Benzimidazole compound as well as preparation method, application and pharmaceutical composition thereof

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to a benzimidazole compound, a preparation method and application thereof and a medicinal composition. The benzimidazole compound is selected from a compound # imgabs0 # shown in the following structural formula, and the benzimidazole compound can inhibit ALK and BET at the same time, so that the benzimidazole compound can play a synergistic role and can treat tumors more efficiently.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to benzimidazole compounds, preparation methods thereof, applications thereof and pharmaceutical compositions thereof. Background Art

[0002] Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor protein-tyrosine kinase family. ALK has been clinically proven to be an effective target for the treatment of lymphoma. Currently, several ALK inhibitors have been approved for marketing, such as Crizotinib, Ceritinib, Brigatinib, etc. However, due to the heterogeneity of tumor cells, these drugs have secondary mutations during use, making tumor cells resistant to these drugs. Therefore, overcoming drug resistance is an urgent problem to be solved in anti-tumor research targeting ALK.

[0003] Resistance to ALK inhibitors caused by abnormalities in the oncogene c-Myc is most common in ALK-targeted anti-lymphoma. Inhibiting ALK while reducing c-Myc expression will produce better anti-tumor effects. Proteins containing the bromodomain and extra-terminal (BET) domain family are epigenetic readers. They include four members: BRD2, BRD3, BRD4, and BRDT. They share two N-terminal bromodomains (BD1 and BD2) and an additional C-terminal domain. Their bromodomains bind to acetylated histones and play an important regulatory role in the transcription of genes such as c-Myc. Simultaneous inhibition of ALK and BET can produce a synergistic anti-tumor effect. The development of ALK / BET dual-target inhibitors can overcome the drug resistance problem of existing ALK inhibitors to a certain extent, providing better drug options for the clinical treatment of lymphoma. However, there are no small molecule compounds in the prior art that can simultaneously inhibit ALK and BET.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a benzimidazole compound, a preparation method thereof, an application thereof and a pharmaceutical composition. The embodiment of the present invention provides a benzimidazole compound that can inhibit ALK and BET simultaneously, thereby exerting a synergistic effect and being able to treat tumors more efficiently.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a benzimidazole compound, which includes any one of the compound represented by the following structural formula I, its salt, its stereoisomer, its solvate, its hydrate and its prodrug,

[0008] Wherein, R1 is selected from any one of the functional groups formed by H, substituted or unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 cycloalkyl;

[0009] R2 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, Any one of the functional groups formed; wherein X is selected from any one of O, S and NH; Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 are independently selected from N or CH; R6 represents mono- or poly-substitution; R6 is selected from any one of the functional groups formed by H, halogen, substituted or unsubstituted C1-C6 alkyl, hydroxyl, substituted or unsubstituted amino and cyano;

[0010] R3 is selected from any one of the functional groups formed by hydrogen, halogen, hydroxyl, substituted or unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 cycloalkyl;

[0011] R4 and R5 are independently selected from any one of the functional groups formed by hydrogen, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 heterocycloalkyl, substituted or unsubstituted aryl and or unsubstituted heteroaryl;

[0012] n and m are independently selected from 0, 1 or 2.

[0013] In a second aspect, the present invention provides a method for preparing the benzimidazole compound described in the aforementioned embodiment, and the synthesis is performed according to the following synthesis route:

[0014]

[0015] In a third aspect, the present invention provides a pharmaceutical composition comprising the benzimidazole compound described in the aforementioned embodiment.

[0016] In a fourth aspect, the present invention provides a use of the benzimidazole compound described in the aforementioned embodiment in the preparation of an inhibitor that simultaneously inhibits ALK and BET.

[0017] In a fifth aspect, the present invention provides a use of the benzimidazole compound described in the above embodiment in the preparation of a drug for treating tumors;

[0018] Preferably, the tumor comprises a lymphoma.

[0019] The present invention has the following beneficial effects: The embodiment of the present invention provides a new benzimidazole compound, which can inhibit ALK and BET simultaneously, thereby achieving dual-target inhibition, and then can produce a synergistic anti-tumor effect, thereby improving the therapeutic effect of tumors. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] The embodiment of the present invention provides a benzimidazole compound, which includes any one of the compound represented by the following structural formula I, its salt, its stereoisomer, its solvate, its hydrate and its prodrug,

[0022] Wherein, R1 is selected from any one of the functional group formed by H, substituted or unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 cycloalkyl; further, R1 is selected from H or unsubstituted C1-C6 alkyl; preferably H or unsubstituted C1-C4 alkyl; more preferably H or unsubstituted C1-C3 alkyl, for example including but not limited to H, methyl or ethyl.

[0023] R2 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, Any one of the functional groups formed; wherein X is selected from any one of O, S and NH; Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 are independently selected from N or CH; R6 is selected from any one of the functional group formed by H, halogen, substituted or unsubstituted C1-C6 alkyl, hydroxyl, substituted or unsubstituted amino and cyano.

[0024] R2 is selected from unsubstituted C3-C6 cycloalkyl, Any one of the functional groups formed, X is selected from O or S, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6are independently selected from CH; R6 represents mono- or poly-substitution; R6 is selected from any one of the functional groups formed by H, halogen, hydroxyl and substituted or unsubstituted C1-C6 alkyl; further preferably, R2 is selected from unsubstituted C3-C6 cycloalkyl, Any one of the functional groups formed, X is selected from S, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 They are independently selected from CH; R6 is selected from any one of the functional groups formed by H, halogen, hydroxyl, unsubstituted C1-C3 alkyl, halogen-substituted C1-C3 alkyl and C1-C3 alkoxy (that is, oxygen-substituted alkyl).

[0025] Wherein, R6 represents mono- or poly-substitution; that is, Y 3 , Y 4 , Y 5 and Y 6 Any one, any two, any three or four of the H groups are replaced by R6.

[0026] For example, R2 includes but is not limited to Any one of the functional groups formed.

[0027] R3 is selected from any one of the functional groups formed by hydrogen, halogen, hydroxyl, substituted or unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 cycloalkyl; further, R3 is selected from H or unsubstituted C1-C6 alkyl; preferably H or unsubstituted C1-C4 alkyl; more preferably H or unsubstituted C1-C3 alkyl, for example including but not limited to H, methyl or ethyl.

[0028] R4 and R5 are independently selected from any one of the functional groups formed by hydrogen, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 heterocycloalkyl, substituted or unsubstituted aryl and or unsubstituted heteroaryl. Preferably, R4 and R5 are independently selected from any one of the functional groups formed by H, unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 heterocycloalkyl; more preferably, R4 and R5 are independently selected from any one of the functional groups formed by H, unsubstituted C1-C3 alkyl, unsubstituted piperidine, and piperidine substituted by C1-C3 alkyl.

[0029] That is, R4 and R5 can be selected from the same group or different groups. For example, R4 can be methyl, R5 can be piperidine, or both can be methyl.

[0030] n and m are independently selected from 0, 1 or 2.

[0031] It should be understood that although the embodiments of the present invention limit n and m to be independently selected from 0, 1 or 2, when both are 2, it is obviously not in compliance with the requirements for the number of chemical bond substitutions. These requirements are well known to those skilled in the art, and the infeasible technical solution when both are 2 can be excluded.

[0032] The unsubstituted alkyl group may be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl alkyl groups, and the present invention does not enumerate them all.

[0033] Similarly, the cycloalkyl group can be selected from cycloalkanes such as cyclopropyl, cyclobutyl and cyclopentyl, and the examples of the present invention will not be exhaustively listed.

[0034] Halogen includes fluorine, chlorine, bromine and iodine.

[0035] Heterocycloalkyl groups include heteroalkyl groups such as piperidine, tetrahydrofuran and hydrogenated pyrimidine, which are not listed in detail in the present embodiment.

[0036] Specifically, the benzimidazole compound is selected from any one of the compounds represented by the following structural formulas:

[0037]

[0038] The acid of the salt of the compound represented by the above structural formula I is selected from any one of hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and ferulic acid.

[0039] In a second aspect, the present invention provides a method for preparing the benzimidazole compound described in the aforementioned embodiment, and the synthesis is performed according to the following synthesis route:

[0040]

[0041] Specifically, the raw material aldehyde A undergoes a reductive amination reaction with (R)-2-aminobutyric acid methyl ester hydrochloride to prepare compound B, B undergoes selective nucleophilic substitution with 2,4-dichloro-5-nitropyrimidine to prepare compound C, C completes intramolecular cyclization under reducing conditions to prepare compound D, compound D undergoes nucleophilic substitution with various iodinated alkanes to prepare compound E, and compound E undergoes nucleophilic substitution with various prepared benzofuran amine intermediates to prepare the product of formula (I).

[0042] In a third aspect, the present invention provides a pharmaceutical composition comprising the benzimidazole compound described in the aforementioned embodiment.

[0043] In a fourth aspect, the present invention provides a use of the benzimidazole compound described in the aforementioned embodiment in the preparation of an inhibitor that simultaneously inhibits ALK and BET.

[0044] In a fifth aspect, the present invention provides a use of the benzimidazole compound described in the above embodiment in the preparation of a drug for treating tumors;

[0045] Preferably, the tumor comprises a lymphoma.

[0046] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0047] Example 1

[0048] The present invention provides a benzimidazole compound, the structural formula of which is shown below:

[0049]

[0050] The synthesis path is as follows:

[0051]

[0052] The details are as follows:

[0053] (1) Preparation of (R)-2-((4-methylthiophene-2-yl)methyl)amino)butyric acid methyl ester (I-3)

[0054] Weigh the commercially available (R)-2-aminobutyric acid methyl ester hydrochloride (I-2, 1.17 g, 10 mmol) into a 250 mL round-bottom flask, add 1,2-dichloroethane (100 mL) to dissolve, then add 4-methylthiophene-2-carboxaldehyde (I-1, 1.05 mL, 8.5 mmol), mix well and cool to 0°C. Add sodium acetate (0.82 g, 10 mmol) and sodium triacetoxyborohydride (4.24 g, 20 mmol) and stir at room temperature for 16 hours. After TLC detection of the reaction is complete, pour the reaction solution into a 10% sodium bicarbonate aqueous solution (200 mL) and extract with dichloromethane (100 mL × 3). Combine the extracts, wash with water (200 mL), add anhydrous sodium sulfate to dry for 1 hour, filter to remove insoluble matter, and concentrate the filtrate to dryness under reduced pressure. The residue was purified by column chromatography to obtain yellow oil I-3 (2.053 g, yield 90%). MS-ESI (m / z): 228 [M+H] + .

[0055] (2) Preparation of (R)-2-((2-chloro-5-nitropyrimidin-4-yl)((4-methylthiophene-2-yl)methyl)amino)butyric acid methyl ester (I-4)

[0056] I-3 (2.053 g, 9 mmol) obtained in the previous step was dissolved in cyclohexane (90 mL), sodium bicarbonate (1.52 g, 18 mmol) was added, and the mixture was stirred at room temperature for 0.5 hours. Then 2,4-dichloro-5-nitropyrimidine (1.93 g, 9.9 mmol) was added, and the reaction solution was heated to 60°C and stirred for 16 hours. After TLC detection, the precipitate was removed by filtration, and the filtrate was collected and concentrated to dryness. The residue was purified by column chromatography to obtain a yellow solid I-4 (3.475 g, yield 100%). MS-ESI (m / z): 385 [M+H] + .

[0057] (3) Preparation of (R)-2-chloro-7-ethyl-8-((4-methylthiophen-2-yl)methyl)-7,8-dihydropteridin-6(5H)-one (I-5)

[0058] Weigh the I-4 (223 mg, 0.58 mmol) obtained in the previous step and dissolve it in glacial acetic acid (2 mL), heat it to 100 ° C, then quickly add reduced iron powder (39 mg, 0.70 mmol), and stir and react at 100 ° C for 6 hours. After TLC detection, the reaction is complete, and the solvent is removed by concentration under reduced pressure. The residue is dissolved in ethyl acetate (100 mL), and the insoluble matter is removed by filtration with diatomaceous earth. The filtrate is collected and concentrated to dryness. The obtained residue is purified by column chromatography to obtain an orange solid I-5 (73 mg, yield 39%). MS-ESI (m / z): 323 [M+H] + .

[0059] (4) Preparation of (R)-2-chloro-7-ethyl-5-methyl-8-((4-methylthiophen-2-yl)methyl)-7,8-dihydropteridin-6(5H)-one (I-6)

[0060] I-5 (73 mg, 0.23 mmol) obtained in the previous step was dissolved in dry N,N-dimethylformamide (3 mL), iodomethane (19 μL, 0.29 mmol) was added, and then cooled to -10 ° C, sodium hydride (12 mg, 0.29 mmol) was slowly added, and the reaction was maintained at -10 ° C for 2 hours. After the reaction was complete by TLC, the reaction solution was poured into saturated brine (20 mL) and extracted with ethyl acetate (30 mL × 2). The combined extracts were washed once with saturated brine, and then dried over anhydrous sodium sulfate for 1 hour, filtered to remove insoluble matter, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography to obtain a white solid I-6 (75 mg, yield 97%). MS-ESI (m / z): 337 [M+H] + .

[0061] (5) Preparation of (R)-2,5-dimethyl-4-(1-methylpiperidin-4-yl)-2,3-dihydrobenzofuran-7-amine (Intermediate A);

[0062]

[0063] For the method of preparing A-8 from A-1, please refer to WO2014071832A1.

[0064] Weigh A-8 (1.21 g, 5 mmol) obtained in the previous step and place it in a 100 mL round-bottom flask, add 1-methyl-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (1.67 g, 7.5 mmol), PdCl2(DPPF) (183 mg, 0.25 mmol), sodium carbonate (1.33 g, 12.5 mmol), N,N-dimethylformamide (25 mL) and water (10 mL), mix well, replace with nitrogen three times, heat to 100 ° C under nitrogen protection and react for 5 hours. After TLC detection, the reaction solution was cooled to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (50 mL × 3). Combine the extracts and concentrate to dryness under reduced pressure. The residue was purified by column chromatography to obtain a light yellow solid A-9 (1.03 g, yield 80%). MS-ESI (m / z): 258 [M+H] + .

[0065] Weigh A-9 (1.03 g, 4 mmol) obtained in the previous step into a 100 mL round-bottom flask, add methanol (40 mL) to dissolve, add palladium carbon (10%, 200 mg), replace with hydrogen three times, and then stir at room temperature overnight under a hydrogen atmosphere. After TLC detection, filter with diatomaceous earth, collect the filtrate, and concentrate to dryness. The residue is purified by column chromatography to obtain a light yellow solid A (0.66 g, yield 63%). 1 H NMR (400MHz, DMSO-d6): δ6.77(s,1H),5.10(s,2H),5.01-4.92(m,1H),3.31-3.24(m,1H),3.04-2.92(m,1H),2.82-2.66(m,1H),2 .49-2.32(m,4H),2.29(s,3H),2.18(s,3H),1.84-1.72(m,2H),1.57-1.48(m,2H),1.44(d,J=6.6Hz,3H).MS-ESI(m / z):260[M+H] + .

[0066] (6) Preparation of (R)-2-(((R)-2,5-dimethyl-4-(1-methylpiperidin-4-yl)-2,3-dihydrobenzofuran-7-yl)amino)-7-ethyl-5-methyl-8-((4-methylthiophen-2-yl)methyl)-7,8-dihydropteridin-6(5H)-one (1)

[0067] Weigh the I-6 (61 mg, 0.18 mmol) obtained in the previous step into a 50 mL thick-walled reaction bottle, dissolve it in n-butanol (5 mL), add intermediate A (47 mg, 0.18 mmol) and p-toluenesulfonic acid (31 mg, 0.18 mmol), and heat to 130 ° C under sealed conditions for 5 hours. After the reaction is complete by TLC, the reaction solution is cooled to room temperature, diluted with 20 mL of water, and extracted with dichloromethane (20 mL × 2). Combine the extracts and concentrate to dryness under reduced pressure. The residue is purified by column chromatography to obtain a light yellow solid product 1 (22 mg, yield 22%). 1 H NMR (400MHz, DMSO-d6): δ8.91(s,1H),8.27(s,1H),7.15(s,1H),6.65(s,1H),6.41(s ,1H),5.08-4.99(m,1H),4.40(s,2H),3.47-3.35(m,4H),3.25-3.18(m,1H),3.03-2.9 4(m,1H),2.77-2.65(m,1H),2.49-2.33(m,4H),2.30(s,3H),2.25(s,3H),2.17(s,3H) ,1.79-1.66(m,4H),1.54-1.42(m,5H),0.90(t,J=2.2Hz,3H).MS-ESI(m / z):561[M+H] + .

[0068] Embodiment 2-8

[0069] Examples 2-8 provide a benzimidazole compound, respectively, and the synthesis method thereof is as described in Example 1. Examples 2-8 in Table 2 are synthesized using different substituted aldehydes, different iodinated alkanes or intermediates B, C, and D. These different substituted aldehydes and different iodinated alkanes can be purchased commercially. The structural formulas and characterizations of intermediates B, C, and D are shown in Table 1.

[0070] Table 1 Structural identification data of intermediates B, C and D

[0071]

[0072]

[0073]

[0074] Table 2 Structural identification data of benzimidazole compounds of Examples 2-8

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Experimental Example 1

[0082] The binding effect of the benzimidazole compounds of Examples 1-8 on the BRD4 (BD1) protein was detected using homogeneous time-resolved fluorescence technology. BRD4 (BD1) time-resolved fluorescence resonance energy transfer (TR-FRET) analysis kit (purchased from BPS Bioscience) and multifunctional microplate reader.

[0083] Experimental process:

[0084] (1) Compound preparation: Compounds were serially diluted using DMSO, with a starting concentration of 1 μM and 3-fold dilutions to 10 concentrations, with each concentration in a single well.

[0085] (2) Prepare the reaction solution: Use the Diluent Buffer in the kit to dilute the BRD4 (BD1) protein and the Biotin-labeled histone H4 peptide.

[0086] (3) Prepare the detection solution: dilute Anti-GST-TB using the Detection Buffer in the kit. 2+ Cryptate and SA-XL-665.

[0087] (4) Take a 384-well plate and arrange it according to the following layout: wells for the test compound, control wells min (high concentration positive drug), and control wells max (DMSO).

[0088] (5) Add 20 nL of the corresponding concentration of compound or DMSO solution to each well of the well plate, then add 10 μL of reaction solution to each well, followed by 10 μL of detection solution.

[0089] (6) After gently shaking to mix, incubate at room temperature for 60 min, and read the fluorescence signal on an Envision multi-function microplate reader.

[0090] Data Analysis:

[0091] Calculation formula

[0092]

[0093] Fitting dose-effect curve

[0094] The log value of the concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The log (inhibitor) vs. response-Variable slope of the analysis software GraphPad Prism 5 was used to fit the dose-effect curve to obtain the IC value of the compound for protein binding inhibition. 50 The results are shown in Table 3.

[0095] Table 3 Activity of benzimidazole compounds on BRD4 (BD1) protein

[0096] Compound <![CDATA[IC 50 (nM)]]> Compound <![CDATA[IC 50 (nM)]]> 1 32 6 46 2 56 7 53 3 44 8 47 4 38 5 226

[0097] As shown in Table 3, the benzimidazole compounds provided in the embodiments of the present invention have a good inhibitory effect on BRD4 (BD1) protein, among which benzimidazole compounds 1 to 4 and 6 to 8 have relatively better effects.

[0098] Experimental Example 2

[0099] Affinity testing of ALK protein

[0100] Mobility shift assay was used to detect the binding effect of the compounds on ALK protein. Instruments, reagents and consumables used: centrifuge (manufacturer: Eppendorf, model: 5430), microplate reader (manufacturer: Perkin Elmer, model: Caliper EZ ReaderII), Echo 550 (manufacturer: Labcyte, model: Echo 550), ALK protein (purchased from Carna, item number 08-518), Kinase substrate 22 (purchased from GL, item number 112393), DMSO (purchased from Sigma), OptiPlate-384F (PerkinElmer).

[0101] Experimental process:

[0102] (1) Compound preparation

[0103] The compound was dissolved in 100% DMSO to a 10 mM stock solution and stored at -20°C in the dark.

[0104] (2) Kinase reaction process

[0105] ①Prepare 1× Kinase buffer.

[0106] ② Preparation of compound concentration gradient: The test compound concentration is 1000nM starting, 3-fold dilution, 10 concentrations, single-well detection. Prepare 10 solutions of 100 times the final concentration in a 384-well plate. Then use Echo550 to transfer 250nL to the 384 reaction plate for later use. Add 250nL of 100% DMSO to the negative control well and the positive control well respectively.

[0107] ③ Use 1× Kinase buffer to prepare a kinase solution with a final concentration of 2.5 times.

[0108] ④ Add 10 μL of kinase solution at 2.5 times the final concentration to the compound wells and positive control wells respectively; add 10 μL of 1× Kinase buffer to the negative control wells.

[0109] ⑤ Centrifuge at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 10 minutes.

[0110] ⑥ Use 1× Kinase buffer to prepare a mixed solution of ATP and Kinase substrate 22 with a final concentration of 25 / 15.

[0111] ⑦ Add 15 μL of a mixed solution of ATP and substrate at 25 / 15 times the final concentration to start the reaction.

[0112] ⑧ Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 25 minutes.

[0113] ⑨Add 30 μL of stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and oscillate to mix.

[0114] ⑩Read the conversion rate using Caliper EZ Reader.

[0115] (3) Data analysis

[0116] Calculation formula

[0117]

[0118] Among them, Conversion%_sample is the conversion rate reading of the sample; Conversion%_min: the mean of the negative control wells, representing the conversion rate reading of the wells without enzyme activity; Conversion%_max: the mean of the positive control wells, representing the conversion rate reading of the wells without compound inhibition.

[0119] Fitting dose-effect curve

[0120] The log value of the concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The log (inhibitor) vs. response-Variable slope of the analysis software GraphPad Prism 5 was used to fit the dose-effect curve to obtain the IC value of each compound on the enzyme activity. 50 value.

[0121] The calculation formula is:

[0122] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSllope))

[0123] See Table 4 for the results.

[0124] Table 4 Affinity test results for ALK protein

[0125] Compound <![CDATA[IC 50 (nM)]]> Compound <![CDATA[IC 50 (nM)]]> 1 60 6 316 2 276 7 522 3 98 8 637 4 66 5 79

[0126] According to Table 4, the benzimidazole compounds provided in the embodiments of the present invention have a good inhibitory effect on ALK protein, among which the benzimidazole compounds 1 and 3 to 5 have relatively better effects.

[0127] From Table 3 and Table 4, it can be seen that the benzimidazole compounds provided in the embodiments of the present invention can inhibit ALK and BET simultaneously.

[0128] Experimental Example 3

[0129] Inhibitory effect on proliferation of lymphoma cells SU-DHL-4

[0130] Method: The compound was dissolved in 100% DMSO to prepare a stock solution of the compound at a concentration of 1 mM or 0.1 mM.

[0131] 100 μL of RPMI 1640 complete medium and 0.2 μL of 1 mM or 0.1 mM compound stock solution were added to each well of a 96-well plate, and 0.2 μL of DMSO was added to the control group.

[0132] ③After counting the SU-DHL-4 cells, dilute them to 60,000 cells / mL with RPMI 1640 medium to prepare the cell suspension.

[0133] ④ Add 100 μL of diluted cell suspension to each well of the 96-well plate containing the compound.

[0134] ⑤ Place the culture plate in a 37°C, 5% CO2 incubator and culture for 72 hours.

[0135] ⑥Add 100 μL CellTiter-Lumi TM (Biyuntian Bio) luminescence detection reagent.

[0136] ⑦ Shake at room temperature for 2 minutes to promote cell lysis.

[0137] ⑧ Leave at room temperature away from light for 10 minutes.

[0138] ⑨Use a multifunctional microplate reader (BioTek Cytation5) to detect chemiluminescence (Luminescence, RLU).

[0139] ⑩ According to the formula, cell inhibition rate

[0140] See Table 5 for the results.

[0141] Table 5 Inhibition rate of lymphoma cell SU-DHL-4

[0142]

[0143]

[0144] It can be seen from Table 5 that the benzimidazole compounds provided by the present invention have a good inhibitory effect on lymphoma cells, among which compound 1 has the best inhibitory effect on lymphoma cells.

[0145] In summary, the present invention provides a class of benzofuran compounds, which have good inhibitory effects on both BRD4 (BD1) and ALK proteins, and can be used as new BET / ALK inhibitors for preventing and / or treating various diseases related to BET and ALK, such as lymphoma, etc., with excellent effects and good application prospects.

[0146] 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 benzimidazole compound, characterized in that: It includes any one of the compounds represented by the following structural formula I, their salts, stereoisomers, solvates, hydrates and prodrugs thereof, Wherein, R1 is selected from any one of the functional groups formed by H, substituted or unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 cycloalkyl; R2 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, Any one of the functional groups formed; wherein X is selected from any one of O, S and NH; Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 are independently selected from N or CH; R6 represents mono- or poly-substitution; R6 is selected from any one of the functional groups formed by H, halogen, substituted or unsubstituted C1-C6 alkyl, hydroxyl, substituted or unsubstituted amino and cyano; R3 is selected from any one of the functional groups formed by hydrogen, halogen, hydroxyl, substituted or unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 cycloalkyl; R4 and R5 are independently selected from any one of the functional groups formed by hydrogen, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 heterocycloalkyl, substituted or unsubstituted aryl and or unsubstituted heteroaryl; n and m are independently selected from 0, 1 or 2.

2. The benzimidazole compound according to claim 1, characterized in that R1 is selected from H or unsubstituted C1-C6 alkyl; preferably H or unsubstituted C1-C4 alkyl; more preferably H or unsubstituted C1-C3 alkyl, most preferably H, methyl or ethyl.

3. The benzimidazole compound according to claim 1, characterized in that R2 is selected from unsubstituted C3-C6 cycloalkyl, Any one of the functional groups formed, X is selected from O or S, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 are independently selected from CH; R6 represents mono- or poly-substitution; R6 is selected from any one of the functional groups formed by H, halogen, hydroxyl and substituted or unsubstituted C1-C6 alkyl; Preferably, R2 is selected from unsubstituted C3-C6 cycloalkyl, Any one of the functional groups formed, X is selected from S, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 are independently selected from CH; R6 is selected from any one of the functional groups formed by H, halogen, hydroxyl, unsubstituted C1-C3 alkyl, halogen-substituted C1-C3 alkyl and C1-C3 alkoxy; More preferably, R2 is selected from Any one of the functional groups formed.

4. The benzimidazole compound according to claim 1, characterized in that R3 is selected from H or unsubstituted C1-C6 alkyl; preferably H or unsubstituted C1-C4 alkyl; more preferably H or unsubstituted C1-C3 alkyl, most preferably H, methyl or ethyl; Preferably, R4 and R5 are independently selected from any one of the functional group formed by H, unsubstituted C1-C6 alkyl and substituted or unsubstituted C3-C6 heterocycloalkyl; Preferably, R4 and R5 are independently selected from any one of the functional group consisting of H, unsubstituted C1-C3 alkyl, unsubstituted piperidine, and piperidine substituted with C1-C3 alkyl.

5. The benzimidazole compound according to any one of claims 1 to 4, characterized in that: The benzimidazole compound is selected from any one of the compounds represented by the following structural formula:

6. The benzimidazole compound according to any one of claims 1 to 4, characterized in that The acid for forming the salt of the compound shown in structural formula I is selected from any one of hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and ferulic acid.

7. A method for preparing the benzimidazole compound according to claim 1, characterized in that: The synthesis was carried out according to the following synthesis route:

8. A pharmaceutical composition, characterized in that It comprises the benzimidazole compound described in claim 1.

9. Use of the benzimidazole compound according to claim 1 in the preparation of an inhibitor that simultaneously inhibits ALK and BET.

10. Use of the benzimidazole compound according to claim 1 in preparing a drug for treating tumors; Preferably, the tumor comprises a lymphoma.

Images