Antibody-auristatins drug conjugate and use thereof

Abstract

An antibody-drug conjugate as shown in formula I, a preparation method therefor, and a use thereof in prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to a use in prevention and / or treatment of tumor diseases.

Description

Antibody-auristatin drug conjugate and its use

[0001] This application claims priority to Chinese Patent Application No. 2024103704626 filed on March 29, 2024, and claims priority to Chinese Patent Application No. 2024116231705 filed on November 14, 2024. This application incorporates the entire contents of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention belongs to the field of medical technology and relates to an antibody-drug conjugate containing auristatin-type bioactive compound and a preparation method thereof, as well as its use in preventing and / or treating diseases related to abnormal cell activity, including but not limited to preventing and / or treating tumor diseases. Background Art

[0003] Auristatins are decapeptide derivatives isolated from the Indian Ocean sea hare. They inhibit tumor growth primarily by targeting tubulin, with activity approximately 1,000 times greater than that of traditional chemotherapy drugs. MMAE and MMAF are the two most commonly used auristatin compounds in ADC development. Currently, six marketed ADCs (Adcetris, Polivy, Padcev, Blenrep, Vedicizumab, and TIVDAK) use MMAE or MMAF as payloads. Over 40 ADCs using auristatin as a payload are in clinical development, accounting for over 50% of all ADCs under development.

[0004] Existing ADCs with auristatin derivatives as payloads generally bind to tumor cell surface antigens through antibody binding, followed by endocytosis into endosomes, and then conversion from endosomes to lysosomes. Lysosomal hydrolases then dissociate the bioactive molecule (toxin or payload) from the ADC. The dissociated bioactive molecule then enters the cytoplasm from the lysosomes and kills the tumor cells. After killing the tumor cells, the bioactive molecule that escapes can further kill surrounding tumor cells that do not express or express low levels of the antigen (the so-called bystander effect). Any change in this entire process can cause the ADC to develop drug resistance and lose its therapeutic effect, such as changes in antigen expression during treatment, weakened or even lost endocytosis, or changes in endosome or lysosome function.

[0005] In addition, the coupling of ADC drugs with auristatin derivatives as payloads that are currently on the market or in the clinical research stage is mainly achieved through the Michael addition reaction between the free thiol groups on the antibody and maleimide. However, there are many literature reports that 30% or more of the ADCs obtained by the thiol Michael addition method will undergo reverse Michael addition in the systemic circulation, resulting in premature shedding of toxins and toxic reactions.

[0006] Therefore, it is necessary to find an ADC that can be stable in the blood circulation system in the body, and at the same time, this ADC can kill tumors by extracellular lysis in the tumor microenvironment without the need for cellular endocytosis to dissociate and release toxins. This ADC will be able to overcome the various drug resistance mechanisms of traditional ADCs, expand the treatment window, and improve the treatment effect, and therefore will be of great significance in clinical treatment. Summary of the Invention

[0007] In a first aspect, the present invention provides an antibody drug conjugate of formula I:

[0008] or a stereoisomer, pharmaceutically acceptable salt or solvate thereof,

[0009] in,

[0010] Tb is an antibody or an antigen-binding fragment thereof;

[0011] q is any value between 0.1 and 12.0;

[0012] L is a linker that covalently binds Tb and D;

[0013] D is a bioactive compound fragment.

[0014] In a second aspect, the present invention provides a drug-linker conjugate of Formula II, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof,

[0015] in,

[0016] Lg is a group that reacts with an antibody; L and D are as defined in the first aspect of the present invention.

[0017] In a third aspect, the present invention provides a biologically active compound represented by formula III, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof,

[0018] Wherein, R1, R2, R3' and Ra' are defined as described in any embodiment of the present invention.

[0019] In a fourth aspect, the present invention provides a method for preparing the antibody-drug conjugate according to the first aspect.

[0020] In a fifth aspect, the present invention provides a conjugate population comprising the antibody-drug conjugate according to the first aspect.

[0021] In a sixth aspect, the present invention provides a composition comprising the antibody-drug conjugate of the first aspect, or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof, or the drug-linker conjugate of the second aspect, or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof, or the biologically active compound of the third aspect, or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof.

[0022] In a seventh aspect, the present invention provides uses of the antibody-drug conjugate, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, of the first aspect, or the drug-linker conjugate, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, of the second aspect, or the bioactive compound, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, of the third aspect.

[0023] Detailed Description of the Invention

[0024] definition

[0025] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0026] As used herein, the term "pharmaceutically acceptable salt" is exemplified by organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formates, acetates, propionates, benzoates, maleates, fumarates, succinates, tartrates, citrates, ascorbates, α-ketoglutarate, α-glycerophosphates, alkylsulfonates, or arylsulfonates; preferably, the alkylsulfonate is methylsulfonate or ethylsulfonate; and the arylsulfonate is benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts may also be formed, including but not limited to hydrochlorides, hydrobromides, hydroiodides, nitrates, bicarbonates, carbonates, sulfates, or phosphates.

[0027] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid affording a pharmaceutically acceptable anion.

[0028] As used herein, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is understood that the scope of the present invention encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0029] Solid lines (-), solid wedges, or dashed wedges may be used herein to depict carbon-carbon bonds of the compounds of the present invention. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the indicated stereoisomers exist. When present in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0030] In the present invention, Indicates the connection point of the connected parts.

[0031] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0032] For the purposes of this invention, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and formulations. These substances, in addition to the active ingredient, have been rationally evaluated for safety and are included in pharmaceutical preparations. In addition to imparting shape, acting as a carrier, and enhancing stability, pharmaceutical excipients also perform important functions such as solubilization, dissolution enhancement, and sustained-release control. They are crucial components that may impact the quality, safety, and efficacy of pharmaceuticals. Based on their source, they can be categorized as natural, semi-synthetic, or fully synthetic. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0033] The pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc. The pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1000 mg of the compound of the present invention or a pharmaceutically acceptable salt or conjugate thereof, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5 to 350 mg, and particularly preferably 1-250 mg.

[0034] The pharmaceutical composition can be administered in the form of an injection, including an injection solution, sterile powder for injection, and concentrated solution for injection. Usable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspending media. The pharmaceutical composition can be administered in the form of an infusion.

[0035] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. This effect can be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) relieving the symptoms of a disease, i.e., causing the disease or symptom to regress.

[0036] In the present invention, the term "individual" includes humans and non-human animals. Exemplary human individuals include human individuals suffering from a disease (e.g., a disease described herein) (referred to as a patient) or normal individuals. The term "non-human animal" in the present invention includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0037] As used herein, the term "effective dose" refers to an amount of an antibody drug conjugate, drug-linker conjugate, compound, or composition that, after administration, will alleviate to some extent one or more symptoms of the condition being treated.

[0038] In the present invention, the terms "antibody drug conjugate" and "ADC" refer to a substance obtained by linking a bioactive compound fragment (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of the present invention, the bioactive compound fragment is connected to the targeting portion via a linker. The linker can be broken in a specific environment (such as a low pH environment in the cell) or under a specific action (such as the action of a lysosomal protease), thereby separating the bioactive compound fragment from the targeting portion or the antibody or its antigen-binding fragment. In some embodiments of the present invention, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present invention, the bioactive compound fragment is directly linked to the targeting portion or the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0039] In the present invention, the terms "bioactive substance", "bioactive compound" and "drug" refer to substances that inhibit or prevent the function of cells and / or cause cell death or destruction.

[0040] In the present invention, when it is said that “the 1-position of L1 is connected to Tb via an S atom”, it can be understood by those skilled in the art that the 1-position of L1 is connected to the sulfhydryl group contained in Tb (e.g., an antibody) after the disulfide bond is opened (for example, the disulfide bond can be opened by reducing the disulfide bond with TCEP to generate a sulfhydryl group -SH). In other words, the -S- between L1 and Tb is not an additional external sulfur atom. For example, the -S- is not an additional external sulfur atom, but rather the sulfhydryl group contained in Tb after the disulfide bond is opened and connected to L1, for example -S- is formed by connecting the 1 bits of

[0041] In the present invention, the terms "linker" and "linker" refer to a fragment that connects a biologically active compound fragment (drug molecule) to an antibody portion.

[0042] In the present invention, the term "natural amino acid residue" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Non-natural amino acids are not particularly limited, and examples include β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids whose side chains differ from those of natural amino acids.

[0043] In the present invention, the "bioactive compound fragment" refers to a portion (fragment or group) of an antibody-drug conjugate (ADC) known in the art, which, after cleavage / degradation / enzymatic cleavage of the linker between tumor tissues or within tumor cells, can form a bioactive drug (e.g., a small molecule cytotoxic drug, including a group resulting from the loss of an atom or group of atoms) or its derivative (e.g., a precursor). For the avoidance of doubt, "drug" does not refer solely to "drugs" approved by pharmaceutical regulatory authorities but also includes any compound with potential therapeutic biological activity in clinical practice, or in research and development and academic research.

[0044] In the present invention, the term "antibody" is interpreted in its broadest sense, including intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed by at least two intact antibodies, as long as they have the desired biological activity. In the present invention, "antibody" and "immunoglobulin" can be used interchangeably.

[0045] In the present invention, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for a small number of naturally occurring mutations that may be present. Monoclonal antibodies have high specificity for a single determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, comprise different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage of being synthesized without contamination by other antibodies. The modifier "monoclonal" herein indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring production by a specific method.

[0046] In some embodiments of the present invention, monoclonal antibodies also specifically include chimeric antibodies, that is, a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, and the remaining portion is identical or homologous to another type, class, or subclass of antibody, as long as they have the desired biological activity (see, for example, US 4,816,567). Chimeric antibodies that can be used in the present invention include primatized antibodies, which contain variable region antigen-binding sequences from non-human primates (e.g., monkeys, orangutans, etc.) and human constant region sequences.

[0047] The term "antibody fragment" refers to a portion of an antibody, preferably the antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single-chain antibody molecules.

[0048] The term "bispecific antibody", also known as "bifunctional antibody conjugate", refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a coupling arm. The conjugate retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0049] The term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.

[0050] The term "intact antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody having one or more effector functions.

[0051] The term "probody" refers to a modified antibody, including an antibody or an antibody fragment, that can specifically bind to its target and can be coupled to a masking group, wherein the masking group refers to a cleavage constant for the binding ability of the antibody or antibody fragment to its target that is at least 100 times, 1000 times, or 10,000 times greater than the cleavage constant for the binding ability of the antibody or antibody fragment to its target without the coupled masking group.

[0052] In the present invention, the "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of a human recipient immunoglobulin are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may also contain residues that are not present in the recipient antibody or the donor antibody. These modifications are intended to further optimize the performance of the antibody. Humanized antibodies generally contain at least one, usually two, variable regions, in which all or nearly all of the hypervariable loops correspond to those of a non-human immunoglobulin, while the FRs are entirely or almost entirely human immunoglobulin sequences. The humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc, usually a human immunoglobulin Fc).

[0053] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the constant region of their heavy chains. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes are further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different antibody classes are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0054] In the present invention, although amino acid substitutions in antibodies are in most cases substituted with L-amino acids, this is not limiting. In some embodiments, the antibody peptide chain may include one or more D-amino acids. Peptides containing D-amino acids are more stable and less susceptible to degradation in the oral cavity, intestinal tract, or plasma than peptides containing only L-amino acids.

[0055] Monoclonal antibodies used in the present invention can be produced by many methods. For example, monoclonal antibodies used in the present invention can be obtained by hybridoma methods using cells from many species (including mice, hamsters, rats and people), or by recombinant DNA technology (see, for example, US 4,816,567), or separated from phage antibody libraries.

[0056] In the present invention, unless otherwise explicitly stated, the descriptions “each independently selected from” and “each independently selected from” used throughout the present invention can be interchanged and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, or that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0057] Throughout this specification, substituents of the compounds of the present invention are described in terms of group classes or ranges. It is specifically noted that the invention includes each independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-6 alkyl" specifically refers to the independently invented methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl groups.

[0058] In the present invention, the term "direct bond" means that the indicated substituent does not exist, and both end portions of the substituent are directly connected to form a bond.

[0059] In the present invention, the terms "include", "comprising", "having", "containing" or "involving" and other variations thereof herein are inclusive or open-ended and do not exclude other unrecited elements or method steps.

[0060] In the present invention, the term "optionally substituted" means that a substituent may be present or absent.

[0061] In the present invention, the term "C1-8 alkyl" refers to a straight-chain or branched alkyl group containing 1 to 8 carbon atoms, including, for example, "C1-3 alkyl", "C1-4 alkyl", or "C1-6 alkyl", methyl, ethyl, etc. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0062] In the present invention, the term "alkylene" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched saturated hydrocarbon group, such as "C1-6 alkylene" and "C1-3 alkylene". Specific examples include but are not limited to: methylene, ethylene, 1,3-propylene, etc.

[0063] In the present invention, the term "C2-6 alkenyl" refers to a linear, branched or cyclic alkenyl group containing at least one double bond and having 2 to 6 carbon atoms, including, for example, "C2-4 alkenyl". Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, and the like.

[0064] In the present invention, the term "C2-6 alkynyl" refers to a straight or branched alkynyl group containing at least one triple bond and having 2 to 6 carbon atoms, including, for example, "C2-4 alkynyl". Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like.

[0065] In the present invention, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0066] In the present invention, the term "C3-10 cycloalkyl" refers to a saturated cyclic alkyl group containing 3-10 carbon atoms. Examples include "C3-6 cycloalkyl" and "C3-8 cycloalkyl." Optionally, the carbon atoms in the cyclic structure may be oxo-substituted. Specific examples include, but are not limited to, cyclopropane (i.e., cyclopropyl), cyclobutane (i.e., cyclobutyl), cyclopentane (i.e., cyclopentyl), and cyclohexane.

[0067] In the present invention, the term "C1-6 alkoxy" refers to an alkyl group as defined above attached to the parent molecular moiety through an oxygen atom, including, for example, "C1-3 alkoxy" or "C1-4 alkoxy". Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0068] In the present invention, the term "C1-6 alkylthio" refers to an alkyl group as defined above attached to the parent molecular moiety through a sulfur atom, including, for example, "C1-3 alkylthio" or "C1-4 alkylthio".

[0069] In the present invention, the term "halogenated C1-6 alkyl" refers to an alkyl group as defined above that is substituted by one or more halogens as defined above, including, for example, "halogenated C1-3 alkyl" or "halogenated C1-4 alkyl". Specific examples include, but are not limited to, chloromethyl, fluoroethyl, bromopropyl, and the like.

[0070] In the present invention, the term "halogenated C1-6 alkoxy" refers to an alkoxy group as defined above that is substituted by one or more halogens as defined above, including, for example, "halogenated C1-3 alkoxy" or "halogenated C1-4 alkoxy". Specific examples include, but are not limited to, chloromethoxy, fluoroethoxy, bromopropoxy, and the like.

[0071] In the present invention, the term "halogenated C1-6 alkylthio" refers to an alkylthio group as defined above substituted by one or more halogens as defined above, including, for example, "halogenated C1-3 alkylthio" or "halogenated C1-4 alkylthio".

[0072] In the present invention, the term "4-10 membered heterocycle" refers to a ring containing 4-10 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "3-6 membered heterocycle" refers to a ring containing 3-6 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "3-7 membered heterocycle" refers to a ring containing 3-7 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "5-6 membered heterocycle" refers to a ring containing 5-6 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure may be oxo-substituted, and specific examples include but are not limited to rings such as pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, and pyrrolidone.

[0073] In the present invention, the term "3-10 membered carbocycle" refers to a ring having 3-10 carbon atoms. Optionally, the carbon atoms in the ring structure may be oxo-substituted. Examples include 3-6 membered carbocycles, 3-7 membered carbocycles, 5-8 membered carbocycles, and the like. Specific examples include, but are not limited to, cyclopentane and cyclohexane.

[0074] In the present invention, the term "4-10 membered heterocyclic radical" refers to a cyclic group containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom, such as an oxygen atom, a nitrogen atom or a sulfur atom). The term "4-6 membered heterocyclic radical" refers to a cyclic group containing 4-6 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "3-6 membered heterocyclic radical" refers to a cyclic group containing 3-6 ring atoms (wherein at least one ring atom is a heteroatom, such as an oxygen atom, a nitrogen atom or a sulfur atom). Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxoed. “4-8 membered heterocyclic group” includes, for example, “4-8 membered nitrogen-containing heterocyclic group”, “4-8 membered oxygen-containing heterocyclic group”, “4-7 membered heterocyclic group”, “4-7 membered oxygen-containing heterocyclic group”, “4-6 membered heterocyclic group”, “5-7 membered heterocyclic group”, “5-6 membered heterocyclic group”, “5-6 membered nitrogen-containing heterocyclic group”, and specific examples include but are not limited to oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl and the like.

[0075] In the present invention, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromatic properties, such as a 6-10 membered aryl group, a 5-8 membered aryl group, and the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, and phenanthrenyl. The "6-10 membered aryl" refers to an aryl group containing 6-10 ring atoms. The "C6-10 aryl" refers to an aryl group containing 6-10 carbon atoms.

[0076] In the present invention, term " heteroaryl " refers to a cyclic group with aromaticity, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulphur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulphur atoms) in the ring structure can be oxoed. Specific examples include but are not limited to 5-10 yuan heteroaryl, 5-6 yuan heteroaryl, 5-10 yuan nitrogen-containing heteroaryl, 6-10 yuan oxygen-containing heteroaryl, 6-8 yuan nitrogen-containing heteroaryl, 5-8 yuan oxygen-containing heteroaryl, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3, 4-oxadiazolyl, pyridinyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0077] In the present invention, the term "Linker Unit" refers to a component of an antibody-drug conjugate or drug-linker conjugate or linker that connects the target-binding antibody or antigen-binding fragment thereof to the remainder of the antibody-drug conjugate. Linker Units can connect the Tb unit to L2. Specific examples include, but are not limited to, (wherein position 1 is connected to the antibody or antigen-binding fragment thereof, and position 2 is connected to L2 or L3):

[0078] In the present invention, the term "Linker" refers to a component of an antibody-drug conjugate or drug-linker conjugate or linker that is used to bind the Linker to an amino acid residue or a short peptide consisting of 2-10 amino acid residues. When present, the Linker can connect L1 to L3. Specific examples include, but are not limited to, (wherein position 1 is connected to the Linker and position 2 is connected to L3):

[0079] In the present invention, the term "spacer unit" refers to a component of an antibody-drug conjugate or a drug-linker conjugate or a linker, which serves to space an amino acid residue or a short peptide consisting of 2-10 amino acid residues from a bioactive compound fragment (drug molecule).

[0080] Antibody Drug Conjugates

[0081] In a first aspect, the present invention provides an antibody-drug conjugate as shown in Formula I:

[0082] or a stereoisomer, pharmaceutically acceptable salt or solvate thereof,

[0083] in,

[0084] Tb is an antibody or an antigen-binding fragment thereof;

[0085] q is any value between 0.1 and 12.0;

[0086] L is a linker that covalently binds Tb and D;

[0087] D is a bioactive compound fragment.

[0088] In some embodiments, D is a structure as shown in Formula I-1:

[0089] or a stereoisomer, pharmaceutically acceptable salt or solvate thereof,

[0090] in,

[0091] R1 is selected from hydrogen, methyl,

[0092] R2 is selected from hydrogen, deuterium or hydroxyl;

[0093] R3 is selected from hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole),

[0094] Ra is selected from hydrogen, deuterium,

[0095] R 4 Selected from hydrogen, C1-6 alkyl (preferably C1-3 alkyl) or hydroxy-substituted C1-6 alkyl;

[0096] s is selected from 1, 2, 3, 4, 5 or 6;

[0097] m and n are each independently selected from 0, 1, 2 or 3;

[0098] It represents the connection site between L and D, where a hydrogen atom or hydroxyl group on D is replaced by L.

[0099] In some embodiments, a hydrogen atom or a hydroxyl group on R1, R2, R3, or Ra in D is replaced by L.

[0100] In some embodiments, a hydrogen atom or a hydroxyl group on R1, R3, or Ra in D is replaced by L.

[0101] In some embodiments, a hydrogen atom or a hydroxyl group on R1 in D is substituted by L.

[0102] In some embodiments, a hydrogen atom or a hydroxyl group on R2 in D is substituted by L.

[0103] In some embodiments, a hydrogen atom or a hydroxyl group on R3 in D is substituted by L.

[0104] In some embodiments, a hydrogen atom or a hydroxyl group on Ra in D is replaced by L.

[0105] In some embodiments, D is a structure as shown in Formula I-2:

[0106] or a stereoisomer, pharmaceutically acceptable salt or solvate thereof,

[0107] in,

[0108] R1, R2, R3, and Ra meet one of the following conditions:

[0109] (1) R1 is selected from hydrogen, methyl, R2 is selected from hydrogen, deuterium or hydroxyl; R3 is selected from 1 is connected to L; Ra is selected from hydrogen, deuterium,

[0110] (2) R1 is selected from hydrogen, methyl, R2 is selected from hydrogen, deuterium or hydroxyl; R3 is selected from hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole), Ra is selected from 1 bit is connected to L; or

[0111] (3) R1 is present or absent, and when R1 is present, it is selected from The 1 position is connected to L, and when R1 is absent, L is directly connected to the nitrogen atom; R2 is selected from hydrogen, deuterium or hydroxyl; R3 is selected from hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole),

[0112] Ra is selected from hydrogen, deuterium,

[0113] R 4 Selected from hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl) or hydroxy-substituted C1-6 alkyl;

[0114] s is selected from 1, 2, 3, 4, 5 or 6;

[0115] m and n are each independently selected from 0, 1, 2 or 3.

[0116] In some embodiments, R1 is selected from hydrogen, methyl, R2 is selected from hydrogen or hydroxy; R3 is selected from Position 1 is connected to L; Ra is selected from hydrogen.

[0117] In some embodiments, R1 is selected from hydrogen, methyl, R2 is selected from hydrogen or hydroxy; R3 is selected from hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole); Ra is selected from 1 bit is connected to L.

[0118] In some embodiments, R1 is present or absent, and when present, R1 is selected from 1 is connected to L, when R1 is absent, L is directly connected to the nitrogen atom; R2 is selected from hydrogen or hydroxyl; R3 is selected from hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole), Ra is selected from hydrogen.

[0119] In some embodiments, R1 is present or absent, and when present, R1 is selected from The 1 position is connected to L, and when R1 is absent, L is directly connected to the nitrogen atom; R2 is selected from hydrogen or hydroxy; R3 is selected from hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole); Ra is selected from hydrogen,

[0120] In some embodiments,

[0121] R1 is selected from hydrogen or methyl;

[0122] R2 is selected from hydrogen, deuterium or hydroxyl;

[0123] R3 and Ra meet one of the following conditions:

[0124] (1) R3 is selected from

[0125] Ra is selected from hydrogen, deuterium, amino or C1-6 alkylhydroxy; or

[0126] (2) R3 is selected from hydrogen, deuterium, C1-6 alkylhydroxyl, 5-12 membered heteroaryl (preferably thiazole) or

[0127] Ra is selected from

[0128] R 4 Selected from hydrogen or C1-6 alkyl (preferably methyl);

[0129] s is selected from 1, 2, 3, 4, 5 or 6;

[0130] m and n are each independently selected from 0, 1, 2 or 3;

[0131] 1 bit is connected to L.

[0132] In some embodiments, R1 is selected from hydrogen or methyl; R2 is selected from hydrogen or hydroxy.

[0133] In some embodiments, R3 is selected from Ra is selected from hydrogen, and the 1 position is connected to L.

[0134] In some embodiments, R3 is selected from Ra is selected from hydrogen, and the 1 position is connected to L.

[0135] In some embodiments, R3 is selected from hydrogen or Ra is selected from 1 bit is connected to L.

[0136] In some embodiments, R3 is selected from hydrogen, Ra is selected from 1 bit is connected to L.

[0137] In some embodiments, s is selected from 1, 2, or 3.

[0138] In some embodiments, m is selected from 1 and n is selected from 1, 2, or 3.

[0139] In some embodiments, R3 is selected from Ra is selected from 1 bit is connected to L.

[0140] In some embodiments, R4 is hydrogen.

[0141] In some embodiments, D is a structure as shown in Formula I-2-1:

[0142] wherein R1 is selected from hydrogen or methyl;

[0143] R3 is selected from One of them is connected to L;

[0144] s is selected from 1, 2 or 3; m is selected from 1, and n is selected from 1, 2 or 3.

[0145] In some embodiments, D is a structure as shown in Formula I-2-2:

[0146] wherein R1 is selected from hydrogen or methyl;

[0147] R3 is selected from hydrogen or

[0148] Ra is selected from 1 bit is connected to L;

[0149] s is selected from 1, 2 or 3.

[0150] In some embodiments, D is selected from the following structures:

[0151] , bit 1 is connected to L.

[0152] In some embodiments, D is selected from the following structures:

[0153] , bit 1 is connected to L.

[0154] In some embodiments, D is selected from the following structures: 1 bit is connected to L.

[0155] In some embodiments, D is selected from the following structures: 1 bit is connected to L.

[0156] In some embodiments, the L is covalently linked to the amino group or thiol group of Tb; preferably, the L is covalently linked to the thiol group of Tb; more preferably, the L is covalently linked to the thiol group formed after the interchain disulfide bond of Tb is opened.

[0157] In some embodiments, the L is a cleavable linker or a non-cleavable linker; preferably, the L is a cleavable linker.

[0158] In some embodiments, L is

[0159] in,

[0160] L1 is a linker unit that covalently binds Tb and L2;

[0161] L2 is a linker unit that covalently binds L1 and L3;

[0162] L3 is an amino acid residue, a short peptide consisting of 2-10 amino acid residues or It covalently binds to L2 and L4;

[0163] L4 is a direct bond or a spacer unit, which covalently binds L3 and D.

[0164] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom or a N atom, and position 2 is connected to L2.

[0165] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom or a N atom, and position 2 is connected to L2.

[0166] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2.

[0167] In some embodiments, L1 is selected from Position 1 is connected to Tb via a N atom, and position 2 is connected to L2.

[0168] In some embodiments, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0169] In some embodiments, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0170] In some embodiments, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0171] In some embodiments, L2 is selected from the structures shown in the following table: Bit 1 is connected to L1 and bit 2 is connected to D.

[0172] In some embodiments, L3 is selected from

[0173] Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 At the same time, it is methyl, ethyl or propyl.

[0174] In some embodiments, L3 is selected from

[0175] Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 At the same time, it is methyl, ethyl or propyl.

[0176] In some embodiments, L3 is selected from

[0177] Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 At the same time, it is methyl, ethyl or propyl.

[0178] In some embodiments, L3 is selected from Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 At the same time it is methyl or propyl.

[0179] In some embodiments, L3 is selected from

[0180] In some embodiments, L4 is selected from

[0181] In some embodiments, L4 is selected from a direct bond or

[0182] In some embodiments, L is selected from:

[0183] Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 It is also a methyl, ethyl or propyl group; the 1 position is connected to Tb through the S atom, and the 2 position is connected to D.

[0184] In some embodiments, L is selected from:

[0185] Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 It is a methyl or propyl group at the same time; the 1 position is connected to Tb through the S atom, and the 2 position is connected to D.

[0186] In some preferred embodiments, L is selected from:

[0187] The 1 position is connected to Tb through an S atom or a N atom, and the 2 position is connected to D.

[0188] In some preferred embodiments, L is selected from:

[0189] The 1 position is connected to Tb through an S atom or a N atom, and the 2 position is connected to D.

[0190] In some preferred embodiments, L is selected from: Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 It is also a methyl, ethyl or propyl group; the 1 position is connected to Tb through the S atom, and the 2 position is connected to D.

[0191] In some preferred embodiments, L is selected from: Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0192] In some preferred embodiments, L is selected from: Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0193] In some preferred embodiments, L is: Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0194] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof having endocytic or non-endocytic activity.

[0195] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof having endocytic activity.

[0196] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has the activity of binding to tumor cell surface antigens.

[0197] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has tumor cell surface antigen binding activity and tumor cell endocytosis activity.

[0198] In some embodiments, the antibody or antigen-binding fragment thereof has antigen-binding activity and no or weak tumor cell endocytosis activity.

[0199] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that binds to a non-endocytosed antigen on the surface of a tumor cell.

[0200] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that does not have tumor cell endocytosis activity.

[0201] In some preferred embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has the activity of binding to tumor cell surface antigens and has the activity of tumor cell endocytosis.

[0202] In some embodiments, the antibody or antigen-binding fragment thereof comprises Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, bispecific antibody or multispecific antibody.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof is a non-human antibody, a humanized antibody, a chimeric antibody, or a fully human antibody.

[0204] In some embodiments, the antibody or antigen-binding fragment thereof is a probody, a bispecific antibody, or a multispecific antibody.

[0205] In some embodiments, the antibody or antigen-binding fragment thereof includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (eg, scFv).

[0206] In some embodiments, the target of Tb is selected from the group consisting of: CD19, CD20, CD21, CD22, CD30, CD33, CD123, B7H3, Her2, Napi2b, and Trop-2.

[0207] In some embodiments, Tb is an anti-Her2 antibody or an antigen-binding fragment thereof, an anti-B7H3 antibody or an antigen-binding fragment thereof, or an anti-Trop-2 antibody or an antigen-binding fragment thereof.

[0208] In some embodiments, Tb is an anti-Trop-2 antibody or an antigen-binding fragment thereof, or an anti-Her2 antibody or an antigen-binding fragment thereof.

[0209] In some embodiments, Tb is an anti-Her2 antibody or an antigen-binding fragment thereof, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab or an antigen-binding fragment thereof; preferably, Tb is Trastuzumab or Pertuzumab; for example, Tb is Trastuzumab.

[0210] In some embodiments, Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, such as 1D1-01, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab or an antigen-binding fragment thereof; preferably, Tb is 2E3-02 antibody.

[0211] In some embodiments, Tb is an anti-Trop-2 antibody or an antigen-binding fragment thereof, such as datopotamab, sacituzumab, or an antigen-binding fragment thereof.

[0212] In some embodiments, Tb is an anti-Napi2b antibody or an antigen-binding fragment thereof.

[0213] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0214] The heavy chain variable region (VH) comprises:

[0215] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 3 in WO2024012524A1 (66C12D12-hz1 Kabat HCDR1);

[0216] (ii) a HCDR2 comprising the amino acid sequence of SEQ ID NO: 5 in WO2024012524A1 (66C12D12-hz1 Kabat HCDR2); and

[0217] (iii) a HCDR3 comprising the amino acid sequence of SEQ ID NO: 6 in WO2024012524A1 (66C12D12-hz1 Kabat HCDR3); and

[0218] The light chain variable region (VL) comprises:

[0219] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 8 in WO2024012524A1 (66C12D12-hz1 Kabat LCDR1);

[0220] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 9 in WO2024012524A1 (66C12D12-hz1 Kabat LCDR2); and

[0221] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 10 in WO2024012524A1 (66C12D12-hz1 Kabat LCDR3);

[0222] The CDRs were determined according to the Kabat definition scheme.

[0223] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 17 in WO2024012524A1 (66C12D12-hz1VH), and the light chain variable region comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 18 in WO2024012524A1 (66C12D12-hz1VL);

[0224] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17 in WO2024012524A1 (66C12D12-hz1VH), and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 18 in WO2024012524A1 (66C12D12-hz1VL).

[0225] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 83 in WO2024012524A1 (66C12D12-hz1 HC heavy chain), and the light chain shown in SEQ ID NO: 84 in WO2024012524A1 (66C12D12-hz1 LC light chain).

[0226] In some embodiments, the antibody drug conjugate is selected from the following structures:

[0227] Wherein, q and Tb are defined as described in any embodiment of the present invention.

[0228] In some embodiments, the antibody drug conjugate is selected from the following structures:

[0229] Wherein, q and Tb are defined as described in any embodiment of the present invention.

[0230] In some embodiments, q is selected from any number between 1-12.

[0231] In some embodiments, q is selected from any number between 1-8.

[0232] In some embodiments, q is selected from any number between 2-8.

[0233] In some embodiments, q is selected from any number between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, and 7-8.

[0234] In some embodiments, q is selected from any number between 2-3.

[0235] In some embodiments, q is selected from any number between 3-4.

[0236] In some embodiments, q is selected from any number between 7-8.

[0237] In some embodiments, q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0238] In some embodiments, q is selected from 2, 4, 6, and 8.

[0239] In some embodiments, the antibody drug conjugate is selected from:

[0240] In some embodiments, the antibody drug conjugate is selected from:

[0241] Among them, NaPi2b is the 66C12D12-hz1 antibody (see WO2024012524A1).

[0242] Drug-Linker Conjugates

[0243] In a second aspect, the present invention provides a drug-linker conjugate represented by formula II:

[0244] or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof;

[0245] in,

[0246] Lg is a group that reacts with antibodies;

[0247] The definitions of L and D are as described in any embodiment of the present invention.

[0248] In some embodiments, L is

[0249] in,

[0250] L1 is a linker unit that covalently binds Lg and L2;

[0251] L2 is a linking unit, and L2 covalently binds L1 and L3;

[0252] L3 is an amino acid residue, a short peptide consisting of 2-10 amino acid residues or It covalently binds to L2 and L4;

[0253] L4 is present or absent. When L4 is present, L4 is a spacer unit that covalently binds L3 and P.

[0254] In some embodiments, Lg is selected from hydrogen, halogen, sulfone, tertiary amine salt (Me3N + 、Et3N + ), diazonium salt, -OMs, MeSO2-, CF3SO3-, p-toluenesulfonyl, Substituted phenoxy, the substituents include halogen and nitro.

[0255] In some embodiments, Lg is selected from F, Cl, Br, MeSO2-, pentafluorophenoxy; more preferably, Lg is MeSO2-.

[0256] In some embodiments, the drug-linker conjugate is as shown in Formula II-1,

[0257] or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof;

[0258] in,

[0259] Lg is a group that reacts with an antibody; L1, L2, L3, L4 and D are defined as described in any embodiment of the present invention.

[0260] In some embodiments, when L1 is When Lg-L1 is Position 2 is connected to L2; the definitions of L2, L3, L4 and D are as described in any embodiment of the present invention.

[0261] In some embodiments, L1 is The 1 position is connected to Lg, and the 2 position is connected to L2; Lg is selected from halogen, sulfone, tertiary amine salt (Me3N + 、Et3N + ), -OMs, MeSO2-, CF3SO3-, p-toluenesulfonyl or pentafluorophenoxy; more preferably, Lg is selected from F, Cl, Br, MeSO2- or pentafluorophenoxy; further preferably, Lg is MeSO2- or pentafluorophenoxy.

[0262] In some embodiments, L1 is Position 1 is connected to Lg, and position 2 is connected to L2; Lg is MeSO2-.

[0263] In some embodiments, L1 is The 1 position is connected to Lg, and the 2 position is connected to L2; Lg is selected from pentafluorophenoxy.

[0264] In some embodiments, the drug-linker conjugate is selected from the group consisting of:

[0265] Bioactive molecules

[0266] In a third aspect, the present invention provides a biologically active compound represented by formula III:

[0267] or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof,

[0268] in,

[0269] R1 is selected from hydrogen, methyl,

[0270] R2 is selected from hydrogen, deuterium or hydroxyl;

[0271] R3' is selected from hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole), C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 、

[0272] Ra' is selected from hydrogen, deuterium, -C1-6 alkylene hydroxyl, -NHR 4 or -C1-6 alkylene-NHR 4 ;

[0273] R 4 Selected from hydrogen, C1-6 alkyl (preferably C1-3 alkyl) or hydroxy substituted C1-6 alkyl;

[0274] s is selected from 1, 2, 3, 4, 5 or 6;

[0275] m and n are each independently selected from 0, 1, 2 or 3.

[0276] In some embodiments,

[0277] R1, R2, R3', Ra' meet one of the following conditions:

[0278] (1) R1 is selected from hydrogen, methyl; R2 is selected from hydrogen, deuterium or hydroxyl; R3' is selected from C2-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 、 Ra' is selected from hydrogen, deuterium, -NHR 4 , C1-6 alkylene hydroxyl or -C1-6 alkylene-NHR 4 ;

[0279] (2) R1 is selected from hydrogen, methyl; R2 is selected from hydrogen, deuterium or hydroxyl; R3' is selected from hydrogen; Ra' is selected from -C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 ;or

[0280] (3) R1 is selected from hydrogen, methyl; R2 is selected from hydrogen, deuterium or hydroxyl; R3' is selected from 5-12 membered heteroaryl (preferably thiazole); Ra' is selected from C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 or -NHR 4 ;

[0281] (4) R1 is selected from R2 is selected from hydrogen, deuterium or hydroxyl; R3' is selected from hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 membered heteroaryl (preferably 5-6 membered heteroaryl, more preferably thiazole), C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 、 Ra' is selected from hydrogen, deuterium, C1-6 alkylene hydroxyl, -NHR 4 or -C1-6 alkylene-NHR 4 ;

[0282] R 4 Selected from hydrogen or C1-6 alkyl (preferably methyl);

[0283] s is selected from 1, 2, 3, 4, 5 or 6;

[0284] m and n are each independently selected from 0, 1, 2 or 3.

[0285] In some embodiments,

[0286] R1 is selected from hydrogen or methyl;

[0287] R2 is selected from hydrogen, deuterium or hydroxyl;

[0288] R3' and Ra' meet one of the following conditions:

[0289] (1) R3' is selected from C2-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 or Ra' is selected from hydrogen, deuterium, -NHR 4 (preferably amino) or C1-6 alkylhydroxyl;

[0290] (2) R3' is selected from hydrogen; Ra' is selected from -C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 ;or

[0291] (3) R3' is selected from 5-12 membered heteroaryl (preferably thiazole); Ra' is selected from C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 or -NHR 4 (preferably amino);

[0292] R 4 Selected from hydrogen or C1-6 alkyl (preferably methyl);

[0293] m and n are each independently selected from 0, 1, 2 or 3.

[0294] In some embodiments, R3' is selected from C2-6 alkylene hydroxy or Ra' is selected from hydrogen.

[0295] In some embodiments, R3' is selected from Ra' is selected from C2-3 alkylene hydroxy or amino.

[0296] In some embodiments, R3' is selected from hydrogen; Ra' is selected from C1-6 alkylene hydroxy (preferably C1-3 alkylene hydroxy).

[0297] In some embodiments, R3' is selected from Ra' is selected from -NH2.

[0298] In some embodiments, R1 is selected from hydrogen or methyl; R2 is selected from hydrogen or hydroxy.

[0299] In some embodiments, R1 is R2 is selected from hydroxy; R3' is selected from methyl; Ra' is selected from hydrogen.

[0300] In some embodiments, R4 is hydrogen.

[0301] In some embodiments, s is selected from 1, 2, or 3.

[0302] In some embodiments, m is selected from 1 and n is selected from 1, 2, or 3.

[0303] In some embodiments, the biologically active compound is selected from the following structures:

[0304] In some embodiments, the present invention provides the use of the bioactive compound in preparing ADC, wherein the bioactive compound has the following structure:

[0305] In some embodiments, the present invention provides the use of the bioactive compound in preparing a drug-linker conjugate, wherein the bioactive compound is selected from the following structures:

[0306] Method for preparing antibody-drug conjugates

[0307] In a fourth aspect of the present invention, the present invention provides a method for preparing an antibody drug conjugate of Formula I, comprising:

[0308] Conjugate Tb with the drug linker shown in formula II performing a coupling reaction;

[0309] Wherein, Tb, Lg, L and D are defined as described in any embodiment of the present invention.

[0310] Specifically, the method comprises conjugating Tb with a drug linker shown in formula II The step of forming a CS bond or a CN bond by coupling reaction is carried out in a solvent.

[0311] In some preferred embodiments, the present invention provides a method for preparing an antibody drug conjugate of Formula I, comprising:

[0312] Conjugate Tb with the drug linker shown in formula II-1 performing a coupling reaction;

[0313] Wherein, Tb, Lg, L1, L2, L3, L4 and D are defined as described in any embodiment of the present invention.

[0314] Specifically, the method comprises conjugating Tb with the drug linker shown in II-1 The step of forming a CS bond or a CN bond by coupling reaction is carried out in a solvent.

[0315] In some embodiments, the molar ratio of Tb to the drug-linker conjugate is 1:(1-20), such as 1:(2-16), 1:(2-14), 1:(2-12), 1:(2-10).

[0316] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0317] In some embodiments, the organic solvent is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, and nitriles (eg, acetonitrile).

[0318] In some embodiments, the method further comprises the step of purifying the coupling product.

[0319] In some embodiments, the coupled product is purified by chromatography.

[0320] In some embodiments, the chromatography method comprises one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, or affinity chromatography.

[0321] In some embodiments, the method is performed at -20-100°C, such as 0-50°C, preferably room temperature.

[0322] Preparation method of drug-linker conjugate

[0323] In a fifth aspect of the present invention, a method for preparing the drug-linker conjugate represented by Formula II is provided.

[0324] Specifically, taking DL-1 as an example, the compound INT1 shown in the general formula is subjected to a condensation reaction with the compound INT4 to obtain the target compound (DL-1).

[0325] Drug-linker conjugates of various structures can be obtained by reacting INT1 analogs with INT4 analogs.

[0326] Conjugate Group

[0327] In a sixth aspect, the present invention provides an antibody drug conjugate group comprising the above-mentioned antibody drug conjugate, its stereoisomers, pharmaceutically acceptable salts or solvates or a combination thereof, wherein the antibody drug conjugate has one, two or more q values.

[0328] In some embodiments, when one q value of the antibody drug conjugates in the population of antibody drug conjugates accounts for the majority (e.g., 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99%), the q value and the average DAR are close.

[0329] In some embodiments, when there is only one q-valued ADC in the population of ADCs, the q-value and the average DAR are equal.

[0330] In some embodiments, when the antibody drug conjugates of the antibody drug conjugate population have two or more q values, the proportion of antibody drug conjugates with a specific q value in all antibody drug conjugates in the composition is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0331] In some embodiments, the average drug to antibody ratio (average DAR) in the antibody drug conjugate population is an integer or decimal selected from 1-12, preferably an integer or decimal selected from 1-10.

[0332] In some embodiments, the average drug to antibody ratio (average DAR) of the population of antibody drug conjugates is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5;

[0333] In some embodiments, the average drug to antibody ratio (average DAR) of the population of antibody drug conjugates is selected from about 2.0, 4.0, 6.0, or 8.0;

[0334] In some embodiments, the average drug to antibody ratio (average DAR) in the population of antibody drug conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9, or 9.7.

[0335] In some embodiments, the antibody drug conjugate population contains ADCs with a distribution of DARs of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8 drug loading species). It is worth noting that degradation products can be produced such that the mixture may also contain DARs of 1, 3, 5, and 7. In addition, the antibody drug conjugate population may also have an average DAR greater than 8. The antibody drug conjugate is produced by reducing the interchain disulfide and then coupling. In some embodiments, the antibody drug conjugate comprises both: an antibody drug conjugate having a DAR of 4 or less (i.e., a drug loading species of 4 or less) and an antibody drug conjugate having a DAR of 6 or more (i.e., a drug loading species of 6 or more).

[0336] Pharmaceutical composition

[0337] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate, or its stereoisomer, pharmaceutically acceptable salt or solvate; or the aforementioned drug-linker conjugate, or its stereoisomer, pharmaceutically acceptable salt or solvate; or the aforementioned antibody-drug conjugate group and optionally one or more pharmaceutical excipients.

[0338] The term "drug to antibody ratio" or "DAR" refers to the amount of drug, e.g., a small molecule toxin, attached to the antibody of an ADC. The DAR of an ADC can range from 1 to 16, but higher loadings (e.g., 20) are possible depending on the number of attachment sites on the antibody. The term DAR can be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. In some embodiments, the ADC comprises ADCs having a distribution of DARs from 1 to 8, e.g., 1.5, 2, 4, 6, and 8 (i.e., drug loading species of 1.5, 2, 4, 6, and 8). Notably, degradation products can be produced such that the ligand drug conjugate can also contain DARs of 1, 3, 5, and 7. In addition, the ADC can also have a DAR greater than 8. The ADC is produced by interchain disulfide reduction followed by conjugation. In some embodiments, the ADC comprises both: the ADC having a DAR of 4 or less (ie, a drug loading species of 4 or less) and the ADC having a DAR of 6 or more (ie, a drug loading species of 6 or more).

[0339] use

[0340] In an eighth aspect, the present invention provides the use of the aforementioned antibody-drug conjugate, or its stereoisomer, pharmaceutically acceptable salt or solvate; or the aforementioned drug-linker conjugate, or its stereoisomer, pharmaceutically acceptable salt or solvate; or the aforementioned antibody-drug conjugate group; or the aforementioned pharmaceutical composition in the preparation of a drug for treating and / or preventing diseases associated with abnormal cell activity (e.g., cancer diseases).

[0341] The present invention provides the aforementioned antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof; or the aforementioned drug-linker conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof; or the aforementioned antibody-drug conjugate population; or the aforementioned pharmaceutical composition, for use in treating and / or preventing diseases associated with abnormal cell activity (e.g., cancer).

[0342] The present invention provides a method for preventing and / or treating diseases associated with abnormal cell activity (e.g., cancer), comprising: administering to an individual in need thereof a preventive and / or therapeutically effective amount of the aforementioned antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof; or the aforementioned drug-linker conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof; or the aforementioned antibody-drug conjugate group; or the aforementioned pharmaceutical composition.

[0343] In some embodiments, the cancer disease of the present invention is selected from esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0344] In some embodiments, the cancer disease of the present invention is selected from ovarian cancer, breast cancer, gastric cancer, lung cancer, colorectal cancer, and esophageal cancer (eg, esophageal adenocarcinoma or esophageal squamous cell carcinoma).

[0345] In some embodiments, the breast cancer is selected from ductal carcinoma.

[0346] In some embodiments, the cancer disease is a cancer disease associated with HER2, TROP2, B7H3, or Napi2b targets.

[0347] In some embodiments, the cancer disease is a solid tumor or a hematological tumor.

[0348] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0349] The reagents and raw materials used in the present invention are commercially available.

[0350] Beneficial effects of the present invention:

[0351] Through extensive research, the present invention has developed an antibody-drug conjugate with an auristatin-like bioactive compound as the payload, which can achieve one or more of the following effects:

[0352] (1) Antibody-drug conjugates with high anti-tumor activity can kill tumor cells more effectively and have better in vivo or in vitro anti-tumor effects than antibody-drug conjugates linked to similar small molecule drugs in the prior art;

[0353] (2) Antibody-drug conjugates have high stability in the circulation, which can reduce the shedding of bioactive molecules in non-target cells and increase the effective release of bioactive compounds in cells, thus having a higher therapeutic index;

[0354] (3) Antibody-drug conjugates use linkers that are cleaved in the tumor microenvironment (both inside and outside tumor cells), thus producing good anti-tumor effects in tumors with low or no antigen expression;

[0355] (4) By increasing the hydrophilicity of the linker, the physicochemical properties of the entire ADC molecule are improved, and the exposure of the entire ADC molecule in the relatively acidic tumor environment is increased. Therefore, the ADC has better tumor tissue targeting, that is, the ability to be enriched in the tumor microenvironment, increasing the ratio of the concentration of bioactive molecules in the tumor and blood, and reducing the mechanism-related toxicity of the ADC molecule (toxicity caused by ADC binding to cell surface antigens in non-tumor tissues and internalization, also known as "on-target toxicity"), thus having a higher therapeutic index;

[0356] (5) The bioactive compound (Payload) used in the antibody-drug conjugate has good liver microsomal stability and can be rapidly metabolized in the body, which can reduce the toxicity caused by exposure of the bioactive compound to the circulatory system and normal tissues, thereby improving the safety of the entire antibody-drug conjugate;

[0357] (6) The bioactive molecules of antibody-drug conjugates have higher anti-tumor cell activity and therefore have an excellent bystander effect. ADCs can more effectively kill tumor cells with high antigen expression as well as tumor cells with low or no antigen expression in tumor tissues.

[0358] (7) The toxin linker of the antibody-drug conjugate has higher stability and better in vivo and in vitro stability than the toxin linkers of similar small molecule drugs in the prior art. The ADC prepared by the method is not easily decomposed / degraded, has excellent physical and chemical stability, is suitable for industrial preparation, and has good drugability.

[0359] Therefore, the antibody-drug conjugate provided by the present invention has high clinical application value. DETAILED DESCRIPTION

[0360] The present invention will be further described below by the description of specific embodiments, but this is not limitation of the present invention. Those skilled in the art can make various modifications or improvements according to the teachings of the present invention without departing from the basic idea and scope of the present invention. Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained commercially.

[0361] The abbreviations used in this invention have the following meanings:

[0362] Preparation plan

[0363] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0364] Nuclear magnetic resonance (NMR) 1H NMR was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the measurement solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0365] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0366] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0367] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0368] The ultra-performance liquid chromatography (UPLC) instrument used was AB SCIEX, model ExionLC.

[0369] The high-resolution mass spectrometer used was AB SCIEX, model X500B.

[0370] Example 1. Synthesis of intermediates

[0371] Example 1.1: Synthesis of INT1

[0372] Step 1: Dissolve compound INT1-1 (1.0 g, 2.65 mmol) in 1,4-dioxane (5 mL). Add HCl / 1,4-dioxane solution (5 mL, 4 M). Stir at room temperature for 3 h. The reaction mixture is concentrated under reduced pressure to obtain the hydrochloride salt of the target compound INT1-2 (0.83 g), which is used directly in the next reaction.

[0373] LCMS (ESI) [M+H] + =278.31.

[0374] Step 2: Dissolve compound INT1-2 (803.35 mg, 2.56 mmol), compound INT1-3 (1.10 g, 2.56 mmol), and HATU (1.17 g, 3.07 mmol) in DMF (2 mL). Add DIPEA (992.56 mg, 7.68 mmol) and stir at room temperature for 16 h. The reaction solution is purified by reverse-phase column chromatography (acetonitrile:H2O (containing 0.5% HCl) = 0%-40%) to obtain the hydrochloride salt of the target compound INT1-4 (1.4 g).

[0375] LCMS (ESI) [M+Na] + =711.79.

[0376] 1 H NMR(400MHz,DMSO-d6)δ8.91(d,J=8.0Hz,1H),7.44-7.25(m,5H),5.20-4.98 (m,2H),4.71-4.48(m,2H),4.02-3.83(m,3H),3.73-3.64(m,1H),3.53-3.51( m,1H),3.35-3.31(m,2H),3.24(s,2H),3.16(s,3H),3.00(s,3H),2.78-2.74( m,6H),2.55-2.50(m,1H),2.47-2.43(m,1H),2.39-2.22(m,2H),2.02-2.00(m 1H),1.88-1.87(m,2H),1.76-1.70(m,3H),1.35-1.25(m,1H),1.15-1.13(m,3H),0.98-0.82(m,16H),0.78-0.75(m,3H).

[0377] Step 3: Dissolve compound INT1-4 (1.4 g, 2.03 mmol) and palladium on carbon (212.84 mg, 10% content) in DCM (12 mL) and MeOH (3 mL). Replace the mixture with hydrogen three times. Stir the reaction mixture at room temperature under a hydrogen atmosphere for 16 hours. Filter the reaction mixture, and concentrate the filtrate under reduced pressure to obtain the target compound INT1 (1.1 g).

[0378] LCMS (ESI) [M+H] + =599.63.

[0379] 1H NMR (400MHz, DMSO-d6) δ12.26 (s, 1H), 8.93 (d, J = 8.0Hz, 1H), 4.68-4.64 (m, 1H), 4.59-4.55 (m, 1H), 4.02 -3.96(m,2H),3.84-3.82(m,1H),3.71-3.69(m,1H),3.67-3.46(m,1H),3.28(s,3H),3.18-3.17(m,3H),3 .03-3.00(m,3H),2.97-2.85(m,2H),2.78-3.73(m,7H),2.35-2.28(m,2H),2.02-1.88(m,2H),1.80-1.70 (m,2H),1.28-1.23(m,3H),1.18-1.16(m,1H),1.11-1.09(m,3H),0.94-0.83(m,15H),0.78-0.74(m,3H).

[0380] Example 1.2: Synthesis of INT2

[0381] Step 1: Dissolve compound INT2-1 (5.0 g, 50.9 mmol) in toluene (170 mL), add tetrabutylammonium bromide (6.3 g, 19.6 mmol), cool to 0°C, add sodium hydroxide (61.2 g, 535.5 mmol), and then add tert-butyl bromoacetate (34.78 g, 178.32 mmol). Incubate at room temperature overnight. Add water (80 mL) to the reaction solution, then extract three times with ethyl acetate (80 mL x 3). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and dried to obtain the target compound INT2-2 (10.2 g).

[0382] 1 H NMR (400MHz, DMSO-d6) δ8.67(s,2H),3.98(s,2H),3.57(t,J=6.2Hz,2H),2.57–2.54(m,2H),2.52(s,3H),1.83–1.76(m,2H),1.42(s,9H).

[0383] Step 2: Dissolve compound INT2-2 (10 g, 50.5 mmol) in tetrahydrofuran (100 mL). Add INT2-3 (10.35 g, 50.5 mmol), bistriphenylphosphine palladium dichloride (3.5 g, 5.05 mmol), cuprous iodide (1.92 g, 10.1 mmol), and triethylamine (15.3 g, 151.5 mmol). Stir and react overnight at 70°C under a nitrogen atmosphere. The reaction mixture was cooled, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain the target compound INT2-4 (5.2 g).

[0384] LCMS (ESI) [M+H] + =323.0;

[0385] 1 H NMR (400MHz, DMSO-d6) δ8.67(s,2H),3.98(s,2H),3.57(t,J=6.2Hz,2H),2.57–2.54(m,2H),2.52(s,3H),1.83–1.76(m,2H),1.42(s,9H).

[0386] Step 3: Dissolve compound INT2-4 (1.0 g, 3.1 mmol) in dichloromethane (50 mL), add trifluoroacetic acid (3.54 g, 31 mmol), and stir at room temperature overnight. The reaction solution is directly concentrated under reduced pressure to obtain compound INT2-5 (1.5 g).

[0387] LCMS (ESI) [M+H] + =267.0;

[0388] 1 H NMR (400MHz, DMSO-d6) δ8.67(s,2H),4.03(s,2H),3.59(t,J=6.2Hz,2H),2.58–2.53(m,2H),2.53(s,3H),1.84–1.77(m,2H).

[0389] Step 4: Dissolve compound INT2-5 (1.5 g, 5.6 mmol) in tetrahydrofuran (20 mL) and water (20 mL), add potassium peroxymonosulfonate (10.3 g, 16.8 mmol), and stir at room temperature for 2 h. The reaction solution is concentrated under reduced pressure to remove tetrahydrofuran. The aqueous phase is extracted three times with ethyl acetate (50 mL). The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product is purified by reverse phase chromatography (acetonitrile: H2O containing 0.05% HCl = 5%-50%) to obtain the target compound INT2 (0.4 g).

[0390] LCMS (ESI) [M+H] + =299.1;

[0391] 1 H NMR (400MHz, DMSO-d6) δ12.60(s,1H),9.11(s,2H),4.03(s,2H),3.60(t,J=6.2Hz,2H),3.41(s,3H),2.63(t,J=7.1Hz,2H),1.87–1.80(m,2H).

[0392] Example 1.3: Synthesis of INT3

[0393] Step 1: Dissolve compound INT3-1 (500.0 mg, 1.0 mmol), compound INT2 (298.31 mg, 1.0 mmol), and HATU (456.28 mg, 1.2 mmol) in DMF (5 mL). Add DIPEA (387.72 mg, 3.0 mmol) and stir at room temperature for 16 h. The reaction mixture is purified by reverse-phase column chromatography (acetonitrile:H₂O = 0%-40%) to obtain the target compound INT3 (610 mg).

[0394] LCMS (ESI) [M+H] + =778.54.

[0395] 1 H NMR(400MHz,DMSO-d6)δ9.11(s,2H),8.86–8.71(m,1H),8.34–8.20(m,2H),7.48(d,J =8.9Hz,1H),4.79–4.56(m,2H),4.34–4.19(m,2H),4.07–3.91(m,4H),3.80–3.71(m,2 H),3.63–3.56(m,2H),3.41(s,3H),2.76–2.57(m,8H),2.05–1.93(m,1H),1.91–1.82 (m,2H),1.77–1.64(m,1H),1.64–1.44(m,7H),1.33–1.23(m,2H),0.92–0.76(m,12H).

[0396] Example 1.4: Synthesis of INT4

[0397] Step 1: Dissolve INT4-1 (100 mg, 0.27 mmol) and INT3 (630.09 mg, 0.81 mmol) in 2 mL of 2M HCl / DMF and stir at room temperature for 3 h. The reaction mixture was directly purified by reverse-phase column chromatography (acetonitrile:H₂O = 0-95%) to obtain the target compound INT4-2 (110 mg).

[0398] LCMS (ESI) [M+H] + =1051.80.

[0399] 1 H NMR(400MHz, DMSO-d6)δ9.13(s,2H),8.69(t,J=8.0Hz,1H),8.32(d,J=8.0Hz,1H),8.28-8.24(m,1H),7.92-7.90(m,2H ),7.68-7.62(m,2H),7.51(d,J=8.0Hz,1H),7.47-7.40(m,2H),7.38-7.31(m,3H),7.29-7.19(m,5H),4.60-4.58(m,2H) ,4.33-4.24(m,3H),4.20-4.14(m,2H),3.98(s,2H),3.85-3.70(m,3H),3.65-3.62(m,2H),3.44(s,3H),3.03-2.97(m, 6H),2.84-2.80(m,1H),2.72-2.62(m,3H),2.07-1.86(m,2H),1.65-1.62(m,8H),1.41-.15(m,3H),0.98-0.78(m,14H).

[0400] Step 2: Dissolve compound INT4-2 (110 mg, 0.10 mmol) in DMF (1 mL), add diethylamine (0.02 mL), and stir at room temperature for 1 h. The reaction solution is directly purified by reverse-phase column chromatography (acetonitrile:H2O = 0-95%) to obtain the target compound INT4 (55.0 mg).

[0401] LCMS (ESI) [M / 2+H] + =829.43.

[0402] Example 1.5: Synthesis of INT5

[0403] Step 1: Dissolve INT3-1 (612.05 mg, 1.23 mmol), INT5-1 (300.0 mg, 1.23 mmol), and HATU (561.22 mg, 1.48 mmol) in DMF (10 mL), then add DIPEA (476.90 mg, 3.96 mmol). Stir the mixture at room temperature for 3 h. The reaction solution is then directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.1% FA = 0-50%) to obtain the target compound INT5-2 (550.0 mg).

[0404] LCMS (ESI) [M+H] = 723.45.

[0405] 1 H NMR(400MHz,DMSO-d6)δ8.75-8.73(m,1H),8.09-8.05(m,1H),7.97-7.94(m,2H),4.71-4.63(m,2H),4.23 -4.14(m,2H),4.11-4.06(m,1H),4.03-3.96(m,2H),3.82-3.79(m,1H),3.73-3.69(m,2H),3.61(s,3H),3 .34-3.32(m,2H),3.31-3.25(m,2H),3.11-3.05(m,1H),2.74-2.51(m,3H),2.49-2.47(m,3H),2.44-2.32 (m,2H),2.06-1.95(m,1H),1.89-1.72(m,3H),1.65-1.45(m,8H),1.38-1.26(m,3H),0.91-0.84(m,12H).

[0406] Step 2: Dissolve INT4-1 (50.0 mg, 0.13 mmol) and INT5-2 (281.91 mg, 0.19 mmol) in HCl / DMF (2 mL, 2 M) and stir at room temperature for 3 h. The reaction mixture was directly purified by reverse-phase column chromatography (acetonitrile:H2O = 0-40%) to obtain the target compound INT5-3 (105.0 mg).

[0407] LCMS (ESI) [M+H] + = 996.54.

[0408] Step 3: Dissolve compound INT5-3 (110 mg, 0.10 mmol) in DMF (1 mL), then add diethylamine (0.02 mL). Stir at room temperature for 1 h. The reaction solution is directly purified by reverse phase column chromatography (acetonitrile:H2O=0-60%) to obtain the target compound INT5 (55.0 mg).

[0409] LCMS (ESI) [M / 2+H]+ = 774.54.

[0410] 1 H NMR (400MHz, DMSO-d6) δ8.03(d,J=8.0Hz,1H),7.92-7.90(m,1H),7.36-7.32(m,3H),7.27-7.22(m,4H),5.58(d,J=8.0Hz,1H) ,4.60-4.54(m,2H),4.19-4.17(m,3H),4.07-4.03(m,1H),3.81-3.78(m,1H),3.68-3.66(m,2H),3.60(s,3H),3.45-3.43(m,2H ),3.10-3.04(m,1H),2.82-2.78(m,2H),2.70-2.67(m,2H),2.57-2.55(m,3H),2.40-2.45(m,4H),2.36-2.30(m,2H),1.85-1. 69(m,5H),1.66-1.56(m,2H),1.46-1.52(m,1H),1.35-1.45(m,5H),1.23-1.26(m,3H),1.02-1.07(m,1H),0.87-0.81(m,12H).

[0411] Example 1.6: Synthesis of INT6

[0412] Step 1:

[0413] Compounds INT6-1 (300 mg, 0.80 mmol) and INT4-1 (1.8 g, 2.40 mmol) were dissolved in HCl-DMF (6 mL, 2 M) and stirred at room temperature for 3 h. The reaction solution was directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% TFA = 10%-40%) to obtain the target compound INT6-2 (440.0 mg).

[0414] LCMS (ESI) [M+H] + =1021.57.

[0415] Step 2:

[0416] Compound INT6-2 (440 mg, 0.43 mmol) was dissolved in DMF (5 mL), and then diethylamine (0.25 mL) was added. After the addition was complete, the reaction was stirred at room temperature for 1 h. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-30%) to obtain the trifluoroacetate salt of the target compound INT6 (280.0 mg).

[0417] LCMS (ESI) [M+H] + =799.47.

[0418] Example 1.7: Synthesis of INT9

[0419] Compounds INT3-1 (500.0 mg, 1.13 mmol), INT7-7 (303.17 mg, 1.13 mmol), and HATU (429.66 mg, 1.13 mmol) were dissolved in DMF (5 mL), followed by the addition of DIPEA (438.12 mg, 3.39 mmol). The mixture was stirred at room temperature for 16 h. The reaction solution was purified by reverse-phase column chromatography (acetonitrile:H2O 0.1% TFA = 0%-40%) to obtain the trifluoroacetic acid salt of the target compound INT9 (600 mg).

[0420] LCMS (ESI) [M+H] + =692.34.

[0421] Example 1.8: Synthesis of INT10

[0422] Step 1:

[0423] Compounds INT9 (100 mg, 0.27 mmol) and INT4-1 (373.5 mg, 0.54 mmol) were dissolved in HCl-DMF (2 mL, 2 M) and stirred at room temperature for 3 h. The reaction solution was directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% TFA = 0%-40%) to obtain the target compound INT10-1 (100.0 mg).

[0424] LCMS (ESI) [M+H] + =965.61.

[0425] Step 2:

[0426] Compound INT10-1 (50 mg, 0.052 mmol) was dissolved in DMF (1 mL), and diethylamine (0.05 mL) was added. After the addition, the reaction was stirred at room temperature for 1 h. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-30%) to obtain the target compound INT10 (30.0 mg).

[0427] LCMS (ESI) [M+H] + =743.36.

[0428] Example 1.9: Synthesis of INT11

[0429] Step 1:

[0430] Compound D-3-3 (100 mg, 0.56 mmol) and FmocCl (144.87 mg, 0.56 mmol) were dissolved in DCM (2 mL), followed by the addition of DIPEA (217.12 mg, 1.68 mmol). The mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure, and the crude product was purified by normal phase column chromatography (MeOH:DCM = 0-5%) to obtain the target compound INT11 (150.0 mg).

[0431] LCMS (ESI) [M+H] + =402.24.

[0432] Example 1.10: Synthesis of INT12

[0433] Step 1:

[0434] Compounds INT11 (100 mg, 0.25 mmol) and INT9 (360.89 mg, 0.50 mmol) were dissolved in HCl-DMF (2 mL, 2 M) and stirred at room temperature for 3 h. The reaction solution was directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% TFA = 0%-40%) to obtain the target compound INT12-1 (90.0 mg).

[0435] LCMS (ESI) [M+H] + =993.61.

[0436] Step 2:

[0437] Compound INT12-1 (90 mg, 0.088 mmol) was dissolved in DMF (1 mL), and then diethylamine (0.05 mL) was added. After the addition was complete, the reaction was stirred at room temperature for 1 h. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-30%) to obtain the target compound INT12 (55.0 mg).

[0438] LCMS (ESI) [M+H] + =771.49.

[0439] Example 1.11: Synthesis of INT13

[0440] Step 1:

[0441] Compounds INT4-1 (2.0 g, 5.36 mmol) and INT13-1 (3.0 g, 10.72 mmol) were dissolved in DMF (12 mL) and HCl-DMF (0.8 mL, 4 M). After addition, the mixture was stirred at room temperature under nitrogen for 3 h. The reaction solution was directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% TFA = 0%-80%) to obtain the target compound INT13-2 (2.0 g).

[0442] LCMS (ESI) [M+H] + =594.22.

[0443] 1 H NMR(400MHz, DMSO-d6)δ8.71(t,J=6.6Hz,1H),7.96–7.85(m,2H),7.67–7.65(m,1H ),7.52(t,J=6.1Hz,1H),7.46–7.41(m,2H),7.40–7.29(m,10H),7.28–7.20(m,4H) ,5.06(s,2H),4.60(d,J=6.6Hz,1H),4.31–4.13(m,3H),3.90–3.74(m,1H),3.73–3 .61(m,3H),3.42–3.38(m,1H),2.90–2.77(m,1H),2.74–2.59(m,1H),2.02(d,1H).

[0444] Step 2:

[0445] Compound INT13-2 (2.0 g, 3.36 mmol) was dissolved in methanol (20 mL), and 10% palladium carbon (0.36 g, 55% water content) was added under nitrogen protection. After the addition, the hydrogen gas was replaced three times. The reaction was stirred at room temperature under a hydrogen atmosphere for 3 h. The reaction liquid was filtered and the filtrate was concentrated under reduced pressure to obtain the target compound INT13-3 (1.55 g).

[0446] LCMS (ESI) [M+H] + =460.22.

[0447] Step 3:

[0448] Compounds INT13-3 (1.55 g, 3.31 mmol), INT13-4 (1.35 g, 3.31 mmol), and DMTMM (1.10 g, 3.97 mmol) were dissolved in DMF (15 mL). After addition, the mixture was stirred at room temperature for 4 h. The reaction solution was directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% TFA = 10%-60%) to obtain the trifluoroacetic acid salt of the target compound INT13-5 (2.2 g).

[0449] LCMS (ESI) [M+H] + =849.54.

[0450] Step 4:

[0451] Compound INT13-5 (2.20 g, 2.59 mmol) was dissolved in DMF (20 mL) and diethylamine (1 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction solution was then directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% TFA = 0%-30%) to obtain the target compound INT13 (1.20 g).

[0452] LCMS (ESI) [M+H] + =627.40.

[0453] 1 H NMR(400MHz, DMSO-d6)δ9.45(s,1H),8.74(t,J=6.6Hz,1H),8.21(t,J=5.9Hz,1H),8.06(d,J=7.5Hz,1H),8.01–7 .97(m,2H),7.38–7.32(m,8H),7.28–7.22(m,3H),5.07–4.98(m,2H),4.63–4.55(m,2H),4.33–4.25(m,1H),3.91 –3.86(m,1H),3.74–3.67(m,2H),3.53–3.43(m,2H),3.35–3.28(m,1H),3.02–2.94(m,2H),2.93–2.76(m,3H),2. 77–2.69(m,7H),2.00–1.92(m,1H),1.75–1.65(m,1H),1.63–1.51(m,3H),1.37–1.26(m,2H),0.86–0.81(m,6H).

[0454] Example 2. Synthesis of biologically active compounds

[0455] Example 2.1: Synthesis of D-1

[0456] Step 1: Dissolve compound INT1 (100.0 mg, 0.17 mmol), compound D-1-1 (30.85 mg, 0.20 mmol), and HATU (77.057 mg, 0.20 mmol) in DMF (1 mL). Add DIPEA (65.91 mg, 0.51 mmol) and stir at room temperature for 16 h. The reaction solution is directly purified by preparative HPLC (acetonitrile:H2O containing 0.05% TFA = 10%-60%) to obtain the trifluoroacetic acid salt of the target compound D-1 (87.0 mg).

[0457] LCMS (ESI) [M+H] + =732.71.

[0458] 1 H NMR (400MHz, DMSO-d6) δ9.55 (s, 1H), 8.94 (d, J = 8.0Hz, 1H), 7.91-7.64 (m, 1H), 7.27-7.18 (m, 4H), 7.16-7.15 (m, 1H) ),4.83-4.56(m,2H),4.11-4.01(m,2H),3.82-3.74(m,2H),3.58-3.56(m,1H),3.39-3.31(m,3H),3.41-3.20(m,7H) ,3.12-3.07(m,2H),3.05-3.03(m,1H),2.97-2.87(m,1H),2.85-2.74(m,6H),2.67-2.58(m,1H),2.49-2.37(m,3H), 2.26-2,02(m,3H),1.84-1.73(m,3H),1.55-1.25(m,3H),1.10-1.03(m,3H),1.02-0.85(m,16H),0.83-0.77(m,3H).

[0459] Example 2.2: Synthesis of D-2

[0460] Step 1: Dissolve compound D-2-1 (2.16 g, 7.73 mmol) in anhydrous THF (15 mL), cool to 0°C, and sequentially add N-methylmorpholine (1.02 mL, 9.28 mmol) and methyl chloroformate (0.719 mL, 9.28 mmol). Stir and react at 0°C for 1 h. Filter, wash the filter cake with anhydrous THF (10 mL), cool the filtrate to 0°C, add a solution of NaBH4 (380 mg, 10.1 mmol) in water (4 mL), and continue stirring for 15 min. Then, return to room temperature and stir for an additional 30 min. The reaction mixture is filtered through a Buchner funnel, and the filtrate is concentrated under reduced pressure. The crude product is purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-2-2 (1.07 g).

[0461] LCMS (ESI) [M+H-100] + =166.26.

[0462] 1 H NMR(400MHz, DMSO-d6)δ7.25(d,J=7.3Hz,2H),7.19–7.14(m,3H),6.74–6.61(m,1H),4.39–4.27( m,1H),3.74–3.60(m,1H),3.45–3.34(m,2H),2.73–2.60(m,2H),1.58–1.46(m,2H),1.32(s,9H).

[0463] Step 2: Dissolve compound D-2-2 (500 mg, 1.88 mmol) in HCl / 1,4-dioxane solution (5 mL, 4 M) and stir at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain the hydrochloride salt of the target compound D-2-3 (370 mg), which was used directly in the next reaction.

[0464] LCMS (ESI) [M+H] + =166.31.

[0465] Step 3: Dissolve compound INT1 (30 mg, 0.050 mmol), D-2-3 (12.10 mg, 0.060 mmol), and HATU (22.81 mg, 0.060 mmol) in DMF (3 mL), and add DIPEA (19.39 mg, 0.15 mmol). Stir the mixture at room temperature for 1 h. The reaction solution is purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 10%-90%) to obtain the trifluoroacetic acid salt of the target compound D-2 (25 mg).

[0466] LCMS (ESI) [M+H] +=746.78.

[0467] 1 H NMR(400MHz,DMSO-d6)δ9.54(s,1H),8.95–8.84(m,1H),7.96–7.64(m,1H),7.26–7.10(m,5H),4.77–4.54(m,2H) ),4.36(s,1H),4.22–3.92(m,3H),3.83–3.63(m,2H),3.59–3.53(m,1H),3.27–3.17(m,6H),3.14–3.08(m,2H), 3.02–2.98(m,1H),2.87–2.62(m,8H),2.47–2.18(m,3H),2.14–1.98(m,2H),1.89–1.59(m,4H),1.56–1.40(m,3 H),1.37–1.21(m,4H),1.20–1.15(m,1H),1.05(dd,J=14.5,6.7Hz,3H),0.99–0.83(m,15H),0.81–0.74(m,3H).

[0468] Example 2.3: Synthesis of D-3

[0469] Step 1: Dissolve compound D-3-1 (1 g, 4.11 mmol), propargyl alcohol (0.25 g, 4.39 mmol), cuprous iodide (15 mg, 0.08 mmol), and bis(triphenylphosphine)palladium dichloride (28 mg, 0.04 mmol) in triethylamine (15 mL). Under nitrogen, stir at room temperature for 2 h. The reaction solution was diluted with water (200 mL) and extracted with ethyl acetate (250 mL*3). The combined organic phases were washed with saturated brine (200 mL*3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by normal phase column chromatography (dichloromethane:methanol = 10:1) to obtain the target compound D-3-2 (623 mg).

[0470] LCMS (ESI) [M+Na] + =195.24.

[0471] 1 H NMR (400MHz, DMSO-d6) δ7.48(d,J=8.2Hz,2H),7.38(d,J=8.3Hz,2H),4.33(s,2H),4.08(s,2H).

[0472] Step 2: Dissolve compound D-3-2 (500 mg, 2.92 mmol) in THF (5 mL). Add Raney nickel (100 mg) under nitrogen. Replace the mixture with hydrogen three times. Stir the reaction mixture at room temperature under a hydrogen atmosphere for 2 h. The reaction mixture is diluted with ethyl acetate (300 mL) and filtered through celite. The filter cake is washed with ethyl acetate (300 mL). The combined filtrates are concentrated under reduced pressure. The crude product is purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 10%-90%) to obtain the hydrochloride salt of the target compound D-3-3 (500 mg).

[0473] LCMS (ESI) [M+H] + =180.29.

[0474] 1 H NMR (400MHz, DMSO-d6) δ8.13(s,3H),7.19(s,4H),4.42(t,J=7.2Hz,1H),3.44–3.40(d,J =6.4Hz,2H),3.09–2.95(m,2H),2.94–2.80(m,2H),2.65–2.55(m,2H),1.84–1.64(m,2H).

[0475] Step 3: Dissolve compound INT1 (100 mg, 0.17 mmol) in DMF (2 mL), add HATU (77 mg, 0.2 mmol) and N,N-diisopropylethylamine (65.9 mg, 0.5 mmol), and stir at room temperature for 10 min. Then add compound D-3-3 (50 mg, 0.28 mmol). After addition, stir at room temperature for 2 h. The reaction solution is directly purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-3 (85 mg).

[0476] LCMS (ESI) [M+H] + =760.07.

[0477] 1H NMR(400MHz,DMSO-d6)δ8.24-8.06(m,2H),7.13-7.05(m,4H),4.63–4.50(m,1H),4.05–3.97(m,1H), 3.86–3.74(m,1H),3.66–3.44(m,4H),3.31(s,3H),3.28(s,3H),3.20(d,J=8.5Hz,3H),3.18(s,3H),3 .03(s,3H),2.80–2.63(m,3H),2.48–2.42(m,1H),2.24(s,3H),2.22(s,3H),2.02–1.75(m,6H),1.72– 1.51(m,4H),1.37–1.25(m,1H),1.08(dd,J=12.2,6.7Hz,3H),0.97–0.84(m,15H),0.82–0.70(m,6H).

[0478] Example 2.4: Synthesis of D-4

[0479] Step 1: Dissolve compound D-4-1 (100 mg, 0.31 mmol), compound INT1 (186 mg, 0.31 mmol), and HATU (130 mg, 0.34 mmol) in DMF (3 mL). Add DIPEA (80 mg, 0.62 mmol) and stir at room temperature for 2 h. The reaction solution is purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% FA = 5%-50%) to obtain the target compound D-4-2 (160 mg).

[0480] LCMS (ESI) [M / 2+H] + =451.37.

[0481] Step 2: Dissolve compound D-4-2 (18.0 mg, 0.020 mmol) in HCl / 1,4-dioxane solution (2 mL, 4 M) and stir at room temperature for 1 h. The reaction solution was purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 5%-50%) to obtain the target compound D-4 (10.5 mg).

[0482] LCMS (ESI) [M / 2+H] + =401.18.

[0483] 1H NMR (400MHz, DMSO-d6) δ8.85–8.60(m,1H),8.14–7.98(m,1H),7.80(dd,J=9.5,3.3Hz,1H),7.65(dd,J=10.3,3.3Hz,1H),6.88(dd ,J=13.9,7.4Hz,1H),6.50–6.38(m,3H),5.55–5.35(m,1H),4.95–4.58(m,4H),4.04–4.00(m,1H),3.85(d,J=9.3Hz,1H),3.67–3. 55(m,1H),3.52–3.38(m,2H),3.30–3.12(m,9H),3.02(s,1H),2.91–2.79(m,1H),2.70–2.63(m,1H),2.44–2.40(m,1H),2.38–2.2 5(m,2H),2.23(s,6H),2.09–1.62(m,6H),1.56–1.28(m,3H),1.11(dd,J=15.0,6.7Hz,3H),0.99–0.88(m,12H),0.81–0.71(m,6H).

[0484] Example 2.5: Synthesis of D-5

[0485] Compound D-5-1 (500 mg, 0.69 mmol) and compound D-5-2 (120 mg, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL). HATU (1 g, 2.64 mmol) and DIPEA (680 mg, 5.28 mmol) were then added sequentially. The mixture was stirred at room temperature for 16 h. The crude product was purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-5 (352 mg).

[0486] LCMS (ESI) [M+H] + =837.45.

[0487] 1H NMR(400MHz, DMSO-d6)δ7.92(d,J=8.7Hz,0.5H),7.84–7.73(m,1H),7.66(d,J=8.5Hz,0.5H),7.36–7.26(m,4H) ,7.23–7.10(m,3H),6.65–6.55(m,2H),5.44(m,0.5H),4.77(m,0.5H),4.61(t,J=8.5Hz,1H),4.57–4.40(m,2H), 4.12–3.92(m,2H),3.84–3.78(m,1H),3.66–3.55(m,1H),3.30–3.19(m,8H),3.15(s,2H),3.02–2.93(m,4H),2.4 9–2.39(m,1H),2.38–1.92(m,4H),1.90–1.69(m,3H),1.62–1.26(m,4H),1.11–0.98(m,9H),0.97–0.76(m,18H).

[0488] Example 3. Synthesis of drug-linker conjugates

[0489] Example 3.1: Synthesis of DL-1

[0490] Compound INT4 (25.0 mg, 0.03 mmol), compound INT1 (21.56 mg, 0.036 mmol), and HATU (13.69 mg, 0.036 mmol) were dissolved in DMF (1.5 mL) and DIPEA (7.75 mg, 0.06 mmol) was added. The reaction was stirred at room temperature for 2 h. The reaction solution was directly purified by preparative HPLC (acetonitrile:H2O containing 0.05% TFA = 10%-60%) to obtain the trifluoroacetic acid salt of the target compound DL-1 (7.6 mg).

[0491] LCMS (ESI) [M / 2+H] + =705.88.

[0492] 1H NMR(400MHz, DMSO-d6)δ9.58(s,1H),9.20-9.12(m,3H),8.95(d,J=8.0Hz,1H),8.73-8.67(m,1H),8.31(d,J=8.0Hz,1H),8.26-8.22(m,1H),8.0 0(d,J=8.0Hz,0.5H),7.77(d,J=8.0Hz,0.5H),7.51(d,J=8.0Hz,1H),7. 25-7.14(m,5H),4.82-4.49(m,4H),4.33-4.27(m,2H),4.21-4.11(m,1H ),4.05-3.98(m,3H),3.85-3.69(m,4H),3.66-3.63(m,2H),3.44(s,3H) ,3.27(s,2H),3.23-3.22(m,4H),3.19(s,2H),3.11(s,1H),3.05-2.95( m,8H),2.85-2.75(m,7H),2.74-2.56(m,4H),2.50-1.97(m,6H),1.94-1 .87(m,2H),1.85-1.60(m,11H),1.50-1.30(m,5H),1.08-0.79(m,36H).

[0493] Example 3.2: Synthesis of DL-2

[0494] Step 1: Dissolve compound INT5 (55.0 mg, 0.071 mmol), compound INT1 (42.52 mg, 0.071 mmol), and HATU (32.40 mg, 0.085 mmol) in DMF (1 mL). Add DIPEA (27.53 mg, 0.21 mmol) and stir at room temperature for 2 h. The reaction mixture is directly purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% FA = 0%-50%) to obtain the target compound DL-2-1 (50 mg).

[0495] LCMS (ESI) [M / 2+H] + =678.74.

[0496] Step 2: Dissolve compound DL-2-1 (50.0 mg, 0.037 mmol) and lithium hydroxide monohydrate (7.76 mg, 0.18 mmol) in THF (0.5 mL) and water (0.5 mL). Stir the mixture at room temperature for 2 h. The reaction mixture was directly purified by column chromatography (acetonitrile:H2O containing 0.05% FA = 0%-50%) to obtain the target compound DL-2-2 (30.0 mg).

[0497] LCMS (ESI) [M / 2+H] + =671.75.

[0498] Step 3: Dissolve compound DL-2-2 (30.0 mg, 0.022 mmol), pentafluorophenol (20.25 mg, 0.11 mmol), DMAP (8.06 mg, 0.066 mmol), and EDCI (21.09 mg, 0.11 mmol) in DMF (1.0 mL) and stir at room temperature for 2 h. The reaction mixture was directly purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 10%-60%) to obtain the formate salt of the target compound DL-2 (9.9 mg).

[0499] LCMS (ESI) [M / 2+H] + =754.66.

[0500] 1 H NMR(400MHz,DMSO-d6)δ8.58-8.55(m,1H),8.21(s,1H),8.14-7.74(m,5H),7.30-7.03(m,5H),4.75-4.50(m,4H),4.20-4.1 7(m,3H),4.12-4.05(m,2H),4.02-3.98(m,1H),3.83-3.80(m,2H),3.73-3.71(m,2H),3.32-3.30(m,2H),3.28-3.24(m,4H) ,3.23-3.22(m,4H),3.19(s,2H),3.12-3.10(m,3H),3.06-3.02(m,3H),2.92-2.80(m,2H),2.73-2.65(m,6H),2.46-2.33(m ,3H),2.28-2.13(m,8H),2.10-1.55(m,11H),1.53-1.24(m,13H),1.08-1.03(m,3H),0.96-0.85(m,25H),0.80-0.71(m,6H).

[0501] Example 3.3: Synthesis of DL-3

[0502] Step 1:

[0503] Compounds INT5 (30.0 mg, 0.038 mmol), INT1 (22.75 mg, 0.038 mmol) and HATU (17.34 mg, 0.046 mmol) were dissolved in DMF (1 mL), and then DIPEA (9.82 mg, 0.076 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the trifluoroacetate salt of the target compound DL-3 (21.52 mg).

[0504] LCMS (ESI) [M+H] + =1379.97.

[0505] 1H NMR(400MHz, DMSO-d6)δ9.20-9.04(m,3H),8.90(d,J=8.2Hz,1H),8.71-8.61(m,1H),8.22-8.13(m,1H),8.13-8.06(m,1H),7.99-7.90(m,1.5H),7 .73(d,J=8.0Hz,0.5H),7.22-7.12(m,5H),4.81-4.50(m,5H),4.30-4.22 (m,1H),4.21-4.02(m,2H),4.01-3.95(m,1H),3.82-3.67(m,4H),3.31(s, 1H),3.24(s,2H),3.23-3.18(m,4H),3.16(s,2H),3.08(s,1H),3.03-2.9 6(m,8H),2.90-2.75(m,7H),2.68-2.52(m,4H),2.47-2.18(m,5H),2.17- 1.90(m,3H),1.88-1.70(m,5H),1.66-1.57(m,7H),1.54-.49(m,2H),1.4 9-1.22(m,5H),1.05-1.00(m,3H),0.98-0.82(m,30H),0.80-0.74(m,3H).

[0506] Example 3.4: Synthesis of DL-4

[0507] Step 1:

[0508] Compound D-3-3 (50 mg, 0.28 mmol) and INT3 (435.62 mg, 0.56 mmol) were dissolved in HCl-DMF (2 mL, 2 M) and stirred at room temperature for 3 h. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile:H2O containing 0.05% TFA = 0% to 40% TFA) to obtain the target compound DL-4-1 (50.0 mg).

[0509] LCMS (ESI) [M+H] + =857.82.

[0510] Step 2:

[0511] Compound D-4-1 (50.0 mg, 0.058 mmol), compound INT1 (34.73 mg, 0.058 mmol) and HATU (26.46 mg, 0.070 mmol) were dissolved in DMF (1 mL), and then DIPEA (14.99 mg, 0.12 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the target compound DL-4 (11.12 mg).

[0512] LCMS (ESI) [M / 2+H] + =719.80.

[0513] 1H NMR(400MHz,DMSO-d6)δ9.11(s,2H),8.66-8.55(m,1H),8.24-8.19(m,2H),8.08-7.96(m,2H),7.47(d,J=8.9Hz,1H),7.27-7.07(m,4H) ,6.53-6.42(m,1H),5.82-5.62(m,1H),4.77-4.50(m,4H),4.34-4.14(m,4H),4.00-3.93(m,3H),3.84-3.78(m,1H),3.74-3.68(m,2H),3 .65-3.58(m,3H),3.43-3.40(m,5H),3.30-3.28(m,4H),3.26(s,2H),3.18(d,J=1.7Hz,4H),3.14(s,1H),3.00(s,1H),2.74-2.61(m,5H) ,2.41-2.28(m,8H),2.23-2.18(m,6H),2.05-1.79(m,8H),1.72-1.50(m,5H),1.44-1.30(m,8H),1.10-1.00(m,3H),0.93-0.70(m,33H).

[0514] Example 3.5: Synthesis of DL-6

[0515] Compound INT10 (25.0 mg, 0.034 mmol), compound INT1 (20.36 mg, 0.034 mmol) and HATU (15.51 mg, 0.041 mmol) were dissolved in DMF (1 mL), and then DIPEA (8.79 mg, 0.068 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the trifluoroacetate salt of the target compound DL-6 (23.50 mg).

[0516] LCMS (ESI) [M / 2+H] + =662.84.

[0517] 1H NMR (400MHz, DMSO-d6) δ9.11 (s, 2H), 8.87 (d, J = 8.0Hz, 1H), 8.71-8.59 (m, 1H), 8.22-8.13 (m, 1H), 8.13-8.08 (m, 1H), 8.02-7.88(m,1.5H),7.74-7.72(m,0.5H),7.22-7.11(m,5H),4.78-4.46(m,5H),4.30-3.92(m,4H),3.80-3.55(m,4H ),3.55-3.45(m,2H),3.41(s,3H),3.31-3.27(m,3H),3.24(s,2H),3.22-3.17(m,4H),3.16(s,1H),2.91-2.79(m,1H) ,2.78-2.73(m,12H),2.71-2.52(m,4H),2.46-1.90(m,8H),1.88-1.55(m,9H),1.48-1.22(m,5H),1.06-0.74(m,30H).

[0518] Example 3.6: Synthesis of DL-7

[0519] Compounds INT12 (55.0 mg, 0.069 mmol), INT1 (41.32 mg, 0.069 mmol) and HATU (26.24 mg, 0.069 mmol) were dissolved in DMF (1 mL), and then DIPEA (26.71 mg, 0.21 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was directly purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the trifluoroacetate salt of the target compound DL-7 (7.20 mg).

[0520] LCMS (ESI) [M / 2+H] + =676.31.

[0521] 1H NMR (400MHz, DMSO-d6) δ9.71-9.37(m,2H),9.12-9.10(m,2H),8.89(d,J=8.0Hz,1 H),8.57(t,J=8.0Hz,1H),8.16(t,J=8.0Hz,1H),8.10(d,J=8.0Hz,1H),8.07-8.00 (m,1H),7.93(d,J=8.0Hz,1H),7.85(t,J=4.0Hz,1H),7.11-7.05(m,4H),4.73-4. 48(m,4H),4.28-4.20(m,1H),4.20-4.10(m,1H),4.06-3.96(m,1H),3.85-3.79(m, 1H),3.76-3.67(m,3H),3.60-3.44(m,4H),3.41(s,3H),3.28(s,2H),3.26(s,2H) ,3.19-3.17(m,3H),3.14(s,2H),3.03-2.97(m,3H),2.83-2.63(m,15H),2.59-2.5 2(m,4H),2.45-2.27(m,4H),2.23-2.13(m,1H),2.07-1.88(m,2H),1.85-1.50(m,1 2H),1.39-1.20(m,3H),1.07-1.02(m,3H),0.98-0.82(m,24H),0.80-0.74(m,3H).

[0522] Example 3.7: Synthesis of DL-8

[0523] Step 1:

[0524] Compound INT13 (1.2 g, 1.91 mmol), INT1 (1.14 mg, 1.81 mmol), and HATU (0.87 g, 2.29 mmol) were dissolved in DMF (15 mL), followed by the addition of DIPEA (0.74 g, 5.73 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction solution was purified by reverse-phase column chromatography (acetonitrile:H₂O containing 0.05% TFA = 0%-50%) to obtain the trifluoroacetic acid salt of the target compound DL-8-1 (1.2 g).

[0525] LCMS (ESI) [M+H] + =1207.84.

[0526] Step 2:

[0527] Compound DL-8-1 (1.2 g, 0.99 mmol) was dissolved in methanol (15 mL). 10% palladium on carbon (0.11 g, 55% water content) was added under nitrogen. The atmosphere was replaced with nitrogen three times. The mixture was stirred at room temperature under a hydrogen atmosphere for 3 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the target compound DL-8-2 (1.0 g), which was used directly in the next step.

[0528] LCMS (ESI) [M+H] + =1073.70.

[0529] Step 3:

[0530] Compound DL-8-2 (1000.0 mg, 0.93 mmol), INT2 (277.32 mg, 0.93 mmol) and HATU (353.61 mg, 0.93 mmol) were dissolved in DMF (10 mL), and then DIPEA (360.58 mg, 2.79 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate salt of the target compound DL-8 (550.33 mg).

[0531] LCMS (ESI) [M+H] + =1353.84.

[0532] 1H NMR (400MHz, DMSO-d6) δ9.61 (s, 1H), 9.40 (d, J = 27.0Hz, 1H), 9.13 (s, 2H), 8.95 ( d,J=8.0Hz,1H),8.75–8.64(m,1H),8.31(d,J=7.5Hz,1H),8.28–8.18(m,1H),8.0 0(d,J=8.0Hz,1H),7.76(d,J=8.0Hz,1H),7.51(d,J=8.0Hz,1H),7.25–7.13(m,5H ),4.75–4.56(m,4H),4.32–4.27(m,2H),4.00–3.96(m,4H),3.79–3.72(m,4H),3. 64(t,J=6.2Hz,2H),3.56–3.50(m,1H),3.44(s,3H),3.38–3.35(m,1H),3.34–3. 30(m,1H),3.27(s,1H),3.23–3.22(m,2H),3.18(s,1H),3.11(s,1H),3.07–2.99( m,4H),2.94–2.85(m,1H),2.83–2.76(m,12H),2.72–2.63(m,3H),2.50–1.95(m,8 H),1.93–1.87(m,2H),1.84–1.56(m,7H),1.49–1.27(m,5H),1.08–0.76(m,30H).

[0533] Example 3.9: Synthesis of DL-9

[0534] Compound DL-9 is commercially available and was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., product number: 1039030, batch number: Lg0515249860.

[0535] Example 4. Preparation of antibodies

[0536] The method for preparing anti-NaPi2b antibodies refers to the preparation of 66C12D12-hz1 antibody in WO2024012524A1.

[0537] Example 5. Synthesis of Antibody Drug Conjugates (ADCs)

[0538] Example 5.1: Preparation of Her2-ADC-001

[0539] 0.2 mL of trastuzumab antibody (28.8 mg / mL) was diluted with 0.002 mL of 20 mM PB + 100 mM edetate disodium solution (pH 7.6). The pH was adjusted to 7.5 with 0.5 M Na2HPO4 solution. 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0117 mL, 0.234 μmol, 6.0 times the molar amount of the antibody) was added to the above solution, mixed thoroughly, and incubated in a 25°C water bath for 90 min. Then, a solution of DL-1 (0.785 mg, 12 times the molar amount of the antibody) in dimethyl sulfoxide (0.0479 mL) was added, mixed thoroughly, and incubated in a 37°C water bath for 2 h. After completion, the buffer was exchanged with 20 mM His-HCl, pH 5.9, using a centrifugal ultrafiltration tube (Merck, Amicon Ultra-15). The conjugate product of DL-1 and trastuzumab antibody, Her2-ADC-001, was obtained. The DAR value determined by mass spectrometry was 8.0.

[0540] In the table, LC represents the antibody light chain; HC represents the antibody heavy chain; DAR1 represents a conjugate comprising a light chain or heavy chain coupled to one drug-linker; DAR2 represents a conjugate comprising a light chain or heavy chain coupled to two drug-linkers; and DAR3 represents a conjugate comprising a light chain or heavy chain coupled to three drug-linkers. LC, HC, DAR1, DAR2, and DAR3 are as described above.

[0541] The trastuzumab light chain was conjugated to 0 and 1 toxin molecules (ratios of 1% and 99%, respectively), and the heavy chain was conjugated to 3 toxin molecules (ratio of 100%). Based on this, the antibody drug conjugate ratio (DAR value) of HER2-ADC-001 was calculated to be 8.0.

[0542] Method for determining the antibody drug coupling ratio (DAR value):

[0543] Sample preparation: 200 μg of ADC sample was diluted to 0.2 mg / ml with ultrapure water, and then 10 μl of 1 mol / L DTT was added. The mixture was reacted at 37°C for 30 min and then directly injected for determination.

[0544] Liquid phase parameters:

[0545] Mass spectrometry parameters:

[0546] Example 5.2: Preparation of Her2-ADC-002

[0547] A similar procedure to Example 5.1 was used, with the addition of DL-1 (0.593 mg, 3 times the molar amount of the antibody substance) to obtain the conjugate product of DL-1 and trastuzumab antibody, Her2-ADC-002. The DAR value determined by mass spectrometry was 2.5.

[0548] The DAR value determination results are as follows:

[0549] The DAR value of Her2-ADC-002 was calculated to be 2.5 based on the determination of trastuzumab light chain conjugated with 0-1 drug-linker conjugates (LC, DAR1 ratios were 88% and 12%, respectively) and heavy chain conjugated with 0-3 drug-linker conjugates (HC, DAR1, DAR2, and DAR3 ratios were 29%, 35%, 30%, and 6%, respectively).

[0550] Example 5.3: Preparation of Her2-ADC-003

[0551] Similar procedures were used as in Example 5.1, but 0.737 mg of DL-1 (5 times the molar amount of the antibody substance) was added to obtain the conjugate product of DL-1 and trastuzumab, Her2-ADC-003. The DAR value determined by mass spectrometry was 3.7.

[0552] The DAR value determination results are as follows:

[0553] The DAR value of Her2-ADC-003 was calculated to be 3.7 based on the determination of trastuzumab light chain conjugated with 0-1 drug-linker conjugates (LC, DAR1 ratios of 68% and 32%, respectively) and heavy chain conjugated with 0-3 drug-linker conjugates (HC, DAR1, DAR2, and DAR3 ratios of 15%, 30%, 40%, and 15%, respectively).

[0554] Example 5.4: Preparation of NaPi2b-ADC-001

[0555] 1.5 mL of anti-NaPi2b antibody was diluted with 0.015 mL of 20 mM PB + 100 mM edetate disodium solution (pH 7.6). The pH was adjusted to 7.5 with 0.5 M Na2HPO4 solution. A 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0241 mL, 0.482 μmol, 2.2 times the molar amount of the antibody) solution was added to the solution, mixed thoroughly, and incubated at 25°C in a water bath for 90 min. A solution of DL-1 (1.692 mg, 5.0 times the molar amount of the antibody) in dimethyl sulfoxide (0.111 mL) was then added, mixed thoroughly, and incubated at 25°C in a water bath for 2 h. After completion, the solution was exchanged with 20 mM His-HCl, pH 5.9, using a centrifugal ultrafiltration tube (Merck, Amicon Ultra-15). This yielded the conjugated product, NaPi2b-ADC-001, consisting of DL-1 and the anti-NaPi2b antibody. The DAR value determined by mass spectrometry was 3.8.

[0556] The DAR value determination results are as follows:

[0557] The anti-NaPi2b antibody light chain was conjugated to 0-1 drug-linker conjugates (LC, DAR1 ratios were 59% and 41%, respectively) and the heavy chain was conjugated to 0-3 drug-linker conjugates (HC, DAR1, DAR2, DAR3 ratios were 17%, 32%, 35%, and 15%, respectively). The DAR value of NaPi2b-ADC-001 was calculated to be 3.8.

[0558] Example 5.5: Preparation of NaPi2b-ADC-002

[0559] A similar procedure to Example 5.4 was used, and DL-3 (1.329 mg, 6.0 times the molar amount of the antibody substance) was added to obtain the conjugate product of DL-3 and anti-NaPi2b antibody, NaPi2b-ADC-002. The DAR value determined by mass spectrometry was 4.1.

[0560] The DAR value determination results are as follows:

[0561] The anti-NaPi2b antibody light chain was conjugated to 0-1 drug-linker conjugates (LC, DAR1 ratios were 51% and 49%, respectively) and the heavy chain was conjugated to 0-3 drug-linker conjugates (HC, DAR1, DAR2, DAR3 ratios were 14%, 33%, 37%, and 16%, respectively). The DAR value of NaPi2b-ADC-002 was calculated to be 4.1.

[0562] Example 5.6: Preparation of NaPi2b-ADC-003

[0563] A similar procedure to Example 5.4 was used, and DL-6 (1.397 mg, 6.0 times the molar amount of the antibody substance) was added to obtain the conjugate product of DL-6 and anti-NaPi2b antibody, NaPi2b-ADC-003. The DAR value determined by mass spectrometry was 4.3.

[0564] The DAR value determination results are as follows:

[0565] The anti-NaPi2b antibody light chain was conjugated to 0-1 drug-linker conjugates (LC, DAR1 ratios were 46% and 54%, respectively) and the heavy chain was conjugated to 0-3 drug-linker conjugates (HC, DAR1, DAR2, DAR3 ratios were 12%, 32%, 35%, and 20%, respectively). The DAR value of NaPi2b-ADC-003 was calculated to be 4.3.

[0566] Example 5.7: Preparation of NaPi2b-ADC-005

[0567] Similar procedures were used as in Example 5.4, with the addition of DL-4 (1.409 mg, 6.0 times the molar amount of the antibody substance) to obtain the conjugate product of DL-47 and anti-NaPi2b antibody, NaPi2b-ADC-005. The DAR value determined by mass spectrometry was 4.4.

[0568] The DAR value determination results are as follows:

[0569] The anti-NaPi2b antibody light chain was conjugated to 0-1 drug-linker conjugates (LC, DAR1 ratios were 41% and 59%, respectively) and the heavy chain was conjugated to 0-3 drug-linker conjugates (HC, DAR1, DAR2, DAR3 ratios were 12%, 34%, 34%, and 19%, respectively). The DAR value of NaPi2b-ADC-005 was calculated to be 4.4.

[0570] Example 5.8: Preparation of NaPi2b-ADC-006

[0571] Similar procedures were used as in Example 5.4, with the addition of DL-9 (1.501 mg, 6 times the molar amount of the antibody) to obtain the conjugate product of DL-9 and anti-NaPi2b antibody, NaPi2b-ADC-006. The DAR value determined by mass spectrometry was 4.7.

[0572] The DAR value determination results are as follows:

[0573] The anti-NaPi2b antibody light chain was conjugated to 0-2 drug-linker conjugates (the ratios of LC, DAR1, and DAR2 were 38%, 59%, and 3%, respectively), and the heavy chain was conjugated to 0-4 drug-linker conjugates (the ratios of HC, DAR1, DAR2, DAR3, and DAR4 were 12%, 34%, 30%, 19%, and 4%, respectively). The DAR value of NaPi2b-ADC-006 was calculated to be 4.7.

[0574] Example 5.9: Preparation of NaPi2b-ADC-007

[0575] Similar procedures were used as in Example 5.4, with the addition of DL-6 (2.560 mg, 11 times the molar amount of the antibody) to obtain the conjugate product of DL-6 and anti-NaPi2b antibody, NaPi2b-ADC-007. The DAR value determined by mass spectrometry was 7.9.

[0576] The DAR value determination results are as follows:

[0577] The anti-NaPi2b antibody light chain was conjugated to 0-1 drug-linker conjugates (LC, DAR1 ratios were 3% and 97%, respectively) and the heavy chain was conjugated to 0-3 drug-linker conjugates (HC, DAR1, DAR2, and DAR3 ratios were 0%, 0%, 0%, and 100%, respectively). The DAR value of NaPi2b-ADC-007 was calculated to be 7.9.

[0578] Example 5.10: Preparation of NaPi2b-ADC-009

[0579] Similar procedures were used as in Example 5.4, with the addition of DL-9 (1.925 mg, 11 times the molar amount of the antibody) to obtain the conjugate product of DL-9 and anti-NaPi2b antibody, NaPi2b-ADC-009. The DAR value determined by mass spectrometry was 8.0.

[0580] The DAR value determination results are as follows:

[0581] The anti-NaPi2b antibody light chain was conjugated to 0-1 drug-linker conjugates (LC, DAR1 ratios were 0% and 100%, respectively) and the heavy chain was conjugated to 0-3 drug-linker conjugates (HC, DAR1, DAR2, and DAR3 ratios were 0%, 0%, 0%, and 100%, respectively). The DAR value of NaPi2b-ADC-009 was calculated to be 8.0.

[0582] Example 5.11: Preparation of Her2-ADC-004

[0583] Similar procedures were used as in Example 5.1, with the addition of DL-6 (1.181 mg, 6 times the molar amount of the antibody) to obtain the conjugate product of DL-1 and trastuzumab, Her2-ADC-004. The DAR value determined by mass spectrometry was 4.4.

[0584] The DAR value determination results are as follows:

[0585] The DAR value of Her2-ADC-004 was calculated to be 4.4 based on the determination of trastuzumab light chain conjugated with 0-1 drug-linker conjugates (LC, DAR1 ratios of 51% and 49%, respectively) and heavy chain conjugated with 0-3 drug-linker conjugates (HC, DAR1, DAR2, and DAR3 ratios of 10%, 31%, 40%, and 20%, respectively).

[0586] Example 6. Cellular activity test of bioactive compounds

[0587] Example 6.1 Inhibitory activity test of bioactive compounds on in vitro cells (SK-BR-3)

[0588] 1. Experimental methods:

[0589] SK-BR-3 (McCoy's 5A + 10% FBS + 1% P / S) cells were revived and cultured in 96-well flat-bottom plates at a density of 5000 / well and 142.5 μL / well, and cultured in a 37 ° C incubator overnight. The next day, the bioactive compound was gradiently diluted to 400 × with DMSO, and then diluted to 20 × with serum-free culture medium to a maximum concentration of 1000nM, 4-fold dilution to 9 concentration points; 7.5 μL of diluted bioactive compound sample (20 ×) was added to the cell culture wells. After culturing at 37 ° C for 4 days, 50 μL Cell titer glo substrate was added to each well, and the chemiluminescence value was detected after incubation for 10 minutes. Among them, the sources of key materials are shown in Table 1 below:

[0590] Table 1 Material sources

[0591] 2. Experimental results:

[0592] As shown in Table 2, the bioactive compounds of the present invention have significant proliferation inhibitory activity on the SK-BR-3 tumor cell line.

[0593] Table 2 Inhibitory activity of bioactive compounds on SK-BR-3 tumor cell proliferation in vitro

[0594] Example 6.2 Inhibitory activity test of bioactive compounds on in vitro cells (IGR-OV1, RMG-1, TOV-21G, SK-OV-3)

[0595] 1. Experimental methods:

[0596] IGR-OV1 cells were revived and cultured (RPMI 1640 (Gibco, catalog number: C22400500CP) + 10% FBS (Gibco, catalog number: 10099-141) + 1% P / S), RMG-1 cells (Ham's F-12 (Invitrogen, catalog number: 11765054) + 10% FBS + 1% P / S), TOV-21G cells (MCDB 105 (SIGMA, catalog number: 117-500): Medium 199 (SIGMA, catalog number: M4530-500ML) (1:1) + 15% FBS + 1% P / S), and SK-OV-3 cells (McCoy's 5a (Invitrogen, catalog number: 12330031) + 10% FBS + 1% P / S). After the cells stabilized, they were plated in 96-well flat-bottom plates (Beyotime, Cat. No. FCP963-48 pcs) according to the density listed in the table below and cultured overnight in a 37°C incubator. The cell line sources and plating conditions are shown in Table 3 below:

[0597] Table 3 Cell line sources and plating conditions

[0598] The small molecule to be tested was serially diluted to 1000× with DMSO, and then diluted to 10× with serum-free medium, so that the highest final concentration was 500 nM. 9 concentration points were obtained by 4-fold dilution. 15 μL of the 10× dilution was added to each well.

[0599] After adding the test substance, the 96-well plate was placed at 37°C and 5% CO2 for 6 days. After the incubation, the 96-well plate was placed at room temperature for 20 minutes. 75 μL of Reagent (ADamas Life, Cat. No.: RA-GL11-A) was used. Chemiluminescence was detected after 10 minutes of incubation.

[0600] 2. Experimental results:

[0601] As shown in Table 4, the bioactive compounds of the present invention have significant proliferation inhibitory activity on the four tumor cell lines.

[0602] Table 4 Inhibitory activity of bioactive compounds on proliferation of four tumor cells in vitro

[0603] Example 7. In vitro bioactivity testing of antibody drug conjugates (ADCs)

[0604] Example 7.1: Detection of ADC inhibitory activity on in vitro cells (NCI-N87)

[0605] 1. Experimental methods:

[0606] NCI-N87 (RPMI 1640 + 10% FBS + 1% P / S) cells were revived and cultured in 96-well flat-bottom plates at a density of 7000 / well and 142.5 μL / well, and cultured in a 37 ° C incubator overnight. The next day, the ADC molecule to be tested was diluted to 20 × with serum-free culture medium to a maximum final concentration of 100nM, and 9 concentration points were diluted 4 times. Add 7.5 μL of diluted ADC sample (20 ×) to the cell culture wells. After culturing at 37 ° C for 3 days, 100 μL Cell titer glo substrate was added to each well, and the chemiluminescence value was detected after incubation for 10 minutes. Among them, the sources of key materials are shown in Table 5 below:

[0607] Table 5 Material sources

[0608] 2. Experimental results:

[0609] As shown in Table 6, the antibody-drug conjugate (ADC) of the present invention has significant proliferation inhibitory activity on the NCI-N87 tumor cell line.

[0610] Table 6 In vitro proliferation inhibitory activity of ADC on NCI-N87 tumor cells

[0611] Example 7.2: Detection of ADC inhibitory activity on cells (SKBR3) in vitro

[0612] 1. Experimental methods:

[0613] SK-BR-3 (McCoy's 5A + 10% FBS + 1% P / S) cells were revived and plated in 96-well flat-bottom plates at a density of 6,000 / well and 142.5 μL / well. They were cultured in a 37°C incubator overnight. The next day, the ADC molecule to be tested was diluted to 20× with serum-free culture medium to a maximum final concentration of 100 nM, and diluted 4-fold to 9 concentration points. 7.5 μL of the diluted ADC sample (20×) was added to the cell culture wells. After culturing at 37°C for 3 days, 50 μL of Cell titer glo substrate was added to each well and the chemiluminescence value was detected after incubation for 10 minutes.

[0614] Among them, the sources of key materials are shown in Table 7 below:

[0615] Table 7 Material sources

[0616] 2. Experimental results:

[0617] As shown in Table 8, the antibody drug conjugate (ADC) of the present invention has significant proliferation inhibitory activity on the SK-BR-3 tumor cell line.

[0618] Table 8 In vitro proliferation inhibitory activity of ADC on SK-BR-3 tumor cells

[0619] Example 7.3: Inhibitory activity test of ADC on in vitro cells (BT474)

[0620] 1. Experimental methods:

[0621] BT-474 cells were revived and cultured in DMEM high-glucose medium (10% FBS, 1% P / S, and 10 μg / mL insulin), plated at a density of 7,000 cells / well, and plated in a 96-well flat-bottom plate at 142.5 μL / well. Incubate overnight at 37°C. The next day, the ADC to be tested (20*) was serially diluted in serum-free medium using a 4-fold serial dilution series for a total of nine concentration points. 7.5 μL of the 20* ADC dilution was added to each well to a maximum final concentration of 100 nM. After addition of the test compound, the 96-well plate was incubated at 37°C, 5% CO2 for 4 days.

[0622] After incubation, the 96-well plate was equilibrated at room temperature for 20 minutes. 50 μL of Cell titer Turbo substrate was added to each well and the chemiluminescence value was measured after incubation for 10 minutes. The sources of key materials are shown in Table 9 below:

[0623] Table 9 Material sources

[0624] 2. Experimental results:

[0625] As shown in Table 10, the antibody-drug conjugates (ADCs) of the present invention have significant proliferation inhibitory activity on the BT-474 tumor cell line.

[0626] Table 10 In vitro proliferation inhibitory activity of ADC on BT-474 tumor cells

[0627] Example 7.4: Detection of the inhibitory activity of ADC on cells (OE-19) in vitro

[0628] 1. Experimental methods:

[0629] Resuscitated OE-19 cells (RPMI 1640 + 10% FBS + 1% P / S) were plated at a density of 8,000 cells / well in a 96-well flat-bottom plate, using 142.5 μL / well. Incubate overnight at 37°C. The next day, the ADC to be tested (20*) was serially diluted in serum-free medium using a 4-fold series dilution schedule for a total of nine concentration points. 7.5 μL of the 20* ADC dilution was added to each well to a maximum final concentration of 100 nM. After addition of the test compound, the 96-well plate was incubated at 37°C, 5% CO2 for 4 days.

[0630] After incubation, the 96-well plate was equilibrated at room temperature for 20 minutes. 50 μL of Cell titer Turbo substrate was added to each well and the chemiluminescence value was measured after incubation for 10 minutes. The sources of key materials are shown in Table 11 below:

[0631] Table 11 Material sources

[0632] 2. Experimental results:

[0633] As shown in Table 12, the antibody-drug conjugates (ADCs) of the present invention have significant proliferation inhibitory activity on the OE-19 tumor cell line.

[0634] Table 12 In vitro proliferation inhibitory activity of ADC on OE-19 tumor cells

[0635] Example 7.5: Inhibitory activity test of ADC on in vitro cells (OVAR3, IGR-OV1, RMG-1)

[0636] 1. Experimental methods:

[0637] OVCAR-3 cells were revived and cultured in RPMI 1640 (ADAmas Life, Catalog No. C8016) + 20% FBS + 1% P / S + 10 μg / ml insulin), IGR-OV1 cells (RPMI 1640 (Gibco, Catalog No. C22400500CP) + 10% FBS + 1% P / S), and RMG-1 cells (Ham's F-12 (Invitrogen, Catalog No. 11765054) + 10% FBS + 1% P / S). After the cells stabilized, they were plated in 96-well flat-bottom plates (Beyotime, Catalog No. FCP963-48 pcs) according to the density shown in the table below and cultured in a 37°C incubator overnight. The cell line sources and plating conditions are shown in Table 13 below:

[0638] Table 13 Cell line sources and plating conditions

[0639] The next day, the ADC to be tested on OVCAR-3 cells was serially diluted to 20 times (20×) the final concentration using serum-free culture medium, with the highest final concentration being 100 nM. A 4-fold serial dilution was performed, for a total of 9 concentration points. 7.5 μL of the 20× dilution was added to each well.

[0640] The ADC to be tested on the remaining two cells was serially diluted to 10 times (10×) the final concentration using serum-free culture medium, with the highest final concentration being 100 nM. A 4-fold serial dilution was performed, for a total of 9 concentration points. 15 μL of the 10× dilution was added to each well.

[0641] After adding the test substance, the 96-well plate was incubated at 37°C, 5% CO2 for 6 days. After the incubation, the 96-well plate was equilibrated at room temperature for 20 minutes. 50 μL Cell Titer Turbo Substrate was added to each well of OVCAR-3 cells, and 75 μL Cell Titer Turbo Substrate was added to each well of the remaining two cells. Reagent (ADamas Life, Cat. No.: RA-GL11-A) was used. Chemiluminescence was detected after 10 minutes of incubation.

[0642] 2. Experimental results:

[0643] As shown in Table 14, the ADC molecules of the present invention have significant proliferation inhibitory activity on three tumor cell lines.

[0644] Table 14 In vitro proliferation inhibition activity of ADC on three tumor cells

[0645] Example 8. In vivo activity test of antibody-drug conjugates (ADCs)

[0646] Example 8.1 Antibody-drug conjugate (ADC) efficacy test on NCI-N87 transplanted tumors

[0647] 1. Experimental Materials

[0648] Test compound: Her2-ADC-002, normal saline as negative control.

[0649] Experimental cells: NCI-N87 cells

[0650] Experimental animals: Balb / c nude mice, female, 5-6 weeks old, were purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[0651] 2. Experimental Plan

[0652] Cell treatment

[0653] NCI-N87 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a culture flask. When the cells reached approximately 80-90% confluency, they were trypsinized with EDTA. The cells were washed twice with PBS, centrifuged, and resuspended in pre-chilled PBS. The cells were counted using a cell counter, and the cell suspension was adjusted to the appropriate concentration for plating.

[0654] 2.2. Tumor cell transplantation

[0655] Balb / c nude mice were acclimated to the laboratory environment for 3-5 days and then subcutaneously inoculated with NCI-N87 cells in the right rib cage at a dose of 5 × 10 6 / mouse, the inoculation volume was 0.2mL (containing 50% Matrigel), and the tumor was grown to 200mm 3 Around 3:00 p.m., the drugs were administered in groups.

[0656] 2.3. Animal Dosing and Testing

[0657] The tumor-bearing nude mice were dosed according to the following regimen:

[0658] Table 15 Dosage regimen

[0659] 2.4. Tumor volume and body weight determination

[0660] The day of the first administration was recorded as day 1, and the administration was performed three times in total. The diameter of the tumor and body weight were measured, and the tumor volume, relative tumor proliferation rate T / C (%) and relative tumor inhibition rate TGI (%) were calculated.

[0661] The formula for calculating tumor volume (V) is: V = 1 / 2 × L length × L short 2 , where Llong and Lshort represent the long diameter and short diameter of the tumor, respectively;

[0662] Relative tumor growth rate (T / C) (%) = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the control group; RTV = Vt / V0, V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment);

[0663] Relative tumor inhibition rate TGI% = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively.

[0664] 3. Experimental Results

[0665] The test results showed that the antibody-drug conjugate of the present invention showed a relatively significant tumor inhibition effect. During the administration period, the animals in each group showed no significant weight loss or obvious drug toxicity. Specific results are shown in Table 16.

[0666] Table 16 Results of tumor volume test in NCI-N87 transplanted tumor model

[0667] Example 8.2 Antibody Drug Conjugate (ADC) Efficacy Test on HCC1569 Xenografts

[0668] 1. Experimental Materials

[0669] Test compound: Her2-ADC-003, normal saline as negative control.

[0670] Experimental cells: HCC1569 cells.

[0671] Experimental animals: NOD / SCID mice, female, 6-9 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0672] 2. Experimental Plan

[0673] Cell treatment

[0674] HCC1569 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a culture flask. When the cells reached approximately 80-90% confluency, they were trypsinized with EDTA. The cells were washed twice with PBS, centrifuged, and resuspended in pre-chilled PBS. Counted using a cell counter, the cell suspension was adjusted to the appropriate concentration, and then plated.

[0675] 2.2. Tumor cell transplantation

[0676] NOD / SCID mice were acclimated to the laboratory environment for 3-5 days and HCC1569 cells were subcutaneously inoculated on the right back at a volume of 2 × 10 6 / mouse, the inoculation volume was 0.2mL (containing 50% Matrigel), and the tumor was grown to 150mm 3 Around 3:00 p.m., the drugs were administered in groups.

[0677] 2.3. Animal Dosing and Testing

[0678] The tumor-bearing nude mice were dosed according to the following regimen:

[0679] Table 17 Dosage regimen

[0680] 3. Experimental Results

[0681] The test results showed that the antibody-drug conjugates of the present invention all showed relatively significant tumor inhibition effects. During the administration period, the animals in each group showed no significant weight loss or obvious drug toxicity. Specific results are shown in Table 18.

[0682] Table 18. Tumor volume data of HCC1569 transplant tumor model

[0683] Example 8.3 Efficacy testing of antibody drug conjugates (ADCs) in the NCI-N87 Enhertu-resistant xenograft tumor model

[0684] 1. Experimental Materials

[0685] Test compound: Her2-ADC-003, normal saline as negative control.

[0686] Experimental cells: NCI-N87 Enhertu-resistant cells were purchased from WuXi AppTec.

[0687] Experimental animals: NOD / SCID mice, female, 6-8 weeks old, provided by Weitonglihua or other qualified suppliers.

[0688] 2. Experimental Plan

[0689] Cell treatment

[0690] NCI-N87 Enhertu-resistant cells were cultured as monolayers in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% anti-antibiotic (antimycotic), and 100 ng / ml Enhertu in a 37°C, 5% CO2 incubator. Cells were digested with EDTA-containing trypsin and passaged weekly. When cell saturation reached 80%-90% and the desired cell number was reached, cells were harvested, counted, and plated.

[0691] 2.2. Tumor cell transplantation

[0692] NOD / SCID mice were acclimated to the laboratory environment for 3-7 days and inoculated subcutaneously on the right side of the back with 8×10 NCI-N87 Enhertu-resistant cells. 6 / mouse, the inoculation volume was 0.2mL (containing 50% Matrigel), and the tumor was grown to 150mm 3 Around 3:00 p.m., the drugs were administered in groups.

[0693] 2.3. Animal Dosing and Testing

[0694] The tumor-bearing nude mice were dosed according to the following regimen:

[0695] Table 19 Dosage regimen

[0696] 3. Experimental Results

[0697] Test results demonstrate that the antibody-drug conjugate of the present invention exhibited significant tumor suppression in the Enhertu-resistant model. During dosing, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 20.

[0698] Table 20 Tumor volume test results of NCI-N87 Enhertu resistant transplant tumor model

[0699] Example 8.4 Antibody Drug Conjugate (ADC) Efficacy Test on OVCAR-3 Xenograft Tumors

[0700] 1. Experimental Materials

[0701] Test compounds: NaPi2b-ADC-001, NaPi2b-ADC-003, NaPi2b-ADC-006, and normal saline as a negative control.

[0702] Experimental cells: OVCAR-3 cells.

[0703] Experimental animals: NCG mice, female, 5-6 weeks old, were purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[0704] 2. Experimental Plan

[0705] Cell treatment

[0706] OVCAR-3 cells were cultured adherently in 15 cm culture dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When cells reached the exponential growth phase, they were trypsinized, harvested, counted, and plated.

[0707] 2.2. Tumor cell transplantation

[0708] NCG mice were acclimated to the laboratory environment for 2-5 days and then subcutaneously inoculated with OVCAR-3 cells in the right rib cage. The inoculated cell volume was 5×10 6 The inoculation volume was 0.2 mL per mouse, containing 50% Matrigel. Experiments were performed when the tumors grew to approximately 200-250 mm3.

[0709] 2.3. Animal Dosing and Testing

[0710] The tumor-bearing nude mice were dosed according to the following regimen:

[0711] Table 21. Dosage regimen

[0712] 2.4. Tumor volume and body weight determination

[0713] The first day of administration was recorded as day 1, and the administration was repeated three times in total. Tumor diameter and body weight were measured regularly. Tumor volume, tumor proliferation rate, and tumor inhibition rate were calculated, and a tumor growth curve was drawn. The calculation formulas were:

[0714] The formula for calculating tumor volume (V) is: V = 1 / 2 × L length × L short 2 , where Llong and Lshort represent the long diameter and short diameter of the tumor, respectively.

[0715] Relative tumor growth rate (T / C) (%) = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the control group; RTV = Vt / V0, V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment);

[0716] Relative tumor inhibition rate TGI% = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively.

[0717] 3. Experimental Results

[0718] Test results showed that the antibody-drug conjugates of the present invention all exhibited significant tumor-suppressing effects. At a dose of 2 mg / kg, NaPi2b-ADC-003 demonstrated significantly superior efficacy to the control compound, NaPi2b-ADC-006. During the dosing period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 22.

[0719] Table 22. OVCAR-3 transplant tumor model tumor volume test results

[0720] Example 8.5 Antibody Drug Conjugate (ADC) Efficacy Test on BT474 Xenograft Tumors

[0721] 1. Experimental Materials

[0722] Test compound: Her2-ADC-004, normal saline as negative control.

[0723] Experimental cells: BT-474 cells.

[0724] Experimental animals: BALB / c nude mice, female, 6-8 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. or other qualified suppliers.

[0725] 2. Experimental Plan

[0726] Cell treatment

[0727] BT-474x cells (ATCC-HTB-20) were cultured in ATCC Hybri-Care Medium with 10% FBS at 37°C in a 5% CO2 incubator. Cells were routinely passaged twice weekly using trypsin containing EDTA. Cells were washed twice with PBS, centrifuged, and resuspended in pre-chilled PBS. Counted cells using a cell counter, the cell suspension was adjusted to the appropriate concentration, and then plated.

[0728] 2.2. Tumor cell transplantation

[0729] BALB / c nude mice were allowed to acclimate to the laboratory environment for 3-5 days. Three days before cell inoculation, a 0.36 mg sustained-release estrogen tablet was subcutaneously inoculated into the left back of each mouse. One week after estrogen tablet inoculation, the animals were assisted to urinate three times per week; if necessary, the animals were assisted to urinate daily. 6 BT-474x cells were subcutaneously inoculated on the right back near the upper limb of each mouse with PBS plus matrigel (1:1). The average tumor volume reached approximately 150-200 mm 3 The group dosing started at .

[0730] 2.3. Animal Dosing and Testing

[0731] The tumor-bearing nude mice were dosed according to the following regimen:

[0732] Table 23. Dosage regimen

[0733] 2.4. Tumor volume and body weight determination

[0734] The first day of administration was designated as day 0, and the administration was repeated three times. Tumor diameter and body weight were measured regularly. Tumor volume, relative tumor growth rate, and relative tumor inhibition rate were calculated, and tumor growth curves were drawn. The calculation formulas were:

[0735] The formula for calculating tumor volume (V) is: V = 1 / 2 × Llong × Lshort2, where Llong and Lshort represent the long diameter and short diameter of the tumor, respectively.

[0736] Relative tumor growth rate (T / C) (%): Calculated as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the vehicle control group). RTV, relative tumor volume, is calculated as RTV = Vt / V0, where V0 is the mean tumor volume measured at the time of group dosing (i.e., D0), and Vt is the mean tumor volume at a single measurement. TRTV and CRTV data are collected on the same day.

[0737] Relative tumor inhibition rate TGI% = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively.

[0738] 3. Experimental Results

[0739] The test results showed that the antibody-drug conjugates disclosed herein showed a relatively significant tumor-suppressing effect. During the administration period, the animals in each group showed no significant weight loss or significant drug toxicity. Specific results are shown in Table 24.

[0740] Table 24. Tumor volume data of BT-474 transplant tumor model

[0741] Example 8.6 Liver microsomal stability test of bioactive compounds

[0742] 1. Experimental methods:

[0743] Liver microsomes from rats, mice, monkeys, and humans were mixed with the test compound and placed in a 37°C water bath. Pre-incubated NADPH solution was added to initiate the reaction. The final incubation system contained 2 μM test compound, 0.5 mg / mL liver microsomes, and 1 mM NADPH. At 0, 5, 10, 30, 60, and 120 minutes, 20 μL of the incubation solution was removed and transferred to 200 μL of acetonitrile containing 1% formic acid to terminate the reaction and precipitate the protein. After centrifugation, the supernatant was diluted 1:1 with water and analyzed by LC-MS / MS. Hepatic clearance was calculated based on compound concentrations at different time points.

[0744] 2. Experimental results:

[0745] Test results show that the bioactive compounds of the present invention have high clearance rates in the livers of four different species. When the bioactive compounds (payload) are exposed to the circulatory system and normal tissues, the antibody-drug conjugates formed thereby can be rapidly metabolized and cleared, thereby reducing toxicity and improving the safety of the entire antibody-drug conjugate.

[0746] Example 8.7 Repeated intravenous toxicity study of antibody-drug conjugates in cynomolgus monkeys

[0747] 1. Test objectives:

[0748] Cynomolgus monkeys were given repeated intravenous injections of antibody-drug conjugates (ADCs) to observe the nature, extent, dose-effect, time-effect relationship, and reversibility of possible toxic reactions.

[0749] 2. Experimental design:

[0750] Each experimental group consisted of two cynomolgus monkeys, half male and half female, who were intravenously injected with the drug once every three weeks for six consecutive weeks (three times in total).

[0751] 3. Preparation method of test sample:

[0752] Pipette an appropriate amount of antibody-drug conjugate (ADC) and dilute it to the desired concentration. After preparation, sterilize it with a 0.22 μm syringe filter before use.

[0753] 4. Testing and observation indicators:

[0754] (1) General status observation: moribund and death observation: observe at least once in the morning and afternoon every day; daily observation: observe at least twice after each administration on the day of administration, and observe at least 1-2 times a day at other times; the observation content includes but is not limited to the injection site, mental state, feeding, drinking water, fur, excrement, death, and other conditions of the animal as well as other toxicity-related symptoms; detailed observation: observe 1-2 times a week, and the observation content includes but is not limited to the injection site, physical appearance, general behavior, mental state, glandular secretion, skin and mucous membrane color, respiratory status, genitals, death, and other abnormal symptoms.

[0755] (2) Body weight: Before the first administration, body weight should be measured at least once a week during the trial period.

[0756] (3) Food intake: measured twice a week.

[0757] (4) Hematology: Hematology was tested once on D4, D7, D14, and D21 after each administration.

[0758] (5) Blood biochemistry: The test was performed once on D5, D7, D14, and D21 after each administration.

[0759] (6) Toxicokinetics: Serum and plasma samples were collected before the first and second administrations, and immediately (0-1 minute), 3 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 168 hours, 336 hours, and 504 hours after administration.

[0760] (7) Gross autopsy: Autopsy was performed at the end of the administration, i.e., on the 50th day of the trial period, and histopathological examination was performed if necessary.

[0761] The results showed that the antibody-drug conjugates of the present invention, such as NaPi2b-ADC-003, did not cause significant drug-related changes in clinical symptoms, body weight, food consumption, or gross morphology at all doses. Overall, the animals were well tolerated under the experimental conditions.

[0762] Example 8.8 ADC Plasma Stability Test

[0763] 1. Solution preparation: Dilute the ADC stock solution to be tested with PBS to a working solution with a concentration of 2 mg / mL.

[0764] 2. Sample Incubation: The antibacterial agent ProClin was added to both ADC samples and human plasma at a final concentration of 0.1%, using sterile procedures. ADC samples were added to the sterile plasma at a final concentration of 200 μg / ml and incubated in a 37°C cell culture incubator at 80 shakes / minute. After 0 and 14 days of incubation, ADC samples were removed and 100 μl of Protein A was added to each tube. Adsorption was allowed to proceed for 2 hours, and elution was performed to obtain the incubated ADCs. The DAR values ​​of the incubated ADCs were tested (as in Example 5.1) to determine the plasma stability of the samples.

[0765] The test results show that the DAR value of the ADC of the present invention, such as NaPi2b-ADC-003, is basically maintained during the plasma incubation process (i.e., almost no toxin linker falls off from the antibody). The experimental results prove that the ADC of the present invention has better plasma stability.

[0766] Unless otherwise defined, the terms used in the present invention have the same meanings as those commonly understood by those skilled in the art.

[0767] The embodiments described in the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various other substitutions, changes and improvements within the scope of the present invention. Therefore, the present invention is not limited to the above-mentioned embodiments, but is only limited by the claims.

Claims

1. An antibody-drug conjugate as shown in Formula I: or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, in, Tb is an antibody or an antigen-binding fragment thereof; q is any value between 0.1 and 12.0; L is a linker that covalently binds Tb and D; D is a bioactive compound fragment, wherein D is selected from the following structures: , bit 1 is connected to L.

2. The antibody-drug conjugate according to claim 1, or its stereoisomer, pharmaceutically acceptable salt or solvate, wherein: The L is selected from: Among them, R L1 、R L2 are independently selected from hydrogen and C1-6 alkyl; preferably, R L1 、R L2 It is also a methyl, ethyl or propyl group, with the 1 position connected to Tb via the S atom and the 2 position connected to D.

3. The antibody-drug conjugate according to claim 1, or its stereoisomer, pharmaceutically acceptable salt or solvate, wherein: The L is selected from: Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

4. The antibody-drug conjugate according to claim 1, or its stereoisomer, pharmaceutically acceptable salt or solvate, wherein: The Tb is selected from the following: (1) Tb is an antibody or antigen-binding fragment thereof with or without endocytosis activity; preferably, Tb is an antibody or antigen-binding fragment thereof with endocytosis activity; (2) Tb is an antibody or antigen-binding fragment thereof that has the activity of binding to tumor cell surface antigens; (3) Tb is an anti-Her2 antibody or an antigen-binding fragment thereof, an anti-B7H3 antibody or an antigen-binding fragment thereof, or an anti-Trop-2 antibody or an antigen-binding fragment thereof; (4) Tb is an anti-Her2 antibody or an antigen-binding fragment thereof; preferably, Tb is anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or an antigen-binding fragment thereof; more preferably, Tb is trastuzumab or pertuzumab; more preferably, Tb is trastuzumab; (5) Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof; preferably, Tb is 1D1-01, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab or an antigen-binding fragment thereof; more preferably, Tb is 2E3-02 antibody; (6) Tb is an anti-Trop-2 antibody or an antigen-binding fragment thereof; preferably, Tb is datopotamab, sacituzumab or an antigen-binding fragment thereof; (7) Tb is an anti-Napi2b antibody or an antigen-binding fragment thereof; preferably, the anti-Napi2b antibody or an antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO: 83 in WO2024012524A1 and a light chain shown in SEQ ID NO: 84 in WO2024012524A1.

5. The antibody-drug conjugate according to claim 1, or its stereoisomer, pharmaceutically acceptable salt or solvate, wherein: The antibody drug conjugate is selected from the following structures: Wherein, q is selected from any number between 1 and 12; The following are preferred: Among them, NaPi2b is the 66C12D12-hz1 antibody in WO2024012524A1.

6. A drug-linker conjugate as shown in Formula II, Lg-LD Formula II or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, in, Lg is a group that reacts with antibodies; Preferably, Lg is selected from F, Cl, Br, MeSO2-, pentafluorophenoxy; More preferably, Lg is MeSO2- L and D are as defined in any one of claims 1-3.

7. The drug-linker conjugate according to claim 6, or its stereoisomer, pharmaceutically acceptable salt or solvate, characterized in that: The drug-linker conjugate is selected from:

8. A method for preparing an antibody-drug conjugate of formula I, comprising: Tb is subjected to a coupling reaction with the drug-linker conjugate Lg-LD represented by formula II; wherein Tb, Lg, L and D are defined as any one of claims 1-7.

9. The method according to claim 8, wherein The method comprises the steps of performing a coupling reaction between Tb and a drug-linker conjugate Lg-LD represented by Formula II in a solvent to form a CS bond; The molar ratio of Tb to the drug-linker conjugate is 1:(1-20), such as 1:(2-16), 1:(2-14), 1:(2-12), 1:(2-10); The coupling reaction is carried out in water and / or an organic solvent; the organic solvent is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, and nitriles (such as acetonitrile).

10. A biologically active compound, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, characterized in that: The biologically active compound is selected from the following structures:

11. A population of antibody-drug conjugates comprising the antibody-drug conjugate according to any one of claims 1 to 5, or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof, or a composition thereof, wherein the antibody-drug conjugate has one, two or more q values; Preferably, the average DAR of the antibody drug conjugate population is an integer or decimal selected from 1-16, for example, selected from 1.5-2.5, 3.5-4.5, 5.5-6.5 and 7.5-8.5; More preferably, the average DAR of the population of antibody drug conjugates is selected from the group consisting of about 2.0, 4.0, 6.0, and 8.

0.

12. A pharmaceutical composition comprising the antibody-drug conjugate according to claims 1-5, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof; or the drug-linker conjugate according to claim 6 or 7, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof; or the antibody-drug conjugate group according to claim 11, and optionally one or more pharmaceutical excipients.

13. Use of the antibody-drug conjugate according to claims 1-5, or its stereoisomers, pharmaceutically acceptable salts, or solvates; or the drug-linker conjugate according to claim 6 or 7, or its stereoisomers, pharmaceutically acceptable salts, or solvates; or the antibody-drug conjugate group according to claim 11; or the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating and / or preventing a disease associated with abnormal cell activity; preferably cancer; Preferably, the cancer disease is selected from solid tumors and hematological tumors; more preferably, the cancer disease is selected from esophageal cancer, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, non-Hodgkin's lymphoma, central nervous system tumors, prostate cancer or thyroid cancer; the esophageal cancer is preferably esophageal adenocarcinoma and esophageal squamous cell carcinoma; the lung cancer is preferably small cell lung cancer and non-small cell lung cancer; the central nervous system tumor is preferably glioma, glioblastoma multiforme, glioma and sarcoma.

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