Hydroxamic acid compounds and their use

Abstract

The application discloses a hydroxamic acid compound with a structure shown in formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof, and an application thereof. The application provides a hydroxamic acid compound, and it is found that after the hydroxamic acid group is oxidized by a novel and efficient oxidation induction strategy, the hydroxamic acid group can be covalently combined with a lysine residue of a target protein, can selectively covalently modify the lysine residue in a protein or a polypeptide, has high selectivity and high efficiency for the modification of the lysine residue, can be used for activity research on the protein, and has important significance for chemical proteomics application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical biology molecular probes and chemical medicine, in particular, hydroxamic acid compounds and their application in covalently binding to lysine residues of proteins. BACKGROUND

[0002] In recent years, the development of covalent inhibitors has achieved excellent results and is applied to the treatment of various diseases. Targeted covalent inhibitors can covalently bind to target proteins and have a long-lasting effect on target protein function. Therefore, targeted covalent inhibitors have the advantages of long-lasting drug efficacy, low drug dosage, and high selectivity. Most of the covalent inhibitors marketed in recent years target covalent cysteine residues. For example, the anticancer drug neratinib covalently binds to the 797th cysteine of EGFR through acrylamide. However, cysteine is very low in abundance in the human proteome, accounting for only 1.9% of amino acid residues in the human proteome. Many ligand binding sites lack accessible cysteine; and with further research, we have found that highly active cysteine is prone to acquired drug resistance due to mutation. For example, the C797S cysteine mutation in EGFR is a common drug resistance mechanism.

[0003] Therefore, the development of targeted covalent inhibitors faces two difficulties. One is that the targetable amino acid is limited to cysteine, which is low in abundance. The other is that the mutation of highly active cysteine leads to acquired drug resistance. In order to solve the above problems, in recent years, researchers have turned their attention to lysine residues, which are 3 times more abundant than cysteine residues in the human proteome and are more conservative and less prone to mutation. Researchers have analyzed and quantified more than 9,000 lysine residues using co-crystal technology and isoTOP-ABPP technology to find that AURKA kinase can covalently bind to lysine residue K162. However, compared with the abundant lysine residues in the human proteome, the number of identified covalent lysine residues is relatively small, and no targeted lysine covalent inhibitor has been approved for marketing. Therefore, it is urgent to find and identify other high-abundance covalent amino acid residues to develop more targeted lysine covalent inhibitors.

[0004] In past research, scientists have been committed to developing covalent probes that can highly selectively bind to lysine residues by continuously improving and innovating chemical strategies. However, some existing lysine probes, such as aryl sulfonyl fluoride warheads, have too high chemical reactivity and can bind to lysine, tyrosine, and serine, which are not highly selective and specific for lysine. On the other hand, some existing lysine probes directly covalently bind to non-target protein lysine residues due to their high reactivity after entering the biological environment. The process of existing lysine probes approaching target proteins lacks control, resulting in low selectivity for target proteins. These factors hinder the development of targeted lysine covalent inhibitors. SUMMARY

[0005] Based on this, the present application provides a class of hydroxamic acid compounds which can selectively covalently modify and label lysine residues after oxidation.

[0006] The present application comprises the following technical solutions.

[0007] A hydroxamic acid compound having a structure shown in formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,

[0008]

[0009] wherein R is selected from: R1-substituted or unsubstituted C6-C 10 aryl, R1-substituted or unsubstituted 5-10 membered heteroaryl, R1-substituted or unsubstituted 5-10 membered heteroaroyl, or selected from:

[0010]

[0011] 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 alkylamino, 5-8 membered heterocyclyl, -C(=O)NR4,

[0012] each R2 is independently selected from: halogen, ethynyl, vinyl, C1-C6 alkoxy, nitro, C1-C6 alkylamino, C6-C 10 aryl, R5NH-, R5(CH2) n NH-;

[0013] each R3 is independently selected from: hydrogen, ethynyl, vinyl, trifluoromethyl, C1-C6 alkoxy, nitro, C1-C6 alkylamino;

[0014] each R4 is independently selected from: C1-C6 alkyl, C6-C 10 aryl-substituted C1-C6 alkyl, tolyl-substituted C1-C6 alkyl;

[0015] each R5 is independently selected from: R6-substituted or unsubstituted 5-10 membered heteroaryl, R6-substituted or unsubstituted C6-C 10 aryl;

[0016] each R6 is independently selected from: 5-10 membered heteroaryl, 5-8 membered heterocyclyl;

[0017] n is selected from: 1, 2, 3, 4.

[0018] In some embodiments, R is selected from the group consisting of R1-substituted or unsubstituted phenyl, R1-substituted or unsubstituted indolyl, R1-substituted or unsubstituted naphthyl, R1-substituted or unsubstituted thienyl, R1-substituted or unsubstituted pyridyl, R1-substituted or unsubstituted 4-oxo-4H-chromenyl, R1-substituted or unsubstituted 1,3-dioxoisoindolinyl, R1-substituted or unsubstituted pyrazinyl.

[0019] In some embodiments, each R1is independently selected from the group consisting of hydrogen, ethynyl, propargyl, vinyl, trifluoromethyl, C1-C3 alkyl, R2-substituted C1-C3 alkyl, R3-substituted or unsubstituted phenyl, diethylamino, tetrahydropyrrolidinyl, -C(=O)NR4;

[0020] each R2is independently selected from the group consisting of phenyl, R5NH-, R5CH2NH-;

[0021] each R3is independently selected from the group consisting of hydrogen, ethynyl, vinyl, trifluoromethyl, methoxy, ethoxy, nitro, diethylamino;

[0022] each R4is independently selected from the group consisting of C1-C3 alkyl, phenyl-substituted C1-C3 alkyl, tolyl-substituted C1-C3 alkyl;

[0023] each R5is independently selected from the group consisting of quinolinyl, imidazo[l,2-a]pyridinyl, dibenzo[b,d]furanyl-substituted phenyl, 4-morpholinylphenyl, benzimidazolyl.

[0024] In some embodiments, R is selected from the group consisting of:

[0025]

[0026] The present application also provides uses of the hydroxamic acid compounds, including the following:

[0027] The present application also provides uses of the hydroxamic acid compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, in selective covalent modification, covalent labeling or covalent binding of lysine residues in proteins.

[0028] In some embodiments, the protein is a pure protein, a protein in a cell lysate or a protein in a living cell.

[0029] The present application also provides uses of the hydroxamic acid compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, in identification and characterization of targets of drug molecules.

[0030] The present application also provides uses of the hydroxamic acid compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, in discovery and analysis of drug targets.

[0031] Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer in identifying protein species in tumor cells.

[0032] Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer in identifying kinase species in tumor cells.

[0033] Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer in discovering and identifying ligandable lysine residues in tumor cells.

[0034] Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer in developing covalent inhibitors targeting lysines.

[0035] Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer in preparing a kinase inhibitor.

[0036] In some embodiments, the kinase is EGFR kinase, CDK family kinase, AURKA kinase, PAK1 kinase.

[0037] Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer in preparing an antitumor drug.

[0038] In some embodiments, the tumor is breast cancer, lung cancer, leukemia, lymphoma.

[0039] The present application provides an antitumor drug prepared from an active ingredient and pharmaceutically acceptable adjuvant, wherein the active ingredient comprises the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer.

[0040] The present application also provides a method for covalently labeling lysine residues in proteins or polypeptides, comprising the following steps:

[0041] (1) incubating the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer with pure proteins or cell lysates or living cells;

[0042] (2) adding an oxidizing agent and incubating.

[0043] The present application also provides a method for identifying and characterizing the target of a drug molecule, comprising the following steps:

[0044] (1) introducing a hydroxamic acid group into a drug molecule, and then incubating it with pure protein or cell lysate or living cells; or incubating the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer according to the present application with pure protein or cell lysate or living cells;

[0045] (2) adding an oxidizing agent and incubating;

[0046] (3) post-treatment and analysis of the incubated reaction solution.

[0047] In some embodiments, the drug molecule is a drug molecule having a kinase inhibitory activity and / or a tumor cell proliferation inhibitory activity.

[0048] The present application also provides a method for discovering and analyzing a drug target, comprising the following steps:

[0049] (1) incubating the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer according to the present application with pure protein or cell lysate or living cells;

[0050] (2) adding an oxidizing agent and incubating;

[0051] (3) post-treatment and analysis of the incubated reaction solution.

[0052] The present application also provides a method for identifying protein species, kinase species and / or ligand-lysine residue in tumor cells, comprising the following steps:

[0053] (1) incubating the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer according to the present application with tumor cell lysate or tumor living cells;

[0054] (2) adding an oxidizing agent and incubating;

[0055] (3) post-treatment and analysis of the incubated reaction solution.

[0056] In some embodiments, the living cells are tumor cells, and the cell lysate is a tumor cell lysate.

[0057] In some embodiments, the oxidizing agent is chloramine-T and / or NaIO4.

[0058] In some embodiments, the incubation in step (1) comprises 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.

[0059] In some embodiments, the temperature of the incubation in step (1) is 30-40℃, and the incubation time is 30 min-5h, preferably 1-2h.

[0060] In some embodiments, the incubation in step (2) comprises the following reaction conditions: the concentration of the oxidant is 0.5-10 times, preferably 2-5 times, of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer.

[0061] In some embodiments, the temperature of the incubation in step (2) is 30-40℃, and the incubation time is 30 min-6h, preferably 2-4h.

[0062] The present application has the following beneficial effects:

[0063] The present application provides a class of hydroxamic acid compounds, and it is found that by a novel and efficient oxidation induction strategy, the hydroxamic acid group can be covalently combined with the lysine residues of target proteins after oxidation, which can selectively covalently modify the lysine residues in proteins or polypeptides, has high selectivity and efficiency for lysine residue modification, and can be used for activity research of proteins, and has important significance for chemical proteomics application.

[0064] The hydroxamic acid compound of the present application can be used for discovering and identifying drug targets, and introducing a hydroxamic acid group into an active drug molecule can be used for identifying and identifying the action target and lysine site of the active drug molecule.

[0065] The oxidant induction strategy of the present application can be a method for realizing the combination of ligands and target proteins of hydroxamic acid bioactive molecules, and because the generated nitrosyl carbonyl intermediate can react with protein amino acid residues, the method can be applied to the analysis of ligand amino acid residues of human kinase proteome, and potential covalently modified amino acid sites are developed, which provides a basis for developing novel lysine-targeted covalent inhibitors. The covalent inhibitor designed and synthesized with the nitrosyl carbonyl after oxidation of the hydroxamic acid group has good kinase inhibition activity and good anti-tumor cell proliferation activity, and can be applied to the development of covalent inhibitors for treating tumors.

[0066] The hydroxamic acid compound provided by the present application has high selectivity and specificity for covalent combination of lysine residues after oxidation induction, and the reactivity of the hydroxamic acid compound itself is low, which cannot directly covalently combine with non-target protein lysine residues in the biological environment, and when it is incubated with biological materials containing target proteins for a certain period of time to enter the binding pocket of the target protein, and then oxidized, high selective covalent combination of lysine residues of the target protein can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 Labeling BSA pure protein profile after compound M1 oxidation induction, FL: fluorescence, SDS-PAGE gel fluorescence imaging; CBB: coomassie brilliant blue, coomassie brilliant blue staining gel, indicating the consistency of protein loading amount.

[0068] Figure 2 Labeling profile of BSA pure protein after oxidation of compound M1 by different oxidants.

[0069] Figure 3 Comparison of labeling effects of compounds M1 and M2 on BSA pure protein.

[0070] Figure 4 Protein labeling profile of compound M1 on MDA-MB-231, A549, AGS, Bxpc-3 and Daudi cancer cell lysates.

[0071] Figure 5 Volcano plot of target proteins labeled by compound M1 in MDA-MB-231 cell lysate.

[0072] Figure 6 Verification of VDAC1, BIP, PLK1 protein targets of compound M1 in MDA-MB-231 cell lysate by pull-down / Western blotting.

[0073] Figure 7 Modification site selectivity analysis of compound M1 on HEK293T cell lysate.

[0074] Figure 8 Lysine binding site analysis of compound M1 on HEK293T cell lysate.

[0075] Figure 9 Site mutation and site verification by pull-down / Western blotting.

[0076] Figure 10 Protein labeling profile of compounds EH1 and EH2 in H3255 cell lysate.

[0077] Figure 11 Pull-down / WB experiment results of compound EH1 in H3255 cell lysate.

[0078] Figure 12 Inhibitory activity of compounds EH1 and EH2 on EGFR kinase.

[0079] Figure 13 Labeling profile of compound EH1 on AURKA pure protein.

[0080] Figure 14 Site identification results of compound EH1 and AURKA pure protein.

[0081] Figure 15 Protein labeling and identification of XH1 in cell lysate, wherein A is the labeling spectrum of XH1 in K562 lysate, and B is the kinase analysis Venn diagram of XH1 identified in the lysate.

[0082] Figure 16 Verification of XH1 binding to protein, wherein A is the labeling spectrum of XH1 and AURKA / SRC pure protein, and B is the Pull-down / WB diagram of XH1 and CDK1 in K562 cell lysate.

[0083] Figure 17 Inhibition rate of hydroxamic acid compound on proliferation of three kinds of cancer cells. DETAILED DESCRIPTION

[0084] The technical solutions of the present application will be further described below through specific examples. Those skilled in the art should understand that the examples are only to help understand the present application, and should not be regarded as specific limitations of the present application.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0086] The terms "comprising" and "having" and any variations thereof used in the present application are intended to cover not exclusively containing. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but optionally also includes steps not listed, or optionally also includes other steps inherent to these processes, methods, products or equipment.

[0087] In the present application, "a plurality of" refers to two or more. The "and / or" describes the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can represent the following three cases: A exists alone, A and B exist together, and B exists alone. The character " / " generally represents that the associated objects before and after are in an "or" relationship.

[0088] In the compounds of the present application, when any variable (e.g., R1, etc.) occurs more than one time in any constituent, its definition in each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in chemically stable compounds. A line drawn to a ring atom means that the bond can be attached to any available ring atom. If the ring system is polycyclic, it means that the bond is attached only to any appropriate carbon atom of the adjacent ring. It is understood that one of ordinary skill in the art can select substituents and substitution patterns for compounds of the present application which allow for the synthesis of chemically stable compounds that are readily accessible from readily available starting materials by techniques known in the art and the methods set forth below. If a substituent is itself substituted by more than one group, it is understood that the groups can be on the same carbon atom or on different carbon atoms, as long as the structure is stable.

[0089] The term "alkyl" as used herein means branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. In the "C1-C6alkyl" group, for example, there are groups having 1, 2, 3, 4, 5, or 6 carbon atoms in the straight or branched chain. For example, "C1-C6alkyl" specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl. The term "cycloalkyl" means monocyclic saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the like. The term "alkoxy" means a group having the structure -O-alkyl, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, and the like. The term "heterocycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic ring system substituent in which one or more of the ring atoms is a heteroatom selected from N, O, or S(O)m(where m is an integer from 0 to 2), with the remainder of the ring atoms being carbon, for example: morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotirazolyl, dihydroazetidinyl, tetrahydrofuryl, tetrahydrothiophenyl, and the like, and N-oxides thereof. The attachment of the heterocycloalkyl substituent can be through a carbon atom or through a heteroatom. The term "heteroaryl" means an aromatic ring containing 1 or more heteroatoms selected from O, N, or S. Heteroaryl groups within the scope of the present application include, but are not limited to: quinolinyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridinyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl; "heteroaryl" is also understood to include N-oxide derivatives of any nitrogen-containing heteroaryl groups. The attachment of the heterocycloalkyl substituent can be through a carbon atom or through a heteroatom.

[0090] The term "substituted" means replacement of a hydrogen group in a particular structure with the radical of a specified substituent.

[0091] As understood by those skilled in the art, "halo" or "halogen" as used herein means chlorine, fluorine, bromine, and iodine.

[0092] The present application includes the free form of the compounds of Formula I, as well as pharmaceutically acceptable salts and stereoisomers thereof. Some of the specific exemplary compounds of the present application are amines in the non-salt form, i.e., the "free form." The pharmaceutically acceptable salts of the present application can be conveniently prepared by conventional chemical methods from the free compounds of the present application having basic or acidic moieties. In general, the salts of the basic compounds are prepared either by ion exchange chromatography or by reacting the free base with a chemical equivalent of the desired salt form in a suitable solvent or combination of solvents. Similarly, the salts of the acidic compounds are formed by reaction with the appropriate inorganic or organic base in a suitable solvent or combination of solvents.

[0093] Thus, pharmaceutically acceptable salts of the compounds of this application include the conventional nontoxic salts of the compounds of the present application as formed by reaction with inorganic or organic acids. For example, conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts derived from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic, and the like.

[0094] If the compounds of the present application are acidic, suitable "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including 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, glucamine, glucosamine, histidine, hydroxycobal, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0095] Pharmaceutical Salts, J. Pharm. Sci. 1977: 66: 1-19, describes the above-mentioned pharmaceutically acceptable salts in more detail, as well as the preparation of other typical pharmaceutically acceptable salts.

[0096] The present application relates to the stereoisomers of the compounds of the present application, i.e. (depending on their structure) as enantiomers, diastereomers, syn- / anti-isomers, cis- / trans-isomers, epimers and (E)- / (Z)-isomers. The compounds of the formula I can be used in the context of the present application in the form of pure stereoisomers or in the form of any mixture of stereoisomers, in the latter case preferably as racemates.

[0097] Metabolites of the compounds of the present application and pharmaceutically acceptable salts thereof, as well as those metabolites which can be formed as a result of in vivo conversion of prodrugs of the compounds of the present application and pharmaceutically acceptable salts thereof, are included within the scope of the present application.

[0098] The present application also provides a pharmaceutical composition for preventing and / or treating tumors, which comprises a safe and effective amount of the active ingredient, and pharmaceutically acceptable adjuvants.

[0099] The "active ingredient" of the present application refers to the compound of the formula I of the present application, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug molecule thereof, or a deuteride thereof, or a tritide thereof.

[0100] A "safe and effective amount" refers to the amount of the active ingredient sufficient to substantially improve the condition without causing serious side effects.

[0101] The pharmaceutical composition is used by applying a safe and effective amount of the compound of the present application to a mammal (e.g. human) in need of treatment, wherein the dose is administered in a pharmaceutically effective amount, and the specific dose should also take into account the route of administration, the health condition of the patient, etc., which are within the skill of a skilled medical practitioner.

[0102] The "pharmaceutically acceptable adjuvants" refer to one or more compatible solid or liquid filler or gel materials suitable for human use and must be of sufficient purity and sufficiently low toxicity.

[0103] "Compatibility" here refers to the ability of the components of the composition to be blended with the active ingredient of the present application and among themselves without significantly reducing the efficacy of the active ingredient.

[0104] Examples of pharmaceutically acceptable adjuvants include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween 80®, Span 80®, etc.), wetting agents (e.g., sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0105] The following are specific examples.

[0106] Example 1 Synthesis of 4-ethyl-N-hydroxybenzamide (M1)

[0107]

[0108] In a 10 mL reaction bottle, NH2-OTHP (117.2 mg, 1.5 mmol, 1.5 eq), 4- ethylbenzoic acid (150 mg, 1.0 mmol, 1.0 eq), EDCI (230 mg, 1.2 mmol, 1.2 eq), HOBt-H2O (162 mg, 1.2 mmol) and DMF (3 mL) were added. The reaction mixture was stirred at room temperature for 2 hours, and the reaction process was monitored by thin layer chromatography. After the reaction was completed, the reaction mixture was poured into water. The reaction mixture was extracted with AcOEt. The organic layer was separated, washed with saturated NaHCO3and saturated brine, and dried over Na2SO4. Purification was performed by silica gel flash column chromatography (ethyl acetate / petroleum ether = 15 / 1000) to obtain S1 as a colorless viscous liquid. S1 was used directly in the next step without purification.

[0109] TsOH-H2O (16.9 mg, 0.0891 mmol) was added to a solution of S1 (0.1 eq) in MeOH (10 mL). 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 obtain M1 as an off-white powder (93 mg, yield 62%).

[0110] 1 H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 9.12 (s, 1H), 7.75 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 7.8 Hz, 2H), 4.35 (s, 1H). 13 ​C NMR (151 MHz, DMSO-d6) δ 163.86, 133.33, 132.20, 127.63, 124.85, 83.30, 83.23. ESI-MS calcd for C9H7NO2 [M+H] + m / z = 162.0550; Found 162.0551.

[0111] Example 2 Synthesis of N-hydroxy-3-((4-(prop-2-yn-1-yloxy)phenyl)amino)propanamide (M2)

[0112]

[0113] tert-Butyl (4-hydroxyphenyl)carbamate (S2): 4-aminophenol (4 g, 36.6 mmol, 1.0 equiv), (Boc)20 (8 g, 36.6 mmol, 1.0 equiv) were dissolved in THF and stirred for 2 h. The reaction mixture was filtered and the filtrate was concentrated to give S2. 1 H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.96 (s, 1H), 7.20 (d, J = 8.3 Hz, 2H), 6.64 (d, J = 8.2 Hz, 2H), 1.44 (s, 9H).

[0114] tert-Butyl (4-(prop-2-yn-1-yloxy)phenyl)carbamate (S3): S2 (7.3 g, 34.9 mmol, 1.0 equiv), K2CO3 (10.0 g, 72.3 mmol, 2.0 equiv) and propargyl bromide (4.29 g, 36.14 mmol, 1.0 equiv) were dissolved in DMF (2 mL) and the reaction mixture was stirred at 50 °C overnight. After TLC monitoring that the reaction was complete, 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 (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 7.35 (d, J = 8.5 Hz, 2H), 6.88 (dd, J = 8.9, 1.9 Hz, 2H), 4.72 (q, J = 1.8 Hz, 2H), 3.51 (t, J = 1.8 Hz, 1H), 1.46 (s, 9H).

[0115] 4-(prop-2-yn-l-yloxy)aniline (S4): S3 was dissolved in CH2Cl2and stirred well. Trifluoroacetic acid was added slowly dropwise into the CH2Cl2solution of S3. The mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated under reduced pressure to obtain crude S4 as a brown solid, which was used directly in the next step without further purification.

[0116] Preparation of 3-((4-(prop-2-yn-l-yloxy)phenyl)amino)propanoic acid methyl ester (S5): Tetrahydrofuran was added to a flask containing S4 and a stirrer, and triethylamine was added dropwise while stirring until no white mist was formed in the reaction solution. Methyl 3-bromopropionate (384 mg, 2.3 mmol, 1.0 equivalent) was added to the reaction mixture, which was then heated to reflux. After the completion of the reaction, which was monitored by TLC, the reaction mixture was filtered. The filtrate was evaporated under reduced pressure, and flash column chromatography was performed to purify S5 as a tan solid. 1 H NMR (400 MHz, DMSO-d6) δ 6.88 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 4.69 (d, J = 2.4 Hz, 2H), 3.61 (s, 3H), 3.51 (d, J = 2.6 Hz, 2H), 3.30 (s, 1H), 2.60 (t, J = 6.8 Hz, 2H).

[0117] N-hydroxy-3-((4-(prop-2-yn-l-yloxy)phenyl)amino)propanamide (M2): A solution of hydroxylamine hydrochloride (120 mg, 0.86 mmol, 4.0 equivalents) in anhydrous methanol (2 mL) was added to a solution of NaOH (68.8 mg, 0.86 mmol, 4.0 equivalents) in anhydrous methanol (2 mL) at a temperature of 0 °C, and the mixture was stirred for 10 minutes and the precipitate was filtered. The filtrate was added to a solution of S5 (100 mg, 0.43 mmol, 1.0 equivalent) in anhydrous methanol (2 mL). The mixture was stirred at room temperature for 2 hours, and the completion of the reaction was monitored by thin layer chromatography. After the reaction was completed, the reaction mixture was concentrated under vacuum to remove the solvent. Flash column chromatography was performed to purify M2 as a brown viscous liquid (86.9 mg, yield 86%). 1 H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.75 (s, 1H), 6.79 (d, J = 8.6 Hz, 2H), 6.57 (d, J = 8.4 Hz, 2H), 4.87 (s, 1H), 4.63 (s, 2H), 3.48 (s, 1H), 3.20 (t, J = 7.2 Hz, 2H), 2.22 (t, J = 7.2 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 173.77, 149.11, 143.95, 116.61, 113.44, 80.43, 78.14, 56.43, 34.30, 32.81. ESI-MS calcd for C 12 H 14N2O3 [M+H] + m / z = 235.1082; Found 235.1083.

[0118] Example 3 Synthesis of N-hydroxy-4-vinylbenzamide (M3)

[0119]

[0120] The synthesis was identical to the last step of the synthesis of M2, M3 was a light orange-red solid (yield 87%).

[0121] 1 H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 9.02 (s, 1H), 7.73 (d, J = 7.7 Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H), 6.78 (dd, J = 17.9, 10.8 Hz, 1H), 5.94 (d, J = 16.9 Hz, 1H), 5.36 (d, J = 11.9 Hz, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 164.25, 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.

[0122] Example 4 Synthesis of N1-hydroxy-N3-(4-methylphenethyl)isophthalamide (M4)

[0123]

[0124] Methyl 3-((4-methylphenyl)carbamoyl)benzoate (S6): A flask was charged with HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (855.5 mg, 2.25 mmol, 1.5 equiv), DIPEA (581.58 mg, 4.5 mmol, 3.0 equiv), 3-(methoxycarbonyl)benzoic acid (300 mg, 1.67 mmol, 1.1 equiv) and 20 mL DMF. After stirring the reaction mixture for 10 min, 2-(p-tolyl)ethan-1-amine (204.8 mg, 1.5 mmol, 1.0 equiv) was added to the reaction and stirred overnight. The completion of the reaction was monitored by thin layer chromatography and upon completion the mixture was concentrated under reduced pressure. Purification by flash column chromatography (EA / PE = 20 / 100) gave S6 as a yellowish viscous liquid (yield 48%). 1 HNMR (400 MHz, DMSO-d6) δ 8.78 (d, J = 5.9 Hz, 1H), 8.41 (s, 1H), 8.09 (d, J = 8.1 Hz, 2H), 7.63 (t, J = 7.4 Hz, 1H), 7.11 (t, J = 6.4 Hz, 4H), 3.89 (s, 3H), 3.47 (q, J = 6.4 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H), 2.26 (s, 3H).

[0125] N1-hydroxy-N3-(4-methylphenethyl)isophthalamide (M4): The synthesis was performed according to the last step of the synthesis of M2. The product M4 was obtained as a yellowish solid (yield 32.7%).

[0126] 1 HNMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 9.13 (s, 1H), 8.67 (s, 1H), 8.23 (s, 1H), 7.91 (dd, J = 33.0, 7.9 Hz, 2H), 7.55 (t, J = 7.8 Hz, 1H), 7.13 (q, J = 8.2 Hz, 4H), 2.82 (t, J = 7.6 Hz, 3H), 2.27 (s, 3H). 13 CNMR (151 MHz, DMSO-d6) δ 166.07, 164.21, 136.83, 135.48, 135.30, 133.45, 130.10, 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.

[0127] Example 5 Synthesis of N-hydroxy-2-naphthamide (M6)

[0128]

[0129] 2-naphthoic acid (500 mg, 2.9 mmol, 1.0 eq), CDI (705.5 mg, 4.35 mmol, 1.5 eq) were added to THF (10 mL) and stirred at room temperature for 1 h. NH2OH HCI (403 mg, 5.8 mmol, 2.0 eq) was added and stirred for 4 h. The completion of the reaction was monitored by thin layer chromatography and the mixture was concentrated under reduced pressure after the reaction was completed. Purification by flash column chromatography (EA / PE = 20 / 100) gave M6 as a white powder (447 mg, yield 82%).

[0130] 1 H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 9.11 (s, 1H), 7.76 (s, 1H), 7.62 (s, 1H), 7.13 (q, J = 3.9 Hz, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 164.73, 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; Found 188.0716.

[0131] Example 6 Synthesis of N-hydroxythiophene-2-carboxamide (M7)

[0132]

[0133] The synthesis of M7 was similar to that of Ml. M7 was a powder orange solid with a yield of 77%.

[0134] 1 H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 9.11 (s, 1H), 7.76 (s, 1H), 7.62 (s, 1H), 7.13 (q, J = 3.9 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 160.01, 137.80, 130.79, 128.35, 127.89. ESI-MS calcd for C5H5N2O2S [M+H] + m / z = 144.0114; Found 144.0114.

[0135] Example 7 Synthesis of N-hydroxy-5-(trifluoromethyl)picolinamide (M8)

[0136]

[0137] The synthesis of M8 was similar to that of M1. M8 was a light orange solid with a yield of 77%.

[0138] 1 H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 9.28 (s, 1H), 8.99 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 8.3 Hz, 1H). ESI-MS calcd for C7H5F3N2O2 [M+H] + m / z = 207.0377; Found 207.0376.

[0139] Example 8 Synthesis of N-hydroxy-3-methyl-4-oxo-2-phenyl-4H-chromen-8- carboxamide (M9)

[0140]

[0141] The synthesis of M9 was similar to that of M1. M9 was a white solid with a yield of 47%.

[0142] 1 H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.33 (s, 1H), 8.19 (dd, J = 7.9, 1.8 Hz, 1H), 7.87 (dd, J = 7.3, 1.8 Hz, 1H), 7.80 (dd, J = 6.7, 2.9 Hz, 2H), 7.62 - 7.58 (m, 3H), 7.54 (t, J = 7.6 Hz, 1H), 2.09 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 177.58, 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.

[0143] Example 9 Synthesis of 2-benzyl-N-hydroxy-l,3-dioxoisoindoline-5-carboxamide (M10)

[0144]

[0145] The synthesis method of M10 is similar to that of Ml, and M10 is a white solid with a yield of 45%.

[0146] 1 H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 9.33 (s, 1H), 8.24 - 8.17 (m, 2H), 7.99 (d, J = 7.7 Hz, 1H), 7.37 - 7.26 (m, 5H), 4.79 (s, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 167.64, 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; Found 297.0868.

[0147] Example 10 Synthesis of N-hydroxy-2'-methoxy-3'-nitro-[l,l'-biphenyl]-3-carboxamide (Ml l)

[0148]

[0149] The synthesis method of Ml l is similar to that of Ml, and Ml l is a light yellow solid with a yield of 51.6%.

[0150] 1 H NMR (400 MHz, DMSO-d6) δ 11.31 (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 = 7.7 Hz, 1H), 7.45 (dd, J = 9.1, 6.8 Hz, 1H), 3.45 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 164.24, 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 calcd for C 16 H 132 N2O4[M+H] + m / z = 289.0820; Found 289.0819

[0151] Example 11 Synthesis of N-hydroxypyrazine-2-carboxamide (M12)

[0152]

[0153] The synthesis method of M12 is similar to that of M1. M12 is a white solid with a yield of 50.2%.

[0154] 1 H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H), 9.27 (d, J = 6.5 Hz, 1H), 9.12 (d, J = 5.3 Hz, 1H), 8.84 (dd, J = 6.8, 2.6 Hz, 1H), 8.69 (dd, J = 5.4, 3.3 Hz, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 160.68, 147.79, 145.53, 143.89, 143.69. ESI-MS calcd for C5H5N3O2[M+H] + m / z = 140.0456; Found 140.0455

[0155] Example 12 Synthesis of 4-(diethylamino)-N-hydroxybenzamide (M13)

[0156]

[0157] The synthesis method of M13 is similar to that of M1. M13 is a yellow solid with a yield of 52%.

[0158] 1 H NMR (400 MHz, DMSO-d6) δ 10.79 (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 = 6.9 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 165.38, 149.85, 131.73, 128.93, 118.70, 110.61, 110.57, 44.24, 44.13, 12.83. ESI-MS calcd for C 11 H 16 N2O2[M+H] + m / z = 209.1290; Found 140.0455

[0159] Example 13 Synthesis of N-hydroxy-4-(pyrrolidin-l-yl)benzamide (M14)

[0160]

[0161] The synthesis of M14 was similar to that of Ml. M14 was a light yellow solid with a yield of 58.3%.

[0162] 1 H NMR (400 MHz, DMSO) δ 10.81 (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 = 3.6 Hz, 4H).13C NMR (151 MHz, DMSO-d6) δ 165.43, 157.56, 150.90, 131.50, 118.94, 111.15, 47.68, 47.65, 25.43. ESI-MS calcd for C 11 H 14 N2O2[M+H] + m / z = 207.1133; Found 207.1128.

[0163] Example 14 Synthesis of N-hydroxy-4-((quinolin-8-ylamino)methyl)benzamide (M15)

[0164]

[0165] The synthesis of M15 was similar to that of M2. M15 was a tan solid with a yield of 52%.

[0166] 1 H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.98 (s, 1H), 8.77 (dd, J = 1.8, 4.2 Hz, 1H), 8.20 (dd, J = 7.7, 5.1, 2.5 Hz, 1H), 7.68 (dd, J = 8.5, 2.4 Hz, 2H), 7.50 (dd, J = 4.2, 8.4 Hz, 1H), 7.45 - 7.42 (d, J = 8.1 Hz, 2H), 7.25 (q, J = 6.6, 5.3 Hz, 2H), 7.05 (d, J = 8.0 Hz, 1H), 6.52 (d, J = 7.6 Hz, 1H), 4.58 (s, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 164.68, 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.

[0167] Example 15 Synthesis of N-hydroxy-4-((imidazo[l,2-a]pyridin-8- ylamino)methyl)benzamide (M16)

[0168]

[0169] M16 was synthesized by the same method as M2, M16 was yellow solid, yield 63%.

[0170] 1 H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 8.97 (s, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 1.2 Hz, 1H), 7.76 (dd, J = 5.3, 1.4 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.45 - 7.42 (m, 1H), 6.80 (dt, J = 13.2, 6.5 Hz, 1H), 6.57 (t, J = 7.0 Hz, 1H), 5.93 - 5.85 (m, 1H), 4.52 (dd, J = 13.9, 6.4 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 167.71, 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-MS calcd for C 15 H 14 N4O2[M+H] + = 283.1190, Found 283.1194.

[0171] Example 16 Synthesis of 4-(((4-(dibenzo[b,d]furan-3-yl)phenyl)amino)methyl)-N- hydroxybenzamide (M17)

[0172]

[0173] The synthesis of M17 was similar to M2. M17 was a yellow solid with a yield of 41 %.

[0174] 1 H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 7.6 Hz, 1 H), 8.01 (dd, J = 7.6, 1.2 Hz, 1 H), 7.96 (dd, J = 8.3, 6.4 Hz, 2H), 7.72 (dd, J = 8.5, 4.5 Hz, 1 H), 7.67 (d, J = 8.7 Hz, 1 H), 7.58 (dd, J = 7.7, 1.3 Hz, 1 H), 7.55 (d, J = 8.4 Hz, 2H), 7.41 (td, J = 7.5, 3.7 Hz, 2H), 6.78 - 6.72 (m, 2H), 4.46 (d, J = 6.2 Hz, 2H), 3.85 (d, J = 2.1 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 167.69, 145.59, 143.59, 138.91, 136.97, 131.78, 131.18, 131.1 1, 129.90, 129.74, 129.72, 127.43, 127.29, 127.26, 1 15.12, 1 15.08, 1 14.42, 1 14.35, 1 13.75, 1 13.68, 97.33, 97.31, 46.09. ESI-MS calcd for C 26 H 20 N2O3[M+H] + m / z = 409.1552, Found 409.1557.

[0175] Example 17 Synthesis of N-hydroxy-4-(((4-morpholinophenyl)amino)methyl)benzamide (M18)

[0176] M18 was synthesized in a similar manner to M2. M18 was a yellow solid with a yield of 45%.

[0177] 1 H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 10.32 (s, 2H), 8.97 (s, 1H), 8.65 (d, J = 1.7 Hz, 3H), 7.68 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 6.71 (d, J = 6.8 Hz, 2H), 6.54 - 6.42 (m, 2H), 4.25 (d, J = 5.8 Hz, 2H), 3.68 (dd, J = 5.9, 3.6 Hz, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 166.61, 144.70, 143.01, 142.91, 131.54, 129.84, 128.58, 127.75, 127.57, 127.45, 127.31, 127.21, 126.75, 118.00, 113.61, 66.76, 50.92, 47.21. ESI-MS calcd for C 18 H 21 N3O3[M+H] + m / z = 329.1661, Found 329.1656.

[0178] Example 18 Synthesis of 4-((((lH-benzimidazol-2-yl)methyl)amino)methyl)-N- hydroxybenzamide (M19)

[0179]

[0180] M19 was synthesized in a similar manner to M2. M19 was a yellow solid with a yield of 41%. 1 H NMR (400 MHz, DMSO-d6) δ 11.19 (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, 4.0 Hz, 1H), 3.83 (d, J = 4.5 Hz, 1H), 3.68 (s, 2H), 3.17 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 164.51, 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.

[0181] Example 19 Synthesis of 5-((4-((3-ethynylphenyl)amino)-7-methoxyquinazolin-6- yl)oxy)-N-hydroxyvaleramide (EH1)

[0182]

[0183] The synthesis of EH1 was similar to M2, and EH1 was a white solid with a yield of 56%.

[0184] 1 H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.65 (s, 1H), 8.72 (s, 1H), 8.53 (s, 1H), 7.99 (s, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.89 (s, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 10.5 Hz, 2H), 4.21 (s, 1H), 4.17 (t, J = 6.3 Hz, 2H), 3.95 (s, 3H), 2.07 (t, J = 7.2 Hz, 2H), 1.83 (m, 2H), 1.73 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 174.84, 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.

[0185] Example 20 Synthesis of 2-((4-((3-ethynylphenyl)amino)-7-methoxyquinazolin-6- yl)oxy)-N-hydroxyacetamide (EH2)

[0186]

[0187] EH1 was synthesized in a similar manner to M2 as a white solid in 59% yield.

[0188] 1 H NMR (400 MHz, DMSO) δ 11.39 (s, 1H), 11.08 (s, 1H), 8.86 (s, 1H), 8.43 (s, 1H), 7.90 (d, J = 1.9 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.42 (d, 1H), 7.36 (s, 1H), 4.77 (s, 2H), 4.28 (s, 1H), 4.03 (s, 3H).

[0189] Example 21 Preparation of 6-((5-cyclopropyl-lH-pyrazol-3-yl)amino)-2-(4-(4-(2- (hydroxyamino)-2-oxoethoxy)benzyl)piperazin-l-yl)-N-(prop-2-yn-l-yl)pyrimidine-4- carboxamide (XH1)

[0190]

[0191] XH1 was synthesized in a similar manner to M2 as a yellow solid in 31% yield.

[0192] 1 H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 10.91 (s, 1H), 9.02 (t, J = 6.2 Hz, 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, 2.6 Hz, 3H), 3.11 (t, J = 2.5 Hz, 1H), 3.02 (s, 4H), 1.95 - 1.85 (m, 1H), 0.91 (m, 2H), 0.71 - 0.66 (m, 2H). 13C NMR (151 MHz, DMSO) d 170.46, 164.55, 164.41, 163.88, 160.78, 159.10, 159.06, 135.48, 133.35, 128.91, 115.56, 115.13, 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-MS calcd for C 27 H 31 N9O4[M+H] + m / z = 546.2577, Found 546.2572.

[0193] Example 22 Probe compound M1 oxidation-induced labeling of pure protein

[0194] The probe M1 was prepared into a probe stock solution with a concentration of 1 mM using DMSO, and NaIO4 was prepared into an oxidant stock solution with a concentration of 2 mM using PBS. Bovine albumin (BSA) pure protein was prepared into a solution with a concentration of 0.5 mg / mL using PBS.

[0195] Oxidation scheme A (pre-mixing): The M1 probe stock solution and the oxidant stock solution were mixed, and after 2 hours of reaction, 2 μL of DMSO or 2 μL of the reaction solution was added to the BSA pure protein solution, which was then incubated in a constant temperature shaker at 37°C for 2 hours.

[0196] Oxidation scheme B (incubation first and then oxidation): 1 μL of DMSO or 1 μL of the M1 probe stock solution was added to the pure protein solution and incubated for 2 hours, and then 1 μL of the NaIO4 stock solution was added and incubated in a constant temperature shaker at 37°C for 2 hours.

[0197] After incubation, the sample solution was added with click chemistry reagents (0.5 μL of 10 mM TBTA, 0.5 μL of 100 mM TCEP, 0.5 μL of 100 mM CuSO4, and 0.5 μL of 10 mM TAMRA-N3 solution), and the mixed sample was placed in a rotary shaker for reaction at room temperature for 2 hours. After adding 6 μL of 5x SDS loading buffer, 15 μL of the sample solution was separated by SDS-PAGE polyacrylamide gel electrophoresis, and after electrophoresis at a constant voltage of 90 V for 30 min, the voltage was adjusted to 110 V for electrophoresis for 60 min. Finally, a biomacromolecular interaction imager was used for scanning and imaging.

[0198] The results are shown in Table 1. Figure 1As 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.

[0199] Example 23 Effect of Oxidizing Agent on Marking Effect

[0200] 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.

[0201] 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.

[0202] Example 24: Effect of Hydroxyxamic Acid Structure on Labeling Efficacy

[0203] 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.

[0204] The results are as follows Figure 3As shown, neither probe Ml nor M2 can produce a labeled band without oxidation induction. After oxidation induction, only probe Ml produces a strong labeled band, while probe M2 does not produce a significant labeled band. It can be seen that the specific structure of hydroxamic acid molecules has a great influence on the labeling effect.

[0205] Example 25 Labeling of proteins in cell lysate after oxidation induction of compound Ml

[0206] Cell MDA-MB-231, Bxpc-3, Daudi, A549 and AGS cells were prepared into cell lysate. Taking MDA-MB-231 cells as an example, the specific experimental steps are as follows:

[0207] The culture medium of MDA-MB-231 cells growing to 90% in a 10 cm culture dish was discarded, and the cells were washed twice with 2 mL of PBS added to the wall of the culture dish. Then, 1 mL of trypsin was added to digest the cells until the cells were spherical under a microscope and no longer adhered to each other. Then, 1 mL of serum-containing medium was added to quench the digestion reaction. The cell solution was collected in a 5 mL sample tube, centrifuged at 1000 rpm for 3 min to precipitate the cells, and the supernatant was discarded. Then, 1 mL of PBS was added and gently shaken to resuspend the cells in PBS. The cell solution was transferred to a 1.5 mL sample tube. The sample tube was placed in an ice box and ultrasonically broken in a cell ultrasonic crusher. The parameters were set as 3 s on and 3 s off, power 15%, and ultrasonic time 3 min. After ultrasonic treatment, the sample was centrifuged at 14000 rpm for 15 min at 4°C in a high-speed centrifuge. The obtained supernatant was the cell lysate, and the bottom precipitate was discarded.

[0208] Take 200 μL cell lysate, add 2 μL DMSO or probe Ml (final concentration 100 μM) and react at 37°C for 2 h, then add NaIO4 (final concentration 500 μM) and react at 37°C for 4 h. After filtration by ultrafiltration membrane spin column, add TAMRA-N3, TBTA, TCEP, CuSO4 (final concentrations are 100 μM, 100 μM, 1 mM, 1 mM respectively) and incubate at 37°C for 2 h in a constant temperature shaker to complete the click reaction of protein-probe complex with fluorescent group TAMRA-N3. Add 1 mL pre-cooled methanol and place the sample at -20°C for more than 30 min (or overnight) to precipitate the protein. Then place the sample in a high-speed centrifuge at 4°C, 14000 rpm, and centrifuge at low temperature and high speed for 15 min. Discard the supernatant and invert the sample tube to dry the protein. Then add 40 μL 1x SDS loading buffer, and sonicate the sample in an ultrasonic cleaner for a few minutes to promote protein dissolution, then heat at 95°C for 15 min. After high-speed centrifugation at 14000 rpm for 30 s, take 20 μL sample solution and add it to the gel electrophoresis (SDS-PAGE, 10% separation gel) sample well, and after electrophoresis at 90V for 30 min, adjust to 110V constant voltage electrophoresis for 60 min. Finally, scan and image using a multifunctional biomolecular imager.

[0209] The results are shown in Table 1: Figure 4 Compound Ml has obvious labeling effect on the cell lysate of MDA-MB-231, Bxpc-3, Daudi, A549 and AGS cells after oxidation induction.

[0210] Example 26 Protein identification analysis of compound Ml on MDA-MB-231 cell lysate

[0211] 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.

[0212] 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.

[0213] Example 27 Target Validation by Drop-down / Western Blotting

[0214] To confirm the target proteins that probe M1 binds to, VDAC1, BIP (Endoplasmic reticulum chaperone BIP), PLK1 (Serine / threonine-protein kinase PLK1) were selected for Pull-down / WB experiment verification in this example.

[0215] MDA-MB-231 cells were prepared into cell lysate according to the method in Example 25, and the cell lysate was treated with probe M1 (final concentration 100 μM), and reacted at 37°C for 2 h, and then reacted with NaIO4 (final concentration 500 μM) at 37°C for 4 h. After filtration by ultrafiltration membrane spin column, fresh pre-mixed click chemistry mixture (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, 1 mM CuSO4 from 100 mM freshly prepared deionized water stock solution) was added. The reaction was further incubated for 2 hours under gentle mixing before the addition of pre-cooled methanol (-20°C) for precipitation. The precipitated protein was then collected by centrifugation (14000 rpm x 10 min, 4°C), and dissolved in PBS containing 1% SDS. After incubation with streptavidin beads for 4 hours at room temperature, the beads were washed with PBS containing 1% SDS (3 x 1 mL) and PBS (3 x 1 mL), respectively. 50 μL (2 x) SDS loading buffer was added and heated at 95°C for 30 minutes, and the sample was separated by SDS-PAGE (12% gel), and the target protein was verified by standard Western blotting method.

[0216] As shown in Figure 6 , probe M1 induced by NaIO4 oxidation can successfully pull down VDAC1, BIP and PLK1 in MDA-MB-231 cell lysate, while probe M1 without NaIO4 oxidation induction cannot produce labeled pull-down. This shows that VDAC1, BIP and PLK1 are indeed the target proteins of probe M1, and also shows that M1 can bind to target proteins through oxidation induction strategy, and the oxidation induction strategy can successfully label proteins at the lysate level.

[0217] Example 28 Modification site analysis of compound M1 on HEK293T cell lysate

[0218] (1) Preparation of cell lysate: discard the culture medium of HEK293T cells grown to 90% in a 10 cm culture dish, add 2 mL of PBS to the cell wall to wash the cells, repeat the washing twice. Centrifuge the cells at 1000 rpm for 3 min to collect the cells, add 1 mL of PBS to gently shake the cells, resuspend the cells in PBS, and place the sample tube in an ice box. Ultrasonic cell disrupter is set at 3 s on and 3 s off, power 15%, and ultrasonic time 3 min. After ultrasonic, the sample is centrifuged at high speed in a high-speed centrifuge at 4°C and 14000 rpm for 15 min. The supernatant obtained is the cell lysate.

[0219] (2) Probe and oxidation incubation: The prepared cell lysate is divided into two equal parts, each 500 μL, and 5 μL of 10 mM probe M1 is added to each part, and incubated in a constant temperature homogenizer at 37°C for 2 h. Add 5 μL of 50 mM oxidant NaIO4 solution and oxidize for 4 h.

[0220] (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.

[0221] (4) Protein precipitation and trypsin digestion: Add 540 μL of MeOH and 135 μL of CH3Cl, mix well and stand at -20°C for 30 min. Centrifuge at 1700 g for 20 min, discard the supernatant, and place the sample tube in a 37°C constant temperature homogenizer for a few minutes to dry the protein. Add 400 μL of (NH4)2CO3 solution, place the sample in an ice box, and ultrasonic for 6-15 s until the protein is fully suspended in the solution. Add 5 μL of trypsin and incubate at 37°C in a constant temperature homogenizer for 12 h. Add 2 μL of trypsin and continue to incubate at 37°C in a constant temperature homogenizer for 2 h. High-speed centrifuge at 14000 g for 5 min, and dry the supernatant in a high-temperature vacuum dryer to obtain 200 μL of polypeptide sample.

[0222] (5) Desalination: Activate the HLB SPE chromatographic column with 1 mL of HPLC-acetonitrile and 2 mL of HPLC-H2O, add 200 μL of polypeptide sample and desalt with 1 mL of HPLC-H2O, and finally elute the polypeptide with HLB-A reagent. Dry the polypeptide eluate in a vacuum centrifuge at 45°C until completely dry.

[0223] (6) Click reaction: each polypeptide sample was mixed with 20 μL HPLC-H20 and 10 μL HPLC acetonitrile. One sample was added with 1 μL Light Az-UV-Biotin stock solution, 4 μL sodium ascorbate, 1 μL TBTA and 4 μL CuSO4, respectively. Another sample was added with 1 μL Heavy Az-UV-Biotin stock solution, 4 μL sodium ascorbate, 1 μL TBTA and 4 μL CuSO4, respectively. The samples were incubated at 37 °C for 2 h in the dark, mixed with 120 μL streptavidin binding buffer, and centrifuged at 14,000 g for 5 min. The supernatants of the two samples were combined and transferred to a new sample tube.

[0224] (7) Enrichment of protein: 100 μL streptavidin beads were washed twice with 1 mL streptavidin binding buffer by gentle shaking and centrifugation at 1,700 g for 3 min. The combined sample and washed beads were added to 10 mL streptavidin binding buffer, and incubated at room temperature for 2 h in the dark.

[0225] (8) Washing of beads: the beads were centrifuged at 1,700 g for 3 min, and the supernatant was discarded. 10 mL streptavidin binding buffer was added, and the sample was gently inverted for 5 min before centrifugation. The above step was repeated twice. 10 mL streptavidin washing buffer was added, and the sample was gently inverted for 5 min before centrifugation. The above step was repeated twice. 1.2 mL light release buffer was added, and the beads were dispersed in the solution by gentle shaking. The sample tube was placed under a 365 nm UV light for 2 h. The sample was centrifuged at 2,000 g for 4 min, and the supernatant was concentrated to 200 μL by a vacuum centrifuge.

[0226] (9) Desalting and sample preparation: an HLB SPE column was activated with 1 mL HPLC-acetonitrile and 2 mL HPLC-H20. 200 μL polypeptide sample was added and desalted with 1 mL HPLC-H20. Finally, HLB-A reagent was added to elute the polypeptide. The polypeptide eluate was dried to completion by a vacuum centrifuge at 45 °C. The dried polypeptide was dissolved in 12 μL 0.1% formic acid by ultrasonic for several minutes. The sample was centrifuged at 14,000 g for 15 min, and 10 μL supernatant was used for LC-MS / MS test.

[0227] The results are shown in Figure 7 , Figure 8 and Table 1-Table 2: The binding site heat map of probe M1 in the lysate of HEK293T cells Figure 7 shows that probe M1 only has modification on K, and the selectivity is between 0.8-1, indicating that the binding of probe M1 to lysine residues has high selectivity. The binding site selectivity and analysis effectiveness of probe M1 are shown in the Venn diagram Figure 7The B) in the figure shows that the selectivity of probe M1 to lysine is more than 90% and the profiling efficiency is also more than 90%, and the high profiling efficiency also indicates the high accuracy of the analysis data.

[0228] In addition, the proteins and specific sites bound by probe M1 were analyzed by proteomics analysis software pFind, and the results showed that probe M1 hit a total of 7241 lysines Figure 8 ), most of which were not annotated, and 662 lysines were annotated as functional lysines. Most of these functional lysines were post-translational modification lysines, and about 7% of the sites were binding sites. These functional binding sites include ATP binding sites, GTP binding sites, NADP + binding sites, CoA binding sites, Mn 2+ binding sites, 3', 5'-cyclic AMP binding sites (Table 1), indicating that M1 can enter the pockets of various proteins, and widely enter the binding pockets of various proteins and bind to the active sites. Compared with more than 3000 lysines identified by STP lysine probe reported by Hacker et al., about 6500 lysine sites identified by M1 are not bound by STP, and M1 has a significant complementary effect on identifying lysines in proteome.

[0229] M1 identified 2499 different proteins, of which 101 proteins were included in Drugbank, and most of the proteins identified by M1 were not in Drugbank. These proteins show a more extensive class distribution than the smaller part of DrugBank proteins containing ligand lysine, which are mainly enzymes. Non-DrugBank proteins with ligand lysine include transcription factors and scaffold proteins that are considered difficult to target with small molecules.

[0230] Among the annotated sites hit by M1 (Table 2), DDX6_K141, DTYMK_K19, ALDH18A1_K311, HSP17B12_K72, CDK2_K129, and MTHFD1_K56 are functional sites of the corresponding proteins, which can affect the function of the protein.

[0231] 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, identify their unexplored lysine residues, and facilitate a deeper understanding of protein conserved lysine and the development of covalent inhibitors targeting lysine.

[0232] Table 1 Functional lysine sites of M1 in HEK293T cell lysate (partial)

[0233]

[0234] Table 2M1 hits lysine sites in HEK293T cell lysate

[0235]

[0236] Example 29 Site validation by site mutation and pull-down / western blot

[0237] Wild / mutant proteins were expressed in HEK293T cells, cell lysate was prepared according to the method of Example 28, and the cell lysate was treated with probe M1 (final concentration 100 μΜ) and reacted at 37 °C for 2 h, then reacted with NaIO4 (final concentration 500 μΜ) at 37 °C for 4 h. After filtration by ultrafiltration membrane spin column, fresh pre-mixed click chemistry mixture (100 μΜ biotin-N3, 100 μΜ TBTA, 2 mM sodium ascorbate, 1 mM CuSO4) was added. Before adding pre-cooled methanol (-20 °C) for precipitation, the reaction was further incubated for 2 hours under gentle mixing. Subsequently, the precipitated protein was collected by centrifugation (14000 rpm x 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 containing 1% SDS (3 x 1 mL) and PBS (3 x 1 mL), respectively. 50 μL (2 x) SDS loading buffer was added and heated at 95 °C for 30 min, and the sample was separated by SDS-PAGE, and the target protein was verified by standard western blot method.

[0238] The results are shown in Figure 9 Figure 6. After mutation of PGK1_K131, PGK_K323 and CDK5_K33 to arginine, the labeling of the corresponding proteins by probe M1 was significantly reduced, indicating that the nitrosyl carbonyl intermediate generated after oxidation of probe M1 can effectively label lysine residues in the whole proteome.

[0239] Example 30 Protein labeling of probe EH1 / EH2 in H3255 cell lysate

[0240] According to the experimental method of Example 25, H3255 lysate was treated with 100 μΜ probe EH1 or EH2 for 2 h, 5 x NaIO4 oxidation for 4 h, and then filtered to remove the oxidant and perform click reaction, followed by fluorescence scanning imaging.

[0241] The results are shown in Figure 10 Figure 7. Among the two probes, EH1 has a stronger labeling effect.

[0242] Example 31 Protein identification and Pull-down / WB experiment of probe EH1 in H3255 cell lysate

[0243] This example identified the target proteins labeled by probe EH1, and the experimental method referred to Examples 26 and 27.

[0244] The results (Table 3) showed that some of the proteins hit by EH1 were kinases, including AURKA (Aurora kinase A), PAK1 (Serine / threonine protein kinase PAK1), CDK1 (Cyclin-dependent kinase 1), etc.

[0245] The results of the Pull-down / WB experiment ( Figure 11 ) showed that EH1 had no obvious labeling effect on EGFR without the addition of NaIO4 oxidation induction; while after NaIO4 oxidation, EH1 could successfully label and pull down EGFR. This indicated that after oxidation induction, EH1 could covalently bind and pull down EGFR.

[0246] Table 3. Protein identification of compound EH1 in H3255 cell lysate (partial)

[0247]

[0248]

[0249] Example 32 Inhibition activity test of probes EH1 / EH2 on EGFR kinase

[0250] Z'-LYTE TM The inhibition of the probes on EGFR kinase was tested by the fluorescence resonance energy transfer (FRET) method, and the parent inhibitors were used as control compounds. Z'-LYTE TM The biochemical assay used a FRET-based coupled enzyme format based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate was labeled with two fluorophores - one at each end - to form a FRET pair. Compounds were tested at 5.1 x 10 -9 M diluted three times to 1 x 10 -4 M. The plates were measured on an EnVision Multilabel Reader (Perkin Elmer). Curve fitting and data presentation were performed using Graph Pad Prism version 4.0.

[0251] 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.

[0252] Example 32: Labeling and Site Identification of Probe EH1 at the Pure Protein Level

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] Example 33: Protein Identification and Verification of XH1 in Cell Lysate

[0258] 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.

[0259] 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.

[0260] result( Figure 15 A) shows that XH1 can indeed produce a mark after oxidation.

[0261] Pull-down / LC-MS / MS experiment (probe concentration selected as 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.

[0262] 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.

[0263] 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.

[0264] Example 34 Cell proliferation inhibition rate test

[0265] 96-well plates were seeded with 8000-10000 cells per well (MDA-MB-231 seeding density was 8000 / well, Daudi, K562 cells seeding density was 10000 / well), and incubated in 37°C incubator for 24 hours. Fragment compounds M3-M19 (final concentration 5 μM) were added to each well, each sample well was set up a duplicate well, the last column was a cell-free blank control, and incubated for 48 hours. 30 μL CCK8 reagent was added to each well, and incubated for 4 hours. The 96-well plates were placed on the plate reader of the microplate reader to measure the absorbance at 450 nm and 650 nm (Synergy HI), and the cell growth inhibition rate VR = (A-A0) / (As-A0) x 100% was calculated, wherein A was the absorbance value of the experimental group (containing cells and fragments), As was the absorbance value of the control group (containing cells and DMSO), and A0 represented the absorbance value of the blank group (without cells). Finally, the software Graphpad Pris was used to calculate the cell growth inhibition rate and plot the graph.

[0266] The results are shown in Table 1: Figure 17 The proliferation inhibition activity of the compound on Daudi cells was better than that on MDA-MB-231 and K562 cells.

[0267] The above-described examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. A hydroxamic acid compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that, The hydroxamic acid compound has a structural formula as shown in the following:

2. Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 in selectively covalently modifying, covalently labeling or covalently binding lysine residues in a protein.

3. Use according to claim 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 or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 in identification and characterization of a target of a drug molecule.

5. Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 in discovery and analysis of a drug target.

6. Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 in identification of protein species and / or ligandable lysine residues in a tumor cell.

7. Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 in identification of kinase species in a tumor cell, the kinase being a CDK family kinase, an AURKA kinase, an SRC kinase.

8. Use of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 in development of a lysine-targeted covalent inhibitor.

9. A method for covalently labeling lysine residues in proteins or polypeptides, characterized in that, The method comprises the following steps: (1) incubating the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 with a pure protein or a cell lysate or a living cell; (2) adding an oxidizing agent and incubating.

10. A method for identifying and characterizing drug molecule targets, characterized in that, The method comprises the following steps: (1) incubating the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 with a pure protein or a cell lysate or a living cell; (2) adding an oxidizing agent and incubating; (3) post-treating and analyzing the incubated reaction solution.

11. The method according to claim 9 or 10, characterized in that, The oxidizing agent is chloramine-T and / or NaIO4; and / or, the living cell is a tumor cell and the cell lysate is a lysate of the tumor cell.

12. The method according to claim 9 or 10, characterized in that, The incubation in step (1) comprises the following reaction conditions: the concentration of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof is 20 μM-500 μM; and / or, The temperature of the incubation in step (1) is 30-40°C and the incubation time is 30 min-5 h; and / or, The incubation in step (2) comprises the following reaction conditions: the concentration of the oxidizing agent is 0.5-10 times that of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof; and / or, The temperature of the incubation in step (2) is 30-40°C and the incubation time is 30 min-6 h.

13. The method of claim 12, wherein, The incubation in step (1) comprises the following reaction conditions: the concentration of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof is 50 μM-200 μM.

14. The method of claim 12, wherein, The incubation time in step (1) is 1-2 h.

15. The method of claim 12, wherein, The incubation in step (2) comprises the following reaction conditions: the concentration of the oxidizing agent is 2-5 times that of the hydroxamic acid compound or its pharmaceutically acceptable salt or stereoisomer thereof.

16. The method of claim 12, wherein, The incubation described in step (2) is for a time of 2h-4h.

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