A chromone mTOR inhibitor and its pharmaceutical composition and application

Abstract

The present invention discloses a chromone mTOR inhibitor and a pharmaceutical composition and application thereof, wherein the chromone mTOR inhibitor comprises a substituted compound having a general formula (I), a stereoisomer, a hydrate or a pharmaceutically acceptable salt thereof, and the general formula (I) has the following structure: The chromone mTOR inhibitor and the pharmaceutical composition comprising the chromone mTOR inhibitor provided by the present invention can be used to inhibit mTOR kinase, have excellent inhibitory activity and selectivity for mTOR kinase, have a novel structure and significant anti-tumor activity, can be potentially used as a drug for the prevention, treatment and auxiliary treatment of related tumors, and have a positive effect on anti-proliferative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a chromone mTOR inhibitor and a pharmaceutical composition and application thereof. Background Art

[0002] Tumors are important diseases that affect human health. Tumor treatment mainly includes traditional surgical treatment, radiotherapy, chemotherapy, targeted therapy, immunotherapy and other treatment methods. With the development of tumor molecular biology and genomics, people's understanding of tumor molecular phenotypes has continued to deepen. The strategy of tumor treatment has gradually changed from the previous systemic chemotherapy stage centered on the site of disease and morphological pathology to precise targeted therapy targeting driver gene mutations. Tumor targeted therapy is a method of treating tumors by specifically targeting molecules or signaling pathways that play an important role in tumor occurrence and progression. Giving corresponding targeted therapy based on the specific molecular phenotype of the tumor has become the preferred option for the treatment of advanced tumors. Targeted drugs have become a hot spot in the research and development of anti-tumor drugs.

[0003] The PI3K / AKT / mTOR signaling pathway affects the proliferation, survival, transcription, translation and metabolism of malignant tumor cells. Abnormal activation of the PI3K-Akt-mTOR signaling pathway is associated with a variety of human tumors, such as ovarian, breast cancer, endometrial, colorectal and glioblastoma (Mol.Biol.Cell., 2009, 20, 1981-1991.). Therefore, organic small molecule kinase inhibitors targeting kinases such as PI3K and mTOR in this pathway have become one of the hot spots in the development of small molecule targeted anti-tumor drugs. As an important serine-threonine protein kinase downstream of PI3K / Akt, mTOR is involved in regulating cell growth, proliferation, survival and autophagy. The mTOR signaling pathway controls cell metabolism, growth, proliferation and survival. Continuous overactivation of mTOR will lead to increased cell metabolism, continuous growth and proliferation, extended cell lifespan and even cell immortality, which can directly or indirectly induce metabolic diseases, cancer and aging diseases. Inhibiting this state can effectively delay or treat cancer, cardiovascular damage, and other diseases caused by excessive activation of mTOR. Therefore, mTOR inhibitors are the focus of research, and many mTOR inhibitors have been developed ( Figure 1 ).

[0004] mTOR inhibitors can be divided into allosteric inhibitors and ATP competitive inhibitors according to protein pockets. Allosteric mTOR inhibitors are mainly rapamycin and its derivatives (Rapalogs). However, Rapalogs have not yet achieved the expected efficacy in the clinical treatment of certain cancers. After long-term use of the drug, the negative feedback of mTORC1 on AKT will activate the upstream pathway of the signal, leading to drug resistance. Rapalogs also have the disadvantages of complex structure, difficult synthesis, poor stability and low bioavailability (Eur. J. Med. Chem., 2020, 208 (5022): 112820.). Although allosteric mTOR inhibitors were discovered earlier and research on Rapalogs has matured, Rapalogs have a large molecular weight, many chiral carbons, and are difficult to synthesize. In addition, site modification is limited. ATP-competitive mTOR inhibitors regulate the downstream signaling pathway of mTOR and reduce Akt feedback activation by competing with the upstream molecules of mTOR kinase for the ATP binding site on mTOR kinase. These inhibitors overcome the shortcomings of rapamycin, are more likely to target mTOR binding sites, and have stronger growth inhibitory effects on tumor cells. However, ATP-competitive inhibitors such as PI-103, MLN0128, and AZD8055 still inevitably lead to side effects such as diarrhea, anemia, neutropenia, and non-infectious pneumonia. Long-term use of the same mTOR inhibitor will lead to drug resistance, and its clinical application is severely limited. Therefore, it is still necessary to develop ATP-competitive mTOR inhibitors with novel structural types, enrich the structural types of mTOR inhibitors, provide theoretical references for innovative drug research based on mTOR inhibitors, and provide more possibilities and options for better adapting to clinical application research. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention proposes a chromone mTOR inhibitor and a pharmaceutical composition and application thereof, so as to select compounds with better efficacy and higher safety for the preparation of cancer therapeutic drugs, in order to improve the therapeutic effect and alleviate the pain of patients.

[0006] In order to achieve the above object, according to one aspect of the present invention, a chromone mTOR inhibitor is provided, including a compound, stereoisomer, hydrate or pharmaceutically acceptable salt of the general formula (I), wherein the general formula (I) has the following structure:

[0007]

[0008] Wherein, in the general formula (I): R 1 and R 2 Can be selected from H, C 1 -C 6 Alkyl, C1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C containing one or more substituents 1 -C 6 Alkoxy, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkylsulfonyl, alkylsulfonyl containing one or more substituents, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylamino, C 3 -C 6 Heterocyclic group, C containing one or more substituents 3 -C 6 Heterocyclic group, C 3 -C 6 Heterocyclic acyl, C 3 -C 6 Heterocyclic acyl, C 4 -C 8 A fused heterobicyclic group or a C 4 -C 8 Fused heterobicyclic group, C 4 -C 8 Condensed heterobicyclic acyl or C containing one or more substituents 4 -C 8 A fused heterobicyclic acyl group; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxylic acid, carboxylate, cyano, acyl;

[0009] R 3 Selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C containing one or more substituents 1 -C 6 Alkoxy, C 1 -C 6 Alkyl acyl, C 1 -C6 Alkyl acyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkylsulfonyl, alkylsulfonyl containing one or more substituents, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylamino, C 3 -C 6 Heterocyclic group, C containing one or more substituents 3 -C 6 Heterocyclic group, C 3 -C 6 Heterocyclic acyl, C 3 -C 6 Heterocyclic acyl, C 4 -C 8 A fused heterobicyclic group or a C 4 -C 8 Fused heterobicyclic group, C 4 -C 8 Condensed heterobicyclic acyl or C containing one or more substituents 4 -C 8 A fused heterobicyclic acyl group; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxylic acid, carboxylate, cyano, acyl;

[0010] R 4 Selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C containing one or more substituents 1 -C 6 Alkoxy, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkylsulfonyl, alkylsulfonyl containing one or more substituents, C 1 -C6 Alkylamino, C 1 -C 6 Alkylamino, C 3 -C 6 Heterocyclic group, C containing one or more substituents 3 -C 6 Heterocyclic group, C 3 -C 6 Heterocyclic acyl, C 3 -C 6 Heterocyclic amide, C 4 -C 8 A fused heterobicyclic group or a C 4 -C 8 Fused heterobicyclic group, C 4 -C 8 Condensed heterobicyclic acyl or C containing one or more substituents 4 -C 8 A fused heterobicyclic acyl group; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxylic acid, carboxylate, cyano, acyl;

[0011] Furthermore, in some embodiments, R 1 Independently selected from the following structures:

[0012]

[0013] R 2 Selected from the following structures:

[0014]

[0015] Furthermore, the chromone mTOR inhibitor comprises at least one of the following compounds:

[0016] (1) (E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide;

[0017] (2) (S,E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide;

[0018] (3) (E)-3-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide;

[0019] (4) (E)-N-(2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)morpholine-4-carboxamide;

[0020] (5) (E)-N-(2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)piperazine-1-carboxamide;

[0021] (6) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)morpholine-4-carboxamide;

[0022] (7) (E)-N1-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)pyrrolidine-1,2-dicarboxamide;

[0023] (8) (S,E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3,3-dimethylbutanamide;

[0024] (9) (S,E)-2-amino-N-(2-((2-(benzo-d[1,3]dioxol-5-yl)-7-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)-3,3-dimethylbutanamide;

[0025] (10) (E)-2-amino-N-(2-((8-amino-2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide;

[0026] (11) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide;

[0027] (12) (S,E)-2-amino-N-(2-((8-amino-2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide;

[0028] (13) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)acetamide;

[0029] (14) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)acetamide;

[0030] (15) (E)-3-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)propanamide;

[0031] (16) (E)-3-amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide;

[0032] (17) (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methoxypropanamide;

[0033] (18) (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methylaminopropanamide;

[0034] (19) (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methylsulfonamidopropionamide;

[0035] (20) (E)-3-amino-N-(2-((2-(2-ethylaminobenzo[d]oxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide;

[0036] (21)(E)-3-amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-6-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)propanamide;

[0037] (22) (E)-3-Amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-6-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)propanamide.

[0038] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising at least one pharmaceutically acceptable excipient, adjuvant or carrier, and a therapeutically effective dose of at least one chromone mTOR inhibitor.

[0039] According to another aspect of the present invention, there is provided a use of the above-mentioned chromone mTOR inhibitor or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating and / or assisting in treating a proliferative disease caused by the action of mTOR kinase.

[0040] Furthermore, the proliferative diseases caused by the action of mTOR kinase are colorectal cancer, gastric cancer, breast cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, thyroid cancer, bladder cancer, kidney cancer, brain tumor, cervical cancer, esophageal cancer, gallbladder cancer, CNS cancer, malignant glioma, or myeloproliferative disease, leukemia and lymphoma.

[0041] According to another aspect of the present invention, there is provided a use of the above-mentioned chromone mTOR inhibitor or the above-mentioned pharmaceutical composition in inhibiting the growth of cancer cells in vitro.

[0042] The present invention provides a novel chromone mTOR inhibitor and a pharmaceutical composition containing the same, which can be used to inhibit mTOR kinase and can provide an inhibitor with better effectiveness and selectivity for the treatment of proliferative diseases mediated by mTOR kinase.

[0043] Beneficial Effects

[0044] Compared with the prior art, the present invention can achieve the following technical effects:

[0045] The present invention firstly finds that chromone derivatives can be used as ATP competitive inhibitors.

[0046] The compounds of the present invention have excellent inhibitory activity and selectivity on mTOR kinase.

[0047] The compound of the present invention has a novel structure and significant anti-tumor activity, and can be potentially used as a drug for the prevention, treatment and auxiliary treatment of related tumors.

[0048] Therefore, the compounds of the present invention have positive and predictable anti-proliferative disease effects, and especially have good development prospects and application potentials in anti-tumor clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 : Structural formulas of representative reported mTOR inhibitors;

[0050] Figure 2 : The synthetic route of the compound having the general formula (I) in the present invention. DETAILED DESCRIPTION

[0051] The technical solution of the present invention will be described in detail below in conjunction with the embodiments of the present invention. However, the following embodiments are only used to understand the present invention and cannot limit the present invention. The present invention can be implemented in a variety of different ways as defined and covered by the claims.

[0052] In order to achieve the goal of providing more effective and selective compounds for the treatment of tumors as pointed out in the background technology section, a new type of mTOR inhibitor of chromone is provided in the present invention. This new type of mTOR inhibitor of chromone includes a substituted compound having the general formula (I), its stereoisomers, hydrates or pharmaceutically acceptable salts. The general formula (I) has the following structure:

[0053]

[0054] Wherein, in the general formula (I): R 1 and R 2 Can be selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C containing one or more substituents 1 -C 6 Alkoxy, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkylsulfonyl, alkylsulfonyl containing one or more substituents, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylamino, C 3 -C 6 Heterocyclic group, C containing one or more substituents 3 -C 6 Heterocyclic group, C 3 -C 6 Heterocyclic acyl, C 3 -C 6 Heterocyclic acyl, C 4 -C 8 A fused heterobicyclic group or a C 4 -C8 Fused heterobicyclic group, C 4 -C 8 Condensed heterobicyclic acyl or C containing one or more substituents 4 -C 8 A fused heterobicyclic acyl group; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxylic acid, carboxylate, cyano, acyl;

[0055] R 3 Selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C containing one or more substituents 1 -C 6 Alkoxy, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkylsulfonyl, alkylsulfonyl containing one or more substituents, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylamino, C 3 -C 6 Heterocyclic group, C containing one or more substituents 3 -C 6 Heterocyclic group, C 3 -C 6 Heterocyclic acyl, C 3 -C 6 Heterocyclic acyl, C 4 -C 8 A fused heterobicyclic group or a C 4 -C 8 Fused heterobicyclic group, C 4 -C 8 Condensed heterobicyclic acyl or C containing one or more substituents 4 -C 8 A fused heterobicyclic acyl group; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxylic acid, carboxylate, cyano, acyl;

[0056] R4 Selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C containing one or more substituents 1 -C 6 Alkoxy, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkyl acyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkoxyacyl, C 1 -C 6 Alkylsulfonyl, alkylsulfonyl containing one or more substituents, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylamino, C 3 -C 6 Heterocyclic group, C containing one or more substituents 3 -C 6 Heterocyclic group, C 3 -C 6 Heterocyclic acyl, C 3 -C 6 Heterocyclic amide, C 4 -C 8 A fused heterobicyclic group or a C 4 -C 8 Fused heterobicyclic group, C 4 -C 8 Condensed heterobicyclic acyl or C containing one or more substituents 4 -C 8 A fused heterobicyclic acyl group; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxylic acid, carboxylate, cyano, acyl;

[0057] Furthermore, in some embodiments, R 1 Independently selected from the following structures:

[0058]

[0059] R 2 Selected from the following structures:

[0060]

[0061] The present invention provides a novel chromone mTOR inhibitor and a pharmaceutical composition containing the same, which can be used to inhibit mTOR kinase and can provide an inhibitor with better effectiveness and selectivity for the treatment of proliferative diseases mediated by mTOR kinase.

[0062] The chromone mTOR inhibitors of the present invention may include pharmaceutically acceptable salts. A pharmaceutically acceptable salt refers to a salt form in which a basic group in a parent compound is converted. Pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic groups such as amine (amino) groups. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, i.e., the basic group in the parent compound is reacted with 1-4 equivalents of an acid in a solvent system. Suitable salts are listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0063] The basic groups of the compounds of the present invention can form salts with acids, and examples of these acid salts include: salts formed with inorganic acids, especially hydrohalic acids (such as hydrochloric acid, hydrobromic acid, hydroiodic acid), nitric acid, sulfuric acid, phosphoric acid, carbonic acid, etc.; salts formed with lower alkyl sulfonic acids, such as methanesulfonic acid and trifluoromethanesulfonic acid; salts formed with aryl sulfonic acids, such as benzenesulfonic acid or p-toluenesulfonic acid; salts formed with organic acids, such as acetic acid, fumaric acid, tartaric acid, oxalic acid, citric acid, maleic acid, malic acid or succinic acid; and salts formed with amino acids, such as aspartic acid or glutamic acid.

[0064] The compounds of the present invention and pharmaceutically acceptable salts also include solvates or hydrates. In general, the solvates or hydrates are equivalent to non-solvated or non-hydrated forms and are included within the scope of the present invention. Some compounds of the present invention may exist in polycrystalline or amorphous forms. In general, all physical forms have equivalent uses and are included within the scope of the present invention.

[0065] In addition, unless otherwise indicated, the structural formula of the compounds in the chromone mTOR inhibitors described in the present invention includes all isomeric forms (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, and (Z), (E) conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformational isomers) are all within the scope of the present invention.

[0066] Unless otherwise indicated, all tautomeric forms of the pyrimidine compounds in the chromone mTOR inhibitors of the present invention are included within the scope of the present invention. In addition, unless otherwise indicated, the structural formula of the compounds described in the present invention includes one or more different isotopically enriched atoms.

[0067] Exemplary embodiments of the present invention will be described in detail below. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0068] As used herein, the terms of the present invention have the following meanings if specific definitions are not provided.

[0069] The term "alkyl" is a saturated straight or branched monovalent hydrocarbon group containing 1 to 20 carbon atoms, wherein the alkyl group may be independently optionally substituted with one or more substituents described herein. In some embodiments, the alkyl group contains 1 to 10 carbon atoms, in other embodiments, the alkyl group contains 1 to 8 carbon atoms, in other embodiments, the alkyl group contains 1 to 6 carbon atoms, and in other embodiments, the alkyl group contains 1 to 4 carbon atoms. Further examples of alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl (n-Pr, -CH 2 CH 2 CH 3 ), isopropyl (i-Pr, -CH (CH 3 ) 2 ), n-butyl (n-Bu, -CH 2 CH 2 CH 2 CH 3 ), isobutyl (i-Bu, -CH 2 CH(CH 3 ) 2 ), sec-butyl (s-Bu, -CH(CH 3 )CH 2 CH 3 ), tert-butyl (t-Bu, -C(CH 3 ) 3 ) etc. The term "alkyl" and its prefix "alkane" as used herein include both straight and branched saturated carbon chains.

[0070] The term "alkoxy" refers to an alkyl group having the same meaning as the aforementioned "alkyl" and is formed by an oxygen atom attached to the main carbon chain of the "alkyl" group.

[0071] The term "haloalkyl" or "haloalkoxy" refers to a situation where an "alkyl" or "alkoxy" group can be substituted by one or more identical or different halogen atoms. The alkyl and alkoxy groups have the meanings as described above in the present invention, and such examples include but are not limited to trifluoromethyl, trifluoromethoxy, etc.

[0072] The term "hydroxyalkyl" or "hydroxyalkoxy" refers to the case where an "alkyl" or "alkoxy" group may be substituted by one or more hydroxyl groups. The "alkyl" and "alkoxy" groups have the meanings as described above in the present invention, and such examples include but are not limited to hydroxymethyl, 1-hydroxyethyl, hydroxypropyl, 1,2-dihydroxypropyl, hydroxymethoxy, 1-hydroxyethoxy, and the like.

[0073] The term "acyl" refers to the radical remaining after removing one or more hydroxyl groups from an organic oxygen-containing acid, and has the general formula -M(O)-;

[0074] The term "alkyl acyl" or "alkoxy acyl" refers to the case where "alkyl" or "alkoxy" may be substituted by one or more acyl groups, wherein "alkyl" or "alkoxy" groups have the meanings as described above in the present invention.

[0075] The term "sulfonyl" is represented by -S(=O) 2 -.

[0076] The term "alkylsulfonyl" refers to the case where an "alkyl" group may be substituted with one or more sulfonyl groups, wherein the "alkyl" group has the meaning as described above in the present invention.

[0077] The term "amino" is represented by -NH 2 .

[0078] The term "alkylamino" refers to the case where an "alkyl" group may be substituted by one or more amino groups, wherein the "alkyl" group has the meaning as described above in the present invention.

[0079] The terms "halo", "halogen", "halogen atom" or "halogen atom" include fluorine, chlorine, bromine, iodine.

[0080] The term "heterocyclyl" may be a carbon group or a heteroatom group. "Heterocyclyl" also includes groups formed by the combination of a heterocyclic group with a saturated or partially unsaturated ring or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, thioxanyl, azetidinyl, oxetanyl, thietanyl, piperidinyl, homopiperidinyl, glycidyl, azepanyl, oxetanyl, thietanyl, N-morpholinyl, 2-morpholinyl, 3-morpholinyl, thiomorpholinyl, N-piperazinyl, 2-piperazinyl, 3-piperazinyl, homopiperazinyl, 4-methoxy-piperidin-1-yl, 1,2 , 3,6-tetrahydropyridin-1-yl, oxazepinyl, diazepinyl, thiazepinyl, pyrrolin-1-yl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-dioxolane, pyrazolinyl, dithianyl, dithiolanyl, dihydrothiophenyl, pyrazolidinylimidazolinyl, imidazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,6-thiadiazinane, 1,1-dioxo-2-yl, quinolizinyl and N-pyridylurea. And the heterocyclic group may be substituted or unsubstituted, wherein the substituent may be but is not limited to oxo (=O), hydroxyl, amino, halogen, cyano, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclic group, thiol, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C (=O), alkyl-C (=O), carboxyalkoxy, etc.

[0081] The term "fused bicyclic ring", "fused ring", "fused bicyclic radical" or "fused cyclic radical" refers to a saturated or unsaturated fused ring system, and refers to a non-aromatic bicyclic ring system. Such a system may contain independent or conjugated unsaturation, but its core structure does not contain aromatic rings or heteroaromatic rings (although aromatic rings can be substituted thereon). Each ring in the fused bicyclic ring is either carbocyclic or heteroalicyclic, and such examples include but are not limited to 2,3,3a,4,7,7a-hexahydro-1H-indenyl, 7-azabicyclo[2.2.1]heptyl, fused bicyclo[3.3.0]octyl, fused bicyclo[3.1.0]hexyl, 1,2,3,4,4a,5,8,8a-octahydronaphthyl, all of which are included in the fused bicyclic ring system. And the fused bicyclic group can be substituted or unsubstituted, wherein the substituent can be but is not limited to halogen, hydroxyl, amino, cyano, aryl, heteroaryl, alkoxy, alkyl, alkenyl, alkynyl, heterocyclic group, thiol, nitro, aryloxy and the like.

[0082] The term "fused heterobicyclic group" refers to a saturated or unsaturated fused ring system, which refers to a non-aromatic bicyclic ring system. Such a system may contain independent or conjugated unsaturated states, but its core structure does not contain aromatic rings or aromatic heterocyclic rings (but aromatics can be substituents thereon). At least one ring system contains one or more heteroatoms, wherein each ring system contains 3-7 ring members, that is, contains 1-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, S, wherein S or P is optionally substituted by one or more oxygen atoms to obtain SO, SO 2 ,PO,PO 2 The fused heterobicyclic group may be substituted or unsubstituted, wherein the substituents may be, but are not limited to, halogen, hydroxyl, amino, cyano, aryl, heteroaryl, alkoxy, alkyl, alkenyl, alkynyl, heterocyclic group, mercapto, nitro, aryloxy, etc.

[0083] The above-mentioned novel chromone mTOR inhibitor provided by the present invention can be prepared in a variety of ways, and those skilled in the art can find an appropriate way to prepare it under the inspiration of the structural formula provided in this application. For ease of understanding, the preparation method of the above-mentioned general formula (I) is provided in this application.

[0084] A method for preparing a compound having a general formula (I): using substituted o-hydroxyacetophenone (A) as a raw material, reacting the raw material (A) with substituted formaldehyde to obtain a corresponding intermediate (B), cyclizing the intermediate (B) under iodine catalysis to generate an intermediate (C), reacting the intermediate (C) with o-phenylenediamine to generate an intermediate (D), and condensing the intermediate (D) with various substituted carboxylic acid amides or with various substituted amines through phenyl chloroformate activation reaction to obtain a target compound (E) having a structure of the general formula (I).

[0085] The synthetic route of the above method is as follows Figure 2 As shown;

[0086] In the above preparation steps, the substituent R in formulas A to E is 1 , R 2 , R 3 and R 4 The definition of the substituent R in the general formula (I) 1 , R 2 , R 3 and R 4 same.

[0087] At the same time, in one embodiment of the present invention, a pharmaceutical composition is also provided, which comprises at least one pharmaceutically acceptable excipient, adjuvant or carrier, and an effective therapeutic dose of at least one of the above-mentioned chromone mTOR inhibitors.

[0088] The term "therapeutically effective dose" refers to an amount of a compound of the formula sufficient to be effective for treatment when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending on the specific activity of the therapeutic agent used, the age, physiological condition, presence of other disease states, and nutritional status of the patient. In addition, other drug treatments that the patient may be receiving will affect the determination of the therapeutically effective amount of the therapeutic agent to be administered.

[0089] The term "treat" means any treatment of a disease in a mammal, including: (i) preventing the disease, i.e., causing clinical symptoms of the disease not to develop; (ii) inhibiting the disease, i.e., arresting the development of clinical symptoms; and / or (iii) palliating the disease, i.e., causing regression of clinical symptoms.

[0090] The term "pharmaceutically acceptable excipient, adjuvant or carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0091] The composition is preferably formulated in unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable for use as a single dose for administration to human subjects and other mammals, each unit containing a predetermined amount of active substance calculated to produce the required therapeutic effect and related suitable pharmaceutical excipients (such as tablets, capsules, ampoules). The pyrimidine compound in the mTOR inhibitor is effective in a wide range of dosages and is generally administered in an effective drug amount. Preferably, for oral administration, each dosage unit contains 10 mg to 2 g of the pyrimidine compound in the mTOR inhibitor, more preferably 10 to 700 mg, and for parenteral administration, preferably 10 to 700 mg of the pyrimidine compound in the mTOR inhibitor, more preferably about 50 to 200 mg. However, it should be understood that the amount of the pyrimidine compound in the mTOR inhibitor actually administered will be determined by the physician according to relevant circumstances, including the condition to be treated, the route of administration selected, the actual compound administered and its relative activity, the age, weight, and response of each patient, the severity of the patient's symptoms, etc.

[0092] In order to prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient (or carrier) to form a solid preformulation composition, which contains a uniform mixture of the compound of the present invention. When these preformulation compositions are referred to as uniform, it means that the active ingredient is evenly dispersed throughout the composition, so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

[0093] Tablet or pill of the present invention can be coated or otherwise compounded to provide a dosage form with a prolonged effect advantage, or protect tablet or pill from the effect of acidic conditions in the stomach. For example, tablet or pill can include inner dosage and outer dosage components, the latter having the form of the outer skin on the former. Two kinds of components can be separated with an enteric layer, wherein the enteric layer is used to prevent the disintegration in the stomach and allows the complete entry of the inner component into the duodenum or delayed release. Various materials can be used for such enteric layers or coatings, and the above-mentioned materials include many macromolecular acids and macromolecular acids and such materials as shellac, hexadecanol and cellulose acetate mixture.

[0094] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutical excipients as described above. Preferably, these compositions are administered by oral or nasal respiratory route to obtain local or systemic effects. Compositions in preferred pharmaceutically acceptable solvents may be atomized by the use of inert gases. Atomized solutions may be inhaled directly from an atomizing device, or the atomizing device may be connected to a mask tent, or an intermittent positive pressure breathing machine. Solutions, suspensions, or powder compositions may be administered by a device that delivers the dosage form in an appropriate manner, preferably by oral or nasal route.

[0095] In another aspect, the present invention provides a use of the above-mentioned chromone mTOR inhibitor or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating and / or assisting in treating a proliferative disease caused by the action of mTOR kinase.

[0096] The proliferative diseases caused by the action of mTOR kinase are colorectal cancer, gastric cancer, breast cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, thyroid cancer, bladder cancer, kidney cancer, brain tumor, cervical cancer, esophageal cancer, gallbladder cancer, CNS cancer, malignant glioma, or myeloproliferative disease, leukemia and lymphoma.

[0097] In another aspect, the present invention provides a use of the above-mentioned chromone mTOR inhibitor or the above-mentioned pharmaceutical composition in inhibiting the growth of cancer cells in vitro.

[0098] The present invention will be further described below in conjunction with Examples 1-22; however, these examples should not be construed as limiting the scope of the present invention.

[0099] In the examples described below, all temperatures are in degrees Celsius unless otherwise indicated. Reagents were purchased from commercial suppliers such as AlfaAesar Chemical Company, Bailingwei Technology Co., Ltd., Aladdin Reagent Co., Ltd., Beijing Coupling Technology Co., Ltd., etc., and were used without further purification unless otherwise indicated. General reagents were purchased from Shantou Xilong Chemical Plant, Guangzhou Chemical Reagent Factory, Tianjin Zhiyuan Chemical Reagent Co., Ltd., and Qingdao Ocean Chemical Plant, etc.

[0100] The chromatographic columns in the examples described below used silica gel columns, and the silica gel (200-300 mesh) was purchased from Qingdao Ocean Chemical Factory. 3 or DMSO-d 6 is the solvent (in ppm), using TMS (0 ppm) or chloroform (7.26 ppm) as the reference standard. When multiple peaks appear, the following abbreviations are used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets). Coupling constants are expressed in Hertz (Hz).

[0101] The low-resolution mass spectrometry (MS) data in the examples described below were measured by an Agilent 6120 series LC-MS spectrometer equipped with a G1311B quaternary pump and a G1316BTCC (column temperature maintained at 30°C), a G1329B autosampler and a G1315C DAD detector were used for analysis, and an ESI source was applied to the LC-MS spectrometer.

[0102] In the examples described below, the injection volume was determined by the sample concentration; the flow rate was 0.5 mL / min; the HPLC peaks were recorded and read by UV-Vis wavelengths at 210 nm and 254 nm. The mobile phase was isopropanol / n-hexane (40:60).

[0103] For the convenience of description, some raw materials are described by their abbreviations in the following examples. These abbreviations are compared with their full names as follows: DCM is CH 2 Cl 2 , i.e. dichloromethane; CDC1 3 is deuterated chloroform; PE is petroleum ether; EtOAc and EA are both ethyl acetate; MeOH and CH 3 OH is methanol; NaOH is sodium hydroxide; DMSO-d 6DIPEA is N,N-diisopropylethylamine; HATU is N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl) urea hexafluorophosphate.

[0104] Example 1: (E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide

[0105] Step 1 Synthesis of (E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one:

[0106] Dissolve o-hydroxyacetophenone (5.0 g, 36.7 mmol) in 150 ml of ethanol, slowly add 20% NaOH aqueous solution under ice bath, stir for 10 minutes, add piperonal (5.5 g, 36.7 mmol), replace nitrogen, and react at room temperature for 24 hours. After the reaction is completed by TLC tracking, add dilute hydrochloric acid to the reaction mixture to adjust the pH to about 1-2, precipitate a yellow solid, filter, recrystallize the upper layer with ethanol, and dry under reduced pressure to obtain a light yellow solid. Yield: 85%, HRMS (ESI): m / z[M+H]+calcd.for[C 16 H 13 O 4 ] + :269.0814,found:269.0819.

[0107] Step 2 Synthesis of 2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-one:

[0108] (E)-3-(Benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one was dissolved in DMSO and added. 2 (0.284g, 1.12mmol), 150℃ for 2 hours. After the reaction was completed by TLC tracking, the reaction mixture was cooled to 60℃ and sodium thiosulfate solution was added to quench the iodine element, filtered, the upper layer was washed with ethyl acetate and dried under reduced pressure to obtain an off-white solid. Yield: 82%. 1 H NMR (600 MHz, DMSO-d 6)δ8.04(d,J=7.9Hz,1H),7.83(t,J=7.7Hz,1H),7.78(d,J=8.4Hz,1H),7.71(d,J=9.1Hz,2H), 7.49(t,J=7.4Hz,1H),7.12(d,J=8.1Hz,1H),6.98(s,1H),6.17(s,2H).HRMS(ESI):m / z[M+H] + calcd.for[C 16 H 11 O 4 ] + :267.0657,found:267.0658.

[0109] Step 3 Synthesis of (E)-2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)aniline:

[0110] Dissolve 2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-one (2.0 g, 7.5 mmol) and o-phenylenediamine (1.6 g, 15.0 mmol) in toluene, add tetraethyl titanate (5.1 g, 22.5 mmol), replace nitrogen, and reflux at 120°C for 20 hours. After the reaction was completed by TLC tracking, the reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM:MeOH=20:1) to obtain the target compound. Yield: 72%. HRMS (ESI): m / z[M+H] + calcd.for[C 22 H 17 N 2 O 3 ] + :357.1239,found:357.1240.

[0111] Step 4: Synthesis of (E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide:

[0112] Compound (E)-2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)aniline (0.15 g, 0.36 mmol), Boc-glycine (0.13 g, 0.72 mmol) and HATU (0.20 g, 0.54 mmol) were dissolved in DMF, and DIPEA (0.23 g, 1.8 mmol) was added to replace nitrogen and reacted at room temperature overnight. After TLC tracking, water was added, ethyl acetate was extracted, the organic layer was dried, and then dry dichloromethane was added to dissolve, and 12 times the equivalent of trifluoroacetic acid was added to react at room temperature for 6 hours. After TLC tracking, saturated sodium bicarbonate aqueous solution was added to adjust the pH to 7-8, ethyl acetate was extracted, the organic layer was dried, and the target compound was purified by silica gel column chromatography (DCM:MeOH=10:1). Yellow solid, yield: 56%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.00(s,1H),8.71–8.50(m,1H),8.19(s,3H),8.01(d,J=8.1Hz,1H),7.98–7.93(m,1H),7.68(s,1H),7.62 –7.49(m,2H),7.41(s,1H),7.34(s,2H),7.12(d,J=8.2Hz,1H),6.83–6.70(m,1H),6.18(s,2H),3.78(s,2H). 13 CNMR (151MHz, DMSO-d 6 )δ165.9,159.0,158.8,158.5,158.3,154.2,148.9,137.6,127.9,126.3,125.6,124.6,123.9,1 20.3,119.2,118.3,116.3,114.3,109.0,108.3,107.0,102.9,96.8,41.4.HRMS(ESI):m / z[M+H] + calcd.for[C 24 H 20 N 3 O 4 ] + :414.1454,found:414.1456.

[0113] Example 2: (S,E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0114] Synthesis method: Replace the substituted carboxylic acid fragment (i.e., Boc-glycine) in step 4 of Example 1 with Boc-alanine, and the other steps and operations are similar to those of Example 1. Yellow solid, yield: 52%. 1 HNMR (600 MHz, DMSO-d 6 )δ10.27(s,1H),8.44(d,J=7.9Hz,1H),8.42–8.37(m,1H),7.71(t,J=7.8Hz,1H),7.63(d,J=8.4Hz,1H),7.45(t,J=7 .5Hz,1H),7.42–7.34(m,2H),7.13–7.00(m,4H),6.70(s,1H),6.12(s,2H),3.40–3.35(m,1H),1.19(d,J=7.0Hz,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ174.5,158.4,153.8,152.3,150.3,148.6,139.5,133.0,131.8,126.1,125.9,125.1,124.3,123. 6,122.6,121.4,120.1,119.3,118.4,109.2,106.3,102.4,97.04,51.5,21.6.HRMS(ESI):m / z[M+H] + calcd.for[C 25 H 22 N 3 O 4 ] + :428.1610,found:428.1615.

[0115] Example 3: (E)-3-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0116] Synthesis method: Replace the substituted carboxylic acid fragment (i.e., Boc-glycine) in step 4 of Example 1 with Boc-β-alanine, and the other steps and operations are similar to those of Example 1. Yellow solid, yield: 63%. 1 HNMR (600 MHz, DMSO-d 6)δ10.04(s,1H),8.40(dd,J=8.0,1.7Hz,1H),8.13(dd,J=7.6,2.1Hz,1H), 7.71–7.67(m,1H),7.60(d,J=8.3Hz,1H),7.46–7.41(m,1H),7.34(d,J=7.8 Hz,2H),7.10–7.06(m,2H),7.04(d,J=8.1Hz,1H),6.95(dd,J=7.2,2.1Hz,1 H),6.51(s,1H),6.12(s,2H),2.75(t,J=6.2Hz,2H),2.31(t,J=6.2Hz,2H). 13 CNMR (151MHz, DMSO-d 6 )δ171.3,157.9,153.7,152.2,150.2,148.5,141.2,132.9,131.4,126.2,125.7,125.4,124.3,123 .9,122.6,122.3,121.3,120.5,118.3,109.2,106.2,102.4,97.4,39.5,38.2.HRMS(ESI):m / z[M+H] + calcd.for[C 25 H 22 N 3 O 4 ] + :428.1610,found:428.1612.

[0117] Example 4: (E)-N-(2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)morpholine-4-carboxamide

[0118] Synthesis method: (E)-2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)aniline (0.20 g, 0.6 mmol) was dissolved in dry DCM, phenyl chloroformate (0.13 g, 0.8 mmol) was added at 0°C, reacted at room temperature for 12 hours, the reaction was completed by TLC monitoring, water was added, DCM was extracted, the organic layer was dried by spin drying, DMSO was added to dissolve, morpholine was added, reacted at 65°C for 1 hour, the reaction was completed by TLC monitoring, water was added, solids were precipitated, filtered, the filter cake was washed with EA and dried. Yellow solid, yield: 49%. 1 H NMR (600 MHz, DMSO-d 6)δ8.36(dd,J=7.9,1.7Hz,1H),7.80(s,1H),7.78–7.73(m,1H),7.71–7.67(m,1H),7.62(dd,J=8.4,1.2Hz,1H),7.47–7.44(m,1H) ),7.42–7.38(m,2H),7.09–7.03(m,3H),7.02–6.97(m,1H),6.57(s,1H),6.12(s,2H),3.44–3.40(m,4H),3.26(t,J=4.8Hz,4H). 13 C NMR (151 MHz, DMSO-d 6 )δ157.7,155.4,153.8,152.1,150.2,148.5,141.9,132.9,131.9,126.2,125.8,124.9,124.0,123 .9,123.4,122.5,121.4,120.6,118.4,109.2,106.3,102.4,97.7,66.2,44.3.HRMS(ESI):m / z[M+H] + calcd.for[C 27 H 24 N 3 O 5 ] + :470.1716,found:470.1716.

[0119] Example 5: (E)-N-(2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)piperazine-1-carboxamide

[0120] Synthesis method: Replace the morpholine in Example 4 with piperazine. Yellow solid, yield: 40%. 1 H NMR (600 MHz, DMSO-d 6 )δ8.35(dd,J=7.9,1.7Hz,1H),7.80–7.75(m,1H),7.73–7.66(m,2H),7.62(d,J=8.2Hz,1H),7.48–7.42(m,1 H),7.40–7.37(m,2H),7.09–7.02(m,3H),7.02–6.96(m,1H),6.59(s,1H),6.12(s,2H),3.21(dd,J=6.0,4.1

[0121] Hz,4H),2.54–2.51(m,4H),1.22(s,1H). 13C NMR (151 MHz, DMSO-d 6 )δ157.8,155.1,153.8,152.1,150.2,148.5,141.4,132.9,132.2,126.2,125.8,124.7,124.0,123 .5,122.9,122.5,121.4,120.5,118.5,109.2,106.4,102.4,97.6,45.7,45.1.HRMS(ESI):m / z[M+H] + calcd.for[C 27 H 25 N 4 O 4 ] + :469.1876,found:469.1880.

[0122] Example 6: (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)morpholine-4-carboxamide

[0123] Synthesis method: Change the o-hydroxyacetophenone in step 1 of Example 1 to 4-fluoro-2-hydroxyacetophenone, and the other steps and operations are similar to those of Example 1 and Example 4. Yellow solid, yield: 38%. 1 H NMR (600 MHz, DMSO-d 6 )δ8.39(dd,J=8.9,6.4Hz,1H),7.78(s,1H),7.75(dd,J=5.9,3.5Hz,1H),7.5 8(dd,J=9.6,2.6Hz,1H),7.34(dd,J=6.2,2.4Hz,1H),7.31(dd,J=8.7,2.6Hz, 1H),7.28(dd,J=8.6,2.3Hz,1H),7.11–7.06(m,2H),6.97(dd,J=7.1,3.9Hz,2 H),6.55(s,1H),4.33–4.26(m,4H),3.45–3.41(m,4H),3.27(t,J=4.8Hz,4H). 13 C NMR (151 MHz, DMSO-d 6)δ165.4,163.7,162.8,157.7,155.3,154.7,154.6,151.4,146.6,144.1,141.6,132.0,127.4,127.3,124.9,124.0,123.8 ,123.3,120.6,119.8,119.5,118.2,115.1,114.0,113.9,105.4,105.2,97.5,66.2,64.9,64.5,44.3.HRMS(ESI):m / z[M+H] + calcd.for[C 28 H 25 FN 3 O 5 ] + :502.1778,found:502.1782.

[0124] Example 7: (E)-N1-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)pyrrolidine-1,2-dicarboxamide

[0125] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 4-fluoro-2-hydroxyacetophenone, replace morpholine in Example 4 with L-prolineamide, and other steps and operations are similar to those of Examples 1 and 4. Yellow solid, yield: 33%. 1 HNMR (600 MHz, DMSO-d 6 )δ8.44(dd,J=8.9,6.4Hz,1H),8.16(d,J=8.2Hz,1H),7.59(dd,J=9.5,2.4Hz,1H),7.48 (s,1H),7.38–7.32(m,3H),7.29(dd,J=8.6,2.3Hz,1H),7.06(t,J=8.5Hz,1H),7.02(d,J =7.6Hz,1H),6.98(d,J=8.7Hz,3H),6.77(s,1H),4.33–4.27(m,4H),4.18(dd,J=8.5,2.2 Hz,1H),3.52–3.46(m,1H),2.25(d,J=7.1Hz,1H),2.05–2.01(m,1H),1.86–1.83(m,3H). 13 C NMR (151 MHz, DMSO-d 6)δ174.9,165.5,163.8,158.4,154.8,154.7,153.5,151.6,146.7,144.1,13 7.9,137.7,136.5,133.7,130.1,129.8,129.3,128.6,127.3,126.4,126.2,1 24.8,124.6,121.9,119.8,119.7,119.6,118.5,118.2,115.1,114.3,114.1, 105.5,105.3,96.9,64.9,64.5,60.1,46.3,24.4,19.8.HRMS(ESI):m / z[M+H] + calcd.for[C 30 H 28 FN 4 O 5 ] + :543.2044,found:543.2046.

[0126] Example 8: (S,E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3,3-dimethylbutanamide

[0127] Synthesis method: Replace the substituted carboxylic acid fragment (i.e., Boc-glycine) in step 4 of Example 1 with Boc-tert-leucine, and the other steps and operations are similar to those of Example 1. Yellow solid, yield: 48%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.69(s,1H),8.41(dd,J=8.0,1.7Hz,1H),8.29–8.22(m,1H),7.72–7.68(m,1H),7.62(d,J=8.3Hz,1H),7.46(t,J=7.6Hz,1H),7. 40–7.32(m,2H),7.12–7.06(m,2H),7.05(d,J=8.2Hz,1H),7.03–6.98(m,1H),6.63(s,1H),6.12(s,2H),3.05(s,1H),0.88(s,9H). 13 C NMR (151 MHz, DMSO-d 6)δ172.5,158.2,153.8,152.3,150.3,148.5,140.2,133.0,131.5,126.1,125.8,125.1,124.2,123.9, 122.6,121.4,120.4,120.1,118.4,109.2,106.3,102.4,97.1,64.8,34.4,27.1.HRMS(ESI):m / z[M+H] + calcd.for[C 28 H 28 N 3 O 4 ] + :470.2080,found:470.2085.

[0128] Example 9: (S,E)-2-amino-N-(2-((2-(benzo-d[1,3]dioxol-5-yl)-7-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)-3,3-dimethylbutanamide

[0129] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 4-methoxy-2-hydroxyacetophenone, replace the carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-tert-leucine, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 48%. 1 HNMR (600 MHz, DMSO-d 6 )δ9.68(s,1H),8.29(d,J=8.9Hz,1H),8.26–8.21(m,1H),7.38(d,J=1.9Hz,1H),7.35(dd,J=8.3,1.9Hz,1H),7.20(d,J=2. 5Hz,1H),7.10–7.02(m,4H),6.98(dd,J=5.8,3.4Hz,1H),6.58(s,1H),6.12(s,2H),3.91(s,3H),3.06(s,1H),0.88(s,9H). 13 CNMR (151MHz, DMSO-d 6 )δ166.7,165.6,163.9,158.6,158.4,154.8,154.7,146.8,144.1,127.9,127.8,126.6,125.0,124.6 ,119.8,118.5,118.3,116.6,115.1,105.4,105.2,97.4,64.5,61.1,33.3,26.6.HRMS(ESI):m / z[M+H] +calcd.

[0130] for[C 29 H 30 N 3 O 5 ] + :500.2185,found:500.2187.

[0131] Example 10: (E)-2-amino-N-(2-((8-amino-2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide

[0132] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 3-amino-2-hydroxyacetophenone, and the other steps and operations are similar to those of Example 1. Yellow solid, yield: 47%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.22(s,1H),8.20(s,3H),7.98(d,J=8.2Hz,1H),7.84(s,1H),7.77–7.61(m,2H),7.51(s, 1H),7.41(s,2H),7.26(s,1H),7.10(d,J=8.3Hz,1H),6.80(s,1H),6.19(s,2H),3.76(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ170.8,166.1,162.6,162.2,158.8,158.5,152.1,148.9,142.6,141.0,139.5,139.4,139.3,1 26.5,118.5,116.5,114.5,110.3,109.5,107.8,107.7,102.9,96.3,41.4.HRMS(ESI):m / z[M+H] + calcd.for[C 24 H 21 N 4 O 4 ] + :429.1563,found:429.1565.

[0133] Example 11: (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide

[0134] Synthesis method: Replace piperonal in step 1 of Example 1 with 1,4-benzodioxane-6-carboxaldehyde, and the other steps and operations are similar to those of Example 1. Yellow solid, yield: 66%. 1 HNMR (600 MHz, DMSO-d 6 )δ9.98(s,1H),8.60(d,J=8.1Hz,1H),8.17(s,3H),8.01(d,J=8.1Hz,1H),7.96(s,1H),7.68(s,1H),7.53(s,1H),7.48–7.37( m,2H),7.34(s,2H),7.05(d,J=8.6Hz,1H),6.75(s,1H),4.35(dd,J=5.7,2.9Hz,2H),4.31(dd,J=5.6,2.9Hz,2H),3.77(s,2H). 13 C NMR(151MHz,DMSO-d)δ165.9,159.0,158.8,158.5,158.3,154.2,144.3,131.3,130.6,126.9,126.8,126.3, 125.5,120.3,119.2,118.5,118.2,116.3,115.4,114.3,100.2,96.7,65.1,64.5,41.4.HRMS(ESI):m / z[M+H] + calcd.for[C 25 H 22 N 3 O 4 ] + :428.1610,found:428.1614.

[0135] Example 12: (S,E)-2-amino-N-(2-((8-amino-2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0136] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 3-amino-2-hydroxyacetophenone, replace piperonal with 1,4-benzodioxane-6-carboxaldehyde, replace the substituted carboxylic acid fragment in step 4 with Boc-alanine, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 52%. 1 HNMR (600 MHz, DMSO-d 6)δ10.20(s,1H),8.43–8.37(m,1H),7.59(dd,J=7.8,1.6Hz,1H),7.50(d,J=2.3Hz,1H),7.38(dd,J=8.6,2.2Hz,1H),7.11(t,J=7.8Hz,1H),7.09 –7.04(m,2H),7.03–6.99(m,1H),6.99–6.93(m,2H),6.62(s,1H),5.61( s,2H),4.33–4.26(m,4H),3.37(d,J=6.9Hz,1H),1.18(d,J=7.0Hz,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ174.5,157.8,153.1,146.4,144.1,142.1,139.9,138.6,131.7,125.7,125.4,124.0,123.6,122.8 ,120.1,120.0,119.2,118.0,116.5,115.4,111.1,96.4,64.9,64.4,51.5,21.6.HRMS(ESI):m / z[M+H] + calcd.for[C 26 H 25 N 4 O 4 ] + :457.1876,found:457.1879.

[0137] Example 13: (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)acetamide

[0138] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 3-methoxy-2-hydroxyacetophenone, replace piperonal with 1,4-benzodioxane-6-carboxaldehyde, and other steps and operations are similar to those of Example 1. Yellow solid, yield: 58%. 1 HNMR (600 MHz, DMSO-d 6 )δ10.25(s,1H),8.44–8.39(m,1H),8.32(d,J=8.9Hz,1H),7.37–7.33(m,1H),7.31–7.26(m,1H), 7.23–7.20(m,1H),7.08–6.97(m,5H),6.64(s,1H),4.32–4.26(m,4H),3.91(s,3H),3.19(s,2H).13 C NMR (151 MHz, DMSO-d 6 )δ171.7,163.2,158.2,155.3,152.1,146.5,144.1,139.4,131.8,126.3,125.1,124.1,12 3.5,120.2,119.7,118.9,118.2,115.8,115.0,114.6,101.5,96.5,64.9,64.5,56.4,45.7.

[0139] Example 14: (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)acetamide

[0140] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 3-fluoro-2-hydroxyacetophenone, replace piperonal with 1,4-benzodioxane-6-carboxaldehyde, and other steps and operations are similar to those in Example 1. Yellow solid, yield: 51%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.26(s,1H),8.49–8.41(m,2H),7.58(dd,J=9.5,2.6Hz,1H),7.35–7.29(m,2H),7.27(dd,J=8.6,2.3Hz,1H),7.12

[0141] –7.07(m,2H),7.03(dd,J=7.6,1.8Hz,1H),6.97(d,J=8.5Hz,1H),6.68(s,1H),4.33–4.25(m,4H),3.20(s,2H),2.20(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ

[0142] 171.7,165.5,163.8,158.5,154.8,154.7,151.6,146.7,144.1,138.9,131.8,127.5,127.4,124.7,124.5,123.6,1 20.1,119.8,119.5,119.0,118.2,115.1,114.1,113.9,105.5,105.3,96.8,64.9,64.5,45.7.HRMS(ESI):m / z[M+H] + calcd.

[0143] for[C25 H 21 FN 3 O 4 ] + :446.1516,found:446.1518.

[0144] Example 15: (E)-3-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0145] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 3-fluoro-2-hydroxyacetophenone, replace piperonal with 1,4-benzodioxane-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-β-alanine, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 59%. 1 HNMR (600 MHz, DMSO-d 6 )δ9.70(s,1H),8.43(dd,J=8.9,6.5Hz,1H),8.06(d,J=7.8Hz,1H),7.58(dd,J=9.6,2.6Hz,1H),7.34–7.28(m,2H),7.23(dd,J=8.5,2.3Hz,1H),7. 14–7.05(m,2H),6.97(d,J=8.5Hz,1H),6.94(dd,J=7.6,1.9Hz,1H),6.47 (s,1H),4.31–4.27(m,4H),2.84(t,J=6.5Hz,2H),2.44(t,J=6.5Hz,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ170.3,165.4,163.7,158.5,158.3,157.9,154.7,154.6,151.6,146.6,144.1,141.4,131.0,128.0,127.9,124.8,124.6,123.9,12 2.9,120.7,119.7,119.5,118.7,118.2,116.7,115.0,113.8,113.7,105.3,105.1,97.2,64.9,64.5,37.1,37.0.HRMS(ESI):m / z[M+H] + calcd.for[C 26 H 23 FN 3 O 4 ] +:460.1673,found:460.1676.

[0146] Example 16: (E)-3-amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0147] Synthesis method: replace piperonal in step 1 of Example 1 with 2-aminobenzoxazole-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-β-alanine, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 66%. 1 HNMR (400 MHz, DMSO-d 6 )δ9.17(s,1H),8.41(dd,J=8.0,1.6Hz,1H),7.99–7.92(m,1H),7.81(d,J=1.7 Hz,3H),7.74–7.68(m,1H),7.65(dd,J=8.4,1.3Hz,1H),7.55(dd,J=8.3,1.8H z,2H),7.48–7.43(m,1H),7.28(d,J=8.3Hz,1H),7.14–7.09(m,2H),6.99(dd, J=7.7,1.7Hz,1H),6.54(s,1H),2.96(t,J=7.0Hz,2H),2.63(t,J=7.0Hz,2H). 13 CNMR (101MHz, DMSO-d 6 )δ169.0,164.9,158.7,158.5,158.3,153.8,152.5,148.8,147.4,142.3,132.9,130.3,125.6,125.5,125.3,123 .9,123.7,122.7,122.6,122.2,121.0,119.3,118.3,116.3,115.7,106.4,97.1,35.8,34.0.HRMS(ESI):m / z[M+H] + calcd.for[C 25 H 21 N 5 O 3 ] + :440.1723,found:440.1725.

[0148] Example 17: (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methoxypropanamide

[0149] Synthesis method: replace piperonal in step 1 of Example 1 with 2-aminobenzoxazole-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-3-methoxypropionic acid, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 61%. 1 HNMR (400 MHz, DMSO-d 6 )δ8.92(s,1H),8.41(dd,J=8.0,1.7Hz,1H),8.05(dd,J=7.8,1.8Hz,1H),7.84–7.77(m,3H),7.65(d,J=8.2Hz,1H),7.57(dd,J=8.3,1.8Hz,1H) ,7.51–7.43(m,1H),7.28(d,J=8.3Hz,1H),7.14–7.09(m,2H),7.02–6.9 5(m,1H),6.57(s,1H),3.48(t,J=6.0Hz,2H),3.02(s,3H),2.49(s,2H). 13 C NMR (101 MHz, DMSO-d 6 )δ169.8,164.9,153.9,148.8,147.5,133.0,130.9,125.6,125.4,124.8,123.9, 122.7,120.8,118.3,115.7,106.5,97.0,68.6,58.2,37.5.HRMS(ESI):m / z[M+H] + calcd.for[C 26 H 23 N 4 O 4 ] + :455.1719,found:455.1722.

[0150] Example 18: (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methylaminopropanamide

[0151] Synthesis method: replace piperonal in step 1 of Example 1 with 2-aminobenzoxazole-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-3-methylaminopropionic acid, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 58%. 1 HNMR (600 MHz, DMSO-d 6)δ9.31(s,1H),8.43–8.39(m,1H),7.99(d,J=7.4Hz,1H),7.82–7.78(m,3H),7.73 –7.66(m,1H),7.65(d,J=8.3Hz,1H),7.55(dd,J=8.3,1.8Hz,1H),7.46(t,J=7.5Hz ,1H),7.28(d,J=8.3Hz,1H),7.15(t,J=7.5Hz,1H),7.10(t,J=7.7Hz,1H),6.98(d, J=7.6Hz,1H),6.55(s,1H),2.98(t,J=6.6Hz,2H),2.67–2.61(m,2H),2.41(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ169.1,164.9,158.6,158.4,158.2,153.9,152.6,148.8,147.4,142.2,132.9,130.4,125.6,125.5,125.2,123.9, 123.7,123.4,122.7,122.6,120.9,118.8,118.3,116.8,115.8,106.4,97.0,45.2,33.5,32.9.HRMS(ESI):m / z[M+H] + calcd.for[C 26 H 24 N 5 O 3 ] + :454.1879,found:454.1882.

[0152] Example 19: (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methylsulfonamidopropionamide

[0153] Synthesis method: replace piperonal in step 1 of Example 1 with 2-aminobenzoxazole-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with 3-methylsulfonylaminopropionic acid, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 43%. 1 HNMR (400 MHz, DMSO-d 6)δ8.99(s,1H),8.44(d,J=7.7Hz,1H),7.97–7.93(m,1H),7.82(s,3H),7.74(s,1H),7.68(s,1H),7.59(d,J=8.3Hz,1H),7.48(s,1H),7. 28(d,J=8.3Hz,1H),7.16(d,J=9.3Hz,2H),6.99(q,J=5.9Hz,2H),6.61–6.54(m,1H),3.16–3.11(m,2H),2.81(s,3H),2.52–2.51(m,2H). 13 C NMR (101 MHz, DMSO-d 6 )δ168.9,164.9,158.4,156.9,152.5,148.8,148.4,147.3,142.4,130.2,125.3,124.0,123.8,123 .6,123.3,122.5,121.1,119.8,115.7,106.5,106.3,96.3,46.1,35.7,33.8.HRMS(ESI):m / z[M+H] + calcd.for[C 26 H 24 N 5 O 5 S] + :518.1498,found:518.1499.

[0154] Example 20: (E)-3-amino-N-(2-((2-(2-ethylaminobenzo[d]oxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0155] Synthesis method: replace piperonal in step 1 of Example 1 with 2-ethylaminobenzoxazole-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-β-alanine, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 59%. 1 HNMR (400 MHz, DMSO-d 6)δ9.64(s,1H),8.41(dd,J=7.9,1.7Hz,1H),8.06(dd,J=7.7,1.7Hz,1H),7.81(d,J=1.7Hz, 1H),7.72–7.67(m,1H),7.63(dd,J=8.4,1.3Hz,1H),7.56(dd,J=8.3,1.8Hz,1H),7.45–7.41 (m,1H),7.31(d,J=8.3Hz,1H),7.13–7.07(m,2H),6.97(dd,J=7.5,1.9Hz,1H),6.56(s,1H) ,3.36(q,J=7.2Hz,2H),2.83(t,J=6.6Hz,2H),2.46(t,J=6.6Hz,2H),1.19(t,J=7.2Hz,3H). 13 C NMR (101 MHz, DMSO-d 6 )δ170.2,164.2,158.5,153.8,152.4,148.9,147.1,141.8,132.8,130.9,125.6,125.5,124.7,123.9 ,123.7,122.9,122.6,120.7,118.3,115.8,106.4,97.0,37.7,37.1,37.0,15.0.HRMS(ESI):m / z[M+H] + calcd.for[C 27 H 26 N 5 O 3 ] + :468.2036,found:468.2039.

[0156] Example 21: (E)-3-amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-6-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0157] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 5-fluoro-2-hydroxyacetophenone, replace piperonal with 2-aminobenzoxazole-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-β-alanine, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 31%. 1 HNMR (400 MHz, DMSO-d 6)δ9.27(s,1H),8.11(dd,J=9.3,3.2Hz,1H),8.00(d,J=7.8Hz,1H),7.84–7.79(m,2H),7.74(dd,J=9.1,4.6Hz,1H),7.65–7.52(m,2H ),7.28(d,J=8.3Hz,1H),7.20–7.06(m,3H),7.00(dd,J=7.5,1.8Hz,1H),6.56(s,1H),2.94(t,J=6.8Hz,2H),2.62(t,J=6.8Hz,2H). 13 C NMR (101

[0158] MHz,DMSO-d 6 )δ169.3,164.9,160.7,158.8,158.3,151.9,150.2,148.8,147.6,141.6,130.6,125.1,124.2, 123.9,123.6,123.4,122.7,120.8,120.4,115.7,106.4,96.3,36.0,34.6.HRMS(ESI):m / z[M+H] + calcd.for[C 25 H 20 FN 5 O 3 ] + :458.1628,found:458.1625.

[0159] Example 22: (E)-3-amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-6-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)propanamide

[0160] Synthesis method: replace o-hydroxyacetophenone in step 1 of Example 1 with 5-methoxy-2-hydroxyacetophenone, replace piperonal with 2-aminobenzoxazole-6-carboxaldehyde, replace the substituted carboxylic acid fragment (i.e. Boc-glycine) in step 4 with Boc-β-alanine, and the other steps and operations are similar to those in Example 1. Yellow solid, yield: 29%. 1 HNMR (400 MHz, DMSO-d 6)δ9.14(s,1H),7.94(d,J=8.0Hz,1H),7.83(d,J=3.2Hz,1H),7.81–7.77(m,3H),7 .62(d,J=9.1Hz,1H),7.53(dd,J=8.3,1.8Hz,1H),7.32(dd,J=9.1,3.2Hz,1H),7. 27(d,J=8.3Hz,1H),7.17(t,J=7.4Hz,1H),7.13–7.09(m,1H),7.00(dd,J=7.7,1. 7Hz,1H),6.50(s,1H),3.89(s,3H),2.95(t,J=6.9Hz,2H),2.64(t,J=7.1Hz,2H). 13 C NMR (101 MHz, DMSO-d 6 )δ

[0161] 168.9,164.9,158.4,156.9,152.5,148.8,148.4,147.3,142.4,130.2,125.3,124.0,123.8,123. 6,123.3,122.5,121.1,119.8,115.7,106.5,106.3,96.3,56.2,35.7,33.8.HRMS(ESI):m / z[M+H] + calcd.for[C 26 H 23 N 5 O 4 ] + :470.1828,found:470.1830.

[0162] The structural formulas of the compounds synthesized in Examples 1 to 22 are shown in Table 1:

[0163] Table 1

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] The following experiments are used to illustrate the beneficial effects and applications of the compounds represented by the general formula (I) of the present invention.

[0170] In vitro mTOR kinase inhibition assay:

[0171] The compounds of the present invention inhibit the activity of mTOR kinase, thereby inhibiting the transduction of cell signaling pathways, thereby affecting the cell cycle and cell proliferation. The inhibitory effect of such compounds on mTOR kinase is evaluated by the following Lance Ultra fluorescence test method.

[0172] Detection principle: Lance Ultra fluorescence assay is a homogeneous non-radioactive detection method that quantitatively determines the activity of purified kinase by detecting the ATP content in the system after the kinase reaction. The ATP content is determined by Mg 2+ The quantification is based on the light intensity produced by the oxidation of firefly luciferin catalyzed by ATP and oxygen. A certain amount of ATP is added to the reaction system. The kinase reaction consumes ATP, and the remaining ATP can react with the firefly luciferase in the Kinase Glo reagent to emit light, thereby quantitatively detecting the amount of remaining ATP and indirectly determining the activity of the reaction kinase.

[0173] Detection method: First, prepare 1×kinase buffer, which contains 50mM HEPES, PH 7.5, 1mM EGTA, and 0.01% Tween-20; dissolve the compound with 100% DMSO and perform gradient dilution, transfer 10nL of the diluted compound to the detection plate, and prepare a control group without compound and a blank control group without kinase at the same time. Add 1×kinase buffer to mTOR to prepare kinase solution, take 5μL and add it to the detection plate and vortex to mix. In addition, prepare 1×kinase reaction buffer containing 4E-BP1 (Thr37 / 46, PE) peptide and ATP substrate, take 5μL and add it to the well plate to start the reaction. After reacting for 1h at room temperature, take 10μL of PBS buffer containing EDTA and Eu-anti-P-4E-BP1 (Thr 37 / 46, PE) antibody and add it to the well plate. Incubate for 60min at room temperature, read the well plate and calculate the inhibition rate of the compound on mTOR kinase. Substitute the inhibition rate and the corresponding concentration into

[0174] GraphPadPrism software was used for curve fitting and IC 50 The test results are shown in Table 2. From the activity data in Table 2 below, it can be seen that the compounds of the present invention can effectively inhibit mTOR kinase and have good inhibitory activity against it. The inhibitory activity of mTOR kinase (IC 50) are all at the nanomolar level (1nm-999nm). Among them, the compounds with higher kinase inhibitory activity are Example 16 and Example 22, and their IC 50 The values ​​are 48 nM and 49 nM, respectively. Therefore, the compounds of the present invention have positive and foreseeable clinical application value in anti-proliferative diseases, especially anti-tumor, and have good development prospects.

[0175] Cell viability assay:

[0176] The Cell Counting Kit (CCK-8) method was used to evaluate the inhibitory activity of the compounds on cell proliferation. The half inhibitory concentration IC was determined by single-concentration activity screening and multiple-concentration determination. 50 The detection principle is that the CCK-8 reagent contains WST-8, which is reduced to a highly water-soluble yellow formazan product (Formazan) by the dehydrogenase in the cell mitochondria under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells. The specific operation is as follows: (1) Inoculation of cells: Use culture medium containing 10% fetal bovine serum to prepare cells into a single cell suspension, and inoculate 90μL 5×10 in each well of a 96-well plate. 4 / mL of adherent cells and 9×10 4 / mL suspended cells in 5% CO 2 , pre-cultured at 37°C for 24 h. (2) Add the sample solution to be tested: add 10 μL of sample solution to each well. For the initial screening of activity, set one concentration for each sample and set up 3 replicate wells; IC 50 8 concentrations (including 0 concentration) were measured, and 3 replicate wells were set for each concentration; the cells were placed in an incubator and cultured for 48 hours. The experiment set up a blank group (Blank), a control group (Control), and a drug group (Drug). (3) Color development: For adherent cells, the old culture medium and drug solution were aspirated (10 μL CCK-8 solution was directly added to suspended cells), and 100 μL CCK-8 solution diluted ten times was added to each well. The cells were incubated at 37°C and 5% CO 2 Continue to culture for 1-4 hours (avoid light and observe in real time). (4) Detection: Use an enzyme-labeled instrument to measure the absorbance at 450nm and record the raw data results. (5) Use Excel software to standardize the raw data and calculate the cell proliferation inhibition rate based on the OD value of each well in the initial screening (formula = (OD Control -OD Drug ) / (OD Control -OD Blank )×100%), and the inhibition rate was calculated. IC 50The results were calculated using GraphPad Prism 8 (version 8.0.2, GraphPad Software Inc), and expressed as ±SD. (6) Positive control: Doxorubicin hydrochloride (Dox).

[0177] The compounds Example 16 and Example 22 with the best kinase inhibitory activity were selected for cell activity assay. First, the proliferation inhibitory activity of the compounds Example 16 and Example 22 on 6 cell lines was measured. The results are shown in Table 3. The compounds Example 16 and Example 22 had high inhibitory activity on the six cell lines HCT 116, SF126, PATU8988T, 5637, CAL-62 and A-673, and the inhibition rates were all greater than 90%. Then, the IC values ​​of the compounds Example 16 and Example 22 in the six cell lines were measured. 50 The results are shown in Table 4. The IC values ​​of Example 16 for six cell lines: HCT 116, SF126, PATU8988T, 5637, CAL-62 and A-673 50 The values ​​were 2.34 μM, 1.84 μM, 2.70 μM, 1.74 μM, 1.40 μM and 8.06 μM, respectively. Example 22 IC values ​​for six cell lines: HCT 116, SF126, PATU8988T, 5637, CAL-62 and A-673 50 The values ​​were 3.32 μM, 2.56 μM, 1.96 μM, 4.86 μM, 1.37 μM and 9.11 μM, respectively. This shows that Example 16 and Example 22 have good inhibitory activity against the six cell lines. The above results all show that the novel selective mTOR inhibitor in this study can be used for anti-tumor research.

[0178] Table 2 In vitro inhibitory activity of target compounds against mTOR kinase

[0179]

[0180] Table 3 Antiproliferative activity of Example 16 and Example 22 against 6 cancer cells

[0181]

[0182] aStandard deviation of three measurements.

[0183] b Doxorubicin hydrochloride (Dox) was administered at a concentration of 10 μM.

[0184] c Example 16 and Example 22 were administered at a concentration of 20 μM.

[0185] Table 4 IC values ​​of Example 16 and Example 22 for 6 cell lines 50 value

[0186]

[0187] aStandard deviation of three measurements.

[0188] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A chromone mTOR inhibitor, characterized in that Including compounds, stereoisomers or pharmaceutically acceptable salts of general formula (I), wherein the structure of general formula (I) is as follows: Wherein, in the general formula (I): R 1 Can be independently selected from the following structures: R 2 Can be independently selected from the following structures: R 3 Can be independently selected from the following structures: H, CH3-O-; R 4 Can be independently selected from the following structures: H, CH3-O-.

2. A chromone mTOR inhibitor, characterized in that: The chromone mTOR inhibitor is selected from at least one of the following compounds: (1) (E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide; (2) (S,E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide; (3) (E)-3-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide; (4) (E)-N-(2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)morpholine-4-carboxamide; (5) (E)-N-(2-((2-(Benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)piperazine-1-carboxamide; (6) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)morpholine-4-carboxamide; (7) (E)-N1-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)pyrrolidine-1,2-dicarboxamide; (8) (S,E)-2-amino-N-(2-((2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3,3-dimethylbutanamide; (9) (S,E)-2-amino-N-(2-((2-(benzo-d[1,3]dioxol-5-yl)-7-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)-3,3-dimethylbutanamide; (10) (E)-2-amino-N-(2-((8-amino-2-(benzo[d][1,3]dioxol-5-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide; (11) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)acetamide; (12) (S,E)-2-amino-N-(2-((8-amino-2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide; (13) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)acetamide; (14) (E)-2-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)acetamide; (15) (E)-3-amino-N-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-7-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)propanamide; (16) (E)-3-amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide; (17) (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methoxypropanamide; (18) (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methylaminopropanamide; (19) (E)-N-(2-((2-(2-aminobenzoxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)-3-methylsulfonamidopropionamide; (20) (E)-3-amino-N-(2-((2-(2-ethylaminobenzo[d]oxazol-6-yl)-4H-benzopyran-4-ylidene)amino)phenyl)propanamide; (21)(E)-3-amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-6-fluoro-4H-benzopyran-4-ylidene)amino)phenyl)propanamide; (22) (E)-3-Amino-N-(2-((2-(2-aminobenzoxazol-6-yl)-6-methoxy-4H-benzopyran-4-ylidene)amino)phenyl)propanamide.

3. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a therapeutically effective dose of at least one chromone mTOR inhibitor according to any one of claims 1-2.

4. Use of the chromone mTOR inhibitor according to any one of claims 1 to 2 or the pharmaceutical composition according to claim 3 in the preparation of a drug for preventing and / or treating and / or assisting in treating a proliferative disease caused by the action of mTOR kinase.

5. The use according to claim 4, characterized in that: The proliferative diseases caused by the action of mTOR kinase are colorectal cancer, gastric cancer, breast cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, thyroid cancer, bladder cancer, kidney cancer, brain tumor, cervical cancer, esophageal cancer, gallbladder cancer, CNS cancer, malignant glioma, or myeloproliferative disease, leukemia and lymphoma.

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