A method for preparing a transferase inhibitor and its intermediate

Abstract

The present invention relates to a method for preparing a transferase inhibitor and an intermediate thereof; the present invention provides for the first time a histone lysine N-methyltransferase inhibitor compound 1-(4-(4-methoxybenzoyl)oxy)phenethyl)-2-(4-(trifluoromethyl)benzamide)-1H-benzo[d]imidazole-5-carboxylic acid and a method for preparing an intermediate compound 2-amino-1-(4-(4-methoxybenzoyl)oxy)phenethyl)-1H-benzo[d]imidazole-5-carboxylic acid tert-butyl ester thereof; except for raw materials, the intermediate compounds are all new compounds. The preparation method used in the present invention has mild reaction conditions, good yield, low post-processing difficulty, provides a guarantee for the subsequent preparation of high-quality drugs, is not only suitable for laboratory small-scale preparation, but also suitable for industrial large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis and relates to a method for preparing a transferase inhibitor and an intermediate thereof, and more specifically, relates to a method for preparing a histone lysine methyltransferase inhibitor compound, 1-(4-(4-methoxybenzoyl)oxy)phenethyl)-2-(4-(trifluoromethyl)benzamido)-1H-benzo[d]imidazole-5-carboxylic acid and an intermediate compound, 2-amino-1-(4-(4-methoxybenzoyl)oxy)phenethyl)-1H-benzo[d]imidazole-5-carboxylic acid tert-butyl ester. Background Art

[0002] Euchromatic histone lysine N-methyltransferase G9a, also known as EHMT2 (euchromatic histone-lysine N-methyltransferase 2), can catalyze the methylation of lysine 9 (H3K9) of histone H3 and lysine 373 (K373) of p53. Studies have shown that G9a is closely related to processes or diseases such as the maintenance of the latent period of human immunodeficiency virus-1, cocaine addiction, central nervous system disorders, gene expression and transcription, and differentiation of hematopoietic stem cells. In addition, it can also act as a co-activator of p21 transcription and cause cell apoptosis. G9a is overexpressed in many cancers such as leukemia, prostate cancer, liver cancer, and lung cancer. Knockout of the G9a gene can inhibit the growth of prostate cancer, lung cancer, and other cells. In a mouse model, when G9a was knocked out, a slowdown in the development of acute myeloid leukemia was observed. It can be seen that G9a-mediated histone methylation is closely related to the occurrence and development of tumors, and is considered to be a new anti-tumor target with broad prospects. The development of its inhibitors has also received increasing attention.

[0003] Among them, the compound 1-(4-(4-methoxybenzoyl)oxy)phenethyl)-2-(4-(trifluoromethyl)benzamido)-1H-benzo[d]imidazole-5-carboxylic acid (product code BIX-01338), the structure of which is shown in the following formula, is a histone lysine N-methyltransferase inhibitor and plays a very important role in the field of medicine, but there is no prior art reporting its synthesis method.

[0004]

[0005] Therefore, designing and implementing a synthetic method that is suitable for industrial production, simple to operate and has a high yield has become the focus of research and development for those skilled in the art. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing a transferase inhibitor and an intermediate thereof. Specifically, the object of the present invention is to provide a method for preparing a histone lysine methyltransferase inhibitor compound 1-(4-(4-methoxybenzoyl)oxy)phenethyl)-2-(4-(trifluoromethyl)benzamido)-1H-benzo[d]imidazole-5-carboxylic acid and an intermediate compound 2-amino-1-(4-(4-methoxybenzoyl)oxy)phenethyl)-1H-benzo[d]imidazole-5-carboxylic acid tert-butyl ester, so as to solve the problems mentioned in the above background technology.

[0007] To achieve the above object, the first aspect of the present invention provides a method for preparing an intermediate compound 2-amino-1-(4-(4-methoxybenzoyl)oxy)phenethyl)-1H-benzo[d]imidazole-5-carboxylic acid tert-butyl ester, comprising the following steps:

[0008]

[0009] Step 1: Compound 1 reacts with compound 2 to obtain compound 3;

[0010] Step 2: Compound 3 undergoes reduction reaction to obtain compound 4;

[0011] Step 3: Compound 4 is cyclized with cyanogen bromide to obtain compound 5;

[0012] Step 4: Compound 5 reacts with compound 6 to obtain compound 7.

[0013] As a preferred technical solution of the present invention, the preparation method of step 1 comprises the following steps: dissolving compound 1 in an organic solvent at room temperature, adding a base and continuing stirring, then adding compound 2, stirring and reacting at room temperature for 4 to 6 hours, after the reaction is completed by TLC monitoring, adding water and extracting, combining the organic phases, washing, drying, and concentrating to obtain; preferably, the organic solvent in step 1 is selected from one or more of acetonitrile, tetrahydrofuran, dichloromethane, dioxane, 2-methyltetrahydrofuran, toluene, N,N-dimethylformamide, methyl tert-butyl ether, ether, and dimethyl sulfoxide, more preferably N,N-dimethylformamide; preferably, the base in step 1 is selected from one or more of sodium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and N,N-diisopropylethylamine, more preferably N,N-diisopropylethylamine; preferably, the molar ratio of compound 1 to compound 2 is 1:(1 to 1.5).

[0014] As a preferred technical solution of the present invention, the preparation method of step 2 comprises the following steps: dissolving compound 3 in an organic solvent, adding a noble metal catalyst, cooling to -15 to -10°C, reacting for 6 to 8 hours under hydrogen conditions, filtering, concentrating, and purifying by column chromatography after the reaction is completed by TLC monitoring, to obtain; or, the preparation method of step 2 comprises the following steps: dissolving compound 3 in an organic solvent, adding a reducing agent, reacting for 6 to 8 hours under -15°C to room temperature, filtering, concentrating, and purifying by column chromatography after the reaction is completed by TLC monitoring, to obtain; preferably, the organic solvent described in step 2 is selected from dichloromethane, isobutanol, methanol, n-butanol, butyl acetate, and ethyl acetate. one or more, more preferably a combination of methanol and ethyl acetate; when the organic solvent is selected from a combination of methanol and ethyl acetate, preferably, the volume ratio of methanol to ethyl acetate is 1:(0.8-1.2); preferably, the precious metal catalyst in step 2 is selected from one of palladium carbon, Raney nickel, and platinum carbon; more preferably palladium carbon, preferably, the mass percentage of precious metal palladium or platinum in the palladium carbon or the platinum carbon is 5-20%, preferably, the mass percentage of precious metal palladium or platinum in the palladium carbon or the platinum carbon is 20%; the reducing agent in step 2 is selected from one of lithium aluminum hydride, sodium dithionite, iron powder / ammonium chloride, and zinc powder / ammonium chloride, more preferably iron powder / ammonium chloride or zinc powder / ammonium chloride.

[0015] As a preferred technical solution of the present invention, the preparation method of step 3 comprises the following steps: dissolving compound 4 in an organic solvent, adding cyanogen bromide to control the reaction temperature not exceeding 35°C to react, monitoring the reaction conversion by TLC, removing the organic solvent by reduced pressure concentration, adding saturated sodium bicarbonate aqueous solution to adjust the pH to 7-8, stirring for 1-2 hours, filtering, and drying to obtain; preferably, the organic solvent described in step 3 is selected from one or more of dichloromethane, isobutanol, methanol, n-butanol, butyl acetate, and ethyl acetate, more preferably methanol; preferably, the molar ratio of compound 4 to cyanogen bromide described in step 3 is 1:(1.2-2).

[0016] As a preferred technical solution of the present invention, the preparation method of step 4 comprises the following steps: dissolving compound 5 in an organic solvent 1, adding a base, cooling to -3°C to 3°C, slowly dripping a mixed solution of compound 6 and organic solvent 2, stirring for 8-15 minutes after the dripping is completed, monitoring the reaction by TLC, diluting with water, and adjusting the pH to 5-6 with a dilute hydrochloric acid solution, extracting, combining the organic phases, drying, concentrating, and purifying by column chromatography to obtain; preferably, the organic solvent 1 described in step 4 is selected from one or more of dichloromethane, toluene, tetrahydrofuran, butyl acetate, and ethyl acetate, more preferably dichloromethane; preferably, the base described in step 4 is selected from potassium acetate, pyridine, sodium carbonate, potassium carbonate, triethylamine, N,N-diisopropylethylamine, phosphorus Preferably, the molar ratio of compound 5 to the base in step 4 is 1:(3-3.5); Preferably, the organic solvent 2 in step 4 is an anhydrous solvent selected from one or more of dichloromethane, toluene, tetrahydrofuran, butyl acetate and ethyl acetate, more preferably dichloromethane. The anhydrous solvent of the present invention is obtained by conventional methods well known to those skilled in the art, such as by molecular sieve drying or directly purchasing a solvent that has been dried; Preferably, the molar ratio of compound 5 to compound 6 in step 4 is 1:(0.95-1.05), and more preferably, the molar ratio of compound 5 to compound 6 in step 4 is 1:1.

[0017] The second aspect of the present invention provides a method for preparing compound 10, comprising the following steps:

[0018]

[0019] Step 5: Compound 7 and compound 8 undergo condensation reaction to obtain compound 9;

[0020] Step 6: Compound 9 is deprotected under acidic conditions to obtain compound 10.

[0021] As a preferred technical solution of the present invention, the preparation method of step 5 comprises the following steps: mixing compound 7, compound 8, a condensing agent, a catalyst and an organic solvent, stirring for 14 to 18 hours, and after the reaction is completed by LCMS monitoring, adding water to the reaction system, filtering and drying after all solids are precipitated to obtain; preferably, the condensing agent in step 5 is EDCI, DCC, HATU, TBTU, T3P, PyBOp, HOBt, and more preferably EDCI; preferably, the catalyst in step 5 is DMAP; preferably, the organic solvent in step 5 is selected from one or more of dioxane, 2-methyltetrahydrofuran, toluene, N,N-dimethylformamide, and methyl tert-butyl ether, and more preferably N,N-dimethylformamide; preferably, the molar ratio of compound 7 to compound 8 in step 5 is 1:(1 to 2); preferably, the molar ratio of compound 7 to the condensing agent in step 5 is 1:(1 to 2); preferably, the molar ratio of the condensing agent in step 5 to the catalyst is 1:(0.1 to 0.2).

[0022] As a preferred technical solution of the present invention, the preparation method of step 6 comprises the following steps: dissolving compound 9 in an organic solvent, adding acid, reacting at room temperature for 4 to 8 hours, and after the reaction is completed by LCMS monitoring, concentrating, adding water and acetonitrile for slurrying, filtering, and drying to obtain; preferably, the acid in step 6 is selected from hydrochloric acid solution, hydrochloric acid gas, hydrochloric acid methanol solution, hydrochloric acid dioxane solution, trifluoroacetic acid, and more preferably trifluoroacetic acid; preferably, the organic solvent in step 6 is selected from one or more of dichloromethane, isobutanol, methanol, n-butanol, butyl acetate, and ethyl acetate, and more preferably dichloromethane.

[0023] The third aspect of the present invention provides an intermediate compound 7 for preparing compound 10, the structure of which is as follows:

[0024]

[0025] Compared with the prior art, the beneficial effects of the present invention include:

[0026] 1) The present invention provides for the first time a method for preparing a histone lysine N-methyltransferase inhibitor compound 1-(4-(4-methoxybenzoyl)oxy)phenethyl)-2-(4-(trifluoromethyl)benzamido)-1H-benzo[d]imidazole-5-carboxylic acid and its intermediate compound 2-amino-1-(4-(4-methoxybenzoyl)oxy)phenethyl)-1H-benzo[d]imidazole-5-carboxylic acid tert-butyl ester; except for the raw materials, the intermediate compounds are all new compounds.

[0027] 2) The present invention uses readily available and inexpensive starting materials to obtain the target product through 6 steps of reaction, wherein steps 1 and 2 can be performed in two steps in succession, the reaction of step 3 is very clean, and a pure compound 5 can be obtained by simple post-treatment; after the reaction of step 5 is completed, water can be added to precipitate the pure compound 9, the reaction yield of the entire route is high, and the reaction effect is very good; especially for step 4, the present invention unexpectedly finds that the combination of acyl chloride compounds and their addition amounts, the control of the type and ratio of the reaction solvent, the screened base, and the reaction adding method, etc., based on the characteristics of compound 5, enables the reaction to obtain a single product with a high yield, and the reagents are conventional, environmentally friendly, the reaction time is short, and the atom economy is good.

[0028] 3) The preparation method used in the present invention has mild reaction conditions, good yield, and low post-processing difficulty, which provides a guarantee for the subsequent preparation of high-quality drugs. It is not only suitable for small-scale laboratory preparation, but also suitable for industrial large-scale production. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under normal conditions.

[0030] Unless otherwise specified, the raw materials and reagents used in the examples are commercially available.

[0031] The room temperature in the examples refers to 10-35° C. Unless otherwise specified, the reagents were used directly without purification. All solvents were purchased from commercial suppliers, such as Aldrich, and were used without treatment.

[0032] The reaction was terminated as judged by the consumption of the starting material by TLC analysis and / or by LCMS analysis. Analytical thin layer chromatography (TLC) was performed on glass plates pre-coated with silica gel 60 F254 0.25 mm plates (EMD Chemicals) and visualized with UV light (254 nm) and / or iodine on silica gel and / or heated with a TLC stain such as alcoholic phosphomolybdic acid, ninhydrin solution, potassium permanganate solution or ceric sulfate solution.

[0033] The abbreviations used in the present invention have the conventional meanings in the art, such as: DMF represents N,N-dimethylformamide; EA represents ethyl acetate; DIPEA represents N,N-diisopropylethylamine; PE represents petroleum ether; DCC represents N,N'-dicyclohexylcarbodiimide; EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HATU represents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; TBTU represents 2-(1H-benzotriazol-L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; T3P represents 1-propylphosphoric anhydride; PyBOp represents 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate; HOBt represents 1-hydroxybenzotriazole; DMAP represents 4-dimethylaminopyridine; MeOH represents methanol.

[0034] Example 1

[0035]

[0036] At room temperature, compound 1 (24.9 g, 103.2 mmol) was dissolved in DMF (250 mL), DIPEA (26.7 g, 206.4 mmol) was added and stirring was continued for 5 min, then compound 2 (15.6 g, 113.5 mmol) was added, and the reaction was stirred at room temperature for 5 h. After the reaction was completed by TLC monitoring, water (1250 mL) was added for dilution, and EA (500 mL×3) was used for extraction. The organic phases were combined, washed with saturated brine (1.5 L), and the aqueous phase was washed with EA (600 mL). The organic phases were combined, dried over anhydrous Na2SO4, and concentrated to dryness to obtain a crude compound 3 (37 g).

[0037] Example 2

[0038]

[0039] The crude compound 3 (37 g) obtained in Example 1 was dissolved in a mixed solution of MeOH (400 mL) and EA (400 mL), and palladium carbon (7 g, palladium content of 20%) was added. The temperature was lowered to -15°C, and a hydrogen balloon was added. The gas was replaced three times and stirred for 7 hours. After the reaction was completed by TLC monitoring, the mixture was filtered, concentrated, and purified by column chromatography (PE:EA=10:1) to obtain a pure solid compound 4 (31.9 g, two-step yield 94%, LCMS: ES + m / z=329[M+H] + ; 1H-NMR (400MHz, DMSO-d6) δ9.18(s,1H),7.15(dd,J=8.0,1.6Hz,1H),7.10(d,J=2.0Hz,1H),7.07(d,J=8.4Hz,2H),6.69(d,J=8.4Hz ,2H),6.45(d,J=8.4Hz,1H),5.18(t,J=6.4Hz,1H),4.68(s,2H),3.25(dd,J=13.2,7.2Hz,2H),2.76(t,J=8.0Hz,2H),1.49(s,9H)).

[0040] Example 3

[0041]

[0042] Compound 4 (31.9 g, 97.1 mmol) was dissolved in MeOH (200 mL), and cyanogen bromide (12.3 g, 116.5 mmol) was added. The mixture was reacted at 30°C. After the reaction was completely converted as monitored by TLC, the methanol was removed by concentration under reduced pressure. The pH of the aqueous phase was adjusted to 7-8 with saturated NaHCO3 aqueous solution. After stirring for 1 h, the mixture was filtered and dried in vacuo to obtain a pure solid compound 5 (31.6 g, 92%, LCMS: ES + m / z=354[M+H] + ; 1 H-NMR (400MHz, DMSO-d6) δ9.30(s,1H),9.02(s,2H),7.86(d,J=1.2Hz,1H),7.75(dd,J=8.4,1.6Hz,1H),7.44(d,J=8 .4Hz, 1H), 6.99 (d, J = 8.4Hz, 2H), 6.62 (d, J = 8.4Hz, 2H), 4.37 (t, J = 7.2Hz, 2H), 2.88 (t, J = 7.2Hz, 2H), 1.55 (s, 9H)).

[0043] Example 4

[0044]

[0045] Compound 5 (20 g, 56.6 mmol) was dissolved in DCM (800 mL), and DIPEA (25.6 g, 198.1 mmol) was added. When the temperature dropped to 0°C under ice bath conditions, a solution of compound 6 (9.7 g, 56.6 mmol) in ultra-dry DCM (200 mL) was slowly added dropwise. After the addition was completed, stirring was continued for 10 min. After the reaction was completed by TLC monitoring, water (400 mL) was added, and the pH of the aqueous phase was adjusted to 5-6 with a dilute hydrochloric acid solution (0.6 mol / L). The mixture was extracted with DCM (600 mL×3), and the organic phases were combined, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH=40:1) to obtain pure solid compound 7 (26.2 g, 95%, LCMS: ES + m / z=488[M+H] + ; 1 H-NMR (400MHz, DMSO-d6) δ8.26(s,2H),8.06(d,J=8.8Hz,2H),7.80(d,J=1.2Hz,1H),7.68(dd,J=8.4,1.6Hz,1H),7.41(d,J=8.4Hz,1H),7. 37(d,J=8.4Hz,2H),7.16(d,J=8.4Hz,2H),7.11(d,J=9.2Hz,2H),4.39(t,J=7.2Hz,2H),3.88(s,3H),3.01(t,J=7.2Hz,2H),1.55(s,9H)).

[0046] Example 5

[0047]

[0048] Compound 7 (18.5 g, 37.9 mmol), compound 8 (10.8 g, 56.9 mmol), EDCI (10.9 g, 56.9 mmol), and DMAP (1.39 g, 11.4 mmol) were dissolved in DMF (370 mL) and stirred for 16 h. After the reaction was completed as monitored by LCMS, water (2 L) was added to the reaction solution, and a large amount of solid precipitated. The solid was filtered and dried to obtain a solid pure compound 9 (23.5 g, 94%, LCMS: ES + m / z=660[M+H] + ; 1H-NMR (400MHz, DMSO-d6) δ12.94(s,1H),8.41(d,J=8.0Hz,2H),8.10(d,J=1.2Hz ,1H),8.03(d,J=8.8Hz,2H),7.87(d,J=8.4Hz,2H),7.79(dd,J=8.4,1.2Hz,1H),7 .55(d,J=8.4Hz,1H),7.35(d,J=8.4Hz,2H),7.13(d,J=8.4Hz,2H),7.09(d,J=8.8 Hz, 2H), 4.56 (t, J = 7.2 Hz, 2H), 3.86 (s, 3H), 3.18 (t, J = 7.2 Hz, 2H), 1.57 (s, 9H)).

[0049] Example 6

[0050]

[0051] Compound 9 (20 g, 30.3 mmol) was dissolved in DCM (500 mL), trifluoroacetic acid (100 mL) was added, and the mixture was reacted at room temperature for 6 h. After the reaction was completed by LCMS monitoring, the DCM was removed by concentration under reduced pressure, and then water (200 mL) and acetonitrile (200 mL) were added for slurrying for 16 h, filtered, and dried to obtain pure compound 10 (17.6 g, 96%, LCMS: ES + m / z=604[M+H] + , 1 H-NMR (400MHz, DMSO-d6) δ12.97(m,2H),8.42(d,J=8.4Hz,2H),8.12(d,J=1.2 Hz,1H),8.04(d,J=8.8Hz,2H),7.88(d,J=8.4Hz,2H),7.85(dd,J=8.8,1.6Hz, 1H),7.58(d,J=8.8Hz,1H),7.38(d,J=8.4Hz,2H),7.15(d,J=8.4Hz,2H),7.10 (d, J=8.8Hz, 2H), 4.56 (t, J=6.8Hz, 2H), 3.87 (s, 3H), 3.19 (t, J=6.8Hz, 2H)).

[0052] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing compound 7, characterized in that: The following steps are included: Step 1: Compound 1 reacts with compound 2 to obtain compound 3; Step 2: Compound 3 undergoes reduction reaction to obtain compound 4; Step 3: Compound 4 is cyclized with cyanogen bromide to obtain compound 5; Step 4: Compound 5 reacts with compound 6 to obtain compound 7.

2. The preparation method according to claim 1, characterized in that: The preparation method of step 1 comprises the following steps: dissolving compound 1 in an organic solvent at room temperature, adding a base and continuing stirring, then adding compound 2, stirring and reacting at room temperature for 4 to 6 hours, and after the reaction is completed by TLC monitoring, diluting with water, extracting, combining the organic phases, washing, drying, and concentrating to obtain the product.

3. The preparation method according to claim 1, characterized in that: The preparation method of step 2 comprises the following steps: dissolving compound 3 in an organic solvent, adding a noble metal catalyst, cooling to -15 to -10°C, reacting for 6 to 8 hours under hydrogen conditions, and after the reaction is completed by TLC monitoring, filtering, concentrating, and purifying by column chromatography to obtain the compound 3; the noble metal catalyst is selected from one of palladium carbon, Raney nickel, and platinum carbon.

4. The preparation method according to claim 1, characterized in that: The preparation method of step 3 comprises the following steps: dissolving compound 4 in an organic solvent, adding cyanogen bromide to control the reaction temperature not to exceed 35°C to react, monitoring the reaction conversion by TLC, removing the organic solvent by vacuum concentration, adding saturated NaHCO3 aqueous solution to adjust the pH to 7-8, stirring for 1-2h, filtering, and drying to obtain the product.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the compound 4 to cyanogen bromide is 1:(1.2-2).

6. The preparation method according to claim 1, characterized in that: The preparation method of step 4 comprises the following steps: dissolving compound 5 in organic solvent 1, adding alkali, cooling to -3°C to 3°C, slowly dropping a mixed solution of compound 6 and organic solvent 2, stirring for 8-15 minutes after the addition is completed, and after the reaction is completed by TLC monitoring, diluting with water, adjusting the pH to 5-6 with dilute hydrochloric acid solution, extracting, combining the organic phases, drying, concentrating, and purifying by column chromatography to obtain the product.

7. The preparation method according to claim 6, characterized in that: The base is selected from one or more of potassium acetate, pyridine, sodium carbonate, potassium carbonate, triethylamine, N,N-diisopropylethylamine, potassium phosphate, and sodium hydroxide; the molar ratio of the compound 5 to the base is 1:(3-3.5).

8. The preparation method according to claim 6, characterized in that: The molar ratio of the compound 5 to the compound 6 is 1:(0.95-1.05).

9. A method for preparing compound 10, comprising the following steps: Step 5: Compound 7 and compound 8 undergo condensation reaction to obtain compound 9; Step 6: Compound 9 is deprotected under acidic conditions to obtain compound 10.

10. An intermediate compound 7 for preparing compound 10, having the following structure:

Images