EZH2 and HDAC dual-target inhibitors, pharmaceutical compositions thereof, preparation methods and uses thereof

Abstract

The present invention discloses an EZH2 and HDAC dual-target inhibitor, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same, and a preparation method and use thereof. The cyclic amide structure in the general formula compound structure of the present invention is the key pharmacophore for inhibiting the activity of EZH2, and the hydroxamic acid moiety can effectively inhibit the activity of HDAC, thereby achieving dual inhibition of EZH2 and HDAC, having stronger activity. It can be used for preventing and / or treating diseases related to the dual targets of EZH2 and HDAC, especially tumors, cancers and inflammatory bowel diseases, such as lymphoma, sarcoma, melanoma, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, thyroid cancer, laryngeal cancer, tongue cancer, head and neck cancer, multiple myeloma, B-cell lymphoma, leukemia, inflammatory bowel disease, and can play a synergistic role in the prevention and treatment of tumors, cancers and inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the field of drug synthesis, and particularly relates to an EZH2 and HDAC dual-target inhibitor or a pharmaceutically acceptable salt thereof, and a preparation method and use thereof. Background Art

[0002] Currently, in the field of EZH2 and HDAC dual-target inhibitors, there are only two articles reported. The first one is the first EZH2 and HDAC dual-target inhibitor reported in the ACS MedChem Lett magazine. Although the compound reported in this article has strong inhibitory activity against the two targets, its inhibitory activity against tumor cell proliferation is at a medium level and combination therapy is required. The other one is an article about EZH2 and HDAC dual-target inhibitors published in the Journal of Medicinal Chemistry. The lead compound reported in this work shows strong activity both at the enzyme level and the cell level, but this study did not disclose that the EZH2 and HDAC dual-target inhibitor can treat inflammatory bowel disease. Therefore, it is still of great significance to develop a new type of EZH2 and HDAC dual-target inhibitor in the present invention. Summary of the Invention

[0003] Based on the needs of the above-mentioned prior art, the present invention provides an EZH2 and HDAC dual-target inhibitor.

[0004] The first aspect of the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0005]

[0006] Wherein:

[0007] R1 is an oxygen-containing heterocyclic alkyl, cycloalkyl or L-C(O)-, wherein L is an alkyl, cycloalkyl or aryl;

[0008] R2 is an alkyl or an alkoxy;

[0009] X can be an aryl(alkoxy), (aryl)(amido)alkyl, (alkynyl)alkyl, (aryl)(alkoxy)aryl, (aryl)(alkoxy)(aryl)alkenyl, aryl or aryl(alkenyl).

[0010] Preferably, when R1 is an oxygen-containing heterocyclic group, it is preferably 4-tetrahydropyranyl or 3-tetrahydrofuranyl.

[0011] Preferably, when R1 is L-C(O)-, L in the L-C(O)- is C 1-6 alkyl or C 3-8 cycloalkyl.

[0012] Preferably, R2 is C1-6 alkyl or C 1-6 alkoxy group.

[0013] Preferably, the R2 is methyl or methoxy group.

[0014] Preferably, the X is selected from C 6-10 aryl(C 1-8 alkoxy), (C 6-10 aryl)(amido group)C 1-8 alkyl, (alkynyl)C 1-6 alkyl, (C 6-10 aryl)(C 1-3 alkoxy)C 6-10 aryl, (C 6-10 aryl)(C 1-3 alkoxy)(C 6-10 aryl)alkenyl, C 6-10 aryl or C 6-10 aryl(alkenyl).

[0015] In some embodiments of the present invention, the compound in the compound or its pharmaceutically acceptable salt is selected from Table 1.

[0016] The second aspect of the present invention provides a pharmaceutical composition, which comprises an effective amount of the above compound or its pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

[0017] The third aspect of the present invention provides the use of the above compound or its pharmaceutically acceptable salt, or the above pharmaceutical composition in the preparation of a dual-target inhibitor of EZH2 and HDAC.

[0018] The fourth aspect of the present invention provides the above compound or its pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 6 for preventing and / or treating tumors and / or cancers and / or inflammatory bowel diseases.

[0019] The fourth aspect of the present invention provides the dual-target inhibitor of EZH2 and HDAC for preventing and / or treating tumors and / or cancers and / or inflammatory bowel diseases, especially cancers. Preferably, the cancers include lymphoma, sarcoma, melanoma, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, thyroid cancer, laryngeal cancer, tongue cancer, head and neck cancer, multiple myeloma, B-cell lymphoma, leukemia, inflammatory bowel diseases.

[0020] The beneficial effects of the present invention at least include:

[0021] It has been reported in the literature that the simultaneous use of EZH2 inhibitors and HDAC inhibitors has synergistic anti-tumor activity, which provides a theoretical basis for the development of dual-target drugs for EZH2 and HDAC. The cyclic amide structure in the structure of the dual-target inhibitor of EZH2 and HDAC of the present invention is the key pharmacophore for inhibiting the activity of EZH2, and the hydroxamic acid moiety can effectively inhibit the activity of HDAC, thereby achieving dual inhibition of EZH2 and HDAC, with stronger activity. It can be used for the prevention and / or treatment of diseases related to the dual targets of EZH2 and HDAC, especially tumors and / or cancers and / or inflammatory bowel diseases, such as lymphoma, sarcoma, melanoma, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, thyroid cancer, laryngeal cancer, tongue cancer, head and neck cancer, multiple myeloma, B-cell lymphoma, leukemia, inflammatory bowel diseases, and can play a synergistic role in the prevention and treatment of tumors, cancers and inflammatory bowel diseases.

[0022] The features and advantages of the present invention will be described in detail in the following specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1(a) and 1(b) show the inhibition rate results of 132 representative compounds in the present invention on EZH2 at 100 nM.

[0024] Figures 2(a) and 2(b) show the inhibition rates of 132 representative compounds in the present invention on HDAC mixed enzymes (Hela cell nuclear extract) at 200 nM.

[0025] Figure 3 It is a Western blot test result diagram of EH-19.

[0026] Figure 4 It shows that EH-19 can effectively inhibit ulcerative colitis in mice and significantly extend the colon length of mice.

[0027] Figure 5 It is a bar chart showing that EH-19 can effectively inhibit ulcerative colitis in mice and significantly extend the colon length of mice. DETAILED DESCRIPTION OF THE INVENTION

[0028] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and specific implementation manners. The examples of the embodiments are shown in the drawings. It should be understood that the specific examples described in the following embodiments of the present invention are only illustrative descriptions of the specific implementation manners of the present invention, and are intended to explain the present invention, rather than limiting the present invention.

[0029] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein. In the description of this application, unless otherwise specified, the meanings of terms such as "a" and "plural" are two or more.

[0030]

Term Explanation

[0031] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0032] As used herein, when referring to a specific recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value.

[0033] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed.

[0034] As used herein, definitions of standard chemical terms (such as groups) can be found in the references in this field.

[0035] Unless otherwise specified, conventional methods within the scope of this field are adopted, such as mass spectrometry, NMR, IR, and UV / VIS spectrometry and pharmacological methods. Unless specifically defined, the terms used in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceuticals and medicinal chemistry herein are known in this field. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and the treatment of patients. For example, the instructions of the manufacturer for the use of the kit can be utilized, or the reactions can be carried out and purified in a manner well-known in this field or as described in the present invention. Generally, the above-mentioned techniques and methods can be implemented according to the descriptions in multiple general and more specific documents cited and discussed in this specification, in a conventional method well-known in this field. In this specification, groups and their substituents can be selected by those of ordinary skill in the art to provide stable structural moieties and compounds.

[0036] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left.

[0037] The section headings used herein are for the purpose of organizing the article only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals of operations, and theses, are hereby incorporated by reference in their entirety.

[0038] Certain chemical groups defined herein are preceded by a simplified symbol indicating the total number of carbon atoms present in the group. For example, C 1-6 Alkyl refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms. The total number of carbon atoms in the simplified symbol does not include the carbon present in substituents that may be present on the group.

[0039] Except as otherwise noted, when used in the specification and claims of this application, the following terms have the meanings set forth below, unless otherwise specifically indicated.

[0040] As used herein, the terms "compounds of the invention" or "active ingredients of the invention" are used interchangeably and refer to stereoisomers, enantiomers, or pharmaceutically acceptable salts of the general formula compounds. The term also includes racemates, optical isomers, isotopically labeled compounds (such as deuterated compounds), or prodrugs.

[0041] "Stereoisomers" are compounds that are composed of the same atoms, bonded by the same bonds, but have different three-dimensional structures. The present invention will cover various stereoisomers and their mixtures.

[0042] When the compounds of the invention contain an olefinic double bond, unless otherwise stated, the compounds of the invention are intended to include both E- and Z-geometric isomers.

[0043] "Tautomers" are isomers formed by the transfer of a proton from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the invention are also included within the scope of the present invention.

[0044] The compounds of the invention or their pharmaceutically acceptable salts may contain one or more chiral carbon atoms and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry. The present invention is intended to include all possible isomers, as well as their racemic and optically pure forms. The compounds of the invention can be prepared by selecting racemates, diastereomers, or enantiomers as starting materials or intermediates. Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0045] Conventional techniques for the preparation / isolation of individual isomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography.

[0046] The invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein. However, it is actually common for one or more atoms to be replaced by atoms having a different atomic weight or mass number from them. Examples of isotopes that can be listed as compounds of the invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine isotopes. Compounds of the invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates thereof, which contain the isotopes or other isotopic atoms of the above compounds are within the scope of the invention. Certain isotopically labeled compounds of the invention, such as radioactive isotopes, are also useful in tissue distribution experiments of drugs and substrates. For example, tritium, i.e., 3 3H and carbon-14, i.e., 14 14C, are relatively easy to prepare and detect and are preferred among isotopes. In addition, heavier isotope substitutions such as deuterium, i.e., 2 2H, have advantages in certain therapies due to their good metabolic stability, such as increasing the half-life in vivo or reducing the dosage, and thus can be preferred in certain cases. Isotopically labeled compounds can be prepared by general methods, by replacing non-isotopic reagents with readily available isotopically labeled reagents, using the procedures disclosed in the examples. In this application, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0047] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic acids or organic acids that can retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, perchlorate, etc.; organic acid salts include, but are not limited to, formate, acetate, p-toluenesulfonate, methanesulfonate, benzenesulfonate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, pyroglutamate, citrate, isethionate, succinate, etc. These salts can be prepared by methods known in the art.

[0048] As described herein, the compounds of the present invention can be taken with any number of substituents or functional groups to expand their scope of inclusion. Generally, the term "substituted", whether it appears before or after the term "optional", in the formulations of the present invention includes the general formula of substituents, which means replacing a hydrogen radical with a specified structural substituent. When multiple positions in a specific structure are substituted with multiple specific substituents, each position of the substituent can be the same or different. The term "substituted" as used herein includes all substitutions allowed for organic compounds. Broadly speaking, the allowed substituents include acyclic, cyclic, branched, unbranched, carbocyclic, and heterocyclic, aromatic and non-aromatic organic compounds. In the present invention, a heteroatom such as nitrogen can have a hydrogen substituent or any of the allowed organic compounds described above to supplement its valence. In addition, the present invention is not intended to limit the allowed substituted organic compounds in any way. The present invention believes that the combination of substituents and variable groups is good for the treatment of diseases in the form of stable compounds. Here, the term "stable" refers to a compound that is stable enough to maintain the integrity of the compound structure for a sufficient period of time for detection, preferably for a sufficient period of time to be effective, and is used herein for the above purposes.

[0049] Metabolites of the compounds and their pharmaceutically acceptable salts involved in this application, as well as prodrugs that can be converted in vivo into the structures of the compounds and their pharmaceutically acceptable salts involved in this application, are also included in the claims of this application.

[0050] Pharmaceutical compositions and administration methods

[0051] The pharmaceutical compositions described in the present invention are used for the prevention and / or treatment of cancers, immune diseases, metabolic diseases, etc. In this application, a "pharmaceutical composition" refers to a preparation of the compounds of the present invention and a medium commonly accepted in the art for delivering bioactive compounds to mammals (such as humans). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and thus exert its biological activity. The term "pharmaceutically acceptable" as used herein refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, that is, the substance can be administered to an individual without causing adverse biological reactions or interacting with any component contained in the composition in an adverse manner.

[0052] In this application, "pharmaceutically acceptable excipients" include but are not limited to any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers that are permitted by the relevant government regulatory authorities for use in humans or livestock.

[0053] The present invention can be widely used for preventing and / or treating diseases related to the dual targets of EZH2 and HDAC, especially for preventing and / or treating tumors and / or cancers and / or inflammatory bowel diseases. As used herein, the term "prevention" includes reducing the likelihood of the occurrence or exacerbation of a disease or disorder in a patient.

[0054] As used herein, the terms "treatment" and other similar synonyms include the following meanings:

[0055] (i) Preventing the occurrence of a disease or disorder in a mammal, especially when such a mammal is susceptible to the disease or disorder but has not been diagnosed as having the disease or disorder;

[0056] (ii) Inhibiting a disease or disorder, i.e., curbing its development;

[0057] (iii) Alleviating a disease or disorder, i.e., causing the state of the disease or disorder to subside; or

[0058] (iv) Relieving the symptoms caused by the disease or disorder.

[0059] As used herein, "cancer" or "tumor" includes, but is not limited to, lymphoma, sarcoma, melanoma, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, thyroid cancer, laryngeal cancer, tongue cancer, head and neck cancer, multiple myeloma, B-cell lymphoma, leukemia, etc.

[0060] As used herein, "inflammatory bowel disease" includes ulcerative colitis and Crohn's disease.

[0061] As used herein, the terms "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" refer to the amount of at least one agent or compound that, when administered, is sufficient to alleviate to some extent one or more symptoms of the disease or disorder being treated. The result can be the reduction and / or alleviation of signs, symptoms or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a significant alleviation of the disorder clinically. Techniques such as dose escalation trials can be used to determine the effective amount suitable for any individual case.

[0062] As used herein, the terms "administering", "administered", "administration", etc. refer to methods capable of delivering a compound or composition to a desired site of biological action. These methods include, but are not limited to, oral route, duodenal route, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Those skilled in the art are familiar with the administration techniques applicable to the compounds and methods described herein. In a preferred embodiment, the compounds and compositions of the present invention are administered orally, and the formulations of the compounds disclosed herein suitable for oral administration may be presented in discrete units, such as tablets, capsules or cachets each containing a predetermined amount of the active ingredient. In other preferred embodiments, the compounds and compositions of the present invention are injectables and powders.

[0063] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier) such as sodium citrate or calcium phosphate dibasic, or with the following components: (a) fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., hydroxypropylmethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrants, e.g., agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (h) adsorbents, e.g., kaolin; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents. Solid dosage forms such as tablets, troches, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be delayed and released in a portion of the digestive tract. Examples of embedding components that may be employed are polymeric substances and waxes. If necessary, the active compound may also be in the form of microcapsules formed with one or more of the above excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures.

[0064] In addition to the active compound, the liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers, and emulsifying agents, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil or mixtures of these substances.

[0065] In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and fragrances.

[0066] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures of these substances.

[0067] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0068] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalants.

[0069] The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required, if necessary. As used herein, the terms "drug combination", "drug combination therapy", "combination therapy", "administering additional therapy", "administering additional therapeutic agent", etc. refer to a pharmaceutical treatment obtained by mixing or combining more than one active ingredient, which includes fixed and non-fixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration to a patient of at least one compound described herein and at least one synergistic agent in the form of a single entity or a single dosage form. The term "non-fixed combination" refers to the simultaneous administration, co-administration, or sequential administration at variable intervals to a patient of at least one compound described herein and at least one synergistic agent in the form of separate entities.

[0070] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (including humans) in need of treatment, wherein the dosage administered is a pharmaceutically effective dosage. For a human weighing 60 kg, the daily dosage is generally 100 to 1000 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0071] The present invention also provides a method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutical carrier with the compound of formula (I) according to the present invention or its crystal form, pharmaceutical salt, hydrate, or solvate to form a pharmaceutical composition.

[0072] The present invention also provides a method of treatment, which includes the step of administering to a subject in need of treatment the compound described in the present invention, or its pharmaceutical salt, or administering the pharmaceutical composition described in the present invention for inhibiting EZH2 and HDAC.

[0073] Preparation Method of General Formula Compounds

[0074] It should be noted that various materials and reagents used in the following examples are common materials and reagents in the art and can be obtained commercially; the multiple amounts mentioned in the following examples are molar multiple amounts.

[0075] Example 1

[0076]

[0077] Methods and Reagents: A. Tetrahydropyranone, acetic acid, sodium triacetoxyborohydride, dichloroethane; B. Acetaldehyde, acetic acid, sodium triacetoxyborohydride, dichloroethane; C. 1M lithium hydroxide, tetrahydrofuran; D. HOBt, EDCI, N-methylmorpholine, anhydrous DMSO; E. Pd(PPh3)4, Na2CO3, dioxane, water, 90 °C, overnight; F. Anhydrous potassium carbonate, DMF; G. Potassium hydroxylamine, anhydrous methanol, 1M hydrochloric acid.

[0078] The specific steps include:

[0079] Methyl 2-methyl-3-amino-5-bromobenzoate (1 equivalent) was dissolved in anhydrous dichloroethane. 1.1 equivalents of tetrahydropyranone were added at room temperature, and then 2 equivalents of acetic acid were added. After stirring at room temperature for 3 hours, 3 equivalents of sodium triacetoxyborohydride were added, and the reaction was carried out overnight at room temperature. Dichloroethane was removed by distillation under reduced pressure, and then saturated sodium bicarbonate was added. The mixture was extracted with ethyl acetate, and the ethyl acetate layer was washed twice with saturated sodium chloride aqueous solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain pure product 1.

[0080] Compound 1 (1 equivalent) was dissolved in anhydrous dichloroethane. 1.1 equivalents of an acetaldehyde solution in tetrahydrofuran (1M) were added at room temperature, and then 2 equivalents of acetic acid were added. After stirring at room temperature for 3 hours, 3 equivalents of sodium triacetoxyborohydride were added, and the reaction was carried out overnight at room temperature. Dichloroethane was removed by distillation under reduced pressure, and then saturated sodium bicarbonate was added. The mixture was extracted with ethyl acetate, and the ethyl acetate layer was washed twice with saturated sodium chloride aqueous solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain pure product 2.

[0081] 10 g of Compound 2 was dissolved in 30 mL of tetrahydrofuran, and 30 mL of 1M aqueous lithium hydroxide solution was added. The mixture was stirred overnight at room temperature. Tetrahydrofuran was removed by distillation under reduced pressure, and the residual liquid was adjusted to pH 6 - 7 with 0.5M HCl. The resulting solid was filtered and washed 4 times with double-distilled water to obtain pure product 3.

[0082] Compound 3 (1 equivalent), compound 4 (1.2 equivalents), and HOBt (1.5 equivalents) were dissolved in anhydrous DMSO. At room temperature, EDCI (2 equivalents) and N-methylmorpholine (6 equivalents) were added. After the mixture was stirred at room temperature for 24 hours, a sufficient amount of saturated sodium bicarbonate aqueous solution was added to adjust the pH of the solution to 8 - 9. Stirring was continued for 1 hour, and the pale pink solid was filtered and washed 3 times with double-distilled water and 3 times with petroleum ether to finally obtain the pure product 5.

[0083] Compound 5 (1 equivalent), 4-hydroxyphenylboronic acid (1.5 equivalents), sodium carbonate (3 equivalents), and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. A mixture of dioxane and water (v / v = 5:1) that had been purged with argon was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate until no fluorescence eluted. The filtrate was collected and washed 3 times with saturated sodium chloride aqueous solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain the pure product 6 as a brown solid.

[0084] Compound 6 (1 equivalent) was dissolved in anhydrous DMF, and methyl bromoacetate (1.2 equivalents) and anhydrous potassium carbonate (1.5 equivalents) were added. The mixture was stirred at room temperature overnight. Saturated sodium chloride aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain a series of pure products 7.

[0085] Finally, compound 7 was dissolved in an anhydrous methanol solution of potassium hydroxylamine. The mixture was stirred at room temperature overnight. Methanol was removed by distillation under reduced pressure, and double-distilled water was added to dissolve the residue. The pH was adjusted to 6 - 7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product. The crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0086] Example 2

[0087]

[0088] Methods and reagents: A. Acetaldehyde, acetic acid, sodium triacetoxyborohydride, dichloroethane; B. Triethylamine, anhydrous dichloromethane; C. 1M lithium hydroxide, tetrahydrofuran; D. HOBt, EDCI, methylmorpholine, anhydrous DMSO; E. Pd(PPh3)4, Na2CO3, dioxane, water, 90 °C, overnight; F. Anhydrous potassium carbonate, DMF; G. Potassium hydroxylamine, anhydrous methanol, 1M hydrochloric acid.

[0089] The specific steps include:

[0090] Compound 1 (1 equivalent) was dissolved in anhydrous dichloroethane. A 1.1-fold amount of a solution of acetaldehyde in tetrahydrofuran (1 M) was added at room temperature, followed by a 2-fold amount of acetic acid. After stirring at room temperature for 3 hours, a 3-fold amount of sodium triacetoxyborohydride was added, and the reaction was carried out overnight at room temperature. Dichloroethane was removed by distillation under reduced pressure. Then, saturated sodium bicarbonate was added, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed twice with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and purified by column chromatography (petroleum ether:ethyl acetate = 6:1) to obtain the pure product 1.

[0091] Compound 1 was dissolved in anhydrous dichloromethane. A 1.2-fold amount of triethylamine was added, and a solution of different acyl chlorides in anhydrous dichloromethane was added dropwise under ice bath conditions. The mixture was transferred to room temperature and stirred for 3 hours. It was directly loaded onto a silica gel column for separation (petroleum ether:ethyl acetate = 6:1) and purified to obtain the pure product 2.

[0092] 8 g of Compound 2 was dissolved in 30 mL of tetrahydrofuran. 30 mL of 1 M aqueous lithium hydroxide solution was added, and the mixture was stirred overnight at room temperature. Tetrahydrofuran was removed by distillation under reduced pressure. The residual liquid was adjusted to pH 6 - 7 with 0.5 M HCl. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the pure product 3.

[0093] Compound 3 (1 equivalent), Compound 4 (1.2 equivalents), and HOBt (1.5 equivalents) were dissolved in anhydrous DMSO. EDCI (2 equivalents) and N-methylmorpholine (6 equivalents) were added at room temperature. After the mixture was stirred at room temperature for 24 hours, a sufficient amount of saturated aqueous sodium bicarbonate solution was added to adjust the pH of the solution to 8 - 9. Stirring was continued for 1 hour, and the light pink solid was filtered and washed 3 times with double-distilled water and 3 times with petroleum ether to finally obtain the pure product 5.

[0094] Compound 5 (1 equivalent), 4-hydroxybenzeneboronic acid (1.5 equivalents), sodium carbonate (3 equivalents), and tetrakis(triphenylphosphine)palladium (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. A mixture of dioxane and water (v / v = 5:1) that had been purged with argon was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate until no fluorescence was eluted. The filtrate was collected and washed 3 times with saturated aqueous sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain the pure product 6.

[0095] Compound 6 (1 equivalent) was dissolved in anhydrous DMF. Methyl bromoalkylate (1.2 equivalents) and anhydrous potassium carbonate (1.5 equivalents) were added. The mixture was stirred overnight at room temperature. Saturated aqueous sodium chloride solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain a series of pure products 7.

[0096] The final compound 7 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred overnight at room temperature. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6-7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product, and the crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0097] Example 3

[0098]

[0099] Methods and reagents: A. Pd(PPh3)4, Na2CO3, dioxane, water, 90 °C, overnight; B. HATU, DIPEA, anhydrous DMF; C. potassium hydroxylamine, anhydrous methanol, 1M hydrochloric acid.

[0100] The specific steps include:

[0101] Compound 1 (1 equivalent), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline 2 (1.5 equivalents), sodium carbonate (3 equivalents) and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. A mixed solution of dioxane and water (v / v = 5:1) that had been purged with argon was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence was eluted. The filtrate was collected and washed 3 times with saturated sodium chloride aqueous solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate. Purification by column chromatography (dichloromethane:methanol = 50:1) gave the pure product 3 as a brown solid.

[0102] Compound 3 (1 equivalent) was dissolved in anhydrous DMF, and monomethyl adipate 4 (1.2 equivalents), HATU (1.5 equivalents) and DIPEA (3 equivalents) were added. The mixture was stirred overnight at room temperature. Saturated sodium chloride aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated sodium chloride aqueous solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate. Purification by column chromatography (dichloromethane:methanol = 50:1) gave a series of pure products 5.

[0103] Compound 5 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred overnight at room temperature. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6-7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product, and the crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0104] Example 4

[0105]

[0106] Methods and Reagents: A. Pd(PPh3)4, Na2CO3, dioxane, water, 90 °C, overnight; B. HATU, DIPEA, anhydrous DMF; C. potassium hydroxylamine, anhydrous methanol, 1 M hydrochloric acid.

[0107] The specific steps include:

[0108] Compound 1 (1 equivalent), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline 2 (1.5 equivalents), sodium carbonate (3 equivalents) and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. A mixture of dioxane and water that had been purged with argon (v / v = 5:1) was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence was eluted. The filtrate was collected and washed 3 times with saturated sodium chloride aqueous solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain the pure product 3 as a brown solid.

[0109] Compound 3 (1 equivalent) was dissolved in anhydrous DMF, and dimethyl adipate 4 (1.2 equivalents), HATU (1.5 equivalents) and DIPEA (3 equivalents) were added. The mixture was stirred at room temperature overnight. Saturated sodium chloride aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain a series of pure products 5.

[0110] Compound 5 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6 - 7 with 1 M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product. The crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0111] Example 5

[0112]

[0113] Methods and Reagents: A. Pd(PPh3)4, CuI, triethylamine, DMF, 90 °C, overnight; B. potassium hydroxylamine, anhydrous methanol, 1 M hydrochloric acid.

[0114] The specific steps include:

[0115] Compound 1 (1 equivalent), terminal alkynyl formate (1.1 equivalents), triethylamine (2 equivalents), copper(I) iodide (0.1 equivalent), and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. DMF that had been degassed with argon was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence eluted. The filtrate was collected and washed 3 times with saturated aqueous sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain the pure product 2 as a white solid.

[0116] Compound 2 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH was adjusted to 6 - 7 with 1 M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product. The crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0117] Example 6

[0118]

[0119] Methods and reagents: A. Pd(PPh3)4, CuI, triethylamine, DMF, 90 °C, overnight; B. potassium hydroxylamine, anhydrous methanol, 1 M hydrochloric acid.

[0120] The specific steps include:

[0121] Compound 1 (1 equivalent), terminal alkynyl formate (1.1 equivalents), triethylamine (2 equivalents), copper(I) iodide (0.1 equivalent), and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. DMF that had been degassed with argon was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence eluted. The filtrate was collected and washed 3 times with saturated aqueous sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain the pure product 2 as a white solid.

[0122] Compound 2 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH was adjusted to 6 - 7 with 1 M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product. The crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0123] Example 7

[0124]

[0125] Methods and Reagents: A. Pd(PPh3)4, Na2CO3, dioxane, water, 90 °C, overnight; B. potassium hydroxylamine, anhydrous methanol, 1 M hydrochloric acid. B. HATU, DIPEA, anhydrous DMF; C. anhydrous potassium carbonate, DMF.

[0126] The specific steps include:

[0127] Compound 1 (1 equivalent), compound 2 (1.5 equivalents), sodium carbonate (3 equivalents) and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. A mixture of dioxane and water that had been purged with argon (v / v = 5:1) was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence was eluted. The filtrate was collected and washed 3 times with saturated sodium chloride aqueous solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain the pure product as a brown solid at the 3-position.

[0128] Compound 3 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH was adjusted to 6 - 7 with 1 M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product. The crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0129] Compound 1 (1 equivalent), 4-hydroxyphenylboronic acid (1.5 equivalents), sodium carbonate (3 equivalents) and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was purged with argon. A mixture of dioxane and water that had been purged with argon (v / v = 5:1) was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence was eluted. The filtrate was collected and washed 3 times with saturated sodium chloride aqueous solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain the pure product 4 as a brown solid.

[0130] Compound 4 (1 equivalent) was dissolved in anhydrous DMF, and methyl 4-(bromomethyl)benzoate (1.2 equivalents) and anhydrous potassium carbonate (1.5 equivalents) were added. The mixture was stirred at room temperature overnight. Saturated sodium chloride aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain a series of pure products 5.

[0131] Compound 5 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6 - 7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product, and the crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0132] Compound 4 (1 equivalent) was dissolved in anhydrous DMF, and compound 6 (1.2 equivalents) and anhydrous potassium carbonate (1.5 equivalents) were added. The mixture was stirred at room temperature overnight, saturated sodium chloride aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain a series of pure products 7.

[0133] Compound 7 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6 - 7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product, and the crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0134] Example 8

[0135]

[0136] Methods and reagents: A. Pd(PPh3)4, Na2CO3, dioxane, water, 90 °C, overnight; B. potassium hydroxylamine, anhydrous methanol, 1M hydrochloric acid. B. HATU, DIPEA, anhydrous DMF; C. anhydrous potassium carbonate, DMF.

[0137] The specific steps include:

[0138] Compound 1 (1 equivalent), compound 2 (1.5 equivalents), sodium carbonate (3 equivalents) and tetrakis(triphenylphosphine)palladium (0.01 equivalent) were placed in a two-necked flask. The air in the flask was replaced with argon. A mixed solution of argon-degassed dioxane and water (v / v = 5:1) was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence was eluted. The filtrate was collected and washed 3 times with saturated sodium chloride aqueous solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate. After purification by column chromatography (dichloromethane:methanol = 50:1), the pure product 3 was obtained as a brown solid.

[0139] Compound 3 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6 - 7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product, and the crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0140] Compound 1 (1 equivalent), 4-hydroxyphenylboronic acid (1.5 equivalents), sodium carbonate (3 equivalents) and tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) were placed in a two-necked flask. The flask was flushed with argon. A mixture of dioxane and water (v / v = 5:1) that had been purged with argon was added. The mixture was reacted at 90 °C overnight. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate until no fluorescence eluted. The filtrate was collected and washed 3 times with saturated sodium chloride aqueous solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate. Purification by column chromatography (dichloromethane:methanol = 50:1) gave the pure product 4 as a brown solid.

[0141] Compound 4 (1 equivalent) was dissolved in anhydrous DMF, and methyl 4-(bromomethyl)benzoate (1.2 equivalents) and anhydrous potassium carbonate (1.5 equivalents) were added. The mixture was stirred at room temperature overnight. Saturated sodium chloride aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain a series of pure products 5.

[0142] Compound 5 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6 - 7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product, and the crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0143] Compound 4 (1 equivalent) was dissolved in anhydrous DMF, and compound 6 (1.2 equivalents) and anhydrous potassium carbonate (1.5 equivalents) were added. The mixture was stirred at room temperature overnight. Saturated sodium chloride aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate. The ethyl acetate layer was washed 3 times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 50:1) to obtain a series of pure products 7.

[0144] Compound 7 was dissolved in an anhydrous methanol solution of potassium hydroxylamine and stirred at room temperature overnight. After evaporating methanol under reduced pressure, double-distilled water was added to dissolve the residue, and the pH value was adjusted to 6 - 7 with 1M HCl solution. The resulting solid was filtered and washed 4 times with double-distilled water to obtain the crude final product, and the crude final product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the pure final product.

[0145] The representative compound numbers, names and structures are shown in Table 1 below:

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] The high-resolution mass spectrometry results of the representative compounds are shown in Table 2 below:

[0181]

[0182]

[0183]

[0184] (I) Results of enzyme activity inhibition:

[0185] First, the inhibition rates of 132 compounds against EZH2 at 100 nM were tested. The specific steps were as follows: Each 100 nM compound was incubated with 5 nM recombinant PRC2 complex in a mixture containing 50 mM Tris (pH 8.6), 0.02% Triton X-100, 2 mM MgCl2, and 1 mM TCEP (total volume 40 μL) for 30 minutes. Then, 3.3 μM H3K27me peptide and 100 μM SAM were added, and the mixture was incubated for another 3 hours. Then, the luminescence of the samples was measured in a BioTek Synergy 2, and the inhibition rates were calculated. The results are shown in Figure 1(a) and Figure 1(b). It can be seen from the results that all 132 compounds showed strong inhibitory activity against EZH2 at a concentration of 100 nM.

[0186] Then, the inhibition rates of 132 representative compounds against the HDAC mixed enzyme (Hela cell nuclear extract) at 200 nM were tested. The specific steps were as follows: The solutions of each 200 nM compound were respectively mixed with the HDAC mixed enzyme, the fluorescent substrate (Boc-Lys(Ac)-pNA), BSA, and the buffer solution, and reacted at 37 °C for 30 minutes. After the reaction, HDAC Developer was added, and then incubated at 37 °C for another 15 minutes. The fluorescence intensity was measured (excitation wavelength 390 nm, emission wavelength 460 nm), and the inhibition rate was calculated. The results are shown in Figures 2(a) and 2(b).

[0187] As can be seen from the results, all 132 compounds showed certain inhibitory activities against HDAC at a concentration of 200 nM. Moreover, with the increase in the length of the linker chain, the inhibitory activity also increased. The compounds with a benzene ring or benzyl group as the linker chain had relatively low inhibition rates, and the reason was that these compounds might mainly inhibit the HDAC6 subtype.

[0188] (2) The proliferation inhibitory activities of individual compounds against human osteosarcoma cells HOS and follicular lymphoma cells were also tested. The specific steps were as follows: A single cell suspension was prepared with the culture medium containing 10% fetal bovine serum, and inoculated into a 96-well plate at 10,000 cells per well, with a volume of 200 μl per well; after adding the compound, it was incubated for 72 hours; then 20 μl of MTT solution (5 mg / ml prepared with PBS) was added to each well, and incubation continued for 4 hours; the culture was terminated, and the culture supernatant in the wells was carefully aspirated. For suspension cells, centrifugation was required before aspirating the culture supernatant in the wells. 150 μl of DMSO was added to each well, and shaken for 10 minutes to fully dissolve the crystals; at a wavelength of 490 nm, the light absorption values of each well were measured on an enzyme-linked immunosorbent monitor, the results were recorded, and a cell growth curve was plotted with time as the abscissa and the light absorption value as the ordinate, and the GI 50 value was calculated. The results are shown in Table 3 below:

[0189]

[0190]

[0191] As can be seen from the results, the representative compounds showed strong proliferation inhibitory activities against both types of cancer cells. The GI 50 values of individual compounds were at the submicromolar level. In particular, compound EH-19 had the strongest activity and was superior to the two positive control drugs Tazemetostat and SAHA.

[0192] (3) In the Western blot experiment, HOS cells were first incubated with different concentrations of EH-19 or DMSO for 48 hours; the cells were washed three times with 1 mL of cold PBS and lysed in 50 μL of cold RIPA buffer for 10 minutes; the cell lysates were centrifuged at 12,000 rpm for 15 minutes at 4°C; the protein concentration was determined by the BCA method; the same amount of cell extracts was separated by SDS-PAGE and transferred to a nitrocellulose membrane; and treated with Ac-HH3 antibody and H3K27me3 antibody. The results showed that compound EH-19 could significantly decrease the expression of H3K27me3 and increase the expression of Ac-histone H3 in HOS cells in a dose-dependent manner, as Figure 3 shown, and this result further demonstrated that the compound was a dual-target inhibitor of EZH2 and HDAC.

[0193] (4) The ability of compound EH-19 to relieve colitis in mice was also tested. The specific steps included: mice were randomly assigned to three groups (n = 10 mice per group): Group 1 received normal saline and dimethyl sulfoxide (5%, v / v); Group 2 was given DSS (2%, w / v), normal saline and DMSO; Group 3 was the dosing group, containing EH-19, DSS (2%, w / v), normal saline and DMSO (5%, v / v). The dosing dose of EH-19 was 5 mg / kg, administered orally once a day for 8 consecutive days. After 8 days, the mice were sacrificed, the colon was removed, and its length was measured.

[0194] The results showed that at a dosing dose of 5 mg / kg, EH-19 could also effectively inhibit DSS-induced ulcerative colitis in mice and significantly extend the length of the mouse colon (as Figure 4 shown), among which the average colon length of the normal control group (i.e., normal mice) was 12.3 cm, the DSS group was 7.6 cm, and the DSS+EH-19 group was 10.5 cm, as Figure 5 shown, which proved that compound EH-19 could significantly relieve DSS-induced colitis in mice.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation to the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should be regarded as the content disclosed by the present invention and all fall within the protection scope of the present invention.

Claims

1. An EZH2 and HDAC dual-target inhibitor, characterized in that R1 is an oxygen-containing heterocyclic alkyl or L-C(O)-. When R1 is an oxygen-containing heterocyclic group, it is selected from 4-tetrahydropyranyl. When R1 is L-C(O)-, L in L-C(O)- is C1-6 alkyl or C3-8 cycloalkyl; R2 is methyl or methoxy; X is selected from C6-10 aryl(C1-8 alkoxy), (C6-10 aryl)(amido)C1-8 alkyl, (alkynyl)C1-6 alkyl, (C6-10 aryl)(C1-3 alkoxy)C6-10 aryl, (C6-10 aryl)(C1-3 alkoxy)(C6-10 aryl)alkenyl, C6-10 aryl or C6-10 aryl(alkenyl).

2. A pharmaceutical composition, characterized in that, Comprising an effective amount of the EZH2 and HDAC dual-target inhibitor according to claim 1.

3. The EZH2 and HDAC dual-target inhibitor according to claim 1, characterized in that, The inhibitor is used for treating cancers, including follicular lymphoma, human osteosarcoma, melanoma, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, B-cell lymphoma.

4. The EZH2 and HDAC dual-target inhibitor according to claim 1, wherein The inhibitor is used for inflammatory bowel disease.

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