Linker compounds, ligand-drug conjugates, methods for preparation, and uses thereof.

Abstract

Novel linker compounds and ligand-drug conjugates, and more specifically, linker compounds comprising short polypeptides, ligand-drug conjugates, their use in the preparation of ligand-drug conjugates containing linkers, and their preparation and use are disclosed. Novel linker compounds improve the coupling efficiency of ligand-drug conjugates, as well as the homogeneity and overall stability of the conjugates, further improving the efficacy and safety of the ligand-drug conjugates.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to PCT / CN2023 / 093976 filed on 12 May 2023, PCT / CN2023 / 117368 filed on 7 September 2023, and PCT / CN2023 / 120684 filed on 22 September 2023. The entire contents of the aforementioned applications are incorporated herein by reference.

[0002] Technical field The present invention belongs to the field of medicinal chemistry and relates to novel linker compounds and ligand-drug conjugates, and more specifically, linker compounds comprising short polypeptides, and ligand-drug conjugates, the use thereof in the preparation of ligand-drug conjugates containing linkers, and the preparation and use thereof. [Background technology]

[0003] The basic molecule of an antibody-drug conjugate (ADC) includes an antibody, a linker, and a toxin molecule, in which the antibody is used to deliver the toxin molecule to the tumor site for enrichment, thereby killing tumor cells. Most conventional toxin molecules are highly active tubulin inhibitors or cytotoxic agents that directly target DNA, and these usually have relatively strong toxic side effects, which limits the applications of ADCs. The advantages of antibody-drug conjugates are that they improve the targeting of specific antibody and antigen binding, deliver the drug around the target cell, release the drug near the target cell, and effectively kill tumor cells by reducing toxic side effects. Camptothecin drugs show promising applications in ADCs.

[0004] Recently, Immunomedics developed a novel ADC drug, IMMU-132, using camptothecin SN38 as the warhead molecule, and this drug has shown good antitumor efficacy. Daiichi Sankyo also developed another ADC drug, DS-8201a, using camptothecin DXd as the warhead molecule, and this drug has also shown good antitumor efficacy. In existing ADC technology, camptothecin compounds are primarily linked to antibodies by improving existing linker technology. Generally, an ideal linker for an ADC must meet the following requirements: First, the small molecule must not separate from the antibody in plasma, and upon entry into cells, the linker must be destroyed under the correct conditions to rapidly release the active small molecule. Second, the linker must have good physicochemical properties so that it can link to the antibody and form a conjugate. And third, the linker must be easy to prepare to lay the foundation for large-scale ADC production. IMMU-132 uses a relatively unstable, pH-sensitive linker, while DS-8201a uses a tetrapeptide structure containing glycine-glycine-phenylalanine-glycine (GGFG), which has better stability. However, the toxins released by the aforementioned ADC drugs are SN38 and Dxd, both of which are Pgp substrates and still present challenges such as tumor multidrug resistance. In addition, the GGFG tetrapeptide structure has relatively low water solubility and is subjected to polymerization during coupling with some antibodies, leading to immune-related side effects in the body, such as interstitial lung disease. AstraZeneca has published a class of novel DNA topoisomerase 1 inhibitor antibody-drug conjugates (Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305. Clin Cancer Res 2022:OF1-OF16.), in which the linker toxin AZ'0133 showed superior efficacy in the body.However, toxins may have low metabolic efficiency, and while the linker's hydrophilicity can only be improved by introducing PEG units, thereby improving aggregation during coupling to some extent, the improvement in the overall stability of the antibody conjugate is very limited by the resortment method (Nat Biotechnol 2015, 33:733-5). In addition, Daiichi Sankyo's GGFG-Dxd-based ADC agents have the side effect of interstitial lung disease (ILD), which may be associated with nonspecific killing of immune-related cells, particularly at high drug load levels. Therefore, further improvement in efficacy and / or a better safety profile is likely to be achieved by more effectively increasing the hydrophilicity of the linker, in conjunction with the discovery and availability of camptothecin derivatives with somewhat different metabolic pathways that function as toxins.

[0005] The technical problem to be solved by this invention is the search for superior antitumor camptothecin compounds. Based on a comprehensive understanding of ADC drugs, the inventors designed a series of active antitumor camptothecin derivatives. It has been experimentally discovered that antitumor small molecule compounds exhibit greater antitumor activity in cell assays. In addition, linker modification improves the coupling efficiency of the antibody conjugate, as well as the homogeneity and overall stability of the conjugate, further improving the efficacy and safety of the antibody-drug conjugate. [Overview of the Initiative]

[0006] In one embodiment, the present invention relates to a compound of formula (I). [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, Q and L are as described herein.

[0007] In other embodiments, the present invention relates to the compound of formula (II). [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, Q, L', Z, and TA are as described herein.

[0008] In a further embodiment, the present invention relates to a ligand-drug conjugate of formula (III). [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, Q', L', Z, TA, LG, and n are as described herein.

[0009] In a further embodiment, the present invention relates to a pharmaceutical composition comprising a ligand-drug conjugate of formula (III) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0010] In a further aspect, the present invention relates to the use of a ligand-drug conjugate of formula (III), or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, as provided herein, in the preparation of a drug for treating a tumor in a subject.

[0011] In a further embodiment, the present invention relates to a ligand-drug conjugate of formula (III), or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, provided herein, for use in the treatment of tumors in a subject.

[0012] In a further embodiment, the present invention relates to a method for treating a tumor in a subject, comprising administering to the subject an effective amount of a ligand-drug conjugate of formula (III) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof. [Brief explanation of the drawing]

[0013] [Figure 1] The amino acid sequences discussed in this disclosure are listed below. [Figure 2] This shows the mean tumor volume in various groups of B-NDG mice treated with T-(II')-1, T-(II')-2, T-(II')-2(DAR4), or T-(II')-4 after being injected with tumor fragments derived from gastric cancer patients. PBS was used as a control. [Figure 3] This shows the mean tumor volume in various groups of B-NDG mice treated with Hep-G2 cells and either hIgG1-(II')-1, hIgG1-(II')-2, hIgG1-(II')-4, T-(II')-1, T-(II')-2, T-(II')-2(DAR4), or T-(II')-4. PBS was used as a control. [Figure 4] Figures A-C show the serum concentrations of all antibodies at 0 minutes, 15 minutes, 4 hours, 24 hours, 72 hours, 10 days, 14 days, and 21 days after administration of hIgG1-GGFG-Dxd (Figure 4A), T-GGFG-Dxd (Figure 4A), hIgG1-(II')-4 (Figure 4B), T-(II')-4 (Figure 4B), or trastuzumab analog (Figure 4C). [Figure 4D] This shows the serum concentrations of the free payload at 0 minutes, 15 minutes, 4 hours, 24 hours, and 72 hours after administration of hIgG1-GGFG-Dxd, T-GGFG-Dxd, hIgG1-(II')-4, T-(II')-4, or a trastuzumab analog. [Figure 5] Figures A-C show the ratio of free payload to total payload in the plasma of humans (Figure 5A), monkeys (cynomolgus monkeys) (Figure 5B), or SD rats (Figure 5C) at 0, 1, 2, 6, 8, 11, and 14 days after addition of T-(II')-1, T-(II')-2, or T-(II')-4 to the plasma. T-GGFG-Dxd was used as a positive control. [Figure 6]Figures A and B show the endocytosis activity of anti-TROP2 / EGFR bispecific antibodies and ADCs in A431 cells (Figure 6A) or NCI-H292 cells (Figure 6B). ISO-CPT2 (DAR8) was used as the isotype control. Sacituzumab govitecan and cetuximab were used as positive controls. [Figure 7] This shows the mean tumor volume in different groups of B-NDG mice that were injected with patient-derived breast cancer fragments and treated with PBS or ADC. [Figure 8] This shows the mean tumor volume in different groups of B-NDG mice injected with SKOV-3 cells and treated with PBS or ADC. [Figure 9] This shows the mean tumor volume in different groups of B-NDG mice injected with A431 cells and treated with PBS, antibody, or ADC. [Figure 10] Figures A-F show the mean tumor volumes in various groups of BALB / c nude mice treated with T-6F7-E-6C4-(II')-2(DAR8) after injection of tumor fragments derived from head and neck squamous cell carcinoma patients (Figure 10A), esophageal cancer patients (Figure 10B), colorectal cancer patients (Figures 10C and 10D), or gastric cancer patients (Figures 10E and 10F). Physiological saline was used as a control. [Figure 11] A and B show the concentrations of total antibody (Figure 11A) and CPT2 (Figure 11B) in the serum of B-NDG mice after administration of T-6F7-E-6C4-(II')-2(DAR4) or T-6F7-E-6C4-(II')-2(DAR8). [Figure 11] C to D show the concentrations of total antibody (Figure 11C) and CPT2 (Figure 11D) in tumor tissue after administration of T-6F7-E-6C4-(II')-2(DAR4) or T-6F7-E-6C4-(II')-2(DAR8) to B-NDG mice. [Figure 12]Figures A and B show the ratio of free CPT2 to total ADCs in the plasma of humans, monkeys (cynomolgus monkeys), or SD rats at 0, 1, 2, 6, 8, 11, and 14 days after the addition of T-6F7-E-6C4-(II')-2(DAR4) (Figure 12A) or T-6F7-E-6C4-(II')-2(DAR8) (Figure 12B) to plasma. PBS was used as a control. Detailed description of the invention

[0014] Herein, certain embodiments are referenced in detail, examples of which are shown in the attached detailed description. Although embodiments are listed, it will be understood that these are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may fall within the scope of the invention as defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in carrying out the invention. The invention is not limited to the methods and materials described herein. If one or more of the referenced documents and similar materials differ from or conflict with this application (including, but not limited to, the terms defined, the usage of terms, the techniques described, etc.), this application shall prevail.

[0015] For clarity, it is understood that certain features of the invention described in relation to separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may be provided separately or in any preferred partial combination.

[0016] definition Terms used herein but not defined have their usual meanings, and the meanings of such terms are independent in each of their respective appearances. Unless otherwise stated, the following definitions apply throughout this specification and the claims.

[0017] As used herein, the singular forms "a," "an," and "the" refer to multiple subjects unless the opposite meaning is explicitly stated.

[0018] As used herein, the terms “comprise” and “include” are intended to identify the presence of a feature, integer, component, or step described herein, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

[0019] As used herein, the term “approximately” means roughly, within a range, roughly, or around. When the term “approximately” is used in conjunction with a numerical range, the term modifies the range by extending its boundary to include the range before and after the given number. Generally, the term “approximately” is used herein to modify a number with a variation of 20%, typically 10%, more typically 5%, and even more typically 1% above and below a given number. In some cases, such a range may fall within experimental error, or within the types of standard methods used to measure and / or determine a given value or range.

[0020] The definitions of specific functional groups and chemical terms are described in detail below. For the purposes of this invention, chemical elements are identified based on the Periodic Table, CAS edition, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are defined as generally described herein. Furthermore, general principles of organic chemistry, as well as specific functional groups and reactivity, are referenced in *Organic Chemistry*, Thomas Sorrell, University Science Books, Sausalito, 1999, and *Smith and March*, *March's Advanced Organic Chemistry*, 5 thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, as described.

[0021] All ranges cited herein are inclusive, unless expressly stated to the contrary.

[0022] When a range of values is recited, each value and subrange within that range is intended to be included. For example, "C 1-6 " includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 are intended to be included.

[0023] In any component, or in any of the formulas (I)-(IV), or in any other formula depicting and describing the compounds of the present invention, if any variable appears more than once, its definition is independent of its definition in each other appearance. Also, combinations of substituents and / or variables are permitted only if such combinations result in stable compounds.

[0024] As used herein, the term "alkyl" refers to an acyclic linear or branched saturated hydrocarbon group that may be independently and optionally substituted (i.e., unsubstituted or substituted) with one or more substituents as described below, whether used as part of other terms or independently. i-j The term "alkyl" refers to an alkyl group having i to j carbon atoms. In certain embodiments, an alkyl group contains 1 to 12 carbon atoms. In certain embodiments, an alkyl group contains 1 to 11 carbon atoms. In certain embodiments, an alkyl group contains 1 to 11 carbon atoms, 1 to 10 carbon atoms, 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl and isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl, neopentyl, and the like. Alkyl groups can be optionally substituted with four or more substituents, in the case of alkyl groups of 1, 2, 3, or two or more carbon atoms, independently selected from the group consisting of alkoxy, acyloxy, amino, aryl, aryloxy, azide, cycloalkyl, cycloalkoxy, halo, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, silyl, cyano, =O, =S, and =NR' [wherein R' is H, alkyl, aryl, or heterocyclyl], as long as the valency is permissible. In certain embodiments, alkyl groups can be optionally substituted with halo, amino, hydroxy, methoxy, nitro, cyano, etc. Each substituent can be unsubstituted by itself, or, as long as the valency is permissible, each corresponding group can be substituted with one or more unsubstituted substituents as defined herein.

[0025] As used herein, the term "alkylidene," whether used as part of another term or independently, refers to a divalent substituent that is a monovalent alkyl having one valence-substituted hydrogen atom. The alkylidene group may be unsubstituted or substituted. An optionally substituted alkylidene is an alkylidene in which the alkyl is optionally substituted as described herein.

[0026] As used herein, the term “alkenyl” refers to a linear or branched hydrocarbon radical having at least one carbon-carbon double bond, which can be independently and optionally substituted (i.e., unsubstituted or substituted) with one or more substituents described in the specification, and which includes radicals having “cis” and “trans” configurations, or alternatively, “E” and “Z” configurations. In certain embodiments, the alkenyl group contains 2 to 12 carbon atoms. In certain embodiments, the alkenyl group contains 2 to 11 carbon atoms. In certain embodiments, the alkenyl group contains 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. In certain embodiments, the alkenyl group contains 2 carbon atoms. Non-limiting examples of alkenyl groups include etylene (or vinyl), propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, and 5-hexenyl. Optionally substituted alkenyls are alkenyls in which the alkyl group is optionally substituted as described herein.

[0027] As used herein, the term “alkenylidene,” whether used as part of another term or independently, refers to a divalent substituent that is a monovalent alkenyl having one hydrogen atom substituted with a valence. The alkenylidene group may be unsubstituted or substituted. Optionally substituted alkenylidene is an alkenylidene in which the alkyl group is optionally substituted as described herein.

[0028] As used herein, the term "cycloalkyl," whether used as part of other terms or independently, refers to non-aromatic saturated monocyclic and polycyclic ring systems in which all ring atoms are carbon and which contain at least three ring-forming carbon atoms. In certain embodiments, cycloalkyls may contain 3 to 12 ring-forming carbon atoms, 3 to 10 ring-forming carbon atoms, 3 to 9 ring-forming carbon atoms, 3 to 8 ring-forming carbon atoms, 3 to 7 ring-forming carbon atoms, 3 to 6 ring-forming carbon atoms, 3 to 5 ring-forming carbon atoms, 4 to 12 ring-forming carbon atoms, 4 to 10 ring-forming carbon atoms, 4 to 9 ring-forming carbon atoms, 4 to 8 ring-forming carbon atoms, 4 to 7 ring-forming carbon atoms, 4 to 6 ring-forming carbon atoms, and 4 to 5 ring-forming carbon atoms. In particular, cycloalkyls may contain 3 to 10 ring-forming carbon atoms (i.e., C 3-10It may contain cycloalkyl groups. In particular, the cycloalkyl group may be monocyclic or bicyclic. The bicyclic cycloalkyl group may be of the bicyclo[pq0]alkyl type, where p and q are independently 1, 2, 3, 4, 5, 6, or 7, and the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, a cross-linked cycloalkyl structure, such as bicyclo[pqr]alkyl, can be given as an example of a bicyclic cycloalkyl group, where r is 1, 2, or 3, and p and q are independently 1, 2, 3, 4, 5, or 6, and the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. The cycloalkyl group can be a spirocyclic group, for example, a spiro[pq]alkyl group, where p and q are independently 2, 3, 4, 5, 6, or 7, and the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and dekalinyl. Cycloalkyl groups can be optionally substituted (i.e., unsubstituted or substituted) with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkyl, alkoxy, acyloxy, amino, aryl, aryloxy, azide, cycloalkyl, cycloalkoxy, halo, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, silyl, cyano, =O, =S, =NR' [wherein R' is H, alkyl, aryl, or heterocyclyl]. Each substituent can be unsubstituted or substituted with each corresponding group with an unsubstituted substituent(s) as defined herein.

[0029] As used herein, the term “cycloalkenyl,” whether used as part of other terms or independently, refers to non-aromatic unsaturated monocyclic and polycyclic ring systems having at least one carbon-carbon double bond, containing at least three ring-forming carbon atoms, and all ring atoms being carbon. Cycloalkenyls can be optionally substituted (i.e., unsubstituted or substituted) independently with one or more substituents described herein. In certain embodiments, cycloalkenyls may contain 3 to 12 ring-forming carbon atoms, 3 to 10 ring-forming carbon atoms, 3 to 9 ring-forming carbon atoms, 3 to 8 ring-forming carbon atoms, 3 to 7 ring-forming carbon atoms, 3 to 6 ring-forming carbon atoms, 3 to 5 ring-forming carbon atoms, 4 to 12 ring-forming carbon atoms, 4 to 10 ring-forming carbon atoms, 4 to 9 ring-forming carbon atoms, 4 to 8 ring-forming carbon atoms, 4 to 7 ring-forming carbon atoms, 4 to 6 ring-forming carbon atoms, and 4 to 5 ring-forming carbon atoms. In particular, the cycloalkenyl group consists of 5-6 ring-forming carbon atoms (i.e., C 5-6 It may contain a cycloalkenyl. In particular, the cycloalkenyl group may be monocyclic or bicyclic. In certain embodiments, the cycloalkenyl may have 1, 2, 3, 4, 5, 6 or more double bonds. In certain embodiments, the cycloalkenyl may have 1, 2, or 3 double bonds. Non-limiting examples of the cycloalkenyl group include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, and cycloheptadienyl. An optionally substituted cycloalkenyl is a cycloalkenyl in which the alkenyl is optionally substituted as described herein.

[0030] As used herein, the term "aryl," whether used as part of another term or independently, refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring having at least one aromatic ring. Aryl groups can be 6- to 12-membered, e.g., 8- to 12-membered, 6- to 10-membered, or 6-membered. All atoms in an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, and indenyl. The aryl group can be optionally substituted (i.e., unsubstituted or substituted) with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkyl, alkoxy, acyloxy, amino, aryl, aryloxy, azide, cycloalkyl, cycloalkoxy, halo, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, silyl, and cyano. Each substituent can be unsubstituted by itself, or substituted with each corresponding group with an unsubstituted substituent(s) as defined herein.

[0031] As used herein, the term "alkoxy" means, whether used as part of another term or independently, the formula -OR [wherein R is an alkyl group, in particular C]. 1-12 Alkyl, C 1-10 Alkyl, C 1-6 This refers to chemical substituents such as alkyl groups. The alkoxy group may be unsubstituted or substituted. An optionally substituted alkoxy is an alkoxy group in which the alkyl group is optionally substituted as defined herein.

[0032] As used herein, the term “heteroalkyl” means an alkyl group (e.g., an alkyl group as defined herein) that is interrupted once or more times by one or two heteroatoms, whether as part of another term or independently. Each heteroatom is independently O, N, or S. No heteroalkyl group contains two consecutive oxygen atoms. Heteroalkyl groups may be unsubstituted or substituted (e.g., optionally substituted heteroalkyl groups). If a heteroalkyl group is substituted and the substituent is bonded to a heteroatom, the substituent is selected according to the properties and valency of the heteroatom. Thus, the substituent bonded to the heteroatom can be =O, -N(R) as long as the valency is acceptable. N2 )2, -SO2OR N3 , -SO2R N2 -SOR N3 ,-COOR N3 A selection from the group consisting of an N protecting group, alkyl, aryl, cycloalkyl, heterocyclyl, or cyano, in the formula, each R N2 Each R is independently H, alkyl, cycloalkyl, aryl, or heterocyclyl, and each R N3 These substituents are independently alkyl, cycloalkyl, aryl, or heterocyclyl. Each of these substituents may be unsubstituted by itself, or substituted with each corresponding group with an unsubstituted substituent(s) as defined herein. If the heteroalkyl is substituted and the substituent is bonded to a carbon, the substituent is selected from those described for alkyl, except that the substituent on the carbon atom bonded to the heteroatom is not Cl, Br, or I. In certain embodiments, the carbon atom is found at the terminus of the heteroalkyl. In certain embodiments, the heteroalkyl is PEG.

[0033] As used herein, the term "heteroaryl," whether used as part of another term or independently, refers to a monocyclic or fused or bridging bicyclic, tricyclic, or tetracyclic cyclic system, the system containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one of the rings being an aromatic ring. Heteroaryl groups can have 5 to 12 members, e.g., 8 to 12 members, 5 to 10 members, or 5 to 6 members. Unless otherwise specified, heteroaryl groups have 1 to 16 carbon atoms. Certain heteroaryl groups may have up to 9 carbon atoms. Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, quinazolinyl, quinolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl (e.g., 1H-1,2,3-triazolyl), tetrazolyl, dihydroindolyl, tetrahydroquinolyl, and tetrahydroisoquinolyl. The terms bicyclic, tricyclic, and tetracyclic heteroaryl groups include at least one ring and at least one aromatic ring having at least one heteroatom as described above. For example, a ring having at least one heteroatom can be fused to one, two, or three carbocyclic rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or other monocyclic or heterocyclic rings. Non-limiting examples of fused heteroaryl groups include 1,2,3,5,8,8a-hexahydroindolidine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, and 2,3-dihydrobenzothiophene.Heteroaryl groups include alkyl, alkoxy, acyloxy, aryloxy, amino, arylalkoxy, cycloalkyl, cycloalkoxy, halogen, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxyl, nitro, thiol, cyano, =O, -NR2 [wherein each R independently is hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl], and -COOR. A [In the formula, R A [is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl], and -CON(R B )2[where each R B Each of the substituents can be independently and optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl. Each substituent can be unsubstituted by itself, or substituted with each corresponding group with an unsubstituted substituent(s) as defined herein.

[0034] As used herein, the term “heterocyclyl” means, unless otherwise specified, a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having condensed or bridged 4, 5, 6, 7, or 8-membered rings, whether used as part of other terms or independently, and the ring system contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The heterocyclyl group can be 3 to 12-membered, for example, 3 to 10-membered, 4 to 12-membered, 4 to 10-membered, 5 to 12-membered, 5 to 10-membered, or 5 to 8-membered. Heterocyclyls can be aromatic or non-aromatic. Aromatic heterocyclyls are heteroaryls as described herein. Non-aromatic 5-membered heterocyclyls have 0 or 1 double bonds, non-aromatic 6- and 7-membered heterocyclyls have 0 to 2 double bonds, and non-aromatic 8-membered heterocyclyl groups have 0 to 2 double bonds and / or 0 or 1 carbon-carbon triple bond. Heterocyclyl groups have 1 to 16 carbon atoms unless otherwise specified. Certain heterocyclyl groups can have up to 9 carbon atoms. Examples of non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridadinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyranyl, dihydropyranyl, and dithiazolyl. The term "heterocyclyl" also refers to heterocyclic compounds having a bridging polycyclic structure in which one or more carbons and / or heteroatoms bridging two non-adjacent members of a monocyclic ring, such as quinuclidine, tropane, or diaza-bicyclo[2.2.2]octane. The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused with one, two, or three carbocyclic rings, such as a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another heterocyclic ring.Non-limiting examples of condensed heterocyclyls include 1,2,3,5,8,8a-hexahydroindidine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, and 2,3-dihydrobenzothiophene. The heterocyclyl group may be unsubstituted, alkyl, alkoxy, acyloxy, aryloxy, amino, arylalkoxy, cycloalkyl, cycloalkoxy, halogen, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxyl, nitro, thiol, cyano, =O, =S, -NR2 [wherein each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl], -COOR. A [In the formula, R A [is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl], and -CON(R B )2[where each R B [The substituents can be independently and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl.]

[0035] As used herein, the term “heterocyclylalkyl” refers to an alkyl group substituted with a heterocyclyl group, whether used as part of another term or independently. Heterocyclylalkyls may be unsubstituted or substituted. In the case of an optionally substituted heterocyclil, the heterocyclyl and alkyl portions are optionally substituted as described for heterocyclil and alkyl, respectively.

[0036] As used herein, the terms "halogen" or "halo" refer to fluorides, chlorides, bromides, and iodides, in particular fluorides and chlorides, and more specifically fluorides.

[0037] As used herein, the term “heteroatom” means nitrogen (N), oxygen (O), and sulfur (S), and unless otherwise specified, may include oxidized forms of nitrogen and sulfur, as well as any quaternized form of basic nitrogen.

[0038] As used herein, the term “substituted” when referring to a chemical group means that the chemical group has one or more hydrogen atoms that have been removed and replaced by substituents. As used herein, the term “substituent” has the common meaning known in the art and refers to a chemical part that is covalently bonded to or, where appropriate, condensed to the parent group. It should be understood that substitutions at a given atom are limited by their valence. Examples of substituents include, but are not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic groups, and aliphatic groups. It is understood that substituents can be further substituted.

[0039] Where a portion is described as being "optionally substituted" in formulas (I) to (IV), or in any embodiment thereof, formula (I) or its embodiment means that it encompasses both compounds in which the portion is substituted with one or more substituents described, and compounds in which the portion does not contain one or more substituents described (i.e., the portion is unsubstituted).

[0040] When used in this specification, the wavy line "

[0041] [ka]

[0042] The symbol '' indicates a connection point between one part and another part.

[0043] The compounds of formulas (I) to (IV), or any other formula illustrating and describing the compounds of the present invention, may have one or more chiral centers. The present invention encompasses all stereoisomers of the compounds of formulas (I) to (IV), or any other formula illustrating and describing the compounds of the present invention. The chiral centers present in the compounds of formulas (I) to (IV), or any other formula illustrating and describing the compounds of the present invention, may all independently have either (R) or (S) configurations. When the bonds to the chiral carbons are depicted as straight lines in the structural formulas of the present invention, or when the compound name is written without a (R) or (S) chiral designation for the chiral carbons, it is understood that both the (R) and (S) configurations of each chiral carbon, and therefore each enantiomer or diastereomer and mixtures thereof, are encompassed in the formula or name. The production of specific stereoisomers or mixtures thereof can be identified in the examples in which such stereoisomers or mixtures thereof are obtained, but this does not in any way limit the inclusion of stereoisomers and mixtures thereof within the scope of the present invention.

[0044] The present invention includes all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers, e.g., mixtures of enantiomers and / or diastereomers in any proportion. Thus, enantiomers are subject to the present invention in the form of a racemic mixture, as both enantiomerically pure forms, levorotatory and dextrorotatory anticellars, and as mixtures of two enantiomers in any proportion. In the case of cis / trans isomerism, the present invention includes both cis and trans forms, as well as mixtures of these forms in any proportion. Preparation of individual stereoisomers can be carried out, as necessary, by separation of mixtures by conventional methods such as chromatography or crystallization, by the use of stereochemically homogeneous starting materials for synthesis, or by stereoselective synthesis. Optionally, derivatization can also be carried out before separation of stereoisomers. Separation of mixtures of stereoisomers can be carried out as an intermediate step in the synthesis of the compound of formula I, or on the final racemic product. Absolute stereochemistry can be determined, if necessary, by X-ray crystallography of crystalline products or intermediates derivatized with reagents containing stereocenters of known configurations. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0045] Unless otherwise specified, the structures described herein include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the atomic mass or mass number that is dominant in nature. These compounds are called “isotope variants.” The present invention is intended to include all pharmaceutically acceptable isotopic variants of the compounds of formulas (I) to (IV) or any other compounds of formulas that describe and explain the compounds of the present invention. Examples of isotopes suitable for inclusion in the compounds of the present invention include: 2 H (i.e., D) and 3 Hydrogen isotopes such as H, 11 C, 13 C, and 14 Isotopes of carbon such as C, 36 Chlorine isotopes such as Cl, 18Fluorine isotopes such as F, 123 I, and 125 Iodine isotopes such as I 13 N and 15 Nitrogen isotopes such as N, 15 O, 17 O, and 18 Oxygen isotopes such as O, 32 Phosphorus isotopes such as P, and 35 Examples of sulfur isotopes include, but are not limited to, S. Specific isotopic variants of the compounds of formulas (I) to (IV), or any other formulas that describe and explain the compounds of the present invention, such as those incorporating radioactive isotopes, may be useful in tissue distribution studies of drugs and / or substrates. In particular, hydrogen is replaced with deuterium ( 2 Compounds having the described structures, differing only in that they can be replaced with heavier isotopes, such as by substitution with H or D, may be useful in several situations because they may offer certain therapeutic benefits, such as improved metabolic stability, extended in vivo half-life, or reduced dose requirements. Compounds of formulas (I) to (IV), or any other isotopic variants of the compounds of the present invention, can generally be prepared by prior art known to those skilled in the art, or by processes similar to those described in the accompanying examples and synthesis, using appropriate isotope-labeled reagents instead of previously used unlabeled reagents.

[0046] As used herein, the term “deuterium substitution” means the substitution of one or more hydrogen atoms of a compound or group with deuterium. When a compound or group is deuterated, one, two, three, or more hydrogen atoms of the compound or group may be substituted with deuterium until all of the hydrogen atoms of the compound or group are replaced with deuterium, at which point the compound or group can be said to be “fully deuterated.”

[0047] In some embodiments, the isotopic abundance of deuterium at the deuterium substitution site is greater than the isotopic abundance of natural deuterium (0.015%), preferably greater than 50%, more preferably greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100%.

[0048] In some cases, such as when the expressions "hydrogen" and "deuterium" appear as parallel alternative expressions, or when the expression "hydrogen" is replaced by "deuterium," the term "hydrogen" refers to the hydrogen isotope " 1 The term "deuterium" represents the hydrogen isotope " 2 This represents "H". Alternatively, it can be understood that at this position in a compound, hydrogen, which is present at other positions in the natural abundance of various isotopes, is replaced by a state in which deuterium is present in greater quantities than the natural deuterium isotope (for example, the amount of deuterium is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100%).

[0049] The compounds provided herein are described with reference to both general formulas and specific compounds. Furthermore, all compounds of the present invention may exist in numerous different forms or derivatives within the scope of the invention. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites.

[0050] As used herein, the term “pharmaceutically acceptable salt” includes, unless otherwise specified, salts that retain the biological efficacy of the free acid / base form of a particular compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts may include salts formed with inorganic bases or acids and organic bases or acids. If a compound of the present invention contains one or more acidic or basic groups, the present invention also includes corresponding pharmaceutically acceptable salts thereof. Thus, compounds of the present invention containing acidic groups such as carboxyl groups can exist in the form of salts and can be used according to the present invention, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts, or ammonium salts. More non-limiting examples of these salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts can be readily obtained, for example, by reacting a compound having an acidic group with a suitable base, such as lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide. Other base salts of the compounds of the present invention include, but are not limited to, copper(I), copper(II), iron(II), iron(III), manganese(II), and zinc salts. Compounds of the present invention containing one or more basic groups, such as protonable groups, can exist in the form of salts and can be used according to the present invention in the form of addition salts with inorganic or organic acids.Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, and other acids known to those skilled in the art. The salts formed include, in particular, hydrochlorides, chlorides, hydrobroms, bromides, iodides, sulfates, phosphates, methanesulfons (mesylates), tosylates, carbonates, bicarbonates, formates, acetates, sulfoacetates, triflates, oxalates, malons, maleates, succinates, tartrates, malates, emponates, mandelates, fumarates, lactates, citrates, glutarates, stearates, aspartates, and glutamates. The stoichiometry of the salts formed from the compounds of the present invention may further be an integer multiple of 1 or a non-integer multiple.

[0051] The compounds of the present invention containing a basic nitrogen-containing group include, for example, methyl, ethyl, isopropyl, and C such as tert-butyl chloride, bromide, and iodide. 1-4 Alkyl halides, such as dimethyl, diethyl, and diamyl sulfates, are diC 1-4 Alkyl sulfates, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides, etc. 10-18 Alkyl halides, and aryl C compounds such as benzyl chloride and phenethyl bromide. 1-4 Quaternization can be performed using reagents such as alkyl halides.

[0052] When the compounds of the present invention contain both acidic and basic groups in their molecules, the present invention also includes internal salts or betaines (amphoteric ions) in addition to the salt forms described above. Each salt can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the compounds of the present invention that are unsuitable for direct use in pharmaceuticals due to their poor physiological compatibility, but can be used, for example, as intermediates in chemical reactions or in the preparation of pharmaceutically acceptable salts. For a more appropriate review of salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0053] Compounds of formulas (I) to (IV), or any other formulas illustrating and describing the compounds of the present invention and their pharmaceutically acceptable salts, may exist in non-solvated and solvated forms. As used herein, the term “solvate” refers to a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules. For example, if the solvent is water, the term “hydrate” is used.

[0054] The pharmaceutically acceptable solvates according to the present invention include those in which the crystallization solvent is isotope-substituted, such as D2O, d6-acetone, and d6-DMSO.

[0055] One method of carrying out the present invention is to administer a compound of formulas (I) to (IV), or any other compound illustrating and describing the compound of the present invention, in the form of a prodrug. Therefore, certain derivatives of compounds of formulas (I) to (IV), or any other compound illustrating and describing the compound of the present invention, which may themselves have little or no pharmacological activity, can be converted into a compound of formulas (I) to (IV), or any other compound illustrating and describing the compound of the present invention, having desired activity, when administered into or onto the body, by hydrolytic cleavage, for example, facilitated by ester or peptidase enzymes. Such derivatives are called “prodrugs.” Further information regarding the use of prodrugs can be found, for example, in T. Higuchi and W. Stella, “Pro-drugs as Novel Delivery Systems”, Vol. 14, ACS Symposium Series, and E.B. Roche (Ed.), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987, American Pharmaceutical Association. You can also refer to Nature Reviews / Drug Discovery, 2008, 7,355, and Current Opinion in Drug Discovery and Development, 2007, 10,550.

[0056] The prodrugs in this invention are, for example, as described in H. Bundgaard, “Design of Prodrugs”, Elsevier, 1985, and YM Choi-Sledeski and C.G. Wermuth, “Designing Prodrugs and Bioprecursors”, Practice of Medicinal Chemistry, 4. thAs described in Edition, Chapter 28, 657-696, Elsevier, 2015, the compounds of formulas (I) to (IV), or any other formulas illustrating and describing the compounds of the present invention, can be prepared by replacing a suitable functional group with a specific part known to those skilled in the art as a “pro part.” Therefore, the prodrugs in the present invention include, but are not limited to, (a) ester or amide derivatives of carboxylic acids in compounds of formulas (I) to (IV), or any other formulas illustrating and describing the compounds of the present invention, if present; (b) amide, imine, carbamate, or amine derivatives of an amino group in a compound of formula (I); (c) oxime or imine derivatives of a carbonyl group in compounds of formulas (I) to (IV), or any other formulas illustrating and describing the compounds of the present invention, if present; or (d) methyl, primary alcohol, or aldehyde groups in compounds of formulas (I) to (IV), or any other formulas illustrating and describing the compounds of the present invention, that are oxidizable to carboxylic acids by metabolism.

[0057] Binder compounds As used herein, the term “binding compound” refers to a compound that, for example, by a coupling reaction, connects a ligand compound to a therapeutic compound, thereby linking the ligand and the therapeutic compound together and forming a ligand-drug conjugate.

[0058] In the first aspect, the present invention relates to a compound of formula (I). [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, Q represents a junction that can be coupled to a ligand by a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds. L represents a linker portion to which Q can be connected to the therapeutic agent, having the following structure: [ka] Here, L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue having a side-chain carboxyl group, such as a glutamic acid residue or an aspartic acid residue, where "-COOH" indicates the carboxyl group of the C-terminal amino acid residue of the polypeptide residue. L2 is absent, or is a monodentate, bidentate, or tricate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of polypeptide residue L1, and L2 is -NHC(R L2a )(R L2b )(R L2c ) has the structure, and here R L2a , R L2b , and R L2c These are H and -(CH2O)(CH2CH2O), respectively. m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d Independently selected from the group consisting of R L2d C is optionally substituted with H or 1 to 6 hydroxyl groups. 1-6 It is an alkyl group, where each m is an independent integer from 0 to 10, preferably from 0 to 4, for example, 0, 1, 2, 3, or 4, and particularly preferably m is 0, and each p is an independent integer from 1 to 4, for example, 1, 2, 3, or 4.

[0059] [ka]

[0060] This indicates the N-terminal side of the polypeptide residue covalently bonded to the junction Q.

[0061] The present invention also relates to the use of a ligand-drug conjugate, for example, the compound of formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the preparation of a ligand-drug conjugate as described herein.

[0062] The present invention also relates to a compound of formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, for use in the preparation of a ligand-drug conjugate, such as the ligand-drug conjugates described herein.

[0063] In some embodiments, the linking moiety Q has the following structure:

Chemical formula

[0064]

Chemical formula

[0065] indicates the site covalently bonded to the linker moiety L.

[0066] In some preferred embodiments, the functional group Q a is selected from the group consisting of

Chemical formula

[0067] It should be understood that when the group "Ar" is present inside the molecular structure of the compound, it can also refer to a divalent substituent.

[0068] In some preferred embodiments, Ar is selected from the group consisting of optionally substituted cyclopentadienyl, optionally substituted phenyl, and optionally substituted 5- to 6-membered heteroaryl.

[0069] In some preferred embodiments, the joining moiety Q is selected from the group consisting of

Chemical formula

[0070] [ka]

[0071] During the ceremony, each AA 1 AA 2 AA 3 ,...AA p These are independently and optionally substituted amino acid residues, AA 1 AA 2 AA 3 ,...AA p At least one of these is an amino acid residue having a side chain carboxyl group, preferably Glu or Asp. p is an integer between 3 and 8, preferably between 3 and 5, for example, 3, 4, or 5. "NH-" indicates the N-terminal side of a polypeptide residue. "-COOH" indicates the C-terminal side of a polypeptide residue.

[0072] As used herein, the term “amino acid” refers to an organic compound containing a basic amino group and an acidic carboxyl group. Examples of amino acids include naturally occurring and synthetic α, β, γ, or δ amino acids, provided that the amino group and side-chain reactive group are adequately protected. Naturally occurring amino acids include, but are not limited to, those arising in natural proteins, namely glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. Other examples of amino acids include unnatural amino acids, such as amino acid variants and derivatives. Examples of synthetic amino acids include citrulline, norvaline, isoleucine, ortholeucine, β-alanine, ornithine, α-methyl amino acids, D-amino acids, histidine-like amino acids, N-alkyl amino acids, and amino acids with an excess of methylene groups in their side chains.

[0073] Amino acids can exist in the form of a single enantiomer or in the form of a racemic or enantiomixture. Preferred amino acids are naturally occurring amino acids with an L structure.

[0074] In this specification, amino acids may be represented by commonly known three-letter symbols or by single-letter symbols as recommended by IUPAC.

[0075] The term "amino acid residue" refers to the corresponding residue obtained when a hydrogen atom is removed from the N-terminal amine group and / or terminal carboxyl group of an amino acid.

[0076] When referring to substituted amino acids or substituted amino acid residues, this term refers to reactive groups on the side chain of an amino acid or amino acid residue, such as carboxyl, mercapto, and amine groups, which are chemical groups, for example, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6Cycloalkyl, C 3-10 Heterocyclyl, C 6-10 Aryl, and C 5-10 This means that it can be further substituted with heteroaryl compounds.

[0077] In some preferred embodiments, each AA 1 AA 2 AA 3 ,...AA p Each is independently and optionally substituted with an amino acid residue selected from the group consisting of Glu, Asp, Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, Gly, and N-alkyl amino acids, and at least one of AA1, AA2, AA3, ... AAp is Glu or Asp. In some preferred embodiments, the unsubstituted or substituted Lys is, for example, "-Lys(NR Lys1 R Lys2 It can contain amino acid residues represented by ) and in the formula, R Lys1 and R Lys2 Each of these is independently either H or C1-6 alkyl; that is, if substituted, the hydrogen of the amine group on the side chain of the lysine residue is substituted with at least one C1-6 alkyl.

[0078] In some preferred embodiments, AA 1 This is an amino acid residue having a side chain carboxyl group, preferably Glu or Asp, and each AA 2 AA 3 ,...AA p These are independently and optionally substituted with amino acid residues selected from the group consisting of Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, and Gly. In some preferred embodiments, unsubstituted or substituted Lys are, for example, "-Lys(NR Lys1 R Lys2 It can contain amino acid residues represented by ) and in the formula, R Lys1 and RLys2 Each of these is independently H or C 1-6 If alkyl, i.e., substituted, then at least one hydrogen atom of the amine group on the side chain of the lysine residue is C 1-6 It is substituted with alkyl.

[0079] In some preferred embodiments, polypeptide residue L1 is NH -Glu-Phe-Lys(NR Lys1 R Lys2 )- COOH , NH -Glu-Val-Lys(NR Lys1 R Lys2 )- COOH , NH -Glu-Ala-Ala-Ala- COOH , NH -Glu-Ala-Ala- COOH , NH -Glu-Val-Ala- COOH , NH -Glu-Val-Cit- COOH , NH -Glu-Gly-Gly-Phe-Gly- COOH , NH -Asp-Phe-Lys- COOH , NH -Asp-Ala-Ala-Ala- COOH , NH -Asp-Val-Ala- COOH , NH -Asp-Val-Cit- COOH , NH -Asp-Gly-Gly-Phe-Gly- COOH , and NH -Asp-Val-Lys(NR Lys1 R Lys2 )- COOH Selected from the group consisting of, in the formula, R Lys1 and R Lys2 Each of these is independently H or C 1-6 Alkyl, preferably C 1-3 Alkyl, more preferably C3 alkyl, for example, propyl or isopropyl. In some preferred embodiments, R Lys1 and RLys2 They are the same, for example, C 1-3 Alkyl, preferably C3 alkyl, for example, propyl or isopropyl.

[0080] In some preferred embodiments, the hydrophilic group L2 is -NHC(R L2a )(R L2b )(R L2c ) has the structure, in the formula R L2a , R L2b , and R L2c These are H and -(CH2O)(CH2CH2O), respectively. m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d Independently selected from the group consisting of R L2a , R L2b , and R L2c Of these, up to two are H at the same time.

[0081] In some embodiments, in the hydrophilic group L2, R L2d However, C is substituted with 1 to 6 hydroxyl groups. 1-6 If they are alkyl groups, these hydroxyl groups can be substituted with the same or different carbon atoms, provided that valence bonding is permitted.

[0082] In some preferred embodiments, in the hydrophilic group L2, R L2d C is substituted with 3 to 5 hydroxyl groups. 4-6 It is an alkyl group, where at most one hydroxyl group is substituted on each carbon atom.

[0083] In some preferred embodiments, in the hydrophilic group L2, R L2d The following group is selected: [ka] In some preferred embodiments, the hydrophilic group L2 is -NHC(R L2a )(RL2b )(R L2c ) has the structure, in the formula R L2a , R L2b , and R L2c These are H, -CH2O(CH2)2C(O)OH, and -CH2O(CH2)2C(O)NHR, respectively. L2d Independently selected from the group consisting of R L2a , R L2b , and R L2c Of these, up to two are H and R at the same time. L2d C is substituted with 3 to 5 hydroxyl groups. 4-6 It is alkyl.

[0084] In some preferred embodiments, the hydrophilic group L2 is selected from the group consisting of: [ka] The asterisk (*) indicates a site covalently bound to polypeptide residue L1.

[0085] In some preferred embodiments, the hydrophilic group L2 can be a group having a specific stereochemistry, such as, but is not limited to, the following: [ka]

[0086] In some preferred embodiments, the compound of formula (I) is a compound selected from the group consisting of: [ka] [ka] [ka] [ka] [ka]

[0087] Linker - therapeutic compound As used herein, the term "linker-therapeutic compound" refers to a compound formed by binding the binder compound of the present invention, that is, by binding the compound of formula (I) to a therapeutic agent.

[0088] In a second aspect, the present invention relates to a compound of formula (II). [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, TA indicates a therapeutic agent. Z is either absent or indicates an auxiliary portion that binds L' to the therapeutic agent TA via a bond selected from the group consisting of disulfide, thioether, thioester, hydrazone, ester, ether, carbamate, and amide bonds. Q represents a junction that can be coupled to a ligand by a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds. L' represents a linker portion that binds Q to the therapeutic agent TA and has the following structure: [ka] In the formula, L'1 is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue having a side-chain carboxyl group, for example, a glutamic acid residue or an aspartic acid residue. L2 is either absent or is a monodentate, bidentate, or tridentate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of polypeptide residue L1, and L2 is -NHC(R L2a )(R L2b )(R L2c ) has the structure, in the formula R L2a , R L2b , and R L2c These are H and -(CH2O), respectively. m (CH2) pC(O)OH and -(CH2O) m (CH2) p C(O)NHR L2d Independently selected from the group consisting of R L2d C is optionally substituted with 1 to 6 hydroxyl groups. 1-6 It is an alkyl group, where each m is an integer between 1 and 6, and each p is an integer between 1 and 4.

[0089] [ka]

[0090] This indicates the N-terminal side of the polypeptide residue covalently bonded to the junction Q.

[0091] [ka]

[0092] This indicates the C-terminal side of the polypeptide residue covalently bonded to the therapeutic agent TA.

[0093] In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, meitansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as its analogues). Useful classes of cytotoxic, cell proliferation inhibitor, or immunomodulatory agents include, for example, antitubulins, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0094] In some embodiments, the therapeutic agent may be, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent, an immunotherapeutic agent, etc.), an antiviral agent, or an antibacterial agent. In some embodiments, the conjugateable therapeutic agent may be, but is not limited to, selected from MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0095] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of drastatin-10), or a derivative thereof. Auristatin can be, for example, an ester formed of auristatin E with a keto acid. For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Publication No. 2003-0083263, International Patent Publication No. WO 04 / 010957, and International Patent Publication No. WO U.S. Patent Nos. 02 / 088172, and U.S. Patent Nos. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5, These are described in Patent Nos. 521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, each of which is incorporated herein by reference for all purposes.

[0096] Auristatin has been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and has been shown to possess anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell proliferation inhibitory effects in cancer cells. Numerous well-known assays exist in the art that can be used to measure whether auristatin or the resulting antibody-drug conjugate exerts cell proliferation inhibitory or cytotoxic effects in desired cells.

[0097] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include thiotepa and cyclosphosphamide (CYTOXAN). TMAlkylating agents such as busulfan, improsulfan, and biposulfan, alkyl sulfonates, aziridines such as benzodopa, carbocone, metsuredopa, and uredopa, ethyleneimines and methylamelamamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamamine, chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, f Nitrogen mustards such as enesterine, prednimastine, trophosphamide, uracil mustard, carmastine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, nitrosoureas, acrasinomycin, actinomycin, anthramycin, azaserine, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophiline, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epi Antibiotics such as rubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; fludarabine, 6-mercaptopri Purine analogs such as thiamiprine and thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU, androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone, anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane, folic acid supplements such as folinic acid, acegraton, aldofamide glycoside, aminolevulinic acid, amsacrine,Best Love Sil, Bisanthren, Edatrexate, Defofamine, Demecoltin, Diadiquan, Elfomitin, Elptinium Acetate, Etoglucid, Gallium Nitrate, Hydroxyurea, Lentinan, Ronidamin, Mitoguazone, Mytoxantrone, Mopidamol, Nitracrine, Pentostatin, Fenamet, Pirarubicin, Podophyllic Acid, 2-Ethylhydrazide, Procarbazine, PSK 7. Lazoxane, schizophyllan, spirogermanium, tenuazonic acid, triadiquan, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobromane, gasitosine, arabinoside ("Ara-C"), cyclophosphamide, taxane, e.g., paclitaxel (TAXOL®, Bristol-Myers Examples include Squibb Oncology (Princeton, New Jersey), doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, platinum analogs such as cisplatin or carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, esperamicin, capecitabine, and any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition also includes antihormone agents that modulate or inhibit hormonal activity in tumors, such as tamoxifen, raloxifene, aromatase inhibitor 4(5)imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston), as well as antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and antiestrogens including any pharmaceutically acceptable salts, acids, or derivatives of the above. A detailed description of chemotherapeutic agents is available at:For example, it can be found in US20180193477A1, and the entire thing is incorporated by reference.

[0098] In some preferred embodiments, the cytotoxic agent is a camptothecin compound, its analogue, or derivative. In some preferred embodiments, the camptothecin compound is a compound having the following structure: [ka] Here, X is selected from the group consisting of -CH2-, O, and S, and Y is selected from the group consisting of H, D, and F.

[0099] In some embodiments, the auxiliary portion Z is selected from the group consisting of mercapto, disulfide, amino, carboxyl, aldehyde, maleimide, haloacetyl, hydrazide, and hydroxyl groups.

[0100] In some embodiments, the joint portion Q is defined as described in the first embodiment.

[0101] In some embodiments, polypeptide residue L'1 has the following sequence:

[0102] [ka]

[0103] During the ceremony, each AA 1 AA 2 AA 3 ,...AA p These are independently and optionally substituted amino acid residues, AA 1 AA 2 AA 3 ,...AA p At least one of these is an amino acid residue having a side chain carboxyl group, preferably Glu or Asp. p is an integer between 3 and 8, preferably between 3 and 5, for example, 3, 4, or 5. "NH-" indicates the N-terminal side of a polypeptide residue. "-C(=O)" indicates the C-terminal side of the polypeptide residue.

[0104] In some embodiments, polypeptide residue L'1 is defined as described in the first embodiment, except that the C-terminus of polypeptide residue L is -COOH (carboxyl group), while the C-terminus of polypeptide residue L'1 is -C(O)- (carbonyl group).

[0105] In some embodiments, the linker portion L' is coupled to the therapeutic agent via a bond selected from the group consisting of carbonyl, amide, and ester bonds, preferably an amide bond.

[0106] In some embodiments, the linker portion L' first binds to the auxiliary portion Z, and then couples to the therapeutic agent via a bond selected from the group consisting of disulfide, thioether, thioester, hydrazone, ester, ether, carbamate, and amide bonds.

[0107] In some embodiments, the hydrophilic group L2 is defined as described in the first embodiment.

[0108] In some preferred embodiments, the present invention relates to the compound of formula (II'). [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, X is selected from the group consisting of -CH2-, O, and S, and Y is selected from the group consisting of H, D, and F. Q represents a junction that can be coupled to a ligand by a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds. L' connects Q to the therapeutic agent and shows a linker portion having the following structure: [ka] In the formula, L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue having a side-chain carboxyl group, for example, a glutamic acid residue or an aspartic acid residue. L2 is either absent or a monodentate, bidentate, or tridentate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of polypeptide residue L1, and L2 is -NHC(R L2a )(R L2b )(R L2c ) has a structure, where R L2a , R L2b and R L2c These are H and -(CH2O)(CH2CH2O), respectively. m (CH2) p C(O)OH and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d Independently selected from the group consisting of R L2d C is optionally substituted with H or 1 to 6 hydroxyl groups. 1-6 It is an alkyl group, where each m is independently an integer from 0 to 10, preferably from 0 to 4, for example, 0, 1, 2, 3 or 4, and particularly preferably m is 0, and each p is independently an integer from 1 to 4, for example, 1, 2, 3 or 4, and

[0109] [ka]

[0110] This indicates the N-terminal side of the polypeptide residue covalently bonded to the junction Q.

[0111] [ka]

[0112] This indicates the C-terminal side of the polypeptide residue covalently bonded to the therapeutic agent TA.

[0113] In some embodiments, the joining portion Q is defined as described for formula (II).

[0114] In some embodiments, the polypeptide residue L'1 is defined as described for formula (II).

[0115] In some embodiments, the hydrophilic group L2 is defined as described for formula (II).

[0116] In some preferred embodiments, the compound of formula (II') of the present invention is selected from the group consisting of: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0117] Ligand-drug conjugate As used herein, the term “ligand-drug conjugate” refers to a compound obtained by linking a ligand and a therapeutic agent together using a conjugate compound, for example, a compound of formula (I). In this disclosure, “ligand-drug conjugate” is preferably an antibody-drug conjugate (ADC), which refers to a conjugate obtained by linking an antibody (e.g., a monoclonal antibody) or antibody fragment to a therapeutic agent (e.g., a cytotoxic agent) via a conjugate.

[0118] In a third aspect, the present invention relates to a ligand-drug conjugate of formula (III): [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, LG indicates the ligand, TA indicates a therapeutic agent. Z is either absent or indicates an auxiliary portion that binds L' to the therapeutic agent TA via a bond selected from the group consisting of disulfide, thioether, thioester, hydrazone, ester, ether, carbamate, and amide bonds. Q' represents the junction that is coupled to ligand LG by a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds. L' represents a linker portion that binds Q to the therapeutic agent TA and has the following structure: [ka] In the formula, L'1 is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue having a side-chain carboxyl group, for example, a glutamic acid residue or an aspartic acid residue. L2 is either absent or is a monodentate, bidentate, or tridentate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of polypeptide residue L1, and L2 is -NHC(R L2a )(R L2b )(R L2c) has a structure, where R L2a , R L2b , and R L2c These are H and -(CH2O)(CH2CH2O), respectively. m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d Independently selected from the group consisting of R L2d C is optionally substituted with H or 1 to 6 hydroxyl groups. 1-6 It is an alkyl group, where each m is an independent integer from 0 to 10, preferably from 0 to 4, for example 0, 1, 2, 3, or 4, and particularly preferably m is 0, and each p is an independent integer from 1 to 4, for example 1, 2, 3, or 4.

[0119] [ka]

[0120] This indicates the N-terminal side of the polypeptide residue covalently bonded to the junction Q'.

[0121] [ka]

[0122] This indicates the C-terminal side of the polypeptide residue covalently bonded to the therapeutic agent TA, and n is a number in the range of 1 to 8.

[0123] As used herein, the term “ligand” refers to a macromolecule compound that recognizes and binds to an antigen or receptor associated with a target cell. The role of a ligand is to present a therapeutic agent (drug) to a population of target cells bound to a ligand, which may include, but is not limited to, a protein-like hormone, lectin, growth factor, antibody, or other cell-binding molecule. In some embodiments, the ligand can form a linkage with a binder via a heteroatom on the ligand, preferably an antibody or its antigen-binding fragment, and the antibody may be selected from chimeric antibodies, humanized antibodies, fully human antibodies, or mouse-derived antibodies, and is preferably a monoclonal antibody.

[0124] In some embodiments, the ligand is coupled to the binder via a mercapto group (-SH) or an amine group (-NH2).

[0125] In some preferred embodiments, the antibody or its antigen-binding fragment is coupled to the binder via a mercapto group (-SH) or amine group (-NH2) on the side chain of an amino acid residue.

[0126] As used herein, the term “antibody” means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining regions (CDRs) (e.g., any of three CDRs derived from an immunoglobulin light chain or any of three CDRs derived from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, antibodies may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0127] As used herein, the term "antigen-binding fragment" means a part of a full-length antibody, and this part of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., the variable domain of the heavy chain or the variable domain of the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab’, F(ab’)2, and Fv fragments.

[0128] As used herein, the term "human antibody" means an antibody encoded by endogenous nucleic acids derived from humans (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, the human antibody is recovered from a human or produced in a human cell culture (e.g., within human hybridoma cells). In some embodiments, the human antibody is produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, the human antibody is produced in bacterial or yeast cells. In some embodiments, the human antibody is produced in a transgenic non-human animal (e.g., bovine) that contains unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0129] As used herein, the term "chimeric antibody" means an antibody that contains sequences present in at least two different species (e.g., antibodies derived from two different mammalian species such as human and mouse antibodies). Non-limiting examples of chimeric antibodies are antibodies that contain the variable domain sequences (e.g., all or part of the light chain and / or heavy chain variable domain sequences) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0130] As used herein, the term “humanized antibody” means a non-human antibody that contains minimal sequences derived from non-human (e.g., mouse) immunoglobulin and sequences derived from human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (recipient antibody) in which residues in the hypervariable (e.g., CDR) region of the recipient antibody are replaced by residues in the hypervariable (e.g., CDR) region of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody having desired specificity, affinity, and capability. In some embodiments, Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can further refine the performance of the antibody. In some embodiments, the humanized antibody contains substantially all, at least one, and typically two, variable domains, with all or substantially all of the hypervariable loop (CDR) corresponding to the hypervariable loop of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework region being a human immunoglobulin sequence. The humanized antibody may also contain an immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. The humanized antibody can be produced using molecular biological methods well known in the art. Non-limiting examples of methods for producing the humanized antibody are described herein.

[0131] As used herein, the term “single-chain antibody” means a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are specifically capable of binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0132] Preferably, the antibodies described herein refer to immunoglobulins, which have a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the composition and order of amino acids in the constant region of the heavy chain, and therefore have different antigenicities. Accordingly, immunoglobulins can be divided into five classes, or isotypes, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains can be divided into κ or λ chains depending on their constant region. Each of the five classes of Ig may have either a κ chain or a λ chain.

[0133] Preferably, the antibodies described herein are specific antibodies against cell surface antigens on target cells, and non-limiting embodiments include the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis antibody Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-integrin antibody, anti-PSMA antibody, anti-tenascin-C antibody, anti-SLC44A4 antibody, anti-CTLA4 antibody, anti-OX40 antibody, or one or more of the anti-mesoserin antibodies, preferably trastuzumab, pertuzumab, nimotuzumab, enobrituzumab, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, vibatuzumab, rorbotuzumab, cBR96, and grematsumab.

[0134] In some embodiments, the antibodies are HER2, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD79b, CD137, CD138, CD147, CD223, EpCAM, Mucin 1. An antibody capable of binding to tumor-associated antigens such as STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, nectin 4, AGS-16, guanylyl cyclase C, mesoserine, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein 3, IL2RA, PTK7, or claudin 18.2.

[0135] In some embodiments, the therapeutic agent TA is defined as described in the second embodiment.

[0136] In some embodiments, the joint portion Q' has the following structure: [ka] In the formula, Q' a This is a functional group coupled to a ligand, A is C which is optionally substituted. 3-8 Alkylidene, optionally substituted with C 3-8 Alkenylidene, optionally substituted with C 3-6 Cycloalkenylidene, optionally substituted C 3-8 Selected from cycloalkylides, optionally substituted diglycol-octaglycolacyls, where alkylidene, alkenylidene, cycloalkenylidene, cycloalkylidene, diglycol-octaglycolacyls are halogens, -CN, R Q’a1 , -OR Q’a1 , -SR Q’a1 , and -N(R Q’a1)1 to 4 substituents independently selected from the group consisting of 2, which are arbitrarily substituted, and in the formula, each R Q’a1 C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclyl, C 6-10 Independently selected from the group consisting of aryls and 5-10 membered heteroaryls,

[0137] [ka]

[0138] This indicates the site covalently bonded to the linker portion L'.

[0139] In some preferred embodiments, joint portion Q ’a The following group is selected: [ka]

[0140] In the formula, Ar is optionally substituted with C. 5-6 Selected from the group consisting of cycloalkenyls, optionally substituted C6 aryls, and optionally substituted 5- to 6-membered heteroaryls, where cycloalkenyls, aryls, and heteroaryls are halogens, -CN, and R Qa2 , -OR Qa2 , -SR Qa2 , and -N(R Qa2 )1 to 4 substituents independently selected from the group consisting of 2, which are arbitrarily substituted, and in the formula, each R Qa2 C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclyl, C 6-10 Independently selected from the group consisting of aryls and 5-10 membered heteroaryls, "*" indicates a site covalently bonded to A, and

[0141] [ka]

[0142] This indicates the site of covalent binding to ligand LG.

[0143] It should be understood that when the group "Ar" is located inside the molecular structure of a compound, this can also refer to a divalent substituent.

[0144] In some preferred embodiments, Ar is selected from the group consisting of optionally substituted cyclopentadienyl, optionally substituted phenyl, and optionally substituted 5- to 6-membered heteroaryls.

[0145] In some embodiments, the polypeptide residue L' is defined as described in the second embodiment.

[0146] In some embodiments, n represents the average amount of therapeutic agent (e.g., cytotoxic agent) loaded onto each ligand (e.g., antibody) in the molecule of formula (III). When the ligand is an antibody, it is also expressed as the ratio of the amount of drug to the amount of antibody, i.e., the drug-antibody ratio (DAR). In some embodiments, n may be, exemplary, a number such as 1, 2, 3, 4, 5, 6, 7, or 8. The average amount of drug per ADC molecule after the coupling reaction can be identified by conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA, and HPLC characterization.

[0147] In some preferred embodiments, the present invention relates to a ligand-drug conjugate of formula (III'). [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, X is selected from the group consisting of -CH2-, O, and S, and Y is selected from the group consisting of H, D, and F. Ab indicates an antibody. Q and L' are as defined in the prior claims, n is a number in the range of 1 to 8.

[0148] In some preferred embodiments, compounds of formula (III') of the present invention are selected from the group consisting of: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] In the formula, n is a number in the range of 1 to 8.

[0149] Camptothecin compounds In a fourth aspect, the present invention relates to a camptothecin compound of formula (IV): [ka] Or, with respect to a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the formula, X is selected from the group consisting of -CH2-, O, and S, and Y is selected from the group consisting of H, D, and F.

[0150] Method of Use and Administration The compounds of the present invention, or mixtures thereof in any proportion, as well as their pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants, can be used as pharmaceuticals. These have been found to exhibit tumor-inhibiting pharmacological activity.

[0151] Therefore, the compounds of the present invention, which are antitumor agents, are particularly useful in the treatment of cancer, especially tumors including solid tumors, brain tumors, lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cancer, pancreatic cancer, liver cancer, gastric cancer, or breast cancer. While we do not wish to adhere to any particular logic or explanation, it can be assumed that the compounds may achieve tumor-inhibiting pharmacological activity by direct effect on cancer cells and / or indirectly by controlling the immune system's response to the tumor.

[0152] The compounds of the present invention can be administered in amounts effective for treating the diseases or conditions described herein. The compounds of the present invention can be administered as the compounds themselves or, alternatively, as pharmaceutically acceptable salts. For the purposes of administration and drug delivery, the compounds of the present invention themselves, or their pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants, are simply referred to as the compounds of the present invention.

[0153] The compounds of the present invention are administered by any preferred route, in the form of a pharmaceutical composition adapted to such route, in a dose effective for the intended treatment. The compounds of the present invention can be administered orally, rectally, vaginally, parenterally, or topically.

[0154] As used herein, the terms “administer” and “to administer” mean to introduce a compound of the present invention, or a pharmaceutical composition thereof, by absorption, digestion, injection, inhalation, implantation, or otherwise. The terms “treatment” and “to treat” mean to reverse, alleviate, delay the onset of, or inhibit the progression of a “medical condition” (e.g., a disease, disorder, or state, or one or more signs or symptoms thereof) as described herein. In certain embodiments, treatment may be administered after one or more signs or symptoms of a disease or condition have developed or been observed. In other embodiments, treatment may be performed even without signs or symptoms of a disease or condition. For example, treatment may be performed on a susceptible person before the onset of symptoms (e.g., considering a history of symptoms and / or considering genetic or other susceptibility factors). Treatment may be continued after the symptoms have subsided, for example, to delay or prevent recurrence. As used herein, the terms “disease,” “disorder,” “state,” and “medical condition” are interchangeable.

[0155] The dose level for administration can be determined by those skilled in the art through routine experimentation. The administration regimen for the compounds of the present invention and / or compositions containing such compounds is based on various factors, including the patient's type, age, weight, sex, and medical condition, the severity of the condition, the route of administration, and the activity of the specific compound being used. Therefore, the administration regimen can vary considerably. For example, the dose level of the compounds of the present invention can be about 0.001 to about 100 mg / kg (i.e., mg / kg of body weight) per day. In certain embodiments, the total daily dose of the compounds of the present invention, administered as a single dose or in divided doses, may be about 0.001 to about 10 mg / kg. It is not uncommon for the administration of the compounds of the present invention to be repeated multiple times a day.

[0156] In some embodiments, the compounds of the present invention can be administered in combination with one or more additional therapeutic agents. In certain embodiments, non-limiting examples of additional therapeutic agents include antitumor agents. The additional therapeutic agent(s) may be administered before, after, or concurrently with the administration of the compounds of the present invention.

[0157] As used herein, the term “antitemoid agent” refers to any agent administered to a patient with cancer for the purpose of treating cancer. Conventional surgery, or radiation therapy, or drug therapy may be used in combination with the compounds of the present invention for the treatment of cancer.

[0158] Pharmaceutical composition In some embodiments, the present invention relates to a pharmaceutical composition comprising a compound of formula (III) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, and at least one pharmaceutically acceptable diluent, carrier, or excipient.

[0159] As used herein, the term “pharmaceutically acceptable diluent, carrier, or excipient” refers to a diluent, carrier, or excipient that is generally safe and non-toxic and useful for preparing a desirable pharmaceutical composition, both biologically and under other conditions, and includes diluents, carriers, or excipients acceptable for veterinary and human pharmaceutical uses. As used herein, pharmaceutically acceptable diluents, carriers, or excipients include both one such diluent, carrier, or excipient and multiple such diluents, carriers, or excipients. The specific diluent, carrier, or excipient used will depend on the means and purpose to which the compound of the present invention is applied. Suitable diluents, carriers, and excipients are well known to those skilled in the art and are described in detail, for example, Ansel, Howard C, et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. To impart a refined appearance to a drug (i.e., a compound or pharmaceutical composition provided herein) or to assist in the manufacture of a pharmaceutical product (i.e., a drug), one or more of the following known additives may also be included: buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, lubricants, processing aids, colorants, sweeteners, flavorings, flavoring additives, and other known additives.

[0160] The compositions of the present invention can be formulated in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., solutions for injection and infusion), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form depends on the intended mode of administration and therapeutic use.

[0161] The pharmaceutical compositions of the present invention can be prepared by any well-known pharmaceutical technique, such as effective formulation and administration procedures. The above considerations regarding effective formulation and administration procedures are well known in the art and are described in standard textbooks. Formulation of pharmaceutical products can be found, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, 3 rd This is discussed in Edition, American Pharmaceutical Association, Washington, 1999.

[0162] In certain embodiments, the pharmaceutical composition comprises a compound of formula (III) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in combination with one or more additional therapeutic agents, such as an antitumor agent, and at least one pharmaceutically acceptable diluent, carrier, or excipient.

[0163] In further embodiments, the present invention relates to a kit for treating tumors, comprising a compound of formula (III) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, or a compound of formula (IIII) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, a container, and optionally, a package insert or label indicating the treatment. In certain embodiments, the kit may further contain one or more additional therapeutic agents, such as antitumor agents.

[0164] Treatment method In a further embodiment, the present invention relates to a method for treating a tumor in a subject requiring treatment of the tumor, comprising administering to the subject a therapeutically effective amount of a compound of formula (III) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

[0165] As used herein, the term “subjects requiring ~” refers to subjects having tumors, or subjects, as a whole, having an increased risk of developing tumors compared to the population. In certain embodiments, subjects are warm-blooded animals. In certain embodiments, warm-blooded animals are mammals. In certain embodiments, warm-blooded animals are humans.

[0166] The tumor treatment methods described herein can be used as monotherapy. As used herein, the term “monotherapy” refers to the administration of a single active or therapeutic compound to a subject requiring treatment. In certain embodiments, monotherapy involves the administration of a therapeutically effective amount of one of the compounds of the present invention, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, to a subject requiring such treatment.

[0167] Depending on the specific disease or condition being treated, the tumor treatment methods described herein may involve combination therapy with one or more additional therapeutic agents, such as antitumor agents, in addition to the administration of the compound of formula (III). As used herein, the term “combination therapy” refers to the administration of a combination of multiple active therapeutic agents. In certain embodiments, the compound of the present invention, or its pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants, may be administered separately or sequentially with treatment by one or more additional therapeutic agents. For example, the additional therapeutic agent(s) may be administered separately from the compound of the present invention / as part of a multiple dosing regimen. Alternatively, the additional therapeutic agent(s) may be mixed with the compound of the present invention in a single composition, which may be part of a single dosing form.

[0168] In a further embodiment, the present invention relates to the use of a compound of formula (III) provided herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the manufacture of a drug for treating tumors in subjects requiring treatment of tumors.

[0169] synthesis The compounds of the present invention can be prepared by general and specific methods described below, using the common general knowledge possessed by those skilled in synthetic organic chemistry. Such common general knowledge can be found in standard reference books, such as Barton and Ollis (Ed.), Comprehensive Organic Chemistry, Elsevier; Richard Larock, Comprehensive Organic Transformations: A Guide to Functional Group Preparations, John Wiley and Sons; and Compendium of Organic Synthetic Methods, Vol. I-XII, Wiley-Interscience.

[0170] The schemes described below in this specification are intended to provide a general description of the methodology used in the preparation of the compounds of the present invention. Some of the compounds of the present invention may contain one or more chiral centers having the stereochemical designation (R) or (S). It will be apparent to those skilled in the art that all synthesis information can be carried out in similar manner, regardless of whether the material is enantioenriched or racemic. Furthermore, the separation into the desired optically active material can be carried out at any desired point in the procedure using well-known methods, such as those described herein and in the chemical literature.

[0171] In some embodiments, the present invention relates to the following a) A step of reacting the ligand with a reducing agent in a buffer to obtain a reduced ligand, b) In a mixture of buffer and organic solvent, the compound of formula (II) of the present invention, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, is coupled with the reduced ligand obtained in step (a) to obtain a ligand-drug conjugate; Alternatively, see below. The present invention involves coupling a compound of formula (II), or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, with a ligand in a mixture of a buffer and an organic solvent to obtain a ligand-drug conjugate. The present invention relates to a method for preparing a ligand-drug conjugate of formula (III), or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, including the present invention.

[0172] Examples Examples are provided below to allow for a more comprehensive understanding of the present invention as described herein. The examples described herein are provided to illustrate the methods and compositions provided herein and should not be construed in any way as limiting their scope.

[0173] All reagents and materials can be purchased from commercial vendors or prepared promptly by those skilled in the art. A list of abbreviations used can be found in the table below.

[0174] [Table 3]

[0175] Example 1. Preparation of the compound 1 1H NMR spectra were recorded using a Bruker Ascend 400 spectrometer. Chemical shifts are expressed in parts per million (ppm, in δ units). Coupling constants are expressed in Hertz (Hz). The redistribution pattern represents the apparent multiplicity and is expressed as s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), and br (broad).

[0176] Using gradient elution, low-resolution analytical mass spectra (MS) were recorded on an Agilent 1290 equipped with SQ detectors, using a Waters Xbridge C18, 4.6 × 50 mm, 3.5 μm.

[0177] Solvent A: 0.1% TFA in water, Solvent B: 0.1% TFA in acetonitrile 1.3 minutes, 5-95% B.

[0178] Preparation Example 1.1. Synthesis of (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione) (CPT-1) [ka] [ka] Step 1: Synthesis of N-(3-chloro-4-nitrophenyl)acetamide (compound 2a) [ka] compound 2a Ac2O (39.185 mL, 417.222 mmol) was added to a solution of 3-chloro-4-nitroaniline (compound 1a, 24 g, 139.074 mmol) in AcOH (50 mL), and the resulting mixture was stirred at 120°C for 2 hours. After the reaction was complete, as confirmed by TLC (PE:EA = 4:1), AcOH was removed by vacuum distillation. The residue was then dissolved in water, and the resulting solution was neutralized with Na2CO3 to reduce its alkalinity. After extraction with EA, compound 2a was obtained as a yellow solid (28 g, 130.475 mmol, yield: 93.82%), as confirmed by LC-MS.

[0179] LC-MS:(ESI)m / z(M+H):215.0.

[0180] Step 2: Synthesis of 3-{[5-(acetylamino)-2-nitrophenyl]sulfanyl}propanoic acid (compound 3a) [ka] compound 3a 3-Sulfanylpropanoic acid (16.62 g, 156.570 mmol) was added to a solution of compound 2a (28 g, 130.475 mmol) and K2CO3 (54.09 g, 391.425 mmol) in EtOH (100 mL), and the resulting mixture was refluxed overnight. The mixture was then cooled to RT, poured into ice water, and the precipitate was filtered. The resulting solid product was then washed with water and MeOH to obtain compound 3a as a yellow solid (28 g, 98.491 mmol, yield: 75.49%), as confirmed by LC-MS.

[0181] LC-MS: (ESI)m / z(M+Na): 307.0.

[0182] Step 3: Synthesis of N-(8-nitro-4-oxothiochroman-5-yl)acetamide (compound 4a) [ka] compound 4a A solution of compound 3a (5 g, 17.588 mmol) in [(dioxo-λ5-phosphanyl)oxy]dioxo-λ5-phosphanmethanesulfonic acid (20 mL) was stirred under an N2 atmosphere at RT for 1.5 hours. After LC-MS analysis showed that 30% DP (desired product) had formed, the resulting mixture was poured into ice water, the precipitate was filtered, and washed with an aqueous Na2CO3 solution to obtain compound 4a as a pale yellow solid (680 mg, 2.554 mmol, yield: 14.52%), as confirmed by LC-MS.

[0183] LC-MS: (ESI)m / z(M+Na): 289.0.

[0184] Step 4: Synthesis of N-(8-amino-4-oxothiochroman-5-yl)acetamide (compound 5a) [ka] compound 5a Compound 4a (640 mg, 2.404 mmol), Fe (536.86 mg, 9.614 mmol), and NH4Cl (1285.67 mg, 24.036 mmol) were mixed with EtOH (8 mL) and water (4 mL) and stirred at 80°C for 2 hours. After the reaction was complete, as confirmed by LC-MS, the resulting product was obtained by filtration and extraction with EA, and compound 5a was obtained as an orange solid (567.94 mg, 2.404 mmol, quantitative yield), as confirmed by LC-MS.

[0185] LC-MS:(ESI)m / z(M+H):237.0.

[0186] Step 5: Synthesis of 5,8-diaminothiochroman-4-one (compound 6a) [ka] compound 6a Compound 5a (400 mg, 1.693 mmol) was stirred in a hydrochloric acid (2.0 N in water) solution at 90°C for 2 hours until the completion of the reaction was confirmed by LC-MS analysis. The mixture was then poured into water, and the resulting solution was neutralized with Na2CO3 to reduce its alkalinity. After extraction by EA, compound 6a was obtained as an orange solid (328.83 mg, 1.693 mmol, quantitative yield), as confirmed by LC-MS.

[0187] LC-MS:(ESI)m / z(M+H):195.2.

[0188] Step 6: Synthesis of CPT-1 Compound 6a (200 mg, 1.030 mmol) and (4S)-4-ethyl-4-hydroxy-3,4,6,7,8,10-hexahydro-1H-pyrano[3,4-f]indolidine-3,6,10-trione (Compound 1, 352.21 mg, 1.338 mmol) were dissolved in toluene (10 mL), to which PPTS (336.22 mg, 1.338 mmol) was added. The resulting mixture was then stirred overnight at 120 °C under an N2 atmosphere. After the reaction was complete, toluene was removed by vacuum distillation as detected by LC-MS, the residue was dissolved in DMF, and then purified using preparative liquid chromatography with TFA as the eluent. CPT-1 was obtained as a dark brown solid (80 mg, 0.19 mmol, yield: 18.44%) as confirmed by LC-MS and NMR.

[0189] LC-MS:(ESI)m / z(M+H):422.2.

[0190] 1HNMR(400MHz,DMSO):δ7.77(d,J=9.2Hz,1H),7.38(d,J=9.2Hz,1H),7.25(s,1H),6.52(s,1H),5.74(s,2H) ,5.47(s,2H),5.24(s,2H),3.52-3.43(m,2H),3.27-3.23(m,2H),1.96-1.87(m,2H),0.94(t,J=7.2Hz,3H).

[0191] Preparation Example 1.2. Synthesis of (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13(2H)-dione (CPT-2) [ka] [ka] Step 1: Synthesis of N-(2-(3-hydroxypropoxy)-4-nitrophenyl)acetamide (compound 2b) [ka] compound 2b N-(2-hydroxy-4-nitrophenyl)acetamide (compound 1b, 10 g, 50.979 mmol) was added to a suspension of NaH (1.25 g, 52.083 mmol) in DMF (200 mL), and the resulting mixture was stirred for 20 minutes. Then, 3-chloropropan-1-ol (6.372 mL, 76.158 mmol) and 18-crown-6 (0.1 g, 0.378 mmol) were added to the mixture, and these were heated at 80°C for 2 days. After the reaction was complete, the resulting mixture was poured into ice water and extracted with EA. The organic layer was then washed with 10% NaOH aqueous solution and brine, dried over Na2SO4, filtered, and concentrated. As confirmed by LC-MS, the product, compound 2b, was obtained as a brown solid (6.34 g, 24.937 mmol, yield: 48.92%) by recrystallization (EtOH / PE).

[0192] LC-MS:(ESI)m / z(M+H):255.2.

[0193] Step 2: Synthesis of 3-(2-acetamido-5-nitrophenoxy)propanoic acid (compound 3b) [ka] compound 3b Jones' reagent (19.937 mL, 39.874 mmol) was added dropwise to a solution of compound 2b (6.336 g, 24.921 mmol) in acetone (125 mL) at 0°C, and the resulting mixture was stirred overnight in RT. After the reaction was complete, the mixture was diluted with water and extracted with EAT. The organic layer was then washed with brine, dried over Na2SO4, filtered, and concentrated. 1 As confirmed by 1H NMR, the product, compound 3b, was obtained as a yellow solid by recrystallization (MeOH / H2O) (5.819 g, 21.694 mmol, yield: 87.05%).

[0194] 1 HNMR(400MHz,DMSO):δ12.50(s,1H),9.37(s,1H),8.39(d,J=8.8Hz,1H),7.90(dd,J=8.8,2 .4Hz,1H),7.87(d,J=2.4Hz,1H),4.35(t,J=6.0Hz,2H),2.85(t,J=6.0Hz,2H),2.18(s,3H).

[0195] Step 3: Synthesis of 3-(2-acetamido-5-aminophenoxy)propanoic acid (compound 4b) [ka] compound 4b The suspension of compound 3b (5 g, 18.641 mmol) and PtO2 (0.59 g, 2.610 mmol) in EtOH (325 mL) and water (75 mL) was stirred overnight in an H2 atmosphere (1 atm). After the reaction was complete, the catalyst was removed by filtration and the solution was concentrated. As confirmed by NMR, the product, compound 4b, was obtained as a brown solid (4.10 g, 17.210 mmol, yield: 92.32%) by recrystallization (MeOH-H2O).

[0196] 1 HNMR(400MHz,DMSO):δ8.60(s,1H),7.37(d,J=8.4Hz,1H),6.28(d,J=2.4Hz,1H),6. 11(dd,J=8.8,2.4Hz,1H),4.06(t,J=6.4Hz,2H),2.71(t,J=6.4Hz,2H),1.97(s,3H).

[0197] Step 4: Synthesis of N-(5-amino-4-oxochroman-8-yl)acetamide (compound 5b) [ka] compound 5b Compound 4b (500 mg, 2.099 mmol) was added to a solution of Eaton's reagent (10 mL, 63.015 mmol), and the mixture was heated to 60°C for 6 hours under an N2 atmosphere. After the reaction was complete, the resulting mixture was basicized with aqueous NaOH solution and extracted with EA. The combined organic phase was then washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The product, compound 5b, was obtained as a yellow solid (73 mg, 0.331 mmol, yield: 15.79%) by flash (PE:EA = 1:9), as confirmed by LC-MS.

[0198] LC-MS:(ESI)m / z(M+H):221.2.

[0199] Step 5: Synthesis of (S)-N-(9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)acetamide (compound 6b) [ka] compound 6b A solution of compound 5b (73 mg, 0.331 mmol) and compound 1 (95.99 mg, 0.365 mmol) in AcOH (3 mL) was heated under reflux in an N2 atmosphere for 5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The product, compound 6b, was obtained as a yellow solid (42.3 mg, 0.104 mmol, yield: 31.48%) by flash (DCM:MeOH = 95:5), as confirmed by LC-MS.

[0200] LC-MS:(ESI)m / z(M+H):448.2.

[0201] Step 6: Synthesis of CPT-2 Compound 6b (138 mg, 0.308 mmol) was added to a solution of 12 M HCl (15 mL) and H2O (15 mL). The mixture was then heated under reflux under a N2 atmosphere for 1 hour. After the reaction was complete, the mixture was concentrated under reduced pressure. The product, CPT-2, was obtained as an orange solid (52 mg, 0.128 mmol, yield: 41.59%) by preparative liquid chromatography (TFA), as confirmed by LC-MS and NMR.

[0202] LC-MS:(ESI)m / z(M+H):406.2.

[0203] 1HNMR (400MHz, DMSO): δ7.56(d,J=9.2Hz,1H),7.35(d,J=9.2Hz,1H),7.20(s,1H),6.46(s,1H),5.41(s ,4H),5.20(s,2H),4.42(t,J=5.6Hz,2H),3.30-3.27(m,2H),1.92-1.80(m,2H),0.87(t,J=7.2Hz,3H).

[0204] Preparation Example 1.3. Synthesis of (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione (CPT-3) [ka] [ka]

[0205] Preparation Example 1.4. Synthesis of (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13(2H)-dione (CPT-4) [ka] [ka] Step 1: Synthesis of 4-fluorobenzo[d]oxazole-2(3H)-one (compound 2d) [ka] compound 2d Di(imidazole-1-yl)methanone (21.05 g, 129.799 mmol) was slowly added at 0°C to a solution of 2-amino-3-fluorophenol (compound 1d, 15 g, 117.999 mmol) in THF (300 mL). The reaction mixture was then stirred at 60°C for 2 hours under an N2 atmosphere. After the reaction was complete, the reaction mixture was cooled to room temperature and diluted with H2O (300 mL). The resulting solution was then concentrated to remove most of the solvent, and the residue was extracted with EA (300 mL x 3). The combined organic phase was then washed with 2 M HCl (300 mL x 2) and brine (900 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the residue. The residue (TLC-T2:DCM:MeOH=20:1, UV, product Rf=0.40) was purified by silica gel column (DCM:MeOH, 2%~4%) to obtain the desired product, compound 2d. This compound was a brown solid (16.13g, 105.349 mmol, yield: 89.28%) and was confirmed by NMR (N230872-011-P1A, DMSO).

[0206] 1 HNMR(400MHz,DMSO):δ12.25(s,1H),7.21-7.13(m,1H),7.13-7.05(m,2H)

[0207] Step 2: Synthesis of 4-fluoro-6-nitrobenzo[d]oxazole-2(3H)-one (compound 3d) [ka] compound 3d HNO3 (7.659 mL, 110.617 mmol) was slowly added at -10°C to a solution of compound 2d (16.13 g, 105.349 mmol) in H2SO4 (160 mL), and the reaction mixture was stirred at -10°C for 0.5 hours under an N2 atmosphere. After the reaction was complete, the reaction mixture was poured into ice water (300 mL), stirred at 0°C for 30 minutes, and then filtered. The filtered cake was then washed continuously with H2O (100 mL x 3), recovered, diluted with MeOH (160 mL), and the resulting solution was concentrated to obtain the desired product, 4-fluoro-6-nitro-2,3-dihydrobenzo[d][1,3]oxazole-2-one (compound 3d, 20.87 g, 105.346 mmol, yield: 100.00%), which was a light brown solid. 1 Confirmed by 1H NMR (N230872-016-P1A, DMSO).

[0208] 1 HNMR (400MHz, DMSO): δ13.08 (s, 1H), 8.29-7.99 (m, 2H).

[0209] Step 3: Synthesis of 2-amino-3-fluoro-5-nitrophenol (compound 4d) [ka] compound 4d When NaOH (210.691 mL, 526.728 mmol) was added at 20°C to a solution of compound 3d (20.87 g, 105.346 mmol) in EtOH (800 mL), the reaction mixture became viscous and turned yellow. The reaction mixture was then stirred at 100°C for 2 hours under an N2 atmosphere. After the reaction was complete, the reaction mixture was cooled to room temperature and then acidified by adding 2 M HCl to a pH of 2-3. The reaction mixture turned yellow. The reaction mixture was then neutralized with saturated NaHCO3 to a pH of 8, filtered, and the resulting filtrate was concentrated to remove the solvent. The resulting residue was extracted with EA (400 mL x 3), washed with brine (1 L x 3), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 4d (10.88 g, 63.212 mmol, yield: 60.00%), which was an orange solid. 1 Confirmed by 1H NMR (N230872-020-P1B, DMSO).

[0210] 1 HNMR (400MHz, DMSO): δ10.52(s, 1H), 7.55(dd, J=11.1, 2.4Hz, 1H), 7.42(d, J=1.5Hz, 1H), 6.12(s, 2H).

[0211] Step 4: Synthesis of N-(2-fluoro-6-hydroxy-4-nitrophenyl)acetamide (compound 5d) [ka] compound 5d Acetic anhydride (7.718 mL, 82.175 mmol) was slowly added at 0°C to a solution of compound 4d (10.88 g, 63.212 mmol) in AcOH (150 mL), and the reaction mixture was stirred at 50°C for 1 hour under an N2 atmosphere. After the reaction was complete, the reaction mixture was cooled to room temperature and H2O (300 mL) was added. The resulting mixture was then acidified with 1 M HCl to a pH of 3-4, stirred at 20°C for 30 minutes, and then filtered. The filtered cake was then washed repeatedly with H2O (50 mL x 3), recovered, and diluted with MeOH (200 mL). The resulting mixture was concentrated to obtain the product, compound 5d (9.91 g, 46.276 mmol, yield: 73.19%), which is a light brown solid. 1 Confirmed by 1H NMR (N230872-026-P1A, DMSO).

[0212] 1 HNMR (400MHz, DMSO): δ11.02(s,1H),9.62(s,1H),7.60(dd,J=9.6,2.5Hz,1H),7.55(dd,J=2.4,1.4Hz,1H),2.06(s,3H).

[0213] Step 5: Synthesis of N-(2-fluoro-6-(3-hydroxypropoxy)-4-nitrophenyl)acetamide (compound 6d) [ka] compound 6d NaH (2.06 g, 51.623 mmol) was slowly added to a solution of compound 5d (10.05 g, 46.930 mmol) in DMF (200 mL) at 20°C under an N2 atmosphere, and the resulting mixture was stirred at 20°C for 20 minutes under an N2 atmosphere. Then, 3-chloropropan-1-ol (5.889 mL, 70.395 mmol) and 18-crown-6 (0.62 g, 2.346 mmol) were slowly added to the mixture at 20°C under an N2 atmosphere, and the reaction mixture was stirred at 80°C for 48 hours under an N2 atmosphere. After the reaction was complete, the reaction mixture was cooled to room temperature and H2O (200 mL) was added. Subsequently, the obtained mixture was extracted with EA (200 mL × 3), the organic layer was washed with 10% NaOH (500 mL × 2), and then with brine (500 mL × 3), and then dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the residue. The residue was purified by silica gel column (DCM:MeOH, 2%~4%) to obtain the desired product, compound 6d (3.71 g, 13.628 mmol, yield: 29.04%), which was a pale yellow solid, and LC-MS (N230872-029-P1) and 1 Confirmed by 1H NMR (N230872-029-P1A, DMSO).

[0214] LC-MS: m / z(ES+)(M+H)+=273.2, Rt=0.575min.

[0215] 1 HNMR(400MHz,DMSO)δ9.62(s,1H),7.79(dd,J=9.5,2.3Hz,1H),7.72(s,1H),4.5 7(s,1H),4.22(t,J=6.3Hz,2H),3.57(s,2H),2.06(s,3H),1.89(p,J=6.2Hz,2H).

[0216] Step 6: Synthesis of 3-(2-acetamido-3-fluoro-5-nitrophenoxy)propanoic acid (compound 7d) [ka] compound 7d Jones' reagent (10.221 mL, 20.442 mmol) was slowly added at 0°C to a solution of compound 6d (3.71 g, 13.628 mmol) in acetone (60 mL), and the reaction mixture was stirred at 0-20°C for 2 hours under an N2 atmosphere. After the reaction was complete, the reaction mixture was quenched with saturated Na2SO3 to pH 8, filtered through Celite, and the filter cake was washed with H2O (20 mL x 3). The resulting filtrate was extracted with EA (100 mL x 3), and the organic layer was discarded. The aqueous layer was acidified with 1M HCl to a pH of 3-4, extracted with EA (100 mL x 3), washed with brine (300 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the product compound 7d (2.26 g, 7.896 mmol, yield: 57.94%), which is a pale yellow solid. LC-MS (N230872-040-P1-1) and 1 Confirmed by 1H NMR (N230872-040-P1A, DMSO).

[0217] LC-MS: m / z(ES+)(M+H)+=287.2, Rt=0.573min.

[0218] 1 HNMR (400MHz, DMSO) δ12.44(s,1H),9.61(s,1H),7.82(dd,J=9.5,2.3Hz,1H),7.76(s,1H),4.36(t,J=6.1Hz,2H),2.74(t,J=6.1Hz,2H),2.05(s,3H).

[0219] Step 7: Synthesis of 3-(2-acetamido-5-amino-3-fluorophenoxy)propanoic acid (compound 8d) [ka] compound 8d Platinum dioxide (0.17 g, 0.737 mmol) was added at 20°C to a solution of compound 7d (2.11 g, 7.372 mmol) in EtOH (64 mL) and H2O (16 mL). The reaction mixture was stirred under H2 at 20°C for 3 hours. After the reaction was complete, the reaction mixture was filtered through Celite, and the resulting filtrate was concentrated to obtain a residue. The residue was diluted with MeOH (2 mL), and H2O (6 mL) was added. The resulting mixture was then stirred under an N2 atmosphere at 20°C for 1 hour. Subsequently, the resulting mixture was filtered, the filter cake was collected, and concentrated to obtain the product, compound 8d (950 mg, 3.708 mmol, yield: 50.29%), which is a pale yellow solid. 1 Confirmed by 1H NMR (N230872-050-P1A, DMSO).

[0220] 1 HNMR(400MHz,DMSO)δ12.34(s,1H),8.63(s,1H),6.07(s,1H),5.96(dd,J=12.0,2 .1Hz,1H),5.34(s,2H),4.04(t,J=6.4Hz,2H),2.63(t,J=6.4Hz,2H),1.91(s,3H).

[0221] Step 8: Synthesis of N-(5-amino-7-fluoro-4-oxochroman-8-yl)acetamide (compound 9d) [ka] compound 9d A molecular sieve (11.33 mg, 0.039 mmol) was added at 20°C to a solution of compound 8d (490 mg, 1.912 mmol) in Eaton's reagent (30 mL), and the reaction mixture was stirred at 50°C for 3 hours under an N2 atmosphere. After the reaction was complete, the reaction mixture was poured into ice water (100 mL), the pH was neutralized to 8-9 with 10% NaOH, and extracted with EA (200 mL x 3). The combined organic layers were washed with saturated NaHCO3 (500 mL x 2) and brine (500 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the product compound 9d (200 mg, 0.840 mmol, yield: 43.90%), which was a yellow solid. LC-MS (N230872-057-P1-1) and 1 Confirmed by 1H NMR (N230872-057-P1A, DMSO).

[0222] LC-MS: m / z(ES+)(M+H)+=239.4, Rt=0.674min.

[0223] 1 HNMR (400MHz, DMSO) δ8.89(s,1H),7.55(s,2H),6.10(d,J=12.5Hz,1H),4.41(s,2H),2.68(s,2H),1.95(s,3H).

[0224] Step 9: Synthesis of (S)-N-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)acetamide (compound 10d) [ka] compound 10d Compound 1 (243.12 mg, 0.924 mmol) and PPTS (221.53 mg, 0.882 mmol) were slowly added at 20°C to a solution of N-(5-amino-7-fluoro-4-oxo-3,4-dihydro-2H-chromen-8-yl)acetamide (200 mg, 0.840 mmol) in toluene (15 mL). The reaction mixture was stirred at 130°C for 2 hours under an N2 atmosphere. After the reaction was complete, the reaction mixture was concentrated, the solvent was removed, and the mixture was used directly for the next step.

[0225] Step 10: Synthesis of CPT-4 [ka] A mixture of N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,9,10,12,15-hexahydropyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]acetamide (390.76 mg, 0.840 mmol) and 6 M HCl (10 mL) was stirred at 100 °C for 1 hour under an N2 atmosphere. After the reaction was complete, the reaction mixture was concentrated and most of the solvent was removed. The resulting residue was purified using a reversed-phase column (80 g C18 gel column, A / B = H2O / MeOH, 50% → 80% B) to obtain the crude product. The crude product (TLC-T1:DCM:MeOH = 10:1, UV, Rf of the product = 0.35) was purified by silica gel column chromatography (DCM:MeOH, 4% → 6%) to obtain the desired product, CPT-4 (0.6 mg, 0.001 mmol), which is a brown solid. 1 This was confirmed by 1H NMR (N230872-061-P1-3, DMSO), LC-MS (N230872-061-P1-3), and HPLC (N230872-061-P1-3-254NM, N230872-061-P1-3-214NM).

[0226] LC-MS: m / z(ES+)(M+H)+=424.2, Rt=0.618min.

[0227] 1HNMR(400MHz,DMSO)δ7.48(d,J=12.3Hz,1H),7.22(s,1H),6.47(s,1H),5.45(s,1H),5.41(s,2) H),5.20(s,2H),4.47(t,J=5.7Hz,2H),3.30(s,2H),1.94-1.74(m,2H),0.87(t,J=7.4Hz,3H).

[0228] Preparation Example 1.5. (S)-N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-8,18-dioxo-13-((3-oxo-3-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propoxy)methyl)-11,15-dioxa-7,19-diazapentacosan-13-yl)-2-(6-(2,5-dioxo-2,5-dihydro- Synthesis of 1H-pyrrole-1-yl)hexanamide)-N1-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (compound (II')-1) [ka] [ka] Step 1: Synthesis of tert-butyl(S)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (compound 2e) [ka] compound 2e 1-Methylimidazole (0.020 mL, 0.249 mmol) was added to a mixture of N-[(2S,5S)-12-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-{3-[(2-methylprop-2-yl)oxy]-3-oxopropyl}-1,4,7-trioxo-2-(prop-2-yl)-3,6-diazadodeca-1-yl]-L-alanine (compound 1e, 67.22 mg, 0.119 mmol) and CPT-1 (50 mg, 0.119 mmol) in DMF (2.2 mL). Then, TCFH (39.93 mg, 0.142 mmol) was added to the mixture, and the resulting mixture was stirred at 25°C for 12 hours. As confirmed by LC-MS, after the reaction was complete, saturated brine (10 mL) was added to the reaction solution to extract a large amount of solid. The resulting mixture was filtered through a cloth funnel, the solid was washed with water, and the resulting residue was dissolved in DCM (30 mL). The organic layer was then dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2e (70 mg, brown solid, yield: 60.82%), which was used in the next step without purification.

[0229] LC-MS: (ESI)m / z(M+H), 971.2.

[0230] Step 2: Synthesis of (S)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (compound 3e) [ka] compound 3e TFA (0.5 mL, 0.067 mmol) is mixed with 2-methylpropan-2-yl(4S)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-5-{[(2S)-1-{[(2S)-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,12,1 A solution of 5-hexahydrothiino[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-1-oxoprop-2-yl]amino}-3-methyl-1-oxobuto-2-yl]amino}-5-oxopentanoate (65 mg, 0.067 mmol) in DCM (2.5 mL) was added at 0°C for 5 minutes. The resulting mixture was then allowed to rise naturally to 15°C and stirred for 1 hour. After the reaction was complete, as confirmed by LC-MS, the resulting mixture was concentrated to obtain compound 3e, which was used in the next step without purification (61.24 mg, yellow solid, quantitative yield).

[0231] LC-MS: (ESI)m / z(M+H), 914.4.

[0232] Step 3: Synthesis of tert-butyl(2S,5S,8S)-13,13-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-oate (compound 4e) [ka] compound 4e A mixture of compound 3e (61 mg, 0.067 mmol), HATU (30.45 mg, 0.080 mmol), HOBT (10.82 mg, 0.080 mmol), and DIEA (0.033 mL, 0.200 mmol) with DMF (2 mL) was stirred at 0°C for 5 minutes. Then, 2-methylpropan-2-yl 3-{[9-amino-9-(7,7-dimethyl-5-oxo-2,6-dioxaocta-1-yl)-2,2-dimethyl-4-oxo-3,7-dioxadeca-10-yl]oxy}propanoate (compound 2, 33.75 mg, 0.067 mmol) was added to the reaction mixture. The resulting mixture was allowed to rise naturally to 15°C and stirred for 1 hour until the reaction was complete, as confirmed by LC-MS. Subsequently, saturated saline solution (3 mL) was added to the reaction solution, and a large amount of solid was extracted. The resulting mixture was then filtered through a cloth funnel, the solid was washed with water, and the residue was dissolved in DCM (10 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4e (90 mg, brown solid, yield: 96.2%), which was used in the next step without purification.

[0233] LC-MS: (ESI)m / z(M+H), 617.3.

[0234] Step 4: Synthesis of (2S,5S,8S)-13,13-bis((2-carboxyethoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-onic acid (compound 5e) [ka] compound 5e TFA (0.5 mL, 0.064 mmol) in DCM (2.5 mL) is added to 2-methylpropan-2-yl3-{[(2S,5S,8S)-13,13-bis(7,7-dimethyl-5-oxo-2,6-dioxaocta-1-yl)-8-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-1-{[(9S)-9-ethyl-9-hydroxy-10,13-diox So-1,2,9,10,12,15-hexahydrothiino[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-2-methyl-1,4,7,11-tetraoxo-5-(prop-2-yl)-3,6,12-triazatetradeca-14-yl]oxy}propanoate (90 mg, 0.064 mmol) was added to a 2.5 mL solution of DCM at 0°C for 5 minutes. The resulting mixture was then allowed to rise naturally to 15°C and stirred for 1 hour, after which the solvent was removed under vacuum. The resulting residue was used in the next step without purification (80 mg, yellow solid, quantitative yield).

[0235] LC-MS:(ESI)m / z(1 / 2M+H),617.2.

[0236] Step 5: Synthesis of Compound (II')-1 3-{[(2S,5S,8S)-13,13-bis{[(2-carboxyethyl)oxy]methyl}-8-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,12,15-hexahydrothino[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-2-methyl-1,4,7,11-tetraoxo-5-(p A mixture of rop-2-yl)-3,6,12-triazatetradeca-14-yl]oxy}propanoic acid (80 mg, 0.065 mmol), HATU (76.46 mg, 0.201 mmol), HOBT (27.17 mg, 0.201 mmol), and DIEA (0.064 mL, 0.389 mmol) with DMF (2 mL) was stirred at 0°C for 20 minutes. Then, (2R,3R,4R,5S)-6-aminohexane-1,2,3,4,5-pentol (compound 3, 36.43 mg, 0.201 mmol) was added to the mixture. The resulting mixture was allowed to rise naturally to 15°C and stirred for 1 hour. The solvent was removed under vacuum, and the residue was purified by preparative HPLC (TFA) to obtain the title compound (II')-1 (11 mg, yellow solid, yield: 9.84%).

[0237] LC-MS:(ESI)m / z(1 / 2M+H),862.2.

[0238] 1HNMR(400MHz,DMSO):δ9.69(s,1H),8.35(d,J=6.4Hz,1H),8.02(d,J=8.0Hz,1H),7.91(d,J=9.2Hz,1H),7.78-7.76(m,4H),7.58(d,J =9.2Hz,1H),7.32(s,1H),7.13(s,1H),6.99(s,2H),6.52(s,1H),5.44(s,2H),5.29(s,2H) ,4.62-4.54(m,2H),4.29-4.21(m,4H),3.58-3.55(m,22H),3.50-3.43(m,11H),3.28-3.23( m,11H),3.13-2.96(m,5H),2.33(t,J=6.4Hz,6H),2.25-1.92(m,8H),1.91-1.83(m,3H),1.6 9-1.65(m,1H),1.55-1.46(m,4H),1.42-1.40(m,3H),1.32-1.20(m,4H),0.90-0.83(m,9H).

[0239] Preparation Example 1.6. (S)-N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-8,18-dioxo-13-((3-oxo-3-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propoxy)methyl)-11,15-dioxa-7,19-diazapentacosan-13-yl)-2-(6-(2,5-dioxo-2,5-dihydro- Synthesis of 1H-pyrrole-1-yl)hexanamide)-N1-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (compound (II')-2) [ka] [ka] Step 1: Synthesis of tert-butyl(S)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (compound 2f) [ka] 1-Methylimidazole (0.043 mL, 0.537 mmol) was added to a mixture of N-[(2S,5S)-12-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-{3-[(2-methylprop-2-yl)oxy]-3-oxopropyl}-1,4,7-trioxo-2-(prop-2-yl)-3,6-diazadodeca-1-yl]-L-alanine (compound 1f, 166.82 mg, 0.294 mmol) and CPT-2 (115 mg, 0.285 mmol) with DMF (1.9 mL), and then TCFH (86.04 mg, 0.307 mmol) was added to the mixture. The resulting mixture was then stirred at 25°C for 12 hours. As confirmed by LC-MS, after the reaction was complete, saturated brine (6 mL) was added to the reaction solution to extract a large amount of solid. The resulting mixture was then filtered through a cloth funnel, and the solid was washed with water. The residue was then dissolved in DCM (30 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound 2f, which was used in the next step without further purification (155 mg, brown solid, yield 63.6%).

[0240] LC-MS: (ESI)m / z(M+H), 954.5.

[0241] Step 2: Synthesis of (S)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (compound 3f) [ka]

[0242] 2,2,2-trifluoroacetic acid (0.85 mL, 8.1 mmol) was added to a solution of compound 2f (155 mg, 0.162 mmol) in DCM (4.25 mL) at 0°C for 5 minutes. The resulting mixture was allowed to rise naturally to 15°C and stirred for 2 hours. After the reaction was complete, as confirmed by LC-MS, the mixture was concentrated to obtain compound 3f, which was used in the next step without purification (145.88 mg, yellow solid, quantitative yield).

[0243] LC-MS: (ESI)m / z(M+1), 898.6.

[0244] Step 3: Synthesis of tert-butyl(2S,5S,8S)-13,13-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-oate (compound 4f) [ka] A mixture of compound 3f (145.88 mg, 0.162 mmol), HATU (74.13 mg, 0.195 mmol), HOBT (26.34 mg, 0.195 mmol), and DIEA (0.081 mL, 0.487 mmol) with DMF (4 mL) was stirred at 0°C for 5 minutes. Then, 2-methylpropan-2-yl 3-{[9-amino-9-(7,7-dimethyl-5-oxo-2,6-dioxaocta-1-yl)-2,2-dimethyl-4-oxo-3,7-dioxadeca-10-yl]oxy}propanoate (compound 2, 82.15 mg, 0.162 mmol) was added to the mixture, and the resulting mixture was allowed to rise naturally to 15°C and stirred for 1 hour. As confirmed by LC-MS, after the completion of the reaction, saturated saline solution (12 mL) was added to the reaction solution to extract a large amount of solid. The mixture was then filtered through a cloth funnel, the solid was washed with water, and the resulting residue was dissolved in DCM (10 mL). The organic layer was then dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound 4f, which was used in the next step without further purification (188 mg, brown solid, yield 83.5%).

[0245] LC-MS: (ESI)m / z(M+1), 1386.6.

[0246] Step 4: Synthesis of (2S,5S,8S)-13,13-bis((2-carboxyethoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-onic acid (compound 5f) [ka] 2,2,2-trifluoroacetic acid (0.769 mL, 7.327 mmol) was added to a solution of compound 4f (188 mg, 0.122 mmol) in DCM (3.18 mL) at 0°C for 5 minutes. The resulting mixture was allowed to rise naturally to 15°C and stirred for 2 hours. After the reaction was complete, as confirmed by LC-MS, the mixture was concentrated to obtain compound 5f, which was used in the next step without purification (148.65 mg, yellow solid, quantitative yield).

[0247] LC-MS: (ESI)m / z(M+1), 1217.6.

[0248] Step 5: Synthesis of Compound (II')-2 Compound 5f (133.785 mg, 0.110 mmol), HATU (158.81 mg, 0.418 mmol), HOBT (56.44 mg, 0.418 mmol), and DIEA (0.145 mL, 0.879 mmol) were mixed with DMF (2.8 mL) and stirred at 0°C for 20 minutes. Then, (2R,3R,4R,5S)-6-aminohexane-1,2,3,4,5-pentol (compound 3, 75.67 mg, 0.418 mmol) was added to the mixture. The resulting mixture was allowed to rise naturally to 15°C and stirred for 1 hour. The solvent was then removed under vacuum, and the residue was purified by preparative HPLC (TFA) to obtain the title compound (II')-2 (20.0 mg, yellow solid, yield: 12.0%).

[0249] LC-MS: (ESI) m / z 1706.8. 1707.8 (M+H).

[0250] 1HNMR(400MHz,DMSO):δ9.51(s,1H),8.48-8.44(m,2H),8.02(d,J=7.6Hz,1H),7.78(t,J=5.2Hz,3H),7.73(d,J=9.6Hz,1H),7.59(d,J=8.8Hz ,1H),7.32(s,1H),7.13(s,1H),6.99(s,2H),6.49(s,1H),5.44(s,2H ),5.28(s,2H),4.64-4.60(m,1H),4.54(t,J=5.6Hz,2H),4.30-4.20(m ,4H),3.61-3.46(m,12H),3.49-3.45(m,10H),3.42-3.36(m,17H),3. 33-3.24(m,8H),3.07-2.97(m,5H),2.35-2.32(m,6H),2.13-2.10(m,4 H),2.04-2.00(m,1H),1.91-1.84(m,3H),1.71-1.65(m,1H),1.51-1.4 6(m,5H),1.37(d,J=7.2Hz,3H),1.24-1.18(m,2H),0.91-0.84(m,9H).

[0251] Preparation Example 1.7. Synthesis of (2S,5S,8S)-13,13-bis((2-carboxyethoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-onic acid (compound (II')-3) [ka] [ka] Step 1: Synthesis of benzyl(tert-butoxycarbonyl)-L-valyl-L-alaninate (3g of compound) [ka] 3g of compound A mixture of N-{[(2-methylprop-2-yl)oxy]carbonyl}-L-valine (1 g, 5 g, 23.014 mmol of compound), DCC (5.70 g, 27.617 mmol), and HOBT (3.27 g, 24.165 mmol) with THF (40 mL) was stirred at 0°C for 30 minutes. Then, benzyl DL-alaninate hydrochloride (2 g, 4.47 g, 20.713 mmol of compound) and NMM (3.793 mL, 34.521 mmol) from THF (40 mL) were added to the mixture. The resulting mixture was allowed to rise naturally to 15°C and stirred for 12 hours. After the reaction was complete, as confirmed by TLC and LC-MS, the mixture was concentrated under reduced pressure, the residue was dissolved in DCM (200 mL), and the resulting solution was filtered. Next, the filtrate was partitioned into DCM (100 × 2 mL) and water (50 mL), and the aqueous layer was then extracted with DCM (50 mL). Subsequently, the combined organic layer was dried over sodium sulfate and filtered, and the resulting filtrate was concentrated under reduced pressure. Finally, the residue was purified by flash chromatography (160 g silica gel column, PE / DCM with DCM from 0 to 100%, and DCM / MeOH with MeOH from 0 to 5%) to obtain 3 g of the compound (6.0 g, white solid, yield: 68.9%).

[0252] LC-MS: (ESI)m / z(M+Na), 401.2.

[0253] Step 2: Synthesis of benzyl L-valyl-L-alaninate (4g of compound) [ka] 4g of compound Hydrogen chloride (61.564 mL, 246.255 mmol) was added at 0°C to a solution of 3 g (4.66 g, 12.313 mmol) of the compound in 1,4-dioxane (62 mL), and the mixture was then stirred at 0°C for 2 hours. After the reaction was complete, as confirmed by LC-MS, the resulting mixture was concentrated to obtain 4 g of the compound, which was used in the next step without purification (3.42 g, white solid, quantitative yield).

[0254] Step 3: Synthesis of benzyl(5S,8S,11S)-5-(3-(tert-butoxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-8-isopropyl-11-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-oate (6g of compound) [ka] 6g of compound A mixture of (2S)-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-5-[(2-methylprop-2-yl)oxy]-5-oxopentanoic acid (compound 4g, 5.20g, 12.215 mmol), HATU (4.88g, 12.826 mmol), and DIEA (6.057 mL, 36.645 mmol) with DMF (40 mL) was stirred at 0°C for 30 minutes, and then benzyl N-[(2S)-2-amino-3-methyl-1-oxobutyl]-L-alaninate (compound 5g, 3.4g, 12.215 mmol) was added to the mixture. The resulting mixture was allowed to rise naturally to 15°C and stirred for 12 hours. Upon completion of the reaction as confirmed by LC-MS, the mixture was concentrated under reduced pressure, and the resulting residue was partitioned into DCM (120 mL) and water (40 mL). Subsequently, the aqueous layer was extracted with DCM (40 mL). The combined organic layer was then dried over sodium sulfate and filtered, and the resulting filtrate was concentrated under reduced pressure. Finally, the residue was purified by flash chromatography (30 g silica gel column, PE / DCM with 0 → 100% DCM, and DCM / MeOH with 0 → 2% MeOH) to obtain 6 g of the title product, compound (6.5 g, white solid, yield: 77.6%).

[0255] LC-MS: (ESI)m / z(M+Na), 708.4.

[0256] Step 4: Synthesis of tert-butyl(S)-4-amino-5-(((S)-1-(((S)-1-(benzyloxy)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (7g of compound) [ka] 7g of compound Diethylamine (12.641 mL, 122.190 mmol) was added at 0°C to a solution of benzyl N-[(5S,8S)-1-(9H-fluoren-9-yl)-5-{3-[(2-methylprop-2-yl)oxy]-3-oxopropyl}-3,6,9-trioxo-8-(prop-2-yl)-4,7-diaza-2-oxanone-9-yl]-L-alaninate (8.38 g, 12.219 mmol) in DMF (12 mL), and the resulting mixture was stirred at 0°C for 2 hours. After the reaction was complete, as confirmed by LC-MS, the mixture was concentrated under reduced pressure, and the resulting residue was dissolved in DMF (6 mL). Next, the obtained solution was purified using a reversed-phase column (120g C18 gel column, H2O / CH3CN with CH3CN content reduced from 0% to 54%) to obtain 7g of the compound (3.6g, white solid, yield: 37.2%).

[0257] LC-MS: (ESI)m / z(M+Na), 464.2.

[0258] Step 5: Synthesis of ((2S,5S)-5-amino-8-(tert-butoxy)-2-isopropyl-4,8-dioxooctanoyl)-L-alanine (8g of compound) [ka] 8g of compound Palladium(0) (1.03 g, 0.971 mmol) was added to a solution of benzyl N-[(7S,10S)-7-amino-2,2-dimethyl-4,8,11-trioxo-10-(prop-2-yl)-9-aza-3-oxaoundec-11-yl]-L-alaninate (1.5 g, 3.236 mmol) in MeOH (60 mL), and the mixture was stirred at 15°C for 2 hours. After the reaction was complete, as confirmed by LC-MS, the mixture was filtered, and the resulting filtrate was concentrated to obtain 8 g of the title compound, which was used in the next step without purification (1.2 g, white solid, quantitative yield).

[0259] LC-MS: (ESI)m / z(M+H), 374.2.

[0260] Step 6: Synthesis of ((S)-5-(tert-butoxy)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-oxopentanoyl)-L-valyl-L-alanine (10g of compound) [ka] 10g of compound A mixture of N-[(7S,10S)-7-amino-2,2-dimethyl-4,8,11-trioxo-10-(prop-2-yl)-9-aza-3-oxaundeca-11-yl]-L-alanine (400 mg, 1.071 mmol) and DIEA (0.531 mL, 3.213 mmol) with DMF (8 mL) was stirred at 0°C for 5 minutes. 1-{6-[(2,5-dioxotetrahydro-1H-pyrrole-1-yl)oxy]-6-oxohexyl}pyrrole-2,5-dione (compound 9 g, 495.31 mg, 1.607 mmol) was added to the mixture, and the resulting mixture was stirred at 0°C for 1 hour. As confirmed by LC-MS, after the reaction was complete, the reaction solvent was removed under vacuum, and the residue was purified by preparative HPLC (C18, H2O containing 0.1% formic acid, with 20-60% acetonitrile) to obtain 10 g of the compound (265 mg, white solid, yield 44.5%).

[0261] LC-MS: (ESI)m / z(M+1), 567.4.

[0262] Step 7: Synthesis of tert-butyl(S)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (11g of compound) [ka] Compound 11g 1-Methylimidazole (0.048 mL, 0.599 mmol) is mixed with N-[(2S,5S)-12-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-{3-[(2-methylprop-2-yl)oxy]-3-oxopropyl}-1,4,7-trioxo-2-(prop-2-yl)-3,6-diazadodeca-1-yl]-L-alanine (228. Compound 4 (115 mg, 0.285 mmol) and (9S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,10,12,13,15-octahydrocyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione (compound 4, 115 mg, 0.285 mmol) were added to a mixture with DMF (2.2 mL). Then, TCFH (95.97 mg, 0.342 mmol) was added to the mixture, and the resulting mixture was stirred at 25°C for 12 hours. After the reaction was complete, as confirmed by LC-MS, saturated brine (10 mL) was added to the reaction solution to extract a large amount of solid. The resulting mixture was then filtered through a cloth funnel, the solid was washed with water, and the residue was dissolved in DCM (30 mL). Finally, the organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 11 g of the title compound, which was used in the next step without further purification (240 mg, brown solid, yield: 88.4%).

[0263] LC-MS: (ESI)m / z(M+H), 952.6.

[0264] Step 8: Synthesis of (S)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (12g of compound) [ka] Compound 12g 2,2,2-trifluoroacetic acid (0.132 mL, 1.260 mmol) is mixed with 2-methylpropan-2-yl(4S)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-5-{[(2S)-1-{[(2S)-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,12 ,15-Hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-1-oxoprop-2-yl]amino}-3-methyl-1-oxobuto-2-yl]amino}-5-oxopentanoate (20 mg, 0.021 mmol) was added to a solution of DCM (0.65 mL) at 0°C for 5 minutes. The resulting mixture was then heated naturally to 15°C and stirred for 1 hour. After the completion of the reaction, as confirmed by LC-MS, the mixture was concentrated to obtain 12 g of the compound, which was used in the next step without purification (18.82 mg, yellow solid, quantitative yield).

[0265] LC-MS: (ESI)m / z(M+1), 896.6.

[0266] Step 9: Synthesis of tert-butyl(2S,5S,8S)-13,13-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-oate (13g of compound) [ka] Compound 13g (4S)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-5-{[(2S)-1-{[(2S)-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline- A mixture of 4-yl]amino}-1-oxoprop-2-yl]amino}-3-methyl-1-oxobuta-2-yl]amino}-5-oxopentanoic acid (18.82 mg, 0.021 mmol), HATU (9.58 mg, 0.025 mmol), HOBT (3.41 mg, 0.025 mmol), and DIEA (0.010 mL, 0.063 mmol) with DMF (0.5 mL) was stirred at 0°C for 5 minutes. Compound 2 (10.62 mg, 0.021 mmol) was then added to the mixture, and the mixture was allowed to rise naturally to 15°C and stirred for 1 hour. After the reaction was complete, as confirmed by LC-MS, saturated brine (3 mL) was added to the reaction solution to extract a large amount of solid. The resulting mixture was filtered through a cloth funnel, and the solid was washed with water. The residue was then dissolved in DCM (10 mL), the organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 13 g of the title compound, which was used in the next step without further purification (28 mg, brown solid, yield: 96.3%).

[0267] Step 10: Synthesis of (II')-3 2,2,2-trifluoroacetic acid (0.057 mL, 0.540 mmol) is used to prepare 2-methylpropan-2-yl3-{[(2S,5S,8S)-13,13-bis(7,7-dimethyl-5-oxo-2,6-dioxaocta-1-yl)-8-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,12,15- Hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-2-methyl-1,4,7,11-tetraoxo-5-(prop-2-yl)-3,6,12-triazatetradeca-14-yl]oxy}propanoate (12 mg, 0.009 mmol) was added to a solution of DCM (0.345 mL) at 0°C for 5 minutes, and the mixture was then heated naturally to 15°C and stirred for 1 hour. The solvent was then removed under vacuum, and the residue was purified by preparative HPLC (TFA) to obtain compound (II')-3 (6.51 mg, yellow solid, yield: 26.5%).

[0268] LC-MS:(ESI)m / z(1 / 2M+H),608.4.

[0269] 1HNMR(400MHz,DMSO):δ12.15(s,3H),9.77(s,1H),8.34(d,J=6.8Hz,1H),8.00-7.95(m,2H),7.82(d,J=9.2Hz,1H),7.59(d,J =8.8Hz,1H),7.31(s,1H),7.09(s,1H),6.99(s,2H),6.50(s,1H),5.43(s,2H),5.26(s,2H),4.55-4.52(m,1H),4.28-4.19(m ,2H),3.57-3.54(m,9H),3.18-3.15(m,3H),2.99-2.95(m,3H),2.40(d,J=6.4Hz,6H),2.13-2.07(m,5H),2.04-2.01(m,3H), 1.89-1.84(m,4H),1.69-1.63(m,1H),1.51-1.45(m,4H),1.42-1.39(d,J=6.8Hz,3H),1.24-1.14(m,3H),0.90-0.84(m,9H).

[0270] Preparation Example 1.8. (S)-N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-8,18-Dioxo-13-((3-Oxo-3-(((2S,3R,4R,5R)-2,3,4,5,6-Pentahydroxyhexyl)amino)propoxy)methyl)-11,15-Dioxa-7,19-Diazapentacosan-13-yl)-2-(6-(2,5-Dioxo-2,5-Dihydro Synthesis of -1H-pyrrole-1-yl)hexaneamide)-N1-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (compound (II')-4) [ka] [ka] Step 1: Synthesis of (II')-4 3-{[(2S,5S,8S)-13,13-bis{[(2-carboxyethyl)oxy]methyl}-8-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-2-methyl-1,4,7,11-tetra A mixture of oxo-5-(prop-2-yl)-3,6,12-triazatetradeca-14-yl]oxy}propanoic acid (122.97 mg, 0.101 mmol), HATU (146.21 mg, 0.385 mmol), HOBT (51.96 mg, 0.385 mmol), and DIEA (0.134 mL, 0.809 mmol) with DMF (2 mL) was stirred at 0°C for 20 minutes. Compound 3 (69.67 mg, 0.385 mmol) was then added to the mixture, and the mixture was allowed to rise naturally to 15°C and stirred for 1 hour. The solvent was removed under vacuum, and the residue was purified by preparative HPLC (TFA) to obtain compound (II')-4 (35.2 mg, yellow solid, yield: 20.4%).

[0271] LC-MS:(ESI)m / z(1 / 2M+H),853.2.

[0272] 1HNMR(400MHz,DMSO):δ9.78(s,1H),8.35(d,J=6.8Hz,1H),8.02(d,J=7.2Hz,1H),7.96(d,J=9.2Hz,1H),7.82(d,J=9.2Hz,1H),7.77(t,J=5.6 Hz,3H),7.60(d,J=8.8Hz,1H),7.31(s,1H),7.13(s,1H),6.99(s,2H), 6.50(s,1H),5.43(s,2H),5.27(s,2H),4.58-4.49(m,2H),4.28-4.18(m ,4H),3.60-3.48(m,14H),3.49-3.45(m,8H),3.42-3.34(m,13H),3.33 -3.24(m,6H),3.18-3.15(m,3H),3.07-2.93(m,7H),2.33(t,J=6.8Hz, 6H),2.15-1.97(m,8H),1.91-1.82(m,3H),1.70-1.65(m,1H),1.52-1. 45(m,4H),1.40(d,J=6.8Hz,3H),1.24-1.18(m,4H),0.90-0.83(m,9H).

[0273] Preparation Example 1.9. Synthesis of Compound LD-12((2S,5S,8S)-13-((2-carboxyethoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-onic acid) (Compound (II')-5) [ka] [ka] Step 1: Synthesis of (S)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (12g of compound) [ka] Compound 12g 2,2,2-trifluoroacetic acid (0.132 mL, 1.260 mmol) is mixed with 2-methylpropan-2-yl(4S)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-5-{[(2S)-1-{[(2S)-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,12 ,15-Hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-1-oxoprop-2-yl]amino}-3-methyl-1-oxobuto-2-yl]amino}-5-oxopentanoate (20 mg, 0.021 mmol) was added to a solution of DCM (0.65 mL) at 0°C for 5 minutes. The resulting mixture was then allowed to rise naturally to 15°C and stirred for 1 hour. After the reaction was complete, as confirmed by LC-MS, the mixture was concentrated to obtain 12 g of the compound, which was used in the next step without purification (18.82 mg, yellow solid, quantitative yield).

[0274] LC-MS: (ESI)m / z(M+1), 896.6.

[0275] Step 2: Synthesis of tert-butyl(2S,5S,8S)-13-((3-(tert-butoxy)-3-oxopropoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-oate (compound 1h) [ka] compound 1h (4S)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-5-{[(2S)-1-{[(2S)-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline A mixture of -4-yl]amino}-1-oxoprop-2-yl]amino}-3-methyl-1-oxobuta-2-yl]amino}-5-oxopentanoic acid (18.82 mg, 0.021 mmol), HATU (9.58 mg, 0.025 mmol), HOBT (3.41 mg, 0.025 mmol), and DIEA (0.010 mL, 0.063 mmol) with DMF (1 mL) was stirred at 0°C for 5 minutes. Compound 5 (7.30 mg, 0.021 mmol) was then added to the mixture, and the resulting mixture was allowed to rise naturally to 15°C and stirred for 1 hour. After the reaction was complete, as confirmed by LC-MS, saturated brine (3 mL) was added to the reaction solution to extract a large amount of solid. The mixture was then filtered through a cloth funnel, and the solid was washed with water. Subsequently, the obtained residue was dissolved in DCM (10 mL), and the organic layer was then dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1h, which was used in the next step without further purification (24 mg, brown solid, yield 93.2%).

[0276] LC-MS: (ESI)m / z(M+1), 1226.6.

[0277] Step 3: Synthesis of (2S,5S,8S)-13-((2-carboxyethoxy)methyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-onic acid ((II')-5) 2,2,2-trifluoroacetic acid (0.126 mL, 1.200 mmol) is used to prepare 2-methylpropan-2-yl3-{[(2S,5S,8S)-13-(7,7-dimethyl-5-oxo-2,6-dioxaocta-1-yl)-8-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-oxohexyl]amino}-1-{[(9S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3, 9,10,12,15-Hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl]amino}-2-methyl-1,4,7,11-tetraoxo-5-(prop-2-yl)-3,6,12-triazatetradeca-14-yl]oxy}propanoate (24 mg, 0.020 mmol) was added to a solution of DCM (0.63 mL) at 0°C for 5 minutes. The resulting mixture was then allowed to rise naturally to 15°C and stirred for 1 hour, after which the solvent was removed under vacuum. The resulting residue was then purified by preparative HPLC (TFA) to obtain compound (II')-5 (5.13 mg, yellow solid, yield: 23.5%).

[0278] LC-MS: (ESI)m / z(M+H), 1113.6.

[0279] 1HNMR(400MHz,DMSO)δ12.17(s,2H),9.78(s,1H),8.30(d,J=6.4Hz,1H),8.00-7.95(m,2H),7.82(d,J=9.2Hz,1H),7.67(d,J =8.4Hz,2H),7.31(s,1H),6.99(s,2H),6.50(s,1H),5.43(s,2H),5.26(s,2H),4.58-4.50(m,1H),4.27-4.21(m,2H),3.95- 3.93(m,1H),3.59-3.51(m,5H),3.18-3.15(m,2H),2.99-2.96(m,2H),2.44-2.41(m,4H),2.17-2.07(m,5H),2.03-2.01(m, 4H),1.91-1.84(m,3H),1.73-1.70(m,1H),1.51-1.45(m,4H),1.39(d,J=7.2Hz,3H),1.23-1.20(m,5H),0.89-0.83(m,9H).

[0280] Example 2. Antibody preparation Preparation of anti-HER2 antibodies The antibodies described herein can be prepared according to conventional antibody methods. For example, the variable region sequence was cloned into a vector containing a sequence encoding the human IgG1 constant region for antibody expression. The binding affinity of the expressed antibody was verified using FACS.

[0281] The light and heavy chain sequences of trastuzumab are shown below.

[0282] Light chain (Sequence ID 1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0283] Heavy chain (SEQ ID NO: 2) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0284] Preparation of anti-TROP2 / EGFR bispecific antibodies Anti-TROP2 / EGFR bispecific antibodies possess an anti-TROP2 antigen-binding domain (T-6F7, VH SEQ ID NO: 3, VL SEQ ID NO: 4) and an anti-EGFR antigen-binding domain (E-6C4, VH SEQ ID NO: 5, VL SEQ ID NO: 4). These antigen-binding domains can pair to form a bispecific antibody (e.g., T-6F7-E-6C4).

[0285] Vectors encoding the light and heavy chains of anti-TROP2 / EGFR antibodies were constructed. Three vectors were co-transduced into CHO-S cells: a first vector encoding the heavy chain of the anti-TROP2 binding arm, a second vector encoding the heavy chain of the anti-EGFR binding arm, and a third vector encoding a common light chain. After 14 days of culture, the cell supernatant was collected and purified by protein A affinity chromatography. Knob-in-hole mutations were introduced into the Fc region of the heavy chains of the anti-TROP2 and anti-EGFR arms. In T-6F7-E-6C4, the knob mutation is contained in the constant region of the heavy chain of T-6F7, and the hole mutation is contained in the constant region of the heavy chain of E-6C4.

[0286] The sequences of the light chain constant region, the heavy chain constant region with a knob mutation, and the heavy chain constant region with a hole mutation are shown in SEQ ID NOs: 6, 7, and 8, respectively.

[0287] Example 3. Preparation of ADC The prepared tris(2-carboxyethyl)phosphine aqueous solution was added to the trastuzumab antibody analog in 1X PBS buffer solution (pH 7.4) (TCEP:antibody = 8:1 (molar ratio)). The mixture was placed in a water bath shaker and shaken at 22°C for 2.5 hours, after which the reaction was terminated.

[0288] Compounds (II')-1, (II')-2, (II')-3, (II')-4, (II')-5, VA-AZ0132 (Mal-PEG8-amide-Val-Ala-(4-NH2)-Exatecan, MedChemExpress, Catalog No.: HY-145399), GGFG-Dxd (Deruxtecan, MedChemExpress, Cat#: HY-13631E), or VC-MMAE (MedChemExpress, HY-15575) were dissolved in DMSO (the molar ratio of the compound to the antibody is shown in the table below), and the resulting solutions were added to the respective solutions. The resulting mixtures were then placed in a water bath shaker and shaken at 25°C for 1.5 hours, after which the reaction was terminated. Subsequently, the reaction solution was desalted, and excess, unconjugated compounds were removed by changing the buffer solution using a Sephadex G25 gel column or UF / DF. Finally, T-(II')-1, T-(II')-2, T-(II')-2(DAR4), T-(II')-3, T-(II')-4, T-(II')-5, T-VA-AZ0132, and T-GGFG-Dxd were obtained and stored at -20°C / -80°C. The average n was calculated by LC-MS. In the control group, the trastuzumab antibody analog was replaced with human IgG1 protein (hIgG1) to obtain hIgG1-(II')-1, hIgG1-(II')-2, hIgG1-(II')-3, hIgG1-(II')-4, hIgG1-(II')-5, hIgG1-VA-AZ0132, or hIgG1-GGFG-Dxd, respectively. Details are shown in the table below.

[0289] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0290] In another similar experiment, purified antibody T-6F7-E-6C4 was coupled with compounds (II')-1, (II')-2, (II')-3, (II')-4, or (II')-5. The antibody-drug conjugate name is preceded by (II')-1, (II')-2, (II')-3, (II')-4, or (II')-5. For example, when T-6F7-E-6C4 is coupled with compound (II')-1, it is named T-6F7-E-6C4-(II')-1. As another example, when T-6F7-E-6C4 is coupled with (II')-2, it is named T-6F7-E-6C4-(II')-2. Examples of ADCs obtained by this method include T-6F7-E-6C4-(II')-1, T-6F7-E-6C4-(II')-2, T-6F7-E-6C4-(II')-3, T-6F7-E-6C4-(II')-4, and T-6F7-E-6C4-(II')-5.

[0291] Mass spectrometry (MS) was used to detect the coupling between antibodies and drug molecules. Human IgG1 molecules were coupled to (II')-1, (II')-2, (II')-3, (II')-4, or (II')-5 to form isotype controls: isotype-(II')-1 (ISO-(II')-1), isotype-(II')-2 (ISO-(II')-2), isotype-(II')-3 (ISO-(II')-3), isotype-(II')-4 (ISO-(II')-4), or isotype-(II')-5 (ISO-(II')-5). The results showed that the drug-antibody ratio (DAR) of the ADCs was approximately 4 or 8. Regarding ADC naming, if the DAR of T-6F7-E-6C4-(II')-2 is approximately 4, the ADC will be named T-6F7-E-6C4-(II')-2(DAR4). If the DAR of T-6F7-E-6C4-(II')-2 is approximately 8, the ADC will be named T-6F7-E-6C4-(II')-2(DAR8).

[0292] In another similar experiment, the purified antibody T-6F7-E-6C4 was coupled to MMAE (monomethyl auristatin E) using a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. When an antibody couples with MMAE, "ADC" is added immediately after the antibody name to the antibody-drug conjugate name. For example, if T-6F7-E-6C4, which has a constant IgG1 region, couples with MMAE, it is named T-6F7-E-6C4-ADC.

[0293] Example 4. In vitro lethal activity Various human tumor cell lines, mouse tumor cell lines, rat tumor cell lines, and canine tumor cell lines cultured in cell culture plates were treated with different concentrations of compounds CPT-1, CPT-2, CPT-3, CPT-4, Dxd (MedChem Express, catalog number: HY-13631D), and SN38 (MedChem Express, catalog number: HY-13704). The killing activity was detected 72–120 hours after incubation using IncuCyte (Sartorius AG, IncuCyte® S3) or a MicroplateReader. The results are summarized in the table below.

[0294] [Table 5] ("N / A" means no detection, and "-" means no killings.) The results showed that CPT-1 and CPT-2 exhibited good in vitro killing activity in multiple cell lines and showed higher tumor cell inhibitory effects than Dxd and SN38.

[0295] Example 5. Antitumor activity in a human pancreatic PDX model The effect of ADC on tumor growth in a human pancreatic tumor PDX model was tested. Immunofluorescence staining was performed on patient-derived tumor fragments, and images were analyzed using the HALO3.2 version. The results showed that Her2-positive cells constitute 86.34% of all cells in human pancreatic tumor tissue.

[0296] Specifically, B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., catalog number: B-CM-002) were transplanted into the right flank of each mouse along with a patient-derived pancreatic tumor fragment (2 mm × 2 mm × 2 mm). When the tumor in the mice reached a volume of approximately 200-300 mm³, the mice were randomly assigned to different groups based on tumor volume. Next, the mice were injected intravenously (iv) with PBS or ADC at doses of 3 mg / kg or 6 mg / kg (a total of one dose). Details are shown in the table below.

[0297] [Table 6]

[0298] The long and short axis lengths of the tumors were measured, and the tumor volume was calculated as 0.5 × (long axis) × (short axis)². Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0. A t-test was performed for statistical analysis. A TGI of over 60% indicates a clear suppression of tumor growth. P < 0.05 is the threshold for statistical significance. Mouse body weight was also measured throughout the entire administration period.

[0299] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and the statistical difference (P-value) of tumor volume between the treatment group and the control group on the day of group assignment (Day 0), 14 days after group assignment (Day 14), and 21 days after group assignment (Day 21).

[0300] [Table 7]

[0301] The results showed that T-(II')-4 has a favorable inhibitory effect on human pancreatic tumors, inhibiting tumor growth at a higher TGI% than the positive control T-GGFG-Dxd.

[0302] Furthermore, T-VC-MMAE was also used as a control in the experiment, and the TGI% at dose levels of 3 mg / kg and 6 mg / kg were 44.9% and 60.7%, respectively, on day 21. This indicates that T-(II')-4 showed a better tumor inhibitory effect than T-VC-MMAE.

[0303] Example 6. Antitumor activity in a human milk PDX model The effect of ADC on tumor growth in a human breast tumor PDX model was tested. Immunofluorescence staining was performed on patient-derived tumor fragments, and images were analyzed using the HALO3.2 version. The results showed that Her2-positive cells constitute 0.65% of all cells in human breast tumor tissue.

[0304] Specifically, tumor fragments (2 mm × 2 mm × 2 mm) derived from patients were transplanted into the right flank of B-NDG mice. When the tumors in the mice reached a volume of approximately 200-300 mm³, the mice were randomly assigned to different groups based on tumor volume. Next, the mice were injected intravenously (iv) with PBS or ADC at a dose of 6 mg / kg. The administration frequency was once every two weeks (a total of two doses). Details are shown in the table below.

[0305] [Table 8]

[0306] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and the statistical difference (P-value) of tumor volume between the treatment group and the control group at the day of group assignment (Day 0), 18 days after group assignment (Day 18), and 28 days after group assignment (Day 28).

[0307] [Table 9]

[0308] The results showed that T-(II')-4 has a good inhibitory effect on human breast tumors, inhibiting tumor growth at a higher TGI% than the positive control T-GGFG-Dxd.

[0309] Example 7. Antitumor activity in a human PDX model The effects of ADCs on tumor growth in human lung, colorectal, or gastric tumor PDX models were tested. Immunofluorescence staining was performed on patient-derived tumor fragments, and images were analyzed using the HALO3.2 version.

[0310] Specifically, patient-derived lung, colorectal, or gastric cancer tumor fragments (2mm x 2mm x 2mm) were transplanted into the right flank of B-NDG mice. When the tumors in the mice reached a volume of approximately 200-300 mm³, the mice were randomly assigned to different groups based on their tumor volume. Next, mice were injected intravenously (iv) at doses of 3 mg / kg or 6 mg / kg with T-(II')-1, T-(II')-2, T-(II')-3, T-(II')-4, T-(II')-5, T-VA-AZ0132, T-GGFG-Dxd, hIgG1-(II')-1, hIgG1-(II')-2, hIgG1-(II')-3, hIgG1-(II')-4, hIgG1-(II')-5, hIgG1-VA-AZ0132, or hIgG1-GGFG-Dxd.

[0311] T-(II')-1, T-(II')-2, T-(II')-3, T-(II')-4, and T-(II')-5 demonstrated good tumor inhibitory effects.

[0312] Antitumor activity in a human lung cancer PDX model Immunofluorescence staining of patient-derived tumor fragments showed that HER2-positive cells constituted 89.49% of all cells in human lung tumor tissue. Mice were injected intravenously (iv) with PBS or ADC at a dose of 6 mg / kg (a total of one dose). Details are shown in the table below.

[0313] [Table 10]

[0314] The table below shows tumor volume, TGI (%), and the statistical difference in tumor volume (P-value) between the treatment group and the control group on the day of group assignment (day 0), 13 days after group assignment (day 13), and 23 days after group assignment (day 23). These results indicate that T-(II')-1, T-(II')-2, T-(II')-2(DAR4), and T-(II')-4 all exhibit dose-dependent tumor inhibitory effects in human lung tumors. Furthermore, T-(II')-1, T-(II')-2, and T-(II')-4 showed better tumor inhibitory effects than T-GGFG-Dxd at both 3 mg / kg and 6 mg / kg doses.

[0315] [Table 11]

[0316] Antitumor activity in a human colorectal cancer PDX model Immunofluorescence staining of patient-derived tumor fragments showed that HER2-positive cells constituted 13.28% of all cells in human colon tumor tissue. Mice were administered PBS or ADC intravenously (iv) at a dose of 6 mg / kg (a total of one dose). Details are shown in the table below.

[0317] [Table 12]

[0318] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and the statistical difference (P-value) of tumor volume between the treatment group and the control group at the day of group assignment (Day 0), 14 days after group assignment (Day 14), and 28 days after group assignment (Day 28).

[0319] [Table 13]

[0320] The results showed that T-(II')-1, T-(II')-2, T-(II')-2(DAR4), and T-(II')-4 all exhibited good tumor inhibitory effects in human colorectal tumors. Furthermore, T-(II')-1, T-(II')-2, and T-(II')-4 showed better tumor inhibitory effects than T-GGFG-Dxd at a dose level of 6 mg / kg.

[0321] Antitumor activity in a human gastric cancer PDX model Immunofluorescence staining of patient-derived tumor fragments showed that HER2-positive cells constitute 0.08% of all cells in human gastric tumor tissue. Mice were injected intravenously (iv) with PBS or ADC at a dose of 6 mg / kg (a total of one dose). The tumor sizes in the groups treated with PBS or ADC are shown in Figure 2. These results indicate that T-(II')-1, T-(II')-2, T-(II')-2(DAR4), and T-(II')-4 all show good tumor inhibitory effects in human gastric cancer.

[0322] Example 8. Antitumor activity in a Hep-G2 xenograft model In a xenograft model of human liver cancer, ADCs were tested for their effect on tumor growth in vivo. Specifically, approximately 1 × 10⁻⁶ 7Hep-G2 cells (ATCC, catalog number: HB-8065) ​​were subcutaneously injected into B-NDG mice. Tumors in the mice were approximately 300 mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on their tumor volume. Next, the mice were injected with either PBS or ADC intravenously (iv). Further details are shown in the table below.

[0323] [Table 14]

[0324] The tumor sizes in the groups treated with PBS or ADC are shown in Figure 3. These figures demonstrate that T-(II')-1, T-(II')-2, T-(II')-2(DAR4), and T-(II')-4 all show significant tumor inhibitory effects in human liver cancer. However, hIgG1-(II')-1, hIgG1-(II')-2, and hIgG1-(II')-4 did not show significant tumor inhibitory effects. Furthermore, T-(II')-2(DAR4) showed a good tumor inhibitory effect in a dose-dependent manner.

[0325] Example 9. Pharmacokinetic profile and plasma stability The pharmacokinetic clearance rates of anti-HER2 ADCs were measured in C57BL / 6 mice. Specifically, the mice were divided into nine groups (8 mice per group), and administered intravenously either hIgG1-GGFG-Dxd (G1, 3 mg / kg), T-GGFG-Dxd (G2, 3 mg / kg, G3, 10 mg / kg), hIgG1-(II')-4 (G4, 3 mg / kg, G5, 10 mg / kg), T-(II')-4 (G6, 3 mg / kg, G7, 10 mg / kg), or a trastuzumab analog (G8, 3 mg / kg, G9, 10 mg / kg). Blood samples were collected before administration and at 15 minutes, 4 hours, 24 hours, 72 hours, 10 days, 14 days, and 21 days after administration. Serum concentrations of all antibodies were detected by sandwich ELISA, and the free payload was detected by mass spectrometry (MS).

[0326] The results are shown in the table below and in Figures 4A-4D, which indicate that T-(II')-4 has a longer half-life than T-GGFG-Dxd. Furthermore, at a dose level of 3 mg / kg, the free payload in group G6 was detectable only 15 minutes after administration, while in group G2, the free payload was still detectable 4 hours after administration. At a dose level of 10 mg / kg, the free payload in group G7 was also detectable 4 hours after administration. On the other hand, in group G3, the payload was still detectable 72 hours after administration. These results indicate that T-(II')-4 exhibits better stability than T-GGFG-Dxd.

[0327] [Table 15]

[0328] In another experiment, the plasma stability of T-(II')-1, T-(II')-2, and T-(II')-4 was measured in human plasma, monkey (cynomolgus monkey) plasma, and rat (SD rat) plasma. Specifically, T-(II')-1, T-(II')-2, T-(II')-4, or T-GGFG-Dxd was added to human, monkey, or rat plasma to a final concentration of 100 μg / mL, respectively. In the control group, the plasma was replaced with PBS containing 0.5% BSA. The content of free payload and ADC was measured at 0, 1, 2, 6, 8, 11, and 14 days after ADC addition, and the ratio of free payload to total payload was calculated. The results are shown in Figures 5A-5C. These studies showed that T-(II')-1, T-(II')-2, and T-(II')-4 are relatively stable in human, monkey, and rat plasma, and have a maximum release rate of free CPT2 of 2.0% or less.

[0329] Example 10. Antibody-drug conjugate Lethal activity outside the body HCC827 cells, NCI-H292 cells, A431 cells, or Panc 02.03 cells cultured in cell culture plates were treated with purified antibodies or ADCs at various concentrations. After 7 days of incubation, the killing activity was detected using the CellCounting-Lite 2.0 Kit Luminescent Cell Viability Assay (Vazyme Biotech Co., Ltd., catalog number: DD1101-02). The results are shown in the table below.

[0330] Sacituzumab govitecan, manufactured by Immunomedics, Inc., is a humanized anti-TROP2 monoclonal antibody drug conjugate.

[0331] Cetuximab is an EGFR-targeted chimeric monoclonal IgG1 antibody manufactured by Merck.

[0332] [Table 16] ("NA" means it does not have in vitro lethal activity, and "-" means it has not been tested.) The above results demonstrate that T-6F7-E-6C4-(II')-2(DAR8) exhibits good in vitro killing activity in HCC827 cells, NCI-H292 cells, A431 cells, and Panc 02.03 cells.

[0333] Internalization of anti-TROP2 / EGFR bispecific antibodies and ADCs A431 cells or NCI-H292 cells cultured in cell culture plates were treated with anti-TROP2 / EGFR bispecific antibodies and ADCs (as shown in the table below). Endocrine activity was monitored using IncuCyte (Sartorius AG, IncuCyte® S3) for 24 hours after incubation, with images captured every hour. The results are shown in Figures 6A-6B, which indicate that the endocytosis activity of T-6F7-E-6C4-(II')-2(DAR4), T-6F7-E-6C4-(II')-2(DAR8), and T-6F7-E-6C4 was better than that of sacituzumab govitecan and cetuximab.

[0334] [Table 17]

[0335] The binding activity of anti-TROP2 / EGFR bispecific antibodies and ADCs. This experiment was conducted to test the binding activity of anti-TROP2 / EGFR bispecific antibodies and ADCs to tumor cell lines.

[0336] Specifically, A431 cells or human lung cancer HCC827 cells (ATCC, catalog number: CRL-2868) are divided into 2 × 10⁻¹⁶ cells. 5Cells were transferred to 96-well plates at a cell / well density. Serially diluted anti-TROP2 / EGFR bispecific antibody or ADC (maximum concentration: 130 nM, 2-fold serial dilution for 9 gradients) was added to the 96-well plates and incubated at 4°C for 25-30 minutes. Subsequently, the cells were incubated with the secondary antibody Alexa Fluor® 647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specificity (Jackson Immuno Research Laboratories, Inc., catalog number: 109-606-170) at 4°C in a dark room for 25-30 minutes, followed by flow cytometry analysis. The results shown in the table below demonstrate that T-6F7-E-6C4-(II')-2(DAR4), T-6F7-E-6C4-(II')-2(DAR8), and T-6F7-E-6C4 can bind to A431 cells and HCC827 cells with high affinity.

[0337] [Table 18]

[0338] Example 11. Antitumor activity in a patient-derived xenograft model of breast cancer Tumor tissue fragments (2mm x 2mm x 2mm) derived from breast cancer patients were transplanted into the right flank of B-NDG mice. Immunofluorescence staining of the patient-derived breast tumor fragments was performed, and images were analyzed using HALO 3.2. The results showed that EGFR-positive cells and TROP2-positive cells accounted for 96.92% and 49.87% of the tumor fragments, respectively. The tumor size in the mice was approximately 200-300mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on their tumor volume. Subsequently, the mice were injected with either PBS or ADC via intravenous administration. Details of the administration schedule, route, and frequency are shown in the table below.

[0339] [Table 19]

[0340] The table below summarizes the results of this experiment, including tumor volume, TGI (%), the percentage of tumor-free mice at the day of group assignment (day 0), 17 days after group assignment (day 17), and 35 days after group assignment (day 35), as well as the statistical difference (P-value) in tumor volume between the treatment group and the control group at day 35.

[0341] [Table 20] The tumor sizes in the groups treated with PBS or ADC are shown in Figure 7. The results showed that both T-6F7-E-6C4-(II')-2 with DAR4 and DAR8 exhibited good tumor inhibitory effects in a dose-dependent manner. Furthermore, T-6F7-E-6C4-(II')-2 with DAR4 showed better tumor inhibitory effects than T-6F7-E-6C4-ADC at a dose of 3 mg / kg.

[0342] Example 12. Antitumor activity in a patient-derived pancreatic cancer xenograft model. Tumor tissue fragments (2mm x 2mm x 2mm) derived from pancreatic cancer patients were transplanted into the right flank of B-NDG mice. Immunofluorescence staining revealed that EGFR-positive cells and TROP2-positive cells accounted for 71.08% and 89.09% of the pancreatic tumor fragments, respectively. The tumor size in the mice was approximately 200-300mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on their tumor volume. Subsequently, the mice were injected with either PBS or ADC intravenously. Further details are shown in the table below.

[0343] [Table 21]

[0344] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and the statistical difference (P-value) of tumor volume between the treatment group and the control group at the day of group assignment (Day 0), 14 days after group assignment (Day 14), and 32 days after group assignment (Day 32).

[0345] [Table 22]

[0346] The results showed that T-6F7-E-6C4-(II')-2, which possesses both DAR4 and DAR8, exhibits a dose-dependent tumor inhibitory effect.

[0347] Example 13. Antitumor activity in the SKOV-3 xenograft model In a xenograft model of ovarian adenocarcinoma, ADC was tested for its effect on tumor growth in vivo. Specifically, approximately 5 × 10⁻⁶ 6 SKOV-3 cells (ATCC, catalog number: HTB-77) were subcutaneously injected into B-NDG mice. Tumors in the mice were approximately 300 mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on their tumor volume. Next, the mice were injected with either PBS or ADC intravenously (iv). Further details are shown in the table below.

[0348] [Table 23]

[0349] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and the statistical difference (P-value) of tumor volume between the treatment group and the control group at the day of group assignment (Day 0), 17 days after group assignment (Day 17), and 35 days after group assignment (Day 35).

[0350] [Table 24]

[0351] The tumor sizes in the groups treated with PBS or ADC are shown in Figure 8. The results showed that both T-6F7-E-6C4-(II')-2 with DAR4 and DAR8 exhibited dose-dependent tumor inhibitory effects in the ovarian adenocarcinoma model, and that T-6F7-E-6C4-(II')-2(DAR8) showed better tumor inhibitory effects than T-6F7-E-6C4-(II')-2(DAR4).

[0352] Example 14. Antitumor activity in A431 xenograft model In a xenograft model of epidermoid carcinoma, ADC was tested for its effect on tumor growth in vivo. Specifically, approximately 1 × 10⁻⁶ 6 A431 cells were subcutaneously injected into B-NDG mice. Tumors in the mice were approximately 200 mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on their tumor volume. Next, the mice were injected intravenously (iv) with PBS, antibody, or ADC. Further details are shown in the table below.

[0353] [Table 25]

[0354] Body weight was measured twice a week. During the experiment, body weight increased in all groups, and there were no significant differences in body weight between groups, indicating that the test ADC was well-tolerated and clearly non-toxic to mice.

[0355] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and the statistical difference (P-value) of tumor volume between the treatment group and the control group on the day of group assignment (Day 0), 17 days after group assignment (Day 17), and 31 days after group assignment (Day 31).

[0356] [Table 26]

[0357] The tumor sizes in the groups treated with PBS, antibody, or ADC are shown in Figure 9. The results showed that both T-6F7-E-6C4-(II')-2 with DAR4 and DAR8 demonstrated better tumor inhibition than sacituzumab govitecan or cetuximab, in a dose-dependent manner. Furthermore, when the experiment was continued up to 49 days after group assignment (day 49), T-6F7-E-6C4-(II')-2 with DAR4 and DAR8 at doses of 6 mg / kg or 10 mg / kg still showed tumor inhibition.

[0358] Example 15. Antitumor activity in the NCI-H292 xenograft model In a xenograft model of lung cancer, antibodies or ADCs were tested for their effects on tumor growth in vivo. Specifically, approximately 5 × 10⁻⁶ 6 NCI-H292 cells were subcutaneously injected into B-NDG mice. Tumors in the mice were approximately 300 mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on their tumor volume. Next, the mice were injected intravenously (iv) with PBS, antibody, or ADC. Further details are shown in the table below.

[0359] [Table 27]

[0360] The table below summarizes the results of this experiment, including tumor volume, mouse survival rate, TGI (%), and statistical difference (P-value) of tumor volume between the treatment group and the control group at the day of group assignment (Day 0), 21 days after group assignment (Day 21), and at the end of the experiment (Day 39).

[0361] [Table 28] The results showed that both T-6F7-E-6C4-(II')-2, possessing DAR4 and DAR8, exhibited favorable tumor inhibitory effects in a lung cancer model, depending on the dose. Furthermore, both T-6F7-E-6C4-(II')-2, possessing DAR4 and DAR8, showed favorable tumor inhibitory effects at a dose level of 10 mg / kg, with a higher TGI than sacituzumab govitecan or cetuximab.

[0362] Example 16. Antitumor activity in a patient-derived xenograft model The effect of T-6F7-E-6C4-(II')-2(DAR8) on tumor growth was tested in head and neck squamous cell carcinoma models, esophageal cancer models, colorectal cancer models, and gastric cancer models. Specifically, BALB / c nude mice were transplanted into patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm). The tumor size in the mice was approximately 100-200 mm. 3 Once the tumor volume reached a certain level, the mice were randomly assigned to different groups based on tumor volume (3 mice per group). The mice were then injected with either physiological saline (G1, control) or 6 mg / kg of T-6F7-E-6C4-(II')-2(DAR8)(G2) (a total of 1 injection).

[0363] Immunohistochemical (IHC) staining was performed on tumor tissues from various patients. The table below shows the histochemical scores for EGFR or TROP2 expression levels in patient-derived tumor tissues. Table 26 below also summarizes the TGI (%) for various patient-derived xenograft models.

[0364] [Table 29]

[0365] The tumor sizes in the groups treated with physiological saline or T-6F7-E-6C4-(II')-2(DAR8) are shown in Figures 10A-10F. These figures demonstrate that T-6F7-E-6C4-(II')-2(DAR8) exhibits favorable tumor growth inhibitory effects in head and neck squamous cell carcinoma, esophageal cancer, colorectal cancer, and gastric cancer.

[0366] Example 17. Pharmacokinetic profile and plasma stability The pharmacokinetic clearance rate of anti-EGFR / TROP2 bispecific ADCs was measured in B-NDG mice. Specifically, approximately 1 × 10⁻⁶ 6 A431 cells were subcutaneously injected into B-NDG mice. Tumors in the mice were approximately 300 mm. 3 Once the tumor volume was reached, the mice were randomly divided into different groups (3 mice per group) based on tumor volume, and then administered intravenously with PBS (G2), T-6F7-E-6C4-(II')-2(DAR4) (G3-G10, 10 mg / kg), or T-6F7-E-6C4-(II')-2(DAR8) (G11-G18, 10 mg / kg) (a total of one dose). Group G1 was used as a blank control. Blood and tumor tissue samples from mice in groups G3-G10 and G11-G18 were collected 15 minutes, 2 hours, 6 hours, 1 day, 3 days, 5 days, 7 days, and 14 days after administration. Blood samples and tumor tissue from mice in Group G1 were collected one hour before administration, while blood samples and tumor tissue from mice in Group G2 were collected 14 days after administration. Using these collected samples, total antibody levels in serum and tumor tissue were detected by sandwich ELISA, and the free payload was detected by mass spectrometry (MS).

[0367] Total antibody levels were measured by sandwich ELISA. Briefly, goat anti-human IgG(H+L) (Jackson ImmunoResearch Inc., catalog number: 109-005-088) was diluted to a final concentration of 2000 ng / mL, added to a 96-well plate (ELISA plate) at 100 μL / well, and then incubated overnight at 2-8°C. After incubation, the plate was refrigerated in PBS-T buffer (Tween TMThe plates were washed four times with PBS-T buffer (replenished with 20). Regions without antibody binding were blocked with 2% BSA (bovine serum albumin) for 2 hours at 37°C. The plates were then washed four times with PBS-T buffer. After washing, 100 μL of blocking buffer (2% BSA) was added to each well. The wells were sealed and incubated at 37°C for 1 hour. After washing the plates in a plate washer, 100 μL / well of peroxidase AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specificity (Jackson ImmunoResearch Inc., catalog number: 109-036-098) was added to each well of the plate, and incubated at 37°C for 1 hour to measure the concentrations of total antibody and payload CPT2. After washing the plates, tetramethylbenzidine (TMB) solution was added as a substrate to a 96-well plate at 100 μL / well. After incubation in the dark at room temperature, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. The emission signal of the plate was measured at 450 nm and 630 nm, and the concentration was calculated. Using the absorbance values ​​of the calibration samples prepared with each test product, and the corresponding concentrations, four parameters (i.e., T) were calculated. 1 / 2 , C max AUC 0~21日 A standard curve with , and CL) was constructed. The antibody or ADC concentration of each serum sample was calculated using the standard curve. A drug concentration-time curve was constructed using the calculated sample concentration at each time point. Phoenix TM Pharmacokinetic parameters were calculated using WinNolin 8.3.

[0368] The results are shown in the table below and in Figures 11A-11D. These show that T-6F7-E-6C4-(II')-2(DAR4) and T-6F7-E-6C4-(II')-2(DAR8) exhibit the expected PK behavior.

[0369] [Table 30]

[0370] In another experiment, the plasma stability of T-6F7-E-6C4-(II')-2(DAR4) and T-6F7-E-6C4-(II')-2(DAR8) was measured in human plasma, monkey (cynomolgus monkey) plasma, and rat (SD rat) plasma. Specifically, T-6F7-E-6C4-(II')-2(DAR4) or T-6F7-E-6C4-(II')-2(DAR8) was added to human, monkey, or rat plasma to a final concentration of 100 μg / mL, respectively. In the control group, the plasma was replaced with PBS containing 0.5% BSA. The content of free payload CPT2 and ADC was measured at 0, 1, 2, 6, 8, 11, and 14 days after ADC addition, and the ratio of free CPT2 to total ADC was calculated. The results are shown in Figures 12A-12B. These results indicate that T-6F7-E-6C4-(II')-2(DAR4) and T-6F7-E-6C4-(II')-2(DAR8) are relatively stable in human, monkey, and rat plasma, and have a maximum free CPT2 release rate of 2.0% or less.

[0371] Example 18. Toxicity Evaluation In preliminary experiments, T-6F7-E-6C4-(II')-2(DAR8) was administered to cynomolgus monkeys by IV injection three times at 3-week intervals (days 1, 22, and 43) to investigate safety and toxicokinetic (TK) profiles. The administered formulations are shown in the table below. The animals were then euthanized on day 50 for overall and histopathological investigation. Mortality / mortality rates, overall observations, body weight, food intake, clinical condition (hematology, coagulation, serological chemistry, and urinalysis), and overall injury were evaluated. Blood samples were also collected for TK analysis, and key TK parameters for payload, total antibodies, and ADCs, such as Tmax, Cmax, and AUC(0-t), were calculated. As a result, T-6F7-E-6C4-CPT2(DAR8) was found to have a favorable safety profile.

[0372] [Table 31]

[0373] Example 19. Biophysical analysis of antibody-drug conjugates The biophysical properties of ADCs were evaluated. Specifically, the following tests were performed: (1) Measurement and quantification of aggregate and fragment levels of purified antibody / ADC by size exclusion ultrahigh performance liquid chromatography (SEC-UPLC) (shown as retention time of the main peak (SEC, min)); (2) Detection of apparent hydrophobicity of antibody / ADC using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (shown as retention time of the main peak (HIC, min)); (3) Comparison of hydrophilicity of various antibody / ADCs using reversed-phase high performance liquid chromatography (RP-HPLC) (shown as retention time of the heavy and light chains of antibody / ADCs).

[0374] In the SEC-UPLC experiment, the sample was diluted to 1 mg / mL with purified water and an Agilent 1290 chromatograph system (connected to an XBridge Protein BEH SEC column (200 Å, Waters Corporation)) was used. The parameters used were as follows: mobile phase: 25 mM phosphate buffer (PB) + 300 mM NaCl, pH 6.8, flow rate: 1.8 mL / min, column temperature: 25°C, detection wavelengths: 280 nm, 370 nm, injection volume: 10 μL, sample tray temperature: approximately 4°C, and run time: 7 minutes.

[0375] In the HIC-HPLC experiment, the sample was diluted to 1 mg / mL with purified water and an Agilent 1260 chromatograph system (connected to a Proteomix HIC phenyl column (4.6 × 250 mm, 5 μm, Sepax Technologies)) was used. The following parameters were used: Mobile phase A: 1.5 M ammonium sulfate, 20 mM phosphate buffer (PB) (pH 7.0), Mobile phase B: 25% isopropanol, 20 mM phosphate buffer (PB) (pH 7.0), Flow rate: 0.6 mL / min, Gradient: 0 min 100% A, 2 min 100% A, 20 min 100% B, 25 min 100% B, 25.1 min 100% A, and 30 min 100% A, Column temperature: 25°C, Detection wavelengths: 280 nm, 370 nm, Injection mass: 20 μg, Run time: 40 min.

[0376] In the RP-HPLC experiments, samples were treated with Tris-HCl and dithiothreitol (DTT), and an Agilent 1260 chromatograph system (UPLC: connected to a PLRP-S column (8 μm, 2.1 × 150 mm, 1000 A, Agilent)) was used. The following parameters were used: mobile phase A: 0.05% aqueous trifluoroacetic acid solution, mobile phase B: 0.05% trifluoroacetic acid in acetonitrile solution, flow rate: 0.8 mL / min, gradient: 0 min 70.5%A-29.5%B, 3 min 70.5%A-29.5%B, 17 min 58%A-42%B, 19 min 5%A-95%B, 20 min 70.5%A-29.5%B, and 22 min 70.5% A-29.5% B, column temperature: 80°C, detection wavelengths: 280 nm, 370 nm, injection mass: 8 μg.

[0377] The results at a wavelength of 280 nm are summarized in the table below. These results show that T-(II')-2 exhibits superhydrophilicity similar to that of trastuzumab analogs and significantly better than that of T-GGFG-Dxd. Furthermore, after conjugation of compound (II')-2 with both DAR4 and DAR8, T-6F7-E-6C4-(II')-2 did not show aggregation and exhibited good hydrophilicity similar to that of T-6F7-E-6C4.

[0378] Table 32

Claims

1. Compound of formula (I) 【Chemistry 1】 or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the formula, Q represents a junction site capable of binding to a ligand via a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds. L represents a linker portion to which Q can be connected to the therapeutic agent, having the following structure: 【Chemistry 2】 In the formula, L １ This is a polypeptide residue consisting of 3 to 8 amino acid residues, and includes at least one amino acid residue having a side chain carboxyl group, where "-COOH" indicates the carboxyl group of the C-terminal amino acid residue of the polypeptide residue. L ２ is absent or is a monodentate, bidentate, or tridentate hydrophilic group bonded to the side-chain carboxyl group on the amino acid residue of the polypeptide residue L １ and L ２ has the structure of -NHC(R Ｌ２ａ )(R Ｌ２ｂ )(R Ｌ２ｃ ), where R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ are each independently selected from the group consisting of H, -(CH ２ O)(CH ２ CH ２ O) ｍ (CH ２ ) ｐ C(O)OH, and -(CH ２ O)(CH ２ CH ２ O) ｍ (CH ２ ) ｐ C(O)NHR Ｌ２ｄ , R Ｌ２ｄ is H or C １－６ alkyl optionally substituted with 1 to 6 hydroxy groups, each m is independently an integer from 0 to 10, and each p is independently an integer from 1 to 4 【Transformation 3】 This indicates the N-terminal side of the polypeptide residue covalently bonded to the junction Q. Compounds, or their pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants.

2. The aforementioned joint portion Q has the following structure: 【Chemistry 4】 In the formula, Q ａ This is a functional group that can bind to a ligand, A is C which is arbitrarily substituted. ３－８ Alkylidene, optionally substituted C ３－８ Alkenylidene, optionally substituted C ３－６ Cycloalkenylidene, optionally substituted C ３－８ Selected from cycloalkylides and optionally substituted diglycol-octaglycolacyls, where alkylidene, alkenylidene, cycloalkenylidene, cycloalkylidene, and diglycol-octaglycolacyls are halogens, -CN, and R. Ｑａ１ , -OR Ｑａ１ ,-SR Ｑａ１ , and -N(R Ｑａ１ ) ２ The substituents are arbitrarily substituted with 1 to 4 substituents independently selected from the group consisting of the following, and in the formula, each R Ｑａ１ C １－６ Alkyl, C １－６ Haloalkyl, C ３－６ Cycloalkyl, 3-10 membered heterocyclyl, C ６－１０ Independently selected from the group consisting of aryls and 5- to 10-membered heteroaryls, 【Transformation 5】 This indicates the portion covalently bonded to the linker portion L. The compound described in claim 1, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

3. The aforementioned functional group Q ａ This refers to a compound according to claim 2, selected from the group consisting of the following, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, 【Transformation 6】 In the formula, Ha is a halogen selected from the group consisting of Cl, Br, and I. Ar is a C that has been optionally substituted. ５－６ Cycloalkenyl, optionally substituted C ６ Selected from the group consisting of aryls and optionally substituted 5- to 6-membered heteroaryls, where cycloalkenyl, aryl, and heteroaryl are halogen, -CN, R Ｑａ２ , -OR Ｑａ２ ,-SR Ｑａ２ , and -N(R Ｑａ２ ) ２ It is optionally substituted with 1 to 4 substituents independently selected from the group consisting of the following, where each R Ｑａ２ C １－６ Alkyl, C １－６ Haloalkyl, C ３－６ Cycloalkyl, 3-10 membered heterocyclyl, C ６－１０ Independently selected from the group consisting of aryls and 5- to 10-membered heteroaryls, The asterisk (*) indicates a site that is covalently bonded to A. Compounds, or their pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants.

4. The polypeptide residue L １ It has the following sequence: 【Transformation 7】 In the formula, each AA １ AA ２ AA ３ ,...AA ｐ AA is an amino acid residue that is independently and optionally substituted. １ AA ２ AA ３ ,...AA ｐ At least one of these is an amino acid residue having a side-chain carboxyl group, preferably Glu or Asp. p is an integer between 3 and 8, preferably between 3 and 5. "NH-" indicates the N-terminal side of the polypeptide residue. "-COOH" indicates the C-terminal side of the polypeptide residue. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

5. Each AA1, AA2, AA3, ... AAp is independently and optionally substituted with an amino acid residue selected from the group consisting of Glu, Asp, Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, Gly, and N-alkyl amino acids, and at least one of AA1, AA2, AA3, ... AAp is either Glu or Asp. The compound described in claim 4, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

6. AA １ is an amino acid residue having a side chain carboxyl group, preferably Glu or Asp, and each AA ２ AA ３ ,...AA ｐ These are independently and optionally substituted with amino acid residues selected from the group consisting of Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, and Gly. The compound according to claim 4 or 5, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

7. The polypeptide residue L １ is ＮＨ -Glu-Phe-Lys(NR Ｌｙｓ１ R Ｌｙｓ２ )- ＣＯＯＨ , ＮＨ -Glu-Val-Lys(NR Ｌｙｓ１ R Ｌｙｓ２ )- ＣＯＯＨ , ＮＨ -Glu-Ala-Ala-Ala- ＣＯＯＨ , ＮＨ -Glu-Ala-Ala- ＣＯＯＨ , ＮＨ -Glu-Val-Ala- ＣＯＯＨ , ＮＨ -Glu-Val-Cit- ＣＯＯＨ , ＮＨ -Glu-Gly-Gly-Phe-Gly- ＣＯＯＨ , ＮＨ -Asp-Phe-Lys- ＣＯＯＨ , ＮＨ -Asp-Ala-Ala-Ala- ＣＯＯＨ , ＮＨ -Asp-Val-Ala- ＣＯＯＨ , ＮＨ -Asp-Val-Cit- ＣＯＯＨ , ＮＨ -Asp-Gly-Gly-Phe-Gly- ＣＯＯＨ , and ＮＨ -Asp-Val-Lys(NR Ｌｙｓ１ R Ｌｙｓ２ )- ＣＯＯＨ selected from the group consisting of, wherein R Ｌｙｓ１ and R Ｌｙｓ２ are each independently H or C １－６ alkyl. A compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

8. The hydrophilic group L ２ is -NHC(R Ｌ２ａ ) (Caution Ｌ２ｂ ) (Caution Ｌ２ｃ ) has the structure, in the formula R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ These are H and -CH, respectively. ２ O(CH ２ ) ２ C(O)OH and -CH ２ O(CH ２ ) ２ C(O)NHR Ｌ２ｄ Independently selected from the group consisting of, R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ Of these, up to two are H and R at the same time. Ｌ２ｄ C is a carbon atom substituted with 3 to 5 hydroxyl groups. ４－６ It is alkyl, preferably R Ｌ２ｄ C is a carbon atom substituted with 3 to 5 hydroxyl groups. ４－６ If it is an alkyl group and at most one hydroxyl group is substituted on each carbon atom, for example, R Ｌ２ｄ teeth, 【Transformation 8】 A compound according to any one of claims 1 to 7, selected from the group consisting of the above, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

9. The hydrophilic group L ２ teeth, 【Chemistry 9】 Selected from the group consisting of, "*" represents polypeptide residue L １ This represents the site of covalent bonding. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

10. The aforementioned compound, 【Chemistry 10-1】 【Chemistry 10-2】 【Chemistry 10-3】 [Chemistry 10-4] 【Transformation 10-5】 A compound according to any one of claims 1 to 9, selected from the group consisting of the above, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

11. Compound of formula (II) 【Chemistry 11】 or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the formula, TA indicates a therapeutic agent. Z is either absent or represents an auxiliary portion that binds L' to the therapeutic agent TA via a bond selected from the group consisting of disulfide, thioether, thioester, hydrazone, ester, ether, carbamate, and amide bonds. Q represents a junction site capable of binding to a ligand via a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds. L' represents a linker portion having the following structure, where Q is bound to the therapeutic agent TA. 【Chemistry 12】 In the formula, L' １ This is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue having a side-chain carboxyl group. L ２ Either it does not exist, or the polypeptide residue L １ A monodentate, bidentate, or tridentate hydrophilic group bonded to the side chain carboxyl group on the amino acid residue of L ２ is -NHC(R Ｌ２ａ ) (Caution Ｌ２ｂ ) (Caution Ｌ２ｃ ) has a structure, where R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ These are H and -(CH), respectively. ２ O) (CH ２ CH ２ O) ｍ (CH ２ ) ｐ C(O)OH and -(CH ２ O) (CH ２ CH ２ O) ｍ (CH ２ ) ｐ C(O)NHR Ｌ２ｄ Independently selected from the group consisting of, R Ｌ２ｄ C is optionally substituted with H or 1 to 6 hydroxyl groups. １－６ It is an alkyl group, where each m is an integer between 0 and 10, and each p is an integer between 1 and 4. 【Chemistry 13】 This indicates the N-terminal side of the polypeptide residue covalently bonded to the junction Q, 【Chemistry 14】 This indicates the C-terminal side of the polypeptide residue covalently bonded to the auxiliary portion Z or the therapeutic agent TA. Compounds, or their pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants.

12. The aforementioned joint portion Q has the following structure: 【Chemistry 15】 In the formula, Q ａ This is a functional group that can bind to a ligand, A is C which is arbitrarily substituted. ３－８ Alkylidene, optionally substituted C ３－８ Alkenylidene, optionally substituted C ３－６ Cycloalkenylidene, optionally substituted C ３－８ Selected from cycloalkylides and optionally substituted diglycol-octaglycolacyls, where alkylidene, alkenylidene, cycloalkenylidene, cycloalkylidene, and diglycol-octaglycolacyls are halogens, -CN, and R. Ｑａ１ , -OR Ｑａ１ ,-SR Ｑａ１ , and -N(R Ｑａ１ ) ２ The substituents are arbitrarily substituted with 1 to 4 substituents independently selected from the group consisting of the following, and in the formula, each R Ｑａ１ C １－６ Alkyl, C １－６ Haloalkyl, C ３－６ Cycloalkyl, 3-10 membered heterocyclyl, C ６－１０ Independently selected from the group consisting of aryls and 5- to 10-membered heteroaryls, 【Chemistry 16】 This indicates the site covalently bonded to the linker portion L'. The compound according to claim 11, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

13. The aforementioned functional group Q ａ teeth, 【Chemistry 17】 Selected from the group consisting of In the formula, Ha is a halogen selected from the group consisting of Cl, Br, and I. Ar is a C that has been optionally substituted. ５－６ Cycloalkenyl, optionally substituted C ５－６ Selected from the group consisting of aryls and optionally substituted 5- to 6-membered heteroaryls, where cycloalkenyl, aryl, and heteroaryl are halogen, -CN, R Ｑａ２ , -OR Ｑａ２ ,-SR Ｑａ２ , and -N(R Ｑａ２ ) ２ The substituents are arbitrarily substituted with 1 to 4 substituents independently selected from the group consisting of the following, and in the formula, each R Ｑａ２ C １－６ Alkyl, C １－６ Haloalkyl, C ３－６ Cycloalkyl, 3-10 membered heterocyclyl, C ６－１０ Independently selected from the group consisting of aryls and 5- to 10-membered heteroaryls, The asterisk (*) indicates a site that is covalently bonded to A. The compound according to claim 12, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

14. The polypeptide residue L' １ It has the following sequence: [Chemistry 18] In the formula, each AA １ AA ２ AA ３ ,...AA ｐ AA is an amino acid residue that is independently and optionally substituted. １ AA ２ AA ３ ,...AA ｐ At least one of these is an amino acid residue having a side-chain carboxyl group, preferably Glu or Asp. p is an integer between 3 and 8, preferably between 3 and 5. "NH-" indicates the N-terminal side of the polypeptide residue. "-C (=O)" indicates the C-terminal side of the polypeptide residue. A compound according to any one of claims 11 to 13, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

15. Each AA １ AA ２ AA ３ ,...AA ｐ These are independently and optionally substituted with amino acid residues selected from the group consisting of Glu, Asp, Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, Gly, and N-alkyl amino acids, AA １ AA ２ AA ３ ,...AA ｐ At least one of them is Glu or Asp. The compound described in claim 14, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

16. AA １ is an amino acid residue having a side chain carboxyl group, preferably Glu or Asp, and each AA ２ AA ３ ,...AA ｐ These are independently and optionally substituted with amino acid residues selected from the group consisting of Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, and Gly. A compound according to claim 14 or 15, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

17. The polypeptide residue L' １ teeth, ＮＨ -Glu-Phe-Lys (NR Ｌｙｓ１ R Ｌｙｓ２ ) - Ｃ（＝Ｏ） , ＮＨ -Glu-Val-Lys (NR Ｌｙｓ１ R Ｌｙｓ２ ) - Ｃ（＝Ｏ） , ＮＨ -Glu-Ala-Ala-Ala- Ｃ（＝Ｏ） , ＮＨ -Glu-Ala-Ala- Ｃ（＝Ｏ） , ＮＨ -Glu-Val-Ala- Ｃ（＝Ｏ） , ＮＨ -Glu-Val-Cit- Ｃ（＝Ｏ） , ＮＨ -Glu-Gly-Gly-Phe-Gly- Ｃ（＝Ｏ） , ＮＨ -Asp-Phe-Lys- Ｃ（＝Ｏ） , ＮＨ -Asp-Ala-Ala-Ala- Ｃ（＝Ｏ） , ＮＨ -Asp-Val-Ala- Ｃ（＝Ｏ） , ＮＨ -Asp-Val-Cit- Ｃ（＝Ｏ） , ＮＨ -Asp-Gly-Gly-Phe-Gly- Ｃ（＝Ｏ） , and ＮＨ -Asp-Val-Lys(NR Ｌｙｓ１ R Ｌｙｓ２ ) - Ｃ（＝Ｏ） Selected from the group consisting of, in the formula, R Ｌｙｓ１ and R Ｌｙｓ２ Each of these is independently H or C １－６ It is alkyl. A compound according to any one of claims 11 to 16, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

18. The hydrophilic group L ２ is -NHC(R Ｌ２ａ ) (Caution Ｌ２ｂ ) (Caution Ｌ２ｃ ) has the structure, in the formula R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ These are H and -CH, respectively. ２ O(CH ２ ) ２ C(O)OH and -CH ２ O(CH ２ ) ２ C(O)NHR Ｌ２ｄ Independently selected from the group consisting of, R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ Of these, up to two are H and R at the same time. Ｌ２ｄ C is a carbon atom substituted with 3 to 5 hydroxyl groups. ４－６ It is alkyl, preferably R Ｌ２ｄ C is a carbon atom substituted with 3 to 5 hydroxyl groups. ４－６ If it is an alkyl group and at most one hydroxyl group is substituted on each carbon atom, for example, R Ｌ２ｄ teeth, 【Chemistry 19】 A compound according to any one of claims 11 to 17, selected from the group consisting of the above, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

19. The hydrophilic group L ２ teeth, 【Chemistry 20】 Selected from the group consisting of, "*" represents the polypeptide residue L' １ This indicates the site of covalent bonding. , the compound according to any one of claims 11 to 18, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

20. The auxiliary portion Z is selected from the group consisting of mercapto, disulfide, amino, carboxyl, aldehyde, maleimide, haloacetyl, hydrazide, and hydroxyl groups. A compound according to any one of claims 11 to 19, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

21. The therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor, such as a camptothecin compound, its analogue or derivative. A compound according to any one of claims 11 to 20, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

22. The aforementioned compound is a compound of formula (II'), 【Chemistry 21】 X is -CH ２ - is selected from the group consisting of O and S, and Y is selected from the group consisting of H, D and F. Q and L' are as defined in the prior claims, A compound according to any one of claims 10 to 19, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

23. The compound is selected from the group consisting of the following, and is a compound according to any one of claims 10 to 20, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof. 【Chemistry 22-1】 【Chemistry 22-2】 【Chemistry 22-3】 【Chemistry 22-4】 【Chemistry 22-5】 【Chemistry 22-6】 【Chemistry 22-7】 【Chemistry 22-8】 【Chemistry 22-9】 【Chemistry 22-10】

24. Ligand-drug conjugate of formula (III) 【Chemistry 23】 or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the formula, LG indicates the ligand, TA indicates a therapeutic agent. Z is either absent or represents an auxiliary portion that binds L' to the therapeutic agent TA via a bond selected from the group consisting of disulfide, thioether, thioester, hydrazone, ester, ether, carbamate, and amide bonds. Q' represents a junction portion that is bound to ligand LG by a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds. L' represents a linker portion having the following structure, where Q is bound to the therapeutic agent TA. 【Chemistry 24】 In the formula, L' １ This is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue having a side-chain carboxyl group. L ２ Either it does not exist, or the polypeptide residue L １ A monodentate, bidentate, or tridentate hydrophilic group bonded to the side chain carboxyl group on the amino acid residue of L ２ is -NHC(R Ｌ２ａ ) (Caution Ｌ２ｂ ) (Caution Ｌ２ｃ ) has a structure, where R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ These are H and -(CH), respectively. ２ O) (CH ２ CH ２ O) ｍ (CH ２ ) ｐ C(O)OH and -(CH ２ O) (CH ２ CH ２ O) ｍ (CH ２ ) ｐ C(O)NHR Ｌ２ｄ Independently selected from the group consisting of, R Ｌ２ｄ C is optionally substituted with H or 1 to 6 hydroxyl groups. １－６ It is an alkyl group, where each m is an integer between 0 and 10, and each p is an integer between 1 and 4. 【Chemistry 25】 This indicates the N-terminal side of the polypeptide residue covalently bonded to the junction Q', 【Chemistry 26】 This indicates the C-terminal side of the polypeptide residue covalently bonded to the therapeutic agent TA, and n is a number in the range of 1 to 8. Ligand-drug conjugates, or pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants thereof.

25. The aforementioned joint portion Q' has the following structure: 【Chemistry 27】 In the formula, Q' ａ This is a functional group that can bind to a ligand, A is C which is arbitrarily substituted. ３－８ Alkylidene, optionally substituted C ３－８ Alkenylidene, optionally substituted C ３－６ Cycloalkenylidene, optionally substituted C ３－８ Selected from cycloalkylides and optionally substituted diglycol-octaglycolacyls, where alkylidene, alkenylidene, cycloalkenylidene, cycloalkylidene, and diglycol-octaglycolacyls are halogens, -CN, and R. Ｑ’ａ１ , -OR Ｑ’ａ１ ,-SR Ｑ’ａ１ , and -N(R Ｑ’ａ１ ) ２ The substituents are arbitrarily substituted with 1 to 4 substituents independently selected from the group consisting of the following, and in the formula, each R Ｑ’ａ１ C １－６ Alkyl, C １－６ Haloalkyl, C ３－６ Cycloalkyl, 3-10 membered heterocyclyl, C ６－１０ Independently selected from the group consisting of aryls and 5- to 10-membered heteroaryls, 【Chemistry 28】 This indicates the portion covalently bonded to the linker portion L'. A ligand-drug conjugate according to claim 24, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

26. The aforementioned joint portion Q' ａ teeth, 【Chemistry 29】 A ligand-drug conjugate according to claim 25, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, selected from the group consisting of the following: In the formula, Ar is a C that can be optionally substituted. ５－６ Cycloalkenyl, optionally substituted C ５－６ Selected from the group consisting of aryls and optionally substituted 5- to 6-membered heteroaryls, where cycloalkenyl, aryl, and heteroaryl are halogen, -CN, R Ｑａ２ , -OR Ｑａ２ ,-SR Ｑａ２ , and -N(R Ｑａ２ ) ２ The substituents are arbitrarily substituted with 1 to 4 substituents independently selected from the group consisting of the following, and in the formula, each R Ｑａ２ C １－６ Alkyl, C １－６ Haloalkyl, C ３－６ Cycloalkyl, 3-10 membered heterocyclyl, C ６－１０ Independently selected from the group consisting of aryls and 5- to 10-membered heteroaryls, The asterisk (*) indicates a site that is covalently bonded to A. 【Transformation 30】 This indicates the site covalently bound to the ligand LG. Ligand-drug conjugates, or pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants thereof.

27. The polypeptide residue L' １ It has the following sequence: 【Chemistry 31】 In the formula, each AA １ AA ２ AA ３ ,...AA ｐ AA is an amino acid residue that is independently and optionally substituted. １ AA ２ AA ３ ,...AA ｐ At least one of these is an amino acid residue having a side-chain carboxyl group, preferably Glu or Asp. p is an integer between 3 and 8, preferably between 3 and 5. "NH-" indicates the N-terminal side of the polypeptide residue. "-C (=O)" indicates the C-terminal side of the polypeptide residue. A ligand-drug conjugate according to any one of claims 24 to 26, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

28. Each AA １ AA ２ AA ３ ,...AA ｐ These are independently and optionally substituted with amino acid residues selected from the group consisting of Glu, Asp, Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, Gly, and N-alkyl amino acids, AA １ AA ２ AA ３ ,...AA ｐ At least one of them is Glu or Asp. A ligand-drug conjugate according to claim 27, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

29. AA １ is an amino acid residue having a side chain carboxyl group, preferably Glu or Asp, and each AA ２ AA ３ ,...AA ｐ These are independently and optionally substituted with amino acid residues selected from the group consisting of Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, and Gly. A ligand-drug conjugate according to claim 27 or 28, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

30. The polypeptide residue L' １ teeth, ＮＨ -Glu-Phe-Lys (NR Ｌｙｓ１ R Ｌｙｓ２ ) - Ｃ（＝Ｏ） , ＮＨ -Glu-Val-Lys (NR Ｌｙｓ１ R Ｌｙｓ２ ) - Ｃ（＝Ｏ） , ＮＨ -Glu-Ala-Ala-Ala- Ｃ（＝Ｏ） , ＮＨ -Glu-Ala-Ala- Ｃ（＝Ｏ） , ＮＨ -Glu-Val-Ala- Ｃ（＝Ｏ） , ＮＨ -Glu-Val-Cit- Ｃ（＝Ｏ） , ＮＨ -Glu-Gly-Gly-Phe-Gly- Ｃ（＝Ｏ） , ＮＨ -Asp-Phe-Lys- Ｃ（＝Ｏ） , ＮＨ -Asp-Ala-Ala-Ala- Ｃ（＝Ｏ） , ＮＨ -Asp-Val-Ala- Ｃ（＝Ｏ） , ＮＨ -Asp-Val-Cit- Ｃ（＝Ｏ） , ＮＨ -Asp-Gly-Gly-Phe-Gly- Ｃ（＝Ｏ） , and ＮＨ -Asp-Val-Lys(NR Ｌｙｓ１ R Ｌｙｓ２ ) - Ｃ（＝Ｏ） Selected from the group consisting of, in the formula, R Ｌｙｓ１ and R Ｌｙｓ２ Each of these is independently H or C １－６ It is alkyl. A ligand-drug conjugate according to any one of claims 24 to 29, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

31. The hydrophilic group L ２ is -NHC(R Ｌ２ａ ) (Caution Ｌ２ｂ ) (Caution Ｌ２ｃ ) has the structure, in the formula R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ These are H and -CH, respectively. ２ O(CH ２ ) ２ C(O)OH and -CH ２ O(CH ２ ) ２ C(O)NHR Ｌ２ｄ Independently selected from the group consisting of, R Ｌ２ａ , R Ｌ２ｂ , and R Ｌ２ｃ Of these, up to two are H and R at the same time. Ｌ２ｄ C is a carbon atom substituted with 3 to 5 hydroxyl groups. ４－６ It is alkyl, preferably R Ｌ２ｄ C is a carbon atom substituted with 3 to 5 hydroxyl groups. ４－６ If it is an alkyl group and at most one hydroxyl group is substituted on each carbon atom, for example, R Ｌ２ｄ teeth, 【Chemistry 32】 A ligand-drug conjugate according to any one of claims 24 to 30, selected from the group consisting of the above, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

32. The hydrophilic group L ２ teeth, 【Transformation 33】 Selected from the group consisting of, "*" represents the polypeptide residue L' １ This indicates the site of covalent bonding. A ligand-drug conjugate according to any one of claims 24 to 31, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

33. The auxiliary portion Z is selected from the group consisting of mercapto, disulfide, amino, carboxyl, aldehyde, maleimide, haloacetyl, hydrazide, and hydroxyl groups, and is a ligand-drug conjugate according to any one of claims 24 to 32, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

34. The ligand LG is an antibody or an antigen-binding fragment thereof, as described in any one of claims 24 to 34, the ligand-drug conjugate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

35. The therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor, as described in any one of claims 24 to 34, a ligand-drug conjugate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

36. The therapeutic agent is a camptothecin compound, an analog or derivative thereof, a ligand-drug conjugate according to any one of claims 24 to 35, or a pharmaceutically acceptable salt, solvate, stereoisomer or isotopic variant thereof.

37. The ligand-drug conjugate is a compound of formula (III'), 【Transformation 34】 or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the formula, X is -CH ２ - is selected from the group consisting of O and S, and Y is selected from the group consisting of H, D and F. Ab indicates an antibody. Q and L' are as defined in the prior claims, n is a number in the range of 1 to 8. A ligand-drug conjugate according to any one of claims 24 to 36, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

38. The ligand-drug conjugate is 【Chemistry 35-1】 【Chemistry 35-2】 【Chemistry 35-3】 【Chemistry 35-4】 【Transformation 35-5】 【Chemistry 35-6】 【Chemistry 35-7】 【Transformation 35-8】 【Chemistry 35-9】 【Chemistry 35-10】 A ligand-drug conjugate according to any one of claims 24 to 37, selected from the group consisting of, In the formula, n is a number in the range of 1 to 8. Ligand-drug conjugates, or pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants thereof.

39. The antibody is selected from the group consisting of mouse-derived antibodies, chimeric antibodies, humanized antibodies, and fully humanized antibodies, and is a ligand-drug conjugate according to any one of claims 34 to 38, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

40. The ligand-drug conjugate according to any one of claims 34 to 39, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the antibody is an mAb.

41. The ligand-drug conjugate according to any one of claims 34 to 40, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the antibody is a bispecific antibody or a multispecific antibody.

42. The ligand-drug conjugate according to any one of claims 34 to 41, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the antibody is an antibody fragment, nanobody, or fusion protein.

43. The antibodies mentioned above are CTLA4, CD137, OX40, HER2, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD79b, CD137, CD138, CD147, CD223, EpCAM, and Mucin.

1. A ligand-drug conjugate according to any one of claims 34 to 42, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, which is an antibody capable of binding to STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, nectin 4, AGS-16, guanylyl cyclase C, mesoserine, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein 3, or claudin 18.2 tumor-associated antigen.

44. A pharmaceutical composition comprising a ligand-drug conjugate according to any one of claims 24 to 43, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

45. The pharmaceutical composition according to claim 44, wherein the ligand-drug conjugate has an ADC DAR value in the range of 1 to 8.

46. Use of a ligand-drug conjugate according to any one of claims 24 to 43, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, in the preparation of a drug for treating a tumor in a subject.

47. The use according to claim 46, wherein the tumor is selected from the group consisting of solid tumors, brain tumors, lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cancer, pancreatic cancer, liver cancer, gastric cancer, or breast cancer.

48. A ligand-drug conjugate according to any one of claims 24 to 43, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, for use in the treatment of tumors in a subject.

49. A ligand-drug conjugate according to any one of claims 24 to 43, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, for use according to claim 48, wherein the tumor is selected from the group consisting of solid tumors, brain tumors, lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cancer, pancreatic cancer, liver cancer, gastric cancer, or breast cancer.

50. A method for treating a tumor in a subject, comprising administering to the subject an effective amount of a ligand-drug conjugate according to any one of claims 24 to 43, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof.

51. The method according to claim 50, wherein the tumor is selected from the group consisting of solid tumors, brain tumors, lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cancer, pancreatic cancer, liver cancer, gastric cancer, or breast cancer.

52. A method for preparing a ligand-drug conjugate according to any one of claims 24 to 43, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, the following: a) A step of reacting a ligand with a reducing agent in a buffer to obtain a reduced ligand, b) A step of conjugating the compound described in any one of claims 11 to 23, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, with the reduced ligand obtained in step a) in a mixture of buffer and organic solvent to obtain the ligand-drug conjugate, Alternatively, see below. The method comprises the step of conjugating a compound according to any one of claims 11 to 23, or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, with a ligand in a mixture of a buffer and an organic solvent to obtain the ligand-drug conjugate. method.

53. Compound of formula (IV), 【Transformation 36】 or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein the formula, X is -CH ２ - Selected from the group consisting of O and S, and Y is selected from the group consisting of H, D, and F. Compounds, or their pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants.

54. The compound according to claim 53, for use in the treatment of tumors in a subject.

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