Regagliptin intermediates, their preparation methods and applications

Abstract

This invention discloses a regagliptin intermediate, its preparation method, and its applications. Specifically, this invention provides a method for preparing the compound shown in Figure 4, comprising the following steps: in the presence of a transaminase and an amino donor, the compound shown in Figure 3 undergoes a transamination reaction to obtain the compound shown in Figure 4, wherein R is H or a C1-C4 alkyl group. This invention employs an enzymatic method for preparing regagliptin, which has low raw material pollution, low cost, and is suitable for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a regagliptin intermediate, its preparation method and application. Background Technology

[0002] Diabetes mellitus is a multifactorial metabolic disease characterized by chronic hyperglycemia, accompanied by disorders of carbohydrate, fat, and protein metabolism caused by defects in insulin secretion and / or action. According to WHO data, diabetes ranks third among non-communicable diseases in terms of prevalence, disability, mortality, and overall health. It, along with cancer and cardiovascular disease, has become one of the three major threats to human health.

[0003] Diabetes is generally classified into type 1 and type 2 diabetes. Currently, there are over 240 million people with diabetes worldwide, more than 90% of whom have type 2 diabetes. The number of type 2 diabetes cases is increasing at a rate of 1% annually, making it a major growth driver for the diabetes drug market. Many types of antidiabetic drugs are available on the market, including injectable insulin, metformin, rosiglitazone, and pioglitazone. However, to date, no drug has been able to maintain HbA1c levels within the target range for type 2 diabetes patients in the long term. In addition, many oral hypoglycemic agents, such as sulfonylureas, alpha-glucosidase inhibitors, and thiazolidinediones, can induce weight gain in patients, and some drugs may also cause cardiovascular disease.

[0004] GLP-1 is a product expressed by the proglucagon gene after eating and mainly secreted by L-cells in the intestinal mucosa. It stimulates insulin secretion from pancreatic β-cells and plays an important role in stabilizing blood glucose. GLP-1 can effectively control blood glucose in animal models and patients with type 2 diabetes through multiple mechanisms. However, GLP-1 is rapidly degraded by DPP-IV in vivo and loses its biological activity, with a half-life of less than 2 minutes, which greatly limits its clinical application.

[0005] Dipeptidyl peptidase-IV (DPP-IV) is a serine protease that cleaves N-terminal dipeptidase in a peptide chain containing a proline residue at the next end. Studies have shown that DPP-IV can inhibit the secretion of glucagon-like peptide (GLP)-1, specifically by cleaving the N-terminal histidine dipeptidase in GLP-1, degrading it from the active form GLP-1(7-36)NH2 to the inactive GLP-1(9-36)NH2 (Endocrinology, 1999, 140:5356–5363). Furthermore, DPP-IV inhibitors can stimulate the regeneration of pancreatic β-cells, improve glucose tolerance and insulin sensitivity, thereby delaying the onset of diabetes.

[0006] Retagliptin phosphate is a 3R-amino-substituted butyramide derivative with important physiological activities and pharmaceutical value. Retagliptin is a DPP-IV inhibitor independently developed by Hengrui Medicine and is currently in phase III clinical trials. Results show that it has excellent inhibitory effects on dipeptidyl peptidase-IV (DPP-IV). The structural formula of retagliptin phosphate is as follows:

[0007]

[0008] Regarding the synthesis methods of regagliptin, there are many reports in the existing technology, which can be roughly divided into the following three methods:

[0009] (1) WO2009082881A discloses a method for preparing regagliptin hydrochloride.

[0010]

[0011] In the first step, compound A and compound B undergo a condensation reaction in dichloromethane under the conditions of triethylamine, bis(2-oxo-3-oxazolyl)phosphine chloride, and stirring at room temperature. The third step requires the use of cobalt octacarbonyl as a catalyst, which is expensive and the reaction conditions are difficult to scale up for production.

[0012] (2) CN106892926B discloses the following synthetic route for regagliptin.

[0013]

[0014] In this reaction, the dichloromethane solution of the reaction product from the second step is cooled to -15°C, a toluene solution of diethylaluminum chloride is added, the resulting mixture is stirred at -10°C, and then the compound is added dropwise. The reaction was initiated with a dichloromethane solution. The resulting mixture was heated to 10°C and stirred for 40 hours. Hydrochloric acid solution was added dropwise to quench the reaction. The organic phase was separated, washed, concentrated, and dried to obtain the product of the third step. In the fourth step, 10% wet palladium on carbon and concentrated sulfuric acid were added to a methanol solution of the product from the third step. Hydrogenation was performed, and the mixture was stirred at 40-50°C. The catalyst was removed by filtration, and the filtrate was neutralized with saturated sodium bicarbonate, concentrated, and extracted with dichloromethane. The organic phase was concentrated and dried to obtain regagliptin. This process requires the use of palladium on carbon for hydrogenation and deamination of the protecting group, and the reaction conditions are relatively dangerous and costly.

[0015] (3) CN113773323A discloses the following synthetic route for regagliptin.

[0016]

[0017] The reaction occurs in an organic solvent in the presence of N,N'-carbonyldiimidazole and imidazole hydrochloride. Compound (I) is dissolved in tetrahydrofuran, followed by the addition of N,N'-carbonyldiimidazole and imidazole hydrochloride. The mixture is heated for a period of time, then compound (II) is added, and the reaction continues. After the reaction is complete, post-treatment is performed to obtain compound (III).

[0018]

[0019] Compound (III) was reacted at room temperature in an organic solvent / HCl environment, and concentrated under reduced pressure to dryness to give compound (IV). Compound (III) was then released and reacted in an organic solvent / H3PO4 environment to give compound (IV').

[0020] Current methods are all chemical methods, requiring the use of large amounts of organic solvents, which are environmentally unfriendly. Therefore, there is an urgent need to find a more environmentally friendly preparation method. Summary of the Invention

[0021] The technical problem to be solved by this invention is to find a safe, low-cost, and environmentally friendly method for preparing regagliptin. To this end, this invention provides regagliptin intermediates, their preparation method, and applications. This preparation method has the following advantages: low raw material pollution, low cost, and suitability for industrial application.

[0022] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0023] The present invention provides a method for preparing the compound shown in 4, the method comprising the following steps: in the presence of transaminase and amino donor, the compound shown in 3 undergoes a transamination reaction to obtain the compound shown in 4;

[0024]

[0025] Wherein, R is H or a C1-C4 alkyl group.

[0026] In one scheme, R is methyl or ethyl.

[0027] The amino acid sequence of the transaminase may be SEQ ID NO:1 or have at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:1; preferably, the amino acid sequence of the transaminase is selected from one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; more preferably, the amino acid sequence of the transaminase is as shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0028] The nucleotide sequence encoding the transaminase may be selected from one or more of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; preferably, the nucleotide sequence encoding the transaminase is as shown in SEQ ID NO:5 and / or SEQ ID NO:6.

[0029] The temperature of the transamination reaction can be 25-60°C, for example 35°C or 45°C, preferably 45°C.

[0030] The amino donor may be a conventional amino donor in the art, such as one or more selected from isopropylamine, phenylethylamine, and alanine, preferably isopropylamine, such as isopropylamine hydrochloride, more preferably 4M isopropylamine hydrochloride. The molar ratio of the amino donor to the compound shown in 3 may be (1-10):1, for example 3:1.

[0031] The transamination reaction may further include a cofactor for the transaminase, such as pyridoxal phosphate, preferably 50 mM pyridoxal phosphate (12.5 g / L). The molar ratio of the compound shown in 3 to the cofactor for the transaminase may be (1-100):1, for example 20:1.

[0032] The transamination reaction may further include a reaction solvent, which is water and / or an organic solvent. The organic solvent may be a sulfoxide solvent and / or an alcohol solvent, such as one or more selected from DMSO, methanol, ethanol, and isopropanol, for example, DMSO. The water may be double-distilled water. The volume ratio of the organic solvent to the total reaction system is, for example, (0.05-1):1; when the reaction solvent is water and an organic solvent, the volume ratio of the organic solvent to the total reaction system may be (0.05-0.3):1, for example, 0.1:1 or 0.2:1.

[0033] The initial concentration of the compound shown in 3 may be 0.01-0.1 mmol / mL, for example 0.02 mmol / mL.

[0034] The transaminase may be in the form of crude enzyme solution, pure enzyme solution, crude enzyme powder, pure enzyme powder, enzyme cells, or immobilized enzyme, preferably in the form of crude enzyme solution.

[0035] The mass ratio of the transaminase cells to the compound shown in Figure 3 is (2-10):1, for example, 4.3:1.

[0036] The pH of the transamination reaction can be a pH conventional for this type of reaction in the art, such as pH 7.0-9.0, or pH 8.5. The pH is controlled by a buffer solution, which can be conventional in the art, such as one or more selected from phosphate buffer solution, Tris-HCl buffer solution and triethanolamine buffer solution, preferably triethanolamine, more preferably 0.5M triethanolamine.

[0037] The transamination reaction can take 2-48 hours, preferably 18 hours.

[0038] The preparation method of the compound shown in 4 may further include the preparation method of the compound shown in 3, wherein the preparation method of the compound shown in 3 includes the following steps: compound 1 and the compound shown in 2 are reacted in a solvent to obtain the compound shown in 3, wherein R is as described in any embodiment of the present invention.

[0039]

[0040] In the method for preparing the compound as shown in 3, the reaction can be carried out under the protection of an inert gas. The inert gas can be a conventional inert gas for this type of reaction in the art, such as one or more selected from nitrogen, helium and argon, with nitrogen being preferred.

[0041] In the preparation method of the compound shown in Figure 3, the solvent can be a conventional solvent for this type of reaction in the art, such as ester solvents and / or alcohol solvents, and for example, one or more selected from ethyl acetate, methanol, ethanol, and isopropanol, or ethyl acetate. The amount of solvent used is not limited, as long as it does not affect the reaction; for example, the molar volume ratio of compound 1 to the solvent can be 0.04-0.8 mmol / mL, for example, 0.23 mmol / mL. The molar volume ratio of the compound shown in Figure 2 to the solvent can be 0.04-0.8 mmol / mL, for example, 0.2 mmol / mL.

[0042] In the preparation method of the compound shown in 3, the molar ratio of compound 1 to the compound shown in 2 can be a conventional molar ratio for this type of reaction in the art, for example (0.5-2.0):1, or for example 1.15:1.

[0043] In the preparation method of the compound shown in Figure 3, the reaction temperature can be a conventional temperature for this type of reaction in the art, such as 20-80°C, or 60°C.

[0044] In the preparation method of the compound shown in Figure 3, the reaction progress can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), and the reaction endpoint is generally defined as the disappearance or cessation of reaction of compound 1 or the compound shown in Figure 2. The reaction time can be 4-24 hours, for example, 20 hours.

[0045] In the preparation method of the compound shown in Figure 3, the reaction may further include a post-treatment. The post-treatment operation is a conventional post-treatment operation in this type of preparation method in the art, and includes the following steps: concentration and purification to obtain the compound shown in Figure 3. The concentration can be a conventional concentration method in this type of reaction in the art, such as vacuum concentration (vacuum degree <3 kPa, 50°C). The purification can be a conventional purification method in this type of compound in the art, such as column chromatography (petroleum ether: ethyl acetate = 3:2).

[0046] The present invention also provides a compound as shown in 3, wherein R is as described in any embodiment of the present invention:

[0047]

[0048] The present invention also provides a method for preparing the compound shown in Figure 3, wherein the preparation method is as described in any of the preceding embodiments.

[0049] The present invention also provides an application of the compound shown in 3, the application being for the preparation of compound 4, comprising the following steps: in the presence of the transaminase and the amino donor, the compound shown in 3 undergoes a transamination reaction to obtain the compound shown in 4, wherein R is as described in any embodiment of the present invention;

[0050]

[0051] The transamination reaction can be performed as described in any of the preceding schemes.

[0052] The application may also include a method for preparing the compound shown in Figure 3, wherein the method for preparing the compound shown in Figure 3 is as described in any of the preceding embodiments.

[0053] The present invention also provides an application of a transaminase; said application is for preparing a compound as shown in 4, wherein R is as described in any embodiment of the present invention, and the amino acid sequence of the transaminase is SEQ ID NO:1 or has at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:1; preferably, the amino acid sequence of the transaminase is selected from one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; more preferably, the amino acid sequence of the transaminase is as shown in SEQ ID NO:1 and / or SEQ ID NO:2;

[0054] Further, the nucleotide sequence encoding the transaminase is selected from one or more of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; for example, the nucleotide sequence encoding the transaminase is shown in SEQ ID NO:5 and / or SEQ ID NO:6;

[0055]

[0056] The application of the transaminase, and the preparation method of the compound shown in 4 are as described in any of the preceding schemes.

[0057] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0058] The reagents and raw materials used in this invention are all commercially available.

[0059] The positive and progressive effects of this invention are as follows: This invention employs an enzymatic method for preparing regagliptin, which has low raw material pollution, low cost, and is suitable for industrialization. Detailed Implementation

[0060] The present invention is further illustrated below by way of examples, but these examples do not limit the invention to the scope of the embodiments described. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions, or as selected according to the product instructions. Reagents not specified as originating from are commercially available, conventional reagents.

[0061] Compound 1 was prepared in our laboratory by referring to the method in WO2004083212A1.

[0062] Compound 2-I was purchased from Shanghai Kalulan Technology Co., Ltd.

[0063] Example 1: Synthesis of the compound shown in 3-I

[0064]

[0065] Under nitrogen protection, 5-[1-hydroxy-2-(2,4,5-trifluorophenyl)ethylene]-2,2-dimethyl-1,3-dioxane-4,6-dione (730 mg, 2.3 mmol) and 5 mL of ethyl acetate were added to a 25 mL single-necked flask and stirred to dissolve. Methyl 3-(trifluoromethyl)-5H,6H,7H,8H-imidazo[1,5-a]pyrazine-1-carboxylic acid (0.5 g, 2.0 mmol) was dissolved in 5 mL of ethyl acetate and added to the reaction solution. The mixture was heated to 60 °C and refluxed for 20 h. The reaction was completed by TLC.

[0066] After the reaction was completed, the solution was concentrated under vacuum (vacuum <3 kPa, 50℃) to give 1.2 g of a yellow oil. Column chromatography purification yielded 0.65 g of a yellow solid, 7-[3-oxo-4-(2,4,5-trifluorophenyl)butyryl]-3-(trifluoromethyl)-5H,6H,7H,8H imidazo[1,5-a]pyrazine-1-carboxylic acid methyl ester. The yield was 70.12%, and the purity (HPLC) was 93.69%.

[0067] Column chromatography method: Flash Column Silica column chromatography, elution with petroleum ether:ethyl acetate = 3:2 (v:v).

[0068] The structure and molecular weight of the product were confirmed by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry, respectively, and the characterization results are shown below.

[0069] 1 H NMR (400MHz, DMSO) δ7.56-7.45(m,1H),7.44-7.31(m,1H),4.96&4.88(2s,2H),4.24&4.15(2t,2H),3.96(m,5H),3.87(t,1H),3.81(2s,3H).

[0070] LCMS m / z 464.10 [M+H] + ,m / z 486.10[M+Na] + m / z 462.10 [MH] - .

[0071] Example 2: Preparation of the compound as shown in 4-I

[0072]

[0073] The transaminase (AT) genes of transaminase 1 (SEQ ID NO:1), transaminase 2 (SEQ ID NO:2), transaminase 3 (SEQ ID NO:3), and transaminase 4 (SEQ ID NO:4) and the pET28a vector were digested with the same restriction endonucleases NdeⅠ and EcoRI. The digested fragments were recovered and ligated with T4 DNA ligase to form the recombinant expression plasmid pET28a-AT. The recombinant expression plasmid was transformed into E. coli BL21(DE3) to obtain a genetically engineered strain. The genetically engineered strain was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 12 h. The inoculum was then transferred to 150 mL of fresh LB liquid medium containing 50 μg / mL kanamycin at a 2% (v / v) inoculation rate and cultured at 37°C with shaking until the OD600 reached approximately 0.8. IPTG was then added to a final concentration of 0.5 mM and induced at 18°C ​​for 16 h. After culturing, the culture medium was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, the bacterial cells were collected, and stored in an ultra-low temperature freezer at -80℃ for later use. The obtained bacterial cells were homogenized with 100 mM phosphate buffer (pH 8.5) at a ratio of 1:10 (w / v, g / mL), and the supernatant was used to obtain crude transaminase solution 1-4.

[0074] In the reaction, 9.3 mg of the compound shown in 3-I (M = 463.34) (0.02 mmol), 200 μL of 0.5 M triethanolamine (0.1 mmol), 15 μL of 4 M isopropylamine hydrochloride (0.06 mmol), 20 μL of 50 mM pyridoxal phosphate (PLP) (12.5 g / L) (0.001 mmol), 200 μL of DMSO, 400 μL of the above crude enzyme solutions, and 165 μL of double-distilled water (ddH2O) were prepared into a 1 mL reaction system.

[0075] The reaction was carried out overnight at pH 8.5 and temperatures of 35°C and 45°C. The conversion rate and ee value were determined by HPLC. The conversion rates of different transaminases reached their highest values ​​after 18 hours, and the conversion rates of different transaminases are shown in Table 1.

[0076] Table 1: 18-hour conversion rates of different transaminases at 35℃ and 45℃

[0077]

[0078] HPLC method for detecting the conversion rate in Example 2:

[0079] Chromatographic column: ZORBAX Eclipse plus C18 (4.6mm*150mm, 3.5um); mobile phase: 0.1% TFA aqueous solution as mobile phase A, 0.1% TFA acetonitrile solution as mobile phase B; flow rate: 1mL / min; gradient elution program: 90%A+10%B (0.00min), 0%A+100%B (10.00min), 0%A+100%B (11.00min), 90%A+10%B (11.5min), 90%A+10%B (16.00min); column temperature: 35℃; injection volume: 10μL; detection wavelength: 210nm.

[0080] The retention time of the compound shown in 3-I is 7.916 min, and the retention time of the compound shown in 4-I is 5.960 min.

[0081] HPLC method for detecting the ee value of the compound shown in Example 2 as 4-I:

[0082] Column: Daicel Chiralpak AD-H (4.6mm*250mm, 5μm); Mobile phase: n-heptane: ethanol: diethylamine = 40:60:0.1 (v / v / v); Detection wavelength: 226nm; Flow rate: 0.8mL / min; Injection volume: 5μL; Column temperature: 25℃; Run time: 30min.

[0083] The retention time of the compound shown in 3-I is 13.227 min, the retention times of the racemic mixture of the compound shown in 4-I are 16.291 min and 20.435 min, and the retention time of the reference standard of the compound shown in 4-I (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) is 16.308 min.

[0084] Example 3: Scale-up of the reaction system

[0085] Accurately weigh 463.34 mg of the substrate (as shown in 3-I) and dissolve it in 5 mL of DMSO (dimethyl sulfoxide). In a 500 mL Erlenmeyer flask, add 10 mL of 0.5 M triethanolamine (pH 8.5), 0.75 mL of 4 M isopropylamine hydrochloride (pH 8.5), 1 mL of 50 mM PLP, and 5 mL of DMSO. Adjust the pH to 8.5, then add 5 mL of the prepared compound solution as shown in 3-I, 20 mL of crude transaminase solution 1 from Example 2, and add water to bring the total volume to 50 mL. Place the flask in a shaker at 45 °C and react at 220 rpm for 18 h. Take 100 μL of the reaction solution, dilute it 20 times with methanol, filter it through an organic membrane, and detect the conversion rate by HPLC. Add approximately 3 mL of flocculant to the remaining reaction solution, stir to precipitate, then add 20 mL of methyl tert-butyl ether and 10 g of diatomaceous earth, stir to mix, filter, and separate the filtrate into layers. Extract the aqueous phase with 20 mL of methyl tert-butyl ether, combine the organic phases, and wash with 2 x 15 mL of 0.1 mol / L hydrochloric acid. Adjust the pH of the aqueous phase to 7-8 with saturated sodium bicarbonate, extract the aqueous phase with 2 x 20 mL of methyl tert-butyl ether, combine the organic phases, concentrate under vacuum to obtain approximately 50 mg of crude product, dissolve in ethanol, and determine the ee value.

[0086] Table 2: Conversion rate of the scaled-up reaction system at 45℃ after 18 hours

[0087]

[0088] Example 4: Preparation of the racemic mixture of the compound shown in 4-I

[0089]

[0090] In a 100 mL single-necked flask, 1.7 g (3.67 mmol) of the compound shown in 3-I was dissolved in 50 mL of anhydrous methanol by stirring. Then, 2.83 g (36.69 mmol) of ammonium acetate was added, and the mixture was heated to 60 °C and refluxed for 2 h. The reaction was monitored by TLC until complete. After cooling to room temperature, 50 mL of water was added dropwise through a constant-pressure dropping funnel with stirring. The solid crystallized, filtered under vacuum, and dried under vacuum to give 1.3 g of compound 6, with a yield of 76%.

[0091] 1 H NMR (400MHz, CDCl3) δ7.11(dd,1H),6.97(td,1H),5.03(s,2H),4.87(s,1H),4.19(t,2H),4.01(t,2H),3.94(s,3H),3.47(s,2H).

[0092] Under nitrogen protection, in a 100 mL three-necked flask, 117 mg (3.09 mmol) of sodium borohydride was added to 20 mL of ethylene glycol dimethyl ether. The mixture was stirred and cooled to -50 °C in a dry ice-ethanol bath. 756 mg (7.87 mmol) of methanesulfonic acid was added dropwise using a syringe. The mixture was kept at this temperature and stirred for 60 min. 1.3 g (2.81 mmol) of compound 6 was dissolved in 1 mL of isopropanol and 20 mL of ethylene glycol dimethyl ether, and this solution was added to the reaction mixture using a syringe. The reaction was kept at -50 °C for 5 h. TLC showed a small amount of reactant remaining, which was quenched by slowly adding 20 mL of water. The mixture was concentrated under vacuum (<3 kPa) at 40 °C to remove ethylene glycol dimethyl ether. 30 mL of ethyl acetate was added for extraction to remove impurities. The pH of the aqueous phase was adjusted to 7–8 using saturated sodium bicarbonate. The aqueous phase was extracted with 2*25 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum (<3 kPa) at 40 °C to give 0.9 g of compound 7 with an HPLC purity of 98.39% and a yield of 69%. Compound 7 is a racemic mixture of the compound shown in 4-I.

[0093] The structure and molecular weight of the product were confirmed by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry, respectively, and the characterization results are shown below.

[0094] LCMS = m / z 465.20[M+H] + ,m / z 506.20[M+H+CH3CN] + m / z 509.15

[0095] [M+HCOO] - .

[0096] 1 H NMR (400MHz, CDCl3) δ7.08(dd,1H),6.92(dd,1H),5.11(m,2H),4.18(m,2H),4.07(m,2H),3.93(2s,3H),3.56(m,1H),2.74(m,2H),2.51(m,2H).

[0097] Example 5: Synthesis of the compound shown in 3-1

[0098]

[0099] 2.0 g of the compound shown in 3-I was added to a 100 mL three-necked flask; 1 mL MeOH and 4 mL H2O were added and stirred to dissolve; 1.0 g LiOH was added at room temperature and the reaction was stirred for 3 h; TLC showed no residual starting material; the pH was adjusted to 3.0–4.0 with 1 M dilute hydrochloric acid at room temperature; a white solid precipitated; the filter cake was collected after filtration and dried to give 1.7 g of the compound shown in 3-I ([3-oxo-4-(2,4,5-trifluorophenyl)butyryl]-3-(trifluoromethyl)-5H,6H,7H,8H imidazo[1,5-a]pyrazine-1-carboxylic acid), with a molar yield of 88.1%.

[0100] LCMS = m / z 450.15[M+H] + m / z 448.10 [MH] - ;

[0101] 1 H NMR (400MHz, CDCl3) δ7.54-7.32(m,2H), 4.91-4.88(2s,2H), 4.22-4.15(m,2H), 3.94-3.86(m,6H).

[0102] Example 6: Synthesis of the compound shown in 3-2

[0103]

[0104] 0.7 g of the compound shown in 3-1 was added to a 100 mL three-necked flask; 12 mL of EtOH was added and stirred to dissolve; 0.63 g of concentrated H2SO4 was added dropwise at room temperature; the mixture was heated to 70 °C and stirred for 5 h; TLC showed no remaining starting material, and the reaction solution was cooled to room temperature; 50 mL of water was slowly added and stirred for 5 min, followed by extraction with EA (40 mL * 2); the organic phases were combined and concentrated, and column chromatography was used to separate 200 mg of the compound shown in 3-2 ([3-oxo-4-(2,4,5-trifluorophenyl)butyryl]-3-(trifluoromethyl)-5H,6H,7H,8H imidazo[1,5-a]pyrazine-1-carboxylic acid ethyl ester), with a molar yield of 31.4%.

[0105] Column chromatography method: Flash Column Silica column chromatography, MeOH:DCM = 1:20 (v:v).

[0106] LCMS = m / z 478.15 [M+H] + m / z 476.10 [MH] - ;

[0107] 1H NMR (400MHz, CDCl3) δ7.55-7.39(m,2H), 4.96-4.88(2s,2H), 4.31-4.26(q,2H), 4.23-4.15(m,2H), 3.97-3.86(m,6H).

[0108] Example 7 Preparation of Compound 4-II

[0109]

[0110] The crude transaminase solutions 1-4 were derived from Example 2. In the reaction, 9.5 mg of substrate 3-2 (M = 477.36) (0.02 mmol), 200 μL of 0.5 M triethanolamine (0.1 mmol), 15 μL of 4 M isopropylamine hydrochloride (0.06 mmol), 20 μL of 50 mM pyridoxal phosphate (PLP) (12.5 g / L) (0.001 mmol), 200 μL of DMSO, 400 μL of the above crude enzyme solution, and 165 μL of double-distilled water (ddH2O) were prepared to form a 1 mL reaction system.

[0111] The reaction was carried out overnight at pH 8.5 and 45°C. The reaction conversion rate was determined by HPLC. The product was then hydrolyzed and methylated, and the ee value was measured.

[0112] HPLC method for detecting conversion rate:

[0113] Chromatographic column: ZORBAX Eclipse plus C18 (4.6mm*150mm, 3.5μm); mobile phase: 0.1% TFA aqueous solution as mobile phase A, 0.1% TFA acetonitrile solution as mobile phase B; flow rate: 1mL / min; gradient elution program: 90%A+10%B (0.00min), 0%A+100%B (10.00min), 0%A+100%B (11.00min), 90%A+10%B (11.5min), 90%A+10%B (16.00min); column temperature: 35℃; injection volume: 10μL; detection wavelength: 210nm.

[0114] The retention time of compound 3-2 is 8.342 min, and the retention time of compound 4-II is 6.320 min.

[0115] The conversion rates of different transaminases are shown in Table 3.

[0116] Table 3: Conversion rates of different transaminases at 45℃ over 18 hours

[0117]

[0118] Example 8: Scale-up of the reaction system

[0119] Accurately weigh 477.36 mg of the substrate (as shown in 3-2) and dissolve it in 5 mL of DMSO (dimethyl sulfoxide). In a 500 mL Erlenmeyer flask, add 10 mL of 0.5 M triethanolamine (pH 8.5), 0.75 mL of 4 M isopropylamine hydrochloride (pH 8.5), 1 mL of 50 mM PLP, and 5 mL of DMSO. Adjust the pH to 8.5, then add 5 mL of the prepared compound solution as shown in 3-2, 20 mL of the crude transaminase solution 2 from Example 2, and add water to bring the total volume to 50 mL. Place the flask in a shaker at 45 °C and react at 220 rpm for 18 h. Take 100 μL of the reaction solution, dilute it 20 times with methanol, filter it through an organic membrane, and detect the conversion rate by HPLC. Add approximately 3 mL of flocculant to the remaining reaction solution, stir to precipitate, then add 20 mL of methyl tert-butyl ether and 10 g of diatomaceous earth, stir to mix, filter, and separate the filtrate into layers. Extract the aqueous phase with 20 mL of methyl tert-butyl ether, combine the organic phases, and wash with 2 x 15 mL of 0.1 mol / L hydrochloric acid. Adjust the pH of the aqueous phase to 7-8 with saturated sodium bicarbonate, extract the aqueous phase with 2 x 20 mL of methyl tert-butyl ether, combine the organic phases, concentrate under vacuum to obtain approximately 50 mg of crude product, dissolve in ethanol, and then perform hydrolysis and methyl esterification before measuring the ee value.

[0120] Table 4: Conversion rate of the scaled-up reaction system at 45℃ after 18 hours

[0121]

[0122]

[0123] Example 9 Preparation of Compound 5

[0124]

[0125] The crude transaminase solutions 1-4 were derived from Example 2, and substrate 3-1 was derived from Example 5. In the reaction, 9.0 mg of substrate 3-1 (M = 449.31) (0.02 mmol), 200 μL of 0.5 M triethanolamine (0.1 mmol), 15 μL of 4 M isopropylamine hydrochloride (0.06 mmol), 20 μL of 50 mM pyridoxal phosphate (PLP) (12.5 g / L) (0.001 mmol), 200 μL of DMSO, 400 μL of the above crude enzyme solution, and 165 μL of double-distilled water (ddH2O) were prepared to form a 1 mL reaction system.

[0126] The reaction was carried out overnight at pH 8.5 and 45°C. The reaction conversion was determined by HPLC, and the ee value of the product after methyl esterification was measured.

[0127] HPLC method for detecting conversion rate:

[0128] Chromatographic column: ZORBAX Eclipse plus C18 (4.6mm*150mm, 3.5um); mobile phase: 0.1% TFA aqueous solution as mobile phase A, 0.1% TFA acetonitrile solution as mobile phase B; flow rate: 1mL / min; gradient elution program: 90%A+10%B (0.00min), 0%A+100%B (10.00min), 0%A+100%B (11.00min), 90%A+10%B (11.5min), 90%A+10%B (16.00min); column temperature: 35℃; injection volume: 10μL; detection wavelength: 210nm.

[0129] The retention time of compound 3-1 is 6.986 min, and the retention time of compound 5 is 5.389 min.

[0130] The conversion rates of different transaminases are shown in Table 5.

[0131] Table 5: Conversion rates of different transaminases at 45℃ over 18 hours

[0132]

[0133]

Claims

1. A process for the preparation of a compound as shown in 4, characterized by, The preparation method includes the following steps: in the presence of transaminase and amino donor, the compound shown in 3 undergoes a transamination reaction to obtain the compound shown in 4; ， The amino acid sequence of the transaminase is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; R is a C1-C4 alkyl group.

2. The process for the preparation of compounds as in claim 1, characterized by, R is methyl or ethyl.

3. The process for the preparation of compounds as in claim 1, characterized by, The nucleotide sequence encoding the transaminase is shown in SEQ ID NO: 5 and / or SEQ ID NO:

6.

4. The process for the preparation of compounds as in claim 1, characterized by, The preparation method satisfies one or more of the following conditions: (1) The temperature of the transamination reaction is 25-60℃; (2) The amino donor is selected from one or more of isopropylamine, phenylethylamine and alanine; (3) The molar ratio of the amino donor to the compound shown in 3 is (1-10):1; (4) The transamination reaction also includes cofactors of transaminase; (5) The transamination reaction also includes a reaction solvent; (6) The initial concentration of the compound shown in 3 is 0.01-0.1 mmol / mL; (7) The transaminase is in the form of crude enzyme solution, pure enzyme solution, crude enzyme powder, pure enzyme powder, enzyme cells or immobilized enzyme. (8) The mass ratio of the transaminase cells to the compound shown in 3 is (2-10):1; (9) The pH of the transamination reaction is 7.0-9.0; The time for the transamination reaction described in (10) is 2-48 hours.

5. The process for the preparation of a compound as in claim 4 according to claim 4, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The temperature of the transamination reaction is 35℃ or 45℃; (2) The amino donor is selected from isopropylamine; (3) The molar ratio of the amino donor to the compound shown in 3 is 3:1; (4) The transamination reaction also includes pyridoxal phosphate; (5) The molar ratio of the compound shown in 3 to the cofactor of the transaminase is (1-100):1; (6) The reaction solvent is water and / or an organic solvent; (7) The initial concentration of the compound shown in 3 is 0.02 mmol / mL; (8) The transaminase is in the form of crude transaminase solution; (9) The mass ratio of the transaminase cells to the compound shown in 3 is 4.3:1; (10) The pH of the transamination reaction is 8.5; (11) pH is controlled by a buffer solution; The time for the transamination reaction described in (12) is 18 hours.

6. The process for the preparation of a compound as in claim 4 according to claim 5, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The temperature of the transamination reaction is 45℃; (2) The amino donor is selected from the hydrochloride salt of isopropylamine; (3) The transamination reaction also includes 50 mM pyridoxal phosphate; (4) The molar ratio of the compound shown in 3 to the cofactor of the transaminase is 20:1; (5) The organic solvent is a sulfoxide solvent and / or an alcohol solvent; (6) The water is double-distilled water; (7) The volume ratio of the organic solvent to the total reaction system is (0.05-1):1; The buffer solution described in (8) is selected from one or more of phosphate buffer solution, Tris-HCl buffer solution and triethanolamine buffer solution.

7. The process for the preparation of a compound as in claim 6, characterized by, The preparation method satisfies one or more of the following conditions: (1) The amino donor is selected from 4M isopropylamine hydrochloride; (2) The organic solvent is one or more of DMSO, methanol, ethanol and isopropanol; (3) When the reaction solvent is water and an organic solvent, the volume ratio of the organic solvent to the total reaction system is (0.05-0.3):1; The buffer solution described in (4) is selected from triethanolamine.

8. The process for the preparation of a compound as in claim 7, characterized by, The preparation method satisfies one or more of the following conditions: (1) The organic solvent is DMSO; (2) When the reaction solvent is water and an organic solvent, the volume ratio of the organic solvent to the total reaction system is 0.1:1 or 0.2:1; The buffer solution described in (3) is selected from 0.5M triethanolamine.

9. The process for the preparation of compounds as in claim 1, according to formula 4, characterized by, The preparation method further includes a method for preparing the compound shown in Figure 3, wherein the method for preparing the compound shown in Figure 3 includes the following steps: Compound 1 and the compound shown in Figure 2 are reacted in a solvent as follows to obtain the compound shown in Figure 3. ， Wherein, R is as described in claim 1 or 2.

10. The process for the preparation of a compound as in claim 9, characterized by, The method for preparing the compound shown in Figure 3 satisfies one or more of the following conditions: (1) The solvent is an ester solvent and / or an alcohol solvent; (2) The molar volume ratio of compound 1 to the solvent is 0.04-0.8 mmol / mL; (3) The molar volume ratio of the compound shown in 2 to the solvent is 0.04-0.8 mmol / mL; (4) The molar ratio of compound 1 to the compound shown in 2 is (0.5-2.0):1; (5) The reaction temperature is 20-80℃; (6) The reaction time is 4-24 hours; (7) The reaction is carried out under the protection of an inert gas; The reaction described in (8) also includes post-processing, which includes the steps of concentration and purification.

11. The process for the preparation of the compound as claimed in claim 10 wherein, The method for preparing the compound shown in Figure 3 satisfies one or more of the following conditions: (1) The solvent is selected from one or more of ethyl acetate, methanol, ethanol and isopropanol; (2) The molar volume ratio of compound 1 to the solvent is 0.23 mmol / mL; (3) The molar volume ratio of the compound shown in 2 to the solvent is 0.2 mmol / mL; (4) The molar ratio of compound 1 to the compound shown in 2 is 1.15:1; (5) The reaction temperature is 60℃; (6) The reaction time is 20 hours; (7) The inert gas is selected from one or more of nitrogen, helium and argon; The concentration described in (8) is vacuum concentration; the purification is column chromatography.

12. The process for the preparation of a compound as in claim 11, characterized by, The solvent is ethyl acetate; the inert gas is nitrogen.

13. A compound as shown in 3: ， wherein, R stands for ethyl.

14. A process for the preparation of a compound as shown in 3 according to claim 13, characterized by, The method for preparing the compound shown in claim 3 is as described in any one of claims 9-12.

15. Use of a compound of Formula 3 as defined in claim 14 for the preparation of compound 4, comprising the steps of: In the presence of transaminase and amino donor, the compound shown in 3 undergoes a transamination reaction to give the compound shown in 4. ； The amino acid sequence of the transaminase is shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; R is ethyl.

16. The use of the compound as shown in claim 15, wherein the transamination reaction is as described in any one of claims 3-8.

17. The application of the compound shown in claim 16 as described in claim 3, the application further comprising a method for preparing the compound shown in claim 3, the method for preparing the compound shown in claim 3 as described in any one of claims 9-12.

18. An application of a transaminase, said application being for the preparation of a compound as shown in 4, said transaminase as described in claim 1; R as described in claim 1 or 2; 。 19. The application of the transaminase as described in claim 18, wherein the method for preparing the compound shown in claim 4 is as described in any one of claims 1-12.

Images