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. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of chemical biological molecular probe and chemical medicine technology, in particular to a hydroxamic acid compound and its application in covalently binding to a lysine residue of a protein. Background Art

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

[0003] Therefore, the development of targeted covalent inhibitors faces two difficulties. First, the targetable amino acids are limited to cysteine, which has low abundance. Second, mutations in highly active cysteine ​​lead to acquired drug resistance. In order to solve the above problems, in recent years, researchers have turned their attention to lysine residues with higher abundance, which are three times more abundant than cysteine ​​residues in the human proteome and are more conservative and less prone to mutation. The researchers used cocrystal technology and isoTOP-ABPP technology to analyze and discover the covalent lysine residue K162 of AURKA kinase and quantified more than 9,000 lysine residues. However, compared with the abundant lysine residues in the human proteome, the number of covalent lysine residues identified so far is relatively small, and no covalent inhibitors targeting lysine have been approved for marketing. Therefore, the discovery and identification of other high-abundance covalent amino acid residues to develop more covalent inhibitors targeting lysine is an urgent problem to be solved.

[0004] In past studies, 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 warheads such as arylsulfonyl fluoride, have too high chemical reactivity and can bind to lysine, tyrosine and serine, but their selectivity and specificity for lysine are not high. On the other hand, after entering the biological environment, some existing lysine probes directly covalently bind to non-target protein lysine residues due to their high reactivity. The process of existing lysine probes approaching target proteins lacks control, resulting in low selectivity for target proteins. These have hindered the development of covalent inhibitors targeting lysine. Summary of the invention

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

[0006] The present invention includes the following technical solutions.

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

[0008]

[0009] Wherein, R is selected from: R 1 Substituted or unsubstituted C 6 -C 10 Aryl, R 1 Substituted or unsubstituted 5-10 membered heteroaryl, R 1 A substituted or unsubstituted 5-10 membered heteroaryl ketone group, or selected from the following groups:

[0010]

[0011] Each R 1 are independently selected from: hydrogen, ethynyl, vinyl, R 2 Substituted or unsubstituted C 1 -C 6 Alkyl, R 3 Substituted or unsubstituted C 6 -C 10 Aryl, C 1 -C 6 Alkoxy, nitro, C 1 -C 6 Alkylamino, 5-8 membered heterocyclic group, -C(=O)NR 4 ,

[0012] Each R 2 are independently selected from: halogen, ethynyl, vinyl, C 1 -C 6 Alkoxy, nitro, C 1 -C 6 Alkylamine, C 6 -C 10 Aryl, R 5 NH-, R 5 (CH 2 ) n NH-;

[0013] Each R 3 are independently selected from: hydrogen, ethynyl, vinyl, trifluoromethyl, C 1 -C 6 Alkoxy, nitro, C1 -C 6 Alkylamine;

[0014] Each R 4 Each independently selected from: C 1 -C 6 Alkyl, C 6 -C 10 Aryl substituted C 1 -C 6 Alkyl, tolyl substituted C 1 -C 6 alkyl;

[0015] Each R 5 Each independently selected from: R 6 Substituted or unsubstituted 5-10 membered heteroaryl, R 6 Substituted or unsubstituted C 6 -C 10 Aryl;

[0016] Each R 6 Each 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: 1 Substituted or unsubstituted phenyl, R 1 Substituted or unsubstituted indolyl, R 1 Substituted or unsubstituted naphthyl, R 1 Substituted or unsubstituted thienyl, R 1 Substituted or unsubstituted pyridyl, R 1 Substituted or unsubstituted 4-oxo-4H-benzopyranyl, R 1 Substituted or unsubstituted 1,3-dioxoisoindolinyl, R 1 a substituted or unsubstituted pyrazinyl group.

[0019] In some embodiments, each R 1 are independently selected from: hydrogen, ethynyl, propargyl, vinyl, trifluoromethyl, C 1 -C 3 Alkyl, R 2 Replace C 1 -C 3 Alkyl, R 3 Substituted or unsubstituted phenyl, diethylamino, tetrahydropyrrolidinyl, -C(=O)NR 4 ;

[0020] Each R 2 are independently selected from: phenyl, R 5NH-, R 5 CH 2 NH-;

[0021] Each R 3 Each of the following is independently selected from the group consisting of hydrogen, ethynyl, vinyl, trifluoromethyl, methoxy, ethoxy, nitro, and diethylamino;

[0022] Each R 4 Each independently selected from: C 1 -C 3 Alkyl, phenyl substituted C 1 -C 3 Alkyl, tolyl substituted C 1 -C 3 alkyl;

[0023] Each R 5 Each of the following is independently selected from the group consisting of quinolinyl, imidazo[1,2-a]pyridinyl, dibenzo[b,d]furanyl substituted phenyl, 4-morpholinylphenyl, and benzimidazolyl.

[0024] In some embodiments, R is selected from:

[0025]

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

[0027] The hydroxamic acid compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer is used 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 hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer described in the present invention is used in the recognition and identification of drug molecule action targets.

[0030] Use of the hydroxamic acid compound or a pharmaceutically acceptable salt or a stereoisomer thereof in the discovery and analysis of drug targets.

[0031] The hydroxamic acid compound or the pharmaceutically acceptable salt or the stereoisomer thereof of the present invention is used in identifying the types of proteins in tumor cells.

[0032] The hydroxamic acid compound or the pharmaceutically acceptable salt or the stereoisomer thereof of the present invention is used for identifying the type of kinase in tumor cells.

[0033] The hydroxamic acid compound or the pharmaceutically acceptable salt or the stereoisomer thereof of the present invention is used in discovering and identifying ligandable lysine residues in tumor cells.

[0034] The hydroxamic acid compound or the pharmaceutically acceptable salt or the stereoisomer thereof of the present invention is used in developing a covalent inhibitor targeting lysine.

[0035] The hydroxamic acid compound or the pharmaceutically acceptable salt or the stereoisomer thereof of the present invention is used in the preparation of kinase inhibitors.

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

[0037] The invention relates to the use of the hydroxamic acid compound or its pharmaceutically acceptable salt or its stereoisomer in the preparation of anti-tumor drugs.

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

[0039] The present invention provides an anti-tumor drug prepared from active ingredients and pharmaceutically acceptable excipients. The active ingredient comprises the hydroxamic acid compound of the present invention or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0040] The present invention also provides a method for covalently labeling lysine residues in a protein or polypeptide, comprising the following steps:

[0041] (1) incubating the hydroxamic acid compound of the present invention or a pharmaceutically acceptable salt thereof or a stereoisomer thereof with pure protein or cell lysate or living cells;

[0042] (2) Add oxidant and incubate.

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

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

[0045] (2) adding an oxidant and incubating;

[0046] (3) Post-processing and analyzing the reaction solution after incubation.

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

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

[0049] (1) incubating the hydroxamic acid compound of the present invention or a pharmaceutically acceptable salt thereof or a stereoisomer thereof with pure protein or cell lysate or living cells;

[0050] (2) adding an oxidant and incubating;

[0051] (3) Post-processing and analyzing the reaction solution after incubation.

[0052] 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:

[0053] (1) incubating the hydroxamic acid compound of the present invention or a pharmaceutically acceptable salt thereof or a stereoisomer thereof with a tumor cell lysate or living tumor cells;

[0054] (2) adding an oxidant and incubating;

[0055] (3) Post-processing and analyzing the reaction solution after incubation.

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

[0057] In some embodiments, the oxidant is chloramine-T and / or NaIO 4 .

[0058] In some embodiments, the incubation in step (1) comprises the following reaction conditions: the concentration of the hydroxamic acid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof is 20 μM-500 μM, preferably 50 μM-200 μM.

[0059] 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.

[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 its stereoisomer.

[0061] 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.

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

[0063] The present invention provides a class of hydroxamic acid compounds, and finds that through a novel and efficient oxidation induction strategy, the hydroxamic acid group can be oxidized to covalently bind to the lysine residues of the target protein, and can selectively covalently modify the lysine residues in the protein or polypeptide, and has high selectivity and efficiency for the modification of lysine residues, can be used for the activity study of proteins, and is of great significance for the application of chemical proteomics.

[0064] The hydroxamic acid compound of the present invention can be used to discover and identify drug targets, and the hydroxamic acid group is introduced into the active drug molecule, which can be used to identify and verify the action target and lysine site of the active drug molecule.

[0065] The oxidant induction strategy of the present invention can be a method for realizing the binding of ligands to target proteins by hydroxamic acid bioactive molecules, and because the generated nitrosocarbonyl intermediates can react with protein amino acid residues, the method can be applied to the analysis of ligandable amino acid residues of human kinase proteome, and potential amino acid sites that can be covalently modified can be developed, providing a basis for the development of new lysine-targeted covalent inhibitors. The covalent inhibitor designed and synthesized using the nitrosocarbonyl group after oxidation of the hydroxamic acid group as a covalent warhead has good kinase inhibitory 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 invention has high selectivity and specificity in covalent binding to lysine residues after oxidation induction. In addition, the hydroxamic acid compound itself has low reactivity and will not directly covalently bind to lysine residues of non-target proteins after entering the biological environment. After incubating it with a biological material containing a target protein for a certain period of time to enter the binding pocket of the target protein, it is then oxidatively induced, thereby achieving highly selective covalent binding to the lysine residues of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the spectrum of pure BSA protein labeled after oxidation induction by compound M1. FL: fluorescence, SDS-PAGE gel fluorescence imaging; CBB: coomassie brilliant blue, Coomassie brilliant blue stained gel, indicating the consistency of protein loading amount.

[0068] Figure 2 This is the labeling spectrum of BSA pure protein after compound M1 was oxidized by different oxidants.

[0069] Figure 3 This is a comparison chart of the labeling effects of compounds M1 and M2 on BSA pure protein.

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

[0071] Figure 5 This is the volcano map of the target protein labeled by compound M1 in MDA-MB-231 cell lysate.

[0072] Figure 6 To verify the effects of compound M1 on VDAC1, BIP, and PLK1 protein targets in MDA-MB-231 cell lysates by pull-down / western blotting.

[0073] Figure 7 This is the 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] Fig. 9 Perform site validation for site mutations with pull-down / Western blotting.

[0076] Fig.10 This is the protein labeling spectrum of compounds EH1 and EH2 in H3255 cell lysate.

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

[0078] Fig.12 The inhibitory activity of compounds EH1 and EH2 on EGFR kinase.

[0079] Fig.13 This is the labeling spectrum of compound EH1 on AURKA pure protein.

[0080] Fig.14 The results of site identification of compound EH1 and AURKA pure protein.

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

[0082] Fig.16This is the verification of XH1 binding to proteins, where A is the labeling spectrum of XH1 and AURKA / SRC pure proteins, and B is the Pull-down / WB diagram of XH1 and CDK1 in K562 cell lysate.

[0083] Fig.17 is the proliferation inhibition rate of hydroxamic acid compounds on three cancer cells. DETAILED DESCRIPTION

[0084] The technical scheme of the present invention is further illustrated by specific examples below. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0085] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0086] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0087] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0088] In the compounds described in the present invention, when any variable (such as R 1 If a (, etc.) compound occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permitted so long as such combinations result in stable compounds. A line drawn from a substituent into the ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds are attached only to any suitable carbon atom of an adjacent ring. It is to be understood that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the invention to provide compounds that are chemically stable and that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

[0089] The term "alkyl" as used herein is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. 1 -C 6 "C" in "alkyl" 1 -C 6 The definition of " includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched chain arrangement. For example, "C 1 -C 6 The term "alkyl" specifically 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 -OCH 3 、-OCH 2 CH 3 、-OCH 2 CH 2 CH 3 、-O-CH 2 CH(CH 3 ) 2 、-OCH 2 CH 2 CH 2 CH 3 、-O-CH(CH 3 ) 2 The term "heterocycloalkyl" is a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent, wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon, for example: morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, etc., and N-oxides thereof, and the connection of the heterocyclic substituent can be achieved through a carbon atom or through a heteroatom. The term "heteroaryl" refers to an aromatic ring containing one or more heteroatoms selected from O, N or S. Heteroaryl within the scope of the present invention includes, but is not limited to, quinolyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl; "heteroaryl" is also understood to include any N-oxide derivatives of heteroaryl containing nitrogen. The connection of the heterocyclic substituent can be achieved through a carbon atom or through a heteroatom.

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

[0091] As will be appreciated by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0092] The present invention includes free forms of compounds of formula I, as well as pharmaceutically acceptable salts and stereoisomers thereof. Some specific exemplary compounds of the present invention are amine compounds in non-salt form, i.e., "free forms". Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic or acidic moiety by conventional chemical methods. Typically, salts of basic compounds are prepared by ion exchange chromatography or by reacting a free base with a stoichiometric amount or excess of an inorganic or organic acid in the desired salt form in an appropriate solvent or a combination of multiple solvents. Similarly, salts of acidic compounds are formed by reacting with an appropriate inorganic or organic base.

[0093] Therefore, the pharmaceutically acceptable salts of the compounds of this invention include conventional non-toxic salts of the compounds of this invention formed by reacting alkaline compounds of this invention with inorganic or organic acids. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic 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, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxy-benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc.

[0094] If the compound of the present invention is acidic, then suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases 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, manganic salts, manganous 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, 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, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, guaiac, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[0095] Berg et al., "Pharmaceutical Salts," J. Pharm. Sci. '1977: 66: 1-19, describes in more detail the preparation of the above-mentioned pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts.

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

[0097] The metabolites of the compounds involved in the present invention and their pharmaceutically acceptable salts, as well as prodrugs that can be converted into the structures of the compounds involved in the present invention and their pharmaceutically acceptable salts in vivo, are also included in the claims of the present invention.

[0098] The present invention also provides a pharmaceutical composition for preventing and / or treating tumors, which comprises active ingredients within a safe and effective amount range and pharmaceutically acceptable excipients.

[0099] The "active ingredient" described in the present invention refers to the compound of formula I described in the present invention, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its prodrug molecule, or its deuteride, or its tritiated product.

[0100] "Safe and effective amount" means that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.

[0101] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage at the time of administration is a pharmaceutically effective dosage. The specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skills of a skilled physician.

[0102] "Pharmaceutically acceptable excipients" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must be sufficiently pure and sufficiently low in toxicity.

[0103] "Compatibility" herein means that the components in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

[0104] Examples of pharmaceutically acceptable 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0105] The following are specific embodiments.

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

[0107]

[0108] Add NH 2 -OTHP (117.2 mg, 1.5 mmol, 1.5 equiv), 4-ethylbenzoic acid (150 mg, 1.0 mmol, 1.0 equiv), EDCI (230 mg, 1.2 mmol, 1.2 equiv), HOBt·H 2 O (162 mg, 1.2 mmol) and DMF (3 mL). Stir at room temperature for 2 hours and monitor the reaction process by thin layer chromatography. After the reaction is completed, the reaction mixture is poured into water. The reaction mixture is extracted with AcOEt. The organic layer is separated and washed with saturated NaHCO 3 The organic layer was washed with saturated brine, and Na 2 SO 4 Dry. Purify by silica gel flash column chromatography (ethyl acetate / petroleum ether=15 / 1000) to obtain S1 as a colorless viscous liquid. S1 is used directly in the next step without purification.

[0109] TsOH·H 2 O (16.9 mg, 0.0891 mmol) was added to a solution of S1 (0.1 equivalent) 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-d 6 )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). 13C NMR (151 MHz, DMSO-d 6 )δ163.86,133.33,132.20,127.63,124.85,83.30,83.23.ESI-MS calcd for C 9 H 7 NO 2 [M+H] + m / z=162.0550; Found162.0551.

[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 equivalent), (Boc 2 O (8 g, 36.6 mmol, 1.0 equiv) was 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-d 6 )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).

[0114] Tert-butyl (4-(prop-2-yn-1-yloxy)phenyl)carbamate (S3): S2 (7.3 g, 34.9 mmol, 1.0 equivalent), K 2 CO 3 (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 the reaction was completed by TLC monitoring, the mixture was added with water and extracted with ethyl acetate. All organic layers were combined, washed with water and saturated brine, and washed with Na 2 SO 4 The crude product was dried 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-d 6)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).

[0115] 4-(Propan-2-yn-1-yloxy)aniline (S4): Add S3 to CH 2 Cl 2 Slowly drip trifluoroacetic acid into the CH 2 Cl 2 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 a crude product S4 as a brown solid, which was used directly in the next step without purification.

[0116] Preparation of methyl 3-((4-(prop-2-yn-1-yloxy)phenyl)amino)propanoate (S5): Tetrahydrofuran was added to an eggplant-shaped bottle equipped with 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 eq) was added to the reaction mixture, which was then heated to reflux. After the reaction monitored by TLC was completed, the reaction mixture was filtered. The filtrate was evaporated under reduced pressure and purified by flash chromatography to give S5 as a tan solid. 1 H NMR (400 MHz, DMSO-d 6 )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).

[0117] N-Hydroxy-3-((4-(prop-2-yn-1-yloxy)phenyl)amino)propanamide (M2): A solution of hydroxylamine hydrochloride (120 mg, 0.86 mmol, 4.0 equiv) in anhydrous methanol (2 mL) was added to a solution of NaOH (68.8 mg, 0.86 mmol, 4.0 equiv) in anhydrous methanol (2 mL) at 0°C, 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 equiv) in anhydrous methanol (2 mL). Stir at room temperature for 2 hours and monitor the completion of the reaction by thin layer chromatography. After the reaction was completed, 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 (400 MHz, DMSO-d6 )δ10.46(s,1H), 8.75(s,1H), 6.79(d,J=8.6Hz,2H), 6.57(d,J=8.4Hz,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 (151 MHz, DMSO-d 6 )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 H14N 2 O 3 [M+H] + m / z=235.1082; Found 235.1083.

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

[0119]

[0120] The synthesis method is the same as the last step of the synthesis of M2. M3 is a light orange-red solid (yield 87%).

[0121] 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6 )δ164.25,140.19,136.37,132.41,127.67,126.53,116.63.ESI-MS calcd for C 9 H 9 NO 2 [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): HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylammonium bromide hexafluorobromide) (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 of DMF were added to an eggplant-shaped flask. After stirring the reaction mixture for 10 minutes, 2-(p-tolyl)ethan-1-amine (204.8 mg, 1.5 mmol, 1.0 equiv) was added to the reaction solution and stirred overnight. 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 S6 as a light yellow viscous liquid (yield 48%). 1 HNMR (400 MHz, DMSO-d 6 )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).

[0125] N1-Hydroxy-N3-(4-methylphenethyl)isophthalamide (M4): The synthesis method is the same as the last step of the synthesis of M2. The product M4 is a light yellow solid (yield 32.7%).

[0126] 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6)δ166.07,164.21,136.83,135.48,135.30,133.45,130.10,129.67,129.40,129.00,128.90,126.47,41.52,35.11,21.11.ESI-MS calcd for C 17 H 18 N 2 O 3 [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.) and CDI (705.5 mg, 4.35 mmol, 1.5 eq.) were added to THF (10 mL) and stirred at room temperature for 1 h. NH 2 OH·HCl (403 mg, 5.8 mmol, 2.0 equiv) was 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. M6 was purified by flash column chromatography (EA / PE=20 / 100) to obtain M6 as a white powder (447 mg, yield 82%).

[0130] 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6 )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 H 9 NO 2 [M+H] + m / z=188.0711; Found188.0716.

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

[0132]

[0133] The synthesis method of M7 is similar to that of M1. M7 is a pink-orange solid with a yield of 77%.

[0134] 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6 )δ160.01,137.80,130.79,128.35,127.89.ESI-MS calcd for C 5 H 5 N 2 O 2 S[M+H] + m / z=144.0114; Found 144.0114.

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

[0136]

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

[0138] 1 H NMR (400 MHz, DMSO-d 6 )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 C 7 H 5 F 3 N 2 O 2 [M+H] + m / z=207.0377; Found207.0376.

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

[0140]

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

[0142] 1 H NMR (400 MHz, DMSO-d 6 )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 NO 4 [M+H] + m / z=296.0923; Found 296.0917.

[0143] Example 9 Synthesis of 2-benzyl-N-hydroxy-1,3-dioxoisoindoline-5-carboxamide (M10)

[0144]

[0145] The synthesis method of M10 is similar to that of M1. M10 is a white solid with a yield of 45%.

[0146] 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6 )δ167.64,167.59,162.67,138.65,136.96,133.78,132.36,129.07,128.27,1 27.93,127.86,127.73,124.95,123.99,121.67,119.58,110.10,41.54.ESI-MS calcd for C 16 H 132 N2 O 4 [M+H] + m / z=297.0877; Found297.0868.

[0147] Example 10 Synthesis of N-hydroxy-2'-methoxy-3'-nitro-[1,1'-biphenyl]-3-carboxamide (M11)

[0148]

[0149] The synthesis method of M11 is similar to that of M1. M11 is a light yellow solid with a yield of 51.6%.

[0150] 1 H NMR (400 MHz, DMSO-d 6 )δ11.31(s,1H),9.11(s,1H),7.97–7.90(m,2H),7.83(dt,J=7.8,1.5Hz,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 (151 MHz, DMSO-d 6 )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 N 2 O 4 [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-d 6)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 (151 MHz, DMSO-d 6 )δ160.68,147.79,145.53,143.89,143.69.ESI-MS calcd for C 5 H 5 N 3 O 2 [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-d 6 )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 N 2 O 2 [M+H] + m / z=209.1290; Found 140.0455

[0159] Example 13 Synthesis of N-hydroxy-4-(pyrrolidin-1-yl)benzamide (M14)

[0160]

[0161] The synthesis method of M14 is similar to that of M1. M14 is a light yellow solid with a yield of 58.3%.

[0162] 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-d 6 )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 N 2 O 2 [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 method of M15 is the same as that of M2. M15 is a brown solid with a yield of 52%.

[0166] 1 H NMR (400 MHz, DMSO-d 6 )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). 13 C NMR (151 MHz, DMSO-d 6 )δ164.68,147.48,144.59,143.85,138.03,136.45,131.79,129.93,128.7 8,128.10,127.47,127.36,126.75,122.23,113.94,105.32,46.30.ESI-MS calcd for C17 H 18 N 3 O 2 [M+H] + m / z=294.1243,Found 294.1217.

[0167] Example 15 Synthesis of N-hydroxy-4-((imidazo[1,2-a]pyridin-8-ylamino)methyl)benzamide (M16)

[0168]

[0169] The synthesis method of M16 is the same as that of M2. M16 is a yellow solid with a yield of 63%.

[0170] 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6 )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 N 4 O 2 [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 method of M17 is similar to that of M2. M17 is a yellow solid with a yield of 41%.

[0174] 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6 )δ167.69,145.59,143.59,138.91,136.97,131.78,131.18,131.11,129.90,129.74,129.72,127. 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 N 2 O 3 [M+H] + m / z=409.1552,Found 409.1557.

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

[0176] The synthesis method of M18 is similar to that of M2. M18 is a yellow solid with a yield of 45%.

[0177] 1 H NMR (400 MHz, DMSO-d 6)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 (151 MHz, DMSO-d 6 )δ166.61,144.70,143.01,142.91,131.54,129.84,128.58,127.75,127.57, 127.45,127.31,127.21,126.75,118.00,113.61,66.76,50.92,47.21.ESI-MS calcd forC 18 H 21 N 3 O 3 [M+H] + m / z=329.1661,Found 329.1656.

[0178] Example 18 Synthesis of 4-((((1H-benzimidazol-2-yl)methyl)amino)methyl)-N-hydroxybenzamide (M19)

[0179]

[0180] The synthesis method of M19 is similar to that of M2. M19 is a yellow solid with a yield of 41%. 1 H NMR (400 MHz, DMSO-d 6 )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 (151 MHz, DMSO-d 6 )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 N 4 O2 [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-hydroxypentanamide (EH1)

[0182]

[0183] The synthesis method of EH1 is similar to that of M2. EH1 is a white solid with a yield of 56%.

[0184] 1 H NMR (400 MHz, DMSO-d 6 )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,1 H),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 (151 MHz, DMSO-d 6 )δ174.84,169.41,156.90,155.21,152.53,148.93,139.96,129.36,127.22,125.71,123.5 3,122.20,109.18,106.60,103.47,83.93,81.12,69.05,56.43,32.35,28.50,22.38.ESI-MS calcd for C 22 H 22 N 4 O 4 [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] The synthesis method of EH1 is similar to that of M2. It is a white solid with a yield of 59%.

[0188] 1 H 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).

[0189] 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)

[0190]

[0191] The synthesis method of XH1 is similar to that of M2. It is a yellow solid with a yield of 31%.

[0192] 1 H NMR (400 MHz, DMSO-d 6 )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 N 9 O 4 [M+H]+ m / z=546.2577, Found 546.2572.

[0193] Example 22 Labeling of pure protein after oxidation induction by probe compound M1

[0194] The probe M1 was prepared into a 1 mM probe stock solution with DMSO, and NaIO 4 Prepared as a 2 mM oxidant stock solution. Prepared as a 0.5 mg / mL solution of pure bovine serum albumin (BSA) protein using PBS.

[0195] Oxidation solution A (premix): Mix the M1 probe mother solution and the oxidant mother solution. After reacting for 2 hours, take 2 μL of DMSO or 2 μL of the reaction solution and add it to the BSA pure protein solution. Incubate at 37°C in a constant temperature mixer for 2 hours.

[0196] Oxidation solution B (incubation before oxidation): 1 μL DMSO or 1 μL M1 probe stock solution was added to the pure protein solution and incubated for 2 hours, followed by the addition of 1 μL NaIO 4 The mother solution was placed in a thermomixer and incubated at 37°C for 2 hours.

[0197] Click chemistry reagents (10 mM TBTA, 100 mM TCEP, 100 mM CuSO 4 and 10 mM TAMRA-N 3 The mixed samples were placed in a rotating mixer at room temperature for 2 hours. After adding 6 μL of 5×SDS loading buffer, 15 μL of the sample solution was taken to separate the protein by SDS-PAGE polyacrylamide gel electrophoresis. After 30 minutes of electrophoresis at 90V, it was adjusted to 110V for 60 minutes. Finally, the biopolymer interaction imaging instrument was used to scan and image.

[0198] The results are as follows Figure 1 As shown in Figure 1, in the oxidation scheme A, after the probe M1 was pre-mixed with the oxidant and then added to the BSA pure protein, the probe M1 did not produce an obvious labeled band. 4In the oxidation-induced oxidation scheme B, the M1 probe can have a significant labeling effect on BSA pure protein, indicating that the hydroxamic acid molecules can only react with the amino acid residues of the protein after being induced by oxidation, and the hydroxamic acid molecules are first incubated with the protein for a period of time, and then close to the amino acid residues of the protein, and then induced by the oxidant to achieve covalent connection with the amino acid residues. It is speculated that the reason for this phenomenon is that the nitrosocarbonyl intermediate generated after the oxidation of hydroxamic acid has the characteristics of short life and high activity. If there are no amino acid residues nearby that react with the nitrosocarbonyl intermediate when it is generated, it will immediately react with the surrounding water molecules and become inactivated, and can no longer react with the amino acid residues.

[0199] Example 23 Effect of oxidants on labeling effect

[0200] In this example, five oxidants were selected: tert-butyl hypochlorite (BuOCl), H 2 O 2 , NaIO 4 , [Bis(trifluoroacetoxy)iodo]benzene (PIFA), chloramine T, and the oxidation scheme B described in Example 22 were used to induce oxidation of probe M1 and react with BSA pure protein, and the oxidation effect was visualized by fluorescence imaging.

[0201] The results are as follows Figure 2 As shown in Figure 2, under the same concentration (final concentration of oxidant is 200 μM, final concentration of M1 is 100 μM) and oxidation time, NaIO 4 and chloramine T had a better oxidation effect on probe M1, among which NaIO 4 The effect is best, and the labeling effect of BSA pure protein is the best.

[0202] Example 24 Effect of Hydroxamic Acid Structure on Labeling Effect

[0203] This example compares the difference in the labeling effect of M1 and M2 on pure protein after oxidation induction. The same concentration (final concentration of 100 μM) of probes M1 and M2 were added to the BSA pure protein solution and incubated for 2 h. Then, NaIO2, which is twice the concentration of the probes, was added. 4 After 2 h of oxidation incubation, click reagent was added and fluorescence imaging was performed after polyacrylamide gel electrophoresis. The detailed method was the same as in Example 22.

[0204] The results are as follows Figure 3As shown in the figure, before oxidation induction, both probes M1 and M2 could not produce labeling bands. After oxidation induction, only probe M1 produced a strong labeling band, while probe M2 did not produce an obvious labeling band. It can be seen that the specific structure of the hydroxamic acid molecule has a great influence on the labeling effect.

[0205] Example 25: Protein labeling of cell lysate after oxidation induction by compound M1

[0206] Prepare cell lysates from 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:

[0207] Discard the culture medium of MDA-MB-231 cells grown to 90% in a 10 cm culture dish, add 2 mL PBS to wash the cells against the wall of the culture dish, and repeat the washing twice. Then add 1 mL of trypsin to digest the cells until the cells are spherical under the microscope and no longer adhere to each other, and then add 1 mL of serum-containing culture medium to quench the digestion reaction. Collect the cell fluid in a 5 mL sample tube, centrifuge at 1000 rpm for 3 min to precipitate the cells, discard the supernatant, add 1 mL PBS and shake gently to resuspend the cells in PBS, and transfer the cell fluid to a 1.5 mL sample tube. Centrifuge at 1000 rpm for 3 min to precipitate the cells, discard the supernatant, add 1 mL PBS and shake gently to resuspend the cells in PBS, place the sample tube in an ice box, and ultrasonically disrupt the cells in a cell ultrasonic disruptor, setting the parameters to 3s on, 3s off, 15% power, and 3 min ultrasonic duration. After the ultrasonication, the sample was centrifuged at 4°C and 14,000 rpm for 15 min in a high-speed centrifuge. The obtained supernatant was the cell lysate, and the bottom precipitate was discarded.

[0208] Take 200 μL of cell lysate, add 2 μL of DMSO or probe M1 (final concentration 100 μM) and react at 37°C for 2 h, then use NaIO 4 (final concentration 500 μM) was reacted at 37°C for 4 h. After filtration through an ultrafiltration membrane spin column, TAMRA-N 3 , TBTA, TCEP, CuSO 4(The final concentrations were 100μM, 100μM, 1mM, and 1mM, respectively) and placed in a constant temperature mixer at 37°C for 2h to allow the protein-probe complex to connect to the fluorescent group TAMRA-N3 to complete the click reaction. Add 1mL of pre-cooled methanol and place the sample at -20°C for more than 30min (or overnight) to allow the protein to precipitate. Then place the sample in a high-speed centrifuge at 4°C, centrifuge at 14000rpm for 15min, discard the supernatant, and turn the sample tube upside down to dry the protein. Then add 40μL of 1×SDS loading buffer, place the sample in an ultrasonic cleaner for several minutes to promote protein dissolution, and then heat at 95°C for 15min. After high-speed centrifugation at 14000rpm for 30s, take 20μL of sample solution and add it to the sample channel of gel electrophoresis (SDS-PAGE, 10% separation gel), and after 90V constant voltage electrophoresis for 30min, adjust to 110V constant voltage electrophoresis for 60min. Finally, the images were scanned using a multifunctional biomolecular imager.

[0209] The results are as follows Figure 4 As shown: Compound M1 has a significant labeling effect on the cell lysates of MDA-MB-231, Bxpc-3, Daudi, A549 and AGS cells after oxidative induction.

[0210] Example 26 Protein Identification Analysis of Compound M1 on MDA-MB-231 Cell Lysate

[0211] 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) at 37° C. for 2 h, and then NaIO 4 (final concentration 500 μM) at 37°C for 4 h. 3 , TBTA, sodium ascorbate, CuSO 4 (The final concentrations were 100 μM, 100 μM, 1 mM, and 1 mM, respectively) and the reaction was allowed to proceed after gentle shaking. Pre-cooled methanol was added and incubated at -20°C for 2 hours. The precipitated protein was then collected, centrifuged and dissolved. After incubation with streptavidin beads at room temperature for 2 hours, it was washed with 1% SDS and PBS. The beads were suspended in 500 μL 6M urea PBS, and 25 μL 200mM DTT and 25 μL 25mM NH 4 HCO 3 , incubate at 37℃ for 30min. Add 25μL 500 mM IAA and incubate at room temperature in the dark for 30min. Then remove the supernatant and wash the beads with 1ml PB. 150μL 2M urea in 50mM NH 4 HCO 3 Add 1 mM CaCl 2and 1 μL of trypsin (1 μg / μL), incubated at 37°C overnight, desalted on a C18 column, and analyzed by LC-MS / MS.

[0212] The knot is like this Figure 5 As shown: Probe M1 binds to a total of 43 high-confidence target proteins. Among the 43 high-confidence proteins bound by probe M1, three proteins belonging to the eukaryotic mitochondrial porin family were hit, namely voltage-dependent anion-selective channel protein 1 (VDAC1), voltage-dependent anion-selective channel protein 2 (VDAC2), and voltage-dependent anion-selective channel protein 3 (VDAC3). VDACs play a central role in cell lifespan and apoptosis. VDACs are expressed more in cancer cells than in normal cells, so they can be used as targets for cancer chemotherapy.

[0213] Example 27 Target Validation by Pull-Down / Western Blotting

[0214] In order to confirm the target protein bound by probe M1, this example selected VDAC1, BIP (Endoplasmicreticulum chaperone BIP, endoplasmic reticulum chaperone protein), and PLK1 (Serine / threonine-protein kinase PLK1, serine-threonine protein kinase PLK1) for Pull-down / WB experimental verification.

[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) at 37° C. for 2 h, and then NaIO 4 (final concentration 500 μM) was reacted at 37°C for 4 h. After filtration through an ultrafiltration membrane spin column, fresh premixed click chemistry mixture (100 μM biotin-N 3 from a 10 mM DMSO stock solution, 100 μM TBTA from a 10 mM freshly prepared DMSO stock solution, 2 mM sodium ascorbate from a 200 mM freshly prepared deionized water stock solution, 1 mM CuSO 4From a freshly prepared 100 mM stock solution in deionized water). The reaction was further incubated with gentle mixing for 2 h before precipitation by adding pre-cooled methanol (-20 °C). The precipitated protein was subsequently collected by centrifugation (14000 rpm × 10 min, 4 °C) and dissolved in PBS containing 1% SDS. After incubation with streptavidin beads for 4 h at room temperature, the beads were washed with PBS containing 1% SDS (3 × 1 mL) and PBS (3 × 1 mL), respectively. 50 μL (2×) SDS loading buffer was added and heated at 95 °C for 30 min. The samples were separated by SDS-PAGE (12% gel) and the target protein was verified by standard western blotting methods.

[0216] like Figure 6 As shown, probe M1 was treated with NaIO 4 After oxidation induction, VDAC1, BIP, and PLK1 in MDA-MB-231 cell lysate were successfully pulled down without NaIO 4 The oxidation-induced probe M1 did not produce labeling pull-down. This indicates that VDAC1, BIP, and PLK1 are indeed the target proteins of probe M1, and that M1 can bind to the target proteins through the oxidation-induced strategy, and that the oxidation-induced strategy can successfully label proteins at the lysate level.

[0217] Example 28 Analysis of modification sites 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 wall of the culture dish to wash the cells, and repeat the washing twice. Collect the cells by centrifugation at 1000 rpm for 3 min, add 1 mL of PBS and shake gently to resuspend the cells in PBS, place the sample tube in an ice box, and ultrasonically disrupt the cells in a cell ultrasonic disruptor. Set the parameters to 3s on, 3s off, 15% power, and 3 minutes of ultrasonication. After the ultrasonication is completed, the sample is centrifuged at 4°C, 14000 rpm for 15 min in a high-speed centrifuge, and the resulting supernatant is the cell lysate.

[0219] (2) Probe and oxidation incubation: The prepared cell lysate was divided into two equal parts, 500 μL each, and 5 μL of 10 mM probe M1 was added to each part. The mixture was incubated at 37°C for 2 h in a thermostatic homogenizer. 5 μL of 50 mM oxidant NaIO 4 Solution was oxidized and incubated 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 a dark place for 30 min.

[0221] (4) Protein precipitation and trypsin digestion: Add 540 μL MeOH and 135 μL CH 3 Cl, mix well and let stand at -20℃ for 30min. Centrifuge at 1700g for 20min, discard the supernatant, and place the sample tube in a 37℃ constant temperature homogenizer for a few minutes to dry the protein. Add 400μL (NH 4 ) 2 CO 3 Solution, place the sample in an ice box, ultrasonicate for 6-15s until the protein is fully suspended in the solution, add 5μL pancreatin, and incubate in a constant temperature mixer at 37℃ for 12h. Add 2μL pancreatin and continue incubating in a constant temperature mixer at 37℃ for 2h. Centrifuge at 14000g for 5min, and dry the supernatant in a high temperature vacuum dryer to obtain 200μL polypeptide sample.

[0222] (5) Desalting: HLB SPE column was passed through 1 mL HPLC-acetonitrile, 2 mL HPLC-H 2 O activation, add 200 μL of peptide sample and precipitate with 1 mL HPLC-H 2 O desalting, and finally adding HLB-A reagent to elute the peptide. The peptide eluate was dried in a vacuum centrifuge at 45°C until complete.

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

[0224] (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. Repeat twice to wash the beads. The combined sample and washed beads were added to 10 mL of streptavidin binding buffer and rotated for enrichment at room temperature in the dark for 2 h.

[0225] (8) Washing 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, gently invert the sample for 5 min, centrifuge again, and repeat the above steps twice. Add 1.2 mL of photorelease buffer, gently shake to disperse the beads in the solution, place the sample tube under 365 nm ultraviolet light for 2 h; centrifuge at 2000g for 4 min; concentrate the supernatant to 200 μL using a vacuum centrifuge.

[0226] (9) Desalting and sample delivery: HLB SPE column was passed through 1 mL HPLC-acetonitrile, 2 mL HPLC- 2 O activation, add 200 μL of peptide sample and precipitate with 1 mL HPLC-H 2 O desalting, and finally adding HLB-A reagent to elute the peptide. The peptide eluate was dried completely in a vacuum centrifuge at 45°C. 12μL 0.1% formic acid was added to the dried peptide, and the peptide was dissolved by ultrasound for several minutes. After high-speed centrifugation at 14000g for 15 minutes, 10μL of the supernatant was taken for LC-MS / MS testing.

[0227] The results are as follows Figure 7 , Figure 8 As shown in Tables 1 and 2: Heat map of binding sites of probe M1 in HEK293T cell lysate ( Figure 7 A) in Figure 2 shows that probe M1 only modifies K, and the selectivity is between 0.8 and 1, indicating that probe M1 is highly selective for binding to lysine residues. Venn diagram of probe M1 binding site selectivity and analytical effectiveness ( Figure 7 B) in the figure shows that the selectivity (Residue selectivity) and profiling efficiency (Profiling efficiency) of probe M1 to lysine are both over 90%. The high profiling efficiency also indirectly indicates the high accuracy of the analysis data.

[0228] In addition, the proteomics analysis software pFind was used to analyze the proteins and specific sites bound by probe M1. The results showed that probe M1 hit a total of 7241 lysine residues ( Figure 8 ). Most of the functions are not annotated. The remaining 662 lysines are annotated as functional lysines. Most of these functional lysines are post-translationally modified lysines. About 7% of the sites are binding sites. These functional binding sites cover ATP binding sites, GTP binding sites, NADP + Binding site, CoA binding site, Mn 2+Binding sites, 3',5'-cyclicAMP binding sites (Table 1), indicating that M1 can enter the pockets of various proteins, widely enter the binding pockets of various proteins and bind to the active sites. Compared with the more than 3000 lysines identified by the 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 supplementary effect on the identification of lysine in the proteome.

[0229] M1 identified a total of 2,499 different proteins, 101 of which were included in the Drugbank library of drug targets, while most of the proteins identified by M1 were not in Drugbank, showing a wider distribution of categories than the smaller portion 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 proteins.

[0231] In summary, the simple structure of M1 enables it to easily enter the binding pockets of various proteins and bind to difficult-to-target proteins, identifying their unstudied lysine residues, which is conducive to a deeper understanding of protein conserved lysines and the development of covalent inhibitors targeting lysines.

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

[0233]

[0234] Table 2 Lysine sites hit by M1 in HEK293T cell lysate

[0235]

[0236] Example 29 Site Verification by Site Mutation and Pull-Down / Western Blotting

[0237] The wild-type / mutant protein was expressed in HEK293T cells, and cell lysate was prepared according to the method of Example 28. The cell lysate was treated with probe M1 (final concentration 100 μM) and reacted at 37° C. for 2 h. Then, NaIO 4 (final concentration 500 μM) was reacted at 37°C for 4 h. After filtration through an ultrafiltration membrane spin column, fresh premixed click chemistry mixture (100 μM biotin-N3 , 100 μM TBTA, 2 mM sodium ascorbate, 1 mM CuSO 4 ). The reaction was further incubated for 2 h with gentle mixing before precipitation by adding pre-cooled methanol (-20°C). 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 for 4 h at room temperature, 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 heated at 95°C for 30 min. The samples were separated by SDS-PAGE and the target protein was verified by standard protein blotting methods.

[0238] The results are as follows Fig. 9 As shown, after PGK1_K131, PGK_K323 and CDK5_K33 mutated to arginine, the labeling of the corresponding proteins by probe M1 was significantly reduced, indicating that the nitrosocarbonyl intermediates produced by the oxidation induction of probe M1 can effectively label the lysine residues in the entire 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 μM probe EH1 or EH2 for 2 hours, 5× NaIO 4 After 4 hours of oxidation, the oxidant was filtered out and the click reaction was performed, followed by fluorescence scanning imaging.

[0241] The results are as follows Fig.10 As shown: Of 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 identifies the target protein labeled with probe EH1. The experimental method is referenced 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] Pull-down / WB Experiment Results( Fig.11 ) shows: Without adding NaIO 4 When oxidatively induced, EH1 had no significant labeling effect on EGFR; however, after NaIO 4 After oxidation, EH1 can successfully label and pull down EGFR. This indicates that after oxidation induction, EH1 can covalently bind to and pull down EGFR.

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

[0247]

[0248]

[0249] Example 32 Test of the inhibitory activity of probe EH1 / EH2 on EGFR kinase

[0250] Using Z'-LYTE TM The inhibitory effect of the probe on EGFR kinase was tested by fluorescence resonance energy transfer (FRET) method, and the parent inhibitor was used as a control compound. TM The biochemical assay uses a FRET-based coupled enzyme format based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores - one at each end - to form a FRET pair. Compounds are expressed in DMSO from 5.1 x 10 -9 M was diluted three-fold to 1x 10 -4 M. Plates were measured on an EnVision Multilabel Reader (Perkin Elmer). Curve fitting and data presentation were performed using Graph PadPrism version 4.0.

[0251] The results are as follows Fig.12 As shown: the inhibitory effect of probe EH1 on EGFR (L858R) mutant (IC50≈2.3nM) is better than Erlotinib (IC50≈3.9nM); the inhibitory effect of probe EH2 on EGFR (L858R) mutant (IC50≈3.3nM) is comparable to Erlotinib. This indicates that the hydroxamic acid group can retain the inhibitory activity of the parent compound on EGFR kinase after being inserted into the Erlotinib core, and because EH1 has a better covalent binding effect on EGFR kinase, it can further improve 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] EH1 identified AURKA in H3255 lysate. This example verifies the binding of EH1 to AURKA. According to the method of Scheme B in Example 22, 1 μM and 10 μM EH1 (5× NaIO 4 ) performed labeling experiments on AURKA protein.

[0254] result( Fig.13 ) showed that EH1 could label AURKA protein at a concentration as low as 1 μM, and produced strong labeling 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 site of AURKA, this example used 100 μM EH1 (5× NaIO 4 ) and AURKA purified protein were used for site identification.

[0256] result( Fig.14 ) showed that EH1 binds to K258 of AURKA. In AURKA, D256 is annotated as the proton receptor active site, E260 and N261 are annotated as ATP binding sites, and K258 identified by EH1 is located in the active pocket formed by AURKA_D256-N261, indicating that EH1 can covalently bind to lysine 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 structural modification of the broad-spectrum kinase ligand XO44. This example verifies the protein identification experiment of XH1 in cell lysate according to the aforementioned method.

[0259] The probe XH1 (100 μM) was incubated with K562, AGS, and MDA-MB-231 cell lysates for 2 hours, and the oxidant NaIO was filtered out after 4 hours of oxidation. 4 The click reaction was carried out for 2 hours, followed by fluorescence scanning imaging.

[0260] result( Fig.15 A) in the figure shows that XH1 can indeed produce labeling after oxidation.

[0261] Pull-down / LC-MS / MS experiment (probe concentration selected 100 μM) results ( Fig.15B) 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. In addition, XH1 identified 73 unique kinases, reflecting the uniqueness of XH1 at the kinase binding level, which can be used to expand our coverage of kinases.

[0262] Analysis of the identified proteins revealed that CDK family kinases such as CDK1, CDK2, CDK6, and CDK9 were detected, indicating that these probes have good labeling capabilities for CDK family kinases. CDK1 plays a key role in the eukaryotic cell cycle by regulating the centrosome cycle and the initiation of mitosis; CDK1 can also promote the G2-M transition, while regulating G1 progression and G1-S transition by binding to multiple interphase cell cycle proteins; abnormal CDKs can lead to cell proliferation, genomic and chromosomal instability, leading to the occurrence of human cancers, promoting cancer progression and invasiveness, so CDKs can be used as a new effective target for cancer treatment.

[0263] This example further uses pure protein labeling experiments to confirm the binding of XH1 to the target proteins AURKA and SRC, and uses Pull-down / WB experiments to further confirm that XH1 successfully binds to the target protein CDK1 ( Fig.16 ), these experimental results indicate that the oxidative induction strategy can be used to analyze kinases in the entire proteome after the hydroxamic acid group is embedded in the backbone of a broad-spectrum kinase inhibitor, thereby providing a global map of the kinome's ligandable catalytic lysines.

[0264] Example 34 Cell proliferation inhibition rate test

[0265] 8000-10000 cells were inoculated in each well of a 96-well plate (MDA-MB-231 inoculation density was 8000 / well, and Daudi and K562 cells were inoculated at a density of 10000 / well), and the cells were cultured in a 37°C incubator for 24 hours. Fragment compounds M3-M19 (final concentration 5 μM) were added to each well, and a duplicate well was set for each sample well. The last column was a cell-free blank control and incubated for 48 hours. 30 μL of CCK8 reagent was added to each well and incubated for another 4 hours. The 96-well plate was 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-A 0 ) / (As–A 0 )×100%, where A is the absorbance value of the experimental group (including cells and fragments), As is the absorbance value of the control group (including cells and DMSO), and A 0The absorbance value represents the blank group (group without cells). Finally, the cell growth inhibition rate was calculated and plotted using the software Graphpad Pris.

[0266] The results are as follows Fig.17 As shown: The proliferation inhibitory activity of the compound on Daudi cells is better than that on MDA-MB-231 and K562 cells.

[0267] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A hydroxamic acid compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, in, 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 heteroaryl ketone, or selected from the following groups: Each R1 is independently selected from the group consisting of 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 heterocyclic group, -C(=O)NR4, 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-; Each R3 is independently selected from the group consisting of hydrogen, ethynyl, vinyl, trifluoromethyl, C1-C6 alkoxy, nitro, and C1-C6 alkylamino; Each R4 is independently selected from: C1-C6 alkyl, C6-C 10 Aryl-substituted C1-C6 alkyl, tolyl-substituted C1-C6 alkyl; Each R5 is independently selected from: R6 substituted or unsubstituted 5-10 membered heteroaryl, R6 substituted or unsubstituted C6-C 10 Aryl; Each R6 is independently selected from: 5-10 membered heteroaryl, 5-8 membered heterocyclyl; n is selected from: 1, 2, 3, 4.

2. The hydroxamic acid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: R is selected from: 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-benzopyranyl, R1 substituted or unsubstituted 1,3-dioxoisoindolyl, R1 substituted or unsubstituted pyrazinyl.

3. The hydroxamic acid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1 or 2, characterized in that: Each R1 is 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; Each R2 is independently selected from: phenyl, R5NH-, R5CH2NH-; Each R3 is independently selected from the group consisting of hydrogen, ethynyl, vinyl, trifluoromethyl, methoxy, ethoxy, nitro, and diethylamino; Each R4 is independently selected from: C1-C3 alkyl, phenyl-substituted C1-C3 alkyl, tolyl-substituted C1-C3 alkyl; Each R5 is independently selected from the group consisting of quinolinyl, imidazo[1,2-a]pyridinyl, dibenzo[b,d]furanyl substituted phenyl, 4-morpholinylphenyl, and benzimidazolyl.

4. The hydroxamic acid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: R is selected from:

5. The hydroxamic acid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The hydroxamic acid compound is selected from:

6. Use of the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in selective covalent modification, covalent labeling, or covalent binding to lysine residues in proteins; Preferably, the protein is a pure protein, a protein in a cell lysate, or a protein in living cells.

7. Use of the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the recognition and identification of drug molecule targets.

8. Use of the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the discovery and analysis of drug targets.

9. Use of the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in identifying protein types, kinase types and / or ligandable lysine residues in tumor cells.

10. Use of the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in developing a covalent inhibitor targeting lysine.

11. Use of the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the preparation of a kinase inhibitor.

12. The use according to claim 11, characterized in that: The kinases are EGFR kinase, CDK family kinase, AURKA kinase, and PAK1 kinase.

13. Use of the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the preparation of an anti-tumor drug.

14. The use according to claim 13, characterized in that The tumor is breast cancer, lung cancer, leukemia, or lymphoma.

15. An anti-tumor drug, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the hydroxamic acid compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

16. A method for covalently labeling lysine residues in proteins or polypeptides, characterized in that: The steps include: (1) incubating the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, with pure protein, cell lysate, or living cells; (2) Add oxidant and incubate.

17. A method for identifying and verifying a drug molecule target, characterized in that: The steps include: (1) introducing a hydroxamic acid group into a drug molecule, and then incubating the drug molecule with a pure protein, a cell lysate, or living cells; or incubating the hydroxamic acid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, with a pure protein, a cell lysate, or living cells; (2) adding an oxidant and incubating; (3) Post-processing and analyzing the reaction solution after incubation.

18. The method according to claim 17, characterized in that The drug molecule is a drug molecule having kinase inhibitory activity and / or tumor cell proliferation inhibitory activity.

19. The method according to claim 16 or 17, characterized in that: The oxidant is chloramine-T and / or NaIO4; and / or, the living cells are tumor cells, and the cell lysate is a lysate of tumor cells.

20. The method according to claim 16 or 17, 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 its stereoisomer is 20 μM-500 μM, preferably 50 μM-200 μM; and / or, The incubation temperature in step (1) is 30°C-40°C, and the incubation time is 30min-5h, preferably 1h-2h; and / or, The incubation in step (2) comprises 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°C-40°C, and the incubation time is 30min-6h, preferably 2h-4h.

Images