Hydroximic acid compound and application thereof

Abstract

The invention discloses a hydroximic acid compound with a structure as shown in a formula (I) or a pharmaceutically acceptable salt or a stereoisomer of the hydroximic acid compound, and application of the hydroximic acid compound or the pharmaceutically acceptable salt or the stereoisomer. The invention provides a hydroximic acid compound, and discovers that a hydroximic acid group can be covalently bound with a lysine residue of a target protein after being oxidized through a novel efficient oxidation induction strategy, the lysine residue in the protein or polypeptide can be selectively and covalently modified, and the modification of the lysine residue has high selectivity and high efficiency; the method can be used for protein activity research, and has important significance on chemical proteomics application.

Description

[0001] This invention is a divisional application of Chinese patent application filed on August 6, 2024, with application number 202411072951X and application title "Hydroxamic acid compounds and their applications". Technical Field

[0002] This invention belongs to the fields of chemical biological molecular probes and chemical medicine, particularly hydroxamic acid compounds and their application in covalently bound lysine residues of proteins. Background Technology

[0003] In recent years, significant progress has been made in the development of covalent inhibitors, which have been applied to the treatment of various diseases. Targeted covalent inhibitors, because they can covalently bind to target proteins and thus inhibit their function for extended periods, offer advantages such as prolonged duration of action, low dosage, and high selectivity. Most covalent inhibitors marketed in recent years target covalent cysteine ​​residues; for example, the anticancer drug neratinib covalently binds to cysteine ​​at position 797 of EGFR via acrylamide. However, cysteine ​​is very rare in the human proteome, accounting for only 1.9% of amino acid residues, and many ligand binding sites lack accessible cysteine ​​residues. Furthermore, with in-depth research, we have found that highly active cysteine ​​residues are prone to acquired resistance due to mutations; for example, the C797S cysteine ​​mutation in EGFR is a common resistance mechanism.

[0004] Therefore, the development of targeted covalent inhibitors faces two major challenges: first, the targetable amino acids are limited to cysteine, which has a low abundance; second, mutations in highly active cysteine ​​residues lead to acquired resistance. To address these issues, researchers have recently turned their attention to lysine residues, which are more abundant than cysteine ​​residues in the human proteome and are more conserved and less prone to mutation. Using cocrystallization and isoTOP-ABPP techniques, researchers discovered the covalently available lysine residue K162 for AURKA kinase and quantified over 9,000 lysine residues. However, compared to the abundant lysine residues in the human proteome, the number of currently identified covalently available lysine residues is relatively small, and no covalent inhibitors targeting lysine have yet been approved for marketing. Therefore, discovering and identifying other highly abundant covalently available amino acid residues to develop more covalent inhibitors targeting lysine is an urgent problem to be solved.

[0005] In past research, scientists have been working to develop covalent probes that can bind highly selectively to lysine residues by continuously improving and innovating chemical strategies. However, some existing lysine probes, such as arylsulfonyl fluoride probes, are chemically too reactive, binding to lysine, tyrosine, and serine, resulting in low selectivity and specificity for lysine. Furthermore, upon entering the biological environment, some existing lysine probes, due to their high reactivity, directly covalently bind to lysine residues of non-target proteins. The process by which existing lysine probes approach target proteins lacks control, leading to low selectivity for target proteins. These factors hinder the development of covalent inhibitors targeting lysine. Summary of the Invention

[0006] Based on this, the present invention provides a class of hydroxamic acid compounds that can be selectively covalently modified and labeled with lysine residues after oxidation.

[0007] The present invention includes the following technical solutions.

[0008] Hydroxyxamic acid compounds having the structure shown in formula (I), or pharmaceutically acceptable salts thereof, or stereoisomers thereof,

[0009]

[0010] Wherein, R is selected from: C6-C with or without R1 substitution. 10 aryl, R1-substituted or unsubstituted 5-10-membered heteroaryl, R1-substituted or unsubstituted 5-10-membered heteroaryl ketone, or selected from the following groups:

[0011]

[0012] Each R1 is independently selected from: hydrogen, ethynyl, vinyl, R2-substituted or unsubstituted C1-C6 alkyl, R3-substituted or unsubstituted C6-C 10 Aryl, C1-C6 alkoxy, nitro, C1-C6 alkylamine, 5-8 membered heterocyclic, -C(=O)NR4,

[0013] Each R2 is independently selected from: halogen, ethynyl, vinyl, C1-C6 alkoxy, nitro, C1-C6 alkylamine, C6-C 10 Aryl, R5NH-, R5(CH2) n NH-;

[0014] Each R3 is independently selected from: hydrogen, ethynyl, vinyl, trifluoromethyl, C1-C6 alkoxy, nitro, and C1-C6 alkylamine;

[0015] Each R4 is independently selected from: C1-C6 alkyl, C6-C 10Aryl-substituted C1-C6 alkyl groups, tolyl-substituted C1-C6 alkyl groups;

[0016] Each R5 is independently selected from: 5-10 heteroaryl groups with or without R6 substitution, or C6-C groups with or without R6 substitution. 10 Aryl;

[0017] Each R6 is independently selected from: 5-10 membered heteroaryl groups and 5-8 membered heterocyclic groups;

[0018] n is selected from: 1, 2, 3, 4.

[0019] In some of these embodiments, R is selected from: R1-substituted or unsubstituted phenyl, R1-substituted or unsubstituted indolyl, R1-substituted or unsubstituted naphthyl, R1-substituted or unsubstituted thiophenyl, R1-substituted or unsubstituted pyridyl, R1-substituted or unsubstituted 4-oxo-4H-benzopyranyl, R1-substituted or unsubstituted 1,3-dioxoisoindolinyl, and R1-substituted or unsubstituted pyrazinyl.

[0020] In some of these embodiments, each R1 is independently selected from: hydrogen, ethynyl, propynyl, vinyl, trifluoromethyl, C1-C3 alkyl, R2-substituted C1-C3 alkyl, R3-substituted or unsubstituted phenyl, diethylamino, tetrahydropyrrolyl, -C(=O)NR4.

[0021] Each R2 is independently selected from: phenyl, R5NH-, R5CH2NH-;

[0022] Each R3 group is independently selected from: hydrogen, ethynyl, vinyl, trifluoromethyl, methoxy, ethoxy, nitro, and diethylamino.

[0023] Each R4 is independently selected from: C1-C3 alkyl, phenyl-substituted C1-C3 alkyl, and tolyl-substituted C1-C3 alkyl;

[0024] Each R5 is independently selected from: quinolinyl, imidazo[1,2-a]pyridyl, dibenzo[b,d]furanyl-substituted phenyl, 4-morpholinylphenyl, and benzimidazolyl.

[0025] In some embodiments, R is selected from:

[0026]

[0027] The present invention also provides uses of the hydroxamic acid compound, including the following:

[0028] The use of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention in selective covalent modification, covalent labeling, or covalent binding of lysine residues in proteins.

[0029] In some embodiments, the protein is a pure protein, a protein in a cell lysate, or a protein in a living cell.

[0030] The use of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention in the identification and characterization of drug molecular targets.

[0031] The use of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention, in the discovery and analysis of drug targets.

[0032] The use of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention in the identification of protein types in tumor cells.

[0033] The use of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention in the identification of kinase types in tumor cells.

[0034] The use of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, described in this invention in the discovery and identification of ligandable lysine residues in tumor cells.

[0035] The application of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention in the development of covalent inhibitors targeting lysine.

[0036] The application of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention, in the preparation of kinase inhibitors.

[0037] In some embodiments, the kinase is EGFR kinase, CDK family kinase, AURKA kinase, or PAK1 kinase.

[0038] The application of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer, as described in this invention, in the preparation of antitumor drugs.

[0039] In some embodiments, the tumor is breast cancer, lung cancer, leukemia, or lymphoma.

[0040] This invention provides an antitumor drug prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes the hydroxamic acid compound described in this invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0041] The present invention also provides a method for covalently labeling lysine residues in proteins or polypeptides, comprising the following steps:

[0042] (1) The hydroxamic acid compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer is incubated with pure protein or cell lysate or live cells.

[0043] (2) Add oxidant and incubate.

[0044] This invention also provides a method for identifying and characterizing drug molecular targets, comprising the following steps:

[0045] (1) Introduce a hydroxamic acid group into a drug molecule and then incubate it with pure protein, cell lysate or live cells; or incubate the hydroxamic acid compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer with pure protein, cell lysate or live cells.

[0046] (2) Add oxidant and incubate;

[0047] (3) Post-processing and analysis of the incubated reaction solution.

[0048] In some embodiments, the drug molecule is a drug molecule having kinase inhibitory activity and / or tumor cell proliferation inhibitory activity.

[0049] This invention also provides a method for discovering and analyzing drug targets, comprising the following steps:

[0050] (1) The hydroxamic acid compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer is incubated with pure protein or cell lysate or live cells.

[0051] (2) Add oxidant and incubate;

[0052] (3) Post-processing and analysis of the incubated reaction solution.

[0053] The present invention also provides a method for identifying protein types, kinase types, and / or ligandable lysine residues in tumor cells, comprising the following steps:

[0054] (1) The hydroxamic acid compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer is incubated together with tumor cell lysate or live tumor cells.

[0055] (2) Add oxidant and incubate;

[0056] (3) Post-processing and analysis of the incubated reaction solution.

[0057] In some embodiments, the living cells are tumor cells, and the cell lysis buffer is a lysis buffer of tumor cells.

[0058] In some embodiments, the oxidant is chloramine-T and / or NaIO4.

[0059] In some embodiments, the incubation in step (1) includes the following reaction conditions: the concentration of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer is 20 μM-500 μM, preferably 50 μM-200 μM.

[0060] In some embodiments, the incubation temperature in step (1) is 30°C-40°C, and the incubation time is 30 min-5 h, preferably 1 h-2 h.

[0061] In some embodiments, the incubation in step (2) includes the following reaction conditions: the concentration of the oxidant is 0.5-10 times, preferably 2-5 times, that of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer.

[0062] In some embodiments, the incubation temperature in step (2) is 30°C-40°C, and the incubation time is 30 min-6 h, preferably 2 h-4 h.

[0063] The present invention has the following beneficial effects:

[0064] This invention provides a class of hydroxamic acid compounds and discovers that by using a novel and efficient oxidation-inducing strategy, the oxidized hydroxamic acid group can covalently bind to the lysine residues of the target protein, enabling selective covalent modification of lysine residues in proteins or peptides. This modification of lysine residues exhibits high selectivity and efficiency, and can be used for the study of protein activity, which is of great significance for the application of chemical proteomics.

[0065] The hydroxamic acid compounds of the present invention can be used to discover and identify drug targets. Introducing the hydroxamic acid group into an active drug molecule can be used to identify and characterize the target of action of the active drug molecule and its lysine site.

[0066] The oxidant-induced strategy of this invention can serve as a method for ligands of hydroxamic acid bioactive molecules to bind to target proteins. Furthermore, since the generated nitrosocarbonyl intermediate can react with protein amino acid residues, this method can be applied to the analysis of ligandable amino acid residues in the human kinase proteome, developing potential covalently modifiable amino acid sites and providing a foundation for the development of novel lysine-targeting covalent inhibitors. Covalent inhibitors designed and synthesized using the nitrosocarbonyl group (derived from the oxidation of the hydroxamic acid group) as a covalent warhead exhibit good kinase inhibitory activity and excellent anti-tumor cell proliferation activity, and can be applied to the development of covalent inhibitors for tumor treatment.

[0067] The hydroxamic acid-like compounds provided by this invention exhibit high selectivity and specificity for covalent binding to lysine residues after oxidative induction. Furthermore, the hydroxamic acid compounds themselves have low reactivity and do not directly covalently bind to non-target protein lysine residues after entering the biological environment. When they are incubated with biological materials containing target proteins for a certain period of time to enter the binding pocket of the target proteins, they are then oxidatively induced, thereby achieving highly selective covalent binding to lysine residues of the target proteins. Attached Figure Description

[0068] Figure 1 The protein spectrum of compound M1 after oxidation induction and labeling with BSA is shown. FL: fluorescence, SDS-PAGE gel fluorescence imaging; CBB: Coomassie brilliant blue, Coomassie brilliant blue stained gel, indicating the consistency of protein loading.

[0069] Figure 2 The labeling patterns of purified BSA protein after oxidation of compound M1 by different oxidants.

[0070] Figure 3 A comparative spectrum showing the labeling effects of compounds M1 and M2 on purified BSA protein.

[0071] Figure 4 This is a diagram showing the protein labeling of compound M1 on the lysates of MDA-MB-231, A549, AGS, Bxpc-3, and Daudi cancer cells.

[0072] Figure 5 Volcano diagram of target proteins labeled with compound M1 in MDA-MB-231 cell lysate.

[0073] Figure 6 To verify the effects of compound M1 on the VDAC1, BIP, and PLK1 proteins in MDA-MB-231 cell lysates using pull-down / Western blotting.

[0074] Figure 7 Selectivity analysis of the modification sites of compound M1 on HEK293T cell lysates.

[0075] Figure 8 Analysis of the lysine binding sites of compound M1 in HEK293T cell lysates.

[0076] Figure 9 Site mutations were validated using pull-down / Western blot methods.

[0077] Figure 10 The protein labeling maps of compounds EH1 and EH2 in H3255 cell lysates.

[0078] Figure 11 The results of the pull-down / WB experiment of compound EH1 in H3255 cell lysate are shown.

[0079] Figure 12 The inhibitory activities of compounds EH1 and EH2 on EGFR kinase are shown.

[0080] Figure 13 This is a labeling map of compound EH1 on purified AURKA protein.

[0081] Figure 14 The results show the site identification of compound EH1 and purified AURKA protein.

[0082] Figure 15 The images show the protein labeling and identification of XH1 in cell lysates. In the images, A is the labeling spectrum of XH1 in K562 lysates, and B is the Venn diagram of kinase analysis of XH1 identified in the lysates.

[0083] Figure 16 To verify the binding of XH1 to the protein, A is the protein labeling spectrum of XH1 with AURKA / SRC, and B is the pull-down / WB spectrum of XH1 with CDK1 in K562 cell lysate.

[0084] Figure 17 The value represents the inhibition rate of proliferation of hydroxamic acid compounds in three types of cancer cells. Detailed Implementation

[0085] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the present invention and should not be construed as limiting the invention.

[0086] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0087] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0088] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0089] In the compounds of this invention, when any variable (e.g., R1, etc.) appears more than once in any component, the definition of each occurrence is independent of the definitions of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. A line drawn from a substituent into the ring system indicates that the bond referred to can be attached to any substituted ring atom. If the ring system is polycyclic, it means that such a bond is attached only to any suitable carbon atom of a neighboring ring. It will be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is itself substituted by more than one group, it should be understood that these groups can be on the same carbon atom or different carbon atoms, as long as structural stability is achieved.

[0090] As used in this invention, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, including branched and straight-chain groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl. The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. The term "alkoxy" refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc. The term "heterocyclic alkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent in which one or more ring atoms are selected from heteroatoms of N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. Examples include: morpholinyl, piperidinyl, piperazine, pyrrolyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazine, dihydropyridinyl, dihydropyrimidinyl, dihydropyrroleyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophene, dihydrotriazolyl, dihydroazacyclobutane, tetrahydrofuranyl, tetrahydrothiophene, etc., and their N-oxides. The connection of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms. The term "heteroaryl" refers to an aromatic ring containing one or more heteroatoms selected from O, N, or S. Heteroaryl groups within the scope of this invention include, but are not limited to: quinolinyl, pyrazolyl, pyrroleyl, thiophenyl, furanyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, and pyridazinyl. "Heteroaryl" is also understood to include any N-oxide derivative of a nitrogen-containing heteroaryl group. The linkage of heterocyclic substituents can be achieved via carbon atoms or via heteroatoms.

[0091] The term "substituted" refers to the replacement of a hydrogen group in a specific structure with a specified substituent.

[0092] As will be understood by those skilled in the art, the term “halo” or “halogen” as used herein refers to chlorine, fluorine, bromine, and iodine.

[0093] This invention includes the free form of compounds of Formula I, as well as their pharmaceutically acceptable salts and stereoisomers. Certain exemplary compounds in this invention are amine compounds in non-salt form, i.e., "free form". Pharmaceutically acceptable salts of this invention can be synthesized from compounds of this invention containing a basic or acidic moiety using conventional chemical methods. Typically, salts of basic compounds are prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric or excess amount of the desired salt form of an inorganic or organic acid in a suitable solvent or a combination of solvents. Similarly, salts of acidic compounds are formed by reacting with a suitable inorganic or organic base.

[0094] Therefore, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting an alkaline compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc., and also include salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pyric acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethylsulfonic acid, trifluoroacetic acid, etc.

[0095] If the compounds of this invention are acidic, then a suitable "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali, including inorganic and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary, and tertiary amines, wherein substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxycobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, aminobutanetriol, etc.

[0096] Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci. '1977: 66: 1–19, describe in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.

[0097] The stereoisomers described in this invention are (depending on their structure) enantiomers, diastereomers, syn- / anti-isomers, cis- / trans-isomers, epimers, and (E)- / (Z)-isomers. Compounds of Formula I can be used in the context of this invention as pure stereoisomers or as any mixture of stereoisomers, with racemic mixtures preferred in the latter case.

[0098] Metabolites of the compounds and pharmaceutically acceptable salts involved in this invention, as well as prodrugs that can be converted in vivo into structures of the compounds and pharmaceutically acceptable salts involved in this invention, are also included in the claims of this invention.

[0099] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of tumors, comprising an active ingredient within a safe and effective range, and pharmaceutically acceptable excipients.

[0100] The "active ingredient" as described in this invention refers to the compound of formula I, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its prodrug molecule, or its deuteride, or its tritide.

[0101] "Safe and effective dose" means that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.

[0102] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) in need of treatment. The dosage at the time of administration is the pharmaceutically considered effective dosage, and the specific dosage should also take into account factors such as the route of administration and the patient's health condition, all of which are within the scope of the skills of a skilled physician.

[0103] "Pharmaceutical acceptable excipients" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0104] "Compatibility" here refers to the ability of the components in the composition to interact with and blend with the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients.

[0105] Pharmaceutically acceptable examples of excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as...). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0106] The following are specific examples.

[0107] Example 1 Synthesis of 4-ethyl-N-hydroxybenzamide (M1)

[0108]

[0109] Add NH₂-OTHP (117.2 mg, 1.5 mmol, 1.5 equivalents), 4-ethylbenzoic acid (150 mg, 1.0 mmol, 1.0 equivalents), EDCI (230 mg, 1.2 mmol, 1.2 equivalents), HOBt·H₂O (162 mg, 1.2 mmol), and DMF (3 mL) to a 10 mL reaction flask. Stir at room temperature for 2 hours, monitoring the reaction process by thin-layer chromatography. After the reaction is complete, pour the reaction mixture into water. Extract the reaction mixture with AcOEt. Separate the organic layer, wash it with saturated NaHCO₃ and saturated brine, and dry it with Na₂SO₄. Purify by silica gel flash column chromatography (ethyl acetate / petroleum ether = 15 / 1000) to obtain S1, a colorless viscous liquid. S1 does not require purification and can be used directly in the next step.

[0110] TsOH·H₂O (16.9 mg, 0.0891 mmol) was added to a 10 mL solution of MeOH (0.1 equivalent) of S1. The reaction mixture was stirred at room temperature for 7 hours. The solvent was removed, and the residue was purified by silica gel flash column chromatography (ethyl acetate / petroleum ether = 1 / 2) to give M1 as a grayish-white powder (93 mg, yield 62%).

[0111] 1 H NMR (400MHz, DMSO-d6) δ11.31(s,1H),9.12(s,1H),7.75(d,J=7.8Hz,2H),7.55(d,J=7.8Hz,2H),4.35(s,1H). 13 C NMR(151MHz,DMSO-d6)δ163.86,133.33,132.20,127.63,124.85,83.30,83.23.ESI-MS calcd for C9H7NO2[M+H] + m / z=162.0550; Found162.0551.

[0112] Example 2 Synthesis of N-hydroxy-3-((4-(prop-2-yn-1-yloxy)phenyl)amino)propionamide (M2)

[0113]

[0114] (4-Hydroxyphenyl)carbamate tert-butyl ester (S2): 4-aminophenol (4 g, 36.6 mmol, 1.0 equivalent) and (Boc)₂O (8 g, 36.6 mmol, 1.0 equivalent) were dissolved in THF and stirred for 2 h. The reaction mixture was filtered, and the filtrate was concentrated to obtain S2. 1 H NMR (400MHz, DMSO-d6) δ9.06 (s, 1H), 8.96 (s, 1H), 7.20 (d, J = 8.3Hz, 2H), 6.64 (d, J = 8.2Hz, 2H), 1.44 (s, 9H).

[0115] (4-(prop-2-yn-1-yloxy)phenyl)tert-butyl carbamate (S3): S2 (7.3 g, 34.9 mmol, 1.0 equivalent), K2CO3 (10.0 g, 72.3 mmol, 2.0 equivalent), and propargyl bromide (4.29 g, 36.14 mmol, 1.0 equivalent) were dissolved in DMF (2 mL), and the reaction mixture was stirred overnight at 50 °C. After the reaction was complete as monitored by TLC, the mixture was added to water and extracted with ethyl acetate. All organic layers were combined, washed with water and saturated brine, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by flash chromatography (PE:EA = 20:1) to give S3. 1 H NMR (400MHz, DMSO-d6) δ9.15(s,1H),7.35(d,J=8.5Hz,2H),6.88(dd,J=8.9,1.9Hz,2H),4.72(q,J=1.8Hz,2H),3.51(t,J=1.8Hz,1H),1.46(s,9H).

[0116] 4-(Prop-2-yn-1-yloxy)aniline (S4): Add S3 to CH2Cl2 and stir until homogeneous. Slowly add trifluoroacetic acid dropwise to the CH2Cl2 solution of S3. Stir the mixture at room temperature for 2 hours. Monitor the reaction progress by TLC. After the reaction is complete, evaporate the solvent under reduced pressure to obtain crude S4, a brown solid, which can be used directly in the next step without purification.

[0117] Preparation of methyl 3-((4-(prop-2-yn-1-yloxy)phenyl)amino)propionate (S5): Tetrahydrofuran was added to a round-bottom flask containing S4 and a stirrer. Triethylamine was added dropwise while stirring until no white fumes formed in the reaction mixture. Methyl 3-bromopropionate (384 mg, 2.3 mmol, 1.0 equivalent) was added to the reaction mixture, and the mixture was heated to reflux. After the reaction was completed as monitored by TLC, the reaction mixture was filtered. The filtrate was evaporated under reduced pressure and purified by flash chromatography to give S5 as a brown solid. 1 H NMR (400MHz, DMSO-d6) δ6.88(d,J=8.6Hz,2H), 6.79(d,J=8.4Hz,2H), 4.69(d,J=2 .4Hz,2H), 3.61(s,3H), 3.51(d,J=2.6Hz,2H), 3.30(s,1H), 2.60(t,J=6.8Hz,2H).

[0118] N-Hydroxy-3-((4-(prop-2-yn-1-yloxy)phenyl)amino)propionamide (M2): A solution of hydroxylamine hydrochloride (120 mg, 0.86 mmol, 4.0 equivalents) in anhydrous methanol (2 mL) was added at 0 °C to a solution of NaOH (68.8 mg, 0.86 mmol, 4.0 equivalents) in anhydrous methanol (2 mL). The mixture was stirred for 10 minutes, and the precipitate was filtered off. The filtrate was added to a solution of S5 (100 mg, 0.43 mmol, 1.0 equivalents) in anhydrous methanol (2 mL). The mixture was stirred at room temperature for 2 hours, and the reaction was monitored by thin-layer chromatography. After the reaction was complete, the reaction mixture was concentrated under vacuum to remove the solvent. M2 was purified by flash column chromatography as a brown viscous liquid (86.9 mg, yield 86%). 1 HNMR (400MHz, DMSO-d6) δ10.46(s,1H), 8.75(s,1H), 6.79(d,J=8.6Hz,2H), 6.57(d,J=8.4H z, 2H), 4.87 (s, 1H), 4.63 (s, 2H), 3.48 (s, 1H), 3.20 (t, J = 7.2Hz, 2H), 2.22 (t, J = 7.2Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ173.77,149.11,143.95,116.61,113.44,80.43,78.14,56.43,34.30,32.81.ESI-MS calcd for C 12 H14N2O3[M+H] + m / z=235.1082; Found 235.1083.

[0119] Example 3 Synthesis of N-hydroxy-4-vinylbenzamide (M3)

[0120]

[0121] The synthesis method is the same as the last step in the synthesis of M2. M3 is a light orange-red solid (yield 87%).

[0122] 1 H NMR (400MHz, DMSO-d6) δ11.20(s,1H),9.02(s,1H),7.73(d,J=7.7Hz,2H),7.55(d,J=8. 3Hz, 2H), 6.78 (dd, J = 17.9, 10.8Hz, 1H), 5.94 (d, J = 16.9Hz, 1H), 5.36 (d, J = 11.9Hz, 1H). 13 C NMR(151MHz,DMSO-d6)δ164.25,140.19,136.37,132.41,127.67,126.53,116.63.ESI-MS calcd for C9H9NO2[M+H] + m / z=164.0711; Found 164.0716.

[0123] Example 4 Synthesis of N1-hydroxy-N3-(4-methylphenylethyl)isophthalamide (M4)

[0124]

[0125] Methyl 3-((4-methylphenyl)carbamoyl)benzoate (S6): HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylhexafluoroammonium bromide) (855.5 mg, 2.25 mmol, 1.5 equivalents), DIPEA (581.58 mg, 4.5 mmol, 3.0 equivalents), 3-(methoxycarbonyl)benzoic acid (300 mg, 1.67 mmol, 1.1 equivalents), and 20 mL LDM were added to a flask. After stirring the reaction mixture for 10 minutes, 2-(p-tolyl)ethyl-1-amine (204.8 mg, 1.5 mmol, 1.0 equivalents) was added, and the mixture was stirred overnight. The reaction was monitored by thin-layer chromatography, and the mixture was concentrated under reduced pressure after the reaction was complete. S6 was purified by flash column chromatography (EA / PE = 20 / 100) and was obtained as a pale yellow viscous liquid (yield 48%). 1HNMR(400MHz,DMSO-d6)δ8.78(d,J=5.9Hz,1H),8.41(s,1H),8.09(d,J=8.1Hz,2H),7.63(t,J=7.4Hz ,1H),7.11(t,J=6.4Hz,4H),3.89(s,3H),3.47(q,J=6.4Hz,2H),2.81(t,J=7.6Hz,2H),2.26(s,3H).

[0126] N1-Hydroxy-N3-(4-Methylphenylethyl)isophthalamide (M4): The synthesis method is the same as the last step of the M2 synthesis. Product M4 is a pale yellow solid (yield 32.7%).

[0127] 1 H NMR (400MHz, DMSO-d6) δ11.29(s,1H),9.13(s,1H),8.67(s,1H),8.23(s,1H),7.91(dd,J=33.0 ,7.9Hz,2H),7.55(t,J=7.8Hz,1H),7.13(q,J=8.2Hz,4H),2.82(t,J=7.6Hz,3H),2.27(s,3H). 13 C NMR(151MHz,DMSO-d6)δ166.07,164.21,136.83,135.48,135.30,133.45,13 0.10,129.67,129.40,129.00,128.90,126.47,41.52,35.11,21.11.ESI-MS calcd for C 17 H 18 N₂O₃[M+H] + m / z=299.1317; Found 299.1390.

[0128] Example 5 Synthesis of N-hydroxy-2-naphthamide (M6)

[0129]

[0130] 2-Naphthoic acid (500 mg, 2.9 mmol, 1.0 equivalent) and CDI (705.5 mg, 4.35 mmol, 1.5 equivalent) were added to THF (10 mL) and stirred at room temperature for 1 h. NH₂OH·HCl (403 mg, 5.8 mmol, 2.0 equivalent) was then added and stirred for 4 h. The reaction was monitored by thin-layer chromatography, and the mixture was concentrated under reduced pressure after the reaction was complete. M6 was purified by flash column chromatography (EA / PE = 20 / 100) as a white powder (447 mg, yield 82%).

[0131] 1 H NMR (400MHz, DMSO-d6) δ11.21(s,1H),9.11(s,1H),7.76(s,1H),7.62(s,1H),7.13(q,J=3.9Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ164.73,134.54,132.62,130.59,129.25,128.46,128.09,128.03,127.62,127.22,124.21.ESI-MS calcd for C 11 H9NO2[M+H] + m / z=188.0711; Found188.0716.

[0132] Example 6 Synthesis of N-hydroxythiophene-2-carboxamide (M7)

[0133]

[0134] The synthesis method of M7 is similar to that of M1. M7 is a pinkish-orange solid with a yield of 77%.

[0135] 1 H NMR (400MHz, DMSO-d6) δ11.21(s,1H),9.11(s,1H),7.76(s,1H),7.62(s,1H),7.13(q,J=3.9Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ160.01,137.80,130.79,128.35,127.89.ESI-MS calcd for C5H5N2O2S[M+H] + m / z=144.0114; Found 144.0114.

[0136] Example 7 Synthesis of N-hydroxy-5-(trifluoromethyl)pyridine amide (M8)

[0137]

[0138] The synthesis method of M8 is similar to that of M1. M8 is a light orange solid with a yield of 77%.

[0139] 1H NMR (400MHz, DMSO-d6) δ11.69(s,1H),9.28(s,1H),8.99(s,1H),8.41(d,J=8.4Hz,1H),8.17(d,J=8.3Hz,1H).ESI-MS calcd for C7H5F3N2O2[M+H] + m / z=207.0377; Found207.0376.

[0140] Example 8 Synthesis of N-hydroxy-3-methyl-4-oxo-2-phenyl-4H-benzopyran-8-carboxamide (M9)

[0141]

[0142] The synthesis method of M9 is similar to that of M1. M9 is a white solid with a yield of 47%.

[0143] 1 H NMR (400MHz, DMSO-d6) δ11.09(s,1H),9.33(s,1H),8.19(dd,J=7.9,1.8Hz,1H),7.87(dd,J=7.3 ,1.8Hz,1H),7.80(dd,J=6.7,2.9Hz,2H),7.62-7.58(m,3H),7.54(t,J=7.6Hz,1H),2.09(s,3H). 13 C NMR(151MHz,DMSO-d6)δ177.58,162.05,160.89,152.80,134.00,133.05,131.08,129 .63,129.07,128.95,127.41,125.70,125.28,125.21,122.47,117.16,11.94.ESI-MS calcd for C 17 H 13 NO4[M+H] + m / z=296.0923; Found 296.0917.

[0144] Example 9 Synthesis of 2-benzyl-N-hydroxy-1,3-dioxoisoindoline-5-carboxamide (M10)

[0145]

[0146] The synthesis method of M10 is similar to that of M1. M10 is a white solid with a yield of 45%.

[0147] 1H NMR (400MHz, DMSO-d6) δ11.61(s,1H),9.33(s,1H),8.24-8.17(m,2H),7.99(d,J=7.7Hz,1H),7.37-7.26(m,5H),4.79(s,2H). 13 C NMR(151MHz,DMSO-d6)δ167.64,167.59,162.67,138.65,136.96,133.78,132.36,129.07 ,128.27,127.93,127.86,127.73,124.95,123.99,121.67,119.58,110.10,41.54.ESI-MS calcd for C 16 H 132 N₂O₄[M+H] + m / z=297.0877; Found297.0868.

[0148] Example 10 Synthesis of N-hydroxy-2'-methoxy-3'-nitro-[1,1'-biphenyl]-3-carboxamide (M11)

[0149]

[0150] The synthesis method of M11 is similar to that of M1. M11 is a light yellow solid with a yield of 51.6%.

[0151] 1 H NMR (400MHz, DMSO-d6) δ11.31(s,1H),9.11(s,1H),7.97-7.90(m,2H),7.83(dt,J=7.8,1.5 Hz,1H),7.78-7.71(m,2H),7.60(t,J=7.7Hz,1H),7.45(dd,J=9.1,6.8Hz,1H),3.45(s,3H). 13 C NMR (151MHz, DMSO-d6) δ164.24,149.94,145.30,136.47,136.44,135.81,133.69,131.91,129.34,127.69,127.19,125.39,124.54,62.40.ESI-MS calcdfor C 16 H 132 N₂O₄[M+H] + m / z=289.0820; Found 289.0819

[0152] Example 11 Synthesis of N-hydroxypyrazine-2-carboxamide (M12)

[0153]

[0154] The synthesis method of M12 is similar to that of M1. M12 is a white solid with a yield of 50.2%.

[0155] 1 H NMR (400MHz, DMSO-d6) δ11.63(s,1H),9.27(d,J=6.5Hz,1H),9.12(d,J=5.3Hz,1H),8.84(dd,J=6.8,2.6Hz,1H),8.69(dd,J=5.4,3.3Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ160.68,147.79,145.53,143.89,143.69.ESI-MS calcd for C5H5N3O2[M+H] + m / z=140.0456; Found 140.0455

[0156] Example 12 Synthesis of 4-(diethylamino)-N-hydroxybenzamide (M13)

[0157]

[0158] The synthesis method of M13 is similar to that of M1. M13 is a yellow solid with a yield of 52%.

[0159] 1 H NMR (400MHz, DMSO-d6) δ10.79(s,1H),8.67(s,1H),7.62-7.54(m,2H),6.69-6.56(m,2H),3.38(s,4H),1.09(t,J=6.9Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ165.38,149.85,131.73,128.93,118.70,110.61,110.57,44.24,44.13,12.83.ESI-MS calcd forC 11 H 16 N₂O₂[M+H] + m / z=209.1290; Found 140.0455

[0160] Example 13 Synthesis of N-hydroxy-4-(pyrrolidone-1-yl)benzamide (M14)

[0161]

[0162] The synthesis method of M14 is similar to that of M1. M14 is a light yellow solid with a yield of 58.3%.

[0163] 1 H NMR (400MHz, DMSO) δ10.81(s,1H),8.68(s,1H),7.79-7.55(m,2H),6.62-6.41(m,2H),3.30-3.25(m,4H),1.96(h,J=3.6Hz,4H).13C NMR(151MHz,DMSO-d6)δ165.43,157.56,150.90,131.50,118.94,111.15,47.68,47.65,25.43.ESI-MS calcd forC 11 H 14 N₂O₂[M+H] + m / z=207.1133; Found 207.1128.

[0164] Example 14 Synthesis of N-hydroxy-4-((quinoline-8-ylamino)methyl)benzamide (M15)

[0165]

[0166] The synthesis method of M15 is the same as that of M2. M15 is a brownish-red solid with a yield of 52%.

[0167] 1 H NMR (400MHz, DMSO-d6) δ11.12(s,1H),8.98(s,1H),8.77(dd,J=1.8,4.2Hz,1H),8.20(dd,J=7.7,5.1,2.5Hz,1H),7.68(dd,J=8.5,2.4Hz,2H), 7.50(dd,J=4.2,8.4Hz,1H),7.45-7.42(d,J=8.1Hz,2H),7.25(q,J=6.6,5.3Hz,2H),7.05(d,J=8.0Hz,1H),6.52(d,J=7.6Hz,1H),4.58(s,2H). 13C NMR(151MHz,DMSO-d6)δ164.68,147.48,144.59,143.85,138.03,136.45,131.79,129 .93,128.78,128.10,127.47,127.36,126.75,122.23,113.94,105.32,46.30.ESI-MS calcd for C 17 H 18 N3O2[M+H] + m / z = 294.1243, Found 294.1217.

[0168] Example 15 Synthesis of N-hydroxy-4-((imidazo[1,2-a]pyridin-8-ylamino)methyl)benzamide (M16)

[0169]

[0170] The synthesis method of M16 is the same as that of M2. M16 is a yellow solid with a yield of 63%.

[0171] 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),8.97(s,1H),7.89(d,J=8.2Hz,1H),7.82(d,J=1.2Hz,1H),7.76(dd,J=5.3,1.4Hz,1H),7.68(d,J=8.3Hz,1H ),7.48(d,J=8.1Hz,1H),7.45-7.42(m,1H),6.80(dt,J=13.2,6.5Hz,1H) ,6.57(t,J=7.0Hz,1H),5.93-5.85(m,1H),4.52(dd,J=13.9,6.4Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ167.71,145.62,143.64,139.04,137.05,131.29,129.89,127.41,115.07,114.38,113.64,97.10,46.08.ESI-MScalcd for C 15 H 14 N4O2[M+H] + =283.1190,Found 283.1194.

[0172] Example 16 Synthesis of 4-(((4-(dibenzo[b,d]furan-3-yl)phenyl)amino)methyl)-N-hydroxybenzamide (M17)

[0173]

[0174] The synthesis method of M17 is similar to that of M2. M17 is a yellow solid with a yield of 41%.

[0175] 1 H NMR (400MHz, DMSO-d6) δ8.16(d,J=7.6Hz,1H),8.01(dd,J=7.6,1.2Hz,1H),7.96(dd,J=8.3,6.4Hz,2H),7.72(dd,J=8.5,4.5Hz,1H),7.67(d,J=8.7Hz ,1H),7.58(dd,J=7.7,1.3Hz,1H),7.55(d,J=8.4Hz,2H),7.41(td,J=7.5,3 .7Hz,2H),6.78-6.72(m,2H),4.46(d,J=6.2Hz,2H),3.85(d,J=2.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ167.69,145.59,143.59,138.91,136.97,131.78,131.18,131.11,129.90,129.74,12 9.72,127.43,127.29,127.26,115.12,115.08,114.42,114.35,113.75,113.68,97.33,97.31,46.09.ESI-MS calcd for C 26 H 20 N₂O₃[M+H] + m / z = 409.1552, Found 409.1557.

[0176] Example 17 Synthesis of N-hydroxy-4-(((4-morpholinylphenyl)amino)methyl)benzamide (M18)

[0177] The synthesis method of M18 is similar to that of M2. M18 is a yellow solid with a yield of 45%.

[0178] 1H NMR (400MHz, DMSO-d6) δ11.13(s,1H),10.32(s,2H),8.97(s,1H),8.65(d,J=1.7Hz,3H),7.68(d,J=8.2Hz,2H),7. 40(d,J=8.0Hz,2H),6.71(d,J=6.8Hz,2H),6.54-6.42(m,2H),4.25(d,J=5.8Hz,2H),3.68(dd,J=5.9,3.6Hz,4H). 13 C NMR(151MHz,DMSO-d6)δ166.61,144.70,143.01,142.91,131.54,129.84,128.58,127.7 5,127.57,127.45,127.31,127.21,126.75,118.00,113.61,66.76,50.92,47.21.ESI-MS calcd forC 18 H 21 N3O3[M+H] + m / z = 329.1661, Found 329.1656.

[0179] Example 18 Synthesis of 4-((((1H-benzimidazol-2-yl)methyl)amino)methyl)-N-hydroxybenzamide (M19)

[0180]

[0181] The synthesis method of M19 is similar to that of M2. M19 is a yellow solid with a yield of 41%. 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),10.04(s,1H),9.03-8.98(m,2H),7.76-7.71(m,2H),7. 60-7.52(m,4H),7.20(dq,J=7.1,4.0Hz,1H),3.83(d,J=4.5Hz,1H),3.68(s,2H),3.17(s,2H). 13 C NMR(151MHz,DMSO-d6)δ164.51,152.30,141.98,132.08,129.77,129.29,129.12,127.37,123.00,57.44,50.98.ESI-MS calcd for C 16 H 16 N4O2[M+H] + m / z=297.1351, Found 297.1346.

[0182] Example 19 Synthesis of 5-((4-((3-ethynylphenyl)amino)-7-methoxyquinazoline-6-yl)oxy)-N-hydroxypentanamide (EH1)

[0183]

[0184] The synthesis method of EH1 is similar to that of M2. EH1 is a white solid with a yield of 56%.

[0185] 1 H NMR (400MHz, DMSO-d6) δ10.42(s,1H),9.65(s,1H),8.72(s,1H),8.53(s,1H),7.99(s,1H),7.91(d,J=2.4Hz,1H),7.89(s,1H),7.42(t, J=8.0Hz,1H),7.23(d,J=10.5Hz,2H),4.21(s,1H),4.17(t,J=6.3Hz,2H),3.95(s,3H),2.07(t,J=7.2Hz,2H),1.83(m,2H),1.73(m,2H). 13 C NMR(151MHz,DMSO-d6)δ174.84,169.41,156.90,155.21,152.53,148.93,139.96,129.36,127.22,125 .71,123.53,122.20,109.18,106.60,103.47,83.93,81.12,69.05,56.43,32.35,28.50,22.38.ESI-MS calcd for C 22 H 22 N4O4[M+H] + m / z=407.1719,Found 407.1714.

[0186] Example 20 Synthesis of 2-((4-((3-ethynylphenyl)amino)-7-methoxyquinazoline-6-yl)oxy)-N-hydroxyacetamide (EH2)

[0187]

[0188] The synthesis method of EH1 is similar to that of M2. It is a white solid with a yield of 59%.

[0189] 1H NMR (400MHz, DMSO) δ11.39(s,1H),11.08(s,1H),8.86(s,1H),8.43(s,1H),7.90(d,J=1.9Hz,1H),7.80( d,J=8.3Hz,1H),7.50(t,J=7.9Hz,1H),7.42(d,1H),7.36(s,1H),4.77(s,2H),4.28(s,1H),4.03(s,3H).

[0190] Example 21 Preparation of 6-((5-cyclopropyl-1H-pyrazol-3-yl)amino)-2-(4-(4-(2-(hydroxyamino)-2-oxoethoxy)benzyl)piperazin-1-yl)-N-(prop-2-yn-1-yl)pyrimidine-4-carboxamide (XH1)

[0191]

[0192] The synthesis method of XH1 is similar to that of M2. It is a yellow solid with a yield of 31%.

[0193] 1 H NMR (400MHz, DMSO-d6) δ11.04(s,1H),10.91(s,1H),9.02(t,J=6.2Hz,2H ),7.56-7.52(m,2H),7.10-7.01(m,3H),6.99(s,1H),6.11(s,2H),4.73(s ,2H),4.51(s,2H),4.27(s,3H),4.02(dd,J=6.1,2.6Hz,3H),3.11(t,J=2. 5Hz,1H),3.02(s,4H),1.95-1.85(m,1H),0.91(m,2H),0.71-0.66(m,2H). 13 C NMR (151MHz, DMSO) δ170.46,164.55,164.41,163.88,160.78,159.10,159.06,135.48,133.35,128.91,115.56,11 5.13,93.31,81.67,73.15,66.19,64.93,58.70,50.51,45.92,41.03,40.51,28.62,8.96,8.37,7.41.ESI-MScalcd for C 27 H 31 N9O4[M+H] + m / z=546.2577, Found 546.2572.

[0194] Example 22: Labeled pure protein after oxidation induction with probe compound M1

[0195] Probe M1 was prepared into a 1 mM stock solution using DMSO, and NaIO4 was prepared into a 2 mM oxidant stock solution using PBS. Bovine serum albumin (BSA) was purified into a 0.5 mg / mL solution using PBS.

[0196] Oxidation scheme A (premixed): Mix the M1 probe stock solution with the oxidant stock solution and react for 2 hours. Then, add 2 μL of LDMSO or 2 μL of the reaction solution to the BSA pure protein solution and incubate at 37°C for 2 hours in a constant temperature mixer.

[0197] Oxidation scheme B (incubation before oxidation): Add 1 μL LDMSO or 1 μL LM1 probe stock solution to the pure protein solution and incubate for 2 hours. Then add 1 μL NaIO4 stock solution and incubate at 37°C for 2 hours in a constant temperature mixer.

[0198] After incubation, click chemistry reagents (0.5 μL each of 10 mM TBTA, 100 mM TCEP, 100 mM CuSO4, and 10 mM TAMRA-N3 solution) were added to the sample solution. The mixture was then incubated in a rotary mixer at room temperature for 2 h. After adding 6 μL of 5×SDS loading buffer, 15 μL of the sample solution was taken and separated by SDS-PAGE polyacrylamide gel electrophoresis. Electrophoresis was performed at 90 V for 30 min, then adjusted to 110 V for 60 min. Finally, images were obtained using a biopolymer interaction imaging system.

[0199] The results are as follows Figure 1 As shown: When probe M1 was premixed with an oxidant and then added to oxidation scheme A of purified BSA protein, probe M1 did not produce a clear labeled band. Only when probe M1 was first incubated with purified BSA protein and then added to oxidation scheme B (induced by NaIO4) did probe M1 show a clear labeling effect on purified BSA protein. This indicates that hydroxamic acid molecules can only react with protein amino acid residues after oxidation induction, and that the covalent linkage with amino acid residues can only be achieved after the hydroxamic acid molecules are incubated with the protein for a period of time and come into proximity to the amino acid residues, followed by induction with the oxidant. It is speculated that the reason for this phenomenon is that the nitrosocarbonyl intermediate generated after the oxidation of hydroxamic acid has a short lifespan and high reactivity. If there are no nearby amino acid residues for the nitrosocarbonyl intermediate to react with it when it is generated, it will immediately react with surrounding water molecules and become inactive, unable to react with amino acid residues.

[0200] Example 23 Effect of Oxidizing Agent on Marking Effect

[0201] In this embodiment, five oxidants were selected: tert-Butyl Hypochlorite (BuOCl), H2O2, NaIO4, [Bis(trifluoroacetoxy)iodo]benzene (PIFA), and chloramine T. The probe M1 was oxidized and reacted with purified BSA protein according to oxidation scheme B described in Example 22, and the oxidation effect was visualized by fluorescence imaging.

[0202] The results are as follows Figure 2 As shown, under the same concentration (final concentration of oxidant is 200 μM, final concentration of M1 is 100 μM) and oxidation time, NaIO4 and chloramine T have better oxidation effects on probe M1, with NaIO4 showing the best effect and the best labeling effect on pure BSA protein.

[0203] Example 24: Effect of Hydroxyxamic Acid Structure on Labeling Efficacy

[0204] This example compares the differences in labeling effects of M1 and M2 on purified protein after oxidation induction. Probes M1 and M2 at the same concentration (final concentration 100 μM) were added to a BSA purified protein solution and incubated for 2 hours. Then, NaIO4 at twice the probe concentration was added and incubated for another 2 hours. Click reagent was then added, and fluorescence imaging was performed after polyacrylamide gel electrophoresis. The detailed method is the same as in Example 22.

[0205] The results are as follows Figure 3 As shown, without oxidation induction, neither probe M1 nor M2 produced a labeled band. After oxidation induction, only probe M1 produced a strong labeled band, while probe M2 did not produce a significant labeled band. This indicates that the specific structure of the hydroxamic acid molecule has a significant impact on the labeling effect.

[0206] Example 25: Compound M1 was used to label proteins in cell lysates after oxidation induction.

[0207] Cell lysis buffers were prepared for MDA-MB-231, Bxpc-3, Daudi, A549, and AGS cells. Taking MDA-MB-231 cells as an example, the specific experimental steps are as follows:

[0208] Discard the culture medium from the 90% confluence of MDA-MB-231 cells in a 10cm culture dish. Wash the cells with 2mL of PBS, adhering to the dish wall, repeating the washing twice. Then, add 1mL of trypsin to digest the cells until they appear spherical under a microscope and are no longer clumped together. Add 1mL of serum-containing culture medium to quench the digestion reaction. Collect the cell suspension in a 5mL sample tube and centrifuge at 1000rpm for 3 minutes to precipitate the cells. Discard the supernatant, add 1mL of PBS, and gently agitate to resuspend the cells in the PBS. Transfer the cell suspension to a 1.5mL sample tube. Centrifuge again at 1000rpm for 3 minutes to precipitate the cells. Discard the supernatant, add 1mL of PBS, and gently agitate to resuspend the cells in the PBS. Place the sample tube in an ice box and sonicate the cells using a cell sonicator with the following parameters: 3s on, 3s off, 15% power, and 3 minutes sonication time. After sonication, the sample is centrifuged at 4°C and 14,000 rpm for 15 minutes in a high-speed centrifuge. The resulting supernatant is the cell lysis buffer, and the bottom precipitate is discarded.

[0209] Take 200 μL of cell lysate, add 2 μL LDMSO or probe M1 (final concentration 100 μM), and react at 37 °C for 2 h. Then react with NaIO4 (final concentration 500 μM) at 37 °C for 4 h. After ultrafiltration through a spin column, add TAMRA-N3, TBTA, TCEP, and CuSO4 (final concentrations 100 μM, 100 μM, 1 mM, and 1 mM, respectively) and incubate at 37 °C for 2 h in a constant temperature mixer to allow the protein-probe complex to connect to the fluorescent group TAMRA-N3 and complete the click reaction. Add 1 mL of pre-cooled methanol, and incubate the sample at -20 °C for at least 30 min (or overnight) to allow protein precipitation. Then centrifuge the sample at 4 °C and 14000 rpm for 15 min, discard the supernatant, and invert the sample tube to air dry the protein. Then, 40 μL of 1×SDS loading buffer was added, and the sample was sonicated in an ultrasonic cleaner for several minutes to promote protein dissolution, followed by heating at 95°C for 15 min. After centrifugation at 14000 rpm for 30 s, 20 μL of sample solution was added to the loading wells of a gel electrophoresis (SDS-PAGE, 10% separating gel). Electrophoresis was performed at a constant voltage of 90 V for 30 min, then adjusted to a constant voltage of 110 V for 60 min. Finally, the sample was scanned and imaged using a multifunctional biomolecular imaging system.

[0210] The results are as follows Figure 4 As shown, compound M1, after oxidation induction, has a significant labeling effect on cell lysates of MDA-MB-231, Bxpc-3, Daudi, A549, and AGS cells.

[0211] Example 26: Protein identification and analysis of compound M1 in MDA-MB-231 cell lysates

[0212] MDA-MB-231 cells were prepared into cell lysate according to the method in Example 25. The cell lysate was treated with probe M1 (final concentration 100 μM) and reacted at 37°C for 2 h, followed by reaction with NaIO4 (final concentration 500 μM) at 37°C for 4 h. After ultrafiltration through a spin column, the lysate was reacted with Biotin-N3, TBTA, sodium ascorbate, and CuSO4 (final concentrations 100 μM, 100 μM, 1 mM, and 1 mM, respectively) by gentle shaking. Pre-cooled methanol was added, and the mixture was incubated at -20°C for 2 h. Subsequently, the precipitated protein was collected, centrifuged, and dissolved. After incubation with streptavidin beads at room temperature for 2 hours, the protein was washed with 1% SDS and PBS. The beads were suspended in 500 μL of 6M urea PBS, and 25 μL of 200 mM DTT and 25 μL of 25 mM NH4HCO3 were added. The mixture was incubated at 37°C for 30 min. Add 25 μL of 500 mM IAA and incubate in the dark at room temperature for 30 min. Then remove the supernatant and wash the beads with 1 ml of PB. Add 150 μL of 2M urea, 1 mM CaCl2 and 1 μL of trypsin (1 μg / μL) to 50 mM NH4HCO3, incubate overnight at 37°C, desalt on a C18 column, and analyze by LC-MS / MS.

[0213] Jie Ruru Figure 5 As shown, probe M1 bound to a total of 43 highly reliable target proteins. Among these 43 highly reliable proteins, three proteins belonging to the eukaryotic mitochondrial porin family were successfully targeted: voltage-dependent anion-selective channel protein 1 (VDAC1), voltage-dependent anion-selective channel protein 1 (VDAC2), and voltage-dependent anion-selective channel protein 1 (VDAC3). VDACs play a central role in cell lifespan and apoptosis. VDACs are expressed at higher levels in cancer cells than in normal cells, thus making them potential targets for cancer chemotherapy.

[0214] Example 27 Target Validation by Drop-down / Western Blotting

[0215] To confirm the target protein bound by probe M1, VDAC1, BIP (Endoplasmic reticulum chaperone BIP), and PLK1 (Serine / threonine-protein kinase PLK1) were selected for pull-down / WB experiments in this embodiment.

[0216] MDA-MB-231 cells were prepared into cell lysates according to the method in Example 25. The cell lysates were treated with probe M1 (final concentration 100 μM) and reacted at 37°C for 2 h, followed by reaction with NaIO4 (final concentration 500 μM) at 37°C for 4 h. After ultrafiltration through a spin column, a freshly premixed click chemistry mixture was added (100 μM biotin-N3 from 10 mM DMSO stock solution, 100 μM TBTA from 10 mM freshly prepared DMSO stock solution, 2 mM sodium ascorbate from 200 mM freshly prepared deionized water stock solution, and 1 mM CuSO4 from 100 mM freshly prepared deionized water stock solution). The reactants were further incubated for 2 h under gentle mixing before adding pre-cooled methanol (-20°C) for precipitation. The precipitated proteins were then collected by centrifugation (14000 rpm × 10 min, 4°C) and dissolved in PBS containing 1% SDS. After incubating with streptavidin beads at room temperature for 4 hours, the beads were washed with PBS (3×1 mL) and PBS (3×1 mL) containing 1% SDS, respectively. 50 μL (2×) SDS loading buffer was added, and the mixture was heated at 95 °C for 30 minutes. The samples were separated by SDS-PAGE (12% gel), and the target protein was verified by standard Western blotting.

[0217] like Figure 6 As shown, probe M1, after being oxidized with NaIO4, successfully pulled down VDAC1, BIP, and PLK1 in the lysate of MDA-MB-231 cells, while probe M1 without NaIO4 oxidation induction did not produce any labeling pull. This indicates that VDAC1, BIP, and PLK1 are indeed the target proteins of probe M1, and also demonstrates that M1 can bind to target proteins through an oxidation-induction strategy, and that this strategy can successfully label proteins at the lysate level.

[0218] Example 28: Analysis of modification sites of compound M1 on HEK293T cell lysates

[0219] (1) Preparation of cell lysis buffer: Discard the culture medium of HEK293T cells grown to 90% in a 10cm culture dish. Add 2mL of PBS to the wall of the culture dish to wash the cells, repeating the washing twice. Centrifuge at 1000rpm for 3min to collect the cells, add 1mL of PBS and gently shake to resuspend the cells in the PBS. Place the sample tube in an ice box and sonicate in a cell sonicator. Set the parameters to 3s on, 3s off, power 15%, and sonication time 3min. After sonication, centrifuge the sample in a high-speed centrifuge at 4℃ and 14000rpm for 15min. The resulting supernatant is the cell lysis buffer.

[0220] (2) Probe and oxidative incubation: The prepared cell lysate was divided into two equal portions of 500 μL each. 5 μL of 10 mM probe M1 was added to each portion, and the portions were incubated at 37 °C for 2 h in a constant temperature homogenizer. 5 μL of 50 mM NaIO4 oxidant solution was added, and the mixture was incubated for 4 h.

[0221] (3) Alkylation: Add 12.5 μL of DTT stock solution and incubate at 75 °C for 30 min. Add 25 μL of IAA stock solution and incubate in the dark for 30 min.

[0222] (4) Protein precipitation and trypsin digestion: Add 540 μL MeOH and 135 μL CH3Cl, mix well and let stand at -20℃ for 30 min. Centrifuge at 1700g for 20 min, discard the supernatant, and place the sample tube open in a 37℃ constant temperature homogenizer for a few minutes to dry the protein. Add 400 μL (NH4)2CO3 solution, place the sample in an ice box, and sonicate for 6-15 s until the protein is fully suspended in the solution. Add 5 μL trypsin and incubate at 37℃ for 12 h in a constant temperature homogenizer. Add 2 μL trypsin and continue incubating at 37℃ for 2 h in a constant temperature homogenizer. Centrifuge at 14000g for 5 min, and dry the supernatant in a high-temperature vacuum desiccator to obtain 200 μL of peptide sample.

[0223] (5) Desalting: The HLB SPE column was activated with 1 mL HPLC-acetonitrile and 2 mL HPLC-H2O. 200 μL of peptide sample was added and desalted with 1 mL HPLC-H2O. Finally, HLB-A reagent was added to elute the peptide. The peptide eluent was dried completely in a vacuum centrifuge at 45 °C.

[0224] (6) Click reaction: Add 20 μL HPLC-H2O and 10 μL HPLC acetonitrile to each peptide sample and vortex to mix. Add 1 μL Light Az-UV-Biotin stock solution, 4 μL sodium ascorbate, 1 μL TBTA and 4 μL CuSO4 to one sample in sequence; add 1 μL Heavy Az-UV-Biotin stock solution, 4 μL sodium ascorbate, 1 μL TBTA and 4 μL CuSO4 to the other sample. Incubate the samples at 37°C for 2 h in the dark, add 120 μL streptavidin binding buffer and mix well, centrifuge at 14000g for 5 min, and combine the supernatants of the two samples into a new sample tube.

[0225] (7) Protein enrichment: 100 μL of streptavidin beads were gently shaken with 1 mL of streptavidin binding buffer and centrifuged at 1,700 g for 3 min. This process was repeated twice to wash the protective solution from the beads. The combined sample and the washed beads were added to 10 mL of streptavidin binding buffer and enriched by rotation at room temperature in the dark for 2 h.

[0226] (8) Washing the beads: Centrifuge at 1700g for 3 min, discard the supernatant, add 10 mL of streptavidin binding buffer, gently invert the sample for 5 min, centrifuge again, and repeat the above steps twice. Add 10 mL of streptavidin washing buffer, gently invert the sample for 5 min, centrifuge again, and repeat the above steps twice. Add 1.2 mL of light release buffer, gently shake to disperse the beads in the solution, and irradiate the sample tube under a 365 nm ultraviolet lamp for 2 h; centrifuge at 2000g for 4 min; concentrate the supernatant to 200 μL using a vacuum centrifuge.

[0227] (9) Desalting and Sample Delivery: The HLB SPE column was activated with 1 mL HPLC-acetonitrile and 2 mL HPLC-H2O. 200 μL of peptide sample was added and desalted with 1 mL HPLC-H2O. Finally, HLB-A reagent was added to elute the peptide. The peptide eluent was dried completely in a vacuum centrifuge at 45 °C. 12 μL of 0.1% formic acid was added to the dried peptide, and the peptide was dissolved by sonication for several minutes. The peptide was centrifuged at 14000g for 15 min, and 10 μL of the supernatant was sent for LC-MS / MS analysis.

[0228] The results are as follows Figure 7 , Figure 8 And as shown in Tables 1-2: Heatmap of the binding sites of probe M1 in HEK293T cell lysates ( Figure 7 As shown in A), probe M1 is modified only for K, with a selectivity ranging from 0.8 to 1, indicating that probe M1 exhibits high selectivity for binding to lysine residues. Venn diagram illustrating the binding site selectivity and analytical effectiveness of probe M1 (…). Figure 7As shown in B), probe M1 exhibits a lysine selectivity and profiling efficiency exceeding 90%, and this high profiling efficiency also indirectly demonstrates the high accuracy of the analytical data.

[0229] Furthermore, the protein and specific binding sites of probe M1 were analyzed using the proteomics analysis software pFind. The results showed that probe M1 hit a total of 7241 lysine residues. Figure 8 Most of these lysines were not annotated, while 662 were annotated as functional lysines. These functional lysines were mostly post-translational modified lysines, and approximately 7% were binding sites. These functional binding sites included ATP-binding sites, GTP-binding sites, and NADP-binding sites. + Binding site, CoA binding site, Mn 2+ The binding sites and 3',5'-cyclic AMP binding sites (Table 1) indicate that M1 can enter the binding pockets of various proteins, extensively entering these pockets and binding to active sites. Compared to the more than 3,000 lysines identified by the STP lysine probe reported by Hacker et al., approximately 6,500 lysine sites identified by M1 were not bound by STP, demonstrating that M1 has a significant complementary role in identifying lysines in the proteome.

[0230] M1 identified 2499 different proteins, of which 101 are listed in the Drugbank. The majority of the proteins identified by M1 are not in the Drugbank, and these proteins exhibit a broader class distribution than the smaller subset of Drugbank proteins containing ligand lysines, which are primarily enzymes. Non-Drugbank proteins containing ligand lysines include transcription factors and scaffold proteins that are considered difficult to target with small molecules.

[0231] Among the annotation sites hit by M1 (Table 2), DDX6_K141, DTYMK_K19, ALDH18A1_K311, HSP17B12_K72, CDK2_K129, and MTHFD1_K56 are the functional sites of the corresponding proteins, which can affect the function of the proteins.

[0232] In summary, the simple structure of M1 allows it to easily enter the binding pockets of various proteins and bind to proteins that are difficult to target, enabling the identification of unstudied lysine residues. This is beneficial for gaining a deeper understanding of conserved lysine residues in proteins and for developing covalent inhibitors that target lysine.

[0233] Table 1. Functional lysine sites of M1 in HEK293T cell lysates (partial list)

[0234]

[0235] Table 2 Lysine sites hit by M1 in HEK293T cell lysates

[0236]

[0237] Example 29: Site Validation via Site Mutation and Drop-down / Western Blotting

[0238] Wild-type / mutant proteins were expressed in HEK293T cells. Cell lysates were prepared according to the method in Example 28. The cell lysates were treated with probe M1 (final concentration 100 μM) and reacted at 37°C for 2 h, followed by reaction with NaIO4 (final concentration 500 μM) at 37°C for 4 h. After ultrafiltration through a spin column, a freshly premixed click chemistry mixture (100 μM biotin-N3, 100 μM TTBTA, 2 mM sodium ascorbate, 1 mM CuSO4) was added. The reaction mixture was further incubated for 2 h under gentle mixing before adding pre-cooled methanol (-20°C) for precipitation. The precipitated protein was then collected by centrifugation (14000 rpm × 10 min, 4°C) and dissolved in PBS containing 1% SDS. After incubation with streptavidin beads at room temperature for 4 hours, the beads were washed with PBS (3 × 1 mL) and PBS (3 × 1 mL) containing 1% SDS, respectively. Add 50 μL (2×) SDS loading buffer and heat at 95 °C for 30 minutes. Separate the sample by SDS-PAGE and verify the target protein by standard Western blotting method.

[0239] The results are as follows Figure 9 As shown, after PGK1_K131, PGK_K323 and CDK5_K33 were mutated to arginine, the labeling of the corresponding proteins by probe M1 was significantly reduced, indicating that the nitrosocarbonyl intermediate generated by probe M1 after oxidation induction can effectively label lysine residues in the entire proteome.

[0240] Example 30 Protein labeling of probes EH1 / EH2 in H3255 cell lysate

[0241] According to the experimental method in Example 25, the H3255 lysis buffer was treated with 100 μM probe EH1 or EH2 for 2 hours, oxidized with 5×NaIO4 for 4 hours, then the oxidant was filtered out and a click reaction was performed, followed by fluorescence scanning imaging.

[0242] The results are as follows Figure 10 As shown: Of the two probes, EH1 has a stronger labeling effect.

[0243] Example 31 Protein identification and pull-down / WB assay of probe EH1 in H3255 cell lysate

[0244] This embodiment identifies the target protein labeled by probe EH1, and the experimental methods are the same as those in Examples 26 and 27.

[0245] The results (Table 3) show that some of the proteins hit by EH1 are kinases, including AURKA (Aurora kinase A, laser kinase), PAK1 (Serine / threonine protein kinase PAK1, serine / threonine protein kinase), CDK1 (Cyclin-dependent kinase 1, cyclin-dependent kinase 1), etc.

[0246] Pull-down / WB experimental results ( Figure 11 The results showed that without the addition of NaIO4 oxidation induction, EH1 had no significant labeling effect on EGFR; however, after NaIO4 oxidation, EH1 successfully labeled and pulled down EGFR. This indicates that after oxidation induction, EH1 can covalently bind to and pull down EGFR.

[0247] Table 3. Protein identification of H3255 cell lysates by compound EH1 (partial)

[0248]

[0249]

[0250] Example 32: Assay of the inhibitory activity of probes EH1 / EH2 against EGFR kinase

[0251] Using Z'-LYTE TM The fluorescence resonance energy transfer (FRET) method was used to test the inhibitory effect of the probe on EGFR kinase, with the parent inhibitor used as a control compound. Z'-LYTE TM Biochemical assays were performed using FRET-based coupled enzymes, leveraging the different sensitivities of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. Peptide substrates were labeled with two fluorophores—one at each end—forming a FRET pair. Compounds were in DMSO from 5.1 x 10⁻⁶. -9 M diluted three times to 1x 10 -4 M. Measurement board on EnVision MultilabelReader (Perkin Elmer). Curve fitting and data presentation were performed using Graph PadPrism version 4.0.

[0252] The results are as follows Figure 12As shown, probe EH1 exhibited a superior inhibitory effect (IC50 ≈ 2.3 nM) against the EGFR (L858R) mutant compared to Erlotinib (IC50 ≈ 3.9 nM); probe EH2 showed a comparable inhibitory effect (IC50 ≈ 3.3 nM) against the EGFR (L858R) mutant to Erlotinib. This indicates that the addition of the hydroxamic acid group to the Erlotinib core preserves the inhibitory activity of the parent compound against EGFR kinase, and because EH1 has a better covalent binding effect to EGFR kinase, it can further enhance the inhibitory effect of EH1 on EGFR and its mutants.

[0253] Example 32: Labeling and Site Identification of Probe EH1 at the Pure Protein Level

[0254] AURKA was identified in H3255 lysis buffer using EH1. This example verifies the binding of EH1 to AURKA. The AURKA protein was labeled with 1 μM and 10 μM EH1 (5×NaIO4) according to the method of Scheme B in Example 22.

[0255] result( Figure 13 The results showed that EH1 could label AURKA protein at concentrations as low as 1 μM, and produced a strong label at 10 μM, confirming the binding of EH1 to AURKA.

[0256] Furthermore, in order to determine the binding site of EH1 to AURKA and to explore whether EH1 can be used to analyze the conserved lysine sites of AURKA, this example used 100 μM EH1 (5×NaIO4) and purified AURKA protein for site identification.

[0257] result( Figure 14 The results show that EH1 binds to K258 of AURKA. In AURKA, D256 is labeled as the proton acceptor active site, and E260 and N261 are labeled as ATP binding sites. The K258 identified by EH1 is located in the active pocket formed by AURKA_D256-N261, indicating that EH1 can covalently bind to lysine residues in the active pocket after oxidation.

[0258] Example 33: Identification and Validation of XH1 Proteins in Cell Lysate

[0259] XH1 is a hydroxamic acid compound obtained by modifying the structure of the broad-spectrum kinase ligand XO44. In this embodiment, the protein identification experiment of XH1 in cell lysate was verified according to the aforementioned method.

[0260] The cells were incubated with probe XH1 (100 μM) in K562, AGS, and MDA-MB-231 cell lysates for 2 hours, oxidized for 4 hours to remove the oxidant NaIO4, and then subjected to a click reaction for 2 hours, followed by fluorescence scanning imaging.

[0261] result( Figure 15 A) shows that XH1 can indeed produce a mark after oxidation.

[0262] Pull-down / LC-MS / MS experiment (probe concentration 100 μM) results ( Figure 15 B) shows that among the identified proteins, probe XH1 identified 75, 31, and 39 different protein kinases in K562, AGS, and MDA-MB-231 cell lysates, respectively, demonstrating the probe's broad binding ability to protein kinases. Furthermore, XH1 identified 73 unique kinases, highlighting the uniqueness of XH1 at the kinase binding level and its potential to expand our coverage of kinases.

[0263] Analysis of the identified proteins revealed the presence of CDK family kinases such as CDK1, CDK2, CDK6, and CDK9, indicating that these probes possess good labeling capabilities for CDK family kinases. CDK1 plays a crucial role in the eukaryotic cell cycle by regulating the centrosome cycle and mitosis initiation; CDK1 can also promote the G2-M transition and regulate G1 progression and G1-S transition by binding to multiple interphase cyclins; abnormal CDKs can lead to cell proliferation, genomic and chromosomal instability, thereby contributing to the development, progression, and invasiveness of human cancers. Therefore, CDKs could serve as a novel and effective target for cancer treatment.

[0264] This embodiment further confirmed the binding of XH1 to the target proteins AURKA and SRC using a pure protein labeling experiment, and further confirmed the successful binding of XH1 to the target protein CDK1 using a pull-down / WB experiment. Figure 16 These experimental results demonstrate that the oxidative induction strategy following the integration of hydroxamic acid groups with the backbone of a broad-spectrum kinase inhibitor allows for the analysis of kinases throughout the entire proteome, thereby providing a global map of ligand-catalyzed lysines within the kinaseome.

[0265] Example 34 Cell proliferation inhibition rate test

[0266] Seed 8000-10000 cells per well in a 96-well plate (8000 cells / well for MDA-MB-231, 10000 cells / well for Daudi and K562 cells) and incubate at 37°C for 24 hours. Add fragment compounds M3-M19 (final concentration 5 μM) to each well, with one replicate for each sample well. The last column is a cell-free control. Incubate for 48 hours. Add 30 μL of CCK8 reagent to each well and incubate for another 4 hours. The absorbance (Synergy HI) of the 96-well plate was measured at 450 nm and 650 nm using a microplate reader. The cell growth inhibition rate (VR) was calculated as (A-A0) / (As-A0)×100%, where A is the absorbance value of the experimental group (containing cells and fragments), As is the absorbance value of the control group (containing cells and DMSO), and A0 represents the absorbance value of the blank group (containing no cells). Finally, the cell growth inhibition rate was calculated and plotted using Graphpad Pris software.

[0267] The results are as follows Figure 17 As shown, compound Daudi exhibits superior inhibitory activity against cell proliferation compared to MDA-MB-231 and K562 cells.

[0268] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of a hydroxamic acid compound having the structure shown in formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the selective covalent modification, covalent labeling, or covalent binding of lysine residues in proteins. in, R is selected from: ethynyl-substituted C6-C 10 aryl, or selected from the following groups:

2. The use according to claim 1, characterized in that, R is selected from: acetylenic-substituted phenyl. Preferably, the hydroxamic acid compound is selected from:

3. The use according to claim 1 or 2, characterized in that, The protein is a pure protein, a protein in a cell lysate, or a protein in a living cell.

4. Use of the hydroxamic acid compound of claim 1 or 2, or its pharmaceutically acceptable salt, or its stereoisomer, in the identification and characterization of drug molecular targets.

5. Use of the hydroxamic acid compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the discovery and analysis of drug targets.

6. Use of the hydroxamic acid compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the identification of protein types, kinase types, and / or ligandable lysine residues in tumor cells; Preferably, the kinase is EGFR kinase, CDK family kinase, AURKA kinase, PAK1 kinase, or SRC kinase.

7. A method for covalently labeling lysine residues in proteins or polypeptides, characterized in that, Includes the following steps: (1) Incubate the hydroxamic acid compound of claim 1 or 2, or its pharmaceutically acceptable salt or its stereoisomer, with pure protein or cell lysate or live cells. (2) Add oxidant and incubate.

8. A method for identifying and characterizing drug molecular targets, characterized in that, Includes the following steps: (1) Introducing a hydroxamic acid group into a drug molecule and then incubating it with pure protein or cell lysate or live cells; or incubating the hydroxamic acid compound of claim 1 or 2 or its pharmaceutically acceptable salt or its stereoisomer with pure protein or cell lysate or live cells. (2) Add oxidant and incubate; (3) Post-processing and analysis of the incubated reaction solution; Preferably, the drug molecule is a drug molecule with kinase inhibitory activity and / or tumor cell proliferation inhibitory activity.

9. The method according to claim 7 or 8, characterized in that, The oxidant is chloramine-T and / or NaIO4; and / or, the living cells are tumor cells, and the cell lysis buffer is a lysis buffer of tumor cells.

10. The method according to claim 7 or 8, characterized in that, The incubation in step (1) includes the following reaction conditions: the concentration of the hydroxamic acid compound, or its pharmaceutically acceptable salt, or its stereoisomer is 20 μM-500 μM, preferably 50 μM-200 μM; and / or, The incubation temperature in step (1) is 30℃-40℃, and the incubation time is 30min-5h, preferably 1h-2h; and / or, The incubation in step (2) includes the following reaction conditions: the concentration of the oxidant is 0.5-10 times, preferably 2-5 times, the concentration of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer; and / or, The incubation temperature in step (2) is 30℃-40℃, and the incubation time is 30min-6h, preferably 2h-4h.

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