Antibody-drug conjugate and use thereof

Abstract

The present invention relates to an antibody-drug conjugate and the use thereof. Specifically, provided are an antibody-drug conjugate represented by formula I and the use thereof in the prevention and / or treatment of diseases related to abnormal cell activity, including, but not limited to, the prevention and / or treatment of tumor diseases.

Description

An antibody-drug conjugate and its uses

[0001] This application claims priority to Chinese patent application 2024116231705, filed on November 14, 2024; Chinese patent application 2025103888770, filed on March 28, 2025; Chinese patent application 2025106297429, filed on May 15, 2025; and Chinese patent application 2025113994511, filed on September 28, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology and relates to an antibody-drug conjugate and its use, particularly to an antibody-drug conjugate containing olistatin-like bioactive compounds and its preparation method, as well as its use in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to its use in the prevention and / or treatment of tumor diseases. Background Technology

[0003] Auristatins are peptide derivatives isolated from the Indian Ocean truncated sea hare. They primarily inhibit tumor growth by acting on microtubules, exhibiting activity approximately 1000 times greater than 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, Vidicetumab, and TIVDAK) use MMAE or MMAF as the payload. In clinical trials, there are over 40 ADCs using auristatin as the payload, accounting for more than 50% of all ADCs in development.

[0004] Existing adjuvant inhibitors (ADCs) using olistatin derivatives as payloads typically work by binding antibodies to tumor cell surface antigens, followed by endocytosis into the endosome, and then conversion from the endosome to the lysosome. In the lysosome, hydrolytic enzymes dissociate the bioactive molecules (toxins or payload) from the ADC. These dissociated bioactive molecules enter the cytoplasm from the lysosome and kill tumor cells. Bioactive molecules escaping after killing tumor cells can further kill surrounding tumor cells that do not express or express low levels of the antigen (the so-called by-stander effect). Changes at any stage of this process can lead to ADC resistance and loss of therapeutic effect, such as changes in antigen expression, weakened or absent endocytosis, or alterations in endosome or lysosomal function.

[0005] In addition, the conjugation of ADC drugs with olistatin derivatives as payloads that are currently on the market or in clinical research is mainly achieved through the Michael addition reaction between the free thiol group on the antibody and maleimide. However, many studies have reported 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 detachment of toxins and toxic reactions.

[0006] Therefore, finding an ADC that can be stable in the bloodstream and that can kill tumors by breaking down toxins through extracellular lysis without endocytosis in the tumor microenvironment would overcome the various drug resistance mechanisms of traditional ADCs, expand the therapeutic window, and improve the therapeutic effect. This would 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 its stereoisomers or pharmaceutically acceptable salts.

[0009] in,

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

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

[0012] L is the linker, which covalently bonds Tb and D;

[0013] D represents a fragment of a bioactive compound.

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

[0015] Lg-LD

[0016] Formula II

[0017] in,

[0018] Lg is a group that reacts with the antibody; L and D are defined as described in the first aspect of this invention.

[0019] In a third aspect, the present invention provides a bioactive compound of Formula III, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0020] The definitions of R1, R2, R3', and Ra' are as described in any embodiment of the present invention.

[0021] In a fourth aspect, the present invention provides a method for preparing the antibody-drug conjugate described in the first aspect.

[0022] In a fifth aspect, the present invention provides a method for preparing the drug linker conjugate described in the second aspect.

[0023] In a sixth aspect, the present invention provides a group of conjugates comprising the antibody-drug conjugates described in the first aspect.

[0024] In a seventh aspect, the present invention provides compositions comprising the antibody-drug conjugate of the first aspect, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the drug linker conjugate of the second aspect, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the bioactive compound of the third aspect, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0025] In an eighth aspect, the present invention provides the use of the antibody-drug conjugate of the first aspect, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the drug-linked conjugate of the second aspect, or a stereoisomer thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, or the bioactive compound of the third aspect, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0026] Invention Details

[0027] definition

[0028] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this invention are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0029] As used in this invention, examples of the term "pharmaceutically acceptable salt" are organic acid adduct salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α-ketoglutarate, α-glycerophosphate, alkyl sulfonates, or aryl sulfonates; preferably, the alkyl sulfonate is a methanesulfonate or ethyl sulfonate; and the aryl sulfonate is a benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts may also be formed, including but not limited to hydrochlorides, hydrobromides, hydroiodates, nitrates, bicarbonates and carbonates, sulfates, or phosphates.

[0030] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

[0031] As used herein, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules may 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-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of the invention covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0032] In this invention, solid lines (—), solid wedges, or dashed wedges may be used to depict the carbon-carbon bonds of the compounds of this invention. Using solid lines to depict bonds to asymmetric carbon atoms indicates that all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) are included at that carbon atom. Using solid or dashed wedges to depict bonds to asymmetric carbon atoms indicates the presence of the indicated stereoisomers. When present in racemic mixtures, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of this 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, trans-isomers, and mixtures thereof). The compounds of this invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0033] In this invention, This indicates the connection point of the connected part.

[0034] The compounds of the present invention can exist as solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0035] In this invention, pharmaceutical excipients refer to the excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical formulation. Besides acting as a formulator, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified into natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0036] The pharmaceutical composition can be formulated into various suitable dosage forms depending on the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound of the present invention or its pharmaceutically acceptable salt or conjugate, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5-350 mg, and particularly preferably 1-250 mg.

[0037] The pharmaceutical composition can be administered in injectable form, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media. The pharmaceutical composition can also be administered via intravenous infusion.

[0038] The term "treatment" as used in this invention generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. The term "treatment" as used in this invention covers any treatment of a patient's disease, including: (a) preventing the occurrence of a disease or symptoms in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, i.e., preventing its progression; or (c) alleviating the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0039] In this invention, the term "individual" includes humans or non-human animals. Exemplary human individuals include individuals suffering from a disease (such as the disease described in this invention) (referred to as patients) or normal individuals. The term "non-human animal" in this 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.).

[0040] In this invention, the term "effective dose" refers to the amount of an antibody-drug conjugate, drug-linked conjugate, compound, or composition that, when administered, will alleviate one or more symptoms of the treated condition to a certain extent.

[0041] In this invention, the terms "antibody-drug conjugate" or "ADC" refer to substances obtained by linking a bioactive compound fragment (drug molecule) with an antibody or its antigen-binding fragment. In some embodiments of this invention, the bioactive compound fragment and the target portion are linked via a linker. The linker is capable of cleaving under specific conditions (e.g., intracellular low pH) or specific actions (e.g., lysosomal proteases), thereby separating the bioactive compound fragment from the target portion or the antibody or its antigen-binding fragment. In some embodiments of this invention, the linker comprises cleavable or cleavable units, such as peptides or disulfide bonds. In some embodiments of this invention, the bioactive compound fragment and the target portion or the antibody or its antigen-binding fragment are directly linked by a covalent bond, which is capable of cleaving under specific conditions or actions, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0042] In this invention, the terms "bioactive substance," "bioactive compound," and "drug" refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0043] In this invention, regarding "position 1 of L1 is connected to Tb via an S atom," those skilled in the art will understand that position 1 of L1 is connected to the thiol group inherent in Tb (such as an antibody) after the disulfide bond is opened (e.g., by reducing the disulfide bond with the reducing agent TCEP to generate a thiol 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 thiol group inherent in Tb itself after the disulfide bond is opened, and the connection between L1 and Tb is, for example... -S- is formed by concatenating the 1st bit.

[0044] In this invention, the terms "linker" or "linker" refer to a fragment that links a bioactive compound fragment (drug molecule) to an antibody portion.

[0045] In this 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 specifically limited, but examples include β-amino acids, γ-amino acids, D-type amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids with side chains different from natural amino acids.

[0046] In this invention, the term "...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 digestion of the linker in tumor tissues or within tumor cells, can form a bioactive drug (e.g., a small molecule cytotoxic drug, which includes a group after the loss of an atom or group of atoms) or its derivative (e.g., its precursor). To avoid ambiguity, "drug" does not only refer to "medicines" approved by pharmaceutical regulatory authorities, but also includes any compound with potential therapeutic bioactivity in clinical practice, or in research and development and academic studies.

[0047] In this invention, the term "antibody" is used in its broadest sense, including intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, provided they possess the desired biological activity. In this invention, "antibody" and "immunoglobulin" are used interchangeably.

[0048] In this invention, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the antibodies constituting this cluster are identical except for a small number of possible natural mutations. Monoclonal antibodies possess high specificity against a single determinant (epitope) of an antigen, while polyclonal antibodies, in contrast, comprise different antibodies targeting different determinants (epitopes). Besides specificity, the advantage of monoclonal antibodies is that their synthesis is unaffected by contamination from other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by originating from a substantially homogeneous group of antibodies, and should not be construed as requiring special methods for preparation.

[0049] In some embodiments of the invention, monoclonal antibodies further include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to one, a class, or a subclass of antibody, while the remainder is identical or homologous to another, a different class, or a different subclass of antibody, provided they possess the desired biological activity (see, for example, US 4,816,567). Chimeric antibodies that can be used in the present invention include primatized antibodies comprising a variable region antigen-binding sequence from a non-human primate (e.g., ancient monkey, chimpanzee, etc.) and a human constant region sequence.

[0050] The term "antibody fragment" refers to a portion of an antibody, preferably an antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity-determining region fragments, diabody, linear antibody, and single-chain antibody molecules.

[0051] The term "bispecific antibody," also known as "bifunctional antibody-drug conjugate," refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a conjugate arm. This conjugate retains the activity of each antibody and thus has both bifunctionality and bispecificity.

[0052] The term "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies. The former is an antibody that has the binding specificity of three different antigens, while the latter is an antibody that has the binding specificity of four different antigens.

[0053] The term "intact antibody" refers to an antibody that contains an antigen-binding variable region and a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant regions can be natural sequences (e.g., human natural constant region sequences) or amino acid sequence variants thereof. Intact antibodies are preferably intact antibodies with one or more effector functions.

[0054] The term "probody" is a modified antibody, including an antibody or antibody fragment that is specifically designed to bind to its target and can be coupled with a masking group, wherein the masking group is defined as having 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 10000 times greater than the cleavage constant for the binding ability of an antibody or antibody fragment without a coupled masking group to its target.

[0055] In this invention, the “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody containing a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which hypervariable region residues of a human recipient immunoglobulin have been replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibodies) having the desired specificity, affinity, and function. In some embodiments, framework region (FR) residues of human immunoglobulins are also replaced with non-human residues. Furthermore, humanized antibodies may also contain residues not present in the recipient or donor antibody. These modifications are intended to further optimize antibody performance. Humanized antibodies generally contain at least one, typically two, variable regions, where all or almost all hypervariable loops correspond to those of the non-human immunoglobulin, while the FR is entirely or almost entirely a sequence of human immunoglobulin. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc, typically human immunoglobulin Fc).

[0056] Intact antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant regions. The five main classes are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different antibody classes are referred to as α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known in the art.

[0057] In this invention, although in most cases the amino acid substitutions in the antibody are replaced by L-amino acids, this is not the only possibility. 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 prone to degradation in the oral cavity, intestines, or plasma than peptides containing only L-amino acids.

[0058] The monoclonal antibodies used in this invention can be produced by many methods. For example, the monoclonal antibodies used in this invention can be obtained by hybridoma methods using cells from many species, including mice, hamsters, rats, and humans, or by recombinant DNA technology (see, for example, US 4,816,567), or isolated from a phage antibody library.

[0059] In this invention, unless otherwise explicitly stated, the descriptive phrases “each…independently selected” and “…independently selected” used throughout this invention are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

[0060] In various parts of this specification, the substituents of the compounds of this invention are disclosed according to the type or scope of the groups. In particular, this invention includes every independent secondary combination of the members of these types and scopes. For example, the term "C1-6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl groups.

[0061] In this invention, the term "direct bond" refers to the absence of a substituent, where the two ends of the substituent are directly connected to form a bond.

[0062] In this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other elements or method steps not listed.

[0063] In this invention, the term "optional substitution" refers to the presence or absence of substituents.

[0064] In this invention, the term "C1-8 alkyl" refers to a straight-chain or branched alkyl group containing 1-8 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, hexyl.

[0065] In this 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" or "C1-3 alkylene", and specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, etc.

[0066] In this invention, the term "C2-6 alkenyl" refers to a straight-chain, branched, or cyclic alkenyl group containing at least one double bond and having 2-6 carbon atoms, including, for example, "C2-4 alkenyl". Examples include, but are not limited to, vinyl, 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, etc.

[0067] In this invention, the term "C2-6 ynyl" refers to a straight-chain or branched ynyl group containing at least one triple bond and having 2-6 carbon atoms, including, for example, "C2-4 ynyl". 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, etc.

[0068] In this invention, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0069] In this invention, the term "C3-10 cycloalkyl" refers to a saturated cycloalkyl group containing 3-10 carbon atoms. This includes, for example, "C3-6 cycloalkyl" or "C3-8 cycloalkyl". Optionally, the carbon atoms in the cyclic structure may be oxidized. Specific examples include, but are not limited to, cyclopropane (i.e., cyclopropyl), cyclobutane (i.e., cyclobutyl), cyclopentane (i.e., cyclopentyl), and cyclohexyl.

[0070] In this invention, the term "C1-6 alkoxy" refers to an alkyl group as defined above, which is attached to the parent molecule via 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, pentoxy, hexoxy, etc.

[0071] In this invention, the term "C1-6 alkylthio" refers to an alkyl group as defined above, which is attached to a parent molecule portion by a sulfur atom, including, for example, "C1-3 alkylthio" or "C1-4 alkylthio".

[0072] In this invention, the term "halogenated C1-6 alkyl" refers to an alkyl group as defined above that is substituted with 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, etc.

[0073] In this invention, the term "halogenated C1-6 alkoxy" refers to an alkoxy group as defined above that is substituted with 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, etc.

[0074] In this invention, the term "halogenated C1-6 alkylthio" refers to an alkylthio group as defined above that is substituted with one or more halogens as defined above, including, for example, "halogenated C1-3 alkylthio" or "halogenated C1-4 alkylthio".

[0075] In this invention, the term "4-10 membered heterocycle" refers to a ring containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). The term "3-6 membered heterocycle" refers to a ring containing 3-6 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). The term "3-7 membered heterocycle" refers to a ring containing 3-7 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). The term "5-6 membered heterocycle" refers to a ring containing 5-6 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the ring structure can be oxidized, specific examples including but not limited to pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, pyrrolidone, and other rings.

[0076] In this invention, the term "3-10 membered carbon ring" refers to a ring with 3-10 carbon atoms. Optionally, the carbon atoms in the ring structure can be replaced by oxygen. Examples include 3-6 membered carbon rings, 3-7 membered carbon rings, 5-8 membered carbon rings, etc., and specific examples include, but are not limited to, cyclopentane and cyclohexane.

[0077] In this invention, the term "4-10 membered heterocyclic group" refers to a cyclic group containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom, such as an oxygen atom, nitrogen atom, or sulfur atom). The term "4-6 membered heterocyclic group" refers to a cyclic group containing 4-6 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). The term "3-6 membered heterocyclic group" refers to a cyclic group containing 3-6 ring atoms (wherein at least one ring atom is a heteroatom, such as an oxygen atom, nitrogen atom, or sulfur atom). Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure may be oxidized. "4-8 membered heterocyclic groups" include, for example, "4-8 membered nitrogen-containing heterocyclic groups", "4-8 membered oxygen-containing heterocyclic groups", "4-7 membered heterocyclic groups", "4-7 membered oxygen-containing heterocyclic groups", "4-6 membered heterocyclic groups", "5-7 membered heterocyclic groups", "5-6 membered heterocyclic groups", and "5-6 membered nitrogen-containing heterocyclic groups". Specific examples include, but are not limited to, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, and homopiperazinyl.

[0078] In this invention, the term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon group, such as 6-10 aryl, 5-8 aryl, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracene, phenanthrene, etc. "6-10 aryl" refers to an aryl group containing 6-10 ring atoms. "C6-10 aryl" refers to an aryl group containing 6-10 carbon atoms.

[0079] In this invention, the term "heteroaryl" refers to an aromatic cyclic group wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxidized. Specific examples include, but are not limited to, 5-10-membered heteroaryl, 5-6-membered heteroaryl, 5-10-membered nitrogen-containing heteroaryl, 6-10-membered oxygen-containing heteroaryl, 6-8-membered nitrogen-containing heteroaryl, 5-8-membered oxygen-containing heteroaryl, etc., such as furanyl, thiophene, pyrrole, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazole, 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, pyridyl, 2-pyridoneyl, 4-pyridoneyl, pyrimidinyl, 1,4-dioxazadienyl, 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, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetraazinyl, aziridine-heptanetrienyl, 1,3-diazacycloheptanetrienyl, aziridine-octatetraenyl, etc.

[0080] In this invention, the term "connector unit" refers to a component of an antibody-drug conjugate or drug-linked conjugate or linker, which functions to link an antibody or its antigen-binding fragment that binds to a target site to the remainder of the antibody-drug conjugate. The connector unit can link a Tb unit to L2, and specific examples include, but are not limited to (where position 1 is linked to the antibody or its antigen-binding fragment, and position 2 is linked to L2 or L3):

[0081] In this invention, the term "linker unit" refers to a component of an antibody-drug conjugate or drug-linker conjugate or linker, which functions to bind a linker unit to an amino acid residue or a short peptide consisting of 2-10 amino acid residues. The presence of the linker unit enables the linker unit to connect L1 to L3. Specific examples include, but are not limited to (where position 1 is connected to the linker unit and position 2 is connected to L3):

[0082] In this invention, the term "spacer unit" refers to a component of an antibody-drug conjugate or drug-linked conjugate or linker, which serves to separate amino acid residues or short peptides consisting of 2-10 amino acid residues from fragments of bioactive compounds (drug molecules).

[0083] Antibody-drug conjugates

[0084] In a first aspect, the present invention provides an antibody-drug conjugate of Formula I:

[0085] Or its stereoisomers or pharmaceutically acceptable salts.

[0086] in,

[0087] Tb is an antibody or its antigen-binding fragment;

[0088] q is any value between 0.1 and 12.0;

[0089] L is the linker, which covalently bonds Tb and D;

[0090] D represents a fragment of a bioactive compound.

[0091] In some implementations, D has the structure shown in Equation I-1:

[0092] Or its stereoisomers or pharmaceutically acceptable salts.

[0093] in,

[0094] R1 is hydrogen, methyl,

[0095] R2 is hydrogen, deuterium, or hydroxyl;

[0096] R3 is hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), or 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole).

[0097] Ra represents hydrogen, deuterium, and...

[0098] R 4 It is a C1-6 alkyl group substituted with hydrogen, C1-6 alkyl (preferably C1-3 alkyl) or hydroxyl;

[0099] s can be 1, 2, 3, 4, 5, or 6;

[0100] m and n are each independently 0, 1, 2 or 3;

[0101] This indicates the connection site between L and D, where a hydrogen atom or hydroxyl group on D is replaced by L.

[0102] In some embodiments, the heteroaryl group has 1, 2, 3 or 4 heteroatoms, and the heteroatoms are independently N, O or S.

[0103] In some embodiments, the heteroaryl group has one or two heteroatoms, and the heteroatoms are independently N or S.

[0104] In some embodiments, a hydrogen atom or hydroxyl group on R1, R2, R3, or Ra in D is replaced by L.

[0105] In some embodiments, a hydrogen atom or hydroxyl group on R1, R3, or Ra in D is replaced by L.

[0106] In some embodiments, a hydrogen atom or hydroxyl group on R1 in D is replaced by L.

[0107] In some embodiments, a hydrogen atom or hydroxyl group on R2 in D is replaced by L.

[0108] In some embodiments, a hydrogen atom or hydroxyl group on R3 in D is replaced by L.

[0109] In some embodiments, a hydrogen atom or hydroxyl group on Ra in D is replaced by L.

[0110] In some implementations, D has the structure shown in Equation I-2:

[0111] Or its stereoisomers or pharmaceutically acceptable salts.

[0112] in,

[0113] R1, R2, R3, and Ra satisfy one of the following conditions:

[0114] (1) R1 is hydrogen, methyl, R2 is hydrogen, deuterium, or hydroxyl; R3 is... 1 is connected to L; Ra represents hydrogen, deuterium,

[0115] (2) R1 is hydrogen, methyl, R2 is hydrogen, deuterium, or hydroxyl; R3 is hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), or 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole). Ra is 1 bit connected to L; or

[0116] (3) Whether R1 exists or not, if R1 exists, then... Position 1 is connected to L; when R1 is absent, L is directly connected to the nitrogen atom; R2 is hydrogen, deuterium, or hydroxyl; R3 is hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), or 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole).

[0117] Ra represents hydrogen, deuterium, and...

[0118] R 4 C1-6 alkyl groups substituted with hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), or hydroxyl;

[0119] s can be 1, 2, 3, 4, 5, or 6;

[0120] m and n are each independently 0, 1, 2 or 3.

[0121] In some implementations, R1 is hydrogen, methyl, R2 is hydrogen or hydroxyl; R3 is... 1 is connected to L; Ra is hydrogen.

[0122] In some implementations, R1 is hydrogen, methyl, R2 is hydrogen or hydroxyl; R3 is hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole); Ra is... 1 is connected to L.

[0123] In some implementations, R1 may or may not exist; when R1 exists, it is... Position 1 is connected to L; if R1 is absent, L is directly connected to the nitrogen atom; R2 is hydrogen or hydroxyl; R3 is hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), or 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole). Ra represents hydrogen.

[0124] In some implementations, R1 may or may not exist; when R1 exists, it is... Position 1 is connected to L; when R1 is absent, L is directly connected to the nitrogen atom; R2 is hydrogen or hydroxyl; R3 is hydrogen, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl) or 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole); Ra is hydrogen,

[0125] In some implementation schemes,

[0126] R1 is hydrogen or methyl;

[0127] R2 is hydrogen, deuterium, or hydroxyl;

[0128] R3 and Ra satisfy one of the following conditions:

[0129] (1) R3 is

[0130] Ra is hydrogen, deuterium, amino, or C1-6 alkyl hydroxyl; or

[0131] (2) R3 is hydrogen, deuterium, C1-6 alkyl hydroxyl, 5-12 heteroaryl (preferably thiazole) or

[0132] Ra is

[0133] R 4 It is hydrogen or C1-6 alkyl (preferably methyl);

[0134] s can be 1, 2, 3, 4, 5, or 6;

[0135] m and n are each independently 0, 1, 2 or 3;

[0136] 1 is connected to L.

[0137] In some implementations, R1 is hydrogen or methyl; R2 is hydrogen or hydroxyl.

[0138] In some implementations, R3 is Ra represents hydrogen, with position 1 connected to L.

[0139] In some implementations, R3 is Ra represents hydrogen, with position 1 connected to L.

[0140] In some implementations, R3 is hydrogen or Ra is 1 is connected to L.

[0141] In some implementations, R3 is hydrogen, and Ra is... 1 is connected to L.

[0142] In some implementations, s is 1, 2, or 3.

[0143] In some implementations, m is 1, and n is 1, 2, or 3.

[0144] In some implementations, R3 is Ra is 1 is connected to L.

[0145] In some implementations, R4 is hydrogen.

[0146] In some implementations, D has the structure shown in Equation I-2-1:

[0147] Wherein, R1 is hydrogen or methyl;

[0148] R3 is One of these bits is connected to L;

[0149] s is 1, 2, or 3; m is 1, and n is 1, 2, or 3.

[0150] In some implementations, D has the structure shown in Equation I-2-2:

[0151] Wherein, R1 is hydrogen or methyl;

[0152] R3 is hydrogen or

[0153] Ra is 1 bit is connected to L;

[0154] s can be 1, 2, or 3.

[0155] In some implementations, D has the following structure:

[0156] 1 is connected to L.

[0157] In some implementations, D has the following structure:

[0158] 1 is connected to L.

[0159] In some implementations, D has the following structure: 1 is connected to L.

[0160] In some embodiments, L is covalently linked to the amino or thiol group of Tb; preferably, L is covalently linked to the thiol group of Tb; more preferably, L is covalently linked to the thiol group formed after the interchain disulfide bond of Tb is opened.

[0161] In some embodiments, L is a detachable connector or a non-detachable connector; preferably, L is a detachable connector.

[0162] In some implementations, L is

[0163] in,

[0164] L1 is the connector unit, which covalently bonds Tb and L2;

[0165] L2 is a connecting unit, which is covalently bonded to L1 and L3;

[0166] L3 is a short peptide composed of amino acid residues, or 2-10 amino acid residues. It covalently binds L2 and L4;

[0167] L4 is a direct bond or spacer unit that covalently bonds L3 and D.

[0168] In some implementations, L1 is Position 1 is connected to Tb via an S or N atom, and position 2 is connected to L2.

[0169] In some implementations, L1 is Position 1 is connected to Tb via an S or N atom, and position 2 is connected to L2.

[0170] In some implementations, L1 is Position 1 is connected to Tb via the S atom, and position 2 is connected to L2.

[0171] In some implementations, L1 is Position 1 is connected to Tb via the N atom, and position 2 is connected to L2.

[0172] In some implementations, L2 is Bit 1 is connected to L1, and bit 2 is connected to L3.

[0173] In some implementations, L2 is Bit 1 is connected to L1, and bit 2 is connected to L3.

[0174] In some implementations, L2 is Bit 1 is connected to L1, and bit 2 is connected to L3.

[0175] In some implementations, L2 has the structure shown in the table below: Bit 1 is connected to L1, and bit 2 is connected to D.

[0176] In some implementations, L3 is

[0177] Among them, R L1 R L2 Each is independently hydrogen, C1-6 alkyl, or r is any integer between 0 and 24; preferably, R L1 R L2 Simultaneously methyl, ethyl, propyl or

[0178] In some implementations, L3 is Among them, R L1 R L2 Each is independently hydrogen, C1-6 alkyl, and r is any integer between 0 and 24; preferably, R L1 R L2 Simultaneously methyl, propyl or

[0179] In some implementations, L3 is Among them, R L1 R L2 Each independently r is any integer between 0 and 24; preferably, R L1 R L2 At the same time

[0180] In some implementations, L3 is

[0181] In some implementations, L3 is

[0182] In some implementations, L4 is

[0183] In some implementations, L4 is a direct key or

[0184] In some implementations, L is Bit 1 is connected to Tb, and bit 2 is connected to D; preferably, L is:

[0185] Among them, R L1 R L2 Each is independently hydrogen, C1-6 alkyl, or r is any integer between 0 and 24; preferably, R L1 R L2 Simultaneously methyl, propyl or Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0186] In some implementations, L is:

[0187] R L1 R L2 Each independently r is any integer between 0 and 24; preferably, R L1 R L2 At the same time More preferably, R L1 R L2 At the same time

[0188] In some preferred embodiments, L is:

[0189] Position 1 is connected to Tb via an S or N atom, and position 2 is connected to D.

[0190] In some preferred embodiments, L is:

[0191] Position 1 is connected to Tb via an S or N atom, and position 2 is connected to D.

[0192] In some preferred embodiments, L is: Among them, R L1 R L2 Each is independently hydrogen, C1-6 alkyl, or r is any integer between 0 and 24; preferably, R L1 R L2 Simultaneously methyl, ethyl, propyl or Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0193] In some preferred embodiments, R L1 RL2 Each independently r is any integer between 0 and 24.

[0194] In some preferred embodiments, R L1 R L2 At the same time

[0195] In some preferred embodiments, R L1 R L2 At the same time

[0196] In some preferred embodiments, R L1 R L2 At the same time

[0197] In some preferred embodiments, L is: Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0198] In some preferred embodiments, L is Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0199] In some implementations, Tb is an antibody or its antigen-binding fragment that may or may not have endocytic activity.

[0200] In some implementations, Tb is an antibody or its antigen-binding fragment that has endocytic activity.

[0201] In some embodiments, the endocytic activity is tumor cell endocytic activity.

[0202] In some implementations, Tb is an antibody or its antigen-binding fragment that has tumor cell surface antigen-binding activity.

[0203] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that has tumor cell surface antigen-binding activity and tumor cell endocytosis activity.

[0204] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that has tumor cell surface antigen-binding activity and has little or no tumor cell endocytic activity.

[0205] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that has tumor cell surface antigen-binding activity but does not have tumor cell endocytosis activity.

[0206] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that does not have tumor cell endocytosis activity.

[0207] In some preferred embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that has tumor cell surface antigen-binding activity and tumor cell endocytosis activity.

[0208] In some embodiments, the antibody or its antigen-binding fragment includes 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.

[0209] In some implementations, the antibody is a bispecific antibody or a multispecific antibody.

[0210] In some embodiments, the antibody or its antigen-binding fragment is a non-human antibody, a humanized antibody, or a fully human antibody.

[0211] In some embodiments, the antibody or its antigen-binding fragment probody, bispecific antibody, or multispecific antibody is used.

[0212] In some embodiments, the antibody or its antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies (e.g., scFv), and chimeric antibodies.

[0213] In some implementations, the targets for Tb are selected from CD19, CD20, CD21, CD22, CD30, CD33, CD123, Her2, Napi2b, and Trop-2.

[0214] In some implementations, Tb is an anti-Her2 antibody or its antigen-binding fragment, or an anti-Trop-2 antibody or its antigen-binding fragment.

[0215] In some implementations, Tb is an anti-Trop-2 antibody or its antigen-binding fragment, or an anti-Her2 antibody or its antigen-binding fragment.

[0216] In some embodiments, Tb is an anti-Her2 antibody or its antigen-binding fragment, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or its antigen-binding fragment; preferably, Tb is trastuzumab or pertuzumab; for example, Tb is trastuzumab.

[0217] In some implementations, Tb is an anti-Trop-2 antibody or its antigen-binding fragment, such as datopotamab, sacituzumab or its antigen-binding fragment.

[0218] In some implementations, Tb is an anti-Napi2b antibody or its antigen-binding fragment.

[0219] In some embodiments, the anti-Napi2b antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein:

[0220] The heavy chain variable region (VH) includes:

[0221] (i)HCDR1, which contains the amino acid sequence (66C12D12-hz1Kabat HCDR1) of SEQ ID NO:3 in WO2024012524A1;

[0222] (ii) HCDR2, comprising the amino acid sequence (66C12D12-hz1Kabat HCDR2) of SEQ ID NO:5 in WO2024012524A1; and

[0223] (iii) HCDR3, which contains the amino acid sequence (66C12D12-hz1Kabat HCDR3) of SEQ ID NO:6 in WO2024012524A1; and

[0224] The light chain variable region (VL) includes:

[0225] (i)LCDR1, which contains the amino acid sequence (66C12D12-hz1Kabat LCDR1) of SEQ ID NO:8 in WO2024012524A1;

[0226] (ii) LCDR2, which contains the amino acid sequence (66C12D12-hz1Kabat LCDR2) of SEQ ID NO:9 in WO2024012524A1; and

[0227] (iii) LCDR3, which contains the amino acid sequence (66C12D12-hz1Kabat LCDR3) of SEQ ID NO:10 in WO2024012524A1;

[0228] The CDR is determined by the Kabat definition scheme.

[0229] In some embodiments, the VH contains the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 5, and 6 of WO2024012524A1, and the VL contains the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:8-10 of WO2024012524A1.

[0230] In some embodiments, in the above-mentioned anti-Napi2b antibody or its antigen-binding fragment, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence (66C12D12-hz1VH) of SEQ ID NO:17 in WO2024012524A1, and the light chain variable region comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence (66C12D12-hz1VL) of SEQ ID NO:18 in WO2024012524A1;

[0231] In some embodiments, in the above-mentioned anti-Napi2b antibody or its antigen-binding fragment, the heavy chain variable region comprises the amino acid sequence (66C12D12-hz1VH) of SEQ ID NO:17 in WO2024012524A1, and the light chain variable region comprises the amino acid sequence (66C12D12-hz1VL) of SEQ ID NO:18 in WO2024012524A1.

[0232] In some embodiments, the anti-Napi2b antibody or its antigen-binding fragment described above comprises a heavy chain (66C12D12-hz1HC heavy chain) with an amino acid sequence as shown in SEQ ID NO:83 of WO2024012524A1, and a light chain (66C12D12-hz1LC light chain) with an amino acid sequence as shown in SEQ ID NO:84 of WO2024012524A1.

[0233] In some embodiments, the above-mentioned anti-Napi2b antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein:

[0234] The heavy chain variable region (VH) includes:

[0235] (i)HCDR1, which contains the amino acid sequence of SEQ ID NO: 1;

[0236] (ii) HCDR2, which contains the amino acid sequence of SEQ ID NO: 2; and

[0237] (iii) HCDR3, which contains the amino acid sequence of SEQ ID NO: 3; and

[0238] The light chain variable region (VL) includes:

[0239] (i)LCDR1, which contains the amino acid sequence of SEQ ID NO: 4;

[0240] (ii) LCDR2, which contains the amino acid sequence of SEQ ID NO: 5; and

[0241] (iii) LCDR3, which contains the amino acid sequence of SEQ ID NO: 6;

[0242] The CDR is determined by the Kabat definition scheme.

[0243] In some embodiments, the anti-Napi2b antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0244] In some embodiments, the anti-Napi2b antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0245] In some embodiments, the anti-Napi2b antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 9 and the light chain shown in SEQ ID NO: 10.

[0246] In some embodiments, the antibody-drug conjugate has the following structure:

[0247] Wherein, q and Tb are defined as described in any of the embodiments of the present invention.

[0248] In some implementations, q is any number between 1 and 12.

[0249] In some implementations, q is any number between 1 and 8.

[0250] In some implementations, q is any number between 2 and 8.

[0251] In some implementations, q is any number between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, and 7-8.

[0252] In some implementations, q is any number between 2 and 3.

[0253] In some implementations, q is any number between 3 and 4.

[0254] In some implementations, q is any number between 7 and 8.

[0255] In some implementations, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0256] In some implementations, q is 2, 4, 6, or 8.

[0257] In some implementations, q is 1, 2, or 4.

[0258] In some implementations, the antibody-drug conjugate is:

[0259] In some implementations, the antibody-drug conjugate is:

[0260] Among them, NaPi2b is the 66C12D12-hz1 antibody (see WO2024012524A1).

[0261] Drug conjugates

[0262] In a second aspect, the present invention provides a drug linker conjugate of Formula II:

[0263] Lg-LD

[0264] Formula II

[0265] Or its stereoisomers or its pharmaceutically acceptable salts;

[0266] in,

[0267] Lg is a group that reacts with antibodies;

[0268] The definitions of L and D are as described in any embodiment of this invention.

[0269] In some implementations, L is

[0270] in,

[0271] L1 is the connector unit, which covalently bonds Lg and L2;

[0272] L2 is a connecting unit, and L2 is covalently bonded to L1 and L3;

[0273] L3 may or may not be present. When L3 is present, L3 is an amino acid residue, a short peptide consisting of 2-10 amino acid residues, or... It covalently binds L2 and L4;

[0274] L4 may or may not exist. When L4 exists, L4 is a spacer unit that is covalently bonded to L3 and P.

[0275] In some implementations, Lg is hydrogen, halogen, sulfone, or a tertiary amine salt (Me3N). + Et3N + Diazonium salts, -OMs, MeSO2-, CF3SO3-, p-toluenesulfonyl groups, Or a substituted phenoxy group, wherein the substituent is a halogen or a nitro group.

[0276] In some embodiments, the substituted phenoxy group is substituted with one or two halogens; for example, it is substituted with F.

[0277] In some embodiments, Lg is F, Cl, Br, MeSO2- or pentafluorophenoxy; more preferably, Lg is MeSO2-.

[0278] In some embodiments, the drug linker conjugate is as shown in Formula II-1.

[0279] Lg-L1-L2-L3-L4-D

[0280] Formula II-1

[0281] Or its stereoisomers or its pharmaceutically acceptable salts;

[0282] in,

[0283] Lg is the group that reacts with the antibody; L1, L2, L3, L4 and D are defined as described in any of the embodiments of this invention.

[0284] In some implementations, when L1 is At that time, Lg-L1 is Two bits are connected to L2; L2, L3, L4 and D are defined as described in any of the embodiments of this invention.

[0285] In some implementations, L1 is Position 1 is connected to Lg, and position 2 is connected to L2; Lg is a halogen, sulfone, or tertiary amine group (Me3N). + Et3N + The following are not specified: Lg is F, Cl, Br, MeSO2- or pentafluorophenoxy; more preferably, Lg is MeSO2- or pentafluorophenoxy.

[0286] In some implementations, L1 is Bit 1 is connected to Lg, and bit 2 is connected to L2; Lg is MeSO2-.

[0287] In some implementations, L1 is Position 1 is connected to Lg, and position 2 is connected to L2; Lg is pentafluorophenoxy.

[0288] In some embodiments, the drug linker conjugate is as follows:

[0289] Bioactive molecules

[0290] Thirdly, the present invention provides a bioactive compound represented by Formula III:

[0291] Or its stereoisomers or pharmaceutically acceptable salts.

[0292] in,

[0293] R1 is hydrogen, methyl,

[0294] R2 is hydrogen, deuterium, or hydroxyl;

[0295] R3' is hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole), C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 ,

[0296] Ra' represents hydrogen, deuterium, -C1-6 alkylene hydroxyl group, or -NHR. 4 or -C1-6 alkylene-NHR 4 ;

[0297] R 4 It is a C1-6 alkyl group substituted with hydrogen, C1-6 alkyl (preferably C1-3 alkyl) or hydroxyl;

[0298] s can be 1, 2, 3, 4, 5, or 6;

[0299] m and n are each independently 0, 1, 2 or 3.

[0300] In some implementation schemes,

[0301] R1, R2, R3', and Ra' satisfy one of the following conditions:

[0302] (1) R1 is hydrogen or methyl; R2 is hydrogen, deuterium or hydroxyl; R3' is C2-6 alkylene hydroxyl or -C1-6 alkylene-NHR 4 , Ra' represents hydrogen, deuterium, and -NHR. 4 C1-6 alkylene hydroxyl or -C1-6 alkylene-NHR 4 ;

[0303] (2) R1 is hydrogen or methyl; R2 is hydrogen, deuterium or hydroxyl; R3' is hydrogen; Ra' is -C1-6 alkylene hydroxyl or -C1-6 alkylene-NHR 4 ;or

[0304] (3) R1 is hydrogen or methyl; R2 is hydrogen, deuterium or hydroxyl; R3' is a 5-12 heteroaryl group (preferably thiazole); Ra' is a C1-6 alkylene hydroxyl group or a -C1-6 alkylene-NHR group. 4 or -NHR 4 ;

[0305] (4) R1 is R2 is hydrogen, deuterium, or hydroxyl; R3' is hydrogen, deuterium, C1-6 alkyl (preferably C1-3 alkyl, more preferably methyl), 5-12 heteroaryl (preferably 5-6 heteroaryl, more preferably thiazole), C1-6 alkylene hydroxyl, -C1-6 alkylene-NHR 4 , Ra' represents hydrogen, deuterium, C1-6 alkylene hydroxyl group, or -NHR. 4 or -C1-6 alkylene-NHR 4 ;

[0306] R 4 It is hydrogen or C1-6 alkyl (preferably methyl);

[0307] s can be 1, 2, 3, 4, 5, or 6;

[0308] m and n are each independently 0, 1, 2 or 3.

[0309] In some implementation schemes,

[0310] R1 is hydrogen or methyl;

[0311] R2 is hydrogen, deuterium, or hydroxyl;

[0312] R3' and Ra' satisfy one of the following conditions:

[0313] (1) R3' is a C2-6 alkylene hydroxyl group, -C1-6 alkylene-NHR 4 or Ra' represents hydrogen, deuterium, and -NHR. 4 (preferably amino) or C1-6 alkyl hydroxyl;

[0314] (2) R3' is hydrogen; Ra' is -C1-6 alkylene hydroxyl or -C1-6 alkylene-NHR 4 ;or

[0315] (3) R3' is a 5-12 membered heteroaryl group (preferably thiazole); Ra' is a C1-6 alkylene hydroxyl group, -C1-6 alkylene-NHR group. 4 or -NHR 4 (Preferably amino);

[0316] R 4 It is hydrogen or C1-6 alkyl (preferably methyl);

[0317] m and n are each independently 0, 1, 2 or 3.

[0318] In some embodiments, R3' is a C2-6 alkylene hydroxyl group or Ra' represents hydrogen.

[0319] In some implementations, R3' is Ra' is a C2-3 alkylene hydroxyl or amino group.

[0320] In some embodiments, R3' is hydrogen; Ra' is a C1-6 alkylene hydroxyl group (preferably a C1-3 alkylene hydroxyl group).

[0321] In some implementations, R3' is Ra' is -NH2.

[0322] In some implementations, R1 is hydrogen or methyl; R2 is hydrogen or hydroxyl.

[0323] In some implementations, R1 is R2 is a hydroxyl group; R3' is a methyl group; Ra' is hydrogen.

[0324] In some implementations, R4 is hydrogen.

[0325] In some implementations, s is 1, 2, or 3.

[0326] In some implementations, m is 1, and n is 1, 2, or 3.

[0327] In some embodiments, the bioactive compound has the following structure:

[0328] In some embodiments, the present invention provides the use of the bioactive compound, or its stereoisomers or pharmaceutically acceptable salts, in the preparation of ADCs, wherein the bioactive compound has the following structure:

[0329] In some embodiments, the present invention provides the use of the bioactive compound, or its stereoisomers or pharmaceutically acceptable salts, in the preparation of drug linker conjugates, wherein the bioactive compound has the following structure:

[0330] Antibody-drug conjugate preparation method

[0331] In a fourth aspect, the present invention provides a method for preparing an antibody-drug conjugate of Formula I, comprising:

[0332] Tb was coupled with the drug linker conjugate Lg-L-D shown in Formula II.

[0333] Wherein, Tb, Lg, L and D are defined as described in any of the embodiments of the present invention.

[0334] Specifically, the method includes the step of coupling Tb with the drug linker conjugate Lg-L-D shown in Formula II in a solvent to form a CS bond or a CN bond.

[0335] In some preferred embodiments, the present invention provides a method for preparing an antibody-drug conjugate of Formula I, comprising:

[0336] Tb is coupled with the drug linker conjugate Lg——L1——L2——L3——L4——D shown in Formula II-1;

[0337] Wherein, Tb, Lg, L1, L2, L3, L4 and D are defined as described in any of the embodiments of the present invention.

[0338] Specifically, the method includes the step of coupling Tb with the drug linker conjugate Lg-L-L2-L3-L4-D shown in II-1 in a solvent to form CS bonds or CN bonds.

[0339] 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), or 1:(2-10).

[0340] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0341] In some embodiments, the organic solvent is one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, and nitrile compounds (e.g., acetonitrile).

[0342] In some embodiments, the method further includes a step of purifying the coupling product.

[0343] In some implementations, the coupling product is purified by chromatography.

[0344] In some embodiments, the chromatography method includes one or more of ion exchange chromatography, hydrophobic chromatography, reversed-phase chromatography, or affinity chromatography.

[0345] In some embodiments, the method is carried out at -20 to 100°C, for example 0 to 50°C, preferably at room temperature.

[0346] Preparation method of drug linker conjugate

[0347] In a fifth aspect, the present invention provides a method for preparing the drug linker conjugate, the general synthetic formula of which is as follows:

[0348] Method 1:

[0349] Wherein, Lg, L and D are defined as described in any of the embodiments of the present invention.

[0350] Specifically,

[0351] Step 1: The compound represented by general formula DL-A reacts with INT4-1 under acidic conditions to give DL-B;

[0352] The acid can be a protic acid or a Lewis acid, including but not limited to hydrochloric acid, sulfuric acid, trifluoroacetic acid, formic acid, etc.; the reaction solvent is an aprotic solvent including but not limited to N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP) or dimethyl sulfoxide (DMSO), etc.; the reaction temperature is 25-120℃.

[0353] Step 2: The compound represented by general formula DL-B is deamino protecting under alkaline conditions to obtain DL-C;

[0354] The base can be an organic base such as DBU, DIEA, diethylamine, dimethylamine, piperidine, etc., or an inorganic base such as Cs2CO3, K2CO3, or various other commonly used bases including but not limited to LDA, LiHMDS, KHMDS, NaHMDS, t-BuOK, K3PO4, etc.; the reaction solvent is selected from, but not limited to, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), or dimethyl sulfoxide (DMSO), etc.; the reaction temperature is 25-120℃.

[0355] Step 3: The compound represented by general formula DL-C and INT1 react under the conditions of a condensing agent to obtain the drug linker conjugate represented by formula II;

[0356] The condensing agent is DIC, DCC, EDCI, HATU, HBTU, HCTU, TBTU, BOP, PyBOP, DPP-Cl, DPPA, MPTA, BOP-Cl, T3P, DMTMM, etc.; the reaction solvent is selected from, but is not limited to, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), or dimethyl sulfoxide (DMSO), etc.; the reaction temperature is 25-120℃.

[0357] Specifically, taking DL-1 as an example, the target compound (DL-1) is obtained by condensing compound INT1, which is represented by the general formula, with compound INT3.

[0358] Drug linker conjugates with various structures can be obtained by reacting analogs of INT1 and INT3.

[0359] In some embodiments, the present invention provides a method for preparing the intermediate described below, the general synthetic formula of which is as follows:

[0360] Wherein, Lg, r, L1 and L2 are defined as described in any of the embodiments of the present invention;

[0361] G is selected from hydroxyl, halogen,

[0362] Specifically,

[0363] Step 1: The compound represented by general formula DL-A'-1 reacts with DL-A'-2 to give intermediate DL-A';

[0364] When G is a hydroxyl group, DL-A'-1 and DL-A'-2 react under alkaline conditions to form DL-A' through the action of a condensation reagent. The condensation reagent includes, but is not limited to, DIC, DCC, EDCI, HATU, HBTU, HCTU, TBTU, BOP, PyBOP, DPP-Cl, DPPA, MPTA, BOP-Cl, T3P, DMTMM, etc. The reaction solvent is selected from, but is not limited to, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), or dimethyl sulfoxide (DMSO), etc. The reaction temperature is 25–120℃.

[0365] When G is a halogen, When DL-A'-1 and DL-A'-2 react under alkaline conditions to generate DL-A', the alkaline base can be an organic base such as DBU, DIEA, diethylamine, dimethylamine, piperidine, etc., or an inorganic base such as Cs2CO3, K2CO3, or various other commonly used bases including but not limited to LDA, LiHMDS, KHMDS, NaHMDS, t-BuOK, K3PO4, etc.; the reaction solvent is selected from, but not limited to, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), or dimethyl sulfoxide (DMSO), etc.; the reaction temperature is 25-120℃.

[0366] Specifically, taking INT2 as an example, the target compound (INT2) is obtained by condensing the compound DL-A'-2, which is represented by the general formula, with the compound DL-A'-1.

[0367] Intermediates with various structures can be obtained by reacting analogs of DL-A'-2 and DL-A'-1.

[0368] In some embodiments, the present invention provides an intermediate of formula DL-A, or a stereoisomer of said intermediate or a pharmaceutically acceptable salt thereof:

[0369] Wherein, Lg and L are defined as described in any of the embodiments of the present invention.

[0370] In some embodiments, the intermediate represented by formula DL-A, or a stereoisomer of the intermediate, or a pharmaceutically acceptable salt thereof, is:

[0371] In some embodiments, the present invention provides an intermediate of formula DL-B, or a stereoisomer of said intermediate or a pharmaceutically acceptable salt thereof:

[0372] Wherein, Lg and L are defined as described in any of the embodiments of the present invention.

[0373] In some embodiments, the intermediate represented by formula DL-B, or a stereoisomer of the intermediate, or a pharmaceutically acceptable salt thereof, is:

[0374] In some embodiments, the present invention provides an intermediate of formula DL-C, or a stereoisomer of said intermediate or a pharmaceutically acceptable salt thereof:

[0375] Wherein, Lg and L are defined as described in any of the embodiments of the present invention.

[0376] In some embodiments, the intermediate represented by formula DL-C, or a stereoisomer of the intermediate, or a pharmaceutically acceptable salt thereof:

[0377] In some embodiments, the present invention provides an intermediate of formula DL-A'-1, or a stereoisomer of said intermediate or a pharmaceutically acceptable salt thereof:

[0378] Method 2:

[0379] in,

[0380] L3' is L3” is -NH-CH2-C(O)-;

[0381] Lg, L1, L2, L4, R L1 R L2 The definitions of D are as described in any embodiment of this invention.

[0382] Specifically, the compound shown in general formula DY-A and the compound shown in general formula DY-B react under the condition of a condensing agent to obtain the drug linker conjugate shown in formula II.

[0383] The condensing agent is DIC, DCC, EDCI, HATU, HBTU, HCTU, TBTU, BOP, PyBOP, DPP-Cl, DPPA, MPTA, BOP-Cl, T3P, DMTMM, etc.; the reaction solvent is selected from, but is not limited to, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), or dimethyl sulfoxide (DMSO), etc.; the reaction temperature is 25-120℃.

[0384] Specifically, taking DL-1 as an example, the target compound (DL-1) is obtained by condensing compound INT5, which is represented by the general formula, with compound INT7.

[0385] Drug linker conjugates with various structures can be obtained by reacting analogs of INT5 and INT7.

[0386] In some embodiments, the present invention provides an intermediate of formula DY-A, or a stereoisomer of said intermediate or a pharmaceutically acceptable salt thereof.

[0387] In some embodiments, the intermediate represented by formula DY-A, or a stereoisomer of the intermediate, or a pharmaceutically acceptable salt thereof, is:

[0388] In some embodiments, the present invention provides an intermediate of formula DY-B, or a stereoisomer of said intermediate or a pharmaceutically acceptable salt thereof.

[0389] In some embodiments, the intermediate represented by formula DY-B, or a stereoisomer of the intermediate, or a pharmaceutically acceptable salt thereof, is:

[0390] In some embodiments, the present invention provides the use of the intermediate, or a stereoisomer of the intermediate, or a pharmaceutically acceptable salt thereof, in the preparation of drug linker conjugates.

[0391] In some embodiments, the present invention provides the use of the intermediate, or a stereoisomer of the intermediate, or a pharmaceutically acceptable salt thereof, in the preparation of antibody-drug conjugates.

[0392] Coupled Groups

[0393] In a sixth aspect, the present invention provides a group of antibody-drug conjugates comprising the antibody-drug conjugates described above, their stereoisomers or pharmaceutically acceptable salts or compositions thereof, wherein the antibody-drug conjugates have one, two or more q values.

[0394] In some implementations, when a q-value of an antibody-drug conjugate accounts for the majority (e.g., 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99%) of the antibody-drug conjugate group, the q-value and the average DAR are close.

[0395] In some implementations, when there is only one q-value antibody-drug conjugate in the said antibody-drug conjugate group, the q-value and the average DAR are equal.

[0396] In some embodiments, when the antibody-drug conjugates of the antibody-drug conjugate group have two or more q values, the proportion of the antibody-drug conjugate with one particular q value among all antibody-drug conjugates in the composition is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0397] In some embodiments, the average drug-to-antibody ratio (average DAR) in the antibody-drug conjugate group is an integer or fractional number between 1 and 12, preferably an integer or fractional number between 1 and 10.

[0398] In some embodiments, the average drug-to-antibody ratio (average DAR) of the antibody-drug conjugate group is 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5;

[0399] In some embodiments, the average drug-to-antibody ratio (average DAR) of the antibody-drug conjugate group is about 2.0, 4.0, 6.0, or 8.0;

[0400] In some embodiments, the average drug-to-antibody ratio (average DAR) in the antibody-drug conjugate group is 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.

[0401] In some embodiments, the antibody-drug conjugate group contains ADCs with a DAR distribution of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., drug loadings of 1.5, 2, 4, 6, and 8). It is noteworthy that degradation products can be generated, such that the mixture may also contain DARs of 1, 3, 5, and 7. Furthermore, the antibody-drug conjugate group may also have an average DAR greater than 8. The antibody-drug conjugates are produced by reduction and subsequent coupling of interchain disulfides. In some embodiments, the antibody-drug conjugates comprise both: antibody-drug conjugates with a DAR of 4 or lower (i.e., drug loadings of 4 or lower) and antibody-drug conjugates with a DAR of 6 or higher (i.e., drug loadings of 6 or higher).

[0402] Pharmaceutical Composition

[0403] In a seventh aspect, the present invention provides a pharmaceutical composition comprising, as described above, a drug conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a drug linker conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or the aforementioned group of antibody-drug conjugates and optionally one or more pharmaceutical excipients.

[0404] The term "drug-to-antibody ratio" or "DAR" refers to the amount of drug, such as a small molecule toxin attached to an antibody in 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 binding sites on the antibody. The term DAR may 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 contains an ADC with a DAR distribution of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., drug loadings of 1.5, 2, 4, 6, and 8). Notably, degradation products can be generated, such that the ligand-drug conjugate may also contain DARs of 1, 3, 5, and 7. Furthermore, the ADC may also have a DAR greater than 8. The ADC is produced by reduction and subsequent coupling of an interchain disulfide. In some implementations, the ADC comprises both of the following: the ADC with a DAR of 4 or lower (i.e., a drug class of 4 or lower) and the ADC with a DAR of 6 or higher (i.e., a drug class of 6 or higher).

[0405] use

[0406] Eighthly, the present invention provides the use of the aforementioned antibody-drug conjugates, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or drug linker conjugates, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or the aforementioned group of antibody-drug conjugates; or the aforementioned pharmaceutical compositions in the preparation of medicaments for treating and / or preventing diseases (e.g., cancer) associated with abnormal cellular activity.

[0407] The present invention provides the aforementioned antibody-drug conjugates, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or the aforementioned drug linker conjugates, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or the aforementioned antibody-drug conjugate groups; or the aforementioned pharmaceutical compositions for the treatment and / or prevention of diseases (e.g., cancer) associated with abnormal cellular activity.

[0408] The present invention provides a method for preventing and / or treating diseases (e.g., cancer) associated with abnormal cellular activity, comprising: administering to an individual in need a preventive and / or therapeutically effective amount of the aforementioned antibody-drug conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a drug-linked conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a group of the aforementioned antibody-drug conjugates; or a pharmaceutical composition thereof.

[0409] In some embodiments, the cancers described in this invention are esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumors, 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.

[0410] In some embodiments, the cancers described in this invention are ovarian cancer, breast cancer, gastric cancer, lung cancer, colorectal cancer, and esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma).

[0411] In some implementations, the breast cancer is ductal carcinoma of the breast.

[0412] In some implementations, the lung cancer is lung adenocarcinoma.

[0413] In some implementations, the cancer is a cancer associated with targets such as HER2, TROP2, and Napi2b.

[0414] In some implementations, the cancer is a solid tumor or a hematologic malignancy.

[0415] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0416] The reagents and raw materials used in this invention are all commercially available.

[0417] The beneficial effects of this invention are:

[0418] Through extensive research, this invention has developed an antibody-drug conjugate using olistatin-like bioactive compounds as the payload, which can achieve one or more of the following effects:

[0419] (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 that link similar small molecule drugs in the prior art.

[0420] (2) Antibody-drug conjugates have high stability in circulation, which can reduce the shedding of bioactive molecules in non-target cells and increase the effective release of bioactive compounds into cells, thus having a higher therapeutic index.

[0421] (3) Antibody-drug conjugates use linkers that cleave in the tumor microenvironment (both inside and outside tumor cells), thus producing good anti-tumor effects in tumors with low antigen expression or no antigen expression.

[0422] (4) By improving the hydrophilicity of the linker, the overall physicochemical properties of the 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 accumulate in the tumor microenvironment, which increases the ratio of the concentration of bioactive molecules in the tumor to the blood, and reduces the mechanism-related toxicity of the ADC molecule (the toxicity produced after the ADC binds to cell surface antigens in non-tumor tissues and endocytoses them, or "on-target toxicity"), thus having a higher therapeutic index.

[0423] (5) The bioactive compound (payload) used in antibody-drug conjugates has good liver microsomal stability and can be rapidly metabolized in vivo, which can reduce the toxicity caused by exposure of bioactive compounds in the circulatory system and normal tissues, thereby improving the overall safety of antibody-drug conjugates.

[0424] (6) The bioactive molecules of antibody-drug conjugates have higher anti-tumor cell activity and therefore have excellent by-stander effect. ADCs can more effectively kill tumor cells with high antigen expression as well as tumor cells with low antigen expression or no antigen expression in tumor tissue.

[0425] Therefore, the antibody-drug conjugate provided by this invention has high clinical application value. Detailed Implementation

[0426] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0427] The abbreviations used in this invention have the following meanings:

[0428] Table 1

[0429] Preparation scheme

[0430] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0431] Nuclear magnetic resonance (NMR) 1 The H NMR (H2N) assay was performed using a Bruker 400MHz NMR spectrometer; the assay solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0432] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0433] 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: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0434] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0435] The ultra-high performance liquid chromatography (UPLC) instrument used was AB SCIEX, model ExionLC.

[0436] The high-resolution mass spectrometer used for the measurements was an AB SCIEX X500B.

[0437] Methods for determining the antibody-drug conjugate ratio (DAR value):

[0438] Sample preparation: Take 200 μg of ADC sample, dilute it with ultrapure water to 0.2 mg / ml, then add 10 μl of 1 mol / L DTT, react at 37℃ for 30 min, and then directly inject the sample for determination.

[0439] Table 2 Liquid phase parameters:

[0440] Table 3 Mass Spectrometry Parameters:

[0441] Example 1. Synthesis of intermediates

[0442] Example 1.1: Synthesis of INT1

[0443] 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), and stir at room temperature for 3 h. Concentrate the reaction solution under reduced pressure to obtain the hydrochloride salt of the target compound INT1-2 (0.83 g), which is directly used in the next step of the reaction.

[0444] LCMS(ESI)[M+H] + =278.31.

[0445] Step 2: 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) were dissolved in DMF (2 mL), and DIPEA (992.56 mg, 7.68 mmol) was added. The mixture was stirred at room temperature for 16 h. The reaction solution was purified by reversed-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).

[0446] LCMS(ESI)[M+Na] + =711.79.

[0447] 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).

[0448] Step 3: Compound INT1-4 (1.4 g, 2.03 mmol) and palladium on carbon (212.84 mg, 10%) were dissolved in DCM (12 mL) and MeOH (3 mL). The mixture was purged with hydrogen three times, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT1 (1.1 g).

[0449] LCMS(ESI)[M+H] + =599.63.

[0450] 1 H 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).

[0451] Example 1.2: Synthesis of INT2

[0452] Step 1:

[0453] Compounds INT2-1 (2.0 g, 4.28 mmol) and INT2-2 (8.57 g, 34.24 mmol) were dissolved in MeOH (30 mL), followed by the addition of sodium cyanoborohydride (2.15 g, 34.24 mmol). After the addition was complete, the mixture was stirred at 50 °C for 2 h. The reaction solution was quenched with saturated ammonium chloride aqueous solution and purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-40%) to obtain the target compound INT2-3 (2.3 g).

[0454] LCMS(ESI)[M+H] + =936.59.

[0455] Step Two:

[0456] Compounds INT2-3 (2.3 g, 2.46 mmol) and INT2-4 (0.55 g, 2.95 mmol) were dissolved in DMF (30 mL), cooled to -15 °C, and then DMTMM (0.82 g, 2.95 mmol) was added. After the addition was complete, the mixture was stirred at -15 °C for 3 h. The reaction solution was then purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-50%) to obtain the target compound INT2-5 (1.6 g).

[0457] LCMS(ESI)[M+H] + =1104.66.

[0458] 1 H NMR (400MHz, DMSO-d6) δ8.91-8.73(m,1H),8.38-8.25(m,1H),8.13-8.06(m,1H),7.91-7.87(m,2 H),7.80-7.67(m,2H),7.46-7.39(m,3H),7.35-7.30(m,2H),4.76-4.59(m,2H),4.34-4.17(m,4H ),4.05-3.95(m,2H),3.94-3.68(m,4H),3.67-3.57(m,3H),3.55-3.47(m,30H),3.45-3.39(m,6H ),3.24-3.20(m,6H),2.05-1.91(m,1H),1.73-1.45(m,4H),1.41-1.17(m,4H),0.91-0.83(m,6H).

[0459] Step 3:

[0460] Compound INT2-5 (1.6 g, 1.45 mmol) was dissolved in DMF (15 mL) and diethylamine (0.75 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was then distilled twice with THF (20 mL * 2) to remove the diethylamine, yielding a DMF solution of the target compound INT2-6 (1.28 g), which was directly used in the next reaction step.

[0461] LCMS(ESI)[M+H] + =882.62.

[0462] Step Four:

[0463] To a DMF (15 mL) solution of compound INT2-6 (1.28 g, 1.45 mmol), compounds INT2-7 (0.47 g, 1.74 mmol), HATU (0.66 g, 1.74 mmol), and DIPEA (0.56 g, 4.35 mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was then purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-50%) to obtain the target compound INT2 (1.10 g).

[0464] LCMS(ESI)[M+H] + =1132.60.

[0465] 1 H NMR (400MHz, DMSO-d6) δ9.17-9.15(m,2H),8.88(t,J=6.8Hz,1H),8.29(t,J=5.7Hz,1H),8.18(d,J=7.4Hz,1H ),8.01(d,J=8.5Hz,1H),4.72–4.67(m,1H),4.62-4.56(m,1H),4.31-4.15(m,2H),4.09-3.98(m,2H),3.85-3 .75(m,6H),3.61-3.52(m,34H),3.46-3.44(m,6H),3.26(s,6H),3.19-3.06(m,2H),2.64-2.56(m,2H),2.50- 2.34(m,2H),2.06-1.93(m,1H),1.91-1.80(m,2H),1.77-1.64(m,3H),1.40-1.28(m,2H),0.91-0.83(m,6H).

[0466] Example 1.3: Synthesis of INT3

[0467] Step 1:

[0468] Compounds INT3-1 (100 mg, 0.27 mmol) and INT2 (420.06 mg, 0.54 mmol) were dissolved in HCl-DMF (2 mL, 2 M) and reacted with the solution at room temperature for 3 h. The reaction solution was then purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-40%) to obtain the target compound INT3-2 (120.0 mg).

[0469] LCMS(ESI)[M+H] + =1405.72.

[0470] Step Two:

[0471] Compound INT3-2 (120 mg, 0.085 mmol) was dissolved in DMF (1 mL) and diethylamine (0.05 mL) and the mixture was stirred at room temperature for 1 h. The reaction solution was then purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-30%) to obtain the target compound INT3 (40 mg).

[0472] LCMS(ESI)[M+H] + =1183.80.

[0473] Example 1.4: Synthesis of INT4

[0474] Step 1: Dissolve compound INT4-1 (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), then add compound tert-butyl bromoacetate (34.78 g, 178.32 mmol), and react overnight at room temperature. Add water (80 mL) to the reaction solution, then extract three times with ethyl acetate (80 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and evaporate to dryness to obtain the target compound INT4-2 (10.2 g).

[0475] 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).

[0476] Step 2: Compound INT4-2 (10 g, 50.5 mmol) was dissolved in tetrahydrofuran (100 mL), and INT4-3 (10.35 g, 50.5 mmol), bis(triphenylphosphine)palladium dichloride (3.5 g, 5.05 mmol), cuprous iodide (1.92 g, 10.1 mmol), and triethylamine (15.3 g, 151.5 mmol) were added. The mixture was stirred overnight at 70 °C under a nitrogen atmosphere. The reaction solution was cooled and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain the target compound INT4-4 (5.2 g).

[0477] LCMS(ESI)[M+H] + =323.0;

[0478] 1H 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).

[0479] Step 3: Dissolve compound INT4-4 (1.0 g, 3.1 mmol) in dichloromethane (50 mL), add trifluoroacetic acid (3.54 g, 31 mmol), and stir overnight at room temperature. The reaction solution is then concentrated under reduced pressure to obtain compound INT4-5 (1.5 g).

[0480] LCMS(ESI)[M+H] + =267.0;

[0481] 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).

[0482] Step 4: Dissolve compound INT4-5 (1.5 g, 5.6 mmol) in tetrahydrofuran (20 mL) and water (20 mL), add potassium peroxide monosulfonate (10.3 g, 16.8 mmol), stir at room temperature for 2 h, concentrate the reaction solution under reduced pressure to remove tetrahydrofuran, extract the aqueous phase three times with ethyl acetate (50 mL), combine the organic phases, dry with anhydrous sodium sulfate, concentrate the organic phase under reduced pressure, and purify the crude product by reversed-phase chromatography (acetonitrile: H2O containing 0.05% HCl = 5%-50%) to obtain the target compound INT4 (0.4 g).

[0483] LCMS(ESI)[M+H] + =299.1;

[0484] 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).

[0485] Example 1.5: Synthesis of INT5

[0486] Step 1: Compound D-1 (400 mg, 0.55 mmol) and INT5-1 (1013.04 mg, 2.75 mmol) were dissolved in 2M HCl-DMF (1.375 mL, 2.75 mmol), and the mixture was stirred at room temperature for 1 h under nitrogen protection. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-50%) to obtain the trifluoroacetate of the target compound INT5-2 (200 mg).

[0487] LCMS(ESI)[M+H] + =1040.62.

[0488] 1 H NMR(400MHz,DMSO-d6)δ8.74-8.70(m,1H),8.27(s,1H),8.07-7.95(m,1H),7.91-7.89(m,2H),7.73-7.71(m,2H),7.62-7.58(m,1H ),7.44-7.41(m,2H),7.35-7.31(m,2H),7.24-7.08(m,5H),4.68-4.52(m,4H),4.30-4.20(m,4H),3.99-3.95(m,1H),3.81-3.56(m, 5H),3.24-3.16(m,9H),3.09(s,2H),3.00(s,2H),2.94-2.74(m,2H),2.70-2.55(m,3H),2.41-2.34(m,1H),2.26-2.12(m,7H),1.9 7-1.90(m,2H),1.85-1.65(m,3H),1.50-1.35(m,1H),1.35-1.23(m,2H),1.05-1.01(m,3H),0.95-0.84(m,12H),0.77-0.70(m,5H).

[0489] Step 2: Dissolve compound INT5-2 (200 mg, 0.19 mmol) in DMF (2 mL) and diethylamine (200 μL), stir at room temperature for 1 h, and purify the reaction solution directly by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.1% FA = 10%-50%) to obtain the formate of the target compound INT5 (53.0 mg).

[0490] LCMS(ESI)[M+H] + =818.64.

[0491] 1H NMR (400MHz, DMSO-d6) δ8.84(t,J=6.4Hz,1H),8.28(s,2H),8.06(d,J=8.7Hz,1H),7.98(d,J=8.7Hz, 1H),7.74(d,J=8.1Hz,1H),7.28–7.11(m,5H),4.78–4.51(m,4H),4.30–3.96(m,3H),3.89–3.75(m,3H ),3.73(s,1H),3.28–3.16(m,9H),3.13–2.79(m,6H),2.59–2.69(m,2H),2.35–2.47(m,1H),2.33–2. 11(m,8H),2.09–1.88(m,2H),1.88–1.63(m,3H),1.42–1.53(m,1H),1.32(s,1H),1.11–0.69(m,22H).

[0492] Example 1.6: Synthesis of INT6

[0493] Step 1:

[0494] Compounds D-2 (16 mg, 0.021 mmol) and INT5-1 (7.74 mg, 0.021 mmol) were dissolved in DMF (0.5 mL). 4 M HCl / DMF (0.5 mL) was added with stirring at room temperature. After the addition was complete, the mixture was stirred at room temperature for 16 h, and the reaction was monitored by LCMS. The reaction solution was directly purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the formate of the target compound INT6-1 (10 mg).

[0495] LCMS(ESI)[M / 2+H] + =528.26.

[0496] Step Two:

[0497] Compound INT6-1 (10 mg, 0.0095 mmol) was dissolved in DMF (2 mL), and diethylamine (0.2 mL) was added with stirring at room temperature. After the addition was complete, the reaction was continued with stirring at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the formate of the target compound INT6 (6.1 mg).

[0498] LCMS(ESI)[M / 2+H] + =417.75.

[0499] 1H NMR(400MHz,DMSO-d6)δ9.11–9.00(m,1H),8.93–8.73(m,1H),8.10–7.97(m,2H),7.88–7.66(m,1H),7.25– 7.10(m,5H),4.80–4.34(m,5H),4.15–3.96(m,2H),3.84–3.76(m,1H),3.62–3.56(m,2H),3.27–3.15(m,6H ),3.13–3.07(m,3H),3.02–2.99(m,1H),2.82–2.57(m,8H),2.47–2.19(m,3H),2.17–1.94(m,3H),1.88–1. 62(m,4H),1.57–1.44(m,3H),1.33–1.22(m,3H),1.20–1.13(m,4H),1.11–1.02(m,3H),0.99–0.73(m,18H).

[0500] Example 1.7: Synthesis of INT7

[0501] Step 1:

[0502] Compound INT2-3 (200 mg, 0.21 mmol) was dissolved in DMF (2 mL) and diethylamine (0.2 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was then distilled twice with THF (10 mL * 2) to remove the diethylamine, yielding a DMF solution of the target compound INT7-1 (152.52 mg), which was directly used in the next reaction step.

[0503] LCMS(ESI)[M+H] + =714.5.

[0504] Step Two:

[0505] Compound INT2-7 (56.23 mg, 0.21 mmol) was dissolved in DMF (8 mL), followed by the addition of HATU (121.6 mg, 0.32 mmol) and triethylamine (32.32 mg, 0.32 mmol). After stirring at room temperature for 20 min, compound INT7-1 (152.52 mg, 0.21 mmol) was added, and stirring was continued at room temperature for another 30 min. After the reaction was completed as detected by LCMS, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile and 0.05% formic acid aqueous solution) to obtain the target compound INT7 (100 mg).

[0506] LCMS(ESI)[M+H] + =964.5.

[0507] Example 1.8: Synthesis of D-1

[0508] Step 1: 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) were dissolved in DMF (1 mL), and then DIPEA (65.91 mg, 0.51 mmol) was added. The mixture was stirred at room temperature for 16 h. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain trifluoroacetate of the target compound D-1 (87.0 mg).

[0509] LCMS(ESI)[M+H] + =732.71.

[0510] 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).

[0511] Example 1.9: Synthesis of D-2

[0512] Step 1: Compound D-2-1 (2.16 g, 7.73 mmol) was dissolved in anhydrous THF (15 mL), cooled to 0 °C, and N-methylmorpholine (1.02 mL, 9.28 mmol) and methyl chloroformate (0.719 mL, 9.28 mmol) were added sequentially. The mixture was stirred at 0 °C for 1 h. After filtration, the filter cake was washed with anhydrous THF (10 mL). The filtrate was cooled to 0 °C, and an aqueous solution of NaBH4 (380 mg, 10.1 mmol) (4 mL) was added. The mixture was stirred for another 15 min, then allowed to return to room temperature and stirred for another 30 min. The reaction mixture was filtered through a Buchner funnel, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-2-2 (1.07 g).

[0513] LCMS(ESI)[M+H-100] + =166.26.

[0514] 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).

[0515] 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. Concentrate the reaction solution under reduced pressure to obtain the hydrochloride salt of the target compound D-2-3 (370 mg), which is directly used in the next step of the reaction.

[0516] LCMS(ESI)[M+H] + =166.31.

[0517] Step 3: Dissolve compounds 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 reaction mixture at room temperature for 1 h. The reaction solution is purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain trifluoroacetate of the target compound D-2 (25 mg).

[0518] LCMS(ESI)[M+H] +=746.78.

[0519] 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).

[0520] Example 2. Synthesis of drug linker conjugates

[0521] Example 2.1: Synthesis Method of DL-1

[0522] Step 1:

[0523] Compounds INT3 (40.0 mg, 0.034 mmol), INT1 (20.07 mg, 0.034 mmol), and HATU (15.51 mg, 0.041 mmol) were added to DMF (1 mL), followed by the addition of DIPEA (13.18 mg, 0.10 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was then purified directly by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound DL-1 (17.35 mg).

[0524] LCMS(ESI)[M / 2+H] + =882.41.

[0525] 1H NMR(400MHz,DMSO-d6)δ9.14(s,2H),8.86(s,1H),8.71-8.61(m,1H),8.20-8.11(m,2H),8.02-7.92(m,1.5H),7.80-7.72(m,0 .5H),7.24-7.16(m,5H),4.69-4.44(m,5H),4.31-3.97(m,5H),3.79-3.74(m,6H),3.61-3.51(m,34H),3.46-3.43(m,8H),3.27 -3.26(m,6H),3.23-3.22(m,2H),3.19(s,1H),3.14-3.10(m,3H),3.02(s,2H),2.93-2.68(m,9H),2.67-2.56(m,4H),2.49-2.3 2(m,4H),2.29-1.97(m,5H),1.91-1.56(m,10H),1.41-1.28(m,4H),1.08-1.03(m,3H),1.00-0.85(m,24H),0.82-0.77(m,3H).

[0526] Example 2.2: Method Two for Synthesizing DL-1

[0527] Step 1:

[0528] Compounds INT5 (42.54 mg, 0.052 mmol) and INT7 (50 mg, 0.052 mmol) were dissolved in DMF (2 mL), cooled to -15 °C, and then DMTMM (23 mg, 0.078 mmol) was added. After the addition was complete, the mixture was stirred at -15 °C for 3 h. The reaction solution was then purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-50%) to obtain the target compound DL-1 (30 mg).

[0529] LCMS(ESI)[M / 2+H] + =882.41.

[0530] 1H NMR(400MHz,DMSO-d6)δ9.14(s,2H),8.86(s,1H),8.71-8.61(m,1H),8.20-8.11(m,2H),8.02-7.92(m,1.5H),7.80-7.72(m,0 .5H),7.24-7.16(m,5H),4.69-4.44(m,5H),4.31-3.97(m,5H),3.79-3.74(m,6H),3.61-3.51(m,34H),3.46-3.43(m,8H),3.27 -3.26(m,6H),3.23-3.22(m,2H),3.19(s,1H),3.14-3.10(m,3H),3.02(s,2H),2.93-2.68(m,9H),2.67-2.56(m,4H),2.49-2.3 2(m,4H),2.29-1.97(m,5H),1.91-1.56(m,10H),1.41-1.28(m,4H),1.08-1.03(m,3H),1.00-0.85(m,24H),0.82-0.77(m,3H).

[0531] Example 2.3: Synthesis of DL-2

[0532] Compound DL-2 is commercially available and was purchased from Shanghai Haohong Biomedical Technology Co., Ltd., product number: 1039030, batch number: Lg0515249860.

[0533] Example 3. Preparation of Antibody

[0534] The preparation method for anti-NaPi2b antibody is as described in WO2024012524A1, specifically the preparation of the 66C12D12-hz1 antibody. The antibody sequence and its specific information are as follows:

[0535] Example 4. Synthesis of Antibody-Drug Conjugates (ADCs)

[0536] Example 4.1 Preparation of NaPi2b-ADC

[0537] Example 4.1.1 Preparation of NaPi2b-ADC-001

[0538] Take 1.5 mL of anti-NaPi2b antibody, dilute with 0.015 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), adjust the pH to 7.5 with 0.5 M Na2HPO4 solution, add 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0241 mL, 0.482 μmol, 2.2 times the molar amount of antibody) solution to the above solution, mix well, and incubate at 25 °C for 90 min. Then add DL-1 (1.318 mg, 5.0 times the molar amount of antibody) in dimethyl sulfoxide (0.111 mL), mix well, and incubate at 25 °C for 2 h. After completion, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to replace the buffer solution with 20 mM His-HCl, pH 5.9 buffer solution. The conjugate product of DL-1 and anti-NaPi2b antibody, NaPi2b-ADC-001, is obtained. The DAR value determined by mass spectrometry was 4.1.

[0539] Table 4 shows the results of the DAR value measurement:

[0540] The DAR value of anti-NaPi2b antibody was determined by measuring the light chain conjugates with 0-1 drug linkers (LC, DAR1 ratio was 51%, 49% respectively) and the heavy chain conjugates with 0-3 drug linkers (HC, DAR1, DAR2, DAR3 ratio was 16%, 31%, 36%, 18% respectively). Based on this, the DAR value of NaPi2b-ADC-001 was calculated to be 4.1.

[0541] Example 4.1.2 Preparation of NaPi2b-ADC-002

[0542] Following a procedure similar to that in Example 4.1.1, DL-1 (2.901 mg, 11 times the molar amount of antibody) was added to obtain the conjugate product NaPi2b-ADC-002 of DL-1 and anti-NaPi2b antibody. The DAR value was determined to be 8.0 by mass spectrometry.

[0543] Table 5 shows the results of the DAR value measurement:

[0544] The DAR value of anti-NaPi2b antibody was determined by measuring the light chain conjugates with 0-1 drug linkers (LC, DAR1 ratio was 4%, 96% respectively) and the heavy chain conjugates with 0-4 drug linkers (HC, DAR1, DAR2, DAR3, DAR4 ratio was 0%, 0%, 0%, 96%, 4% respectively). The DAR value of NaPi2b-ADC-002 was calculated to be 8.0.

[0545] Example 4.1.3: Preparation of NaPi2b-ADC-003

[0546] Following a procedure similar to that in Example 4.1.1, DL-2 (1.501 mg, 6 times the molar amount of antibody) was added to obtain the conjugate product NaPi2b-ADC-003 of DL-2 and anti-NaPi2b antibody. The DAR value was determined to be 4.7 by mass spectrometry.

[0547] Table 6 shows the results of the DAR value measurement:

[0548] The DAR value of anti-NaPi2b antibody was determined by measuring the light chain conjugates with 0-2 drug linkers (LC, DAR1, DAR2 ratios were 38%, 59%, and 3%, respectively) and the heavy chain conjugates with 0-4 drug linkers (HC, DAR1, DAR2, DAR3, DAR4 ratios were 12%, 34%, 30%, 19%, and 4%, respectively). Based on this, the DAR value of NaPi2b-ADC-003 was calculated to be 4.7.

[0549] Example 4.1.4: Preparation of NaPi2b-ADC-004

[0550] Following a procedure similar to that in Example 4.1.1, DL-2 (1.925 mg, 11 times the molar amount of antibody) was added to obtain the conjugate product NaPi2b-ADC-004 of DL-2 and anti-NaPi2b antibody. The DAR value was determined to be 8.0 by mass spectrometry.

[0551] Table 7 shows the results of the DAR value measurement:

[0552] The DAR value of anti-NaPi2b antibody was determined by measuring the light chain conjugates with 0-1 drug linkers (LC, DAR1 ratio 0%, 100%) and the heavy chain conjugates with 0-3 drug linkers (HC, DAR1, DAR2, DAR3 ratio 0%, 0%, 0%, 100%). The DAR value of NaPi2b-ADC-004 was calculated to be 8.0.

[0553] Example 4.1.5: Preparation of NaPi2b-ADC-005

[0554] Take 1.5 mL of anti-NaPi2b antibody (21.5 mg / mL), dilute with 0.015 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), adjust the pH to 7.5 with 0.5 M Na2HPO4 solution, add 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.012 mL, 0.24 μmol, 1.1 times the molar amount of antibody) solution to the above solution, mix well, and incubate at 25 °C for 120 min. Then add DL-1 (1.093 mg, 2.2 times the molar amount of antibody) in dimethyl sulfoxide (0.0549 mL), mix well, and incubate at 25 °C for 2 h. After completion, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to replace the buffer solution with 20 mM His-HCl, pH 5.9 buffer solution. The conjugate product NaPi2b-ADC-005 of DL-1 and anti-NaPi2b antibody was obtained. The DAR value was determined by mass spectrometry to be 2.3.

[0555] Table 8 shows the results of the DAR value measurement:

[0556] The DAR value of anti-NaPi2b antibody was determined by measuring the light chain conjugates with 0-1 drug linkers (LC, DAR1 ratio was 57%, 43%) and the heavy chain conjugates with 0-3 drug linkers (HC, DAR1, DAR2, DAR3 ratio was 47%, 35%, 15%, 2%). Based on this, the DAR value of NaPi2b-ADC-005 was calculated to be 2.3.

[0557] Example 4.2: Preparation of Her2-ADC

[0558] Example 4.2.1: Preparation of Her2-ADC-001

[0559] Take 0.9 mL of Trastuzumab antibody (20.3 mg / mL), dilute with 0.009 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), and adjust the pH to 7.5 with 0.5 M Na2HPO4 solution. Add 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0154 mL, 0.308 μmol, 2.5 times the molar amount of antibody) solution to the above solution and mix well. Incubate at 25 °C for 120 min. Then add DL-1 (1.680 mg, 6 times the molar amount of antibody) in dimethyl sulfoxide (0.0844 mL), mix well, and incubate at 25 °C for 2 h. After completion, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to replace the buffer solution with 20 mM His-HCl, pH 5.9 buffer solution. Her2-ADC-001, a conjugate of DL-1 and Trastuzumab antibody, was obtained. The DAR value was determined to be 4.3 by mass spectrometry.

[0560] Table 9 shows the results of the DAR value measurement:

[0561] The DAR value of Her2-ADC-001 was calculated to be 4.3 based on the determination of the light chain conjugates with 0-1 drug linkers (LC, DAR1 ratio was 51%, 49%, and 3%, respectively) and the heavy chain conjugates with 0-3 drug linkers (HC, DAR1, DAR2, and DAR3 ratio was 11%, 31%, 41%, and 17%, respectively).

[0562] Example 4.2.2: Preparation of Her2-ADC-002

[0563] Following a procedure similar to that in Example 4.2.1, DL-2 (0.977 mg, 6 times the molar amount of antibody) was added to obtain the conjugate Her2-ADC-002 of DL-2 and anti-Trastuzumab antibody. The DAR value was determined to be 4.5 by mass spectrometry.

[0564] Table 10 shows the results of DAR value measurement:

[0565] The DAR value of Her2-ADC-002 was calculated to be 4.5 based on the determination of the light chain conjugates with 0-2 drug linkers (LC, DAR1, DAR2 ratios were 42%, 56%, and 2%, respectively) and the heavy chain conjugates with 0-4 drug linkers (HC, DAR1, DAR2, DAR3, and DAR4 ratios were 10%, 38%, 33%, 16%, and 3%, respectively).

[0566] Example 5. Cell activity test of bioactive compounds

[0567] Example 5.1 Inhibitory activity test of bioactive compound on in vitro cells (SK-BR-3)

[0568] 1. Experimental Method:

[0569] SK-BR-3 cells (McCoy's 5A + 10% FBS + 1% P / S) were revived and seeded at a density of 5000 cells / well and 142.5 μL / well in 96-well plates, and incubated overnight at 37°C. The next day, the bioactive compound was serially diluted to 400× with DMSO, then further diluted to 20× with serum-free medium, resulting in a maximum concentration of 1000 nM. Nine concentration points were obtained through 4-fold dilutions. 7.5 μL of the diluted bioactive compound sample (20×) was added to each cell culture well. After culturing at 37°C for 4 days, 50 μL of Cell titer glo substrate was added to each well, and chemiluminescence values ​​were measured after 10 minutes of incubation. The sources of key materials are shown in Table 11 below.

[0570] Table 11 Material Sources

[0571] 2. Experimental Results:

[0572] As shown in Table 12, the bioactive compounds of the present invention have significant inhibitory activity on the proliferation of SK-BR-3 tumor cell lines.

[0573] Table 12 Inhibitory activity of bioactive compounds against the in vitro proliferation of SK-BR-3 tumor cells.

[0574] Example 5.2 Inhibitory activity test of bioactive compounds on in vitro cells (IGR-OV1, RMG-1, TOV-21G, SK-OV-3)

[0575] 1. Experimental Method:

[0576] IGR-OV1 cells were resuscitated and cultured using 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 seeded into 96-well flat-bottom plates (Beyotime, catalog number FCP963-48pcs) at the density shown in the table below and incubated overnight at 37°C. The cell line source and seeding details are shown in Table 13 below.

[0577] Table 13 Cell line origin and plating status

[0578] The small molecule to be tested was serially diluted to 1000× with DMSO, and then further diluted to 10× with serum-free medium to achieve a final concentration of 500 nM. Nine concentration points were obtained by 4-fold dilution. 15 μL of 10× diluent was added to each well.

[0579] After adding the analyte, the 96-well plate was incubated at 37°C and 5% CO2 for 6 days. After incubation, the plate was allowed to equilibrate to room temperature for 20 minutes. 75 μL of the analyte was then added to each well. Reagent (ADamas Life, catalog number: RA-GL11-A). Detect chemiluminescence value after incubation for 10 minutes.

[0580] 2. Experimental Results:

[0581] As shown in Table 14, the bioactive compounds of the present invention exhibit significant inhibitory activity on the proliferation of four tumor cell lines.

[0582] Table 14. Inhibitory activity of bioactive compounds against the in vitro proliferation of four tumor cells.

[0583] Example 5.3 Stability test of bioactive compounds in liver microsomes

[0584] 1. Experimental Method:

[0585] Liver microsomes from rats, mice, monkeys, and humans were mixed with the test compound and placed in a 37°C water bath. A pre-incubated NADPH solution was added to initiate the reaction. The final incubation system contained 2 μM of the test compound, 0.5 mg / mL of liver microsomes, and 1 mM of NADPH. At 0 min, 5 min, 10 min, 30 min, 60 min, and 120 min, 20 μL of the incubation solution was collected 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 the concentration of each test compound was analyzed by LC-MS / MS. Liver clearance was calculated based on the compound concentrations at different time points.

[0586] 2. Experimental Results:

[0587] Test results show that the bioactive compounds of the present invention, such as D1, have high clearance rates in the livers of four different species. The antibody-drug conjugates formed therefrom can be rapidly metabolized and cleared when the bioactive compound (payload) is exposed to the circulatory system and normal tissues, thereby reducing toxicity and improving the overall safety of the antibody-drug conjugate.

[0588] Example 6. In vitro inhibitory activity assay of antibody-drug conjugates (ADCs)

[0589] Example 6.1: Inhibitory activity test of ADC on in vitro cells (OVAR3, IGR-OV1, RMG-1)

[0590] 1. Experimental Method:

[0591] OVCAR-3 cells (RPMI 1640 (ADamas Life, catalog number: C8016) + 20% FBS + 1% P / S + 10 μg / ml insulin), IGR-OV1 cells (RPMI 1640 (Gibco, catalog number: C22400500CP) + 10% FBS + 1% P / S), and RMG-1 cells (Ham's F-12 (Invitrogen, catalog number: 11765054) + 10% FBS + 1% P / S) were resuscitated and cultured. After the cells stabilized, they were seeded into 96-well plates (Beyotime, catalog number FCP963-48pcs) at the density shown in the table below and incubated overnight at 37°C. The cell line sources and seeding arrangements are shown in Table 15 below.

[0592] Table 15 Cell line origin and plating status

[0593] The following day, the test ADCs on OVCAR-3 cells were serially diluted 20-fold (20×) to their final concentration using serum-free medium, resulting in a maximum final concentration of 100 nM. This was repeated 4-fold for a total of 9 concentration points. 7.5 μL of 20× diluent was added to each well.

[0594] The ADCs on the remaining two cells were serially diluted 10-fold (10×) to their final concentration using serum-free medium, resulting in a maximum final concentration of 100 nM. This was repeated 4-fold for a total of 9 concentration points. 15 μL of the 10× diluent was added to each well.

[0595] After adding the analytes, the 96-well plates were incubated at 37°C and 5% CO2 for 6 days. After incubation, the plates were allowed to equilibrate to room temperature for 20 minutes. 50 μL of Cell Titer Turbo substrate was added to each well for OVCAR-3 cells, and 75 μL of the substrate was added to each of the other two cell lines. Reagent (ADamas Life, catalog number: RA-GL11-A). Detect chemiluminescence value after incubation for 10 minutes.

[0596] 2. Experimental Results:

[0597] As shown in Table 16, the ADCs of the present invention, such as NaPi2b-ADC-001, have significant proliferative inhibitory activity in three tumor cell lines.

[0598] Table 16. Inhibitory activity of ADCs on the in vitro proliferation of three tumor cells.

[0599] .

[0600] Example 6.2: Inhibitory activity test of ADC on in vitro cells (N87, BT474, HCC1569, OE19)

[0601] 1. Experimental Method:

[0602] NCI-N87 (RPMI 1640 + 10% FBS + 1% P / S), OE-19 (RPMI 1640 + 10% FBS + 1% P / S), HCC1569 (RPMI 1640 + 10% FBS + 1% P / S), and BT-474 (DMEM + 10% FBS + 1% P / S + 10 μg / mL insulin) cells were revived and cultured. After the cells stabilized, NCI-N87 / BT-474 cells were seeded at a density of 9000 cells / well, and OE-19 / HCC1569 cells were seeded at a density of 4000 cells / well, with a density of 142.5 μL / well in 96-well plates. The cells were incubated overnight at 37°C. The next day, the ADC molecules to be tested were serially diluted 20-fold (20*) to the final concentration using serum-free medium, resulting in a maximum final concentration of 100 nM. Nine concentration points were obtained by 3-fold dilution. Add 7.5 μL of 20× analyte dilution to each well. After adding the analyte, incubate the 96-well plate at 37°C and 5% CO2 for 4 days.

[0603] After incubation, the 96-well plate was equilibrated at room temperature for 20 min. 100 μL (NCI-N87) / 50 μL (BT-474 / OE-19 / HCC1569) Cell titer Turbo substrate was added to each well, and the chemiluminescence value was measured after 10 minutes of incubation. The sources of key materials are shown in Table 17 below.

[0604] Table 17 Material Sources

[0605] 2. Experimental Results:

[0606] As shown in Table 18, the ADCs of the present invention, such as Her2-ADC-001, have significant proliferative inhibitory activity in four tumor cell lines.

[0607] Table 18. In vitro proliferation inhibitory activity of ADCs against four tumor cell lines.

[0608] Example 6.3: Non-targeted killing activity assay of antibody-drug conjugates (ADCs)

[0609] 1. Experimental Method:

[0610] MDA-MB-468 (DMEM + 10% FBS + 1% P / S), PC-9 (RPMI 1640 + 10% FBS + 1% P / S), and NCI-H1975 (RPMI 1640 + 10% FBS + 1% P / S) cells were resuscitated and cultured. MDA-MB-468 cells were seeded at 2000 cells / well, PC-9 cells at 500 cells / well, and NCI-H1975 cells at 1000 cells / well, with a cell suspension density of 142.5 μL / well in 96-well plates. The plates were incubated overnight at 37°C. The following day, the target ADC molecules were serially diluted 20-fold (20*) and 3-fold using serum-free medium, resulting in a total of 9 concentration points. 7.5 μL of 20* ADC dilution was added to each well to achieve a final concentration of up to 500 nM. After adding the analyte, the 96-well plate was incubated at 37°C and 5% CO2 for 6 days.

[0611] After incubation, the 96-well plate was equilibrated at room temperature for 20 min. 50 μL of Cell titer Turbo substrate was added to each well, and chemiluminescence values ​​were measured after 10 minutes of incubation. The sources of key materials are shown in Table 19 below:

[0612] Table 19 Material Sources

[0613] 2. Experimental Results:

[0614] As shown in Table 20, the antibody-drug conjugates of the present invention, such as NaPi2b-ADC-001 and NaPi2b-ADC-002, have much lower killing activity on target-negative cells than the control compounds NaPi2b-ADC-003 and NaPi2b-ADC-004. Therefore, the ADCs of the present invention can greatly reduce the non-targeted killing toxicity of ADC drugs and improve the therapeutic window.

[0615] Table 20 In vitro proliferative inhibitory activity of ADCs against target-negative tumor cells

[0616] Example 7. In vivo efficacy test of antibody-drug conjugate (ADC)

[0617] Example 7.1 Efficacy test of antibody-drug conjugate (ADC) against OVCAR-3 xenograft tumors

[0618] 1. Experimental Materials

[0619] Test compound: NaPi2b-ADC-001, with physiological saline as a negative control.

[0620] Experimental cells: OVCAR-3 cells.

[0621] Experimental animals: NCG mice, female, 5-6 weeks old, purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[0622] 2. Experimental Design

[0623] 2.1. Cell Treatment

[0624] OVCAR-3 cells were cultured in 15cm culture dishes under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, in an incubator at 37°C with 5% CO2. When the cells reached the exponential growth phase, they were digested with trypsin, collected, counted, and seeded.

[0625] 2.2. Tumor cell transplantation

[0626] NCG mice were acclimatized to the laboratory environment for 2-5 days, and then OVCAR-3 cells were subcutaneously injected into the right rib area at a cell density of 5 × 10⁶ cells. 6 / each, inoculated at a volume of 0.2 mL, containing 50% matrix gel. Wait until the tumor grows to 200-250 mm. 3 The experiment was conducted on the left and right sides.

[0627] 2.3. Animal drug administration and detection

[0628] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[0629] Table 21. Dosing Regimen

[0630] 2.4. Tumor volume and body weight measurement

[0631] Day 1 is the day of the first dose. A total of three doses are administered, with tumor diameter and body weight measured periodically. Tumor volume, tumor proliferation rate, and tumor inhibition rate are calculated, and tumor growth curves are plotted. The calculation formulas are as follows:

[0632] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 Where L_long and L_short represent the long and short diameters of the tumor, respectively.

[0633] Relative tumor proliferation rate T / C (%) = TRTV / CRTV × 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment);

[0634] 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).

[0635] 3. Experimental Results

[0636] The test results showed that the antibody-drug conjugates of the present invention all exhibited significant antitumor effects, and no significant weight loss or drug toxicity was observed in any group of animals during the administration period. Specific results are shown in Table 22.

[0637] Table 22 Results of tumor volume assay in the OVCAR-3 xenograft model

[0638] Example 7.2 Efficacy test of antibody-drug conjugate (ADC) against IGR-OV1 xenografts

[0639] 1. Experimental Materials

[0640] Test compounds: NaPi2b-ADC-001, NaPi2b-ADC-003, and physiological saline were used as negative controls.

[0641] Experimental cells: IGR-OV1 cells.

[0642] Experimental animals: BALB / c nude mice, female, 5-6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0643] 2. Experimental Design

[0644] 2.1. Cell Treatment

[0645] IGR-OV1 cells were cultured in 15cm culture dishes under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, in an incubator at 37°C with 5% CO2 air. When the cells reached the exponential growth phase, they were digested with trypsin, collected, counted, and seeded.

[0646] 2.2. Tumor cell transplantation

[0647] Mice were acclimatized to the laboratory environment for 2-5 days. A certain number of IGR-OV1 cells were subcutaneously inoculated into the right back of the mice, with a cell density of 5 × 10⁶ cells. 6 / mouse, inoculated with 0.1 mL of 50% matrix gel. Tumor growth was observed regularly, and once the tumor reached a certain size, mice were randomly assigned to receive the drug based on tumor size and body weight.

[0648] 2.3. Animal drug administration and detection

[0649] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[0650] Table 23. Dosing Regimen

[0651] 2.4. Tumor volume and body weight measurement

[0652] Day 1 is the day of the first dose. A total of three doses are administered, with tumor diameter and body weight measured periodically. Tumor volume, tumor proliferation rate, and tumor inhibition rate are calculated, and tumor growth curves are plotted. The calculation formulas are as follows:

[0653] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 Where L_long and L_short represent the long and short diameters of the tumor, respectively.

[0654] Relative tumor proliferation rate T / C (%) = TRTV / CRTV × 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment);

[0655] 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).

[0656] 3. Experimental Results

[0657] The test results showed that the antibody-drug conjugate NaPi2b-ADC-001 of the present invention had a significantly better antitumor effect than the control compound NaPi2b-ADC-003; during the administration period, no significant weight loss or drug toxicity was observed in any of the groups of animals. Specific results are shown in Table 24.

[0658] Table 24. Results of tumor volume assay in the IGR-OV1 xenograft model

[0659] Example 7.3 Efficacy test of antibody-drug conjugate (ADC) against BT-474 xenograft tumors

[0660] 1. Experimental Materials

[0661] Test compounds: Her2-ADC-001, Her2-ADC-002, with physiological saline as a negative control.

[0662] Experimental cells: BT-474 cells.

[0663] Laboratory animals: BALB / c nude mice, female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. or other qualified suppliers.

[0664] 2. Experimental Design

[0665] 2.1. Cell Treatment

[0666] The culture conditions for BT-474x cells (ATCC-HTB-20) were as follows: ATCC Hybri-Care Medium + 10% FBS, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin containing EDTA. After washing twice with PBS, the cells were centrifuged and resuspended in pre-chilled PBS. Cells were counted using a cell counter, and the cell suspension was adjusted to an appropriate concentration before seeding.

[0667] 2.2. Tumor cell transplantation

[0668] BALB / c nude mice were acclimatized to the laboratory environment for 3-5 days. Three days before cell inoculation, 0.36 mg of estrogen extended-release tablets were subcutaneously injected into the left posterior back of each mouse. One week after estrogen tablet inoculation, assisted urination was initiated three times per week; if necessary, assisted urination was initiated daily. 0.2 mL (10 x 10⁶ cells, PBS plus matrigel (1:1)) of BT-474x cells were subcutaneously injected into the right posterior back near the upper limb of each mouse. When the average tumor volume reached approximately 150-200 mm³, grouping and administration began.

[0669] 2.3. Animal drug administration and detection

[0670] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[0671] Table 25. Dosing Regimen

[0672] 2.4. Tumor volume and body weight measurement

[0673] The day of the first dose is recorded as day 0. A total of three doses are administered, and tumor diameter and body weight are measured periodically. Tumor volume, relative tumor proliferation rate, and relative tumor inhibition rate are calculated, and tumor growth curves are plotted. The calculation formulas are as follows:

[0674] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 Where L_long and L_short represent the long and short diameters of the tumor, respectively.

[0675] Relative tumor proliferation rate (T / C%): The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the solvent control group). RTV, relative tumor volume, is calculated as RTV = Vt / V0, where V0 is the average tumor volume measured at the time of drug administration (i.e., D0), and Vt is the average tumor volume at a certain measurement. TRTV and CRTV are based on data from the same day.

[0676] 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).

[0677] 3. Experimental Results

[0678] The test results showed that the antibody-drug conjugate Her2-ADC-001 of the present invention had a significantly better antitumor effect than the control compound Her2-ADC-002; during the administration period, no significant weight loss or drug toxicity was observed in any of the groups of animals. Specific results are shown in Table 26.

[0679] Table 26. Tumor volume data of the BT-474 xenograft model

[0680] Example 7.4: Efficacy test of antibody-drug conjugate (ADC) against RMG-1 xenograft tumors

[0681] 1. Experimental Materials

[0682] Test compounds: NaPi2b-ADC-001, NaPi2b-ADC-003, with physiological saline as a negative control.

[0683] Experimental cells: RMG-1 cells.

[0684] Laboratory animals: BALB / c nude, female, 6-10 weeks old, purchased from Beijing Ankai Yibo Biotechnology Co., Ltd.

[0685] 2. Experimental Design

[0686] 2.1. Cell Treatment

[0687] Cells were cultured in Ham's F-12 medium containing 10% FBS, and cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0688] 2.2. Tumor cell transplantation

[0689] Mice were allowed to acclimatize to the laboratory environment for 2-5 days. A certain number of cells (5 × 10⁶ cells) were subcutaneously inoculated into the right back of each mouse. 6 / mouse, inoculated with 0.1 mL of matrix gel. Tumor growth was observed regularly, and once the tumor reached a certain size, mice were randomly assigned to receive the drug based on tumor size and body weight.

[0690] 2.3. Animal drug administration and detection

[0691] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[0692] Table 27. Dosing Regimen

[0693] 2.4. Tumor volume and body weight measurement

[0694] The day of the first dose is recorded as day 0. A total of three doses are administered, and the tumor diameter and body weight are measured periodically. Tumor volume, tumor proliferation rate, and tumor inhibition rate are calculated, and a tumor growth curve is plotted. The calculation formulas are as follows:

[0695] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 Where L_long and L_short represent the long and short diameters of the tumor, respectively.

[0696] Relative tumor proliferation rate T / C (%) = TRTV / CRTV × 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment);

[0697] 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).

[0698] 3. Experimental Results

[0699] Test results show that the antibody-drug conjugate NaPi2b-ADC-001 of this invention has a significantly better antitumor effect than the control compound NaPi2b-ADC-003; during the administration period, no significant weight loss or drug toxicity was observed in any group of animals. Specific results are shown in Table 28.

[0700] Table 28. Tumor volume data of the RMG-1 xenograft model

[0701] Unless otherwise specified, the terms used in this invention have the meanings commonly understood by those skilled in the art.

[0702] The embodiments described in this invention are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various other substitutions, changes and improvements within the scope of this invention. Therefore, this invention is not limited to the above embodiments, but is only defined by the claims.

Claims

1. An antibody drug conjugate of the formula I, ###0001### I Or its stereoisomers or pharmaceutically acceptable salts. in, Tb is an antibody or its antigen-binding fragment; q is any value between 0.1 and 12.0; L is the linker, which covalently bonds Tb and D; The L is the following structure: wherein R L1 , R L2 each independently is r is any integer between 0 and 24; D is a fragment of a biologically active compound, said D being of the structure: 1 is connected to L.

2. The antibody-drug conjugate as shown in Formula I as claimed in claim 1, or its stereoisomer or pharmaceutically acceptable salt, characterized in that, R L1 , R L2 At the same time for Preferably, R L1 , R L2 At the same time, R More preferably, R L1 , R L2 At the same time, R 3. The antibody drug conjugate of Formula I as claimed in claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein, The L is 1 is connected to Tb via an S atom and 2 is connected to D.

4. The antibody drug conjugate of Formula I as claimed in claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein, The Tb is one of the following: (1) Tb is an antibody or an antigen-binding fragment thereof with or without endocytic activity; preferably, Tb is an antibody or an antigen-binding fragment thereof with endocytic activity. (2) Tb is an antibody or its antigen-binding fragment that has the activity of binding to tumor cell surface antigens; (3) Tb is anti-Her2 antibody or its antigen-binding fragment, or anti-Trop-2 antibody or its antigen-binding fragment; (4) Tb is an anti-Her2 antibody or its antigen-binding fragment; preferably, Tb is anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab or its antigen-binding fragment; more preferably, Tb is Trastuzumab or Pertuzumab; more preferably, Tb is Trastuzumab. (5) Tb is an anti-Trop-2 antibody or its antigen-binding fragment; preferably, Tb is datopotamab, sacituzumab or its antigen-binding fragment; (6) Tb is an anti-Napi2b antibody or its antigen-binding fragment; preferably, the anti-Napi2b antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region (VH) includes: (i)HCDR1, which contains the amino acid sequence of SEQ ID NO: 1; (ii) HCDR2, which contains the amino acid sequence of SEQ ID NO: 2; and (iii) HCDR3, which contains the amino acid sequence of SEQ ID NO: 3; and The light chain variable region (VL) includes: (i)LCDR1, which contains the amino acid sequence of SEQ ID NO: 4; (ii) LCDR2, which contains the amino acid sequence of SEQ ID NO: 5; and (iii) LCDR3, which contains the amino acid sequence of SEQ ID NO: 6; Preferably, the anti-Napi2b antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 9 and the light chain shown in SEQ ID NO:

10.

5. The antibody drug conjugate of Formula I as claimed in claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein, The antibody drug conjugate is of the following structure: Wherein, q is any number between 1 and 12.

6. The antibody drug conjugate of Formula I as claimed in claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein, The antibody drug conjugate is of the following structure: Among them, NaPi2b is a 66C12D12-hz1 antibody.

7. A drug linker conjugate as shown in Formula II, Lg-LD Formula II Or its stereoisomers or pharmaceutically acceptable salts. in, Lg is a group that reacts with antibodies; Preferably, Lg is F, Cl, Br, MeSO2-, or pentafluorophenoxy; more preferably, Lg is MeSO2-. The definitions of L and D are as defined in any one of claims 1-3.

8. The drug linker conjugate of claim 7, represented by Formula II, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein, The drug linker conjugate is:

9. A group of antibody-drug conjugates comprising the antibody-drug conjugate of any one of claims 1-6 as shown in Formula I, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has one, two or more q values; Preferably, the average DAR of the antibody-drug conjugate group is an integer or decimal between 1 and 16, for example, 1.5-2.5, 3.5-4.5, 5.5-6.5 or 7.5-8.5; More preferably, the average DAR of the antibody-drug conjugate group is about 2.0, 4.0, 6.0 or 8.

0.

10. A pharmaceutical composition comprising an antibody-drug conjugate as shown in Formula I as claimed in claims 1-6, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a drug linker conjugate as shown in Formula II as claimed in claims 7 or 8, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or the group of antibody-drug conjugates as claimed in claim 9, and optionally one or more pharmaceutical excipients.

11. Use of a substance in the preparation of a medicament for the treatment and / or prevention of diseases associated with abnormal cellular activity; The substance is an antibody-drug conjugate as described in Formula I as claimed in claims 1-6, or a stereoisomer thereof, or a pharmaceutically conjugate thereof. The salt; or the drug linker conjugate of Formula II as described in claim 7 or 8, or its stereoisomer or pharmaceutically acceptable salt; or the antibody-drug conjugate group as described in claim 9; or the pharmaceutical composition as described in claim 10; Preferred cancers; Preferably, the cancer is a solid tumor or a hematologic malignancy; more preferably, the cancer is 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 tumor, prostate cancer, or thyroid cancer; the esophageal cancer is preferably esophageal adenocarcinoma or esophageal squamous cell carcinoma; the lung cancer is preferably small cell lung cancer or non-small cell lung cancer; the central nervous system tumor is preferably glioma, glioblastoma multiforme, glioma, or sarcoma.

12. A method of preparing an antibody drug conjugate of Formula I, comprising: The drug linker conjugate Lg-LD shown in Formula II is coupled together; wherein Tb, Lg, L and D are defined as defined in any one of claims 1-8.

13. The method of claim 12, wherein, The method includes the step of coupling Tb with the drug linker conjugate Lg-LD shown in 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), or 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, dimethylsulfoxide, N-methylpyrrolidone, nitriles (e.g. acetonitrile). 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, dimethylsulfoxide, N-methylpyrrolidone, nitriles (e.g. acetonitrile). 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

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