Preparation method of diacylglycerol kinase inhibitor
The method for preparing the diacylglycerol kinase inhibitor R59949 solves the problem of the lack of synthetic routes in the existing technology, and realizes efficient and low-cost preparation, supporting its application in tumor immunotherapy.
Patent Information
- Application Number
- CN202511039727.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-11-07
AI Technical Summary
Existing technologies have failed to provide an effective synthetic route for the diacylglycerol kinase inhibitor R59949, thus limiting its application in tumor immunotherapy.
The diacylglycerol kinase inhibitor R59949 was prepared by a series of steps including the preparation of Grignard reagents, addition reaction, Heck coupling reaction, substitution reaction, Mitsunobu reaction and cyclization reaction, under an inert gas atmosphere, using iodine as an initiator and N-tert-butyloxycarbonyl-4-piperidinone, N,N'-thiocarbonyldiimidazole and other raw materials.
A preparation method with readily available raw materials, low cost and high yield is provided to meet the needs of biological evaluation and clinical application of R59949.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of organic synthesis, and particularly relates to a preparation method of a diacylglycerol kinase inhibitor. BACKGROUND
[0002] Diacylglycerol kinase (DGK) is a kind of key lipid kinase, and its core function is to catalyze diacylglycerol (DAG) phosphorylation to generate phosphatidic acid (PA). This reaction plays a core role in cell signal transduction, lipid metabolism and membrane dynamics. Diacylglycerol kinase inhibitors regulate the lipid signal network by blocking the conversion of DAG to PA, and show important value in disease treatment.
[0003] R59949 (also known as Ricaradin) is a highly selective diacylglycerol kinase alpha (DGK alpha) inhibitor, and plays a key role in tumor immunotherapy by targeting the regulation of the lipid signal pathway. R59949 represents a new strategy for enhancing anti-tumor immunity from the perspective of lipid signal reprogramming; however, there is no related literature reporting the synthesis route of R59949.
[0004] Therefore, it is an urgent problem to be solved to develop a preparation method of the diacylglycerol kinase inhibitor R59949. SUMMARY
[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a preparation method of a diacylglycerol kinase inhibitor. The preparation method provided by the present application has simple and readily available raw materials, simple preparation method, low cost and high yield.
[0006] To achieve the purpose of the present application, the following technical solutions are adopted:
[0007] A preparation method of a diacylglycerol kinase inhibitor comprises the following steps:
[0008] S1. Under an inert gas atmosphere, 4-fluorobenzyl bromide is reacted with magnesium to obtain Grignard reagent B using iodine as an initiator;
[0009] S2. Under an inert gas atmosphere, Grignard reagent B is subjected to addition reaction with N-tert-butoxycarbonyl-4-piperidone, and then subjected to hydrolysis and deprotection to obtain intermediate C;
[0010] S3. Under an inert gas atmosphere, intermediate C, trifluoromethanesulfonic acid-4-fluorophenyl ester and a catalyst are subjected to Heck coupling reaction under alkaline conditions to obtain intermediate D;
[0011] S4. Under an inert gas atmosphere, intermediate D, chloroethanol is subjected to substitution reaction under alkaline condition to obtain intermediate E;
[0012] S5. Under an inert gas atmosphere, intermediate E is subjected to Mitsunobu reaction to obtain intermediate F;
[0013] S6. Intermediate F is subjected to addition reaction with N,N'-thioformyl diimidazole to obtain intermediate G;
[0014] S7. Intermediate G, methyl 2-aminobenzoate is subjected to cyclization reaction under the action of base to obtain the diacylglycerol kinase inhibitor R59949;
[0015] The reaction route is as follows:
[0016]
[0017] Specifically, in the S1 step, the molar ratio of the 4-fluorobromobenzyl to magnesium is 1: (10-20), the reaction temperature is reflux temperature, and the reaction time is 4-8 h; the magnesium can be selected from magnesium turnings or magnesium powder; preferably, the Grignard reaction time can be 4 h, 5 h, 6 h, 7 h, or 8 h, etc., but is not limited to the above-mentioned values, and other values within the above-mentioned value range are also applicable.
[0018] Preferably, the reaction also needs to add a solvent, and the solvent includes one of anhydrous THF, anhydrous diethyl ether.
[0019] Specifically, in the S2 step, the molar ratio of the 4-fluorobromobenzyl to N-tert-butoxycarbonyl-4-piperidone is 1: (1.1-1.3); the addition reaction temperature is 0-10℃, and the reaction time is 2-6 h; preferably, the addition reaction temperature can be 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, or 10℃, etc., and the reaction time can be 2 h, 3 h, 4 h, 5 h, or 6 h, etc., but is not limited to the above-mentioned values, and other values within the above-mentioned value range are also applicable.
[0020] Preferably, the addition reaction also needs to add a solvent, and the solvent includes one of anhydrous THF, anhydrous diethyl ether.
[0021] Specifically, in the S2 step, concentrated acid is also needed to be added in the hydrolysis and deprotection reaction to a pH value of 1-2, the reaction temperature is reflux temperature, and the reaction time is 3-5 h; preferably, the reaction time can be 3 h, 4 h, or 5 h, etc., but is not limited to the above-mentioned values, and other values within the above-mentioned value range are also applicable.
[0022] Preferably, the concentrated acid includes one of concentrated hydrochloric acid and trifluoroacetic acid.
[0023] Specifically, in the S3 step, the molar ratio of the intermediate C, trifluoromethanesulfonic acid-4-fluorophenyl ester, the catalyst, the base is 1: (1.1-1.2): (0.05-0.2): (1.2-1.3); the temperature of the Heck coupling reaction is 60-90℃, and the time is 8-16h; preferably, the temperature of the coupling reaction can be 60℃, 70℃, 80℃ or 90℃, etc., but is not limited to the above listed values, and other values not listed in the above value range are also applicable.
[0024] Preferably, the catalyst includes one of palladium acetate, palladium chloride, palladium chloride tetraphenylphosphine, and [1,1'-bis (di-tert-butylphosphine) ferrocene] dichloropalladium, and the base includes one of cesium carbonate, potassium carbonate, and potassium phosphate;
[0025] More preferably, a solvent needs to be added in the Heck coupling reaction, and the solvent includes one of anhydrous DMF and anhydrous THF.
[0026] Specifically, in the S4 step, the molar ratio of the intermediate D, chloroethanol and the base is 1: (1.1-1.3): (1.2-1.5), the temperature of the substitution reaction is reflux temperature, and the time is 8-16h; preferably, the temperature of the substitution reaction can be 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h or 16h, etc., but is not limited to the above listed values, and other values not listed in the above value range are also applicable.
[0027] Preferably, the base includes one of potassium carbonate, sodium carbonate, sodium hydroxide, and cesium carbonate;
[0028] More preferably, a solvent needs to be added in the substitution reaction, and the solvent includes one of acetonitrile, acetone, and tetrahydrofuran.
[0029] Specifically, in the S5 step, the intermediate E is converted into an amino group through a Mitsunobu reaction, the Mitsunobu reaction includes adding N-hydroxyphthalimide, triphenylphosphine and a hydroxyl activation reagent to perform a first reaction, and then adding an ammonolysis reagent to perform a second reaction to obtain the intermediate F;
[0030] The temperature of the first reaction is room temperature, and the time is 4-8h, and the temperature of the second reaction is room temperature, and the time is 1-3h; preferably, the time of the first reaction can be 4h, 5h, 6h, 7h or 8h, etc., and the time of the second reaction can be 1h, 2h or 3h, etc., but is not limited to the above listed values, and other values not listed in the above value range are also applicable.
[0031] Preferably, the Mitsunobu reaction also needs a solvent, and the solvent includes one of THF, DCM.
[0032] Preferably, the molar ratio of the intermediate E, N-hydroxyphthalimide, triphenylphosphine, a hydroxyl-activating agent and an aminolysis agent is 1:(1.1-1.3):(1.1-1.3):(1.2-1.4):(1.5-3).
[0033] Preferably, the hydroxyl-activating agent includes one of diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD), and the aminolysis agent includes one of 80% hydrazine hydrate solution or 40% aqueous methylamine solution.
[0034] Specifically, in the S6 step, the molar ratio of the intermediate F and N,N'-thio carbonyl diimidazole is 1:(1.1-1.3), the temperature of the addition reaction is room temperature, and the time is 4-8h; preferably, the time of the addition reaction can be 4h, 5h, 6h, 7h or 8h, but is not limited to the above-mentioned values, and other values not listed in the above-mentioned value range are also applicable.
[0035] Preferably, the addition reaction also needs to add a solvent, and the solvent includes one of THF, 1,4-dioxane.
[0036] Specifically, in the S7 step, the molar ratio of the intermediate G, methyl 2-aminobenzoate and a base is 1:(1.1-1.3):(1.1-1.3), the temperature of the cyclization reaction is room temperature, and the time is 2-8h; preferably, the time of the cyclization reaction can be 2h, 3h, 4h, 5h, 6h, 7h or 8h, but is not limited to the above-mentioned values, and other values not listed in the above-mentioned value range are also applicable.
[0037] Preferably, the base includes one of sodium hydroxide and potassium hydroxide.
[0038] More preferably, the cyclization reaction also needs to add a solvent, and the solvent includes one of 80% THF solution and 80% ethanol solution.
[0039] but is not limited to the above-mentioned values, and other values not listed in the above-mentioned value range are also applicable.
[0040] Compared with the prior art, the present application has the following beneficial effects:
[0041] The present application discloses a synthetic route of diacylglycerol kinase inhibitor R59949 for the first time, and the route has the characteristics of mild reaction condition, reasonable design, cheap and easily available raw materials and high yield, thereby providing theoretical support for the biological evaluation and clinical application of R59949. Detailed Implementation
[0042] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.
[0043] A method for preparing a diacylglycerol kinase inhibitor includes the following steps:
[0044] S1. Under an inert gas atmosphere, 4-fluorobenzyl bromide is reacted with magnesium in a solvent (including anhydrous THF or anhydrous diethyl ether) using iodine as an initiator. The reaction temperature is the reflux temperature, and the reaction time is 4-8 h, to obtain Grignard reagent B. The molar ratio of 4-fluorobenzyl bromide to magnesium is 1:(10-20).
[0045] S2. Under an inert gas atmosphere, Grignard reagent B reacts with N-tert-butyloxycarbonyl-4-piperidinone in a solvent (including anhydrous THF or anhydrous diethyl ether) at a temperature of 0-10℃ for 2-6 h; then, under concentrated acid conditions (including concentrated hydrochloric acid or trifluoroacetic acid), the reaction proceeds through hydrolysis and deprotection at reflux temperature for 3-5 h to obtain intermediate C; wherein the molar ratio of 4-fluorobenzyl bromide to N-tert-butyloxycarbonyl-4-piperidinone is 1:(1.1-1.3).
[0046] S3. Under an inert gas atmosphere, intermediate C, 4-fluorophenyl trifluoromethanesulfonate, and catalyst undergo a Heck coupling reaction under alkaline conditions and in the presence of a solvent (including one of anhydrous DMF and anhydrous THF) at a temperature of 60-90℃ for 8-16 hours to obtain intermediate D; the molar ratio of intermediate C, 4-fluorophenyl trifluoromethanesulfonate, catalyst, and base is 1:(1.1-1.2):(0.05-0.2):(1.2-1.3).
[0047] The catalyst includes one of palladium acetate, palladium chloride, tetra-triphenylphosphine palladium chloride, and [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium dichloride, and the base includes one of cesium carbonate, potassium carbonate, and potassium phosphate.
[0048] S4. Under an inert gas atmosphere, intermediate D and chloroethanol undergo a substitution reaction in the presence of an alkaline condition and a solvent (acetonitrile, acetone, or tetrahydrofuran) at reflux temperature for 8-16 hours to yield intermediate E. The molar ratio of intermediate D, chloroethanol, and base is 1:(1.1-1.3):(1.2-1.5). The base includes one of potassium carbonate, sodium carbonate, sodium hydroxide, or cesium carbonate.
[0049] S5. Under the atmosphere of inert gas, intermediate E and intermediate F are obtained by Mitsunobu reaction, specifically, intermediate E and N-hydroxyphthalimide, triphenylphosphine, hydroxyl-activated reagent are subjected to a first reaction in a solvent (one of THF, DCM) at room temperature for 4-8 h; then, an ammonia-lysis reagent is added to perform a second reaction at room temperature for 1-3 h;
[0050] wherein, the molar ratio of intermediate E, N-hydroxyphthalimide, triphenylphosphine, hydroxyl-activated reagent and ammonia-lysis reagent is 1:(1.1-1.3):(1.1-1.3):(1.2-1.4):(1.5-3); the hydroxyl-activated reagent includes one of diethyl azodicarboxylate or diisopropyl azodicarboxylate, and the ammonia-lysis reagent includes one of 80% hydrazine hydrate solution or 40% methylamine aqueous solution.
[0051] S6. Intermediate F and N,N'-thiocarbonyl diimidazole are subjected to an addition reaction in a solvent (one of THF, 1,4-dioxane) at room temperature for 4-8 h to obtain intermediate G; in the step S6, the molar ratio of intermediate F and N,N'-thiocarbonyl diimidazole is 1:(1.1-1.3).
[0052] S7. Under the action of intermediate G, methyl 2-aminobenzoate and a base, a cyclization reaction occurs in a solvent (one of 80% THF solution, 80% ethanol solution) at room temperature for 2-8 h to obtain diacylglycerol kinase inhibitor R59949; in the step S7, the molar ratio of intermediate G, methyl 2-aminobenzoate and the base is 1:(1.1-1.3):(1.1-1.3), wherein the base includes one of sodium hydroxide and potassium hydroxide.
[0053] The reaction route is as follows:
[0054]
[0055] Example 1
[0056] The embodiment provides a preparation method of a diacylglycerol kinase inhibitor, including the following steps:
[0057] S1. Under the protection of nitrogen, 4-fluorobenzyl bromide (20.0 g, 105.8 mmol), magnesium chips (38.6 g, 1.6 mol) and iodine (3 grains) are added to a reaction bottle containing 200 mL of anhydrous tetrahydrofuran, the reaction liquid is heated to reflux, and the reaction is performed for 6 h, no solid remains after the reaction is completed, to obtain Grignard reagent B;
[0058] S2. Under nitrogen protection, N-tert-butoxycarbonyl-4-piperidone (25.3 g, 127.0 mmol) was added to a reaction flask containing 300 mL of anhydrous tetrahydrofuran, and the temperature was lowered to 0°C. The Grignard reagent B prepared above was slowly added to the reaction solution, and the temperature was controlled to be less than 5°C. After the addition was completed, the temperature was slowly raised to room temperature, and the reaction was continued for 4 h. TLC detection showed that there was no starting material left. The reaction solution was poured into 100 mL of concentrated hydrochloric acid, and heated to reflux for 4 h. When the reaction was complete, the pH was adjusted to 1-2. 4M NaOH aqueous solution was added to adjust the pH to 11-12. 200 mL of ethyl acetate was added, and the extraction was separated. The organic phase was collected, and the aqueous phase was extracted with 100 mL of ethyl acetate for three times. The organic phases were combined, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated, and column chromatography (ethyl acetate: petroleum ether = 1:6) was performed to obtain the intermediate C 17.7 g in the form of a light yellow solid, with a yield of 87.5%.
[0059] S3. Under nitrogen protection, intermediate C (15.0 g, 78.4 mmol), 4-fluorophenyl trifluoromethanesulfonate (22.0 g, 90.2 mmol), cesium carbonate (31.9 g, 98.0 mmol), and palladium acetate (880 mg, 3.9 mmol) were added to a reaction flask containing 300 mL of anhydrous DMF, and heated to 80°C for 12 h. TLC detection showed that there was no starting material left. The reaction solution was poured into 1 L of saturated brine, and 200 mL of ethyl acetate was added. The extraction was separated, and the organic phase was collected. The aqueous phase was extracted with 100 mL of ethyl acetate for three times. The organic phases were combined, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated to obtain a light yellow crude product. The crude product was dissolved in 200 mL of ethyl acetate, and 200 mL of 6M hydrochloric acid was added. The extraction was performed, and the aqueous phase was collected. The aqueous phase was washed with 100 mL of ethyl acetate for three times. The aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, and 100 mL of ethyl acetate was added for extraction three times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated to obtain the intermediate D 19.2 g in the form of a light yellow solid, with a yield of 85.8%.
[0060] S4. Under nitrogen protection, intermediate D (15.0 g, 52.6 mmol), chloroethanol (5.1 g, 63.1 mmol), and potassium carbonate (10.9 g, 78.9 mmol) were added to a reaction flask containing 200 mL of acetonitrile, and heated to reflux for 12 h. The temperature was lowered to room temperature, and the filtration was performed. The organic phase was collected, and the organic phase was concentrated to obtain a crude product. Column chromatography (ethyl acetate: petroleum ether = 1:3) was performed to obtain the intermediate E 16.8 g in the form of a light yellow solid, with a yield of 97.0%.
[0061] S5. Intermediate E (10.0 g, 30.4 mmol), N-hydroxyphthalimide (5.9 g, 36.4 mmol), triphenylphosphine (8.8 g, 33.4 mmol), DIAD (8.0 g, 39.5 mmol) were added into a reaction flask containing 200 mL THF, the first reaction was carried out at room temperature for 4 h, then 40% aqueous methylamine solution (4.7 g, 60.7 mmol) was added dropwise, the second reaction was continued at room temperature for 2 h, the reaction was complete, filtration was carried out, the filtrate was collected, and concentrated to obtain a crude product. The crude product was dissolved in 200 mL ethyl acetate, 200 mL 6M hydrochloric acid was added, extraction was carried out, the aqueous phase was collected, the aqueous phase was washed with 100 mL ethyl acetate for three times, the aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, 100 mL ethyl acetate was added for extraction three times, the organic phase was collected, dried over anhydrous sodium sulfate, filtration was carried out, the filtrate was collected, and concentrated to obtain a light yellow solid, which was intermediate F 8.8 g, the yield was 88.3%.
[0062] S6. Intermediate F (10.0 g, 30.5 mmol) was added into a reaction flask containing 150 mL THF, N,N'-thio carbonyl diimidazole (6.5 g, 36.5 mmol) was slowly added, the reaction was carried out at room temperature for 6 h, the reaction was complete, a large amount of solid was precipitated in the reaction system, filtration was carried out, the filtrate was collected, and concentrated to obtain a crude product, column chromatography (ethyl acetate: petroleum ether = 1:5) was carried out to obtain a light yellow solid, which was intermediate G 10.3 g, the yield was 91.3%.
[0063] S7. Intermediate G (10.0 g, 27.0 mmol), methyl 2-aminobenzoate (4.9 g, 32.4 mmol), sodium hydroxide (1.3 g, 32.4 mmol) were added into a reaction flask containing 150 mL 80% THF solution, the reaction was carried out at room temperature for 6 h, the reaction was complete, TLC detection showed that there was no raw material left, 50 mL ethyl acetate was added, extraction was carried out, the organic phase was collected, the aqueous phase was extracted with 50 mL ethyl acetate for three times, the organic phase was combined, washed with saturated sodium chloride solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtration was carried out, the filtrate was collected, and concentrated to obtain a crude product, which was recrystallized with ethyl acetate to obtain a light yellow solid, which was diacylglycerol kinase inhibitor R599491 1.1 g, the yield was 83.9%, and the purity was 99.9%.
[0064] Example 2
[0065] The embodiment provides a preparation method of a diacylglycerol kinase inhibitor, which comprises the following steps:
[0066] S1. Under nitrogen protection, 4-fluorobenzyl bromide (20.00 g, 105.8 mmol), magnesium turnings (25.7 g, 1.06 mol), iodine (2 grains) were added into a reaction flask containing 200 mL of anhydrous tetrahydrofuran, the reaction liquid was heated to reflux, and the reaction was carried out for 4 h. After the reaction was completed, no solid remained, and Grignard reagent B was obtained.
[0067] S2. Under nitrogen protection, N-tert-butoxycarbonyl-4-piperidone (23.2 g, 116.4 mmol) was added into a reaction flask containing 300 mL of anhydrous tetrahydrofuran, and the temperature was lowered to 0°C. Grignard reagent B was slowly added into the reaction liquid, and the temperature was controlled to be less than 10°C. After the addition was completed, the temperature was slowly increased to room temperature, and the reaction was continued for 2 h. After TLC detection showed that no raw material remained, the reaction liquid was poured into 100 mL of concentrated hydrochloric acid, heated to reflux for 3 h, and the reaction was completed until the pH was 1-2. 4M NaOH aqueous solution was added to adjust the pH to 11-12. 200 mL of ethyl acetate was added, and the extraction was separated. The organic phase was collected, the aqueous phase was extracted with 100 mL of ethyl acetate for three times, the organic phases were combined, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated. Column chromatography (ethyl acetate: petroleum ether = 1:6) was performed to obtain a light yellow solid, which was intermediate C, 17.5 g, with a yield of 86.6%.
[0068] S3. Under nitrogen protection, intermediate C (15.0 g, 78.4 mmol), 4-fluorophenyl trifluoromethanesulfonate (21.1 g, 86.3 mmol), cesium carbonate (28.1 g, 86.3 mmol), and palladium acetate (1.76 g, 7.84 mmol) were added into a reaction flask containing 300 mL of anhydrous DMF, heated to 60°C, and incubated for 16 h. After TLC detection showed that no raw material remained, the reaction liquid was poured into 1 L of saturated brine, 200 mL of ethyl acetate was added, and the extraction was separated. The organic phase was collected, the aqueous phase was extracted with 100 mL of ethyl acetate for three times, the organic phases were combined, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain a light yellow crude product. The crude product was dissolved in 200 mL of ethyl acetate, 200 mL of 6M hydrochloric acid was added, extracted, and the aqueous phase was collected. The aqueous phase was washed with 100 mL of ethyl acetate for three times, the aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, 100 mL of ethyl acetate was added for extraction three times, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain a light yellow solid, which was intermediate D, 19.0 g, with a yield of 84.9%.
[0069] S4. Under nitrogen protection, intermediate D (15.0 g, 52.6 mmol), chloroethanol (4.7 g, 57.9 mmol), potassium carbonate (8.7 g, 63.1 mmol) were added to a reaction flask containing 200 mL of acetonitrile, heated to reflux for 8 h, reduced to room temperature, filtered, collected organic phase, concentrated to get crude product, column chromatography (ethyl acetate: petroleum ether = 1:3) to get yellowish solid, intermediate E 16.3 g, yield: 94.1%.
[0070] S5. Under nitrogen protection, intermediate E (10.0 g, 30.4 mmol), N-hydroxyphthalimide (5.5 g, 33.4 mmol), triphenylphosphine (9.6 g, 36.4 mmol), DIAD (7.4 g, 36.4 mmol) were added to a reaction flask containing 200 mL of THF, the first reaction was carried out at room temperature for 6 h, then 40% aqueous methylamine solution (3.5 g, 45.5 mmol) was added dropwise, and the second reaction was continued at room temperature for 1 h, the reaction was complete, filtered, collected filtrate, concentrated to get crude product. The crude product was dissolved in 200 mL of ethyl acetate, 200 mL of 6M hydrochloric acid was added, extracted, collected aqueous phase, the aqueous phase was washed with 100 mL of ethyl acetate three times, the aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, 100 mL of ethyl acetate was added to extract three times, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, collected filtrate, concentrated to get yellowish solid, intermediate F 8.5 g, yield: 85.2%.
[0071] S6. Intermediate F (10.0 g, 30.5 mmol) was added to a reaction flask containing 150 mL of THF, N,N'-thiocarbonyldiimidazole (6.0 g, 33.5 mmol) was slowly added, and the reaction was carried out at room temperature for 4 h, the reaction was complete, and a large amount of solid was precipitated from the reaction system, filtered, collected filtrate, concentrated to get crude product, column chromatography (ethyl acetate: petroleum ether = 1:5) to get yellowish solid, intermediate G 10.1 g, yield: 89.5%.
[0072] S7. Intermediate G (10.0 g, 27.0 mmol), methyl 2-aminobenzoate (4.5 g, 29.7 mmol), sodium hydroxide (1.2 g, 29.7 mmol) were added to a reaction flask containing 150 mL of 80% THF solution, and the reaction was carried out at room temperature for 2 h, the reaction was complete, TLC detection showed that there was no raw material left, 50 mL of ethyl acetate was added, extracted and separated, the organic phase was collected, the aqueous phase was extracted with 50 mL of ethyl acetate three times, the organic phase was combined, washed with saturated sodium chloride solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, collected filtrate, concentrated to get crude product, recrystallized with ethyl acetate to get yellowish solid, diacylglycerol kinase inhibitor R599491 1.1 g, yield: 84.2%, purity: 99.8%.
[0073] Example 3
[0074] The present example provides a method for preparing a diacylglycerol kinase inhibitor, comprising the following steps:
[0075] S1. Under nitrogen protection, 4-fluorobenzyl bromide (20.0 g, 105.8 mmol), magnesium turnings (51.4 g, 2.12 mol), iodine (3 grains) were added to a reaction flask containing 300 mL of anhydrous tetrahydrofuran, the reaction liquid was heated to reflux, and the reaction was carried out for 8 h. No solid remained after the reaction was complete, and Grignard reagent B was obtained.
[0076] S2. Under nitrogen protection, N-tert-butoxycarbonyl-4-piperidone (27.4 g, 137.5 mmol) was added to a reaction flask containing 300 mL of anhydrous tetrahydrofuran, and the temperature was lowered to 0°C. Grignard reagent B was slowly added to the reaction liquid, and the temperature was controlled to be less than 5°C. After the addition was completed, the temperature was slowly raised to room temperature, and the reaction was continued for 6 h. TLC detection showed that no raw material remained, the reaction liquid was poured into 100 mL of concentrated hydrochloric acid, heated to reflux for 5 h, and the reaction was complete when the pH was 1-2. 4M NaOH aqueous solution was added to adjust the pH to 11-12. 200 mL of ethyl acetate was added, the extraction was separated, the organic phase was collected, the aqueous phase was extracted with 100 mL of ethyl acetate three times, the organic phases were combined, washed with saturated sodium chloride solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated, and column chromatography (ethyl acetate: petroleum ether = 1:6) was performed to obtain a light yellow solid, which was intermediate C, 18.1 g, yield: 89.5%.
[0077] S3. Under nitrogen protection, intermediate C (15.0 g, 78.4 mmol), 4-fluorophenyl trifluoromethanesulfonate (23.0 g, 94.1 mmol), cesium carbonate (33.2 g, 102.0 mmol), and palladium acetate (3.5 g, 15.7 mmol) were added to a reaction flask containing 300 mL of anhydrous DMF, heated to 90°C, and incubated for 8 h. TLC detection showed that no raw material remained, the reaction liquid was poured into 1 L of saturated brine, 200 mL of ethyl acetate was added, the extraction was separated, the organic phase was collected, the aqueous phase was extracted with 100 mL of ethyl acetate three times, the organic phases were combined, washed with saturated sodium chloride solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated to obtain a light yellow crude product. The crude product was dissolved in 200 mL of ethyl acetate, 200 mL of 6M hydrochloric acid was added, extracted, the aqueous phase was collected, the aqueous phase was washed with 100 mL of ethyl acetate three times, the aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, 100 mL of ethyl acetate was added and extracted three times, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated to obtain a light yellow solid, which was intermediate D, 19.5 g, yield: 87.1%.
[0078] S4. Under nitrogen protection, intermediate D (15.0 g, 52.6 mmol), chloroethanol (5.5 g, 68.4 mmol), potassium carbonate (9.8 g, 71.0 mmol) were added to a reaction flask containing 200 mL of acetonitrile, heated to reflux for 16 h, reduced to room temperature, filtered, collected organic phase, concentrated to obtain a crude product, column chromatography (ethyl acetate: petroleum ether = 1:3) to obtain a light yellow solid, intermediate E 16.5 g, yield: 95.3%.
[0079] S5. Under nitrogen protection, intermediate E (10.0 g, 30.4 mmol), N-hydroxyphthalimide (6.4 g, 39.5 mmol), triphenylphosphine (10.4 g, 39.5 mmol), DIAD (8.6 g, 42.5 mmol) were added to a reaction flask containing 200 mL of THF, the first reaction was carried out at room temperature for 8 h, then 40% aqueous methylamine solution (7.1 g, 91.1 mmol) was added dropwise, and the second reaction was continued at room temperature for 3 h, the reaction was complete, filtered, collected the filtrate, concentrated to obtain a crude product. The crude product was dissolved in 200 mL of ethyl acetate, 200 mL of 6M hydrochloric acid was added, extracted, collected the aqueous phase, the aqueous phase was washed with 100 mL of ethyl acetate three times, the aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, extracted with 100 mL of ethyl acetate three times, collected the organic phase, dried over anhydrous sodium sulfate, filtered, collected the filtrate, concentrated to obtain a light yellow solid, intermediate F 9.2 g, yield: 92.3%.
[0080] S6. Intermediate F (10.0 g, 30.5 mmol) was added to a reaction flask containing 150 mL of THF, N,N'-thio carbonyl diimidazole (7.1 g, 39.6 mmol) was slowly added, and the reaction was carried out at room temperature for 8 h, the reaction was complete, and a large amount of solid was precipitated from the reaction system, filtered, collected the filtrate, concentrated to obtain a crude product, column chromatography (ethyl acetate: petroleum ether = 1:5) to obtain a light yellow solid, intermediate G 10.5 g, yield: 93.1%.
[0081] S7. Intermediate G (10.0 g, 27.0 mmol), methyl 2-aminobenzoate (8.0 g, 227.26, 35.1 mmol), sodium hydroxide (1.4 g, 40, 35.1 mmol) were added to a reaction flask containing 150 mL of 80% THF solution, and the reaction was carried out at room temperature for 8 h, the reaction was complete, TLC detection showed that there was no raw material left, 50 mL of ethyl acetate was added, extracted and separated, collected the organic phase, the aqueous phase was extracted with 50 mL of ethyl acetate three times, the organic phases were combined, washed with saturated sodium chloride solution, collected the organic phase, dried over anhydrous sodium sulfate, filtered, collected the filtrate, concentrated to obtain a crude product, recrystallized with ethyl acetate to obtain a light yellow solid, diacylglycerol kinase inhibitor R599491 1.2 g, yield: 84.8%, purity: 99.9%.
[0082] Example 4
[0083] The present embodiment provides a preparation method of a diacylglycerol kinase inhibitor, comprising the following steps:
[0084] S1. Under the protection of nitrogen, 4-fluorobenzyl bromide (20.0 g, 105.8 mmol), magnesium chips (38.6 g, 1.59 mol), iodine (3 grains) were added to a reaction bottle containing 200 mL of anhydrous ether, the reaction liquid was heated to reflux, and the reaction was carried out for 6 h. After the reaction was completed, no solid remained, and a Grignard reagent B was obtained.
[0085] S2. Under the protection of nitrogen, N-tert-butoxycarbonyl-4-piperidone (25.3 g, 127.0 mmol) was added to a reaction bottle containing 300 mL of anhydrous ether, and the temperature was lowered to 0°C. The Grignard reagent B was slowly added to the reaction liquid, and the temperature was controlled to be less than 5°C. After the addition was completed, the temperature was slowly increased to room temperature, and the reaction was continued for 4 h. TLC detection showed that no raw material remained. The reaction liquid was poured into 100 mL of trifluoroacetic acid, heated to reflux for 4 h, and the reaction was completed to pH = 1-2. 4M NaOH aqueous solution was added to adjust the pH to 11-12. 200 mL of ethyl acetate was added, extracted and separated, the organic phase was collected, the aqueous phase was extracted with 100 mL of ethyl acetate for three times, the organic phases were combined, washed with saturated sodium chloride solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated, and column chromatography (ethyl acetate: petroleum ether = 1:6) was performed to obtain a light yellow solid, which was intermediate C, 17.6 g, yield: 87.0%.
[0086] S3. Under the protection of nitrogen, intermediate C (15.0 g, 78.4 mmol), 4-fluorophenyl trifluoromethanesulfonate (22.0 g, 90.2 mmol), potassium carbonate (13.6 g, 98.0 mmol), and palladium chloride (695 mg, 3.92 mmol) were added to a reaction bottle containing 300 mL of anhydrous THF, heated to 80°C, and incubated for 12 h. TLC detection showed that no raw material remained. The reaction liquid was poured into 1 L of saturated brine, 200 mL of ethyl acetate was added, extracted and separated, the organic phase was collected, the aqueous phase was extracted with 100 mL of ethyl acetate for three times, the organic phases were combined, washed with saturated sodium chloride solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated to obtain a light yellow crude product. The crude product was dissolved in 200 mL of ethyl acetate, 200 mL of 6M hydrochloric acid was added, extracted, the aqueous phase was collected, the aqueous phase was washed with 100 mL of ethyl acetate for three times, the aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, 100 mL of ethyl acetate was added to extract three times, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated to obtain a light yellow solid, which was intermediate D, 19.4 g, yield: 86.7%.
[0087] S4. Under nitrogen protection, intermediate D (15.0 g, 52.6 mmol), chloroethanol (5.1 g, 63.1 mmol), sodium hydroxide (3.2 g, 78.9 mmol) were added to a reaction flask containing 200 mL of acetone, heated to reflux for 12 h, reduced to room temperature, filtered, collected organic phase, concentrated to get crude product, column chromatography (ethyl acetate: petroleum ether = 1:3) to get yellowish solid, intermediate E 16.2 g, yield: 93.6%.
[0088] S5. Under nitrogen protection, intermediate E (10.0 g, 30.4 mmol), N-hydroxyphthalimide (5.9 g, 36.4 mmol), triphenylphosphine (8.8 g, 33.4 mmol), DEAD (6.9 g, 39.5 mmol) were added to a reaction flask containing 200 mL of DCM, the first reaction was carried out at room temperature for 4 h, then 80% hydrazine hydrate (3.8 g, 60.7 mmol) was added dropwise, the second reaction was continued at room temperature for 2 h, the reaction was complete, filtered, collected filtrate, concentrated to get crude product. The crude product was dissolved in 200 mL of ethyl acetate, 200 mL of 6M hydrochloric acid was added, extracted, collected aqueous phase, the aqueous phase was washed with 100 mL of ethyl acetate three times, the aqueous phase was adjusted to pH 12-13 with 4M NaOH solution, 100 mL of ethyl acetate was added to extract three times, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, collected filtrate, concentrated to get yellowish solid, intermediate F 8.9 g, yield: 89.3%.
[0089] S6. Intermediate F (10.0 g, 30.5 mmol) was added to a reaction flask containing 150 mL of 1,4-dioxane, N,N'-thio carbonyl diimidazole (6.5 g, 36.5 mmol) was slowly added, the reaction was carried out at room temperature for 6 h, the reaction was complete, a large amount of solid was precipitated from the reaction system, filtered, collected filtrate, concentrated to get crude product, column chromatography (ethyl acetate: petroleum ether = 1:5) to get yellowish solid, intermediate G 10.1 g, yield: 89.5%.
[0090] S7. Intermediate G (10.0 g, 27.0 mmol), methyl 2-aminobenzoate (4.9 g, 32.4 mmol), potassium hydroxide (1.8 g, 32.4 mmol) were added to a reaction flask containing 150 mL of 80% ethanol solution, the reaction was carried out at room temperature for 6 h, the reaction was complete, TLC detection showed that there was no raw material left, 50 mL of ethyl acetate was added, extracted and separated, the organic phase was collected, the aqueous phase was extracted with 50 mL of ethyl acetate three times, the organic phases were combined, washed with saturated sodium chloride solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, collected filtrate, concentrated to get crude product, recrystallized with ethyl acetate to get yellowish solid, diacylglycerol kinase inhibitor R599491 1.0 g, yield: 82.9%, purity: 99.9%.
[0091] The applicant declares that the present application is illustrated by the above examples for the preparation of diacylglycerol kinase inhibitors, but the present application is not limited to the above examples, i.e. it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.
[0092] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.
[0093] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.
Claims
1. A method for the preparation of a diacylglycerol kinase inhibitor, characterized by, The method comprises the following steps: S1. under the atmosphere of inert gas, 4-fluorobenzyl bromide reacts with magnesium to obtain Grignard reagent B by using iodine as initiator; S2. under the atmosphere of inert gas, Grignard reagent B reacts with N-tert-butyloxycarbonyl-4-piperidone to obtain intermediate C by addition reaction, and then hydrolysis and deprotection are carried out; S3. under the atmosphere of inert gas, intermediate C, trifluoromethanesulfonic acid-4-fluorobenzyl ester and catalyst are subjected to Heck coupling reaction under alkaline condition to obtain intermediate D; S4. under the atmosphere of inert gas, intermediate D and chloroethanol are subjected to substitution reaction under alkaline condition to obtain intermediate E; S5. under the atmosphere of inert gas, intermediate E is subjected to Mitsunobu reaction to obtain intermediate F; S6. intermediate F and N,N'-thiocarbonyldiimidazole are subjected to addition reaction to obtain intermediate G; S7. intermediate G and methyl 2-aminobenzoate are subjected to cyclization reaction under the action of alkali to obtain the diacylglycerol kinase inhibitor R59949; The reaction route is as follows:
2. The production method according to claim 1, characterized by, In the step S1, the molar ratio of 4-fluorobenzyl bromide to magnesium is 1: (10-20), the reaction temperature is reflux temperature, and the reaction time is 4-8 h; Preferably, the reaction further needs to add a solvent, and the solvent comprises one of anhydrous THF and anhydrous diethyl ether.
3. The preparation method according to claim 1, characterized in that, In the step S2, the molar ratio of 4-fluorobenzyl bromide to N-tert-butyloxycarbonyl-4-piperidone is 1: (1.1-1.3), the addition reaction temperature is 0-10℃, and the reaction time is 2-6 h; Preferably, the addition reaction further needs to add a solvent, and the solvent comprises one of anhydrous THF and anhydrous diethyl ether.
4. The method of claim 1, wherein, In the step S2, concentrated acid is further added in the hydrolysis and deprotection reaction, the reaction temperature is reflux temperature, and the reaction time is 3-5 h; Preferably, the concentrated acid comprises one of concentrated hydrochloric acid and trifluoroacetic acid.
5. The preparation method according to claim 1, characterized in that, In the step S3, the molar ratio of intermediate C, trifluoromethanesulfonic acid-4-fluorobenzyl ester, catalyst and alkali is 1: (1.1-1.2): (0.05-0.2): (1.2-1.3), the Heck coupling reaction temperature is 60-90℃, and the reaction time is 8-16 h; Preferably, the catalyst comprises one of palladium acetate, palladium chloride, tetrakis triphenylphosphine palladium, palladium chloride and [1,1'-bis (di-tert-butylphosphine) ferrocene] dichloride, and the alkali comprises one of cesium carbonate, potassium carbonate and potassium phosphate; More preferably, the Heck coupling reaction further needs to add a solvent, and the solvent comprises one of anhydrous DMF and anhydrous THF.
6. The method of claim 1, wherein, In the step S4, the molar ratio of intermediate D, chloroethanol and alkali is 1: (1.1-1.3): (1.2-1.5), the substitution reaction temperature is reflux temperature, and the reaction time is 8-16 h; Preferably, the alkali comprises one of potassium carbonate, sodium carbonate, sodium hydroxide and cesium carbonate; More preferably, the substitution reaction further needs to add a solvent, and the solvent comprises one of acetonitrile, acetone and tetrahydrofuran.
7. The preparation method according to claim 1, characterized in that, In step S5, the Mitsunobu reaction includes adding N-hydroxyphthalimide, triphenylphosphine and a hydroxyl-activating reagent for a first reaction, and then adding an ammonolysis reagent for a second reaction to obtain the intermediate F; The first reaction is carried out at room temperature for 4-8 hours, and the second reaction is carried out at room temperature for 1-3 hours. Preferably, the Mitsunobu reaction further needs a solvent, and the solvent includes one of THF and DCM.
8. The production method according to claim 7, characterized by, In step S5, the molar ratio of the intermediate E, N-hydroxyphthalimide, triphenylphosphine, the hydroxyl-activating reagent and the ammonolysis reagent is 1:(1.1-1.3):(1.1-1.3):(1.2-1.4):(1.5-3). Preferably, the hydroxyl-activating reagent includes one of diethyl azodicarboxylate and diisopropyl azodicarboxylate, and the ammonolysis reagent includes one of 80% hydrazine hydrate solution and 40% methylamine aqueous solution.
9. The method of claim 1, wherein, In step S6, the molar ratio of the intermediate F and N,N'-thiocarbonyl diimidazole is 1:(1.1-1.3), and the addition reaction is carried out at room temperature for 4-8 hours. Preferably, the addition reaction further needs to add a solvent, and the solvent includes one of THF and 1,4-dioxane.
10. The method of claim 1, wherein, In step S7, the molar ratio of the intermediate G, methyl 2-aminobenzoate and a base is 1:(1.1-1.3):(1.1-1.3), and the cyclization reaction is carried out at room temperature for 2-8 hours. Preferably, the base includes one of sodium hydroxide and potassium hydroxide. More preferably, the cyclization reaction further needs to add a solvent, and the solvent includes one of 80% THF solution and 80% ethanol solution.