to do) and IC 50(Round to four significant figures) or EC 50 The results were obtained. If necessary, the relative biological activity, i.e., specific activity, of sample A was calculated. Dxd was used as a control sample for calculating specific activity.

[0215] 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 (The puro group is the puro-treated group) Specific activity (%) of the sample = (IC of the control sample) 50 IC value of test sample 50 Value) × 100 Specific activity (%) of the control sample = (IC of the control sample) 50 Value / IC of previous test sample 50 Value) × 100 6. Verification of Results R 2 Value ≥ 0.950, CV% between overlapping wells ≤ 30%.

[0216] result: The results are shown in the table below. [Table 10] Example of effect 2: The effect of HER2-targeting conjugates on cell proliferation. Research Objective: To evaluate the effect of ADCs on the proliferation of two high-HER2 tumor cell lines, SK-BR-3 and NCI-N 87, and the HER2-negative tumor cell line, MDA-MB-468, by measuring detected cell activity using CellTiter-Glo, and to analyze the effect of ADCs on tumor cell proliferation.

[0217] Materials and equipment PBS: Gibco, Cat#10010-023); Trypsin: Gibco, Cat#25200056); FBS: Gibco, Cat#10270-106); CellTiter-Glo(registered trademark) 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 deep-well plate: Thermo / 278743; 15mL microcentrifuge tube: Thermo / 339650; 1.5mL microcentrifuge tube: Beaver.

[0218] Cell inoculation - day 1 (1) Microscopic examination of cells (2) Digestion: Digested with 2 mL of 0.25% trypsin for 3 minutes. (3) Centrifugal separation: 1000 rpm, 5 min; (4) Cell counting A. Cell dilution: B. Cell inoculation: 100 μl / well C. Incubation: 37°C, 5% CO2, overnight.

[0219] Cell examination (1) Microscopic examination (2) Preparation of drugs: A. Preparation of buffer solution for sample preparation: Use the culture medium (10% FBS) of the test cells to prepare the required amount A buffer solution for sample preparation was prepared. B. Sample preparation: The sample was diluted from the first concentration to the desired concentration in a 96-deep-well plate.

[0220] Test samples were prepared by diluting them in cell culture medium to 10 final concentrations: 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0.000512, and 0.0001024 nM.

[0221] A stock solution of 10 mg / ml puromycin was used as a positive control and diluted in cell culture medium. A 100-fold dilution was performed, and then a 10-fold dilution was performed again so that the puromycin test concentration was 5 μg / ml when administering the compound.

[0222] (3) Administration of the compound: The compound was administered so that each concentration in the dilution series had three overlapping values. 100 μl / well.

[0223] (4) After administering the drug, the cells were incubated in an incubator for 120 hours.

[0224] Cell activity test (1) Preparation of detection reagent: The CellTiter-Glo Luminescent Cell Viability Assay reagent was heated to room temperature while protected from light.

[0225] (2) Cell preparation: The test cells were removed from the incubator and allowed to equilibrate at room temperature (25°C) for 30 minutes.

[0226] (3) ATP measurement: Discard the culture medium, add 100 μl / well of DMEM, add 50 μl / well of CTG to a 96-well plate, shield from light with aluminum foil, and shake at 200 rpm for 10 minutes at room temperature using a vortex mixer.

[0227] (4) Measurement program: After setting the program, the black wall transparent bottom plate was placed on the equipment without attaching the cover.

[0228] (5) Data collection 5. Data Processing and Analysis Data processing was performed using GraphPad software. A 4-Lameter curve model was used to plot the cell proliferation inhibition rate against the concentration of the control or test sample, and data fitting was performed to obtain the R-values ​​for the control and test samples, respectively. 2 (Round to four significant figures) (to do) and IC 50 (Round to four significant figures) or EC50 I obtained it.

[0229] 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 (The puro group is the puro-treated group) 6. Verification of Results R 2 Value ≥ 0.950, CV% between overlapping wells ≤ 30%.

[0230] result: The results are shown in the following table, Figure 1, Figure 2, and Figure 3. [Table 11]

[0231] In HER2-highly expressing SK-BR-3 and NCI-N 87, the conjugates (CH-2-589, CH-2-593, CH-2-518, LC302-2-1(4), and LC302-2-4(4)) showed significant effects. IC of the conjugates 50 The value was smaller than that of the small molecule payload Dxd. In HER2-negative cells, the conjugate showed minimal effect. No cytotoxicity was observed in HER2-negative cells MDA-MB-468, demonstrating the stability of the conjugate and reducing the release of the payload outside the target, thus indicating good safety. In summary, the conjugate exhibited dose-dependent HER2-dependent cytotoxic activity, demonstrating good targeting ability, remarkable efficacy, and good safety.

[0232] Example 3: Evaluation of Bystander Killing Effect Study Objective: To measure the bystander killing activity of ADCs (CH-2-589, CH-2-593, CH-2-518 and LC302-2-4(4)) against MDA-MB-468 cells in a co-culture system of HER2-positive tumor cell line SK-BR-3 and HER2-negative tumor cell line MDA-MB-468, and to identify control ADCs with a structure similar to DS8201a. The bystander killing effect was compared with (LC1184 (8)).

[0233] Materials and equipment L15: Hyclone, SH30525.01; McCoy's 5A: Gibco, 16600-082; FBS: Gibco, 10099-141; Firefly luciferase reporter gene assay kit: Beyotime, RG006; 50ml centrifuge tube: Corning, 430829; 15ml centrifuge tube: Corning, 430791; 96-well plate: Corning, 3599; CO2 cell incubator: Th Thermo; Centrifuge: Thermos, L550; Microscope: Nikon, ISZ; Cell counter: Countstar, IC1000; Cytation3 plate reader: BioTek, Cytation3; HTX multimode microplate reader: BioTek, Synergy HTX; SK-BR-3: ATCC, HTB-30; MDA-MB-468-Luc-GFP: Self-produced using lentivirus infection.

[0234] Experimental method 1) Cell collection and counting.

[0235] 2) Cell density 1*10 5 Adjusted to cells / ml 3) A cell mixture was prepared using SKBR3:MDA-MB-468-Luc-GFP=4:1.5 ml.

[0236] Cell inoculation: 1*10 4 Cells / well, 100 μl / well Test samples: Prepared by diluting in cell culture medium to three final concentrations: 50, 10, and 1 nM.

[0237] 4) On the second day, the drug was administered, and the volume was increased to 100 μl / well.

[0238] 5) The samples were incubated in a 5% CO2 incubator at 37°C for 120 hours.

[0239] (1) Wash with PBS to remove dead cells, then detect with a Cytation3 plate reader. (2) Luciferase reporter gene assay kit manufactured by Biyuntian: Dissolved in 100 μl of lysis buffer in a 37°C incubator for 10 min. Centrifuged at 2000 rpm for 5 minutes. 100 μl of supernatant was taken. 100 μl of substrate was added. Chemiluminescence was detected by a microplate reader (gain: 200).

[0240] result: Two methods (GFP fluorescence counting and luciferase substrate luminescence) were used to detect bystander killing effects (Figures 4a, 4b, and 5).

[0241] During detection using these two methods, the conjugates (CH-2-593, CH-2-589, CH-2-518, and LC302-2-4(4)) all showed significant bystander lethality. CH-2-593 showed bystander lethality equivalent to or higher than LC1184(8).

[0242] Example 4: In vivo evaluation of conjugates 4.1 JIMT-1 Human Breast Cancer Xenograft Model 5x10 6 JIMT-1 human breast cancer cells (HER2 medium) in SCID Beige mode The injection was administered subcutaneously to the right side of the patient's armpit. Seven days later, the average volume of the tumor was 142 mm². 3 If it reaches the cancerous stage, The conjugates were distributed and administered intravenously with LC1184(8) and five other different conjugates at a dose of 5 mg / kg. Tumor volume was measured twice weekly using a calipas. LC1184(8) showed higher efficacy than LC1184(4), indicating that a higher DAR results in higher efficacy when the payload is the same. The efficacy of LC302-2-1(4) and LC302-2-4(4) was higher than that of LC1184(8) (Figure 6). The conjugates of the present invention achieve higher efficacy with a lower DAR.

[0243] 4.2 Capan-1 Human Pancreatic Cancer Xenograft Model (1) 5x10 6 Capan-1 human pancreatic cancer cells (HER2 low) were treated with BALB / c nude methane. A subcutaneous injection was administered to the right side of the patient's armpit to obtain a xenograft model. After 8 days, the average volume of the tumor was 178 mm². 3 If it reaches that point, LC1184(8) and five other different condyloma species are used in tumor-bearing mice. The drug was administered intravenously at a dose of 5 mg / kg. Tumor volume was measured twice a week using a caliper. LC1184(8) showed higher efficacy than LC1184(4). LC302-2-1(4) and LC302-2-4(4) showed efficacy equivalent to LC1184(8) (Figure 7).

[0244] 4.3 Capan-1 Human Pancreatic Cancer Xenograft Model (2) Study Objective: The objective of this study is to evaluate the in vivo antitumor efficacy of CH-2-589, CH-2-593, CH-2-518, and LC302-2-4(4) during treatment of a subcutaneous Capan-1 human pancreatic cancer xenograft model in female BALB / c nude mice.

[0245] Research Design: Cell culture: Capan-1 tumor cells (ATCC-HTB-79) were cultured in air at 37°C. IM filled with 20% fetal bovine serum and 1% antibiotic / antifungal agent in a 5% CO2 atmosphere. The cells were maintained in vitro as monolayer cultures using DM. Tumor cells were subjected to standard subculturing twice a week after treatment with trypsin-EDTA. Cells that had grown at the logarithmic growth stage were harvested, counted, and used for tumor inoculation.

[0246] Animals: Female BALB / c nude mice, 6-8 weeks old, weighing approximately 18-22g. The study requires a total of 48 mice (30 plus 60%), to be purchased from Shanghai Lingchang Laboratory Animal Co., LTD or other certified suppliers.

[0247] Tumor inoculation: Each mouse was inoculated with 0.2 mL of PBS containing Matrigel (1:1) and Capan-1 tumor cells (5 × 10⁻¹). 6 The drug was subcutaneously injected into the right armpit to promote tumor growth. The average volume of the tumor is approximately 150-200 mm². 3 If it reaches that point, the animals will be randomly divided into groups. The treatment was initiated, and efficacy studies were conducted. The dosage of the test substance and the number of animals in each group are shown in the experimental design table below.

[0248] Groups and processes [Table 12]

[0249] The experiment duration was adjusted based on tumor volume.

[0250] Animal Care: To allow animals to adapt to the laboratory environment, there is an adaptation period of approximately one week from the time the animals arrive in the laboratory until tumor inoculation. Mice were kept in a pathogen-free environment and in single, ventilated cages (three mice per cage). All cages, bedding, and water were disinfected before use. When working in the mouse room, researchers wore lab coats and latex or ethylene gloves. Each cage was labeled with a card indicating the number of animals, sex, breed, receipt date, treatment, study number, group number, and treatment start date. Cages containing food and water were changed twice a week. The target environmental and photoperiod conditions for the animal room were as follows: Temperature: 20~26°C Humidity: 40~70% Light-dark cycle: 12 hours of light, 12 hours of darkness Dietary materials: All animals were given free access to a certified, commercially available standard test animal diet. The maximum permissible concentration of contaminants in the diet was controlled by the manufacturer and regularly analyzed. The animals were given free access to autoclaved municipal tap water suitable for human consumption. The dietary materials were considered free from known contaminants that could affect tumor growth.

[0251] Grouping: Before initiating treatment, all animals were weighed and their tumor volume was measured. Since tumor volume can influence the effectiveness of any given treatment, mice were grouped using Excel-based randomization software, and stratified randomization was performed based on their tumor volume. This ensured that all groups were equivalent at baseline.

[0252] Observations: The protocols and any corrections or procedures related to the management and use of animals in this study were reviewed and approved by the Institutional Animal Care Committee (IACUC) of WuXi AppTec prior to the commencement of the study. During the study period, animal care and use were carried out in accordance with the regulations of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). After inoculation, the incidence and mortality rates of the animals were checked daily. During routine monitoring, the animals were checked for any effects of tumor growth and treatment on normal behavior, such as motility, feed and water consumption, weight gain / loss, dullness of eyes / hair, and any other abnormal effects. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[0253] Endpoints: The primary endpoint was to observe whether tumor growth could be delayed or whether the mice could be cured. Tumor size was measured twice weekly in two dimensions using a caliper, and volume was calculated using the formula: V = 0.5a × b 2 Calculated in mm 3 This is expressed as follows, where a and b are the longest and shortest diameters of the tumor, respectively. Next, the size of the tumor is used to calculate the TGI (%) and the relative tumor growth rate T / C (%). The TGI for each group is calculated using the formula TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the average tumor volume of the treatment group on a given day, T0 is the average tumor volume of the treatment group on day 1 of treatment, Vi is the tumor volume of the vehicle control group on the same day as Ti, and V0 is the average tumor volume of the vehicle group on day 1 of treatment.

[0254] The T / C (%) value for each group is T / C% = T RTV / C RTV It is calculated using the formula ×100%, (TRTV : Relative mean tumor volume (RTV) of the treatment group, C RTV :T RTV This is in comparison to the vehicle on the same day. The relative mean tumor volume (RTV) of each group is given by: RTV = V t The formula is calculated as / V0, where V0 is the tumor volume on day 1 of treatment, and V t This represents the tumor volume on a specified day.

[0255] end: 1) Weight loss: Animals showing a 20% weight loss in any given day should be humanely slaughtered or contacted to a veterinarian.

[0256] 2) Tumor load: Tumor load is 3,000 mm 3 It must not exceed 3,000. mm 3 Once this is reached, the individual animal is immediately slaughtered.

[0257] Ulceration: If ulceration of the tumor occurs, apply the following procedure. Animals with ulcerative tumors should be monitored at least three times per week, increasing the frequency up to once a day depending on clinical signs.

[0258] Ulcerated tumors without a scab should be cleansed with an appropriate wound cleansing solution (e.g., Novalsan). Antibiotic cream may only be applied to the ulcer / lesion if directed by a veterinarian.

[0259] The criteria for euthanasia include the presence of a disease that meets (one or more of the following criteria): If it does not heal within a week and no scab forms, If the diameter exceeds 5 mm, If it becomes hollow, If there are signs of infection (e.g., presence of pus) or bleeding, or if the animal shows signs of discomfort (e.g., excessive licking and biting of the affected area) or systemic signs of illness (lethargy, decreased activity, loss of appetite), then the animal should be taken into consideration. This includes cases showing decreased energy expenditure, a decline in physical condition, or weight loss. Contact your veterinarian to discuss any possible exceptions.

[0260] 3) Clinical signs: If an animal is found to be near death, it must be euthanized (certified by the IACUC with sufficient justification, as included in the protocol, and with warmed subcutaneous fluids and Diet placed beside the animal so that it can reach its food). Exclude if additional supportive care is provided, such as placing a gel feeding cup or placing the cage on a heating pad for supplemental heat. (Note: "Tyrant" means the animal has little chance of survival). Please contact your veterinarian with any questions regarding these endpoints.

[0261] Clinical cases of the disease may include the following: Shrink your body. Prolonged bed rest and lack of response to handling or other stimuli. Signs of severe organ or system failure. Rui is thin. Hypothermia. CNS deficiency spasms Respiration: Rapid breathing, labored breathing, coughing, rales. GI: Diarrhea lasting more than two days, jaundice.

[0262] Any animal exhibiting the above clinical problems will be humanely slaughtered with CO2.

[0263] In cases of unexpected death, an autopsy will not be performed.

[0264] For statistical analysis, independent sample t-tests were used to compare two groups. For comparisons between three or more groups, one-way analysis of variance (ANOVA) was performed. Multiple comparisons were performed after ANOVA if a significant F-statistic (ratio of treatment variance to error variance) was obtained. All data were analyzed using SPSS 17.0. A p-value of <0.05 was considered statistically significant.

[0265] result: All conjugates showed significant tumor-suppressing effects (Figures 8a and 8b).

[0266] NCI-N87 Human Gastric Cancer Xenograft Model Study Objective: The objective of this study is to evaluate the in vivo antitumor efficacy of CH-2-589, CH-2-593, CH-2-518, and LC302-2-4(4) during treatment of a subcutaneous NCI-N87 human gastric cancer xenograft model in female BALB / c nude mice.

[0267] Research Design: Cell culture: NCI-N87 tumor cells (ATCC, Manassas, VA, cat#CRL-5822) were cultured at 37°C in an atmosphere of 5% CO2 in air, in 10% fetal bovine serum. The cells were maintained in vitro as monolayer cultures in RPMI1640 medium packed with 1% antibiotic and antifungal agents. Tumor cells were subjected to conventional subculturing twice a week after trypsin-EDTA treatment. Cells that had grown at the logarithmic growth stage were harvested and counted for tumor inoculation.

[0268] Animals: Female BALB / c nude mice, 6-8 weeks old, weighing approximately 18-22g. The study requires a total of 36 mice (30 plus 20%) from Shanghai Lingchang Laboratory Animal Co., Ltd. or other certified. Purchase from a supplier that has already been used.

[0269] Tumor inoculation: Each mouse was inoculated with 0.2 mL of PBS containing Matrigel (1:1) and NCI-N87 tumor cells (10 × 10). 6 The drug is subcutaneously injected into the right armpit to promote tumor growth. The average volume of the tumor was approximately 150-200 mm². 3 If it reaches that point, the animals will be randomly divided into groups. The treatment was initiated, and efficacy studies were conducted. The dosage of the test substance and the number of animals in each group are shown in the experimental design table below.

[0270] Groups and processes [Table 13]

[0271] The experiment duration was adjusted based on tumor volume.

[0272] result: All conjugates showed significant tumor-suppressing effects (Figures 9a and 9b).

[0273] Effect Example 5: Stability of Conjugate in In Vitro Serum For the study of in vitro serum stability, the conjugates LC302-2-1(4), LC302-2-4(4), and LC1184(8) were inoculated into mixed human serum at 37°C. At 0, 24, 48, and 96 hours, the conjugates were captured with the antigen, deglycosylated with glycosidase, and dissociated with acid. The supernatant was collected, centrifuged, and detected by high-resolution LC-MS to determine the DAR.

[0274] LC302-2-1(4) and LC302-2-4(4) were highly stable after 96 hours of incubation in serum. No decrease in DAR was observed for LC302-2-1(4) and LC302-2-4(4) after 96 hours of incubation. However, the DAR of LC1184(8) decreased from 7.8 to 2.6 after 96 hours of incubation (Figure 10). Linker stability indicates improved therapeutic index as more payload is delivered to the target tumor and the release of off-target free payload is reduced. [Sequence List] SEQ ID NO: 1: Ab0001-LCCTL-HC LCDR1 RASQDVNTAVA SEQ ID NO: 2: Ab0001-LCCTL-HC LCDR2 SASFLYS SEQ ID NO: 3: Ab0001-LCCTL-HC LCDR3 QQHYTTPPT SEQ ID NO: 4: Ab0001-LCCTL-HCHCDR1 DTYIH SEQ ID NO: 5: Ab0001-LCCTL-HCHCDR2 RIYPTNGYTRYADSVKG SEQ ID NO: 6: Ab0001-LCCTL-HCHCDR3 WGGDGFYAMDY SEQ ID No. 7: Ab0001-LCCT L -HC light chain variable domain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV SEQ ID NO: 8: Ab0001-LCCT L -HC heavy chain variable domain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSSEQ ID No. 9: Ab0001-LCCT L -HC Light Chain Length: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG SEQ ID No. 10: Ab0001-LCCT L -HC Heavy Chain Length: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A compound of formula (I), 【Chemistry 1】 Here, opSu is 【Chemistry 2】 or 【Transformation 3】 And, R 0 is C 1 ~ 10 It is an alkyl group, n is any integer between 2 and 20. k1 and k2 are independent integers between 1 and 7. i is an integer between 1 and 20. j is an integer between 1 and 20. P1 and P2 are independent payloads with the structure shown below. 【Chemistry 4】 It is a compound.

2. Having the structure shown in formula (I-1), 【Transformation 5】 Here, P1, P2, R 0 The compound according to claim 1, wherein opSu, n, i, and j are as defined in claim 1.

3. R 0 is C 1 ~ 6 It is an alkyl group and / or, n is an integer between 2 and 5, and / or k1 and k2 are independently 1, 3, or 5, and / or i is an independent integer between 1 and 20, and / or The compound according to claim 1 or 2, wherein j is an integer from 1 to 20 independently.

4. Having the structure shown in formula (II), 【Transformation 6】 Here, A is an antibody or its antigen-binding fragment, and the antibody or its antigen-binding fragment is modified to form formula (I) 【Transformation 7】 (Gly) in the compound n Combine into parts, z is an integer between 1 and 20. opSu is 【Transformation 8】 or 【Chemistry 9】 And, R0 is a C1-10 alkyl group, n is any integer between 2 and 20. k1 and k2 are independent integers between 1 and 7. i is an integer between 1 and 20. j is an integer between 1 and 20. P1 and P2 are independent payloads with the structure shown below. 【Chemistry 10】 It is a conjugate.

5. A pharmaceutical composition comprising a preventive or therapeutically effective amount of the conjugate according to claim 4 and at least one pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, wherein the drug-to-antibody ratio (DAR) of the conjugate is an integer or non-integer between 1 and 8.

7. Use of the conjugate according to claim 4 or the pharmaceutical composition according to claim 5 or 6 in the manufacture of a drug for treating a disease, wherein the disease is a tumor or an autoimmune disease.

8. The tumor comprises HER2-positive tumor cells and further HER2-low-expressing tumor cells. The use according to claim 7, wherein the HER2 expression level of the HER2-low-expressing tumor cells is lower than that of the HER2-positive tumor cells.

9. The following structure 【Chemistry 11】 Compounds having or pharmaceutically acceptable salts, stereoisomers, solvates, tautomers, or isotopic compounds thereof.

10. A compound of formula (1), 【Chemistry 12】 Here, k is an integer from 1 to 7. P is a payload having the structure shown below. 【Chemistry 13】 It is a compound.

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