Method for synthesizing Itrimod intermediate through asymmetric ester hydrolysis

Abstract

The invention relates to the technical field of medicines, in particular to a method for synthesizing an Itrimod intermediate through asymmetric ester hydrolysis. According to the method, the key chiral intermediate (R)-formula I-a or (R)-formula I-b of the itramod is prepared through biological enzyme resolution, the chiral intermediate with the ee value larger than 98% and high optical purity is obtained through stereoselective hydrolysis of hydrolase, the product yield is increased to 40-45%, and a green synthesis route suitable for large-scale production is developed. # imgabs0 #

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a method for preparing ezetimibe and its key intermediates, particularly a method for preparing (R)-form Ib compounds. Background Art

[0002] Arginine ezetimidate is a receptor agonist that can achieve immunosuppressive, anti-inflammatory, and hemostatic effects by regulating leukocyte transport, isolating lymphocytes in secondary lymphoid tissues, and enhancing lymphatic integrity. Arginine ezetimidate can be used to treat inflammatory bowel diseases, including ulcerative colitis, Crohn's disease, atopic dermatitis, eosinophilic esophagitis, and alopecia areata, with relatively few side effects. The structural formula of ezetimidate is (R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopentano[b]indol-3-yl)acetic acid (II-a):

[0003]

[0004] There are many synthetic routes for ectomod, and the following are some of the more mature processes:

[0005]

[0006] In existing technologies, compound 6 is hydrolyzed and resolved by immobilized Candida antarctica lipase B to obtain compound 7 (R-type), while the larger molecular weight compound 7-1 (S-type) is discarded, resulting in low reaction yield, poor economic efficiency of reaction raw materials, and high cost. Therefore, given the promising market prospects of ezetimibe, it is urgent to develop an economical, safe, simple, high-yield, low-cost method for preparing key chiral intermediates and finished products of ezetimibe suitable for industrial production. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of the prior art and provide a simple, short, high-yield, safe and environmentally friendly bio-enzyme catalysis technology for preparing ectomod with high chiral purity and its key intermediates.

[0008] The technical solution adopted by this invention to solve the above-mentioned technical problems is: to provide ezetimibe and its key intermediate compound I for enzymatic resolution to obtain chiral intermediate Ib, including the following steps:

[0009]

[0010] In this process, under the action of hydrolytic enzymes, buffer solutions, and solubilizers, compound I is hydrolyzed and resolved to obtain chiral intermediates of formula I-b or formula Id.

[0011] R is selected from C1-C6 alkyl, halogenated C1-C6 alkyl, benzyl, etc., and includes but is not limited to these groups. Methyl, ethyl, trifluoromethyl, benzyl, etc. are preferred.

[0012] The hydrolytic enzymes used in this invention include: Novozym 435, Novozym RM, Novozym 51032, Lipozyme TLIM, Lipozyme CALB, Lipozyme TL100L, Savinase 16L, Resinase HT, Novocor ADL, Neutrase 0.8L, Plastase 20000L, and Alcalase 2.4L;

[0013] The hydrolytic enzymes selected in this invention are: Lipase PSSD, Lipase PSIM, Lipase CL, Lipase CLIM, Lipase PSAK, Lipase AK, Lipase AH, Lipase AYS, and Lipase AS; preferably, Lipase PSIM from Amano.

[0014] In this invention, the mass ratio of enzyme to reaction substrate is selected from a mass ratio of hydrolase to compound of formula I of 2%-10%.

[0015] The co-solvent used in this invention is selected from one or more solvents, including alcohol solvents, nitrile solvents, ether solvents, ester solvents, aliphatic hydrocarbon solvents, dimethyl sulfoxide, and N,N-dimethylformamide.

[0016] The alcohol solvents are selected from methanol, ethanol, isopropanol, and tert-butanol;

[0017] The nitrile solvent is selected from acetonitrile;

[0018] The ether solvents are selected from tetrahydrofuran and methyl tert-butyl ether;

[0019] The ester solvent is selected from ethyl acetate;

[0020] Aromatic solvents are selected from toluene and xylene;

[0021] Aliphatic hydrocarbon solvents are selected from cyclohexane and n-hexane;

[0022] The hydrolytic enzyme resolution reaction temperature is 30-60℃, and the reaction time is 20-48 hours, with the preferred reaction temperature being 35-45℃ and the reaction time being 20-24 hours.

[0023] The concentration of the compound of formula I is 50 mg / mL to 100 mg / mL.

[0024] The pH value of the hydrolytic enzyme resolution reaction is between 6 and 8;

[0025] The buffer solution for the hydrolytic enzyme resolution reaction is selected from one of the following: PBS phosphate buffer, sodium phosphate buffer, Tris buffer, and acetate-ammonium acetate buffer.

[0026] The present invention also provides an HPLC method for detecting the conversion rate of product R-form Ib as follows:

[0027] Conversion rate describes the degree to which a reactant is converted in a chemical reaction. The formula for calculating conversion rate is: Conversion rate = (Amount of reactant converted / Amount of initial reactant) × 100%. According to the reaction process of the present invention, the conversion rate of the ester = 100% - the conversion rate of the acid %.

[0028] The liquid chromatography column was selected from: ZORBAX Eclipse XDB-C18 (4.6*150mm, 5-Micron);

[0029] The mobile phase was selected from: 0.5% trifluoroacetic acid / acetonitrile (50:50);

[0030] The flow rate was selected from 1.0 mL / min;

[0031] The detection wavelength was selected from 269nm;

[0032] The column temperature is selected from: 35℃;

[0033] The injection volume was selected as 5 μL;

[0034] The elution time was selected from 20 min;

[0035] The blank solvent was selected from: 0.5% trifluoroacetic acid / acetonitrile (50:50);

[0036] The test solution was selected from 1.0 mg / mL;

[0037] This invention also provides an HPLC method for detecting the purity (ee value) of the S-configuration Ib enantiomer as follows:

[0038] The chromatographic column was selected from Chiralpak-IB (4.6mm*250mm, 5um);

[0039] The mobile phase is selected from: n-hexane / isopropanol (90:10);

[0040] The flow rate was selected from 1.0 mL / min;

[0041] The detection wavelength is selected from 269nm;

[0042] The column temperature is selected from 35℃;

[0043] The injection volume was selected as 5 μL;

[0044] The elution time was selected from 45 min;

[0045] The blank solvent was selected from isopropanol;

[0046] The test solution was selected from 1.0 mg / mL;

[0047] Beneficial effects of this invention:

[0048] 1. The hydrolytic enzyme used in this invention is Lipase PSIM enzyme obtained through screening. In a buffer solvent, substrate formula I co-solvent is added, and a certain amount of Lipase PSIM enzyme is used for hydrolysis to obtain the desired conformational product, formula I-b, with an ee value of 98% and a yield of 40-45%.

[0049] 2. Compared with existing technologies, the enzymes selected in this invention have stronger specificity, selectivity, and better atom economy, making them very suitable for industrial development and application. Attached Figure Description

[0050] Figure 1 R represents the racemic mixture of benzyl-I, as shown in the HPLC chromatogram.

[0051] Figure 2 : ee value detection spectrum of (R)-Ⅰ-b (R is benzyl) Detailed Implementation

[0052] The technical solution of the present invention is illustrated below through specific embodiments, but the scope of protection of the present invention is not limited thereto. In route 1, the enzyme preferentially selects the S configuration of the compound of formula I for hydrolysis to obtain the product formula I-b (R-type ester);

[0053] Example 1---Route 1 Enzyme Screening:

[0054]

[0055] Take 20 mg of compound I (R is benzyl), 1 mL of phosphate-buffered saline (PBS), 200 μL of MTBE (medium-methyl-thionyl alcohol), and 10 mg of hydrolytic enzyme. React at 38 °C and pH 6-8 for 24 h, then sample and measure the conversion rate and ee value. The hydrolysis results of compound I are shown in Table 1.

[0056] Table 1. Route 1 Enzyme Screening

[0057]

[0058] Conclusion: According to the screening results, Lipase PSIM and Lipase AK can hydrolyze compound I. After acid treatment, Amano lipase Lipase PSIM can yield ester I-b (R type, ee = 98%) with higher stereoselectivity.

[0059] Example 2---Route 2 Enzyme Screening:

[0060]

[0061] Using the same experimental procedure as described above, different enzymes were selected for hydrolysis and resolution tests. The hydrolysis results of compound I are shown in Table 2.

[0062] Table 2. Enzyme Screening Process via Route 2

[0063]

[0064]

[0065] Conclusion: The above enzymes can directly hydrolyze the R-configuration substrates in Formula I compounds. For example, using Lipozyme CALB (Candida antarcticis lipase) and Lipase CL lipase can yield I-a (R-type, ee = 95%) lipases with higher stereoselectivity.

[0066] Example 3---Comparative Experiment of Lipozyme CALB (Candida antarcticis lipase) and Lipase PSIM Enzyme

[0067] Weigh 1.0 g of compound I, 10 mL of phosphate buffer (0.1 M PBS), and 10 mL of solubilizer (MTBE). Weigh 100 mg each of CALB and Lipase PSIM and add them to the reaction system. Control the reaction temperature at 35-45℃ and pH = 6-8. Take samples after 24 h of reaction to measure the conversion rate. The results are shown in Table 3.

[0068] Table 3 Comparison Experiment

[0069]

[0070] Conclusion: Both Lipozyme CALB (Candida antarcticis lipase) and Lipase PSIM can yield the desired conformation (R-type), but Lipase PSIM achieves higher conversion rates (56%), better stereoselectivity (ee = 98%), and a shorter synthesis time, making it more economical and meeting the synthesis requirements. Therefore, Lipase PSIM will be used as the hydrolase in subsequent examples.

[0071] Example 4---Enzyme Stability Test

[0072] Stability studies were conducted on enzymes with good selectivity selected from Tables 1 and 2 (Lipase PSIM, Lipase AK, Lipozyme CALB, Novozym435, Lipase CL).

[0073] 1) Change the temperature in the preferred operation step to 50℃, keeping all other variables unchanged. The enzyme separation results are shown in Table 4:

[0074] Table 4. Effects of Temperature on Different Enzymes

[0075]

[0076]

[0077] Conclusion: Lipase PSIM enzyme still maintains good stereoselectivity under high temperature conditions.

[0078] 2) Change the pH in the preferred operation step to 10, keeping other variables unchanged. The enzyme results are shown in Table 5:

[0079] Table 5. Effects of pH on different enzymes

[0080]

[0081] Conclusion: Lipase PSIM enzyme still maintains good stereoselectivity under high pH conditions.

[0082] Example 5 - Screening of Reaction Temperature

[0083] Weigh 1.0 g of compound I (R is benzyl), 10 mL of phosphate buffer (0.1 M PBS), 10 mL of solubilizer (MTBE), pH = 6-8, and 100 mg (10%) of Lipase PSIM. React at different temperatures (30℃, 35℃, 40℃, 45℃, 50℃) for 24 h, then sample and measure the conversion rate. The results of temperature screening are shown in Table 6.

[0084] Table 6: Screening Results for Reaction Temperature

[0085]

[0086]

[0087] Conclusion: The temperature screening results show that the conversion rate and the ee value of product I-b are best when the temperature is between 35-45℃.

[0088] Example 6---Screening of Solvent Volume

[0089] Weigh 1.0 g of compound I (R is benzyl), and use MTBE in volumes of 10.0 mL, 8.30 mL, 7.14 mL, 6.25 mL, 5.56 mL, 5.00 mL, 4.55 mL, and 4.17 mL. Use phosphate buffer (0.1 M PBS) in the same volumes as above. Use 10% of the substrate weight of Lipase PSIM enzyme. Maintain pH 6-8, react at 35-45℃ for 24 h, and measure the conversion rate. The solvent volume selection results are shown in Table 7.

[0090] Table 7: Screening Results of Solvent Volume

[0091]

[0092] Conclusion: It is better to control the conversion rate between 50% and 60%. The results show that the substrate concentration ≤100mg / mL (solvent volume >10mL) yields good results.

[0093] Example 7 - pH Screening

[0094] Weigh 1.0 g of compound I (R is benzyl), 6 mL of buffer solution, 6 mL of solubilizer (MTBE), and 100 mg (10%) of Lipase PSIM enzyme. React at different pH levels (gradients: 5, 6, 7, 8, 9, 10) at 35-45℃ for 24 h. Take samples to measure the conversion rate. The pH screening results are shown in Table 8.

[0095] Table 8. Screening Results at Different pH Levels

[0096]

[0097] Conclusion: The pH screening results show that the effect is best at pH 6-8.

[0098] Example 8

[0099] Weigh 50.0 g of compound I (R is methyl), 300 mL of 0.1 M PBS (pH 6-8), 300 mL of MTBE (solubilizer), 1.0 g (2%) of Liapse PSIM enzyme, and stir the reaction at 35-45 °C with pH controlled between 6 and 8 for 22 h. The conversion rate is 58%, and the ee value of product I-b is 98.3%.

[0100] Example 9

[0101] Weigh 80.0 g of compound I (R is methyl), 400 mL of 0.1 M PBS (pH 6-8), 400 mL of solubilizer (MTBE), 8.0 g (10%) of Lipase PSIM enzyme, stir the reaction at 35 °C with pH controlled between 6 and 8 for 24 h, the conversion rate is 62%, and the ee value of product I-b is 99.5%.

[0102] Example 10

[0103] Weigh 10.0 g of compound I (R is benzyl), add 60 mL of 0.1 M PBS (pH 6-8), 60 mL of MTBE (solubilizer), and 1.0 g (10%) of Lipase PSIM enzyme. Maintain the reaction temperature at 35-45℃ and keep the pH between 6 and 8 while stirring. After 20 hours of reaction, the conversion rate is 57%, and the ee value of the product I-b (R is benzyl) is 98.2%.

[0104] Example 11

[0105] Weigh 10.0 g of compound I (R is benzyl), add 60 mL of 0.1 M PBS (pH 6-8), 60 mL of MTBE (solubilizer), and 0.5 g (5%) of Lipase PSIM enzyme. Stir the reaction at 40 °C for 22 h while maintaining the pH between 6 and 8. The conversion rate is 59%, yielding compound I-b with an ee value of 98.2%.

[0106] Example 12

[0107] Weigh 50.0 g of compound I (R is benzyl), add 300 mL of 0.1 M PBS (pH 6-8), 300 mL of MTBE (a cosolvent), and 1.0 g (2%) of Liapse PSIM enzyme. Stir the reaction at 45 °C for 22 h while maintaining the pH between 6 and 8. The conversion rate is 60%, yielding compound I-b with an ee value of 98.6%.

[0108] In summary, through Examples 8 to 12, we can see that using Liapse PSIM enzyme hydrolysis can scale up the reaction while maintaining good stereoselectivity and conversion rate.

Claims

1. A method for preparing an etramod intermediate (R)-compound of formula Ib, characterized in that The steps include: Under the action of hydrolase, buffer solution and cosolvent, the compound of formula I is hydrolyzed and resolved; The hydrolase used was Lipase PSIM from Amano; Wherein R is selected from C1-C6 alkyl, halogenated C1-C6 alkyl, and benzyl.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the hydrolase to the compound of formula I is 2%-10%.

3. The preparation method according to claim 1, characterized in that: The co-solvent is selected from one or more of methanol, acetonitrile, methyl tert-butyl ether, ethyl acetate, toluene, dimethyl sulfoxide, isopropanol, N,N-dimethylformamide and n-hexane.

4. The preparation method according to claim 1, characterized in that: The hydrolase splitting reaction temperature is 30-60°C and the reaction time is 20-48 hours.

5. The preparation method according to claim 1, characterized in that: The concentration of the compound of formula I is 50 mg / mL-100 mg / mL.

6. The preparation method according to claim 1, characterized in that: The pH value of the hydrolase resolution reaction is between 6-8.

7. The preparation method according to claim 1, characterized in that: The buffer solution includes phosphate buffer solution (PBS), Tris buffer, and acetic acid-ammonium acetate buffer.

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