A benzoxazine-2-thionetobenzimidazole compound, its preparation method and medical use

Abstract

This invention provides a class of benzoxazine-2-thionobenzimidazole compounds, belonging to the fields of medicinal chemistry and pharmacotherapeutic science, specifically compounds, isomers, or pharmaceutically acceptable salts of Formula I. Pharmacological experiments show that the compounds of this invention possess good in vitro anti-AML cell proliferation activity. In this invention, the preparation method of the above compounds is simple, the reaction raw materials are inexpensive and readily available, and the cost is low.

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and pharmacotherapeutic science, specifically relating to a class of benzoxazine-2-thionobenzimidazole compounds. These compounds can be used to prepare drugs against acute myeloid leukemia. This invention also relates to methods for preparing these compounds and pharmaceutical combinations containing them. Background Technology

[0002] 1,3-Benzoxazinone compounds typically possess a wide range of biological activities, such as bactericidal, herbicidal, insecticidal, anti-inflammatory, inhibition of various human proteases, and anticancer effects. Therefore, the synthesis and application of 1,3-benzoxazinones have attracted widespread attention. In 2006, Gurram R. Madhavan et al. synthesized derivatives of 1,3-benzoxazinones and found that they could act as effective dual PPAR-α and PPAR-γ activators, significantly reducing plasma glucose (Bioorganic & Medicinal Chemistry, 2006, 14, 584-591). In 2010, Karel Waisser et al. synthesized 3-benzyl-1,3-benzoxazine-2,4-dione compounds, which exhibited activity against INH-resistant strains *Mycobacterium kansas* CNCTC My 235 / 80 and *Mycobacterium avium* CNCTC My 330 / 88 (European Journal of Medicinal Chemistry, 2010, 45, 2719-2725). In 2016, Jing Sun et al. synthesized 3-benzyl-1,3-benzoxazine-2,4-dione, which exhibits good antiviral activity and can be used as a MEK1 inhibitor (Bioorganic & medicinal chemistry, 2016, 24, 3472-3482). In 2017, Mariateresa Badolato et al. synthesized pyrrolobenzoxazine one derivatives and found that they have the activity of regulating CD1 levels in MCF-7 breast cancer cells (Bioorganic & medicinal chemistry letters, 2017, 27, 3092-3095). In 2018, Eman A et al. synthesized benzoxazine one derivatives and reported that they showed high antitumor and cytotoxic activity against human breast cancer cells (Journal of Heterocyclic Chemistry, 2018, 55, 1223-1231). In 2020, Ahmed Mekabaty et al. synthesized a 1,3-benzoxazinone derivative and found that it exhibited strong cytotoxicity against MCF-7 cells but less cytotoxicity against RPE-1 cells (Journal of Heterocyclic Chemistry, 2020, 57:1123-1132). Also in 2020, Alessio Malacrid et al. reported the discovery of MV1035, a small molecule of imidazole benzoxazine-5-thione, through computer-aided drug design. This small molecule exhibits selective inhibitory activity against ALKBH5.MV1035 can reduce the migration and invasion of U-87MG glioblastoma cells by selectively inhibiting ALKBH5 (Bioorganic & Medicinal Chemistry, 2020, 28, 115300).

[0003] Currently, there are no reports of compounds combining the 1,3-benzoxazinethion ring and the benzimidazole ring, nor are there any studies exploring the biological activities of such compounds. Summary of the Invention

[0004] The purpose of this invention is to provide a class of benzoxazine-2-thionobenzimidazole compounds based on the prior art, using MV1035 as the lead compound. Pharmacological experiments have shown that this class of compounds has good in vitro anti-AML (acute myeloid leukemia) cell proliferation activity, which is superior to the lead compound MV1035.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned benzoxazine-2-thiononebenzimidazole compounds.

[0006] A third object of the present invention is to provide the pharmaceutical use of the above-mentioned benzoxazine-2-thionone-benzimidazole compounds.

[0007] The technical solution of the present invention is as follows:

[0008]

[0009] Compounds, isomers, or pharmaceutically acceptable salts of Formula I

[0010] in,

[0011] R 1 Represents hydrogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 aldehyde, nitro, cyano, hydroxyl, or halogen;

[0012] R 2 It represents hydrogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 aldehyde, nitro, cyano, hydroxyl, or halogen.

[0013] In a preferred embodiment, R 1 It represents hydrogen, methyl, ethyl, methoxy, ethoxy, formaldehyde, acetaldehyde, fluorine, chlorine, or bromine.

[0014] In a more preferred embodiment, R 1 It represents hydrogen, methyl, ethoxy, fluorine, chlorine, or bromine.

[0015] In a preferred embodiment, R 2 It represents hydrogen, methyl, ethyl, methoxy, ethoxy, formaldehyde, acetaldehyde, fluorine, chlorine, or bromine.

[0016] In a more preferred embodiment, R 2 It represents hydrogen or methyl.

[0017] Furthermore, the compound represented by Formula I is selected from the following compounds:

[0018]

[0019] In this invention, the compounds are named as follows: 1,3-benzoxazine-2-thionobenzimidazole (compound 1), 6-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 2), 6-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 3), 6-chloro-1,3-benzoxazine-2-thionobenzimidazole (compound 4), 8-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 5), 6-fluoro-1,3-benzoxazine-2-thionobenzimidazole (compound 6), 6-ethoxy-1,3-benzoxazine-2-thionobenzimidazole (compound 7), and 6-bromo-9-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 8) on cell proliferation ability. 8-Bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 5), 6-bromo-9-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 8).

[0020] This invention utilizes substituted salicylaldehyde and substituted o-phenylenediamine to synthesize substituted-2-benzimidazole phenol intermediate, and then uses substituted-2-benzimidazole phenol and 1,1'-thiocarbonyldiimidazole to synthesize benzoxazine-2-thiononebenzimidazole compounds. Through detection and experiments, pharmacological experiments have shown that it has good in vitro anti-AML cell proliferation activity.

[0021] The present invention also discloses a method for preparing the compound of formula I, which includes the following steps:

[0022] Step 1: In the presence of ammonium acetate catalyst, compound III and compound IV undergo a chemical reaction to prepare intermediate compound II;

[0023] Step 2: Compound II is chemically reacted with 1,1'-thiocarbonyldiimidazole to prepare the compound described in Formula I. The specific synthetic route is as follows:

[0024]

[0025] In this invention, in the first step, the molar ratio of compound III to compound IV is 1:1.0-2.0, preferably 1:1.2-1.5; more preferably 1:1.5.

[0026] In the first step, the molar ratio of compound III to the catalyst is 1:2-6, preferably 1:3-5; more preferably 1:5.

[0027] In the first step, the reaction temperature is 70℃-100℃, preferably 80℃-90℃, and more preferably 80℃.

[0028] In the first step, the reaction time is 4-16 hours, preferably 8-12 hours, and more preferably 8 hours.

[0029] In the first step, during the reaction, the selected organic solvent is one or more of N,N-dimethylformamide, N,N-dimethylacetamide, or methanol, preferably N,N-dimethylformamide.

[0030] In this invention, in the second step, the molar ratio of compound II to 1,1'-thiocarbonyldiimidazole is 1:2-6, preferably 1:3-5; more preferably 1:5.

[0031] In the second step, the reaction temperature is 80℃-110℃, preferably 90℃-100℃, and more preferably 90℃.

[0032] In the second step, the reaction time is 8-18 hours, preferably 8-12 hours, and more preferably 12 hours.

[0033] In the second step, during the reaction, the selected organic solvent is one or more of tetrahydrofuran, toluene, or chloroform, preferably tetrahydrofuran.

[0034] In this invention, these intermediates or target compounds can be purified using conventional separation techniques and, if necessary, converted into addition salts with pharmaceutically acceptable acids.

[0035] The present invention also provides a pharmaceutical composition having the above-mentioned compound, isomer or pharmaceutically acceptable salt thereof as the active ingredient or main active ingredient, supplemented with pharmaceutically acceptable excipients.

[0036] The compound isomers of the present invention or their pharmaceutically acceptable salts can be used in the preparation of drugs against acute myeloid leukemia, and have good in vitro anti-AML (acute myeloid leukemia) cell proliferation activity.

[0037] Unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below:

[0038] "Pharmaceutically acceptable salts" refer to those salts that retain the bioavailability and properties of the parent compound. These salts include:

[0039] (1) It forms salts with acids, which are obtained by reacting the free base of the parent compound with inorganic or organic acids. Inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid and perchloric acid, etc. Organic acids include acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, ascorbic acid, camphoric acid, benzoic acid, hydroxybenzoic acid, γ-hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, dodecyl sulfate, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid, glutaric acid or malonic acid, etc.

[0040] (2) Salts formed by replacing acidic protons in the parent compound with metal ions or by coordinating with organic bases. Examples of metals include alkali metal ions, alkaline earth metal ions, or aluminum ions. Examples of organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, quinine, etc.

[0041] "Pharmaceutical composition" refers to the mixing of one or more of the compounds of this invention, or their pharmaceutically acceptable salts, solvates, hydrates, or prodrugs, with other chemical components, such as a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate the administration of the drug to animals.

[0042] "Pharmaceutical carrier" or "pharmaceuticalally acceptable carrier" refers to an inactive component in a pharmaceutical composition that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound, such as, but not limited to: calcium carbonate, calcium phosphate, various sugars (e.g., lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic polymers or methacrylic polymers, gels, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, etc.

[0043] "alkyl" refers to a saturated aliphatic hydrocarbon group with 1-20 carbon atoms, including straight-chain and branched groups (the numerical range mentioned in this application, such as "1-20", refers to the group, which is an alkyl group and may contain 1, 2, 3, etc., up to 20 carbon atoms). More preferably, the alkyl group is a medium-sized alkyl group with 1-10 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, etc. Preferably, the alkyl group is a lower alkyl group with 1-8 or 1-6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, or tert-butyl, etc. The alkyl group may be substituted or unsubstituted. When it is a substituted alkyl group, the substituent is preferably one or more, more preferably 1-3, and most preferably 1 or 2 substituents.

[0044] “Cyano” represents the -CN group.

[0045] “Nitro” represents the -NO2 group.

[0046] "Hydroxy" represents the -OH group.

[0047] "Alkoxy" refers to -O- (unsubstituted alkyl) and -O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy.

[0048] "Halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine or bromine.

[0049] The advantages of using the technical solution of this invention are as follows:

[0050] This invention provides a class of benzoxazine-2-thionobenzimidazole compounds, which, according to pharmacological experiments, exhibit good in vitro anti-AML cell proliferation activity. The preparation method of these compounds is simple, using inexpensive and readily available reactants, resulting in low cost. Detailed Implementation

[0051] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0052] Example 1: Synthesis of 1,3-benzoxazine-2-thionobenzimidazole (compound 1)

[0053] Salicylic aldehyde (1 mL, 10.04 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and o-phenylenediamine (1.63 g, 15.07 mmol) and ammonium acetate (3.87 g, 50.2 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for about 8 h to complete the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(1-benzimidazol-2-yl)phenol as a white solid. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1-4:1, V / V) yielded the intermediate 2-(1-benzimidazol-2-yl)phenol (1.39 g, 6.34 mmol), with a yield of 63.2%.

[0054] The above-mentioned product, 2-(1-benzimidazol-2-yl)phenol (200 mg, 0.95 mmol), was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF). 1,1'-thiocarbonyldiimidazole (TCDI, 941 mg, 5.28 mmol) was added, and the mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, resulting in the precipitation of a large amount of yellow solid. This was filtered to obtain a yellow crude product. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-8:1, V / V) yielded 161 mg of the target product, 1,3-benzoxazine-2-thionobenzimidazole (compound 1), with a melting point (mp) of 255.3-258.1 °C and a yield of 67.6%.

[0055] 1 H NMR(400MHz,Chloroform-d)δppm:9.08(d,J=7.6Hz,1H),8.40(dd,J=8.0,1.6Hz,1H,Ar-H),7.94-7.87(m ,1H,Ar-H),7.68(ddd,J=8.6,7.2,1.6Hz,1H,Ar-H),7.58(td,J=7.6,1.4Hz,1H,Ar-H),7.57-7.46(m,3H). 13 C NMR(101MHz,Chloroform-d)δppm:174.1,152.1,144.5,143.1,133.4,131.9,12 7.3,126.7,125.6,125.1,120.2,116.8,116.5,113.0.ESI-HRMS(TOF):m / z[M+H] + calcd for C 14 H8N2OS, 253.0430, found 253.0434; HPLC purity95.28%.

[0056] Example 2: Synthesis of 6-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 2)

[0057] 5-Bromosalicylaldehyde (1.01 g, 5.02 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and o-phenylenediamine (815 mg, 7.54 mmol) and ammonium acetate (1.95 g, 25.10 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for approximately 8 h to terminate the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(1-benzimidazol-2-yl)-6-bromophenol as a white solid. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1-4:1, V / V) yielded the intermediate 2-(1-benzimidazol-2-yl)-6-bromophenol (460 mg, 1.59 mmol), with a yield of 31.7%.

[0058] The above product, 2-(1-benzimidazol-2-yl)-6-bromophenol (200 mg, 0.69 mmol), was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF). 1,1'-thiocarbonyldiimidazole (TCDI, 614 mg, 3.45 mmol) was added, and the mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, resulting in the precipitation of a large amount of yellow solid. This was filtered to obtain a crude yellow product. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-8:1, V / V) yielded 7.97 mg of the target product, 6-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 2), with a melting point (mp) of 226.8-230.4 °C and a yield of 3.5%.

[0059] 1 H NMR (400MHz, Chloroform-d) δppm: 9.05 (d, J=8.4Hz, 1H, Ar-H), 8.53 (d, J=2.2Hz, 1H, Ar-H), 7.93-7.86 ( m,1H,Ar-H),7.75(dd,J=8.8,2.4Hz,1H,Ar-H),7.64-7.50(m,2H,2×Ar-H),7.39(d,J=8.8Hz,1H,Ar-H). 13 C NMR(101MHz,Chloroform-d)δppm:173.2,150.9,144.3,141.7,136.3,131.9,12 7.6,127.6,126.1,120.5,119.7,118.2,116.8,114.5.ESI-HRMS(TOF):m / z[M+H] + calcd for C 14H7N2BrOS,330.9541,found330.9555; HPLC purity 96.15%.

[0060] Example 3: Synthesis of 6-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 3)

[0061] 5-Methylsalicylaldehyde (1.30 g, 9.56 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and o-phenylenediamine (1.24 g, 11.47 mmol) and ammonium acetate (3.68 g, 47.80 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for approximately 8 h to complete the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(1-benzimidazol-2-yl)-6-methylphenol as a white solid. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1-4:1, V / V) yielded the intermediate 2-(1-benzimidazol-2-yl)-6-methylphenol (870 mg, 3.88 mmol), with a yield of 40.6%.

[0062] The above product, 2-(1-benzimidazol-2-yl)-6-methylphenol (200 mg, 0.89 mmol), was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF). 1,1'-thiocarbonyldiimidazole (TCDI, 758 mg, 4.25 mmol) was added, and the mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, resulting in the precipitation of a large amount of yellow solid. This was filtered to obtain a yellow crude product. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-8:1, V / V) yielded 130 mg of the target product, 6-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 3), with a melting point (mp) of 263.2-265.1 °C and a yield of 54.9%.

[0063] 1 H NMR (400MHz, Chloroform-d) δppm: 9.05 (d, J = 7.2Hz, 1H, Ar-H), 8.21 -8.08(m,1H,Ar-H),7.90-7.79(m,1H,Ar-H),7.61-7.40(m,3H,3×Ar-H),7.36(d,J=8.4Hz,1HAr-H),2.46(s,3H,-CH3). 13C NMR(101MHz,Chloroform-d)δppm:174.1,150.4,144.4,143.2,136.9,134.4,131.9 ,127.2,125.5,124.8,120.1,116.8,116.2,112.4,21.1.ESI-HRMS(TOF):m / z[M+H] + calcd for C 15 H 10 N2OS, 267.0587, found 267.0574; HPLC purity96.30%.

[0064] Example 4: Synthesis of 6-chloro-1,3-benzoxazine-2-thionobenzimidazole (compound 4)

[0065] 5-Chlorosalicylic acid (1.50 g, 9.61 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and o-phenylenediamine (1.25 g, 11.54 mmol) and ammonium acetate (3.68 g, 47.80 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for approximately 8 h to complete the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(1-benzimidazol-2-yl)-6-chlorophenol as a white solid. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1-4:1, V / V) yielded the intermediate 2-(1-benzimidazol-2-yl)-6-chlorophenol (750 mg, 3.06 mmol), with a yield of 31.8%.

[0066] The above product, 2-(1-benzimidazol-2-yl)-6-chlorophenol (200 mg, 0.82 mmol), was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF). 1,1'-thiocarbonyldiimidazole (TCDI, 727 mg, 4.08 mmol) was added, and the mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, resulting in the precipitation of a large amount of yellow solid. This was filtered to obtain a yellow crude product. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-8:1, V / V) yielded 155 mg of the target product, 6-chloro-1,3-benzoxazine-2-thionobenzimidazole (compound 4), with melting points (mp): 278.4-281.9 °C; yield 66.3%.

[0067] 1H NMR (400MHz, Chloroform-d) δppm: 8.37-8.23 (m, 2H, 2×Ar-H), 7.91-7.80 (m, 1H, Ar-H), 7.63 (ddd, J=8.4, 7.4, 1.6Hz, 1H, Ar-H), 7.55-7.38 (m, 3H, 3×Ar-H). 13 C NMR(101MHz,Chloroform-d)δppm:151.3,146.0,143.9,143.1,133.3,130.5,126.4,126.2,125.7,125.1,120.2,117.1 115.0,112.4.ESI-HRMS(TOF):m / z[M+H] + calcd forC 14 H7ClN2OS, 287.0040, found 287.0058; HPLC purity 97.46%.

[0068] Example 5: Synthesis of 8-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 5)

[0069] 3-Bromosalicylaldehyde (1.01 g, 5.02 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and o-phenylenediamine (815 mg, 7.54 mmol) and ammonium acetate (1.95 g, 25.10 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for approximately 8 h to complete the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(1-benzimidazol-2-yl)-8-bromophenol as a white solid. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1-4:1, V / V) yielded the intermediate 2-(1-benzimidazol-2-yl)-8-bromophenol (740 mg, 2.56 mmol), with a yield of 50.9%.

[0070] The product 2-(1-benzimidazol-2-yl)-8-bromophenol (200 mg, 0.69 mmol) was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF), and 1,1'-thiocarbonyldiimidazole (TCDI, 616 mg, 3.46 mmol) was added. The mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, and a large amount of yellow solid precipitated. The solid was filtered to obtain a yellow crude product. Purification by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1-8:1, V / V) yielded 94 mg of the target product 8-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 5), melting point (mp): 226.8-230.0 °C; yield 40.9%.

[0071] 1 H NMR (400MHz, Chloroform-d) δppm: 9.05 (d, J=7.6Hz, 1H, Ar-H), 8.35 (dd, J=8.0, 1.6Hz, 1H, Ar-H),7.94-7.83(m,2H,2×Ar-H),7.64-7.50(m,2H,2×Ar-H),7.36(t,J=8.0Hz,1H,Ar-H). 13 C NMR(101MHz,Chloroform-d)δppm:172.6,149.2,144.6,142.4,136.8,131.9,12 7.5,127.3,126.0,124.2,120.4,116.8,114.5,109.8.ESI-HRMS(TOF):m / z[M+H] + calcd for C 14 H7N2BrOS, 330.9541, found 330.9556; HPLC purity 99.49%.

[0072] Example 6: Synthesis of 6-fluoro-1,3-benzoxazine-2-thionobenzimidazole (compound 6)

[0073] 5-Fluorosic acid aldehyde (1.01 g, 7.21 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and o-phenylenediamine (940 mg, 8.66 mmol) and ammonium acetate (2.78 g, 36.10 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for approximately 8 h to complete the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(1-benzimidazol-2-yl)-6-fluorophenol as a white solid. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1-4:1, V / V) yielded the intermediate 2-(1-benzimidazol-2-yl)-6-fluorophenol (988 mg, 4.33 mmol), with a yield of 60.1%.

[0074] The above product, 2-(1-benzimidazol-2-yl)-6-fluorophenol (200 mg, 0.88 mmol), was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF). 1,1'-thiocarbonyldiimidazole (TCDI, 781 mg, 4.39 mmol) was added, and the mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, resulting in the precipitation of a large amount of yellow solid. This was filtered to obtain a yellow crude product. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-8:1, V / V) yielded 130 mg of the target product, 6-fluoro-1,3-benzoxazine-2-thionobenzimidazole (compound 6), with a melting point (mp) of 243.2-247.1 °C and a yield of 54.7%.

[0075] 1 H NMR (400MHz, Chloroform-d) δppm: 9.08 (d, 1H, J = 7.6, Ar-H), 8.08 (dd, J = 8.0, 3.0Hz, 1H, Ar-H) ,7.95-7.88(m,1H,Ar-H),7.65-7.48(m,3H,3×Ar-H),7.38(ddd,J=9.0,7.8,3.0Hz,1H,Ar-H). 13 C NMR (101MHz, Chloroform-d) δppm: 173.4, 160.0 (d, J = 248.2Hz), 148.4, 144.4, 142.3, 131.9, 127.5, 126.0, 121.0 (d ,J=24.9Hz),120.5,118.5(d,J=8.7Hz),116.8,114.2(d,J=9.6Hz),110.9(d,J=26.2Hz).ESI-HRMS(TOF):m / z[M+H]+ calcd for C 14 H7N2FOS, 271.0336, found 271.0346; HPLC purity 99.669%.

[0076] Example 7: Synthesis of 6-ethoxy-1,3-benzoxazine-2-thionobenzimidazole (compound 7)

[0077] 5-Ethoxysalicylaldehyde (1.01 g, 7.21 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and o-phenylenediamine (940 mg, 8.66 mmol) and ammonium acetate (2.78 g, 36.10 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for approximately 8 h to complete the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(1-benzimidazol-2-yl)-6-ethoxyphenol as a white solid. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1-4:1, V / V) yielded the intermediate 2-(1-benzimidazol-2-yl)-6-ethoxyphenol (988 mg, 4.33 mmol), with a yield of 60.1%.

[0078] The above product, 2-(1-benzimidazol-2-yl)-6-ethoxyphenol (200 mg, 0.88 mmol), was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF). 1,1'-thiocarbonyldiimidazole (TCDI, 781 mg, 4.39 mmol) was added, and the mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, resulting in the precipitation of a large amount of yellow solid. This was filtered to obtain a yellow crude product. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-8:1, V / V) yielded 130 mg of the target product, 6-ethoxy-1,3-benzoxazine-2-thionobenzimidazole (compound 7), with a melting point (mp) of 230.2-234.8 °C and a yield of 61.8%.

[0079] 1H NMR (400MHz, Chloroform-d) δppm: 9.08 (d, J = 8.0, 1H, Ar-H), 7.93-7.86 (m, 2H, 2×Ar-H), 7.60-7.46 (m, 2H, 2×Ar-H), 7. 36(t,J=8.0Hz,1H,Ar-H),7.16(dd,J=8.2,1.4Hz,1H,Ar-H),4.24(q,J=7.0Hz,2H,-CH2-),1.55(t,J=7.0Hz,3H,-CH3). 13 C NMR(101MHz,Chloroform-d)δppm:173.5,146.2,144.5,143.3,142.4,131.9,127.2,1 26.7,125.5,120.1,116.8,116.1,115.7,113.8,65.4,14.8.ESI-HRMS(TOF):m / z[M+H] + calcdfor C 14 H8N2OS, 297.0692, found 297.0703; HPLC purity 97.63%.

[0080] Example 8: Synthesis of 6-bromo-9-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 8)

[0081] 5-Bromosalicylaldehyde (1.01 g, 5.00 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and 4-methyl-o-phenylenediamine (732 mg, 6.00 mmol) and ammonium acetate (1.92 g, 25.01 mmol) were added. The mixture was heated and stirred in an oil bath at 80 °C, and refluxed for about 8 h to complete the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed once with distilled water and once with saturated sodium chloride solution. The washed organic layer was then dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude 2-(10-methyl-1-benzimidazol-2-yl)-6-bromophenol. Purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1-4:1, V / V) to give intermediate 2-(10-methyl-1-benzimidazol-2-yl)-6-bromophenol (453 mg, 1.5 mmol), yield 30.2%.

[0082] The above product, 2-(10-methyl-1-benzimidazol-2-yl)-6-bromophenol (200 mg, 0.66 mmol), was dissolved in approximately 10 mL of anhydrous tetrahydrofuran (THF). 1,1'-thiocarbonyldiimidazole (TCDI, 589 mg, 3.31 mmol) was added, and the mixture was stirred in an oil bath at 90 °C for approximately 12 h. TLC monitoring showed that the starting material had reacted completely, and the reaction was terminated. Distilled water was added to the reaction solution, resulting in the precipitation of a large amount of yellow solid. This was filtered to obtain a yellow crude product. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-8:1, V / V) yielded 76 mg of the target product, 6-bromo-9-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 8), with a melting point (mp) of 245.8-248.3 °C and a yield of 33.2%.

[0083] 1 H NMR (400MHz, Chloroform-d) δppm: 8.97 (d, J = 9.0Hz, 1H, Ar-H), 8.25 -8.13(m,1H,Ar-H),8.03(d,J=2.0Hz,1H,Ar-H),7.63(dd,J=8.8,1.9Hz,1H,Ar- H),7.52(d,J=2.2Hz,1H,Ar-H),7.42(d,J=8.6Hz,1H,Ar-H),2.51(s,3H,-CH3). 13 C NMR(101MHz,Chloroform-d)δppm:174.0,150.4,144.1,143.2,137.0,134.6,131.8 ,127.4,125.6,124.8,120.0,116.7,116.2,112.3,21.2.ESI-HRMS(TOF):m / z[M+H] + calcd forC 15 H9BrN2OS, 344.9692, found 344.9709; HPLC purity 96.23%.

[0084] The following are some of the pharmacological tests and results of representative compounds of this invention:

[0085] Activity test

[0086] The in vitro anti-AML cell proliferation activity of 1,3-benzoxazine-2-thionobenzimidazole (compound 1), 6-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 2), 6-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 3), 6-chloro-1,3-benzoxazine-2-thionobenzimidazole (compound 4), 8-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 5), 6-fluoro-1,3-benzoxazine-2-thionobenzimidazole (compound 6), 6-ethoxy-1,3-benzoxazine-2-thionobenzimidazole (compound 7), and 6-bromo-9-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 8) was tested using the CCK-8 assay.

[0087]

[0088] MOLM13 cells in the logarithmic growth phase were harvested, and the cell count was set at 1.5 × 10⁻⁶. 4 Cells were seeded at a density of cells / well in 96-well plates. The blank control group received only 200 μL of RPMI-1640 medium, with three replicates for each drug concentration. Cells were divided into seven groups: a negative control group and six drug-treated groups. The negative control group received DMSO, while the other groups received the drug, resulting in final drug concentrations of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. 200 μL of autoclaved PBS was added to the side wells of the 96-well plate, and the plates were incubated at 37°C with 5% CO2. After 72 hours of drug treatment, 10 μL / well of CCK8 solution was added to each well, gently mixed, and the plates were incubated for another 3 hours. The 96-well plates were first shaken on a microplate reader for 5 seconds to adjust the optical density (OD) to 450 nm before measuring the absorbance of each well. Zero the cell count using the blank control wells and calculate the cell growth activity for each group. Each experiment was repeated three times. Cell proliferation curves were constructed based on the detected OD values ​​to evaluate the effects of 1,3-benzoxazine-2-thionobenzimidazole (compound 1), 6-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 2), 6-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 3), 6-chloro-1,3-benzoxazine-2-thionobenzimidazole (compound 4), 8-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 5), 6-fluoro-1,3-benzoxazine-2-thionobenzimidazole (compound 6), 6-ethoxy-1,3-benzoxazine-2-thionobenzimidazole (compound 7), 6-bromo-9-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 8), and the lead compound MV1035 on cell proliferation.

[0089] Table 1. Inhibitory activity of compounds against MOLM13 proliferation.

[0090]

[0091]

[0092] Table 1 shows that 8-bromo-1,3-benzoxazine-2-thionobenzimidazole (compound 5) and 6-bromo-9-methyl-1,3-benzoxazine-2-thionobenzimidazole (compound 8) significantly inhibited the proliferation of MOLM13 cells, with an IC50 of 100%. 50 The values ​​are 2.06 μM and 8.19 μM, respectively.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The compound represented by Formula I or a pharmaceutically acceptable salt thereof, in, R 1 Represents hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen; R 2 It represents hydrogen or C1-C4 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R 1 It represents hydrogen, methyl, ethyl, methoxy, ethoxy, fluorine, chlorine, or bromine.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein, R 1 It represents hydrogen, methyl, ethoxy, fluorine, chlorine, or bromine.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R 2 It represents hydrogen, methyl, or ethyl.

5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein, R 2 It represents hydrogen or methyl.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

7. A pharmaceutical composition comprising, as an active ingredient or main active ingredient, the compound of claim 1 or a pharmaceutically acceptable salt thereof, supplemented by a pharmaceutically acceptable carrier.

8. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-acute myeloid leukemia drug.

Images