Antibody-drug conjugate containing NMT inhibitor, preparation method therefor, and use thereof

Abstract

The present application provides a drug-linker conjugate containing an NMT inhibitor and an antibody-drug conjugate thereof. The present application also provides a preparation method for the drug-linker conjugate and the antibody-drug conjugate thereof, and use of the antibody-drug conjugate in preventing and / or treating diseases related to abnormal cell activity, including but not limited to use thereof in preventing and / or treating tumor diseases.

Description

An antibody-drug conjugate containing an NMT inhibitor, its preparation method and uses

[0001] This application claims priority to Chinese patent application 2024112173804, filed on August 30, 2024; Chinese patent application 2024115882889, filed on November 8, 2024; Chinese patent application 202411843945X, filed on December 14, 2024; Chinese patent application 2025100058320, filed on January 3, 2025; Chinese patent application 2025102773407, filed on March 10, 2025; Chinese patent application 2025105199062, filed on April 24, 2025; and Chinese patent application 2025108993203, filed on July 1, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of pharmaceutical technology and relates to antibody-drug conjugates of NMT inhibitor bioactive compounds, NMT inhibitor bioactive compounds, drug-linked conjugates and their preparation methods, as well as their use in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to their use in the prevention and / or treatment of tumor diseases. Background Technology

[0003] Chemotherapy using cytotoxic agents was once the standard treatment for cancer, but highly lethal cytotoxic molecules can damage normal cells, causing severe toxic side effects. Targeted antitumor drugs, possessing both targeting and antitumor activity, have become a hot topic in current cancer research; however, the selectivity of targeted drugs often leads to significant toxic side effects, thus limiting their therapeutic efficacy. Biological macromolecular drugs, such as antibodies or antibody fragments, while highly targeted, have limited or no therapeutic effect on solid tumors. Antibody-drug conjugates (ADCs), combining the targeting ability of antibodies with the activity of bioactive molecules, act as a biological missile, offering promising advantages in efficacy and safety. Antibodies guide ADCs to bind to target cells, where they are subsequently internalized. The small molecule drug is then released intracellularly through enzymatic decomposition by specific enzymes, treating the disease.

[0004] Various lipids and lipid metabolites bind to and modify proteins in eukaryotic cells; this is known as "protein lipidation." There are four main types of protein lipidation: myristoylation, palmitoylation, isopentenylation, and glycosylphosphatidylinositol anchoring. N-Myristoylation refers to the attachment of the 14-carbon fatty acid myristate ester to the N-terminal glycine of a protein via N-myristyltransferase (NMT). Myristoylation plays an important role in signal transduction, immune regulation, and tumorigenesis and development.

[0005] Currently, upregulated expression and enhanced activity of N-myristyltransferase (NMT) have been observed in various solid tumors. The role of NMT in tumorigenesis was first observed in colorectal cancer, where its expression and activity are directly related to disease progression. Furthermore, significantly elevated NMT mRNA expression has been observed in lung cancer patients; and in breast epithelial cells, cell proliferation is correlated with NMT activity.

[0006] In March 2024, Myricx Pharma published an ADC patent (WO2024052684A1) using a small molecule NMT inhibitor as the payload. Its ADC compound showed good in vitro and in vivo efficacy. According to the published toxicological data, the tolerated dose in cynomolgus monkeys was 20 mpk, and the safety window needs to be further improved.

[0007] NMT small molecule inhibitors, as a core component of ADC efficacy, are typically closely related to both pharmacodynamics and toxicity. Therefore, to further increase the safety window and reduce their half-life in vivo, thereby minimizing toxicity caused by payload system exposure and significantly improving the therapeutic window and efficacy of ADC drugs, searching for a novel NMT inhibitor as an ADC payload is a highly promising research direction. Furthermore, as a compound with a novel mechanism, NMT inhibitors, when applied to ADCs, hold the potential to overcome drug resistance to existing drugs and provide better treatment options for cancer patients. Therefore, this disclosure has high clinical application value. Summary of the Invention

[0008] In a first aspect, this disclosure provides a novel class of NMT inhibitor bioactive compounds, or stereoisomers thereof, or pharmaceutically acceptable salts thereof.

[0009] In a second aspect, this disclosure provides an antibody-drug conjugate of Formula II:

[0010] Or its stereoisomers or pharmaceutically acceptable salts.

[0011] in,

[0012] Tb is an antibody or its antigen-binding fragment;

[0013] q is any value between 0.1 and 12.0;

[0014] L is the linker, which covalently bonds Tb and D;

[0015] D represents a fragment of a bioactive compound that acts as an NMT inhibitor.

[0016] In a third aspect, this disclosure provides a drug linker conjugate of Formula III, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0017] Lg—LD

[0018] Formula III

[0019] in,

[0020] Lg is a group that reacts with the antibody; L and D are defined as described in the second aspect of this disclosure.

[0021] In a fourth aspect, this disclosure provides a method for preparing the antibody-drug conjugate described in the second aspect.

[0022] In a fifth aspect, this disclosure provides a method for preparing the drug linker conjugate described in the third aspect.

[0023] In a sixth aspect, this disclosure provides a group of conjugates comprising the antibody-drug conjugates described in the second aspect.

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

[0025] In an eighth aspect, this disclosure provides the use of the bioactive compound described in the first aspect, or a stereoisomer or pharmaceutically acceptable salt thereof, or the antibody-drug conjugate described in the second aspect, or a stereoisomer or pharmaceutically acceptable salt thereof, or the drug linker conjugate described in the third aspect, or a stereoisomer or pharmaceutically acceptable salt thereof, or the group of conjugates described in the sixth aspect, or the composition described in the seventh aspect.

[0026] Invention Details

[0027] definition

[0028] In this disclosure, 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 disclosure are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0029] As used in this disclosure, 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 in this disclosure, 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 this disclosure 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 this disclosure 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 disclosure, solid lines (—), solid wedges, or dashed wedges may be used to depict the carbon-carbon bonds of the compounds of this disclosure. The use of 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. The use of 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 disclosure 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 disclosure may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

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

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

[0035] In this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, 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 disclosed herein or its pharmaceutically acceptable salts or conjugates, 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] As used in this disclosure, the term "treatment" 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. As used in this disclosure, "treatment" covers any treatment of a patient's disease, including: (a) prevention of disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0039] In this disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described in this disclosure) (referred to as patients) or normal individuals. The term "non-human animal" in this disclosure 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 disclosure, 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 disclosure, the terms "antibody-drug conjugate" and "ADC" refer to substances obtained by linking a bioactive compound fragment (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of this disclosure, 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 disclosure, the linker comprises cleavable or cleavable units, such as peptides or disulfide bonds. In some embodiments of this disclosure, 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 disclosure, 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 disclosure, 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 is connected to L1, for example... -S- is formed by concatenating the 1st bit.

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

[0045] In this disclosure, 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 disclosure, the term "...bioactive compound fragment" refers to a portion (fragment or group) of an antibody-drug conjugate (or 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 biologically active drug (e.g., a small molecule cytotoxic drug, said drug including a group after the loss of an atom or group of atoms) or a derivative thereof (e.g., its precursor). To avoid ambiguity, "drug" does not only refer to "medicines" that have been approved by pharmaceutical regulatory authorities, but also includes any compound with potential therapeutic biological activity in clinical practice, or in research and development and academic studies.

[0047] In this disclosure, the term "antibody" is used in its broadest sense to include 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 disclosure, "antibody" and "immunoglobulin" are used interchangeably.

[0048] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the antibodies constituting the cluster are identical except for a small number of possible naturally occurring 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). In addition to specificity, monoclonal antibodies have the advantage of being synthesized without 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 this disclosure, monoclonal antibodies further include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, while the remainder is identical or homologous to another type, class, or subclass of antibody, provided they possess the desired biological activity. Chimeric antibodies that can be used in this disclosure include primatized antibodies, which comprise 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 disclosure, the “humanized” form of a nonhuman (e.g., mouse) antibody refers to a chimeric antibody containing a minimal amount of nonhuman immunoglobulin sequence. Most humanized antibodies are donor antibodies (e.g., mouse, rat, rabbit, or nonhuman primate) hypervariable region residues of human recipient immunoglobulins that have been replaced with nonhuman (e.g., mouse, rat, rabbit, or nonhuman primate) hypervariable region residues having the desired specificity, affinity, and function. In some embodiments, framework region (FR) residues of human immunoglobulins are also replaced with nonhuman 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 nonhuman 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 disclosure, although in most cases the amino acid substitutions in the antibody are 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 disclosure can be produced by many methods. For example, the monoclonal antibodies used in this disclosure 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 disclosure, unless otherwise expressly indicated, the descriptive phrases “each…independently selected from / as” and “…each independently selected from / as” used throughout this disclosure are interchangeable and should be interpreted broadly. They can mean either 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 disclosed compounds are disclosed according to the type or range of groups. In particular, this disclosure includes every independent secondary combination of the respective members of these group types and ranges. For example, the term "C1-6 alkyl / C..." 1-6 "Alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0061] In this disclosure, the term "direct bond" refers to the absence of a substituent, in which the two ends of the substituent are directly bonded together.

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

[0063] In this disclosure, the term "optional substitution" refers to the presence or absence of a substituent.

[0064] In this disclosure, the term "C1-6 alkyl / C 1-6 "Alkyl" refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, including, for example, "C1-3 alkyl / C". 1-3 Alkyl" "C1-4 alkyl / C 1-4 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 disclosure, the term "C2-6 alkenyl / C 2-6 "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 / C 2-4 Examples of these 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.

[0066] In this disclosure, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0067] In this disclosure, the term "heterocycle" refers to a ring containing at least one ring atom selected from P, N, S, or O, with the remaining reducing atoms being carbon, and the ring may be saturated or have at least one double bond (i.e., partially unsaturated). Specific examples include, but are not limited to, rings such as aziridine, ethylene oxide, dioxane, piperidine, and imidazole.

[0068] In this disclosure, the term "4-6 membered nitrogen heterocycle" refers to an aliphatic ring containing at least one nitrogen heteroatom, which may be saturated or have at least one double bond (i.e., partially unsaturated). Specific examples include, but are not limited to, nitrogen heterocyclic butanes, pyrrolidines, pyrrololine rings, etc.

[0069] In this disclosure, the term "3-6 membered cycloalkyl" refers to a saturated cycloalkyl group containing 3-6 carbon atoms. 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 disclosure, the term "5-6 membered cycloalkenyl" refers to an unsaturated cycloalkyl group containing 5-6 carbon atoms. Specific examples include, but are not limited to, cyclopentenyl and cyclohexenyl.

[0071] In this disclosure, the term "connector unit" refers to a component of an antibody-drug conjugate or drug-linked conjugate or linker that functions to link an antibody or its antigen-binding fragment that binds to a target to the remainder of the antibody-drug conjugate. The connector unit enables the attachment of a Tb unit to L2, and specific examples include, but are not limited to (where one position is attached to an antibody or its antigen-binding fragment, and two positions are attached to L2 or L3):

[0072] In this disclosure, 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 one position is linked to the linker unit and two positions are linked to L3):

[0073] In this disclosure, the term "spacer unit" refers to a component of an antibody-drug conjugate or drug-linked conjugate or linker that serves to space amino acid residues or short peptides consisting of 2-10 amino acid residues from fragments of a bioactive compound (drug molecule).

[0074] Bioactive molecules

[0075] In a first aspect, this disclosure provides NMT inhibitor bioactive compounds of formula ID, or their stereoisomers or pharmaceutically acceptable salts.

[0076] in,

[0077] R1 is -(CH2) t NR d R e ;

[0078] R2 is -C(OH)R a R b , -C(O)R c -C(O)N(R) d )R c -C(O)-4-6 membered nitrogen heterocycle (CH2) t OH, 5-6 membered cycloalkenyl, C 2-6 Alkenyl-OH or -4-6 membered nitrogen heterocycle (CH2) t OH;

[0079] R3 and R4 are each independently hydrogen, methyl, and C. 1-6 Alkylene OH, C 1-4 Halogenated alkyl or 3-6 membered cycloalkyl,

[0080] Alternatively, R3, R4, and the atoms attached to them can form 5-6 membered heterocycles;

[0081] R5 and R6 are independently hydrogen, cyano, and C groups, respectively. 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0082] G1 and G2 are each independently carbon or nitrogen atoms;

[0083] t can be 0, 1, 2, 3, or 4;

[0084] R a R b Each independently is hydrogen, C 1-4 Alkyl, 3-6 membered cycloalkyl, 3-6 membered halocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 2-6 alkenyl or C 2-6 Haloalkenyl;

[0085] Or, R a R b Together with the atoms attached thereto, they form unsubstituted or substituted 3-6 membered cycloalkyl groups; the substitution refers to the substitution by halogens, -C... 1-4 Alkyl or -C 1-4 Alkylene OH substitution;

[0086] R c Unsubstituted or substituted: 3-6 membered cycloalkyl, C 2-6 alkenyl, C 1-4 alkylene OH or C 1-6 Alkyl; the substitution refers to the substitution by halogen, -C 1-4 Alkyl, -C 1-4 Alkylene OH or -OH substitution;

[0087] R d R e Each can be either hydrogen or methyl;

[0088] R f -C(O)C 1-4 Alkyl, -C(O)C 1-4 Alkyl-NH2, -C(O)C 1-4 Alkyl-N(CH3)2 or glycosyl.

[0089] Those skilled in the art will understand that the conjugated double bonds in aromatic rings do not exist like isolated double bonds, but rather manifest as delocalized large π bonds or similar forms. In this disclosure, the Kekulé structure is used to simplify the writing of aromatic compounds. Classical structural formulas of N-containing heteroaromatic rings are often represented by alternating single and double bonds, but the actual structure is a resonance hybrid. In this disclosure, the cyclic delocalized large π bond represented by the Kekulé structure in formula ID can be shown as follows:

[0090] This disclosure also provides NMT inhibitors of bioactive compounds of formula IC, or stereoisomers thereof or pharmaceutically acceptable salts thereof;

[0091] in,

[0092] R1 is -CH2NR d R e ;

[0093] R2 is -C(OH)R a R b , -C(O)R c -C(O)N(R) d )R c -C(O)-4-6 membered nitrogen heterocycle (CH2) t OH, 5-6 membered cycloalkenyl, C 2-6 Alkenyl-OH or -4-6 membered nitrogen heterocycle (CH2) t OH,

[0094] R3 and R4 are each independently hydrogen, methyl, and C. 1-6 Alkylene OH, C 1-4 Halogenated alkyl or 3-6 membered cycloalkyl,

[0095] Alternatively, R3, R4, and the atoms attached to them can form 5-6 membered heterocycles;

[0096] t can be 0, 1, 2, 3, or 4;

[0097] R a R b Each independently is hydrogen, C 1-4 Alkyl, 3-6 membered cycloalkyl, 3-6 membered halocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 2-6 alkenyl or C 2-6 Haloalkenyl;

[0098] Or, R a R bTogether with the atoms attached thereto, they form unsubstituted or substituted 3-6 membered cycloalkyl groups; the substitution refers to the substitution by halogens, -C... 1-4 Alkyl or -C 1-4 Alkylene OH substitution;

[0099] R c Unsubstituted or substituted: 3-6 membered cycloalkyl, C 2-6 alkenyl, C 1-4 alkylene OH or C 1-6 Alkyl; the substitution refers to the substitution by halogen, -C 1-4 Alkyl, -C 1-4 Alkylene OH or -OH substitution;

[0100] R d R e Each can be either hydrogen or methyl;

[0101] R f -C(O)C 1-4 Alkyl, -C(O)C 1-4 Alkyl-NH2 or -C(O)C 1-4 Alkyl-N(CH3)2.

[0102] In some implementations, R1 is -CH2NH(CH3) or -CH2N(CH3)2.

[0103] In some implementations, R1 is -CH2NH(CH3).

[0104] In some implementations, R2 is -C(OH)R a R b -C(O)R c -C(O)N(R) d )R c -C(O)-4-6 membered nitrogen heterocycle (CH2) t OH, 5-6 membered cycloalkenyl, C 2-6 Alkenyl-OH or -4-6 membered nitrogen heterocycle (CH2) t OH;

[0105] R3 and R4 are each independently hydrogen, methyl, and C. 1-6 Alkylene OH, C 1-4 Halogenated alkyl or 3-6 membered cycloalkyl,

[0106] Alternatively, R3, R4, and the atoms attached to them can form 5-6 membered heterocycles;

[0107] t can be 0, 1, 2, 3, or 4;

[0108] R a R bEach independently is hydrogen, C 1-4 Alkyl, 3-6 membered cycloalkyl, 3-6 membered halocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 2-6 alkenyl or C 2-6 Haloalkenyl;

[0109] R c Unsubstituted or substituted 3-6 membered cycloalkyl, C 2-6 alkenyl, C 1-4 alkylene OH or C 1-6 Alkyl; the substitution refers to the substitution by halogen, -C 1-4 Alkyl, -C 1-4 Alkylene OH or -OH substitution;

[0110] R d R e Each can be either hydrogen or methyl.

[0111] In some implementations, R2 is -C(OH)R a R b R a For hydrogen, R b C 1-4 Alkyl, 3-6 membered cycloalkyl, 3-6 membered halocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 2-6 alkenyl or C 2-6 Haloalkenyl groups.

[0112] In some implementations, R2 is -C(OH)R a R b R a For hydrogen, R b C 1-4 Alkyl-substituted 3-6 membered cycloalkyl groups.

[0113] In some implementations, R2 is -C(O)R c R c It is a 3-6 membered cycloalkyl group, C 1-6 Alkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, 3-6 membered halocycloalkyl, C 1-6 Haloalkyl, C 2-6 alkenyl or C 2-6 Haloalkenyl groups.

[0114] In some implementations, R2 is -C(O)R c R c C 1-6 Alkyl, 3-6 membered cycloalkyl, C 1-6 Halogenated alkyl, C 1-4Alkyl-substituted 3-6 membered cycloalkyl or 3-6 membered halocycloalkyl.

[0115] In some implementations, R2 is -C(O)R c R c Cyclopropylidene, C 1-4 Alkyl-substituted cyclopropane or halocyclopropane.

[0116] In some implementations, R2 is

[0117] In some implementations, R2 is

[0118] In some implementations, R2 is

[0119] In some implementations, R2 is

[0120] In some implementations, R2 is

[0121] In some implementations, R2 is

[0122] In some implementations, R2 is

[0123] In some implementations, R2 is

[0124] In some implementations, both R3 and R4 are methyl groups.

[0125] In some implementations, R3, R4, and the atoms attached to them together form a 5-membered heterocycle.

[0126] In some implementations, G1 is independently a nitrogen atom and G2 is independently a carbon atom.

[0127] In some implementations, G1 is independently a carbon atom and G2 is independently a nitrogen atom.

[0128] At this point, the formula ID (represented in Kekulé form) is as follows: In some embodiments, the NMT inhibitor bioactive compound has any of the following structures:

[0129] This disclosure provides the use of the aforementioned bioactive compounds in the preparation of antibody-drug conjugates.

[0130] This disclosure provides the use of the aforementioned bioactive compounds in the preparation of drug linker conjugates.

[0131] Antibody-drug conjugates

[0132] Secondly, this disclosure provides antibody-drug conjugates represented by Formula II:

[0133] Or its stereoisomers or pharmaceutically acceptable salts.

[0134] in,

[0135] Tb is an antibody or its antigen-binding fragment;

[0136] q is any value between 0.1 and 12.0;

[0137] L is the linker, which covalently bonds Tb and D;

[0138] D represents a fragment of a bioactive compound that acts as an NMT inhibitor.

[0139] In some embodiments, D is a fragment of the NMT inhibitor bioactive compound of the first aspect that is missing one hydroxyl hydrogen or amino hydrogen, and the N or O atom on D is attached to L.

[0140] In some implementations, D has the structure shown in Formula IID-1:

[0141] In this case, R'1 is -CH2N(CH3)-, with the N atom bonded to L;

[0142] R2, R3, R4, R5, R6, G1, and G2 are defined as described in any of the embodiments of this disclosure.

[0143] In some implementations, D has the structure shown in Formula IID-2:

[0144] In R'2, the O atom is connected to the L atom;

[0145] R'2 is -C(O)R' c -C(O)N(R) d )R' c -、-C(O)-4-6 membered nitrogen heterocycle (CH2) t O-、-C 2-6 Alkenyl-O-, -4-6 membered nitrogen heterocycle -(CH2) t O-;

[0146] R' c It is a 3-6 membered cycloalkyl-C 1-4Alkylene O-, 3-6 membered cycloalkyl-O-, C 1-4 Alkylene O-;

[0147] R1, R3, R4, R5, R6, R d , t, G1 and G2 are defined as described in any embodiment of this disclosure.

[0148] In some implementations, D has the structure shown in Formula IID-3:

[0149] In R'3, the O atom is bonded to the L atom;

[0150] R'3 is -C 1-6 Alkylene O-; R4 is hydrogen, methyl, or a 3-6 membered cycloalkyl group;

[0151] R1, R2, R5, R6, G1, and G2 are defined as described in any of the embodiments of this disclosure.

[0152] In some implementations, D has the structure shown in Formula IIC-1:

[0153] In this case, R'1 is -CH2N(CH3)-, with the N atom bonded to L;

[0154] R2, R3, and R4 are defined as described in any of the embodiments of this disclosure.

[0155] In some implementations, D has the structure shown in Formula IIC-2:

[0156] In R'2, the O atom is connected to the L atom;

[0157] R'2 is C(O)R' c -C(O)N(R) d )R' c -、-C(O)-4-6 membered nitrogen heterocycle (CH2) t O-、-C 2-6 Alkenyl-O- or -4-6-membered nitrogen heterocycle-(CH2) t O-;

[0158] R' c It is a 3-6 membered cycloalkyl-C 1-4 alkylene-O-, 3-6 membered cycloalkyl-O- or C 1-4 Alkylene O-;

[0159] R1, R3, R4, R d t is defined as described in any embodiment of this disclosure.

[0160] In some implementations, R'2 is The O atom is bonded to the L atom.

[0161] In some implementations, D is any of the following structures:

[0162] 1 is connected to L.

[0163] In some implementations, D is any of the following structures:

[0164] 1 is connected to L.

[0165] 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.

[0166] In some embodiments, L is a detachable connector or a non-detachable connector; preferably, L is a detachable connector.

[0167] In some embodiments, the L comprises amino acid residues, or a short peptide consisting of 2-10 amino acid residues.

[0168] In some embodiments, L comprises an amino acid residue and a short peptide consisting of 2-10 amino acid residues, wherein the amino acid residue and the short peptide consisting of 2-10 amino acid residues are... Among them, R L1 R L2 R L3 and R L4 Each independently is hydrogen, C 1-4 alkyl, This indicates the site where the nitrogen atom is attached; each p is an independent integer between 0 and 20.

[0169] In some implementations, the L includes Among them, R L1 R L2 Each independently is C 1-4 alkyl or R L3 for and R L4 for Or hydrogen; p can be any integer between 1 and 20 independently.

[0170] In some embodiments, L comprises an amino acid residue and a short peptide consisting of 2-10 amino acid residues, wherein the amino acid residue and the short peptide consisting of 2-10 amino acid residues are... Among them, R L1 R L2 Each independently is C 1-4 alkyl or R L3 for and R L4 For hydrogen; p is an independent integer between 1 and 20.

[0171] In some implementations, the L includes Among them, R L1 R L2 Each independently is C 1-4 alkyl or R L3 for and R L4 For hydrogen; p is an independent integer between 1 and 20.

[0172] In some embodiments, the L includes a connector unit, the connector unit being...

[0173] In some implementations, L is

[0174] in,

[0175] L1 is the connector unit, which covalently bonds Tb and L2;

[0176] L2 is a connecting unit, which is covalently bonded to L1 and L3;

[0177] L3 may or may not be present. When L3 is present, it is a short peptide composed of 2-10 amino acid residues, which is covalently bound to L2 and L4.

[0178] L4 may or may not exist. When L4 exists, L4 is a spacer unit that is covalently bonded to L3 and D.

[0179] In some implementations, L is

[0180] in,

[0181] L1 is the connector unit, which covalently bonds Tb and L2;

[0182] L2 is a connecting unit, which is covalently bonded to L1 and L3;

[0183] L3 is a short peptide composed of 2-10 amino acid residues, which covalently binds to L2 and L4;

[0184] L4 is a spacer unit that is covalently bonded to L3 and D.

[0185] In some implementations, L is

[0186] in,

[0187] L1 is the connector unit, which covalently bonds Tb and L2;

[0188] L2 is a connecting unit, which is covalently bonded to L1 and L3;

[0189] L3 does not exist;

[0190] L4 is a spacer unit that is covalently bonded to L3 and D.

[0191] In some implementations, L1 is Position 1 is connected to Tb via the S atom, and position 2 is connected to L2.

[0192] In some implementations, L1 is Position 1 is connected to Tb via the S atom, and position 2 is connected to L2.

[0193] In some implementations, L2 is m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; 1 bit is connected to L1, and 2 bits are connected to L3.

[0194] In some implementations, L2 is m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; 1 bit is connected to L1, and 2 bits are connected to L3.

[0195] In some implementations, L2 is Bit 1 is connected to L1, and bit 2 is connected to L3.

[0196] In some implementations, L2 is m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; 1 bit is connected to L1, and 2 bits are connected to L3.

[0197] In some implementations, m is 3.

[0198] In some implementations, n is 7.

[0199] In some implementations, L3 can be any of the following structures:

[0200] One bit is connected to L1 or L2, and two bits are connected to L4 or D.

[0201] In some implementation schemes, R L1 R L2 R L3 and R L4 Each independently is hydrogen, C 1-4 alkyl, Indicates the site where the nitrogen atom is bonded;

[0202] p can be any integer between 0 and 20.

[0203] In some implementations, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0204] In some implementation schemes, R L1 R L2 Each independently is hydrogen, C 1-4 alkyl or This indicates the site where the nitrogen atom is attached.

[0205] In some implementation schemes, R L1 R L2 Each independently is C 1-4 alkyl or

[0206] In some implementation schemes, R L3 R L4 Each independently of hydrogen, This indicates the site where the nitrogen atom is attached.

[0207] In some implementation schemes, R L3 R L4 At the same time This indicates the site where the nitrogen atom is attached.

[0208] In some implementation schemes, R L3 for and R L4 It is hydrogen.

[0209] In some implementation schemes, R L3 for and RL4 It is hydrogen.

[0210] In some implementations, L3 is Among them, R L1 R L2 Each independently is C 1-4 alkyl or R L3 for and R L4 For hydrogen; p is an independent integer between 1 and 20.

[0211] In some implementations, L3 is Among them, R L1 R L2 Simultaneously methyl, ethyl, propyl, butyl or R L3 for and R L4 For hydrogen; p is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0212] In some implementations, L3 is

[0213] In some implementations, L4 may or may not exist; when L4 exists, L4 is...

[0214] In some implementations, L4 is

[0215] In some implementations, L4 is

[0216] In some implementations, L is One bit is connected to Tb, and two bits are connected to D; preferably, L has any of the following structures:

[0217] Among them, R L1 R L2 Each independently is hydrogen, C 1-4 alkyl, This indicates the site where the nitrogen atom is bonded; position 1 is bonded to Tb via an S atom, and position 2 is bonded to D.

[0218] In some implementation schemes, R L1 R L2 Each is independently methyl, ethyl, propyl, This indicates the site where the nitrogen atom is attached.

[0219] In some implementation schemes, R L1 For hydrogen, C 1-4 Alkyl (preferably methyl); R L2 for This indicates the site where the nitrogen atom is attached.

[0220] In some implementations, L is Among them, R L1 R L2 Each independently is C 1-4 alkyl or p can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, with position 1 connected to Tb via an S atom and position 2 connected to D.

[0221] In some implementations, L is Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0222] In some implementations, L is Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0223] In some implementations, L is Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0224] In some implementations, L is Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0225] In some implementations, L is Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0226] In some embodiments, this disclosure provides a drug linker conjugate of Formula II-1,

[0227] The definitions of Tb, q, L1, L2, L3, L4 and D are as described in any of the schemes in this disclosure.

[0228] In some implementations, Tb is an antibody or its antigen-binding fragment that has endocytic or non-endocytic activity.

[0229] In some implementations, Tb is an antibody or its antigen-binding fragment that has endocytic activity.

[0230] In some embodiments, the endocytic activity is tumor cell endocytic activity.

[0231] In some implementations, Tb is an antibody or its antigen-binding fragment that has tumor cell surface antigen-binding activity.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] In some implementations, the antibody is a bispecific antibody or a multispecific antibody.

[0239] In some embodiments, the antibody or its antigen-binding fragment is a non-human antibody, a humanized antibody, or a fully human antibody.

[0240] In some embodiments, the antibody or its antigen-binding fragment probody, bispecific antibody, or multispecific antibody is used.

[0241] 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.

[0242] In some implementations, Tb targets CD19, CD20, CD21, CD22, CD30, CD33, CD123, CDH17, LIV1, Her2, or Trop-2.

[0243] 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.

[0244] 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.

[0245] In some implementations, Tb is an anti-Trop-2 antibody or its antigen-binding fragment, such as datopotamab, sacituzumab or its antigen-binding fragment.

[0246] In some embodiments, this disclosure provides antibody-drug conjugates of Formula II-2.

[0247] Among them, Tb, q, R2, R3, R4, R L1 R L2 R L3 and R L4 The definition is as described in any of the embodiments in this disclosure.

[0248] In some embodiments, the antibody-drug conjugate has any of the following structures:

[0249] Wherein, q and Tb are defined as described in any of the schemes in this disclosure.

[0250] In some implementations, q is any number between 1 and 12.

[0251] In some implementations, q is any number between 1 and 8.

[0252] In some implementations, q is any number between 2 and 8.

[0253] In some implementations, q is any number between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, and 7-8.

[0254] In some implementations, q is any number between 2 and 3.

[0255] In some implementations, q is any number between 3 and 4.

[0256] In some implementations, q is any number between 7 and 8.

[0257] In some implementations, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0258] In some implementations, q is 2, 4, 6, or 8.

[0259] In some embodiments, the antibody-drug conjugate has any of the following structures:

[0260] Wherein, Tb1 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, Tb1 is Trastuzumab, and q is any number between 7 and 8.

[0261] In some embodiments, the antibody-drug conjugate has any of the following structures:

[0262] Wherein, Tb2 is an anti-Trop-2 antibody or its antigen-binding fragment, such as datopotamab, sacituzumab or its antigen-binding fragment, preferably Tb2 is sacituzumab; q is any number between 7 and 8.

[0263] Drug conjugates

[0264] Thirdly, this disclosure provides a drug linker conjugate as shown in Formula III:

[0265] Lg-LD

[0266] Formula III

[0267] Or its stereoisomers or pharmaceutically acceptable salts;

[0268] in,

[0269] Lg is the leaving group that reacts with the antibody;

[0270] The definitions of L and D are as described in any of the embodiments of this disclosure.

[0271] In some implementations, a drug linker conjugate of Formula III-1 is provided.

[0272] Lg-L1-L2-L3-L4-D

[0273] Formula III-1

[0274] The definitions of L1, L2, L3, L4 and D are as described in any of the embodiments of this disclosure.

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

[0276] In some embodiments, Lg is F, Cl, Br, MeSO2-, or pentafluorophenoxy; more preferably, Lg is MeSO2-.

[0277] 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 schemes in this disclosure.

[0278] In some implementations, L1 is Bit 1 is connected to Lg, and bit 2 is connected to L2; Lg is MeSO2-.

[0279] In some embodiments, a drug linker conjugate of Formula III-2 is provided.

[0280] Among them, R2, R3, R4, R L1 R L2 R L3 and R L4 The definition is as described in any of the embodiments in this disclosure.

[0281] In some embodiments, the drug linker conjugate has any of the following structures:

[0282] This disclosure provides the use of the aforementioned drug linker conjugate in the preparation of antibody-drug conjugates (ADCs).

[0283] Connector

[0284] Furthermore, this disclosure also provides a linker for antibody-drug conjugates. In this configuration, position 1 is linked to Tb, and position 2 is linked to the bioactive molecular fragment; L is defined as described in any of the schemes disclosed herein.

[0285] In some embodiments, the linker in the antibody-drug conjugate has the following structure: Among them, position 1 is linked to Tb, and position 2 is linked to the bioactive molecular fragment; L1, L2, L3, and L4 are defined as described in any of the schemes in this disclosure;

[0286] In some implementations, the connector 1 is connected to Tb and 2 is connected to D.

[0287] This disclosure also provides the use of the aforementioned linker in the preparation of antibody-drug conjugates.

[0288] This disclosure also provides the use of the aforementioned linker in the preparation of drug linker conjugates.

[0289] Antibody-drug conjugate preparation method

[0290] In a fourth aspect, this disclosure provides a method for preparing the antibody-drug conjugate of Formula II, comprising:

[0291] Tb was coupled with the drug linker conjugate Lg-LD shown in Formula III;

[0292] Wherein, Tb, Lg, L and D are defined as described in any of the embodiments of this disclosure.

[0293] Specifically, the method includes the step of coupling Tb with the drug linker conjugate Lg-LD shown in Formula III in a solvent to form CS bonds or CN bonds.

[0294] 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).

[0295] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0296] 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).

[0297] In some embodiments, the method further includes a step of purifying the coupling product.

[0298] In some implementations, the coupling product is purified by chromatography.

[0299] In some embodiments, the chromatography method includes one or more of ion exchange chromatography, hydrophobic chromatography, reversed-phase chromatography, or affinity chromatography.

[0300] In some embodiments, the method is carried out at -20 to 100°C, for example 0 to 50°C, preferably at room temperature.

[0301] Preparation method of drug linker conjugate

[0302] In a fifth aspect of this disclosure, a method for preparing the drug linker conjugate shown in Formula III is provided.

[0303] In some implementation schemes, the preparation route of the drug linker conjugate represented by Formula III is as follows (Method 1):

[0304] Step 1: The compound shown in formula DL-X-1 or its salt reacts with the compound shown in formula IC to obtain the compound shown in formula DL-X-2 or its salt;

[0305] Step 2: Remove the amino protecting group from the compound of formula DL-X-2 or its salt to obtain the compound of formula DL-X-3 or its salt;

[0306] Step 3: The compound shown in formula DL-X-3 or its salt and the compound shown in formula DL-X-4 or its salt are condensed together to obtain the compound shown in formula III-2;

[0307] Among them, R2, R3, R4, R L1 R L2 R L3 and R L4 The definition is as described in any of the embodiments of this disclosure;

[0308] P is an amino protecting group, including but not limited to benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl; preferably, P is 9-fluorenylmethoxycarbonyl (Fmoc).

[0309] This disclosure provides compounds of formula DL-X-1, DL-X-2 and DL-X-3 or salts thereof.

[0310] In some embodiments, the compound represented by formula DL-X-1 has the following structure:

[0311] This disclosure provides the use of compounds of formula DL-X-1, DL-X-2 and DL-X-3 or salts thereof in the preparation of drug linker conjugates.

[0312] This disclosure provides the use of compounds of formula DL-X-1, DL-X-2 and DL-X-3 or salts thereof in the preparation of ADCs.

[0313] 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 INT2.

[0314] Drug linker conjugates with various structures can be obtained by reacting analogs of INT1 and INT2.

[0315] In some implementation schemes, the preparation route of the drug linker conjugate represented by Formula III is as follows (Method 2):

[0316] The compound shown in formula DL-X-5 or its salt reacts with the compound shown in formula IC to give the compound shown in formula III-2.

[0317] Among them, R2, R3, R4, R L1 R L2 R L3 and R L4 The definition is as described in any of the embodiments of this disclosure;

[0318] G is selected from chlorine and

[0319] Specifically, taking DL-021 as an example, the target compound (DL021) is obtained by reacting compound INT10, which is represented by the general formula, with compound D-20.

[0320] Drug linker conjugates with various structures can be obtained by reacting analogs of INT10 and D-20.

[0321] This disclosure provides a compound of formula DL-X-5 or a salt thereof.

[0322] In some embodiments, the compound represented by formula DL-X-5 is selected from the following structures:

[0323] This disclosure provides the use of the compound of formula DL-X-5 or a salt thereof in the preparation of drug linker conjugates.

[0324] This disclosure provides the use of the compound of formula DL-X-5 or a salt thereof in the preparation of ADCs.

[0325] In some embodiments, the preparation route of the compound represented by formula DL-X-5 is as follows:

[0326] Step 1: Remove the amino protecting group from the compound of formula DL-X-6 or its salt to obtain the compound of formula DL-X-7 or its salt;

[0327] Step 2: The compound shown in formula DL-X-7 or its salt and the compound shown in formula DL-X-4 or its salt undergo a condensation reaction to obtain the compound shown in formula DL-X-8;

[0328] Step 3: The compound shown in formula DL-X-8 or its salt is reacted to obtain the compound shown in formula DL-X-5;

[0329] Among them, R L1 R L2 R L3 and R L4 The definition is as described in any of the embodiments of this disclosure;

[0330] G is selected from chlorine or

[0331] P is selected from amino protecting groups, including but not limited to benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl; preferably, P is selected from 9-fluorenylmethoxycarbonyl (Fmoc).

[0332] Specifically, taking INT10 as an example, a similar connector can be obtained through a similar method.

[0333] This disclosure provides compounds of formula DL-X-6, DL-X-7 and DL-X-8 or salts thereof.

[0334] In some embodiments, the compound represented by formula DL-X-8 has the following structure:

[0335] This disclosure provides the use of compounds of formula DL-X-6, DL-X-7 and DL-X-8, or salts thereof, in the preparation of drug linker conjugates.

[0336] This disclosure provides the use of compounds of formula DL-X-6, DL-X-7 and DL-X-8 or salts thereof in the preparation of ADCs.

[0337] In some embodiments, the preparation route of the compound represented by formula IC is as follows:

[0338] Step 1: The compound shown in Formula CX-1 or its salt and the compound shown in Formula CX-2 or its salt are coupled together to obtain the compound shown in Formula CX-3;

[0339] Step 2: The compound shown in formula CX-3 or its salt reacts with a brominating reagent to obtain the compound shown in formula CX-4;

[0340] Step 3: The compound shown in formula CX-4 or its salt is coupled to obtain the compound shown in formula CX-5;

[0341] Step 4: The compound shown in formula CX-5 or its salt is oxidized to obtain the compound shown in formula CX-6;

[0342] Step 5: The compound shown in formula CX-6 or its salt is reduced to obtain the compound shown in formula CX-7;

[0343] Step 6: The compound shown in Formula CX-7 or its salt and the compound shown in Formula CX-8 or its salt are reacted via an etherification reaction to obtain the compound shown in Formula CX-9;

[0344] Step 7: The compound shown in Formula CX-9 or its salt is deamino protecting to obtain the compound shown in Formula IC;

[0345] R2, R3 and R4 are defined as described in any of the embodiments in this disclosure;

[0346] X is selected from halogens, with iodine being the preferred choice;

[0347] Q is an amino protecting group, including but not limited to benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl, or ethoxycarbonyl.

[0348] This disclosure provides compounds of formulas CX-3, CX-4, CX-5, CX-6, CX-7, CX-8 and CX-9, or salts thereof.

[0349] This disclosure provides the use of compounds of formula CX-3, CX-4, CX-5, CX-6, CX-7, CX-8 and CX-9, or salts thereof, in the preparation of drug linker conjugates.

[0350] This disclosure provides the use of compounds of formula CX-3, CX-4, CX-5, CX-6, CX-7, CX-8 and CX-9, or salts thereof, in the preparation of ADCs.

[0351] Coupled Groups

[0352] In a sixth aspect, this disclosure 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.

[0353] 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.

[0354] In some implementations, when there is only one q-value antibody-drug conjugate in the antibody-drug conjugate group, the q-value and the average DAR are equal.

[0355] 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%.

[0356] 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.

[0357] 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;

[0358] 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;

[0359] 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.

[0360] 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).

[0361] Pharmaceutical Composition

[0362] In a seventh aspect, this disclosure provides pharmaceutical compositions comprising the aforementioned bioactive compounds, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or 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 group of antibody-drug conjugates and optionally one or more pharmaceutical excipients.

[0363] 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).

[0364] use

[0365] Eighthly, this disclosure provides the use of the aforementioned bioactive compounds, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or 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 in the preparation of medicaments for treating and / or preventing diseases (e.g., cancer) associated with abnormal cellular activity.

[0366] This disclosure provides the aforementioned bioactive compounds, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or the aforementioned antibody-drug conjugates, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or the aforementioned drug-linked conjugates, or stereoisomers thereof or pharmaceutically acceptable salts thereof; or the aforementioned groups of antibody-drug conjugates; or the aforementioned pharmaceutical compositions for the treatment and / or prevention of diseases (e.g., cancer) associated with abnormal cellular activity.

[0367] This disclosure provides methods for preventing and / or treating diseases associated with abnormal cellular activity (e.g., cancer), comprising: administering to an individual in need a preventive and / or therapeutically effective amount of the aforementioned bioactive compound, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or 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 the aforementioned group of antibody-drug conjugates; or the aforementioned pharmaceutical composition.

[0368] In some implementations, the cancer is a solid tumor or a hematologic malignancy.

[0369] In some embodiments, the cancers described in this disclosure are myeloma, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), breast cancer, colorectal cancer, gastric cancer, urothelial carcinoma, or prostate cancer.

[0370] In some implementations, the cancer is a cancer related to the targets HER2 and TROP2.

[0371] Without violating common sense in the field, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this disclosure.

[0372] All reagents and raw materials used in this disclosure are commercially available.

[0373] The beneficial effects of this disclosure are:

[0374] This disclosure, through extensive research, develops an antibody-drug conjugate with an NMT inhibitor as the payload, capable of achieving one or more of the following effects:

[0375] (1) The payload (NMT inhibitor) of the antibody-drug conjugate disclosed herein has potent and broad-spectrum tumor cell killing activity, and can produce good therapeutic effects when used in ADC drugs.

[0376] (2) The payload (NMT inhibitor) of the antibody-drug conjugate disclosed herein has a short in vivo half-life, which can greatly reduce the toxicity of systemic exposure and expand the therapeutic window of the drug.

[0377] (3) The payload (NMT inhibitor) of the antibody-drug conjugate disclosed herein has excellent membrane permeability, thus it can better penetrate into tumor cells and produce a killing effect.

[0378] (4) The linker disclosed herein has high hydrophilicity, which can improve the overall physicochemical properties of ADC molecules and enhance the pharmacokinetic properties of ADC drugs;

[0379] (5) The antibody-drug conjugate disclosed herein has excellent anti-tumor effects and can kill tumor cells more effectively. It has better in vivo or in vitro anti-tumor effects than antibody-drug conjugates that link similar small molecule drugs in the prior art.

[0380] (6) The antibody-drug conjugate disclosed herein has an excellent bystander effect and can kill tumor cells more effectively;

[0381] (7) The antibody-drug conjugate disclosed herein improves the tumor tissue targeting of the ADC drug, increases the ratio of the concentration of bioactive molecules in the tumor and blood, and reduces its toxicity to normal tissues, thus having a higher therapeutic index.

[0382] Therefore, the antibody-drug conjugates disclosed herein have high clinical application value. Detailed Implementation

[0383] The present disclosure is further illustrated below through a description of specific embodiments, but this is not intended to limit the scope of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this disclosure without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0384] The abbreviations used in this disclosure have the following meanings:

[0385] Table 1

[0386] Preparation scheme

[0387] 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).

[0388] 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).

[0389] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0390] 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 deuteride. δ values ​​are expressed in ppm.

[0391] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0392] The ultra-high performance liquid chromatography (UPLC) instrument used was AB SCIEX, model ExionLC.

[0393] The high-resolution mass spectrometer used for the measurements was an AB SCIEX X500B.

[0394] Methods for determining the antibody-drug conjugate ratio (DAR value):

[0395] Take 50 μg of ADC sample, dilute it with ultrapure water to 0.5 mg / ml, then add 1 μl of 1M DTT, mix well, centrifuge and take the supernatant for injection.

[0396] Table 2 Liquid phase parameters:

[0397] Table 3 Mass Spectrometry Parameters:

[0398] Example 1. Synthesis of NMT inhibitor active compounds

[0399] Example 1.1: Synthesis of D-1

[0400] Step 1:

[0401] Compound D-1-1 (80 mg, 0.14 mmol) was dissolved in THF (2 mL), cooled to 0 °C in an ice bath, and allyl magnesium bromide (0.56 mL, 0.56 mmol, 1 mol / mL) was added dropwise with stirring. After the addition was complete, the mixture was moved to room temperature and stirred for 1 h. 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 target compound D-1-2 (30 mg).

[0402] LCMS(ESI)[M+H] + =622.31.

[0403] 1H NMR (400MHz, DMSO-d6) δ8.94–8.73(m,1H),8.22(s,1H),8.05–7.88(m,2H),7.41–7.26(m,2H),5.63–5.45(m,2H),4.93–4.82(m,6 H),3.98(t,J=7.6Hz,2H),3.55–3.55(m,3H),2.82–2.76(m,5H),2.55–2.52(m,1H),2.37–2.29(m,4H),1.91(s,3H),1.37(s,9H).

[0404] Step Two:

[0405] Compound D-1-2 (15 mg, 0.024 mmol) was dissolved in DCM (2 mL), and trifluoroacetic acid (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 concentrated under reduced pressure, and DCM (3 x 3 mL) was added and distilled to remove trifluoroacetic acid. After dissolving in DMF (1 mL), the solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the formate of the target compound D-1 (2.8 mg).

[0406] LCMS(ESI)[M+H] + =522.25.

[0407] 1 H NMR (400MHz, DMSO-d6) δ8.65 (t, J = 1.3Hz, 1H), 8.19 (s, 1H), 7.66 (s, 1H), 7. 64–7.59(m,1H),7.43(dd,J=9.3,1.7Hz,1H),7.35(dd,J=9.5,6.3Hz,2H),5. 68–5.57(m,2H),4.96–4.87(m,4H),4.62(s,1H),4.30(s,2H),4.02(t,J=7.6 Hz, 2H), 3.56 (s, 3H), 2.86 (t, J = 7.7Hz, 2H), 2.47-2.37 (m, 8H), 1.86 (s, 3H).

[0408] Example 1.2: Synthesis of D-2

[0409] Step 1:

[0410] Mg powder (21 mg, 0.86 mmol) and a small amount of iodine granules were placed in a three-necked flask. Under nitrogen protection, THF (0.5 mL) was added, followed by stirring at room temperature with 1,4-dibromobutane (93 mg, 0.43 mmol). The mixture was heated until the reaction was initiated. After initiation, THF (1 mL) was added for dilution, and the reaction was continued with stirring for 0.5 hours. Then, a THF (0.5 mL) solution of D-1-1 (50 mg, 0.086 mmol) was added, and the reaction was continued with stirring for 1 hour. The reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was directly purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-2-1 (23 mg).

[0411] LCMS(ESI)[M+H] + =596.28.

[0412] 1 H NMR (400MHz, DMSO-d6) δ8.52(s,1H),7.66(s,1H),7.61(d,J=9.3Hz,1H),7.39(dd,J=9.4,1.7Hz,1H),7.34–7.21(m,2H),4.80(s,2H),4.47(s,1H ),3.95(t,J=7.7Hz,2H),3.51(s,3H),2.79(t,J=7.7Hz,2H),2.71(s,3H ),1.89–1.76(m,5H),1.74–1.61(m,4H),1.54–1.46(m,2H),1.36(s,9H).

[0413] Step Two:

[0414] Compound D-2-1 (13 mg, 0.022 mmol) was dissolved in DCM (2 mL), and trifluoroacetic acid (0.2 mL) was added with stirring at room temperature. After the addition was complete, the mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and DCM (3 x 3 mL) was added and distilled to remove trifluoroacetic acid. After dissolving in DMF (1 mL), the solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the formate of the target compound D-2 (6.16 mg).

[0415] LCMS(ESI)[M+H] + =478.21.

[0416] 1H NMR (400MHz, DMSO-d6) δ8.67–8.61(m,1H),7.66–7.59(m,2H),7.43–7.32(m,3H),5.62–5.56(m,1H),4.21(s,2H),3.93(t,2 H),3.59(s,3H),2.81(t,J=7.6Hz,2H),2.57–2.54(m,3H),2.39(s,3H),2.26–2.18(m,2H),1.96(s,3H),1.79–1.71(m,2H).

[0417] Example 1.3: Synthesis of D-3

[0418] Step 1:

[0419] Compound D-3-1 (150 mg, 0.25 mmol) was dissolved in THF (5.0 mL), cooled to 0 °C, and then isopropenyl magnesium bromide (1 mL, 1 N) was slowly added. After the addition was complete, the mixture was allowed to naturally rise to room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride, concentrated under reduced pressure, and purified by reverse-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound D-3-2 (90 mg).

[0420] LCMS(ESI)[M+H] + =580.25.

[0421] Step Two:

[0422] Compound D-3-2 (30 mg, 0.052 mmol) was dissolved in MeOH (2.0 mL), cooled to 0 °C, and then NaBH4 (4 mg, 0.10 mmol) was added. After the addition was complete, the reaction was maintained at 0 °C for 0.5 h. The reaction was monitored by LCMS. The reaction solution was filtered and concentrated under reduced pressure to obtain the target compound D-3-3 (30 mg).

[0423] LCMS(ESI)[M+H] + =582.4.

[0424] Step 3:

[0425] Compound D-3-3 (30 mg, 0.052 mmol) was dissolved in a TFA / DCM (3 mL, 20%) mixed solvent and reacted at room temperature with stirring for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the formate of the target compound D-3 (6.2 mg).

[0426] LCMS(ESI)[M+H] + =482.23.

[0427] 1 H NMR (400MHz, DMSO-d6) δ8.61(s,1H),8.33(s,1H),7.71(s,1H),7.60(d,J=9.4Hz,1H),7.43(dd,J=9.4,1.6Hz,1H),7.37–7.25(m,2H),4.85 (s,1H),4.78(s,1H),4.62(s,1H),4.39(s,2H),4.00–3.90(m,2H),3.49(s,3H),2.72–2.60(m,2H),2.50(s,3H),1.86(s,3H),1.23(s,3H).

[0428] Example 1.4: Synthesis of D-4

[0429] Step 1:

[0430] Compound D-3-1 (50 mg, 0.078 mmol) was dissolved in THF (10 mL). Under nitrogen protection, the mixture was cooled to 0 °C, and cyclobutylmagnesium bromide (22.66 mg, 0.16 mmol, 0.5 min) was added with stirring. After the addition was complete, the reaction was continued to be stirred at room temperature for 2 h, and the reaction was monitored by LCMS. The reaction solution was quenched with methanol (1 mL), filtered, and the filtrate was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-4-1 (20 mg).

[0431] LCMS(ESI)[M+H] + =594.30.

[0432] Step Two:

[0433] Compound D-4-1 (20 mg, 0.034 mmol) was dissolved in THF (3 mL), and sodium cyanoborohydride (3 mg, 0.07 mmol) was added with stirring at room temperature. After the addition was complete, the reaction was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with H2O (0.1 mL), and purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-4-2 (13 mg).

[0434] LCMS(ESI)[M+H] + =596.55.

[0435] Step 3:

[0436] Compound D-4-2 (10 mg, 0.017 mmol) was dissolved in DCM (3 mL), and TFA (0.3 mL) was added with stirring at room temperature. The reaction was continued with stirring at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and then DCM (3*3 mL) was added in portions for further concentration under reduced pressure to remove TFA. The crude product was dissolved in DMF (2 mL) and purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-4 (2.3 mg).

[0437] LCMS(ESI)[M+H] + =496.24.

[0438] 1 H NMR(400MHz,Methanol-d4)δ8.47(s,1H),7.80(s,1H),7.60–7.56(m,1H),7.50–7 .43(m,1H),7.25–7.19(m,1H),7.18–7.09(m,1H),4.53(s,2H),4.42(d,J=8.9Hz,1 H),4.19–4.11(m,1H),4.06–3.97(m,1H),3.37(s,3H),2.84–2.69(m,5H),2.66–2 .58(m,1H),2.06–1.96(m,1H),1.91(s,3H),1.88–1.65(m,4H),1.62–1.52(m,1H).

[0439] Example 1.5: Synthesis of D-5

[0440] Step 1:

[0441] Compound D-1-1 (200 mg, 0.34 mmol) was dissolved in a mixed solution of dichloromethane (2 mL) and trifluoroacetic acid (0.5 mL), and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound D-5-1 (120 mg).

[0442] LCMS(ESI)[M+H] + =484.24.

[0443] Step Two:

[0444] Compound D-5-1 (120 mg, 0.25 mmol) was dissolved in tetrahydrofuran (2 mL), and formaldehyde aqueous solution (0.5 mL, 35% concentration) was added. The mixture was stirred at room temperature for 10 min. Then, sodium cyanoborohydride (31 mg, 0.5 mmol) was added. After the addition was complete, the reaction was continued at room temperature for 30 min, and the reaction was monitored by LCMS. The reaction solution was quenched with water (2 mL), extracted with ethyl acetate (5 mL * 3), the organic liquids were combined, concentrated under reduced pressure, and the crude product was purified by normal phase column chromatography (DCM:MeOH = 10:1) to obtain trifluoroacetate of the target compound D-5-2 (70 mg).

[0445] LCMS(ESI)[M+H]+=498.28.

[0446] 1 H NMR (400MHz, DMSO-d6) δ10.18(s,1H),8.82(s,1H),7.93(s,1H),7.67(d,J=9.3Hz,1H),7.47(d,J=9.3Hz,1H),7.39–7.32(m,2H),4.76(s ,2H),4.10(q,J=7.1Hz,2H),4.01(t,J=7.0Hz,2H),3.67(s,3H),2.87(t,J=7.0Hz,2H),2.79(s,6H),1.98(s,3H),1.17(t,J=7.1Hz,3H).

[0447] Step 3:

[0448] Compound D-5-2 (70 mg, 0.14 mmol) was dissolved in methanol (2 mL) and water (2 mL), and then lithium hydroxide monohydrate (34 mg, 1.4 mmol) was added. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was directly evaporated to dryness to obtain the lithium salt of the target compound D-5-3 (100 mg), which was used directly in the next step of the reaction.

[0449] LCMS(ESI)[M+H] + =470.22.

[0450] Step Four:

[0451] Lithium salt of compound D-5-3 (20 mg, 0.042 mmol) and HATU (15 mg, 0.052 mmol) were added to DMF (1 mL), followed by DIPEA (56 mg, 0.43 mmol) and 3-azacyclobutane methanol hydrochloride.

[0452] (11 mg, 0.086 mmol), after addition, the mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the formate of the target compound D-5 (3 mg).

[0453] LCMS(ESI)[M+H] + =539.28.

[0454] 1 H NMR (400MHz, DMSO-d6) δ8.48(s,1H),8.14(s,1H),7.58–7.50(m,2H),7.33–7.22(m,3H),4.31(t,J=9.1Hz,1H),4.11–4.01(m,3H),3.88( t,J=9.1Hz,1H),3.79(s,2H),3.64–3.57(m,5H),3.50(d,J=6.3Hz,2H),2.92–2.85(m,2H),2.69–2.56(m,1H),2.17(s,6H),1.85(s,3H).

[0455] Example 1.6: Synthesis of D-6

[0456] Step 1:

[0457] Lithium salt of compound D-5-3 (20 mg, 0.042 mmol) and HATU (15 mg, 0.052 mmol) were added to DMF (1 mL), followed by DIPEA (56 mg, 0.43 mmol) and trifluoroacetate of compound D-6-1 (15 mg, 0.15 mmol) in sequence.

[0458] After addition, the mixture was stirred at room temperature for 1 hour, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound D-6 (5.3 mg).

[0459] LCMS(ESI)[M+H] + =553.28.

[0460] 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.96(s,1H),8.10(s,1H),7.79(d,J=9.3Hz,1H),7.67(d,J=9.4Hz,1H),7.45–7.32(m,2 H),4.84(s,2H),4.26–3.95(m,5H),3.68(s,3H),2.97–2.79(m,9H),2.72–2.62(m,2H),2.44–2.18(m,2H),2.06–1.91(m,5H).

[0461] Example 1.7: Synthesis of D-7

[0462] Step 1:

[0463] Lithium salt of compound D-5-3 (20 mg, 0.042 mmol) and HATU (15 mg, 0.052 mmol) were added to DMF (1 mL), followed by DIPEA (56 mg, 0.43 mmol) and trifluoroacetate of compound D-7-1 (15 mg, 0.15 mmol). After addition, the mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound D-7 (2.3 mg).

[0464] LCMS(ESI)[M+H] + =553.28.

[0465] 1H NMR (400MHz, DMSO-d6) δ9.97(s,1H),8.93(s,1H),8.06(s,1H),7.77(d,J=9.3Hz,1H),7.62(d,J=9.1Hz,1H),7.48–7.31(m,2 H),5.12–4.70(m,3H),4.38–3.91(m,4H),3.67(s,3H),2.86(s,9H),2.72–2.62(m,2H),2.44–2.30(m,2H),2.11–1.80(m,5H).

[0466] Example 1.8: Synthesis of D-8

[0467] Step 1:

[0468] Lithium salt of compound D-5-3 (20 mg, 0.042 mmol) and HATU (15 mg, 0.052 mmol) were added to DMF (1 mL), followed by DIPEA (16 mg, 0.13 mmol) and compound D-8-1 (6 mg, 0.084 mmol). After addition, the mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound D-8 (5.2 mg).

[0469] LCMS(ESI)[M+H] + =527.26.

[0470] 1 H NMR (400MHz, DMSO-d6) δ10.47(s,1H),8.95(d,J=5.5Hz,1H),8.08(s,1H),7.75(d,J= 9.3Hz,1H),7.70–7.54(m,1H),7.43–7.30(m,2H),4.81(s,2H),4.05–3.96(m,2H),3.6 4(s,3H),3.53–3.46(m,1H),3.44(s,2H),3.40–3.33(m,1H),3.04–2.94(m,1H),2.86 (s,2H),2.82(s,6H),2.69(t,J=7.0Hz,2H),2.60–2.52(m,1H),1.99(d,J=4.3Hz,3H).

[0471] Example 1.9: Synthesis of D-9

[0472] Step 1:

[0473] Compound D-3-1 (100 mg, 0.17 mmol) was dissolved in THF (2.0 mL), cooled to 0 °C, and then cyclopropylmagnesium bromide (0.5 mL, 2.5 N) was slowly added. After the addition was complete, the mixture was allowed to rise naturally to room temperature and stirred for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride, concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0-10%) to obtain the target compound D-9-1 (60 mg).

[0474] LCMS(ESI)[M+H] + =580.34.

[0475] Step Two:

[0476] Compound D-9-1 (25 mg, 0.043 mmol) was dissolved in DCM (2.0 mL), and then TFA (0.5 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-9 (11.3 mg).

[0477] LCMS(ESI)[M+H] + =480.20.

[0478] 1 H NMR (400MHz, DMSO-d6) δ8.90–8.85(m,2H),8.04(s,1H),7.79–7.76(m,1H),7.68–7.64(m,1H),7.47–7.34(m,2H),4.70(s,2 H),4.02(t,J=6.9Hz,2H),3.80(s,3H),2.93–2.86(m,3H),2.67(s,3H),2.09(s,3H),0.91–0.86(m,2H),0.84–0.80(m,2H).

[0479] Example 1.10: Synthesis of D-10

[0480] Step 1:

[0481] Compound D-3-1 (100 mg, 0.17 mmol) was dissolved in THF (2.0 mL), cooled to 0 °C, and then allyl magnesium bromide (0.5 mL, 2.5 N) was slowly added. After the addition was complete, the mixture was allowed to rise naturally to room temperature and stirred for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride (0.5 mL), concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0-8%) to obtain the target compound D-10-1 (50 mg).

[0482] LCMS(ESI)[M+H] + =580.32.

[0483] Step Two:

[0484] Compound D-10-1 (33 mg, 0.043 mmol) was dissolved in MeOH (2.0 mL), cooled to 0 °C, and then sodium borohydride (10.8 mg, 0.29 mmol) was added. The mixture was stirred at room temperature for 0.5 h. The reaction was monitored by LCMS. DCM (5 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic phase was concentrated under reduced pressure to obtain the target compound D-10-2 (30 mg).

[0485] LCMS(ESI)[M+H] + =582.34.

[0486] Step 3:

[0487] Compound D-10-2 (30 mg, 0.052 mmol) was dissolved in DCM (2.0 mL), and then TFA (0.5 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-10 (14.5 mg).

[0488] LCMS(ESI)[M+H] + =482.20.

[0489] 1 H NMR(400MHz, DMSO-d6)δ8.96(s,3H),8.12(s,1H),7.85–7.76(m,2H),7.44(dd,J=12.2,8.0Hz,2H),5.75–5.64(m,2H),5.00–4.91(m ,2H),4.71(s,2H),4.40(t,J=6.9Hz,1H),4.03(d,J=8.3Hz,2H),3.57(s,3H),2.78–2.65(m,5H),2.36(t,J=6.9Hz,2H),1.96(s,3H).

[0490] Example 1.11: Synthesis of D-11

[0491] Step 1:

[0492] Compound D-10-1 (10 mg, 0.017 mmol) was dissolved in DCM (1.0 mL), and then TFA (0.3 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-11 (4.2 mg).

[0493] LCMS(ESI)[M+H] + =480.18.

[0494] 1H NMR(400MHz, DMSO-d6)δ8.95(s,2H),8.85(s,1H),7.96(s,1H),7.72–7.69(m,1H),7.57–7.54(m,1H),7.45–7.34(m,2H),5.97–5.85(m,1H), 5.15–5.08(m,2H),4.67(s,2H),4.05(t,J=6.8Hz,2H),3.77(s,3H),3.59(d,J=6.9Hz,2H),2.92(t,J=6.8Hz,2H),2.65(s,3H),2.05(s,3H).

[0495] Example 1.12: Synthesis of D-12

[0496] Step 1:

[0497] Compound D-4-1 (10 mg, 0.017 mmol) was dissolved in DCM (2 mL), and then trifluoroacetic acid (0.5 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 30 min, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound D-12 (1.95 mg).

[0498] LCMS(ESI)[M+H] + =494.17.

[0499] 1 H NMR (400MHz, DMSO-d6) δ8.96(s,2H),8.90(s,1H),8.01(s,1H),7.74(d,J=9.4Hz,1H),7.63(d,J=9.3Hz,1H),7.42–7.37(m,2H),4.66(s ,2H),4.03(t,J=7.0Hz,2H),4.01–3.95(m,1H),3.72(s,3H),2.91(t,J=6.9Hz,2H),2.62(s,3H),2.03–2.00(m,5H),1.86–1.64(m,4H).

[0500] Example 1.13: Synthesis of D-13

[0501] Step 1:

[0502] Compound D-3-2 (30 mg, 0.052 mmol) was dissolved in methanol (3 mL), and platinum dioxide (13 mg, 0.052 mmol) was added under nitrogen protection. The mixture was purged with hydrogen three times, and then stirred for 60 min under a hydrogen atmosphere. The reaction was monitored by LC-MS. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the target compound D-13-1 (15 mg).

[0503] LCMS(ESI)[M+H] + =582.30.

[0504] Step Two:

[0505] Compound D-13-1 (10 mg, 0.017 mmol) was dissolved in DCM (2 mL), and then trifluoroacetic acid (0.5 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 30 min, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound D-13 (1.65 mg).

[0506] LCMS(ESI)[M+H] + =482.25.

[0507] 1 H NMR (400MHz, DMSO-d6) δ8.84(s,1H),8.80(s,2H),7.93(s,1H),7.68(d,J=9.4Hz,1H),7.55(d,J=9.4Hz,1H),7.41–7.34(m,2H),4.64(t, 2H),4.00(t,J=6.9Hz,2H),3.74(s,3H),3.56–3.46(m,1H),2.89(t,J=6.9Hz,2H),2.66–2.59(m,3H),2.03(s,3H),0.94(d,J=6.9Hz,6H).

[0508] Example 1.14: Synthesis of D-14

[0509] Step 1:

[0510] Compound D-14-1 (500 mg, 2.77 mmol) and NBS (592 mg, 3.32 mmol) were dissolved in MeCN (5.0 mL), and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0–3%) to obtain the target compound D-14-2 (700 mg).

[0511] LCMS(ESI)[M+H] + =259.07.

[0512] 1 H NMR (400MHz, DMSO-d6) δ4.29(q,J=7.1Hz,2H),4.25–4.20(m,2H),2.90–2.84(m,2H),2.62–2.58(m,2H),1.31(t,J=7.1Hz,3H).

[0513] Step Two:

[0514] Compound D-14-2 (700 mg, 2.77 mmol), potassium ethylene trifluoroborate (542 mg, 4.05 mmol), dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) (191 mg, 0.28 mmol), and potassium phosphate (1.43 g, 6.75 mmol) were dissolved in dioxane (12.0 mL) and water (3.0 mL). The mixture was heated to 80 °C for 2.5 h under nitrogen protection. The reaction was monitored by LCMS. The reaction solution was cooled to room temperature, and extracted with water (30 mL) and EA (15 mL * 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0–3%) to obtain the target compound D-14-3 (290 mg).

[0515] LCMS(ESI)[M+H] + =207.18.

[0516] Step 3:

[0517] Compound D-14-3 (290 mg, 1.41 mmol) and diacetic acid iodobenzene (454 mg, 1.41 mmol) were dissolved in acetonitrile (6.0 mL), cooled to -10 °C, and sulfuric acid (5%, 0.6 mL) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed for 1.0 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure and extracted with EA (10 mL) and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-14-4 (250 mg).

[0518] LCMS(ESI)[M+H] + =223.17.

[0519] Step Four:

[0520] Compound D-14-4 (250 mg, 1.12 mmol) was dissolved in ethanol (4.0 mL), cooled to 0 °C, and then sodium borohydride (93 mg, 2.46 mmol) was added and the reaction was allowed to proceed for 0.5 h. The reaction was monitored by LCMS. DCM (15 mL), methanol (1.5 mL), and water (10 mL) were added to the reaction mixture for extraction. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0–3%) to obtain the target compound D-14-5 (170 mg).

[0521] LCMS(ESI)[M+H] + =225.21.

[0522] 1 H NMR (400MHz, DMSO-d6) δ4.57(t,J=5.4Hz,1H),4.25(q,J=7.1Hz,2H),4.14–4.07(m,2H),3.56–3 .51(m,2H),2.88–2.83(m,2H),2.80(t,J=7.0Hz,2H),2.59–2.54(m,2H),1.29(t,J=7.1Hz,3H).

[0523] Step 5:

[0524] Compounds D-14-6 (240 mg, 0.62 mmol), D-14-5 (167 mg, 0.74 mmol), and cyanomethylenetri-n-butylphosphine (300 mg, 1.24 mmol) were dissolved in toluene (5.0 mL). Under nitrogen protection, the mixture was heated to 110 °C and stirred for 16 h. The reaction was monitored by LC-MS. Extraction was performed by adding DCM (15 mL), methanol (1.5 mL), and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0–5%) to obtain the target compound D-14-7 (165 mg).

[0525] LCMS(ESI)[M+H] + =596.26.

[0526] 1H NMR(400MHz, DMSO-d6)δ8.46(s,1H),7.66(s,1H),7.57(d,J=9.3Hz,1H),7.32–7.26(m,3H),4.78(s,2H),4.18–4.09(m,4H),3.9 1(t,J=7.3Hz,2H),2.92(t,J=6.7Hz,2H),2.70(s,3H),2.52–2.47(m,2H),2.34–2.25(m,2H),1.36(s,9H),1.20(t,J=7.1Hz,3H).

[0527] Step Six:

[0528] Compound D-14-7 (140 mg, 0.24 mmol) was dissolved in methanol (2.0 mL) and water (2.0 mL), and then lithium hydroxide monohydrate (40.3 mg, 0.96 mmol) was added. The mixture was heated to 45 °C and stirred for 4 h. The reaction was monitored by LCMS. The reaction solution was kept at 0 °C, and the pH was adjusted to 2 with dilute hydrochloric acid (1 N). The mixture was extracted with DCM (10 mL), and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-14-8 (100 mg).

[0529] LCMS(ESI)[M+H] + =568.25.

[0530] Step Seven:

[0531] Compound D-14-8 (100 mg, 0.18 mmol), dimethylhydroxylamine hydrochloride (35 mg, 0.36 mmol), EDCI (51.7 mg, 0.27 mmol), HOBT (36.5 mg, 0.27 mmol), and TEA (72.9 mg, 0.72 mmol) were dissolved in THF (5.0 mL). After the addition was complete, the mixture was heated to 40 °C and stirred for 16 h. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH, DCM = 0–8%) to obtain the target compound D-14-9 (100 mg).

[0532] LCMS(ESI)[M+H] + =611.27.

[0533] Step 8:

[0534] Compound D-14-9 (100 mg, 0.16 mmol) was dissolved in THF (2.0 mL), cooled to 0 °C, and cyclopropylmagnesium bromide (0.8 mL, 1 N) was slowly added. After the addition was complete, the mixture was naturally heated to 25 °C and stirred for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride aqueous solution (1 mL), concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0–8%) to obtain the target compound D-14-10 (80 mg).

[0535] LCMS(ESI)[M+H] + =592.38.

[0536] Step Nine:

[0537] Compound D-14-10 (60 mg, 0.10 mmol) was dissolved in TFA / DCM (2 mL, 20%, V / V) and stirred for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-14 (20.5 mg).

[0538] LCMS(ESI)[M+H] + =492.16.

[0539] 1 H NMR (400MHz, DMSO-d6) δ8.54(s,1H),8.46(s,1H),7.78(s,1H),7.56(d,J=9.4Hz,1 H),7.42(dd,J=9.4,1.3Hz,1H),7.30–7.26(m,1H),7.15(dd,J=16.8,9.2Hz,1H),4 .46(s,2H),4.26(t,J=6.2Hz,2H),3.98–3.91(m,2H),3.03(t,J=6.2Hz,2H),2.91– 2.83(m,1H),2.74–2.70(m,2H),2.68(s,3H),2.53–2.43(m,2H),0.97–0.93(m,4H).

[0540] Example 1.15: Synthesis of D-15

[0541] Step 1:

[0542] Compound D-3-1 (60 mg, 0.10 mmol) was dissolved in tetrahydrofuran (2 mL), cooled to 0 °C in an ice bath under nitrogen protection, and then methylmagnesium bromide (60 mg, 0.50 mmol) was added dropwise. After the addition was complete, the reaction mixture was moved to room temperature and stirred for 30 min. The reaction was monitored by LCMS. The reaction mixture was quenched with methanol (1 mL) and then purified by direct reversed-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the target compound D-15-1 (40 mg).

[0543] LCMS(ESI)[M+H] + =554.28.

[0544] 1 H NMR(400MHz, DMSO-d6)δ9.34(s,1H),7.66(s,1H),7.56(d,J=9.2Hz,1H),7.26(dd,J=9.3,1.7Hz,1H),7.21–7.15(m,1H),6.79–6.75 (m,1H),4.79(s,2H),3.99(t,J=6.8Hz,2H),3.69(s,3H),2.85(t,J=6.7Hz,2H),2.70(s,3H),2.29(s,3H),1.91(s,3H),1.35(s,9H).

[0545] Step Two:

[0546] Compound D-15-1 (40 mg, 0.072 mmol) was dissolved in DCM (2 mL), and then trifluoroacetic acid (0.5 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain trifluoroacetate of the target compound D-15 (20.21 mg).

[0547] LCMS(ESI)[M+H] + =454.39.

[0548] 1 H NMR (400MHz, DMSO-d6) δ8.88(s,3H),8.02(s,1H),7.75(d,J=9.3Hz,1H),7.64(d,J=9.3Hz,1H),7.42–7.32(m,2 H),4.66(t,2H),4.03(t,J=6.9Hz,2H),3.73(s,3H),2.88(t,2H),2.65–2.60(m,3H),2.29(s,3H),2.04(s,3H).

[0549] Example 1.16: Synthesis of D-16

[0550] Step 1:

[0551] Compound D-16-1 (30 mg, 0.05 mmol) was dissolved in DCM (3 mL), and TFA (0.3 mL) was added with stirring at room temperature. The reaction was continued with stirring for 1 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and DCM (3 x 3 mL) was added to concentrate and remove TFA. The crude product was dissolved in DMF (2 mL) and purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-90%) to obtain the formate of the target compound D-16 (6.5 mg).

[0552] LCMS(ESI)[M+H] + =496.18.

[0553] 1 H NMR(400MHz,DMSO-d6)δ8.54(s,1H),8.32(s,1H),7.58–7.51(m,2H),7.35–7.26(m,3H),4.05(s,2H ),4.01(t,J=6.9Hz,2H),3.71(s,3H),2.89(t,J=6.9Hz,2H),2.29(s,3H),1.93(s,3H),1.25(s,9H).

[0554] Example 1.17: Synthesis of D-17

[0555] Step 1:

[0556] Compound D-17-1 (8.0 g, 32.38 mmol), NBS (6.05 mg, 34.00 mmol), and azobisisobutyronitrile (0.53 g, 3.24 mmol) were dissolved in carbon tetrachloride (80 mL). After the addition was complete, the mixture was heated to 85 °C and stirred for 2 h under nitrogen protection. The reaction solution was concentrated under reduced pressure, and the crude product was purified by normal-phase column chromatography (EA:PE = 0-30%) to obtain the target compound D-17-2 (8.8 g).

[0557] LCMS(ESI)[M+H] + =324.95.

[0558] 1H NMR (400MHz, DMSO-d6) δ4.78 (s, 2H), 4.28 (q, J = 8.0Hz, 2H), 3.97 (s, 3H), 1.29 (t, J = 8.0Hz, 3H).

[0559] Step Two:

[0560] Compound D-17-2 (5.0 g, 16.03 mmol) was dissolved in DMSO (50 mL) and saturated sodium bicarbonate aqueous solution (20 mL). After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-40%) to obtain the target compound D-17-3 (3.5 g).

[0561] LCMS(ESI)[M+H] + =263.18.

[0562] Step 3:

[0563] Compound D-17-3 (3.50 g, 13.30 mmol), TBDPSCl (4.02 g, 14.63 mmol), and imidazole (2.72 g, 39.90 mmol) were dissolved in DCM (35 mL). After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (EA:PE = 0%-20%) to obtain the target compound D-17-4 (5.30 g).

[0564] LCMS(ESI)[M+H] + =501.11.

[0565] 1 H NMR (400MHz, DMSO-d6) δ7.66–7.57(m,4H),7.51–7.40(m,6H),4.76(s,2H),4.27(q,J=8.0Hz,2H),3.98(s,3H),1.28(t,J=8.0Hz,3H),0.99(s,9H).

[0566] Step Four:

[0567] Compound D-17-4 (5.0 g, 9.97 mmol), potassium trifluoroborate (2.00 g, 14.96 mmol), PdCl2 (dtbpf) (649.79 mg, 1.0 mmol), and potassium phosphate (6.35 g, 29.91 mmol) were dissolved in DMF (50 mL) and water (10 mL). After the addition was complete, the mixture was heated to 80 °C under nitrogen protection and stirred for 2 h. The reaction solution was poured into EA (500 mL), allowed to stand, separated, and the organic phase was washed three times with water (300 mL * 3). The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (EA: PE = 0% - 20%) to obtain the target compound D-17-5 (3.30 g).

[0568] LCMS(ESI)[M+H] + =449.21.

[0569] 1 H NMR (400MHz, DMSO-d6) δ7.66–7.55(m,4H),7.53–7.37(m,6H),6.77(dd,J=20.0,12.0Hz,1H),5.33 –5.10(m,2H),4.77(s,2H),4.25(q,J=8.0Hz,2H),3.79(s,3H),1.27(t,J=8.0Hz,3H),0.98(s,9H).

[0570] Step 5:

[0571] Compound D-17-5 (2.0 g, 4.46 mmol) was dissolved in acetonitrile (40 mL), cooled to 0 °C, and iodophthalic acid (1.51 mg, 4.68 mmol) was added. The mixture was stirred at 0 °C for 10 min, followed by the addition of 5% sulfuric acid aqueous solution (4 mL, V / V). The mixture was stirred at room temperature for 30 min. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution (20 mL), concentrated under reduced pressure to remove acetonitrile, and the aqueous phase was extracted with DCM (40 mL * 3). The organic phase was dried, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-17-6 (1.8 g).

[0572] LCMS(ESI)[M+H] + =465.18.

[0573] Step Six:

[0574] Compound D-17-6 (1.8 g, 3.87 mmol) was dissolved in ethanol (40 mL), cooled to 0 °C, and sodium borohydride (0.59 g, 15.48 mmol) was added. The mixture was stirred at 0 °C for 1 h. The reaction solution was quenched with saturated ammonium chloride aqueous solution (5 mL), concentrated under reduced pressure, and the crude product was directly purified by column chromatography (EA:PE = 0%-60%) to obtain the target compound D-17-7 (1.5 g).

[0575] LCMS(ESI)[M+H] + =467.23.

[0576] 1 H NMR (400MHz, DMSO-d6) δ7.63–7.60(m,4H),7.50–7.43(m,6H),4.70(s,2H),4.43(t,J=8.0Hz,1H),4.23(q ,J=8.0Hz,2H),3.89(s,3H),3.32–3.28(m,2H),2.52(t,J=8.0Hz,2H),1.27(t,J=8.0Hz,3H),0.97(s,9H).

[0577] Step Seven:

[0578] Compounds D-17-7 (100 mg, 0.21 mmol), D-17-8 (81.77 mg, 0.21 mmol), and cyanomethylenetri-n-butylphosphine (101.37 g, 0.42 mmol) were dissolved in toluene (2 mL). The mixture was heated to reflux and stirred for 16 h under nitrogen protection. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (EA:PE = 0%-70%) to obtain the target compound D-17-9 (120.0 mg).

[0579] LCMS(ESI)[M+H] + =838.36.

[0580] 1 H NMR (400MHz, DMSO-d6) δ7.62(s,1H),7.56–7.52(m,4H),7.48–7.34(m,8H),7.29–7.11(m,3H),4.71(s,2H),4.53(s,2H),4. 07(t,J=8.0Hz,2H),3.86(t,J=8.0Hz,2H),3.75(s,3H),2.69–2.61(m,5H),1.30(s,9H),1.12(t,J=8.0Hz,3H),0.91(s,9H).

[0581] Step 8:

[0582] Compound D-17-9 (120.0 mg, 0.14 mmol) was dissolved in DCM (2.0 mL) and TFA (0.5 mL), and the mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain the target compound D-17-10 (105.0 mg), which was directly used in the next step of the reaction.

[0583] LCMS(ESI)[M+H] + =738.31.

[0584] Step Nine:

[0585] Compound D-17-10 (105.0 mg, 0.14 mmol) was dissolved in THF (2.0 mL) and formaldehyde aqueous solution (0.5 mL). Sodium cyanoborohydride (35.19 mg, 0.56 mmol) was added with stirring. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was quenched with saturated ammonium chloride solution (0.5 mL), concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0%-5%) to obtain the target compound D-17-11 (86.0 mg).

[0586] LCMS(ESI)[M+H] + =752.38.

[0587] 1 H NMR (400MHz, DMSO-d6) δ7.69–7.50(m,6H),7.57–7.43(m,4H),7.44–7.28(m,6H),4.79(s,2H),4.62(s,2H),4.06(q,J=8.0 Hz,2H),3.86(t,J=8.0Hz,2H),3.81(s,3H),2.88–2.76(m,6H),2.67(t,J=8.1Hz,2H),1.16(t,J=8.1Hz,3H),0.94(s,9H).

[0588] Step 10:

[0589] Compound D-17-11 (86.0 mg, 0.11 mmol) and lithium hydroxide monohydrate (48.05 mg, 1.10 mmol) were dissolved in methanol (1 mL) and water (0.5 mL). The reaction mixture was stirred at 30 °C for 1 h. The reaction solution was then purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% HCl = 0%-60%) to obtain the target compound D-17-12 (70.0 mg).

[0590] LCMS(ESI)[M+H] + =724.35.

[0591] Step Eleven:

[0592] Compound D-17-12 (70.0 mg, 0.086 mmol), HOBT (23.24 mg, 0.17 mmol), EDCI (49.46 mg, 0.26 mmol), and triethylamine (43.51 mg, 0.43 mmol) were dissolved in THF (1.0 mL), and the mixture was stirred at 35 °C for 16 h. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0%-5%) to obtain the target compound D-17-13 (50.0 mg).

[0593] LCMS(ESI)[M+H] + =767.31.

[0594] Step Twelve:

[0595] Compound D-17-13 (50.0 mg, 0.065 mmol) was dissolved in THF (1.0 mL), cooled to 0 °C, and cyclopropylmagnesium bromide (1 mL, 1 M) was added. After the addition was complete, the mixture was moved to room temperature and stirred for 1 h. The reaction solution was quenched with saturated ammonium chloride aqueous solution (0.5 mL), concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH: DCM = 0%-5%) to obtain the target compound D-17-14 (30.0 mg).

[0596] LCMS(ESI)[M+H] + =748.40.

[0597] Step Thirteen:

[0598] Compound D-17-14 (30.0 mg, 0.040 mmol) and TBAF (52.29 mg, 0.20 mmol) were dissolved in THF (1.0 mL) and stirred at room temperature for 1 h. The reaction solution was then purified directly by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.1% TFA = 0%-50%) to obtain the target compound D-17 (3.32 mg).

[0599] LCMS(ESI)[M+H] + =510.28.

[0600] 1H NMR(400MHz, Methanol-d4)δ8.46(s,1H),7.62(s,1H),7.49(d,J=9.4Hz,1H),7.33(d,J=9.2Hz,1H),7.27–7.18(m,1H),7.17–7.05(m,1H) ,4.44(s,2H),4.02(t,J=6.6Hz,2H),3.97(s,2H),3.88(s,3H),3.02(t,J=6.6Hz,2H),2.98-2.33(m,1H),2.37(s,6H),0.89–0.84(m,4H).

[0601] Example 1.18: Synthesis of D-18

[0602] Step 1:

[0603] Compound D-3-3 (400 mg, 0.69 mmol) was dissolved in DCM (4 mL), followed by the addition of acetic anhydride (414 mg, 6.90 mmol), DIPEA (1.78 g, 13.80 mmol), and DMAP (84 mg, 0.69 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by normal-phase column chromatography (DCM:MeOH = 20:1) to give the target compound D-18-1 (320 mg).

[0604] LCMS(ESI)[M+H] + =624.24.

[0605] 1 H NMR (400MHz, DMSO-d6) δ8.52(s,1H),7.65(s,1H),7.57(d,J=9.3Hz,1H),7.45–7.12(m,3H),5.94(s,1H),4.78(s,2H),4.69(d,J= 8.9Hz,2H),3.89–3.76(m,2H),3.55(s,3H),2.69(s,3H),2.65–2.59(m,1H),1.91(s,3H),1.82(s,3H),1.47(s,3H),1.34(s,9H).

[0606] Step Two:

[0607] Compound D-18-1 (150 mg, 0.24 mmol) was dissolved in DCM (2 mL), and then TFA (0.5 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound D-18 (100 mg).

[0608] LCMS(ESI)[M+H] + =524.15.

[0609] 1 H NMR(400MHz,DMSO-d6)δ9.17(s,2H),8.98(s,1H),8.12(s,1H),7.85–7.74(m,2H),7.48–7.34(m,2H),5.98(s,1H ),4.81–4.79(m,2H),4.72(s,2H),4.03–3.88(m,2H),3.60(s,3H),2.75–2.61(m,5H),1.94(s,6H),1.54(s,3H).

[0610] Example 1.19: Synthesis of D-19

[0611] Step 1:

[0612] Compound 3-iodo-1,5-dimethyl-1H-pyrazole (800 mg, 3.60 mmol) and D-19-1 (740 mg, 5.04 mmol) were dissolved in THF (5.0 mL), cooled to 0 °C, and a solution of isopropyl magnesium bromide and lithium bromide (1.3 N, 5.54 mL) was slowly added. The reaction was continued at 0 °C for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride solution, and extracted with water (30 mL) and EA (15 mL * 2). The organic phase was dried, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (EA:PE = 5–25%) to obtain the target compound D-19-2 (470 mg).

[0613] LCMS(ESI)[M+H] + =183.14.

[0614] 1 H NMR (400MHz, DMSO-d6) δ6.65(s,1H),3.82(s,3H),2.31(s,3H),1.84–1.78(m,2H),1.61–1.52(m,2H).

[0615] Step Two:

[0616] Compound D-19-2 (460 mg, 2.52 mmol) and NBS (492 mg, 2.77 mmol) were dissolved in MeCN (5.0 mL), and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by normal-phase column chromatography (MeOH:DCM = 0–3%) to obtain the target compound D-19-3 (550 mg).

[0617] LCMS(ESI)[M+H] + =261.03.

[0618] Step 3:

[0619] Compound D-19-3 (550 mg, 2.11 mmol), potassium ethylene trifluoroborate (565 mg, 4.22 mmol), dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) (137.5 mg, 0.21 mmol), and potassium phosphate (1.12 g, 5.27 mmol) were dissolved in DMF (12.0 mL) / water (3.0 mL) and reacted at 80 °C for 2 h under nitrogen protection. The reaction was monitored by LCMS. The reaction solution was cooled to room temperature, extracted with water (30 mL) and EA (15 mL * 2), and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal-phase column chromatography (MeOH:DCM = 0–3%) to obtain the target compound D-19-4 (310 mg).

[0620] LCMS(ESI)[M+H] + =209.11.

[0621] Step Four:

[0622] Compound D-19-4 (310 mg, 1.49 mmol) and iodobenzene diacetate (480 mg, 1.49 mmol) were dissolved in acetonitrile (6.2 mL), cooled to -10 °C, and then sulfuric acid (5%, 0.62 mL) was slowly added dropwise. After the addition was complete, the reaction was maintained at -10 °C for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and then extracted with EA (10 mL) and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-19-5 (240 mg).

[0623] LCMS(ESI)[M+H] + =225.08.

[0624] Step 5:

[0625] Compound D-19-5 (160 mg, 0.71 mmol) was dissolved in ethanol (3.0 mL), cooled to 0 °C, and then sodium borohydride (53.7 mg, 1.42 mmol) was added. The reaction was maintained at 0 °C for 0.5 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride solution, and then extracted with DCM (15 mL), methanol (1.5 mL), and water (10 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (MeOH:DCM = 0–8%) to obtain the target compound D-19-6 (80 mg).

[0626] LCMS(ESI)[M+H] + =229.13.

[0627] Step Six:

[0628] Compounds D-17-8 (125 mg, 0.31 mmol), D-19-6 (73 mg, 0.32 mmol), and cyanomethylenetri-n-butylphosphine (154 mg, 0.64 mmol) were dissolved in toluene (3.0 mL). Under nitrogen protection, the mixture was heated to 110 °C and stirred for 16 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography (acetonitrile: H₂O containing 0.05% FA = 10%-70%) to obtain the target compound D-19-7 (15 mg).

[0629] LCMS(ESI)[M+H] + =600.14.

[0630] 1 H NMR (400MHz, DMSO-d6) δ8.52–8.46(m,1H),7.69–7.58(m,2H),7.43–7.22(m,3H),5.39(s,1H),4.81(s,2H),4.57( d,J=16.9Hz,1H),4.00–3.85(m,2H),3.55(s,3H),2.83–2.67(m,5H),1.79(s,3H),1.37(s,9H),0.89–0.66(m,4H).

[0631] Step Nine:

[0632] Compound D-19-7 (12 mg, 0.02 mmol) was dissolved in TFA / DCM (20%, 1 mL). After the addition was complete, the mixture was stirred for 1.5 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain trifluoroacetate of the target compound D-19 (5.0 mg).

[0633] LCMS(ESI)[M+H] + =500.16.

[0634] 1 H NMR (400MHz, DMSO-d6) δ9.12(s,2H),9.03(s,1H),8.20(s,1H),7.92–7.85(m,2H),7.47–7.37(m,2H),4.74(s,2H),4. 52(d,J=18.4Hz,1H),4.08–4.04(m,2H),3.59(s,3H),2.86–2.74(m,2H),2.68(s,3H),1.99(s,3H),0.96–0.68(m,4H).

[0635] Example 1.20: Synthesis of D-20

[0636] Method 1:

[0637] Step 1:

[0638] Compounds 3-iodo-1,5-dimethyl-1H-pyrazole (3.0 g, 13.51 mmol) and D-20-1 (3.09 g, 21.62 mmol) were dissolved in THF (20.0 mL), cooled to 0 °C, and then a solution of isopropyl magnesium bromide and lithium bromide (20.8 mL, 1.3 N) was slowly added. After the addition was complete, the reaction was stirred at 0 °C for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride aqueous solution (10 mL), and then extracted with EA (50 mL) and water (30 mL) sequentially. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (EA:PE = 5-25%) to obtain the target compound D-20-2 (1.3 g).

[0639] LCMS(ESI)[M+H] + =179.16.

[0640] 1 H NMR (400MHz, DMSO-d6) δ6.45(s,1H),3.79(s,3H),2.27(s,3H),1.70(q,J=3.4Hz,2H),1.39(s,3H),0.85(q,J=3.5Hz,2H).

[0641] Step Two:

[0642] Compound D-20-2 (1.3 g, 7.29 mmol) and NBS (1.43 g, 8.02 mmol) were dissolved in MeCN (15.0 mL), and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was directly concentrated under reduced pressure, and the crude product was purified by normal-phase column chromatography (EA:PE = 10-25%) to obtain the target compound D-20-3 (1.8 g).

[0643] LCMS(ESI)[M+H] + =257.03.

[0644] 1 H NMR (400MHz, DMSO-d6) δ3.86(s,3H),2.27(s,3H),1.56(q,J=3.6Hz,2H),1.40(s,3H),0.87(q,J=3.6Hz,2H).

[0645] Step 3:

[0646] Compound D-20-3 (1.8 g, 7.00 mmol), potassium ethylene trifluoroborate (1.88 g, 14 mmol), dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) (456 mg, 0.7 mmol), and potassium phosphate (3.71 g, 17.5 mmol) were dissolved in DMF (20.0 mL) and water (5.0 mL). Under nitrogen protection, the mixture was heated to 80 °C and stirred for 2.0 h. The reaction was monitored by LCMS. The reaction solution was cooled to room temperature, and extracted with water (100 mL) and EA (30 mL x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal-phase column chromatography (MeOH:DCM = 0-3%) to obtain the target compound D-20-4 (950 mg).

[0647] LCMS(ESI)[M+H] + =205.14.

[0648] 1 H NMR (400MHz, DMSO-d6) δ6.88–6.80(m,1H),5.33–5.16(m,2H),3.81(s,3H),2.33(s,3H),1.55(q,J=3.5Hz,2H),1.39(s,3H),0.84(q,J=3.5Hz,2H).

[0649] Step Four:

[0650] Compound D-20-4 (0.95 g, 4.65 mmol) and iodobenzene diacetate (1.5 g, 4.65 mmol) were dissolved in acetonitrile (19 mL), cooled to -10 °C, and sulfuric acid (1.9 mL, 5%) was slowly added dropwise. After the addition was complete, the mixture was stirred for 1.0 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure and extracted with EA (20 mL) and water (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-20-5 (730 mg), which was directly used in the next reaction.

[0651] LCMS(ESI)[M+H] + =221.13.

[0652] Step 5:

[0653] Compound D-20-5 (730 mg, 3.31 mmol) was dissolved in ethanol (5.0 mL), cooled to -0 °C, and then sodium borohydride (100 mg, 2.65 mmol) was added. After the addition was complete, the mixture was stirred for 0.5 h. The reaction was monitored by LCMS. After quenching the reaction mixture, DCM (15 mL), methanol (1.5 mL), and water (10 mL) were added for extraction. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal-phase column chromatography (MeOH:DCM = 0–8%) to obtain the target compound D-20-6 (70 mg).

[0654] LCMS(ESI)[M+H] + =223.13.

[0655] 1 H NMR (400MHz, DMSO-d6) δ4.58(t,J=5.3Hz,1H),3.84(s,3H),3.46(dd,J=12.3,6.9Hz,2H),2. 75(t,J=7.1Hz,2H),2.26(s,3H),1.67(q,J=3.4Hz,2H),1.46(s,3H),0.86(q,J=3.5Hz,2H).

[0656] Step Six:

[0657] Compounds D-17-8 (110 mg, 0.28 mmol), D-20-6 (62 mg, 0.28 mmol), and CMBP (135 mg, 0.64 mmol) were dissolved in toluene (3.0 mL). Under nitrogen protection, the mixture was heated to 110 °C and reacted for 16 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography (acetonitrile: H₂O containing 0.05% FA = 10%-70%) to obtain the target compound D-20-7 (55 mg).

[0658] LCMS(ESI)[M+H] + =594.17.

[0659] 1 H NMR (400MHz, DMSO-d6) δ8.86(s,1H),8.24(s,1H),7.94–7.88(m,2H),7.40–7.31(m,2H),4.90(s,2H),3.94(t,J=6.9Hz,2 H),3.73(s,3H),2.84–2.76(m,5H),2.06(s,3H),1.44(d,J=2.7Hz,2H),1.37(s,9H),1.23(s,3H),0.71(q,J=3.5Hz,2H).

[0660] Step Seven:

[0661] Compound D-20-7 (15 mg, 0.025 mmol) was dissolved in TFA / DCM (1 mL, 20%) and the mixture was stirred for 1.5 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-20 (13.0 mg).

[0662] LCMS(ESI)[M+H] + =494.13.

[0663] 1 H NMR (400MHz, DMSO-d6) δ8.94–8.88(m,3H),8.06(s,1H),7.81–7.78(m,1H),7.71–7.69(m,1H),7.46–7.37(m,2H),4.71(s,2H),4.04(t,J=6. 9Hz, 2H), 3.73 (s, 3H), 2.85 (t, J = 7.0Hz, 2H), 2.67 (t, J = 4.9Hz, 3H), 2.05 (s, 3H), 1.52 (q, J = 3.3Hz, 2H), 1.30 (s, 3H), 0.76 (q, J = 3.6Hz, 2H).

[0664] Method 2:

[0665] Step 1:

[0666] Compound D-3-1 (100 mg, 0.17 mmol) was dissolved in THF (2.0 mL), cooled to 0 °C, and then (1-methylcyclopropyl)magnesium bromide (0.5 mL, 2.5 N) was slowly added. After the addition was complete, the mixture was allowed to rise naturally to room temperature and stirred for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride, concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0-10%) to obtain the target compound D-20-7 (20 mg).

[0667] LCMS(ESI)[M+H]+=594.17.

[0668] 1 H NMR (400MHz, DMSO-d6) δ8.86(s,1H),8.24(s,1H),7.94–7.88(m,2H),7.40–7.31(m,2H),4.90(s,2H),3.94(t,J=6.9Hz,2 H),3.73(s,3H),2.84–2.76(m,5H),2.06(s,3H),1.44(d,J=2.7Hz,2H),1.37(s,9H),1.23(s,3H),0.71(q,J=3.5Hz,2H).

[0669] Step Two:

[0670] Compound D-20-7 (20 mg, 0.034 mmol) was dissolved in TFA / DCM (1 mL, 20%) and stirred for 1.5 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-20 (15.0 mg).

[0671] LCMS(ESI)[M+H] + =494.13.

[0672] 1H NMR (400MHz, DMSO-d6) δ8.94–8.88(m,3H),8.06(s,1H),7.81–7.78(m,1H),7.71–7.69(m,1H),7.46–7.37(m,2H),4.71(s,2H),4.04(t,J=6. 9Hz, 2H), 3.73 (s, 3H), 2.85 (t, J = 7.0Hz, 2H), 2.67 (t, J = 4.9Hz, 3H), 2.05 (s, 3H), 1.52 (q, J = 3.3Hz, 2H), 1.30 (s, 3H), 0.76 (q, J = 3.6Hz, 2H).

[0673] Example 1.21: Synthesis of D-21

[0674] Step 1:

[0675] Compounds 3-iodo-1,5-dimethyl-1H-pyrazole (5.12 g, 20.1 mmol) and D-21-1 (3.4 g, 20.8 mmol) were dissolved in THF (5.0 mL), cooled to 0 °C, and a solution of isopropyl magnesium bromide and lithium bromide (21 mL, 1.3 N) was slowly added. After the addition was complete, the mixture was stirred at 0 °C for 1 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride aqueous solution (2 mL), concentrated under reduced pressure, and the crude product was purified by column chromatography (EA:PE = 5-25%) to obtain the target compound D-21-2 (3.2 g).

[0676] LCMS(ESI)[M+H] + =199.26.

[0677] Step Two:

[0678] Compound D-21-2 (500 mg, 2.5 mmol) and NBS (538 mg, 3 mmol) were dissolved in MeCN (5.0 mL), and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0-3%) to obtain the target compound D-21-3 (580 mg).

[0679] LCMS(ESI)[M+H] + =277.02.

[0680] Step 3:

[0681] Compound D-21-3 (672 mg, 2.42 mmol), potassium ethylene trifluoroborate (648 mg, 4.84 mmol), dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) (158 mg, 0.24 mmol), and potassium phosphate (1.3 g, 6.1 mmol) were dissolved in DMF (12.0 mL) and water (3.0 mL). The reaction mixture was stirred at 80 °C for 2.0 h under nitrogen protection. The reaction was monitored by LCMS. The reaction solution was cooled to room temperature, and extracted with water (30 mL) and EA (15 mL x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0-3%) to obtain the target compound D-21-4 (410 mg).

[0682] LCMS(ESI)[M+H] + =225.12.

[0683] Step Four:

[0684] Compound D-21-4 (150 mg, 0.67 mmol) and diacetic acid iodobenzene (216 mg, 0.67 mmol) were dissolved in acetonitrile (2 mL), cooled to -20 °C, and then sulfuric acid (0.2 mL, 5%) was slowly added dropwise. After the addition was complete, the mixture was stirred for 30 min. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure and extracted with EA (10 mL) and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-21-5 (170 mg), which was directly used in the next reaction.

[0685] LCMS(ESI)[M+H] + =241.10.

[0686] Step 5:

[0687] Compound D-21-5 (161 mg, 0.67 mmol) was dissolved in ethanol (3.0 mL), cooled to -10 °C, and then sodium borohydride (10 mg, 0.27 mmol) was added. After the addition was complete, the mixture was stirred for 0.5 h. The reaction was monitored by LCMS. The reaction solution was extracted with EA (15 mL) and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to column chromatography (MeOH:DCM = 0-8%) to obtain the target compound D-21-6 (85 mg).

[0688] LCMS(ESI)[M+H] + =243.12.

[0689] Step Six:

[0690] Compound D-21-6 (85 mg, 0.35 mmol) was dissolved in dichloromethane (3.0 mL), and triethylamine (106 mg, 1.05 mmol) and MsCl (80 mg, 0.7 mmol) were added under ice bath conditions. The mixture was stirred under ice bath conditions for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (PE:EA = 0-50%) to obtain the target compound D-21-7 (70 mg).

[0691] LCMS(ESI)[M+H] + =321.01.

[0692] Step Seven:

[0693] Compounds D-21-7 (50 mg, 0.16 mmol) and D-17-8 (50 mg, 0.13 mmol) were dissolved in DMF (1 mL), and then cesium carbonate (104 mg, 0.32 mmol) was added. The mixture was stirred at 80 °C for 12 h. The reaction was monitored by LCMS. The reaction solution was extracted with EA (15 mL) and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to column chromatography (MeOH:DCM = 0-8%) to obtain the target compound D-21-8 (50 mg).

[0694] LCMS(ESI)[M+H] + =614.19.

[0695] Step 8:

[0696] Compound D-21-8 (10 mg, 0.016 mmol) was dissolved in DCM (3 mL), and then TFA (0.3 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 30 min, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in DMF (2 mL). The crude product was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain the target compound D-21 (3.3 mg).

[0697] LCMS(ESI)[M+H] + =514.22.

[0698] 1 H NMR (400MHz, DMSO-d6) δ8.89–8.85(m,3H),7.97(s,1H),7.71(d,J=9.3Hz,1H),7.58(d,J=9.3Hz,1H),7.44–7.37(m,2H),4.72–4.66(m ,2H),4.05(t,J=6.8Hz,2H),3.74(s,3H),2.89(t,J=6.9Hz,2H),2.68–2.63(m,3H),2.12–2.08(m,2H),2.05(s,3H),1.56–1.48(m,2H).

[0699] Example 1.22: Synthesis of D-22

[0700] Step 1:

[0701] Compound D-21-8 (25 mg, 0.041 mmol) was dissolved in ethanol (3 mL), cooled to 0 °C, and sodium borohydride (2 mg, 0.053 mmol) was added with stirring. After the addition was complete, the reaction was stirred at room temperature for 30 min. The reaction was monitored by LCMS. The reaction solution was quenched with 5 mL of water, extracted with DCM (5 mL x 3), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-22-1 (30 mg), which was directly used in the next step of the reaction.

[0702] LCMS(ESI)[M+H] + =616.23.

[0703] Step Two:

[0704] Compound D-22-1 (30 mg, 0.05 mmol) was dissolved in DCM (3 mL), and TFA (0.3 mL) was added with stirring at room temperature. After the addition was complete, the reaction was stirred for 1 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and DCM (3 x 3 mL) was added to concentrate and remove TFA. The crude product was dissolved in DMF (2 mL) and purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound D-22 (3.8 mg).

[0705] LCMS(ESI)[M+H] + =516.24.

[0706] 1 H NMR(400MHz,DMSO-d6)δ9.03(s,2H),8.92–8.84(m,1H),8.06–7.95(m,1H),7.82–7.64(m,2H),7.45–7.32(m,2H),4.67(s,2H),4.46(s,1H),4.0 8–3.99(m,2H),3.55–3.54(m,4H),2.90–2.81(m,1H),2.76–2.69(m,1H) ,2.67–2.61(m,3H),1.92(s,3H),1.07–0.97(m,2H),0.92–0.79(m,2H).

[0707] Example 1.23: Synthesis of D-23

[0708] Step 1:

[0709] Compound D-20-7 (20 mg, 0.034 mmol) was dissolved in methanol (2.0 mL), cooled to 0 °C, and then sodium borohydride (2.6 mg, 0.068 mmol) was slowly added. After the addition was complete, the mixture was stirred at 0 °C for 15 min. The reaction was monitored by LCMS. The reaction was quenched by adding saturated ammonium chloride aqueous solution, and extracted with DCM (10 mL) and water (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound D-23-1 (20 mg).

[0710] LCMS(ESI)[M+H] + =596.20.

[0711] Step Two:

[0712] Compound D-23-1 (20 mg, 0.034 mmol) was dissolved in TFA / DCM (1 mL, 20%). After the addition was complete, the mixture was stirred for 1.5 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-23 (5.0 mg).

[0713] LCMS(ESI)[M+H] + =496.26.

[0714] 1 H NMR (400MHz, CD3OD) δ8.90(s,1H),8.29(s,1H),8.02–7.98(m,1H),7.92–7.86(m,1H),7.43–7.29(m,1H),7.27–7.17(m,1H),4.84(s,2H),4. 38–4.05(m,2H),3.87(s,1H),3.63(s,3H),2.94(s,3H),2.85–2.72(m ,2H),2.10(s,3H),0.93(s,3H),0.53–0.44(m,2H),0.35–0.09(m,2H).

[0715] Example 1.24: Synthesis of D-24

[0716] Step 1:

[0717] Compound D-19-5 (450 mg, 2.01 mmol) was dissolved in ethanol (3.0 mL), cooled to 0 °C, and then NaBH4 (38.02 mg, 1.00 mmol) was added. The reaction was stirred for another 0.5 h. The reaction was monitored by LCMS. The reaction solution was quenched with saturated ammonium chloride aqueous solution, extracted with DCM and water, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (MeOH:DCM = 0–5%) to obtain the target compound D-24-1 (190 mg).

[0718] LCMS(ESI)[M+H] + =227.23;

[0719] Step Two:

[0720] Compound D-24-1 (130 mg, 0.57 mmol) was dissolved in DCM (2.0 mL), and MsCl (130.6 mg, 1.14 mmol) was added and the temperature was controlled at 0 °C. Then, DIPEA (173 mg, 1.71 mmol) was added, and the reaction was continued at room temperature for 0.5 h. The reaction was monitored by LCMS. After quenching the reaction solution with saturated ammonium chloride aqueous solution, DCM and water were added for extraction. The organic phase was filtered through anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (MeOH:DCM = 0–5%) to obtain the target compound D-24-2 (130 mg).

[0721] LCMS(ESI)[M+H] + =305.06.

[0722] Step 3:

[0723] Compound D-17-8 (60 mg, 0.15 mmol), compound D-24-2 (50 mg, 0.17 mmol), and cesium carbonate (97.8 mg, 0.30 mmol) were dissolved in DMF (2.0 mL), and the mixture was heated to 80 °C and reacted for 16 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H₂O containing 0.05% TFA = 10%-70%) to obtain the target compound D-24-3 (80 mg).

[0724] LCMS(ESI)[M+H] + =598.38.

[0725] Step Four:

[0726] Compound D-24-3 (80 mg, 0.13 mmol) was dissolved in TFA / DCM (2 mL, 20%), and the reaction was stirred for 1.5 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound D-24 (45.3 mg).

[0727] LCMS(ESI)[M+H] + =498.15.

[0728] 1 H NMR (400MHz, DMSO-d6) δ8.94–8.88(m,3H),8.04(s,1H),7.76(d,J=9.3Hz,1H),7.66(d,J=9.3Hz,1H),7.46–7.36(m,2H),4.70(s,2H),4.0 7(t,J=6.8Hz,2H),3.74(s,3H),2.92(t,J=6.8Hz,2H),2.66(d,J=4.7Hz,3H),2.08(s,3H),1.76(td,J=8.9,5.4Hz,2H),1.53–1.42(m,2H).

[0729] Example 2. Synthesis of intermediates

[0730] Example 2.1: Synthesis of INT1

[0731] Step 1:

[0732] Compound INT1-1 (2.0 g, 6.02 mmol), 5-formylsalicylic acid (1.0 g, 6.02 mmol), EDCI (1.38 g, 7.22 mmol), HOBT (0.98 g, 7.22 mmol), and DIPEA (2.33 g, 18.06 mmol) were dissolved in DMF (10.0 mL) and reacted at room temperature with stirring for 16 h. The reaction was monitored by LCMS. The reaction solution was diluted with water (80 mL), extracted with EA (20 mL * 3), and the combined organic phases were dried over anhydrous sodium sulfate. The mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0-10%) to obtain the target compound INT1-2 (0.9 g).

[0733] LCMS(ESI)[M+Na] + =507.32.

[0734] Step Two:

[0735] Compound INT1-2 (900 mg, 1.86 mmol), methyl α-D-glucuronide (810 mg, 2.05 mmol), and Ag₂O (810 mg, 2.05 mmol) were dissolved in acetonitrile (5.0 mL), and the mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM = 0-10%) to obtain the target compound INT1-3 (1.2 g).

[0736] LCMS(ESI)[M+Na] + =823.26.

[0737] Step 3:

[0738] Compound INT1-3 (600 mg, 0.75 mmol) was dissolved in EA (15.0 mL), and palladium on carbon (80 mg, 10%) was added under nitrogen protection. The mixture was purged three times with hydrogen, and then stirred at room temperature for 16 h under a hydrogen atmosphere. The reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT1-4 (550 mg), which was directly used in the next step of the reaction.

[0739] LCMS(ESI)[M+H] + =803.36.

[0740] Step Four:

[0741] Compound INT1-4 (550 mg, 0.69 mmol) and di(p-nitrobenzene) carbonate (420 mg, 1.38 mmol) were dissolved in DMF (6.0 mL), and then DIPEA (270 mg, 2.07 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 0%-70%) to obtain the target compound INT1 (350 mg).

[0742] LCMS(ESI)[M+H] + =968.34.

[0743] Example 2.2: Synthesis of INT2

[0744] Step 1:

[0745] Compound INT2-1 (1.34 g, 5.02 mmol) was dissolved in N,N-dimethylformamide (10 mL), followed by the sequential addition of HATU (1.9 g, 5.02 mmol) and N,N-diisopropylethylamine (1.61 g, 12.56 mmol). The mixture was stirred at 25 °C for 0.5 h, and then compound INT2-2 (1.9 g, 5.02 mmol) was added. The reaction mixture was stirred at 25 °C for 1 h. The reaction solution was purified by reverse-phase column chromatography (0.1% TFA in water, ACN) to obtain the target compound INT2-3 (2 g).

[0746] LCMS(ESI)[M+H] + =629.6.

[0747] 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),9.12(s,2H),8.16(d,J=7.6Hz,1H),7.95(d,J=8.4Hz,1H),7.54( d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),4.42(s,2H),4.40–4.35(m,1H),4.23–4.18(m,1H),3.41(s,3H ),3.41–3.40(m,1H),3.06–2.98(m,2H),2.75(s,3H),2.74(s,3H),2.56(t,J=7.0Hz,2H),2.41–2.33( m,2H),2.01–1.95(m,1H),1.87–1.80(m,2H),1.68–1.59(m,3H),1.38–1.27(m,2H),0.89–0.82(m,6H).

[0748] Step Two:

[0749] Compound INT2-3 (1.8 g, 2.87 mmol) was dissolved in N,N-dimethylformamide (10 mL), and compound di(p-nitrobenzene) carbonate (1.74 g, 5.74 mmol) and N,N-diisopropylethylamine (740 mg, 5.74 mmol) were added. The mixture was stirred at 25 °C for 2 h, and the reaction was monitored by LCMS. Methyl tert-butyl ether (80 mL) was added to the reaction solution, and a solid precipitated. The solid was filtered, and the filter cake was slurried with anhydrous diethyl ether (100 mL) to obtain the target compound INT2 (1.8 g).

[0750] LCMS(ESI)[M+H] + =794.6.

[0751] 1H NMR (400MHz, DMSO-d6) δ10.08(s,1H),9.11(s,2H),8.31(d,J=9.1Hz,2H),8.17(d,J=7.5Hz,1H),7.93(d,J=8. 5Hz,1H),7.64(d,J=8.5Hz,2H),7.56(d,J=9.1Hz,2H),7.41(d,J=8.5Hz,2H),5.24(s,2H),4.44–4.36(m,1H),4 .25–4.17(m,1H),3.41(s,3H),2.90–2.79(m,2H),2.68–2.57(m,6H),2.58–2.54(m,2H),2.42–2.31(m,2H),2.0 5–1.92(m,1H),1.88–1.78(m,2H),1.77–1.62(m,2H),1.61–1.53(m,2H),1.41–1.26(m,2H),0.89–0.82(m,6H).

[0752] Example 2.3: Synthesis of INT3

[0753] Step 1:

[0754] Compound INT2-2 (5.0 g, 18.6 mmol), N-hydroxysuccinimide (2.57 g, 22.4 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.29 g, 22.4 mmol) were dissolved in dichloromethane (100 mL). The reaction solution was stirred at room temperature for 2 h. The reaction solution was then mixed with silica gel and purified by normal phase column chromatography (methanol:dichloromethane = 0-10%) to obtain the target compound INT3 (5.8 g).

[0755] LCMS(ESI)[M+H] + =366.12.

[0756] Example 2.4: Synthesis of INT4

[0757] Step 1:

[0758] Compound INT4-1 (1.0 g, 1.67 mmol) was dissolved in dry N,N-dimethylformamide (5 mL), and INT4-2 (1.0 g, 3.33 mmol) and N,N-diisopropylethylamine (430 mg, 3.33 mmol) were added. The reaction mixture was stirred at room temperature for 1 h under nitrogen protection. The reaction was monitored by LCMS. The reaction mixture was added dropwise to 0 °C methyl tert-butyl ether (200 mL), stirred for 10 min, and the supernatant was removed. The resulting oily substance was added to dichloromethane (10 mL), and the mixture was repeatedly stirred to obtain the target compound INT4 (1.2 g).

[0759] LCMS(ESI)[M+H] + =766.7.

[0760] 1 H NMR (400MHz, DMSO-d6) δ10.23(s,1H),8.30(d,J=9.1Hz,2H),7.89(d,J=7.5Hz,2H),7.77–7. 72(m,2H),7.69–7.64(m,2H),7.56(d,J=9.1Hz,2H),7.46–7.38(m,6H),7.32(t,J=7.5Hz,2H) .5.25(s,2H),4.63–4.33(m,1H),4.34–4.16(m,3H),4.04–3.88(m,1H),3.00–2.90(m,2H),2. 70–2.61(m,6H),2.02–1.74(m,2H),1.72–1.56(m,3H),1.43–1.30(m,2H),0.91–0.79(m,6H).

[0761] Example 2.5: Synthesis of INT5

[0762] Step 1:

[0763] Compounds INT5-1 (3 g, 8.14 mmol) and INT5-2 (14.67 g, 81.41 mmol) were dissolved in methanol (30 mL), and acetic acid (3 mL) and sodium cyanoborohydride (2.56 g, 40.7 mmol) were added. The reaction mixture was stirred at 50 °C for 6 h. The crude product was purified by reversed-phase silica gel column chromatography (acetonitrile: 0.05% HCOOH aqueous solution) to obtain the target compound INT5-3 (5.2 g).

[0764] LCMS(ESI)[M+H] + =697.35.

[0765] Step Two:

[0766] Compound INT5-3 (100 mg, 0.14 mmol) was dissolved in DMF (1 mL), and then diethylamine (20.48 mg, 0.28 mmol) was added. The reaction mixture was stirred at 30 °C for 2 h. THF (5 mL) was added and distilled three times. The crude INT5-4 was used directly in the next step of the reaction.

[0767] LCMS(ESI)[M+H] + =475.44.

[0768] Step 3:

[0769] Compound INT5-4 (68 mg, 0.14 mmol) was dissolved in DMF (1 ml) and water (1 ml), then INT2-2 (37.56 mg, 0.14 mmol) and DMTMM (38.74 mg, 0.14 mmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: 0.05% TFA aqueous solution) to obtain the target compound INT5 (30 mg).

[0770] LCMS(ESI)[M+H] + =725.27.

[0771] Example 2.6: Synthesis of INT6

[0772] Step 1:

[0773] Compounds INT3 (500 mg, 1.37 mmol) and INT6-1 (370 mg, 1.51 mmol) were dissolved in DMF (5.0 mL), and then DIPEA (350 mg, 2.74 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound INT6-2 (550 mg).

[0774] LCMS(ESI)[M+Na] + =519.22.

[0775] 1H NMR (400MHz, DMSO-d6) δ9.15 (s, 2H), 7.96 (d, J = 7.5Hz, 1H), 6.78 (t, J = 5.3Hz, 1H), 4.12 (dd, J = 12.9, 8.1Hz, 1H), 3.44 (s, 3H), 3.17 (dd, J = 12.8, 6.4Hz, 2H),2.93–2.87(m,2H),2.65–2.56(m,3H),2.38–2.28(m,2H),1.89–1.78(m ,2H),1.75–1.65(m,1H),1.62–1.51(m,1H),1.39(s,9H),1.31–1.25(m,2H).

[0776] Step Two:

[0777] Compound INT6-2 (300 mg, 0.60 mmol) was dissolved in DCM (8.0 mL) and TFA (2.0 mL), and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain trifluoroacetate (300 mg) of the target compound INT6-3.

[0778] LCMS(ESI)[M+H] + =397.18.

[0779] Step 3:

[0780] The compounds dodecaethylene propionate monomethyl ether (272.08 mg, 0.43 mmol), DIPEA (222.3 mg, 1.72 mmol), and HATU (179.9 mg, 0.47 mmol) were dissolved in DMF (5.0 mL) and stirred at room temperature for 15 min. Then, trifluoroacetate of INT6-3 (220 mg, 0.43 mmol) was added, and the mixture was stirred at room temperature for 45 min after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound INT6 (210 mg).

[0781] LCMS(ESI)[M+H] + =1011.56.

[0782] 1H NMR (400MHz, DMSO-d6) δ9.15 (s, 2H), 8.11 (d, J = 7.7Hz, 1H), 7.83 (t, J = 5.5Hz, 1H), 4.20–4.14 (m, 1H), 3.61 (t, J = 6.5Hz, 2H), 3.55–3.45 (m, 48H), 3.44(s,3H),3.27(s,3H),3.06–3.03(m,2H),2.59(t,J=7.1Hz,2H),2.3 8–2.28(m,4H),1.89–1.82(m,2H),1.77–1.53(m,2H),1.45–1.29(m,4H).

[0783] Example 2.7: Synthesis of INT7

[0784] Step 1:

[0785] Compound INT7-1 (500 mg, 1.68 mmol), N-hydroxysuccinimide (250 mg, 2.18 mmol), and EDCI (420 mg, 2.18 mmol) were dissolved in DCM (5 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by reverse-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the target compound INT7 (450 mg).

[0786] LCMS(ESI)[M+H] + =396.12.

[0787] 1 H NMR (400MHz, DMSO-d6) δ9.13(s,2H),4.65(s,2H),3.73(t,J=6.1Hz,2H),3.44(s,3H),2.86(s,4H),2.66(t,J=7.0Hz,2H),1.94–1.87(m,2H).

[0788] Example 2.8: Synthesis of INT8

[0789] Step 1:

[0790] Compounds INT8-1 (1.20 g, 2.63 mmol), INT8-2 (1.06 g, 2.89 mmol), and EEDQ (1.3 g, 5.26 mmol) were dissolved in DCM (5.0 mL) and reacted with the solution at room temperature for 16 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by normal-phase column chromatography (MeOH:DCM = 0%-5%) to obtain the target compound INT8-3 (1.45 g).

[0791] LCMS(ESI)[M+H] + =802.98.

[0792] Step Two:

[0793] Compound INT8-3 (1.40 g, 1.74 mmol) and di-p-nitrophenyl carbonate (1.59 g, 5.22 mmol) were dissolved in DMF (8.0 mL), and then DIPEA (0.96 g, 6.96 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound INT8 (0.8 g).

[0794] LCMS(ESI)[M+Na] + =990.23.

[0795] 1 H NMR(400MHz, DMSO-d6)δ8.82(s,1H),8.38–8.31(m,2H),8.09(d,J=1.3Hz,1H),7.64–7.57(m,2H),7.26–7 .24(m,1H),7.19–7.12(m,1H),6.76(t,J=5.3Hz,1H),5.68(d,J=7.8Hz,1H),5.54(t,J=9.7Hz,1H),5.29–5 .19(m,3H),5.11(t,J=9.7Hz,1H),4.77(d,J=10.0Hz,1H),3.73(t,J=6.4Hz,2H),3.67(s,3H),3.58–3.48 (m,12H),3.38(t,J=6.1Hz,2H),3.07(q,J=5.9Hz,2H),2.65–2.60(m,2H),2.08–2.02(m,9H),1.39(s,9H).

[0796] Example 2.9: Synthesis of INT9

[0797] Step 1:

[0798] Compounds INT9-1 (2.0 g, 4.28 mmol) and INT9-2 (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 INT9-3 (2.3 g).

[0799] LCMS(ESI)[M+H] + =936.59.

[0800] Step Two:

[0801] Compound INT9-3 (700.0 mg, 0.75 mmol) and p-aminobenzyl alcohol (184.73 mg, 1.50 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL), and then EEDQ (556.40 mg, 2.25 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 16 h. The reaction solution was concentrated under reduced pressure, and the crude product was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the target compound INT9-4 (600.0 mg).

[0802] LCMS(ESI)[M+H] + =1041.53.

[0803] Step 3:

[0804] Compound INT9-4 (600.0 mg, 0.58 mmol) and di(p-nitrobenzene) carbonate (352.88 mg, 1.16 mmol) were added to DMF (6 mL), followed by the addition of DIPEA (299.84 mg, 2.32 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the target compound INT9 (440.0 mg).

[0805] LCMS(ESI)[M+H] + =1206.58.

[0806] Example 2.10: Synthesis of INT10

[0807] Step 1:

[0808] Compound INT9-4 (500 mg, 0.48 mmol) was dissolved in DMF (5.0 mL), and then diethylamine (0.5 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound INT10-1 (393 mg), which was directly used in the next step of the reaction.

[0809] LCMS(ESI)[M+H] + =819.5.

[0810] Step Two:

[0811] Compounds INT10-1 (393 mg, 0.48 mmol), INT6 (485.3 mg, 0.48 mmol), and HATU (273.6 mg, 0.72 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (185.8 mg, 1.44 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. 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%-70%) to obtain the target compound INT10-2 formate (200 mg).

[0812] LCMS(ESI)[M+H] + =1812.0.

[0813] Step 3:

[0814] Compound INT10-2 (200 mg, 0.11 mmol) and di(p-nitrobenzene) carbonate (66.88 mg, 0.22 mmol) were dissolved in DMF (2 mL), and then DIPEA (42.57 mg, 0.33 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the target compound INT10 (150 mg).

[0815] LCMS(ESI)[M / 2+H] + =989.0.

[0816] Example 3. Synthesis of drug linker conjugates

[0817] Example 3.1: Synthesis of DL001

[0818] Step 1:

[0819] Compounds D-3 (45 mg, 0.093 mmol), INT1 (108 mg, 0.11 mmol), and HOAT (15.2 mg, 0.11 mmol) were dissolved in DMF (0.5 mL), and then DIPEA (36 mg, 0.28 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL001-1 (46 mg).

[0820] LCMS(ESI)[M / 2+H] + =655.90.

[0821] 1 H NMR(400MHz,DMSO-d6)δ8.61(s,1H),7.94–7.90(m,1H),7.66–7.60(m,3H),7.51–7.36(m,2H),7.31–7.23(m,2 H),7.18–7.12(m,2H),6.75–6.72(m,1H),5.76(d,J=7.9Hz,1H),5.51(t,J=9.6Hz,1H),5.24–5.20(m,1H),5.1 3–5.07(m,4H),4.91–4.73(m,5H),4.54(s,1H),3.95–3.80(m,2H),3.65(s,3H),3.58–3.47(m,17H),3.43–3.3 8(m,7H),3.07(q,J=5.8Hz,2H),2.84(s,3H),2.71–2.58(m,2H),2.05–2.00(m,9H),1.84(s,3H),1.38(s,9H).

[0822] Step Two:

[0823] Compound DL001-1 (46 mg, 0.035 mmol) was dissolved in THF (1.0 mL), followed by the addition of hydrochloric acid (1 mL, 6 N). After the addition was complete, the mixture was heated to 45 °C and stirred for 4 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL001-2 (14 mg).

[0824] LCMS(ESI)[M+H] + =1070.75.

[0825] Step 3:

[0826] Compounds DL001-2 (14 mg, 0.047 mmol), INT3 (5.7 mg, 0.016 mmol), and HOAT (2.1 mg, 0.016 mmol) were dissolved in DMF (0.5 mL), followed by the addition of DIPEA (5.0 mg, 0.039 mmol). The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL001 (7.2 mg).

[0827] LCMS(ESI)[M / 2+H] + =660.75.

[0828] 1 H NMR(400MHz,DMSO-d6)δ9.14(s,2H),8.63(s,1H),8.39(s,1H),7.96(s,1H),7.82(s,1H),7.71–7.58(m,2H),7.47( s,1H),7.41–7.38(m,1H),7.35–7.20(m,3H),5.67(s,1H),5.32(s,1H),5.15–5.05(m,4H),4.90–4.77(m,3H),4.55 (s,1H),3.96–3.80(m,4H),3.60–3.50(m,18H),3.49–3.45(m,2H),3.44–3.41(m,7H),3.24–3.21(m,2H),2.84(s,3 H),2.71–2.68(m,1H),2.65–2.55(m,5H),2.37–2.35(m,2H),2.29(t,J=7.4Hz,2H),1.88–1.80(m,5H),1.39(s,2H).

[0829] Example 3.2: Synthesis of DL002

[0830] Step 1:

[0831] Compounds D-9 (15 mg, 0.025 mmol), INT2 (25.8 mg, 0.033 mmol), and HOBT (5.1 mg, 0.038 mmol) were dissolved in DMF (0.5 mL), followed by the addition of DIPEA (9.7 mg, 0.075 mmol). The reaction was carried out at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL002 (15.5 mg).

[0832] LCMS(ESI)[M+H] + =1134.45.

[0833] 1 H NMR (400MHz, DMSO-d6) δ10.05(s,1H),9.41(s,1H),9.14(s,2H),8.88(s,1H),8.21(d,J=7.5Hz,2H),7.97(d,J=8.4Hz, 1H),7.92–7.82(m,2H),7.60–7.56(m,2H),7.38–7.25(m,4H),5.08(s,2H),4.95(s,2H),4.45–4.38(m,2H),4.26–4.21( m,1H),3.90–3.85(m,2H),3.77(s,3H),3.06–3.01(m,2H),2.87–2.82(m,5H),2.78(d,J=4.4Hz,6H),2.58(t,J=7.2Hz, 3H),2.46–2.35(m,2H),2.08–1.96(m,4H),1.91–1.60(m,7H),1.47–1.28(m,2H),0.98–0.85(m,9H),0.72–0.68(m,2H).

[0834] Example 3.3: Synthesis of DL003

[0835] Step 1:

[0836] Compounds D-9 (74 mg, 0.16 mmol), INT1 (186 mg, 0.19 mmol), and HOAT (26.1 mg, 0.19 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (62 mg, 0.48 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL003-1 (123 mg).

[0837] LCMS(ESI)[M+H] + =1308.51.

[0838] Step Two:

[0839] Compound DL003-1 (100 mg, 0.076 mmol) was dissolved in methanol (2.0 mL) and water (2.0 mL), and then lithium hydroxide monohydrate (31.9 mg, 0.76 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL003-2 (35 mg).

[0840] LCMS(ESI)[M+H] + =1168.57.

[0841] Step 3:

[0842] Compound DL003-2 (35 mg, 0.030 mmol) was dissolved in DCM (1.0 mL), and then TFA (0.3 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain trifluoroacetate (35 mg) of the target compound DL003-3.

[0843] LCMS(ESI)[M+H] + =1068.46.

[0844] Step Four:

[0845] The trifluoroacetate of compound DL003-3 (35 mg, 0.030 mmol) and INT3 (12 mg, 0.033 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (11.6 mg, 0.09 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL003 (16.08 mg).

[0846] LCMS(ESI)[M+H] + =1318.51.

[0847] 1H NMR (400MHz, DMSO-d6) δ9.14(s,2H),8.58(s,1H),8.32(t,J=5.5Hz,1H),7.95(t,J=5.5Hz,1H),7.82(s,1H),7. 70(s,1H),7.59(d,J=9.3Hz,1H),7.47(s,1H),7.35–7.17(m,4H),5.73(d,J=4.0Hz,1H),5.41(s,1H),5.10(s,3 H),4.86(s,2H),3.99–3.94(m,3H),3.71(s,3H),3.56–3.51(m,16H),3.45–3.42(m,9H),3.24–3.21(m,2H),2.9 0–2.81(m,6H),2.57(t,J=7.1Hz,2H),2.29(t,J=7.3Hz,2H),1.94(s,3H),1.87–1.78(m,2H),0.90–0.77(m,4H).

[0848] Example 3.4: Synthesis of DL004

[0849] Step 1:

[0850] The trifluoroacetate of compound D-9 (55 mg, 0.093 mmol), INT4 (85 mg, 0.11 mmol), and HOAT (15.2 mg, 0.11 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (36 mg, 0.28 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL004-1 (33 mg).

[0851] LCMS(ESI)[M+H] + =1106.53.

[0852] 1H NMR (400MHz, DMSO-d6) δ10.10(s,1H),9.34(s,1H),8.87(s,1H),8.20–8.14(m,2H),7.93–7.80(m,3H),7.76(dd,J=7.3, 3.2Hz,3H),7.62–7.23(m,11H),5.08(s,2H),4.94(s,2H),4.47–4.42(m,1H),4.37–4.33(m,1H),4.28–4.20(m,2H),3.9 7–3.86(m,3H),3.76(s,3H),3.03–2.97(m,2H),2.88(s,3H),2.85–2.80(m,3H),2.75(d,J=4.5Hz,5H),2.71–2.69(m,1H ),2.06–1.98(m,4H),1.78–1.59(m,4H),1.42–1.29(m,2H),0.90(t,J=6.7Hz,6H),0.87–0.82(m,2H),0.83–0.70(m,2H).

[0853] Step Two:

[0854] The trifluoroacetate salt of compound DL004-1 (30 mg, 0.025 mmol) was dissolved in DMF (2.0 mL), and then diethylamine (0.1 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound DL004-2 (21.7 mg), which was directly used in the next step of the reaction.

[0855] LCMS(ESI)[M+H] + =884.38.

[0856] Step 3:

[0857] Compound DL004-2 (14 mg, 0.016 mmol), INT5 (13.9 mg, 0.019 mmol), and HATU (7.3 mg, 0.019 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (6.2 mg, 0.048 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. 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%-70%) to obtain the target compound DL004 formate (7.5 mg).

[0858] LCMS(ESI)[M / 2+H] + =795.99.

[0859] 1H NMR(400MHz,DMSO-d6)δ9.62(s,1H),9.09(s,2H),8.54(s,1H),8.30–8.15(m,3H),8.09–7.98(m,1H),7.68–7.56 (m,4H),7.29–7.15(m,5H),5.08–5.03(m,2H),4.85(s,2H),4.55–4.14(m,9H),3.99–3.95(m,4H),3.71–3.67(m, 6H),3.63–3.58(m,7H),3.52–3.48(m,10H),3.43(s,3H),2.90–2.81(m,7H),2.71–2.69(m,1H),2.50–2.47(m,1H ),2.36–2.23(m,4H),2.19–2.06(m,7H),1.91(s,3H),1.80–1.54(m,5H),1.48–1.18(m,8H),0.94–0.78(m,10H).

[0860] Example 3.5: Synthesis of DL005

[0861] Step 1:

[0862] Compound DL004-2 (21 mg, 0.024 mmol), INT6 (26.7 mg, 0.026 mmol), and HATU (11.0 mg, 0.029 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (9.3 mg, 0.072 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL005 formate (24 mg).

[0863] LCMS(ESI)[M / 2+H] + =939.19.

[0864] 1H NMR (400MHz, DMSO-d6) δ10.08(s,1H),9.14(s,2H),8.54(s,1H),8.36(s,1H),8.14(dd,J=14.9,7.6Hz,2H),7.82(dd,J=14.1,7. 3Hz,2H),7.72–7.45(m,4H),7.30–7.18(m,5H),5.06(s,2H),4.85(s,2H),4.38–4.22(m,3H),3.96(s,2H),3.71(s,3H),3.59(s,2 H),3.54–3.50(m,46H),3.43(s,3H),3.26(s,3H),3.04–2.99(m,2H),2.90–2.80(m,6H),2.58(t,J=7.1Hz,2H),2.38–2.28(m,4H) ,2.24(t,J=6.8Hz,2H),2.15(s,6H),2.03–1.97(m,1H),1.93–1.80(m,5H),1.65(s,3H),1.57–1.20(m,10H),0.91–0.78(m,10H).

[0865] Example 3.6: Synthesis of DL006

[0866] Step 1:

[0867] The trifluoroacetate of compound D-9 (40 mg, 0.067 mmol), DL006-1 (56.5 mg, 0.074 mmol), and HOAT (10.9 mg, 0.08 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (34.6 mg, 0.27 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL006-2 (40 mg).

[0868] LCMS(ESI)[M+H] + =1107.56.

[0869] Step Two:

[0870] Compound DL006-2 (40 mg, 0.036 mmol) was dissolved in DMF (2.0 mL), and then diethylamine (0.1 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound DL006-3 (31.97 mg), which was directly used in the next step of the reaction.

[0871] LCMS(ESI)[M+H] + =885.41.

[0872] Step 3:

[0873] Compound DL006-3 (15.5 mg, 0.018 mmol), INT5 (18.1 mg, 0.022 mmol), and HATU (8.2 mg, 0.022 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (9.3 mg, 0.072 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL006 (7.1 mg).

[0874] LCMS(ESI)[M+H] + =1592.28.

[0875] 1 H NMR(400MHz, DMSO-d6)δ9.70(s,1H),9.10(s,2H),8.55(s,1H),8.34–8.28(m,1H),8.18–8.05(m,2H),7.68–7.56(m,4H),7.30–7.18( m,5H),6.04–6.02(m,1H),5.44(s,2H),5.07–5.04(m,2H),4.85(s,2H),4.56–4.15(m,9H),3.96(s,3H),3.74–3.68(m,6H),3.63–3.56 (m,4H),3.55–3.46(m,6H),3.43(s,3H),3.42–3.39(m,10H),3.07–2.93(m,3H),2.90–2.81(m,6H),2.66–2.59(m,1H),2.48–2.45(m, 1H),2.33–2.22(m,2H),2.12–2.08(m,1H),1.91(s,3H),1.82–1.74(m,2H),1.70–1.56(m,3H),1.48–1.36(m,4H),0.96–0.77(m,10H).

[0876] Example 3.7: Synthesis of DL007

[0877] Step 1:

[0878] Compounds DL006-3 (15.5 mg, 0.018 mmol), INT6 (21.8 mg, 0.022 mmol), and HATU (8.2 mg, 0.022 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (9.3 mg, 0.072 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL007 (16.5 mg).

[0879] LCMS(ESI)[M / 2+H] + =939.50.

[0880] 1 H NMR(400MHz,DMSO-d6)δ10.05(s,1H),9.12(s,2H),8.96(s,1H),8.32(s,1H ),8.11(dd,J=26.2,7.5Hz,2H),7.95(s,2H),7.82(s,1H),7.73(d,J=8.6Hz, 1H),7.63–7.19(m,6H),6.01(s,1H),5.43(s,2H),5.08(s,2H),4.96(s,2H), 4.42–4.38(m,1H),4.33–4.28(m,1H),4.25–4.20(m,1H),3.87–3.83(m,3H), 3.78(s,3H),3.60–3.57(m,2H),3.55–3.48(m,46H),3.46–3.44(m,2H),3.4 3(s,3H),3.26(s,3H),3.06–2.96(m,4H),2.89(s,3H),2.85–2.75(m,3H),2. 58(t,J=7.0Hz,2H),2.31(t,J=6.6Hz,3H),2.08(s,3H),2.02–1.97(m,1H),1 .86–1.81(m,2H),1.71–1.60(m,3H),1.55–1.30(m,7H),0.92–0.64(m,10H).

[0881] Example 3.8: Synthesis of DL008

[0882] Step 1:

[0883] The trifluoroacetate of compound D-13 (60 mg, 0.10 mmol), INT1 (106 mg, 0.11 mmol), and HOAT (15.0 mg, 0.11 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (39 mg, 0.30 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL008-1 (123 mg).

[0884] LCMS(ESI)[M+H] + =1310.45.

[0885] Step Two:

[0886] Compound DL008-1 (25 mg, 0.019 mmol) was dissolved in THF (2.0 mL) / HCl solution (1.0 mL, 6 N). After the addition was complete, the mixture was heated to 40 °C and stirred for 4 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL008-2 (14 mg).

[0887] LCMS(ESI)[M+H] + =1070.47.

[0888] Step 3:

[0889] The trifluoroacetate of compound DL008-2 (14 mg, 0.012 mmol) and INT3 (4.8 mg, 0.013 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (4.6 mg, 0.036 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL008 (8.0 mg).

[0890] LCMS(ESI)[M+H] + =1320.45.

[0891] 1H NMR(400MHz,DMSO-d6)δ9.14(s,2H),8.94(s,1H),8.33–8.21(m,2H),7.97–7.78(m,4H),7.51–7.39(m,2 H),7.32–7.14(m,2H),5.76(s,1H),5.47(s,1H),5.15–5.12(m,3H),4.95(s,2H),3.98(d,J=9.5Hz,1H),3 .89(t,J=6.8Hz,2H),3.75(s,3H),3.59–3.45(m,23H),3.43(s,3H),3.22(d,J=5.7Hz,2H),2.91–2.83(m, 5H),2.57(t,J=6.4Hz,2H),2.29(t,J=7.4Hz,2H),2.06(s,3H),1.86–1.79(m,2H),0.89(d,J=6.8Hz,6H).

[0892] Example 3.9: Synthesis of DL009

[0893] Step 1:

[0894] The trifluoroacetate of compound DL003-3 (21.0 mg, 0.02 mmol) and INT7 (8.0 mg, 0.02 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (7.8 mg, 0.06 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL009 (10.6 mg).

[0895] LCMS(ESI)[M+H] + =1348.48.

[0896] 1H NMR (400MHz, DMSO-d6) δ9.13(s,2H),8.90(s,1H),8.32–8.23(m,2H),7.91–7.84(m,3H),7.72(t,J=5.8Hz,1H ),7.54–7.34(m,2H),7.29–7.24(m,2H),5.76(s,1H),5.47(s,1H),5.12(s,3H),4.94(s,2H),3.98(d,J=9.5H z,1H),3.91–3.85(m,4H),3.77(s,3H),3.62–3.51(m,18H),3.46–3.43(m,7H),3.43(s,3H),3.29–3.25(m,2H ),2.89(s,3H),2.85–2.77(m,3H),2.68(t,J=7.2Hz,2H),2.07(s,3H),1.94–1.84(m,2H),0.88–0.66(m,4H).

[0897] Example 3.10: Synthesis of DL010

[0898] Step 1:

[0899] The trifluoroacetate of compound D-15 (62 mg, 0.11 mmol), INT4 (96.8 mg, 0.11 mmol), and HOAT (16.5 mg, 0.12 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (56.8 mg, 0.44 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL010-1 (60 mg).

[0900] LCMS(ESI)[M+H] + =1080.42.

[0901] Step Two:

[0902] The trifluoroacetate salt of compound DL010-1 (60 mg, 0.050 mmol) was dissolved in DMF (2.0 mL) / diethylamine (0.1 mL), and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound DL010-2 (43 mg), which was directly used in the next step of the reaction.

[0903] LCMS(ESI)[M+H] + =858.36.

[0904] Step 3:

[0905] Compound DL010-2 (43.0 mg, 0.05 mmol), INT6 (50.5 mg, 0.02 mmol), and HATU (20.9 mg, 0.055 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (19.4 mg, 0.15 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL010 (25 mg).

[0906] LCMS(ESI)[M / 2+H] + =926.77.

[0907] 1 H NMR (400MHz, DMSO-d6) δ10.04(s,1H),9.14(s,2H),8.52(s,1H),8.09(t,J=7.7Hz,2H),7.82(t,J=5.4Hz,1H),7.76(d,J=8.8Hz,1H),7.68 (s,1H),7.57–7.54(m,3H),7.31–7.08(m,5H),5.05(s,2H),4.84(s,2H),4.40–4.20(m,3H),3.98(s,2H),3.68(s,3H),3.59(t,J=6.5Hz,2 H),3.54–3.50(m,41H),3.50–3.44(m,6H),3.43(s,3H),3.26(s,3H),3.01(d,J=7.2Hz,3H),2.85–2.82(m,5H),2.58(t,J=6.8Hz,2H),2.3 4–2.28(m,6H),2.18–2.14(m,2H),2.10(s,6H),2.02–1.96(m,1H),1. 89(s,3H),1.86–1.81(m,2H),1.65–1.25(m,13H),0.88–0.82(m,6H).

[0908] Example 3.11: Synthesis of DL011

[0909] Step 1:

[0910] Compounds D-15 (50 mg, 0.11 mmol), INT8 (106 mg, 0.11 mmol), and HOAT (16.5 mg, 0.12 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (43 mg, 0.33 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL011-1 (46 mg).

[0911] LCMS(ESI)[M+H] + =1282.29

[0912] Step Two:

[0913] Compound DL011-1 (48 mg, 0.036 mmol) was dissolved in methanol (1.0 mL) / water (1.0 mL), and then lithium hydroxide monohydrate (8.6 mg, 0.376 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL011-2 (37.8 mg).

[0914] LCMS(ESI)[M+H] + =1142.33.

[0915] Step 3:

[0916] Compound DL011-2 (35 mg, 0.033 mmol) was dissolved in DCM (1.0 mL), and then TFA (0.3 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain trifluoroacetate of the target compound DL011-3 (38 mg).

[0917] LCMS(ESI)[M+H] + =1042.29.

[0918] Step Four:

[0919] The trifluoroacetate of compound DL011-3 (38 mg, 0.033 mmol) and INT3 (12 mg, 0.033 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (12.9 mg, 0.10 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL011 (25.8 mg).

[0920] LCMS(ESI)[M+H] + =1292.34.

[0921] 1 H NMR(400MHz, DMSO-d6)δ9.13(s,2H),9.09(s,1H),8.90–8.82(m,1H),8.25–8.20(m,2H),7.95(t,J=5.6Hz,1H),7.90–7.83(m,2H) ,7.38–7.32(m,1H),7.17–7.03(m,3H),5.91(s,1H),5.39(s,2H),5.07(s,2H),4.95(s,2H),4.80(d,J=7.5Hz,1H),3.96(t,J=6.5H z,2H),3.87(d,J=9.6Hz,1H),3.74(s,3H),3.69(t,J=6.3Hz,2H),3.53(s,3H),3.51–3.49(m,8H),3.41–3.39(m,9H),3.24–3.20( m,2H),2.93(s,3H),2.84(t,J=6.9Hz,2H),2.65–2.55(m,4H),2.28(t,J=7.4Hz,2H),2.23(s,3H),2.05(s,3H),1.86–1.80(m,2H).

[0922] Example 3.12: Synthesis of DL012

[0923] Step 1:

[0924] Compounds D-9 (85 mg, 0.16 mmol), INT8 (135.5 mg, 0.14 mmol), and HOAT (20.96 mg, 0.15 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (54 mg, 0.42 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL012-1 (135 mg).

[0925] LCMS(ESI)[M+H] + =1308.31.

[0926] Step Two:

[0927] Compound DL012-1 (48 mg, 0.037 mmol) was dissolved in methanol (1.0 mL) / water (1.0 mL), and then lithium hydroxide monohydrate (8.9 mg, 0.37 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse-phase chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL012-2 (38 mg).

[0928] LCMS(ESI)[M+H] + =1168.38.

[0929] Step 3:

[0930] Compound DL012-2 (35 mg, 0.033 mmol) was dissolved in DCM (1.0 mL), and then TFA (0.3 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain trifluoroacetate (38 mg) of the target compound DL012-3.

[0931] LCMS(ESI)[M+H] + =1068.34.

[0932] Step Four:

[0933] The trifluoroacetate of compound DL012-3 (38 mg, 0.030 mmol) and INT3 (12 mg, 0.033 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (12.8 mg, 0.09 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 10%-70%) to obtain the target compound DL012 (29.3 mg).

[0934] LCMS(ESI)[M+H] + =1318.29.

[0935] 1H NMR(400MHz,DMSO-d6)δ9.13(s,2H),9.09(s,1H),8.94(s,1H),8.32–8.20(m,2H),7.99–7.88(m,3H),7.39–7.32(m,1 H),7.28–6.88(m,3H),5.80–5.40(m,3H),5.08(s,2H),4.96(s,2H),4.80(d,J=7.5Hz,1H),3.87–3.84(m,3H),3.78(s ,3H),3.68(d,J=6.4Hz,2H),3.54–3.51(m,12H),3.43–3.39(m,8H),3.24–3.20(m,2H),2.91(s,3H),2.84(t,J=7.1Hz ,2H),2.80–2.75(m,1H),2.66–2.55(m,4H),2.28(t,J=7.4Hz,2H),2.09(s,3H),1.86–1.77(m,2H),0.90–0.77(m,4H).

[0936] Example 3.13: Synthesis of DL013

[0937] Step 1:

[0938] Compound D-15 (50.0 mg, 0.11 mmol) and INT9 (159.24 mg, 0.13 mmol) were dissolved in DMF (1.0 mL), and then DIPEA (42.65 mg, 0.33 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-50%) to obtain the target compound DL013-1 (70.0 mg).

[0939] LCMS(ESI)[M / 2+H] + =760.84.

[0940] Step Two:

[0941] Compound DL013-1 (70.0 mg, 0.046 mmol) was dissolved in DMF (1.0 mL) and diethylamine (0.05 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 0%-30%) to obtain the target compound DL013-2 (30.0 mg).

[0942] LCMS(ESI)[M / 2+H] + =650.20.

[0943] Step 3:

[0944] Compound DL013-2 (30.0 mg, 0.023 mmol) and INT6 (23.26 mg, 0.023 mmol) were dissolved in DMF (1.0 mL), followed by the addition of HATU (10.49 mg, 0.028 mmol) and DIPEA (8.92 mg, 0.069 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain trifluoroacetate of the target compound DL013 (6.10 mg).

[0945] LCMS(ESI)[M / 2+H] + =1146.35.

[0946] 1 H NMR(400MHz,DMSO-d6)δδ10.10(s,1H),9.38(s,1H),9.16–9.10(m,2H),8.66(s ,1H),8.19(d,J=7.2Hz,1H),8.10(d,J=7.8Hz,1H),7.93–7.77(m,2H),7.74–7.6 4(m,2H),7.62–7.53(m,2H),7.51–7.35(m,1H),7.33–7.19(m,4H),5.06(s,2H), 4.88(s,2H),4.43–4.36(m,1H),4.33–4.18(m,2H),3.98(s,2H),3.78–3.74(m,4 H),3.70(s,3H),3.60–3.57(m,6H),3.54–3.49(m,74H),3.45–3.43(m,10H),3.2 6(s,3H),3.25(s,6H),3.16–3.12(m,2H),3.03–2.99(m,2H),2.85(s,6H),2.62– 2.56(m,3H),2.34–2.26(m,6H),2.05–1.96(m,2H),1.95(s,3H),1.87–1.80(m,2 H),1.73–1.62(m,4H),1.55–1.47(m,1H),1.43–1.33(m,4H),0.89–0.82(m,6H).

[0947] Example 3.14: Synthesis of DL014

[0948] Step 1:

[0949] The trifluoroacetate of compound D-18 (100 mg, 0.19 mmol) was dissolved in DMF (2 mL), followed by the addition of compound INT4 (170 mg, 0.23 mmol) and DIPEA (120 mg, 0.95 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain the trifluoroacetate of the target compound DL014-1 (55 mg).

[0950] LCMS(ESI)[M / 2+H] + =576.17.

[0951] 1 H NMR (400MHz, DMSO-d6) δ10.06(s,1H),8.19(s,1H),8.15–8.07(m,3H),7.88(d,J=7.6Hz,2H),7.73(t,J=6.7Hz,2H),7.58–7.51(m,3H),7.44–7.3 7(m,3H),7.33–7.23(m,7H),6.97–6.87(m,2H),5.92(s,1H),5.04(s,2H ),4.83(s,2H),4.65(s,2H),4.44–4.38(m,1H),4.34–4.26(m,1H),4.25–

[0952] 4.19(m,3H),3.92(t,J=8.0Hz,1H),3.85–3.78(m,2H),3.53(s,3H),2.81(s,3H),2.63–2.55(m,2H),2.35(s,6H),2 .04–1.93(m,1H),1.87(s,3H),1.82(s,3H),1.76–1.57(m,2H),1.45(s,3H),1.40–1.20(m,3H),0.88–0.84(m,6H).

[0953] Step Two:

[0954] The trifluoroacetate salt of compound DL014-1 (55 mg, 0.19 mmol) was dissolved in DMF (2 mL), and then diethylamine (0.2 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound DL014-2 (50 mg), which was directly used in the next step of the reaction.

[0955] LCMS(ESI)[M / 2+H] + =464.84.

[0956] Step 3:

[0957] Compound DL014-2 (50 mg, 0.054 mmol) was dissolved in DMF (2 mL), followed by the addition of compound INT6 (66 mg, 0.065 mmol), HATU (30.8 mg, 0.081 mmol), and DIPEA (35 mg, 0.27 mmol). The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-90%) to obtain trifluoroacetate of the target compound DL014 (8.03 mg).

[0958] LCMS(ESI)[M / 2+H] + =961.49.

[0959] 1 H NMR(400MHz,DMSO-d6)δ10.07(s,1H),9.59(s,1H),9.11(s,2H),8.76(s,1H) ,8.21–8.14(m,1H),8.10–8.04(m,1H),8.00–7.89(m,1H),7.87–7.79(m,1H), 7.78–7.72(m,1H),7.70–7.59(m,2H),7.58–7.44(m,2H),7.42–7.19(m,5H),5 .91(s,1H),5.04(s,2H),4.89(s,2H),4.67(s,2H),4.43–4.30(m,1H),4.30–4 .23(m,1H),4.22–4.15(m,1H),3.85(s,2H),3.56(s,3H),3.51–3.48(m,49H), 3.23(s,3H),3.02–2.97(m,4H),2.85(s,3H),2.74(d,J=4.0Hz,6H),2.64–2.5 3(m,4H),2.35–2.24(m,6H),1.97(s,1H),1.88(d,6H),1.84–1.79(m,2H),1.6 8–1.57(m,5H),1.46(s,3H),1.40–1.21(m,8H),0.83(dd,J=12.9,6.7Hz,6H).

[0960] Example 3.15: Synthesis of DL015

[0961] Step 1:

[0962] Compound DL015-1 (30.0 mg, 0.060 mmol) and INT9 (79.62 mg, 0.066 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (23.26 mg, 0.18 mmol). The mixture was stirred at room temperature for 1 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-50%) to obtain the target compound DL015-2 (50.0 mg).

[0963] LCMS(ESI)[M / 2+H] + =782.91.

[0964] Step Two:

[0965] Compound DL015-2 (50.0 mg, 0.046 mmol) was dissolved in DMF (1.0 mL) and diethylamine (0.05 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was then distilled three times under reduced pressure using THF (10 mL * 3) to obtain a DMF solution of the target compound DL015-3 (40.12 mg), which was directly used in the next step of the reaction.

[0966] LCMS(ESI)[M / 2+H] + =671.81.

[0967] Step 3:

[0968] Compounds DL015-3 (40.12 mg, 0.046 mmol) and INT6 (36.40 mg, 0.036 mmol) were dissolved in DMF (1.0 mL), followed by the addition of HATU (13.69 mg, 0.036 mmol) and DIPEA (11.63 mg, 0.090 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the trifluoroacetate of the target compound DL015 (5.54 mg).

[0969] LCMS(ESI)[M / 2+H] + =1168.81.

[0970] 1H NMR(400MHz,DMSO-d6)δ10.10(s,1H),9.49(s,1H),9.14(s,2H),8.90(s,1H ),8.26–8.13(m,2H),8.12–8.07(m,1H),7.93–7.87(m,2H),7.87–7.81(m,1 H),7.74–7.67(m,1H),7.63–7.51(m,2H),7.41–7.24(m,4H),5.11–5.05(m, 2H),4.99–4.92(m,2H),4.43–4.38(m,1H),4.32–4.27(m,1H),4.25–4.21(m ,1H),4.04(s,1H),3.96–3.86(m,3H),3.64–3.49(m,96H),3.26(s,3H),3.0 7–2.98(m,5H),2.90(s,3H),2.79–2.75(m,6H),2.61–2.57(m,3H),2.35–2. 28(m,3H),2.06–1.97(m,2H),1.94(s,3H),1.90–1.71(m,4H),1.70–1.60(m ,4H),1.59–1.46(m,2H),1.42–1.34(m,4H),0.89–0.83(m,6H),0.73(s,9H).

[0971] Example 3.16: Synthesis of DL016

[0972] Step 1:

[0973] Compound DL015-1 (30.0 mg, 0.060 mmol) and INT4 (50.55 mg, 0.066 mmol) were dissolved in DMF (1.0 mL), and then DIPEA (23.26 mg, 0.18 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-50%) to obtain the target compound DL016-1 (30.0 mg).

[0974] LCMS(ESI)[M+H] + =1124.29.

[0975] Step Two

[0976] Compound DL016-1 (30.0 mg, 0.027 mmol) was dissolved in DMF (1.0 mL) and diethylamine (0.05 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was then distilled three times under reduced pressure using THF (10 mL * 3) to obtain a DMF solution of the target compound DL016-2 (24.07 mg), which was directly used in the next reaction step.

[0977] LCMS(ESI)[M+Na] + =924.50.

[0978] Step 3:

[0979] Compounds DL016-2 (24.07 mg, 0.027 mmol) and INT6 (30.07 mg, 0.030 mmol) were dissolved in DMF (1.0 mL), followed by the addition of HATU (12.32 mg, 0.032 mmol) and DIPEA (10.47 mg, 0.081 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-60%) to obtain the target compound DL016 (6.34 mg).

[0980] LCMS(ESI)[M / 2+H] + =948.20.

[0981] 1H NMR(400MHz,DMSO-d6)δ9.66(s,1H),8.94(s,1H),8.69(s,2H),8.37(s,1H),7.80–7.71(m,1H),7.71–7.48(m,2H),7.45–7.35(m,2H),7.34–7.20 (m,2H),7.20–7.06(m,2H),6.96–6.79(m,4H),4.64(s,2H),4.48(s,2H) ,4.01–3.74(m,4H),3.62(s,1H),3.50–3.43(m,2H),3.35–3.31(m,4H),3 .11–3.07(m,56H),2.83(s,2H),2.82(s,3H),2.71(s,2H),2.60–2.56(m ,2H),2.44(s,3H),2.39–2.29(m,2H),2.18–2.13(m,3H),1.91–1.84(m,3 H),1.64–1.53(m,3H),1.46(s,3H),1.43–1.37(m,2H),1.30–1.19(m,4H ),1.12–1.01(m,2H),0.98–0.89(m,4H),0.46–0.39(m,6H),0.31(s,9H).

[0982] Example 3.17: Synthesis of DL017

[0983] Step 1:

[0984] The trifluoroacetate of compound D-20 (20 mg, 0.033 mmol), INT4 (32 mg, 0.036 mmol), and HOAT (5.4 mg, 0.04 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (25.5 mg, 0.20 mmol). The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL017-1 (23 mg).

[0985] LCMS(ESI)[M+H] + =1120.43.

[0986] Step Two:

[0987] The trifluoroacetate salt of compound DL017-1 (23 mg, 0.050 mmol) was dissolved in DMF (2.0 mL) and diethylamine (0.1 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound DL017-2 (18.4 mg), which was directly used in the next step of the reaction.

[0988] LCMS(ESI)[M+H] + =898.17.

[0989] Step 3:

[0990] Compound DL017-2 (18.0 mg, 0.02 mmol), INT6 (24.3 mg, 0.024 mmol), and HATU (9.1 mg, 0.024 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (7.8 mg, 0.06 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL017 (7.34 mg).

[0991] LCMS(ESI)[M / 2+H] + =946.74.

[0992] 1H NMR(400MHz,DMSO-d6)δ10.05(s,1H),9.60(s,1H),9.10(s,2H),8.80(s,1H) ,8.21–8.02(m,3H),7.80(t,J=8.4Hz,2H),7.67(d,J=8.6Hz,2H),7.53(s,2H ),7.39–7.16(m,4H),5.03(s,2H),4.89(s,2H),4.41–4.15(m,3H),3.89(s,2 H),3.66(s,3H),3.55(d,J=6.5Hz,3H),3.50–3.48(m,40H),3.45(d,J=1.7Hz ,4H),3.39(s,3H),3.22(s,3H),2.98(d,J=6.2Hz,5H),2.84(s,3H),2.73(d, J=3.7Hz,8H),2.55(t,J=7.0Hz,2H),2.35–2.25(m,5H),2.00–1.93(m,4H),1 .85–1.77(m,2H),1.72(s,1H),1.67–1.58(m,4H),1.49(d,J=9.3Hz,1H),1.4 0–1.26(m,9H),1.19(s,3H),0.82(dd,J=12.7,6.7Hz,6H),0.67–0.64(m,2H).

[0993] Example 3.18: Synthesis of DL018

[0994] Step 1:

[0995] Compound D-21 (50 mg, 0.08 mmol) and INT9 (116 mg, 0.096 mmol) were dissolved in DMF (3 mL). DIPEA (52 mg, 0.4 mmol) was added with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10%-90%) to obtain the target compound DL018-1 (70 mg).

[0996] LCMS(ESI)[M+H] + =1580.84.

[0997] Step Two:

[0998] Compound DL018-1 (100 mg, 0.063 mmol) was dissolved in DMF (3 mL), and then diethylamine (0.3 mL) was added. After the addition was complete, the mixture was stirred at 25 °C for 1 h. The reaction was monitored by LCMS, and the reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% TFA = 10%-90%) to obtain the target compound DL018-2 (50 mg).

[0999] LCMS(ESI)[M / 2+H] + =679.69.

[1000] Step 3:

[1001] Compound DL018-2 (45 mg, 0.031 mmol) was dissolved in DMF (3 mL). INT6 (31 mg, 0.31 mmol), HATU (24 mg, 0.062 mmol), and DIPEA (12 mg, 0.093 mmol) were added sequentially with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H₂O containing 0.1% TFA = 10%-90%) to obtain the trifluoroacetate of the target compound DL018 (20.65 mg).

[1002] LCMS(ESI)[M / 2+H] + =1176.20.

[1003] 1H NMR(400MHz, DMSO-d6)δ10.08(s,1H),9.33–9.24(m,1H),9.14(s,2H),8.84–8.54(m,1H),8.25–8.07(m,2H),8.05–7.79(m,2H),7.78–7.65(m, 2H),7.64–7.47(m,3H),7.42–7.20(m,4H),5.07(s,2H),4.91(s,2H),4. 46–4.36(m,1H),4.33–4.19(m,2H),4.00–3.92(m,2H),3.80–3.74(m,4H ),3.70(s,3H),3.65–3.46(m,75H),3.46–3.41(m,16H),3.27–3.25(m, 8H),3.18–3.11(m,2H),3.06–2.98(m,2H),2.90–2.78(m,5H),2.64–2.5 7(m,2H),2.40–2.27(m,4H),2.07–1.97(m,5H),1.90–1.79(m,2H),1.77 –1.61(m,4H),1.59–1.46(m,3H),1.43–1.24(m,7H),0.93–0.80(m,6H).

[1004] Example 3.19: Synthesis of DL019

[1005] Step 1:

[1006] Compound D-21 (50 mg, 0.08 mmol) and INT4 (73 mg, 0.096 mmol) were dissolved in DMF (3 mL). DIPEA (52 mg, 0.4 mmol) was added with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10%-90%) to obtain the target compound DL019-1 (60 mg).

[1007] LCMS(ESI)[M+H] + =1140.45.

[1008] Step Two:

[1009] Compound DL019-1 (60 mg, 0.053 mmol) was dissolved in DMF (3 mL), and then diethylamine (0.3 mL) was added. After the addition was complete, the mixture was stirred at 25 °C for 1 h. The reaction was monitored by LCMS, and the reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% TFA = 10%-90%) to obtain the target compound DL019-2 (40 mg).

[1010] LCMS(ESI)[M+H] + =918.32.

[1011] Step 3:

[1012] Compound DL019-2 (40 mg, 0.044 mmol) was dissolved in DMF (3 mL). INT6 (49 mg, 0.48 mmol), HATU (33 mg, 0.088 mmol), and DIPEA (17 mg, 0.13 mmol) were added sequentially with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H₂O containing 0.1% TFA = 10%-90%) to obtain trifluoroacetate of the target compound DL019 (14.3 mg).

[1013] LCMS(ESI)[M / 2+H] + =957.19.

[1014] 1H NMR (400MHz, DMSO-d6) δ10.08(s,1H),9.44(s,1H),9.14(s,2H),8.90–8.60(m,1H),8.19(d,J=7.4Hz,1H),8.09(d,J=7.9Hz,2H),7.86–7.77(m,2H) ,7.69(d,J=8.6Hz,2H),7.57(s,2H),7.39–7.19(m,4H),5.07(s,2H),4.93 (s,2H),4.44–4.36(m,1H),4.32–4.20(m,2H),3.94(s,2H),3.71(s,3H),3 .60(t,J=6.5Hz,2H),3.56–3.48(m,52H),3.46–3.45(m,2H),3.43–3.43( m,3H),3.26(s,3H),3.04–3.01(m,2H),2.88(s,3H),2.82(s,2H),2.77(d, J=4.2Hz,6H),2.61–2.56(m,2H),2.37–2.28(m,4H),2.07–1.98(m,6H),1. 88–1.81(m,2H),1.72–1.60(m,4H),1.57–1.36(m,6H),0.90–0.82(m,6H).

[1015] Example 3.20: Synthesis of DL020

[1016] Step 1:

[1017] Compounds D-24 (25 mg, 0.041 mmol), INT9 (51.9 mg, 0.043 mmol), and HOAT (6.1 mg, 0.045 mmol) were dissolved in DMF (2.0 mL), followed by the addition of DIPEA (21.20 mg, 0.16 mmol). The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL020-1 (35 mg).

[1018] LCMS(ESI)[M+H] + =1565.83

[1019] Step Two:

[1020] Compound DL020-1 (35 mg, 0.050 mmol) was dissolved in DMF (1.0 mL) / diethylamine (0.05 mL) and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound DL020-2 (30 mg), which was directly used in the next step of the reaction.

[1021] LCMS(ESI)[M+H] + =1342.74

[1022] Step 3:

[1023] Compound DL020-2 (30.0 mg, 0.022 mmol), INT6 (24.5 mg, 0.024 mmol), and HATU (10 mg, 0.026 mmol) were dissolved in DMF (1.5 mL), followed by the addition of DIPEA (8.53 mg, 0.066 mmol). The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL020 (12 mg).

[1024] LCMS(ESI)[M / 2+H] + =1169.16.

[1025] 1 H NMR(400MHz,DMSO-d6)δ10.09(s,1H),9.37(s,1H),9.12(s,2H),8.92(s,1H),8.30–8.05(m,3H),7.92–7.82(m,3H),7.72–7.49(m,3H),7 .44–7.21(m,4H),5.07(s,2H),4.95(s,2H),4.41–4.22(m,5H),3.99–3.90(m,4H),3.77(s,6H),3.72(s,3H),3.62–3.57(m,7H),3.55–3.5 0(m,63H),3.47–3.42(m,9H),3.39–3.33(m,5H),3.28–3.24(m,8H),3.15(s,2H),3.02(d,J=6.2Hz,2H),2.92–2.82(m,5H),2.61–2.57(m ,2H),2.36–2.30(m,5H),2.06(s,3H),2.01(d,J=6.5Hz,1H),1.89–1.80(m,2H),1.70–1.64(m,6H),1.46–1.22(m,9H),0.89–0.82(m,6H).

[1026] Example 3.21: Synthesis of DL021

[1027] Method 1

[1028] Step 1:

[1029] The trifluoroacetate of compound D-20 (250 mg, 0.41 mmol), INT9 (519 mg, 0.43 mmol), and HOAT (61 mg, 0.45 mmol) were dissolved in DMF (5.0 mL), and then DIPEA (212 mg, 1.6 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL021-1 (400 mg).

[1030] LCMS(ESI)[M / 2+H] + =780.85.

[1031] Step Two:

[1032] The trifluoroacetate salt of compound DL021-1 (400 mg, 0.25 mmol) was dissolved in DMF (4.0 mL) / diethylamine (0.2 mL), and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the target compound DL021-2 (200 mg).

[1033] LCMS(ESI)[M / 2+H] + =670.30.

[1034] Step 3:

[1035] Compound DL021-2 (200 mg, 0.149 mmol), INT6 (150.6 mg, 0.149 mmol), and HATU (62.2 mg, 0.164 mmol) were dissolved in DMF (4.0 mL), followed by the addition of DIPEA (57.6 mg, 0.447 mmol). The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain trifluoroacetate of the target compound DL021 (180 mg).

[1036] LCMS(ESI)[M / 2+H] + =1166.49.

[1037] 1H NMR(400MHz,DMSO-d6)δ10.04(s,1H),9.23(s,1H),9.10(s,2H),8.90(s,1H ),8.26–8.10(m,2H),8.04(d,J=7.8Hz,1H),7.88–7.77(m,3H),7.67–7.44( m,3H),7.39–7.17(m,4H),5.03(s,2H),4.92(s,2H),4.36–4.18(m,4H),3.8 7(s,3H),3.74–3.70(m,5H),3.67(s,3H),3.56–3.51(m,20H),3.50–3.46(m, 48H),3.42–3.38(m,12H),3.33(s,6H),3.23–3.21(m,9H),3.13–3.08(m,2H ),3.00–2.95(m,2H),2.85(s,3H),2.74(t,J=7.0Hz,2H),2.55(t,J=7.1Hz, 2H),2.34–2.24(m,4H),2.02–1.91(m,4H),1.82–1.62(m,7H),1.51–1.45(m ,1H),1.41–1.21(m,8H),1.17(s,3H),0.89–0.76(m,6H),0.66–0.65(m,2H).

[1038] Method 2

[1039] Step 1:

[1040] The trifluoroacetate of compound D-20 (30 mg, 0.049 mmol), INT10 (96.87 mg, 0.049 mmol), and HOAT (10 mg, 0.074 mmol) were dissolved in DMF (5.0 mL), followed by the addition of DIPEA (12.64 mg, 0.098 mmol). The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 10%-70%) to obtain the trifluoroacetate of the target compound DL021 (40 mg).

[1041] LCMS(ESI)[M / 2+H] + =1166.49.

[1042] 1H NMR(400MHz,DMSO-d6)δ10.04(s,1H),9.23(s,1H),9.10(s,2H),8.90(s,1H ),8.26–8.10(m,2H),8.04(d,J=7.8Hz,1H),7.88–7.77(m,3H),7.67–7.44( m,3H),7.39–7.17(m,4H),5.03(s,2H),4.92(s,2H),4.36–4.18(m,4H),3.8 7(s,3H),3.74–3.70(m,5H),3.67(s,3H),3.56–3.51(m,20H),3.50–3.46(m, 48H),3.42–3.38(m,12H),3.33(s,6H),3.23–3.21(m,9H),3.13–3.08(m,2H ),3.00–2.95(m,2H),2.85(s,3H),2.74(t,J=7.0Hz,2H),2.55(t,J=7.1Hz, 2H),2.34–2.24(m,4H),2.02–1.91(m,4H),1.82–1.62(m,7H),1.51–1.45(m ,1H),1.41–1.21(m,8H),1.17(s,3H),0.89–0.76(m,6H),0.66–0.65(m,2H).

[1043] Example 4: Preparation of HER2-ADC

[1044] Example 4.1 Preparation of HER2-ADC-001

[1045] Take 1.0 mL of trastuzumab (21.8 mg / mL), dilute with 0.01 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, 0.0405 mL, 0.81 μmol, 5.5 times the molar amount of antibody) solution, mix well, and incubate at 25 °C for 90 min. Then add DL001 (2.099 mg, 10 times the molar amount of antibody) in dimethyl sulfoxide (0.159 mL), mix well, and incubate at 25 °C for 2 h. After completion, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to change the buffer solution to 20 mM His-HCl, pH 5.9 buffer solution. The conjugate product Her2-ADC-001 of DL001 and trastuzumab is obtained. The DAR value determined by mass spectrometry was 7.9.

[1046] Table 4

[1047] In the table, LC represents the antibody light chain; HC represents the antibody heavy chain; DAR1 represents a conjugate containing one drug linker conjugate (either light or heavy chain); DAR2 represents a conjugate containing two drug linkers conjugate (either light or heavy chain); and DAR3 represents a conjugate containing three drug linkers conjugate (either light or heavy chain). LC, HC, DAR1, DAR2, and DAR3 will be explained as above in the following text.

[1048] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-001 was calculated to be 7.9 based on the following measurements: light chain conjugation with 0 toxin molecules (LC+DAR0 ratio of 2%), light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 98%), heavy chain conjugation with 2 toxin molecules (HC+DAR2 ratio of 1%), and heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 99%).

[1049] Example 4.2 Preparation of HER2-ADC-002

[1050] Using the same method as in Example 4.1, DL002 was used to replace DL001 to obtain the conjugate product HER2-ADC-002 of DL002 and trastuzumab. The DAR value was determined to be 8.1 by mass spectrometry.

[1051] Table 5

[1052] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-002 was calculated to be 8.1 based on the following measurements: light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 96%), and heavy chain conjugation with 4 toxin molecules (HC+DAR4 ratio of 4%).

[1053] Example 4.3 Preparation of HER2-ADC-003

[1054] Using the same method as in Example 4.1, DL001 was replaced with DL003 to obtain the conjugate product HER2-ADC-003 of DL003 and trastuzumab, and the DAR value was determined to be 8.1 by mass spectrometry.

[1055] Table 6

[1056] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-003 was calculated to be 8.1 based on the following measurements: light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 97%), and heavy chain conjugation with 4 toxin molecules (HC+DAR4 ratio of 3%).

[1057] Example 4.4 Preparation of HER2-ADC-004

[1058] Using the same method as in Example 4.1, DL004 was used to replace DL001 to obtain the conjugate product HER2-ADC-004 of DL004 and trastuzumab, and the DAR value was determined to be 8.1 by mass spectrometry.

[1059] Table 7

[1060] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-004 was calculated to be 8.1 based on the following measurements: light chain conjugation with 0 toxin molecules (LC+DAR0 ratio of 6%), light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 94%), heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 89%), and heavy chain conjugation with 4 toxin molecules (HC+DAR4 ratio of 11%).

[1061] Example 4.5 Preparation of HER2-ADC-005

[1062] Using the same method as in Example 4.1, DL001 was replaced with DL005 to obtain the conjugate product HER2-ADC-005 of DL005 and trastuzumab, and the DAR value was determined to be 8.1 by mass spectrometry.

[1063] Table 8

[1064] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-005 was calculated to be 8.1 based on the following measurements: light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 99%), light chain conjugated with 2 toxin molecules (LC+DAR2 ratio of 1%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 94%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 6%).

[1065] Example 4.6 Preparation of HER2-ADC-006

[1066] Using the same method as in Example 4.1, DL001 was replaced with DL006 to obtain the conjugate product HER2-ADC-006 of DL006 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1067] Table 9

[1068] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-006 was calculated to be 8.0 based on the following measurements: light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugation with 2 toxin molecules (HC+DAR2 ratio of 2%), heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 94%), and heavy chain conjugation with 4 toxin molecules (HC+DAR4 ratio of 4%).

[1069] Example 4.7 Preparation of HER2-ADC-007

[1070] Using the same method as in Example 4.1, DL001 was replaced with DL007 to obtain the conjugate product HER2-ADC-007 of DL007 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1071] Table 10

[1072] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-007 was calculated to be 8.0 based on the following measurements: light chain conjugation with 0 toxin molecules (LC+DAR0 ratio of 1%), light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 99%), and heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 100%).

[1073] Example 4.8 Preparation of HER2-ADC-008

[1074] Using the same method as in Example 4.1, DL001 was replaced with DL008 to obtain the conjugate product HER2-ADC-008 of DL008 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1075] Table 11

[1076] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-008 was calculated to be 8.0 based on the following measurements: light chain conjugation with 0 toxin molecules (LC+DAR0 ratio of 1%), light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 99%), heavy chain conjugation with 2 toxin molecules (HC+DAR2 ratio of 1%), and heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 99%).

[1077] Example 4.9 Preparation of HER2-ADC-009

[1078] Using the same method as in Example 4.1, DL001 was replaced with DL010 to obtain the conjugate product HER2-ADC-009 of DL010 and trastuzumab, and the DAR value was determined to be 8.2 by mass spectrometry.

[1079] Table 12

[1080] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-009 was calculated to be 8.2 based on the following measurements: light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 99%), light chain conjugated with 2 toxin molecules (LC+DAR2 ratio of 1%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 92%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 8%).

[1081] Example 4.10 Preparation of HER2-ADC-010

[1082] Using the same method as in Example 4.1, DL001 was replaced with DL013 to obtain the conjugate product HER2-ADC-010 of DL013 and trastuzumab, and the DAR value was determined to be 7.6 by mass spectrometry.

[1083] Table 13

[1084] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-010 was calculated to be 7.6 based on the following measurements: light chain conjugated with 1 toxin molecule (LC ratio of 17%), light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 83%), heavy chain conjugated with 2 toxin molecules (HC+DAR2 ratio of 2%), and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 98%).

[1085] Example 4.11 Preparation of HER2-ADC-011

[1086] Using the same method as in Example 4.1, DL001 was replaced with DL015 to obtain the conjugate product HER2-ADC-011 of DL015 and trastuzumab, and the DAR value was determined to be 8.1 by mass spectrometry.

[1087] Table 14

[1088] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-011 was calculated to be 8.1 based on the following measurements: light chain conjugation with 0 toxin molecules (LC ratio of 1%), light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 98%), heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 96%), and heavy chain conjugation with 4 toxin molecules (HC+DAR3 ratio of 4%).

[1089] Example 4.12 Preparation of HER2-ADC-012

[1090] Using the same method as in Example 4.1, DL001 was replaced with DL016 to obtain the conjugate product HER2-ADC-012 of DL016 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1091] Table 15

[1092] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-012 was calculated to be 8.0 based on the following measurements: light chain conjugation with 0 toxin molecules (LC ratio of 2%), light chain conjugation with 1 toxin molecule (LC+DAR1 ratio of 98%), and heavy chain conjugation with 3 toxin molecules (HC+DAR3 ratio of 100%).

[1093] Example 4.13 Preparation of HER2-ADC-013

[1094] Using the same method as in Example 4.1, DL001 was replaced with DL017 to obtain the conjugate product HER2-ADC-013 of DL017 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1095] Table 16

[1096] Trastuzumab light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%) and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 100%) were measured, and the antibody-drug conjugation ratio (DAR value) of HER2-ADC-013 was calculated to be 8.0.

[1097] Example 4.14 Preparation of HER2-ADC-014

[1098] Using the same method as in Example 4.1, DL001 was replaced with DL018 to obtain the conjugate product HER2-ADC-014 of DL018 and trastuzumab, with a DAR value of 7.9 determined by mass spectrometry.

[1099] Table 17

[1100] Trastuzumab light chain conjugated with 0 toxin molecules, 1 toxin molecule (LC ratio of 3%, LC+DAR1 ratio of 97%), and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 100%) were measured. Based on this, the antibody-drug conjugation ratio (DAR value) of HER2-ADC-014 was calculated to be 7.9.

[1101] Example 4.15 Preparation of HER2-ADC-015

[1102] Using the same method as in Example 4.1, DL001 was replaced with DL019 to obtain the conjugate product HER2-ADC-015 of DL019 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1103] Table 18

[1104] Trastuzumab light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugated with 3 toxin molecules, and 4 toxin molecules (HC+DAR3 ratio of 98%) and HC+DAR4 ratio of 2%) were measured. Based on this, the antibody-drug conjugation ratio (DAR value) of HER2-ADC-015 was calculated to be 8.0.

[1105] Example 4.16 Preparation of HER2-ADC-016

[1106] Using the same method as in Example 4.1, DL001 was replaced with DL020 to obtain the conjugate product HER2-ADC-016 of DL020 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1107] Table 19

[1108] The antibody-drug conjugation ratio (DAR value) of HER2-ADC-016 was calculated to be 8.0 based on the determination of trastuzumab light chain conjugated with 0 toxin molecules, 1 toxin molecule (LC ratio of 1%, LC+DAR1 ratio of 99%), and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 100%).

[1109] Example 4.17 Preparation of Her2-ADC-017

[1110] Using the same method as in Example 4.1, DL001 was replaced with DL021 to obtain the conjugate product HER2-ADC-017 of DL021 and trastuzumab, and the DAR value was determined to be 8.0 by mass spectrometry.

[1111] Table 20

[1112] Trastuzumab light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%) and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 100%) were measured, and the antibody-drug conjugation ratio (DAR value) of HER2-ADC-017 was calculated to be 8.0.

[1113] Example 4.2.1 Preparation of Trop2-ADC-001

[1114] Using the same method as in Example 4.1, DL001 was replaced with DL017 and Trastuzumab was replaced with sacituzumab to obtain the coupling product Trop2-ADC-001 of DL017 and sacituzumab. The DAR value was determined to be 8.0 by mass spectrometry.

[1115] Table 21

[1116] The antibody-drug conjugation ratio (DAR value) of Trop2-ADC-001 was calculated to be 8.0 based on the determination of sacituzumab light chain conjugated to 1 toxin molecule (LC+DAR1 ratio of 100%) and heavy chain conjugated to 3 toxin molecules (HC+DAR3 ratio of 100%).

[1117] Example 4.2.2 Preparation of Trop2-ADC-002

[1118] Using the same method as in Example 4.1, DL001 was replaced with DL013 and Trastuzumab was replaced with sacituzumab to obtain the coupling product Trop2-ADC-002 of DL013 and sacituzumab. The DAR value was determined to be 8.0 by mass spectrometry.

[1119] Table 22

[1120] The antibody-drug conjugation ratio (DAR value) of Trop2-ADC-002 was calculated to be 8.0 based on the determination of sacituzumab light chain conjugation to 1 toxin molecule (LC+DAR1 ratio of 99%), conjugation to 2 toxin molecules (LC+DAR2 ratio of 1%), and heavy chain conjugation to 3 toxin molecules (HC+DAR3 ratio of 100%).

[1121] Example 4.2.3 Preparation of Trop2-ADC-003

[1122] Using the same method as in Example 4.1, DL020 was replaced with DL001 and sacituzumab was replaced with trastuzumab to obtain the coupling product Trop2-ADC-003 of DL020 and sacituzumab. The DAR value was determined to be 8.0 by mass spectrometry.

[1123] Table 23

[1124] The antibody-drug conjugation ratio (DAR value) of Trop2-ADC-003 was calculated to be 8.0 based on the determination of sacituzumab light chain conjugated to 1 toxin molecule (LC+DAR1 ratio of 100%) and heavy chain conjugated to 3 toxin molecules (HC+DAR3 ratio of 100%).

[1125] Example 4.2.4 Preparation of Trop2-ADC-004

[1126] Using the same method as in Example 4.1, DL021 was replaced with DL001 and sacituzumab was replaced with trastuzumab to obtain the coupling product Trop2-ADC-004 of DL021 and sacituzumab. The DAR value was determined to be 8.0 by mass spectrometry.

[1127] Table 24

[1128] The antibody-drug conjugation ratio (DAR value) of Trop2-ADC-004 was calculated to be 8.0 based on the determination of sacituzumab light chain conjugated to 1 toxin molecule (LC+DAR1 ratio of 100%) and heavy chain conjugated to 3 toxin molecules (HC+DAR3 ratio of 100%).

[1129] Example for comparison:

[1130] The Payload+Linker structure is as follows (synthesized according to patent WO2024052685A1):

[1131] Example 1: Preparation of control HER2-ADC-1

[1132] Using the same method as in Example 4.1, DL001 was replaced with the control Payload+Linker to obtain the control Payload+Linker and trastuzumab conjugate product HER2-ADC-1, with a DAR value of 7.8 determined by mass spectrometry.

[1133] Table 25

[1134] The antibody-drug conjugation ratio (DAR value) of trastuzumab was determined as follows: light chain conjugated with 0 toxin molecules (LC ratio of 8%), conjugated with 1 toxin molecule (LC+DAR1 ratio of 92%), heavy chain conjugated with 2 toxin molecules (HC+DAR2 ratio of 2%), and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 98%). Based on these results, the antibody-drug conjugation ratio (DAR value) of the control HER2-ADC-1 was calculated to be 7.8.

[1135] Comparative Example 2: Preparation of Trop2-ADC-1

[1136] Using the same method as in Example 4.1, DL001 was replaced with the control Payload+Linker and Trastuzumab was replaced with sacituzumab to obtain the control Payload+Linker and sacituzumab coupling product control Trop2-ADC-1, and the DAR value was determined to be 7.7 by mass spectrometry.

[1137] Table 26

[1138] The antibody-drug conjugation ratio (DAR value) of sacituzumab was determined by measuring the light chain conjugation of 0 toxin molecules, 1 toxin molecule (LC ratio of 5%, LC+DAR1 ratio of 95%), the heavy chain conjugation of 1 toxin molecule, 2 toxin molecules, and 3 toxin molecules (HC+DAR1 ratio of 3%, HC+DAR2 ratio of 7%, HC+DAR3 ratio of 90%). Based on this, the antibody-drug conjugation ratio (DAR value) of the control Trop2-ADC-1 was calculated to be 7.7.

[1139] Example 5. Cell activity assay of NMT inhibitor active compounds

[1140] Example 5.1. Cell activity assay of NMT inhibitory compounds NCI-N87 and BT-474

[1141] 1. Experimental methods for inhibiting cell proliferation activity:

[1142] NCI-N87 (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 cells were seeded at a density of 3000 cells / well and BT-4747 cells at a density of 3000 cells / well, with a density of 142.5 μL / well in 96-well plates. The plates were incubated overnight at 37°C. The next day, the test compounds were serially diluted 400-fold (400×) with DMSO, and then further diluted 20-fold (20×) with serum-free medium to a final concentration of 1000 nM. Nine 4-fold dilutions were performed. 7.5 μL of 20× test compound dilution was added to each well. After adding the test compounds, the 96-well plates were incubated at 37°C and 5% CO2 for 8 days.

[1143] After incubation, the 96-well plate was equilibrated at room temperature for 20 min. 100 μL (NCI-N87) / 50 μL (BT-474) 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 27 below.

[1144] Table 27. Material Sources

[1145] 2. Experimental Results:

[1146] As shown in Table 28, the NMT inhibitor compounds disclosed herein all exhibit significant proliferative inhibitory activity on tumor cells.

[1147] Table 28. Inhibitory activity of NMT inhibitor compounds against tumor cell proliferation in vitro (IC50 value)

[1148] Example 5.2. Cell viability assay of NMT inhibitor compounds OVCAR-3, MCF-7, MDA-MB-468, OE19, HCC1569, PC9, HCC827, SNU-16, and SK-BR-3

[1149] 1. Experimental methods for inhibiting cell proliferation activity:

[1150] Resuscitate and culture cells (cell culture conditions are shown in Table 29 below). Once the cells are stable, seed them into 96-well plates at a density of 142.5 μL / well and incubate overnight at 37°C. The next day, dilute the ADC to be tested 20-fold (20×) with serum-free medium to a final concentration of 500 nM, and perform 3-fold dilutions at 9 concentration points. Serially dilute the small molecule to be tested with DMSO to a final concentration of 400-fold (400×), then further dilute with serum-free medium to a final concentration of 1000 nM, and perform 3-fold dilutions at 9 concentration points. Add 7.5 μL of 20× analyte dilution to each well. After adding the analytes, incubate the 96-well plate at 37°C with 5% CO2 for 6-8 days.

[1151] Table 29. Cell Culture Conditions and Plating Density

[1152] 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 30 below.

[1153] Table 30. Material Sources

[1154] 2. Experimental Results:

[1155] As shown in Table 31, the NMT inhibitor compounds disclosed herein all exhibit significant proliferative inhibitory activity on tumor cells.

[1156] Table 31. Inhibitory activity of NMT inhibitor compounds against tumor cell proliferation in vitro (IC50 value)

[1157] Note: " / " indicates that the activity was not measured.

[1158] Example 6. Determination of the in vitro cell-killing activity of ADC

[1159] Example 6.1 Assay of Her2-ADC's inhibitory activity on the proliferation of BT-474, NCI-N87, and SK-BR-3 tumor cells

[1160] 1. Experimental methods for inhibiting cell proliferation activity:

[1161] Resuscitate and culture NCI-N87 (RPMI 1640 + 10% FBS + 1% P / S), SK-BR-3 (McCoy's 5A + 10% FBS + 1% P / S), and BT-474 (DMEM + 10% FBS + 1% P / S + 10 μg / mL insulin) cells. After cell stabilization, seed NCI-N87 cells at 3000 / well, SK-BR-3 cells at 1000 / well, and BT-474 cells at 3000 / well, at a density of 142.5 μL / well in 96-well plates and incubate overnight at 37°C. The next day, dilute the ADC to be tested 20-fold (20×) with serum-free medium to a final concentration of 200 nM, and perform 4-fold dilutions at 9 concentration points. Add 7.5 μL of 20× analyte diluent to each well. After adding the analyte, the 96-well plate was incubated at 37°C and 5% CO2 for 8 days.

[1162] After incubation, the 96-well plate was equilibrated at room temperature for 20 min. 100 μL (NCI-N87) / 50 μL (SK-BR-3 / BT-474) 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 32 below.

[1163] Table 32. Material Sources

[1164] 2. Experimental Results:

[1165] As shown in Table 33, the Her2-ADC disclosed herein exhibits significant inhibitory activity on tumor cells.

[1166] Table 33. Inhibitory activity of Her2-ADC against tumor cell proliferation in vitro (IC50 value)

[1167] Note: " / " indicates that the activity was not measured.

[1168] Example 6.2 Test of Trop2-ADC's inhibitory activity on the proliferation of MDA-MB-468 and PC-9 tumor cells

[1169] 1. Experimental methods for inhibiting cell proliferation activity:

[1170] MDA-MB-468 (DMEM + 10% FBS + 1% P / S) and PC-9 (RPMI 1640 + 10% FBS + 1% P / S) cells were revived and cultured. After the cells stabilized, MDA-MB-468 cells were seeded at a density of 1500 cells / well and PC-9 cells at a density of 500 cells / well, for a total of 142.5 μL / well in 96-well plates and incubated overnight at 37°C. The next day, the ADC to be tested was diluted 20-fold (20×) with serum-free medium to a final concentration of 500 nM, and 3-fold dilutions were performed at 9 concentration points. 7.5 μL of 20× analyte dilution was added to each well. After adding the analyte, the 96-well plates were incubated at 37°C and 5% CO2 for 8 days.

[1171] 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 was measured after 10 minutes of incubation. The sources of key materials are shown in Table 34 below.

[1172] Table 34. Material Sources

[1173] 2. Experimental Results:

[1174] As shown in Table 35, the Trop2-ADC disclosed herein exhibits significantly stronger proliferative inhibitory activity in tumor cells than the control ADC.

[1175] Table 35. Inhibitory activity of Trop2-ADC against tumor cell proliferation in vitro (IC50 value)

[1176] Example 6.3 Test of Trop2-ADC's inhibitory activity on the proliferation of NCI-H929 and NCI-N87 tumor cells

[1177] 1. Experimental methods for inhibiting cell proliferation activity:

[1178] NCI-H292 and NCI-N87 cells were revived and cultured. After the cells stabilized, both cell types were seeded at a density of 1500 cells / well in 96-well plates and incubated overnight at 37°C. The next day, the ADC to be tested was diluted 20-fold (20×) with serum-free medium to a final concentration of 500 nM. Nine concentration points were obtained by 3-fold dilution. 7.5 μL of 20× analyte diluent was added to each well. After adding the analyte, the 96-well plates were incubated at 37°C and 5% CO2 for 8 days.

[1179] 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 36 below.

[1180] Table 36. Material Sources

[1181] 2. Experimental Results:

[1182] As shown in Table 37, the Trop2-ADC disclosed herein exhibits significantly stronger proliferative inhibitory activity in tumor cells than the control ADC.

[1183] Table 37. Inhibitory activity of Trop2-ADC against tumor cell proliferation in vitro (IC50 value)

[1184] Example 7. In vivo activity assay of ADC

[1185] Example 7.1 Efficacy test of ADC on BT-474 xenograft tumor

[1186] 1. Experimental Materials

[1187] Test compound: Her2-ADC-001, with physiological saline as a negative control.

[1188] Experimental cells: BT-474 cells.

[1189] 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.

[1190] 2. Experimental Design

[1191] 2.1. Cell Treatment

[1192] 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.

[1193] 2.2. Tumor cell transplantation

[1194] BALB / c nude mice were acclimatized to the laboratory environment for 3-5 days. Three days before cell inoculation, 0.36 mg estrogen extended-release tablets were subcutaneously injected into the left posterior back of each mouse. One week after estrogen tablet injection, assisted urination was initiated three times per week; if necessary, assisted urination was initiated daily. 0.2 mL (10 x 10) 6 BT-474x cells were subcutaneously inoculated into the right back near the upper limb of each mouse using PBS plus matrigel (1:1). The average tumor volume reached approximately 150-200 mm. 3 Dosing will begin in groups at that time.

[1195] 2.3. Animal drug administration and detection

[1196] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1197] Table 38. Dosing Regimen

[1198] 2.4. Tumor volume and body weight measurement

[1199] 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:

[1200] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short 2, where L_length and L_short represent the long diameter and short diameter of the tumor, respectively.

[1201] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups)) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1202] 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.

[1203] 3. Experimental Results

[1204] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 39.

[1205] Table 39. Tumor volume data of the BT-474 xenograft model

[1206] Example 7.2 Efficacy test of ADC on NCI-N87 xenograft tumor

[1207] 1. Experimental Materials

[1208] Test compound: physiological saline as negative control, Her2-ADC-001.

[1209] Experimental cells: NCI-N87 cells.

[1210] Experimental animals: Balb / c Nude mice, female, 5-6 weeks old, purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[1211] 2. Experimental Design

[1212] 2.1. Cell Treatment

[1213] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in culture flasks. When the cell confluence reached approximately 80-90%, they were digested with trypsin containing EDTA. The cells were washed twice with PBS, then 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.

[1214] 2.2. Tumor cell transplantation

[1215] Balb / c Nudd mice were acclimatized to the laboratory environment for 3-5 days, and then subcutaneously injected with NCI-N87 cells in the right rib area at a cell density of 5 × 10⁶ cells. 6 / animal, inoculated with 0.2 mL (containing 50% Matrigel), until the tumor grows to 200 mm 3 When the time is around 10:00, administer the medication in groups.

[1216] 2.3. Animal drug administration and detection

[1217] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1218] Table 40. Dosing Regimen

[1219] 2.4. Tumor volume and body weight measurement

[1220] Tumor volume and body weight were measured twice a week, and the relative tumor proliferation rate (T / C) and relative tumor inhibition rate (TGI) were calculated.

[1221] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short²;

[1222] 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);

[1223] Relative tumor inhibition rate (TGI%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1224] 3. Experimental Results

[1225] The test results showed that the antibody-drug conjugates disclosed herein all exhibited significant antitumor effects. 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 41.

[1226] Table 41. Tumor volume data of NCI-N87 xenograft model

[1227] Example 7.3 Efficacy test of ADC on NCI-N87 xenograft tumor

[1228] 1. Experimental Materials

[1229] Test compounds: physiological saline as a negative control, Herceptin, Her2-ADC-009.

[1230] Experimental cells: NCI-N87 cells.

[1231] Experimental animals: Balb / c Nude mice, female, 5-6 weeks old, purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[1232] 2. Experimental Design

[1233] 2.1. Cell Treatment

[1234] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in culture flasks. When the cell confluence reached approximately 80-90%, they were digested with trypsin containing EDTA. The cells were washed twice with PBS, then 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.

[1235] 2.2. Tumor cell transplantation

[1236] Balb / c Nudd mice were acclimatized to the laboratory environment for 3-5 days, and then subcutaneously injected with NCI-N87 cells in the right rib area at a cell density of 5 × 10⁶ cells. 6 / animal, inoculated with 0.2 mL (containing 50% Matrigel), until the tumor grows to 200 mm 3 When the time is around 10:00, administer the medication in groups.

[1237] 2.3. Animal drug administration and detection

[1238] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1239] Table 42. Dosing Regimen

[1240] 2.4. Tumor volume and body weight measurement

[1241] Tumor volume and body weight were measured twice a week, and the relative tumor proliferation rate (T / C) and relative tumor inhibition rate (TGI) were calculated.

[1242] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 ;

[1243] 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);

[1244] Relative tumor inhibition rate (TGI%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1245] 3. Experimental Results

[1246] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 43.

[1247] Table 43. Tumor volume data of NCI-N87 xenograft model

[1248] Example 7.4 Efficacy test of ADC on NCI-N87 xenograft tumor

[1249] 1. Experimental Materials

[1250] Test compounds: physiological saline as negative control, control ADC-1, Her2-ADC-011, Her2-ADC-012.

[1251] Experimental cells: NCI-N87 cells.

[1252] Experimental animals: Balb / c Nude mice, female, 5-6 weeks old, purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[1253] 2. Experimental Design

[1254] 2.1. Cell Treatment

[1255] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in culture flasks. When the cell confluence reached approximately 80-90%, they were digested with trypsin containing EDTA. The cells were washed twice with PBS, then 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.

[1256] 2.2. Tumor cell transplantation

[1257] Balb / c Nudd mice were acclimatized to the laboratory environment for 3-5 days, and then subcutaneously injected with NCI-N87 cells in the right rib area at a cell density of 5 × 10⁶ cells. 6 / animal, inoculated with 0.2 mL (containing 50% Matrigel), until the tumor grows to 200 mm 3 When the time is around 10:00, administer the medication in groups.

[1258] 2.3. Animal drug administration and detection

[1259] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1260] Table 44. Dosing Regimen

[1261] 2.4. Tumor volume and body weight measurement

[1262] Tumor volume and body weight were measured twice a week, and the relative tumor proliferation rate (T / C) and relative tumor inhibition rate (TGI) were calculated.

[1263] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short²;

[1264] 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);

[1265] Relative tumor inhibition rate (TGI%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1266] 3. Experimental Results

[1267] The test results showed that the antibody-drug conjugates disclosed herein all exhibited significant antitumor effects. 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 45.

[1268] Table 45. Tumor volume data of NCI-N87 xenograft model

[1269] Example 7.5 ADC efficacy test on BT-474 xenograft tumor

[1270] 1. Experimental Materials

[1271] Test compounds: Controls ADC-1, Her2-ADC-014, Her2-ADC-016, and physiological saline as a negative control.

[1272] Experimental cells: BT-474 cells.

[1273] 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.

[1274] 2. Experimental Design

[1275] 2.1. Cell Treatment

[1276] 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.

[1277] 2.2. Tumor cell transplantation

[1278] BALB / c nude mice were acclimatized to the laboratory environment for 3-5 days. Three days before cell inoculation, 0.36 mg estrogen extended-release tablets were subcutaneously injected into the left posterior back of each mouse. One week after estrogen tablet injection, assisted urination was initiated three times per week; if necessary, assisted urination was initiated daily. 0.2 mL (10 x 10) 6 BT-474x cells were subcutaneously inoculated into the right back near the upper limb of each mouse using PBS plus matrigel (1:1). The average tumor volume reached approximately 150-200 mm. 3 Dosing will begin in groups at that time.

[1279] 2.3. Animal drug administration and detection

[1280] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1281] Table 46. Dosing Regimen

[1282] 2.4. Tumor volume and body weight measurement

[1283] 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:

[1284] 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.

[1285] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups)) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1286] 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.

[1287] 3. Experimental Results

[1288] Test results showed that the antibody-drug conjugate disclosed herein exhibited better tumor-suppressing effects than the control ADC-1. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 47.

[1289] Table 47. Tumor volume data of the BT-474 xenograft model

[1290] Example 7.6 Efficacy test of ADC on BT-474 xenograft tumor

[1291] 1. Experimental Materials

[1292] Test compounds: Her2-ADC-016, Her2-ADC-017, with physiological saline as a negative control.

[1293] Experimental cells: BT-474 cells.

[1294] 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.

[1295] 2. Experimental Design

[1296] 2.1. Cell Treatment

[1297] 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.

[1298] 2.2. Tumor cell transplantation

[1299] BALB / c nude mice were acclimatized to the laboratory environment for 3-5 days. Three days before cell inoculation, 0.36 mg estrogen extended-release tablets were subcutaneously injected into the left posterior back of each mouse. One week after estrogen tablet injection, assisted urination was initiated three times per week; if necessary, assisted urination was initiated daily. 0.2 mL (10 x 10) 6 BT-474x cells were subcutaneously inoculated into the right back near the upper limb of each mouse using PBS plus matrigel (1:1). The average tumor volume reached approximately 150-200 mm. 3 Dosing will begin in groups at that time.

[1300] 2.3. Animal drug administration and detection

[1301] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1302] Table 48. Dosing Regimen

[1303] 2.4. Tumor volume and body weight measurement

[1304] 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:

[1305] 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.

[1306] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups)) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1307] 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.

[1308] 3. Experimental Results

[1309] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 49.

[1310] Table 49. Tumor volume data of the BT-474 xenograft model

[1311] Example 7.7 Efficacy test of ADC on NCI-N87 xenograft tumor

[1312] 1. Experimental Materials

[1313] Test compounds: physiological saline as negative control, Her2-ADC-016, Her2-ADC-017, and control HER2-ADC-1.

[1314] Experimental cells: NCI-N87 cells.

[1315] Experimental animals: Balb / c Nude mice, female, 5-6 weeks old, purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[1316] 2. Experimental Design

[1317] 2.1. Cell Treatment

[1318] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in culture flasks. When the cell confluence reached approximately 80-90%, they were digested with trypsin containing EDTA. The cells were washed twice with PBS, then 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.

[1319] 2.2. Tumor cell transplantation

[1320] Balb / c Nudd mice were acclimatized to the laboratory environment for 3-5 days, and then subcutaneously injected with NCI-N87 cells in the right rib area at a cell density of 5 × 10⁶ cells. 6 / animal, inoculated with 0.2 mL (containing 50% Matrigel), until the tumor grows to 200 mm 3 When the time is around 10:00, administer the medication in groups.

[1321] 2.3. Animal drug administration and detection

[1322] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1323] Table 50. Dosing Regimen

[1324] 2.4. Tumor volume and body weight measurement

[1325] Tumor volume and body weight were measured twice a week, and the relative tumor proliferation rate (T / C) and relative tumor inhibition rate (TGI) were calculated.

[1326] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 ;

[1327] 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);

[1328] Relative tumor inhibition rate (TGI%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1329] 3. Experimental Results

[1330] The test results showed that the antibody-drug conjugates disclosed herein all exhibited significant antitumor effects. 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 51.

[1331] Table 51. Tumor volume data of NCI-N87 xenograft model

[1332] Example 7.8 Efficacy test of ADC against ENHERTU-induced resistance in NCI-N87 xenografts

[1333] 1. Experimental Materials

[1334] Test compounds: physiological saline as negative control, Her2-ADC-016, Her2-ADC-017, Trop2-ADC-004.

[1335] Experimental cells: NCI-N87 cells.

[1336] Experimental animals: Balb / c Nude mice, female, 5-6 weeks old, purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[1337] 2. Experimental Design

[1338] 2.1. Cell Treatment

[1339] ENHERTU-induced resistant NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in culture flasks. When the cell confluence reached approximately 80-90%, the cells were digested with trypsin containing EDTA. The cells were washed twice with PBS, then 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.

[1340] 2.2. Tumor cell transplantation

[1341] Balb / c Nudd mice were acclimatized to the laboratory environment for 3-5 days, and then subcutaneously injected with ENHERTU-induced NCI-N87 cells at a dose of 5 × 10⁶ cells in the right rib area. 6 / animal, inoculated with 0.2 mL (containing 50% Matrigel), until the tumor grows to 200 mm 3 When the time is around 10:00, administer the medication in groups.

[1342] 2.3. Animal drug administration and detection

[1343] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[1344] Table 52. Dosing Regimen

[1345] 2.4. Tumor volume and body weight measurement

[1346] Tumor volume and body weight were measured twice a week, and the relative tumor proliferation rate (T / C) and relative tumor inhibition rate (TGI) were calculated.

[1347] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 ;

[1348] 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);

[1349] Relative tumor inhibition rate (TGI%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[1350] 3. Experimental Results

[1351] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect and has the potential to overcome drug resistance. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 53.

[1352] Table 53. Tumor volume data of NCI-N87 xenograft model induced by ENHERTU resistance.

[1353] Example 8: Evaluation of the bystander killing effect of Trop2-ADC drug on tumor cell co-culture system

[1354] 1. Cell markers

[1355] (a) Collect negative control cells (NCI-H292) and adjust the cell density to 1×10⁻⁶ cells using serum-free phosphate-buffered saline (PBS). 6 Single-cell suspensions were prepared using cells / mL.

[1356] (b) Add 1 μL of CellTrace per milliliter of cell suspension. TM Fluorescent dyes were incubated at 37°C in the dark for 20 minutes.

[1357] (c) Add 5 times the volume of complete culture medium (RPMI-1640 containing 10% fetal bovine serum) and let stand at room temperature for 5 minutes to quench unbound dye.

[1358] (d) Centrifuge at 300×g for 5 minutes to collect cells, discard the supernatant, resuspend the cells in preheated complete culture medium, and complete the labeling.

[1359] 2. Construction of co-culture system and drug delivery

[1360] (a) Unlabeled target-positive cells (A549) and labeled negative cells (NCI-H292) were seeded in 6-well plates at a ratio:

[1361] -Experimental group: 4×10⁶ seeds per well 4 A549 cells + 2 × 10 4 2:1 labeled NCI-H292 cells

[1362] -Control group 1: Single vaccination of 4×10 4 NCI-H292 cells (100% negative control)

[1363] -Control group 2: Single vaccination of 2×10 4 A549 cells (50% target-positive control)

[1364] (b) The experimental group was given complete culture medium containing gradient concentrations of control Trop2-ADC-1 and Trop2-ADC-004 (1 nM–100 μM), while the control group was given an equal volume of solvent.

[1365] (c) Incubate at 37°C and 5% CO2 for 4 days.

[1366] 3. Flow cytometry detection

[1367] (a) Collect cells from each well, centrifuge at 300×g for 5 minutes, and discard the supernatant.

[1368] (b) Resuspend the cells in pre-cooled FACS buffer (PBS containing 1% BSA).

[1369] (c) Detection using flow cytometry:

[1370] -via CellTrace TM Fluorescent channels distinguish labeled cells (NCI-H292) from unlabeled cells (A549).

[1371] - Analyze the survival rate of each cell subpopulation (e.g., using PI / Annexin V double staining).

[1372] (d) Calculate the specific killing rate of Trop2-ADC-1 and Trop2-ADC-004 on A549 cells using the following formula:

[1373] Specific kill rate (%) = [1 - (A549 survival rate in experimental group / A549 survival rate in control group)] × 100%

[1374] Table 54. List of Key Materials

[1375] 4. Experimental Results

[1376] Test results show that the antibody-drug conjugate disclosed herein exhibits excellent bystander effect. Specific results are shown in Table 55.

[1377] Table 55.

[1378] Example 9: Evaluation of the bystander killing effect of Her2-ADC drug on tumor cell co-culture system

[1379] 1. Cell markers

[1380] (a) Collect negative control cells (PC9) and adjust the cell density to 1×10⁶ cells using serum-free phosphate-buffered saline (PBS). 6 Single-cell suspensions were prepared using cells / mL.

[1381] (b) Add 1 μL of CellTrace per milliliter of cell suspension. TM Fluorescent dyes were incubated at 37°C in the dark for 20 minutes.

[1382] (c) Add 5 times the volume of complete culture medium (RPMI-1640 containing 10% fetal bovine serum) and let stand at room temperature for 5 minutes to quench unbound dye.

[1383] (d) Centrifuge at 300×g for 5 minutes to collect cells, discard the supernatant, resuspend the cells in preheated complete culture medium, and complete the labeling.

[1384] 2. Construction of co-culture system and drug delivery

[1385] (a) Unlabeled target-positive cells (NCI-N87) and labeled negative cells (PC9) were seeded in 6-well plates at a ratio:

[1386] -Experimental group: 4×10⁶ seeds per well 4 PC9 cells + 2 × 10 4 2:1 labeled NCI-N87 cells

[1387] -Control group 1: Single vaccination of 4×10 4 NCI-N87 cells (100% negative control)

[1388] -Control group 2: Single vaccination of 2×10 4 PC9 cells (50% target-positive control)

[1389] (b) The experimental group was given complete culture medium containing gradient concentrations of controls Her2-ADC-1, HER2-ADC-014, and HER2-ADC-016 (1 nM–100 μM), while the control group was given an equal volume of solvent.

[1390] (c) Incubate at 37°C and 5% CO2 for 4 days.

[1391] 3. Flow cytometry detection

[1392] (a) Collect cells from each well, centrifuge at 300×g for 5 minutes, and discard the supernatant.

[1393] (b) Resuspend the cells in pre-cooled FACS buffer (PBS containing 1% BSA).

[1394] (c) Detection using flow cytometry:

[1395] -via CellTrace TM Fluorescent channels distinguish labeled cells (NCI-N87) from unlabeled cells (PC9).

[1396] - Analyze the survival rate of each cell subpopulation (e.g., using PI / Annexin V double staining).

[1397] (d) Calculate the specific killing rate of Her2-NMTi against PC9 cells using the following formula:

[1398] Specific kill rate (%) = [1 - (PC9 survival rate in experimental group / PC9 survival rate in control group)] × 100%

[1399] Table 56. List of Key Materials

[1400] 4. Experimental Results

[1401] Test results show that the antibody-drug conjugate disclosed herein exhibits excellent bystander effect. Specific results are shown in Table 57.

[1402] Table 57.

[1403] Although specific embodiments of this disclosure have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings published, and all such modifications and variations are within the scope of this disclosure. The scope of this disclosure is defined by the appended claims and any equivalents thereof.

Claims

1. An antibody-drug conjugate of Formula II, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, in, Tb is an antibody or its antigen-binding fragment; q is any value between 0.1 and 12.0; L is a linker that covalently bonds Tb and D, and L contains Among them, R L1 R L2 Each independently is C 1-4 alkyl or R L3 for and R L4 for Or hydrogen; p can be any integer between 1 and 20 independently; D represents a fragment of a bioactive compound that acts as an NMT inhibitor.

2. The antibody-drug conjugate as described in claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, The L includes Among them, R L1 R L2 Each independently is C 1-4 alkyl or R L3 for and R L4 For hydrogen, p is an independent integer between 1 and 20; Preferably, L is in, L1 is the connector unit, which covalently bonds Tb and L2; L2 is a connecting unit, which is covalently bonded to L1 and L3; L3 is Among them, R L1 R L2 Each independently is C 1-4 alkyl or R L3 for and R L4 For hydrogen; p is an independent integer between 1 and 20; L4 is a spacer unit that is covalently bonded to L3 and D.

3. The antibody-drug conjugate as described in claim 2, or its stereoisomer or pharmaceutically acceptable salt, wherein, L1 is Position 1 is connected to Tb via an S atom, and position 2 is connected to L2; Preferably, L1 is Position 1 is connected to Tb via the S atom, and position 2 is connected to L2.

4. The antibody-drug conjugate as described in claim 2, or its stereoisomer or pharmaceutically acceptable salt, wherein, The L2 is m and n are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8; 1 bit is connected to L1 and 2 bits are connected to L3; Preferably, L2 is Bit 1 is connected to L1, and bit 2 is connected to L3.

5. The antibody-drug conjugate as described in claim 2, or its stereoisomer or pharmaceutically acceptable salt, wherein, The L3 is Among them, R L1 R L2 Simultaneously methyl, ethyl, propyl, butyl or R L3 for and R L4 For hydrogen; p is an independent integer between 1 and 20; Preferably, L3 is 6. The antibody-drug conjugate as described in claim 2, or its stereoisomer or pharmaceutically acceptable salt, wherein, The L4 is 7. The antibody-drug conjugate as claimed in claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, L is: R L1 R L2 Each independently is C 1-4 alkyl or p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, position 1 is connected to Tb through an S atom, and position 2 is connected to D; Preferably, L is Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

8. The antibody-drug conjugate as claimed in claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, The structure D is shown in Formula IIC-1: in, R'1 is -CH2N(CH3)-, with the N atom bonded to L; R2 is -C(OH)R a R b -C(O)R c -C(O)N(R) d )R c -C(O)-4-6 membered nitrogen heterocycle (CH2) t OH, 5-6 membered cycloalkenyl, C 2-6 Alkenyl-OH or -4-6 membered nitrogen heterocycle (CH2) t OH; R3 and R4 are each independently hydrogen, methyl, and C. 1-6 Alkylene OH, C 1-4 Halogenated alkyl or 3-6 membered cycloalkyl, Alternatively, R3, R4, and the atoms attached to them can form 5-6 membered heterocycles; t is 0, 1, 2, 3, or 4; R a R b Each independently is hydrogen, C 1-4 Alkyl, 3-6 membered cycloalkyl, 3-6 membered halocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 2-6 alkenyl or C 2-6 Haloalkenyl; R c Unsubstituted or substituted 3-6 membered cycloalkyl, C 2-6 alkenyl, C 1-4 alkylene OH or C 1-6 Alkyl; the substitution refers to the substitution by halogen, -C 1-4 Alkyl, -C 1-4 Alkylene OH or -OH substitution; R d R e Each can be either hydrogen or methyl.

9. The antibody-drug conjugate of claim 8, or its stereoisomer or pharmaceutically acceptable salt, wherein, The D satisfies one or more of the following conditions: (1) R2 is -C(OH)R a R b R a For hydrogen, R b C 1-4 Alkyl, 3-6 membered cycloalkyl, 3-6 membered halocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 2-6 alkenyl or C 2-6 Haloalkenyl; Preferably, R2 is (2) R2 is -C(O)R c R c C 1-6 Alkyl, 3-6 membered cycloalkyl, C 1-6 Halogenated alkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl or 3-6 membered halocycloalkyl; Preferably, R2 is (2) R3 and R4 are both methyl groups.

10. The antibody-drug conjugate of claim 8, or its stereoisomer or pharmaceutically acceptable salt, wherein, The D has the following structure: 1 is connected to L.

11. The antibody-drug conjugate of claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, Tb satisfies one or more of the following conditions: (1) Tb is an anti-Her2 antibody or its antigen-binding fragment or an anti-Trop-2 antibody or its antigen-binding fragment; (2) Tb is an anti-Her2 antibody or its antigen-binding fragment, preferably anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or its antigen-binding fragment; preferably, Tb is trastuzumab or pertuzumab; more preferably, Tb is trastuzumab. (3) Tb is an anti-Trop-2 antibody or its antigen-binding fragment, preferably datopotamab, sacituzumab or its antigen-binding fragment; preferably, Tb is sacituzumab.

12. The antibody-drug conjugate of claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, The q is any number between 1 and 8; preferably, the q is any number between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, and 7-8; more preferably, the q is 2, 4, 6, or 8.

13. The antibody-drug conjugate of claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, The antibody-drug conjugate has the structure shown in Formula II-1. Wherein, Tb, q, L1, L2, L3, L4 and D are defined as in any one of claims 1-12; Preferably, the antibody-drug conjugate has the structure shown in Formula II-2. Among them, Tb, q, R2, R3, R4, R L1 R L2 R L3 and R L4 The definition is as defined in any one of claims 1-12.

14. The antibody-drug conjugate of claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, The antibody-drug conjugate has any of the following structures:

15. The antibody-drug conjugate of claim 1, or its stereoisomer or pharmaceutically acceptable salt, wherein, The antibody-drug conjugate has any of the following structures: Wherein, Tb1 is an anti-Her2 antibody or its antigen-binding fragment, preferably anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab or its antigen-binding fragment; preferably, Tb1 is Trastuzumab, and q is any number between 7 and 8; Wherein, Tb2 is an anti-Trop-2 antibody or its antigen-binding fragment, preferably datopotamab, sacituzumab or its antigen-binding fragment, and preferably Tb2 is sacituzumab; q is any number between 7 and 8.

16. The drug-linked conjugate shown in Formula III, or its stereoisomer or pharmaceutically acceptable salt, Lg-LD Formula III Wherein, Lg is a group that reacts with the antibody; 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 1-10.

17. The drug linker conjugate of claim 16, or its stereoisomer or pharmaceutically acceptable salt, wherein, The drug linker conjugate has the structure shown in Formula III-1. Lg-L1-L2-L3-L4-D Formula III-1 in, When L1 is At that time, Lg-L1 is 2 bits are connected to L2; When L1 is At this time, bit 1 is connected to Lg, and bit 2 is connected to L2; Lg is MeSO2-; The definitions of L2, L3, L4 and D are as defined in any one of claims 1-10; Preferably, the drug linker conjugate has the structure shown in Formula III-2. Among them, R2, R3, R4, R L1 R L2 R L3 and R L4 The definition is as defined in any one of claims 1-10.

18. The drug linker conjugate of claim 16, or its stereoisomer or pharmaceutically acceptable salt, wherein, The drug linker conjugate has any of the following structures:

19. The NMT inhibitor bioactive compound of formula IC, or its stereoisomer or pharmaceutically acceptable salt thereof; in, R1 is -CH2NH(CH3); R2 is -C(O)R c , R c C 1-6 Alkyl, 3-6 membered cycloalkyl, C 1-6 Haloalkyl, -C 1-4 Alkyl-substituted 3-6 membered cycloalkyl or 3-6 membered halocycloalkyl, preferably, R c Cyclopropylidene, C 1-4 Alkyl-substituted cyclopropane or halocyclopropane; Preferably, R2 is R3 and R4 are both methyl groups.

20. The NMT inhibitor bioactive compound of claim 19, or its stereoisomer or pharmaceutically acceptable salt, wherein, The NMT inhibitor bioactive compound has any of the following structures:

21. The antibody-drug conjugate shown in Formula II, or its stereoisomer or pharmaceutically acceptable salt, 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; D is a fragment of the NMT inhibitor bioactive compound of claim 19 or 20 that is missing one hydroxyl hydrogen or amino hydrogen, and the N or O atom on D is connected to L.

22. The antibody-drug conjugate of claim 21, or its stereoisomer or pharmaceutically acceptable salt, wherein, The definition of L is as defined in any one of claims 2-7, the definition of Tb is as defined in claim 11, and the definition of q is as defined in claim 12.

23. A group of antibody-drug conjugates comprising any one of claims 1-15, 21 or 22, or a pharmaceutically acceptable salt thereof, or a composition thereof, wherein the antibody-drug conjugate has one, two or more q values; Preferably, the average DAR of the antibody-drug conjugate group is an integer or decimal between 1 and 16, preferably 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.

24. A pharmaceutical composition comprising an antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-15, 21, or 22; or a drug linker conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 16-18; or a bioactive compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in claim 19 or 20; or the group of antibody-drug conjugates as claimed in claim 23; and optionally one or more pharmaceutical excipients.

25. The use of an antibody-drug conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, as described in any one of claims 1-15, 21, or 22; or a drug linker conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, as described in any one of claims 16-18; or a bioactive compound, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, as described in claim 19 or 20; or the antibody-drug conjugate group of claim 23; or the pharmaceutical composition of claim 24, in the preparation of a medicament for treating and / or preventing diseases associated with abnormal cellular activity; preferably, the disease is cancer. Preferably, the cancer is a cancer related to the targets HER2 and TROP2; More preferably, the cancer is myeloma, lung cancer, breast cancer, colorectal cancer, gastric cancer, urothelial carcinoma, or prostate cancer; preferably, the lung cancer is small cell lung cancer or non-small cell lung cancer.

26. A linker in an antibody-drug conjugate, in, Position 1 is linked to Tb, and position 2 is linked to a bioactive molecular fragment; L is defined as in any one of claims 1-10; Preferably, its structure is as follows: Among them, position 1 is linked to Tb, and position 2 is linked to a bioactive molecular fragment; The definitions of L1, L2, L3, and L4 are as defined in any one of claims 1-10; The definition of the bioactive molecular fragment is as defined by D as in any one of claims 1-10.

27. A compound as shown in formula DL-X-5, or a salt thereof, in, G is chlorine or Preferred Preferably, the compound represented by formula DL-X-5 has the following structure:

28. A method for preparing a drug linker conjugate, characterized in that, It includes the following method: reacting the compound of formula DL-X-5 or its salt with the compound of formula IC to obtain the compound of formula III-2. Among them, R2, R3, R4, R L1 R L2 R L3 and R L4 The definition is as defined in any one of claims 1-10; G is chlorine or

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