Method for preparing GLP-1 receptor agonist intermediate

Abstract

The invention discloses a novel method for synthesizing an intermediate (S)-3-aminobutyronitrile of a GLP-1 receptor stimulant. According to the synthesis method, a chemical method and an enzyme method are combined for the first time to realize green and environment-friendly high-selectivity synthesis of the (S)-3-aminobutyronitrile. The catalytic reaction system disclosed by the invention is simple, mild in condition and high in selectivity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology industrial application, and specifically relates to a new synthesis route of an intermediate (S)-3-aminobutyronitrile of a GLP-1 receptor agonist. Background Art

[0002] Compound 101a can be used as a glucagon-like peptide-1 receptor agonist, i.e., a GLP-1 receptor agonist, and has the functions of lowering blood sugar, lowering blood lipids, and protecting cardiovascular system, and is suitable for development into a weight loss drug or a drug for the treatment of type II diabetes (WO2021 / 155841Al). Compound 16 is a key intermediate in the synthesis of Compound 101a. The piperidine-pyrazole structure of its parent core needs to be synthesized using (S)-3-aminobutyronitrile (Compound 13) as the starting substrate ( Figure 1 ).

[0003]

[0004] At present, only chemical synthesis routes have been reported for (S)-3-aminobutyronitrile (Compound 13), and there are two main routes: the general process of one route is to start from (S)-2-aminopropanol, first add Boc to protect the amino group, synthesize sulfonate at the same time, and then use highly toxic sodium cyanide for cyanation, and finally remove the Boc group to obtain (S)-3-aminobutyronitrile. The continuous multi-step reaction makes the total yield of the final product (S)-3-aminobutyronitrile about 30%. Another route is to use (S)-Boc-3-aminopropanol as the starting substrate, and use a variety of chemical oxidation reagents such as HATU (2-(7-azobenzotriazole))-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIPEA (diisopropylethylamine) and TFAA (trifluoroacetic anhydride) to gradually oxidize the carboxylic acid into cyano. Although the product yield of this route is slightly increased to 60%, a large amount of organic reagents are used in the reaction process, which is relatively expensive and not conducive to environmental protection. The starting substrates of the two chemical synthesis routes are both non-natural amino acid reduction products, whose price and supply volume cannot meet the growing market demand for (S)-3-aminobutyronitrile. Both have relatively difficult post-reaction treatment problems, and have the defects of high cost and high pollution.

[0005] The current routes of the prior art lack a route that uses cheap and readily available materials as a starting point and directly synthesizes the product through simple steps. Therefore, it is necessary to find an economical, efficient, green and environmentally friendly synthetic route with mild reaction conditions to achieve the preparation of (S)-3-aminobutyronitrile with high chiral purity. The starting materials involved in this application can be synthesized by cheap bulk chemicals, and its manufacturing cost, economic and environmental benefits are much higher than the currently known routes. Summary of the invention

[0006] In order to overcome the defects of the (S)-3-aminobutyronitrile chemical synthesis route in the current prior art, the present application provides a catalytic route for the efficient synthesis of (S)-3-aminobutyronitrile by a chemical method and an enzyme method. At present, there is no report on the synthesis route of (S)-3-aminobutyronitrile involving biological enzymes.

[0007] First, the first synthetic route of (S)-3-aminobutyronitrile provided in the present application is as follows Figure 2 As shown, the route includes a two-step reaction process, wherein the second step reaction is an enzymatic catalytic reaction, and S-transaminase can directly catalyze cyanoacetone to selectively generate the product (S)-3-aminobutyronitrile. The substrate cyanoacetone of the enzymatic reaction can be obtained by two chemical synthesis methods, one of which is to use the relatively cheap and easily available 3-aminocrotononitrile as a substrate to obtain the product cyanoacetone; the other chemical synthesis method is to use acetonitrile and methyl acetate as substrates, heat reflux in a toluene solution, and obtain the product cyanoacetone after post-treatment.

[0008]

[0009] Secondly, the second synthetic route of (S)-3-aminobutyronitrile provided in the present application is as follows Figure 3 As shown, the route includes a two-step reaction process, wherein the second step reaction is an enzymatic catalytic reaction, and R-aminotransferase can prepare (S)-3-aminobutyronitrile by splitting racemic 3-aminobutyronitrile. Racemic 3-aminobutyronitrile can be prepared by two chemical synthesis methods, one is to use a reducing agent sodium borohydride to reduce the imine structure of the substrate 3-aminocrotonitrile in an ethanol solution to obtain racemic 3-aminobutyronitrile; the other is to use 2-butenenitrile as a substrate, heat reflux in an ammonia solution, and obtain the racemic product 3-aminobutyronitrile after post-treatment.

[0010]

[0011] In addition, the present application can also use cyanoacetone or 3-aminobutyronitrile as the starting material and synthesize (S)-3-aminobutyronitrile only by enzymatic method. The amino acid sequence of the transaminase provided in the present application is shown in SEQ ID No. 1-4.

[0012] At present, whether it is chemical or enzymatic methods, there is a lack of a route that starts with cheap and easily available starting materials and directly synthesizes the product in 1-2 steps. From the perspective of compound structure, direct transamination of cyanoacetone or separation of racemic products is more direct than the above steps, but no transaminase has been reported for similar reactions.

[0013] Compared with the currently used chemical synthesis technology, the transaminase catalytic route provided by the present invention has the following beneficial effects: using relatively economical and readily available compounds as starting substrates, no need for amino protection and deprotection, shortening the reaction process, and improving the product yield; no metal or toxic reagents are required in the transaminase catalytic process, which greatly reduces the cost and is more environmentally friendly; the enzyme catalytic reaction can be carried out at 30°C, the conditions are mild, the energy consumption is low, the reaction system is simple, and the final (S)-3-aminobutyronitrile has a high chiral value. The synthetic route of the present application has important application value for the green enzymatic synthesis of (S)-3-aminobutyronitrile, and can provide high-quality and low-cost raw materials for the synthesis of GLP-1 receptor agonist Compound 101a, which has high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Figure 1 Chemical process route for (S)-3-aminobutyronitrile as a building block for the active product Compound 101a

[0015] Figure 2 Transaminase-catalyzed synthesis of (3)-S-aminobutyronitrile route 1

[0016] Figure 3 Transaminase-catalyzed synthesis of (3)-S-aminobutyronitrile route 2

[0017] Figure 4 SDS-PAGE gel images of the supernatant (1) and precipitate (2) of the homogenized wet bacterial cells of the S-aminotransferase shown in SEQ ID No.1 and the S-aminotransferase shown in SEQ ID No.2

[0018] Figure 5 SDS-PAGE gel images of the supernatant (1) and precipitate (2) of the homogenized and disrupted wet bacterial cells of the R-aminotransferase shown in SEQ ID No.3 and the R-aminotransferase shown in SEQ ID No.4

[0019] Figure 6 Chiral derivatization phase diagram of the reaction solution of the synthesis of (S)-3-aminobutyronitrile from cyanoacetone catalyzed by S-aminotransferase shown in SEQ ID No.1

[0020] Figure 7 Chiral derivatization phase diagram of the reaction solution of the synthesis of (S)-3-aminobutyronitrile from cyanoacetone catalyzed by S-aminotransferase shown in SEQ ID No.2

[0021] Figure 8 Chiral derivatization phase diagram of the reaction solution at 0h of the reaction of the route of preparing (S)-3-aminobutyronitrile from 3-aminobutyronitrile catalyzed by R-aminotransferase shown in SEQ ID No.3

[0022] Fig. 9 Chiral derivatization phase diagram of the reaction solution after 24 hours of reaction of the route of preparing (S)-3-aminobutyronitrile from 3-aminobutyronitrile catalyzed by R-aminotransferase shown in SEQ ID No.3

[0023] Fig.10 Chiral derivatization phase diagram of the reaction solution at 0h of the reaction of the route of preparing (S)-3-aminobutyronitrile from 3-aminobutyronitrile catalyzed by R-aminotransferase shown in SEQ ID No.4

[0024] Fig.11 Chiral derivatization phase diagram of the reaction solution after 24 hours of reaction of the route of preparing (S)-3-aminobutyronitrile from 3-aminobutyronitrile catalyzed by R-aminotransferase shown in SEQ ID No.4

[0025] Fig.12 Chiral derivatization liquid phase diagram of (S)-3-aminobutyronitrile standard sample

[0026] Fig.13 Chiral derivatization liquid phase diagram of (R)-3-aminobutyronitrile standard sample DETAILED DESCRIPTION

[0027] The present invention is further illustrated below with examples, and the technical scheme of the present invention is clearly and completely described, but the present invention is not limited thereto, and the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In addition, the experimental methods involved in the following embodiments but not indicating specific conditions are usually in accordance with the conditions conventional in the field of biochemical technology or recommended by the manufacturer, and the reagents and equipment involved but not particularly indicated are all commercially available.

[0028] Example 1: Chemical synthesis of cyanoacetone by hydrolysis of 3-aminocrotononitrile with hydrochloric acid

[0029] Dissolve 10.0 g of 3-aminocrotononitrile in 1.25 mL of water, slowly add 12.5 mL of HCl, control the temperature below 15 °C, and heat the mixture at 80 °C for 2 hours. Cool the reaction mixture to room temperature, extract it three times with ethyl acetate, and wash it with anhydrous Na 2 SO 4 Drying and concentration under vacuum gave cyanoacetone in 90% yield.

[0030] Example 2: Chemical catalytic synthesis of cyanoacetone from methyl acetate

[0031] Add 10 mL of toluene (containing 5 equivalents of acetonitrile) to a 100 mL round-bottom flask, cool to 0-5 °C, and 2NaH (55%, 2.5 eq.) was gradually introduced under the atmosphere and stirred for 30 minutes. Then 5 mL of toluene (containing 5 times the equivalent of methyl acetate) was added. The reaction solution was refluxed for 17 hours. After the reaction was completed, the mixture was slowly poured into ice water, extracted three times with ethyl acetate, and washed with anhydrous Na 2 SO 4 After drying and distillation under reduced pressure, the yield of cyanoacetone obtained was 93%.

[0032] Example 3: Gene cloning and construction of expression vector

[0033] The corresponding nucleic acids of the amino acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 were synthesized by common techniques in the art and cloned into the expression vector pACYC-Duet-B. The recombinant expression plasmid was transformed into competent cells of E.coil BL21 (DE3) under the conditions of heat shock at 42°C for 90s, ice bath for 5min, then LB culture medium was added to recover in a shaking incubator at 37°C for 1h, and finally coated on an LB plate containing chloramphenicol resistance and incubated at 37°C overnight to obtain a recombinant transformant.

[0034] Example 4: Expression of S-aminotransferase

[0035] A single microbial colony of Escherichia coli BL21 (DE3) containing the expression plasmids of SEQ ID No. 1 and SEQ ID No. 2 was inoculated into a 250 mL conical flask containing 50 mL LB medium (containing 30 μg / mL chloramphenicol) and cultured overnight in a shaking incubator at 30°C. When the OD of the culture solution reached 600 When it reaches 2, press 5% (v / v) The inoculum was inoculated into a 1000mL conical flask containing 250mL TB medium, and IPTG with a final concentration of 1mM was added to induce the expression of S-transaminase, and the cells were placed in a shaker at 30°C. After shaking culture for 20h, the culture solution was centrifuged at 8000rpm, and the supernatant was discarded after 10min. The cells were collected to obtain wet cells. The wet cells successfully expressed soluble SEQ ID No.1 transaminase of about 49.1kDa and soluble SEQ ID No.2 transaminase of about 48.8kDa ( Figure 4 The wet cells can be used directly for enzyme catalysis reactions or can be stored frozen at -20°C until use.

[0036] Example 5: Expression of R-aminotransferase

[0037] A single microbial colony of Escherichia coli BL21 (DE3) containing the expression plasmids of SEQ ID No. 3 and SEQ ID No. 4 was inoculated into a 250 mL conical flask containing 50 mL LB medium (containing 30 μg / mL chloramphenicol) and cultured overnight in a shaking incubator at 30°C. When the OD of the culture solution reached 600 When the cell density reached 2, the inoculum was inoculated into a 1000 mL conical flask containing 250 mL TB medium at a 5% (v / v) inoculation amount, and IPTG with a final concentration of 1 mM was added to induce the expression of R-transaminase, and the cells were placed in a shaker at 30°C. After 20 hours of shaking culture, the culture solution was centrifuged at 8000 rpm, and the supernatant was discarded after 10 minutes. The cells were collected to obtain wet cells. The wet cells successfully expressed the soluble protein SEQ ID No.3 transaminase of about 37.9 kDa, and the soluble protein SEQ ID No.4 transaminase of about 36.1 kDa ( Figure 5 The wet cells can be used directly for enzyme catalysis reactions or can be stored frozen at -20°C until use.

[0038] Example 6: 5 mL reaction of cyanoacetone to (S)-3-aminobutyronitrile catalyzed by S-aminotransferase shown in SEQ ID No.1

[0039] 100 g / L of wet bacteria expressing S-transaminase shown in SEQ ID No. 1 was weighed into a 25 mL glass bottle. The reaction solution used in the reaction system was 0.1 M TEOA, and the pH of the reaction system was 8.5. The final concentration of the substrate cyanoacetone in the reaction was 50 g / L. The final concentration of pyridoxal phosphate (PLP) was 10 mM. The amine donor was isopropylamine, and the final concentration was 35.6 g / L. A magnetic stirring rotor was added to the glass bottle, the reaction temperature was set to 30 ° C, the stirring speed was 400 rpm, and an equal volume of pure acetonitrile was used for quenching after 24 hours. The supernatant of the reaction solution was taken and derivatized with a chiral derivatizing agent N-α-(2,4-dinitro-5-fluorophenyl)-L-valinamide. The treated sample solution was subjected to HPLC detection. The yield of the target product (S)-3-aminobutyronitrile was calculated to be 89%, and the (S)-3-aminobutyronitrile ee% (S) ≥ 99% ( Figure 6 ).

[0040] Example 7: 5 mL reaction of cyanoacetone to (S)-3-aminobutyronitrile catalyzed by S-aminotransferase shown in SEQ ID No.2

[0041] 50 g / L of wet bacteria expressing S-transaminase shown in SEQ ID No. 2 were weighed into a 25 mL glass bottle. The reaction solution used in the reaction system was 0.1 M TEOA, and the pH of the reaction system was 8.5. The final concentration of the substrate cyanoacetone in the reaction was 4.15 g / L. The final concentration of pyridoxal phosphate (PLP) was 10 mM. The amine donor was isopropylamine, and the final concentration was 2.95 g / L. A magnetic stirring rotor was added to the glass bottle, the reaction temperature was set to 30 ° C, the stirring speed was 400 rpm, and an equal volume of pure acetonitrile was used for quenching after 24 hours. The supernatant of the reaction solution was taken and derivatized with a chiral derivatizing agent N-α-(2,4-dinitro-5-fluorophenyl)-L-valinamide. The treated sample solution was subjected to HPLC detection. The yield of the target product (S)-3-aminobutyronitrile was calculated to be 64%, and the (S)-3-aminobutyronitrile ee(S)%≥99%( Figure 7 ).

[0042] Example 8: Synthesis of racemic 3-aminobutyronitrile using 3-aminocrotononitrile as substrate

[0043] 38mg 0.6mmol sodium cyanoborohydride was added to 0.5mmol substrate ethanol (3mL) solution, followed by a drop of 0.5 bromocresol green ethanol solution as an indicator (color change range pH color change range 3.8 yellow ~ 5.4 blue-green), 32% concentrated hydrochloric acid was added dropwise until the system showed a stable yellow color, and the reaction was carried out at room temperature for 3 hours. After the reaction was completed, water was added, and 25% concentrated ammonia water was added to adjust the pH to 11. The reaction solution was extracted with ether three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and column chromatographed to obtain the target product, with a yield of 80%.

[0044] Example 9: Synthesis of racemic 3-aminobutyronitrile using 2-butenenitrile as substrate

[0045] 0.5M substrate 2-butenenitrile (cis-trans molar mass ratio of 1:1) was transferred into a 500mL round-bottom flask, and 8 equivalents of 40% ammonia solution were slowly added. The mixture was heated to reflux at 140°C for 2h, cooled to room temperature, and the reaction solution was extracted with ether three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then column chromatography was performed to obtain the target product. The yield of the racemic product 3-aminobutyronitrile was 97%.

[0046] Example 10: 5 mL reaction of R-aminotransferase catalyzed resolution of 3-aminobutyronitrile as shown in SEQ ID No. 3

[0047] Weigh 100 g / L of wet bacteria expressing R-transaminase in a 25 mL glass bottle. The reaction solution used in the reaction system is 0.1 M TEOA, and the pH of the reaction system is 8.5. The final concentration of 3-aminobutyronitrile in the reaction is 50 g / L. The final concentration of pyridoxal phosphate (PLP) is 10 mM. The final concentration of acetone is 69 g / L. Add a magnetic stirring rotor to the glass bottle, set the reaction temperature to 30 ° C, the stirring speed to 400 rpm, take an equal volume of pure acetonitrile for inactivation at 0 h, and perform the liquid phase analysis as shown in the figure below. Figure 8 As shown, after 24 hours, an equal volume of pure acetonitrile was used for quenching. The supernatant of the reaction solution was taken and derivatized with a chiral derivatizing agent N-α-(2,4-dinitro-5-fluorophenyl)-L-valinamide. The treated sample solution was placed on HPLC for detection. The residual rate of the target product (S)-3-aminobutyronitrile was calculated to be 50%, and the ee% (S) of (S)-3-aminobutyronitrile was ≥99% ( Fig. 9 ).

[0048] Example 11: 5 mL reaction of R-aminotransferase catalyzed resolution of 3-aminobutyronitrile as shown in SEQ ID No. 4

[0049] 100 g / L of wet bacteria expressing R-transaminase shown in SEQ ID No. 4 was weighed into a 25 mL glass bottle. The reaction solution used in the reaction system was 0.1 M TEOA, and the pH of the reaction system was 8.5. The final concentration of 3-aminobutyronitrile in the reaction was 3 g / L. The final concentration of pyridoxal phosphate (PLP) was 10 mM. The final concentration of acetone was 4.14 g / L. A magnetic stirring rotor was added to the glass bottle, the reaction temperature was set to 30 ° C, the stirring speed was 400 rpm, and an equal volume of pure acetonitrile was sampled at 0 h for inactivation, and the liquid phase analysis diagram for detection was shown in the figure below. Fig.10 As shown, after 24 hours, an equal volume of pure acetonitrile was used for quenching. The supernatant of the reaction solution was taken and derivatized with a chiral derivatizing agent N-α-(2,4-dinitro-5-fluorophenyl)-L-valinamide. The treated sample solution was placed on HPLC for detection. The calculated residual rate of (S)-3-aminobutyronitrile was 45%, and the ee% (S) of (S)-3-aminobutyronitrile was ≥99% ( Fig.11 )

[0050] The HPLC instrument used for detecting the above reaction is a commercially available Agilent 1100 chromatograph and a PDA detector.

[0051] The chromatographic column was Agilent ZORBAX SB-C18 4.6*150mm*5μm, the mobile phase was pure acetonitrile and aqueous solution containing 1 / 1000 formic acid, the flow rate was 0.9mL / min, 36% acetonitrile was isocratic eluted for 17min, 36% to 90% acetonitrile gradient elution was 0.01min, 90% to 90% acetonitrile was isocratic elution was 2.89min, 90% to 36% acetonitrile gradient elution was 0.01min, 36% acetonitrile was isocratic elution was 3.09min, the wavelength was 340nm, and the total running time was 23min. The solvent was 50% acetonitrile, the injection volume was 10μL, and the retention time of (S)-3-aminobutyronitrile was about 12.9min( Fig.12 ), the retention time of (R)-3-aminobutyronitrile is about 16.7min ( Fig.13 ).

[0052] It should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0053]

[0054]

[0055]

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[0057]

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[0059]

Claims

1. A route for synthesizing (S)-3-aminobutyronitrile, which comprises preparing cyanoacetone from 3-aminocrotononitrile or methyl acetate, and then using cyanoacetone as a substrate and utilizing S-aminotransferase to catalyze a reaction to asymmetrically generate (S)-3-aminobutyronitrile.

2. The method according to claim 1, characterized in that First, a chemical synthesis method is used to prepare cyanoacetone by using 3-aminocrotononitrile as a substrate and hydrolyzing it with hydrochloric acid at 80°C under reflux; or methyl acetate is used as a substrate and heated under reflux with NaH and acetonitrile in a toluene solution to prepare cyanoacetone, and then transaminase is used for catalysis, and the reaction system includes 0.1M TEOA solution, pH is 8.5, and the reaction temperature is 30°C.

3. The method according to claim 1-2, characterized in that: The loading amount of substrate cyanoacetone is 4.15-50 g / L, the final concentration of pyridoxal phosphate (PLP) is 10 mM, the amine donor is isopropylamine, the final concentration is 2.95-35.6 g / L, and the ee of (S)-3-aminobutyronitrile is ≥99%.

4. The method according to claims 1 to 3, characterized in that: Examples of S-aminotransferases include, but are not limited to, those from Microbacterium and Aspergillus fumigatus, and amine donors include, but are not limited to, isopropylamine, phenylethylamine, and alanine.

5. A synthetic route for (S)-3-aminobutyronitrile, wherein racemic 3-aminobutyronitrile is produced by reduction of 3-aminocrotononitrile imine or amination of 2-butenenitrile, and then (S)-3-aminobutyronitrile is selectively produced by catalyzing the racemic 3-aminobutyronitrile in 0.1 M TEOA solution using R-type aminotransferase.

6. The method according to claim 5, characterized in that Using 3-aminocrotononitrile as a substrate, sodium cyanoborohydride or other reducing agents are used in an ethanol solution to carry out imine reduction to prepare racemic 3-aminobutyronitrile; or using 2-butenenitrile as a substrate, heating and reflux in an ammonia solution to prepare racemic 3-aminobutyronitrile, and then carrying out transaminase catalysis to generate (S)-3-aminobutyronitrile.

7. The method according to claims 5-6, characterized in that The substrate 3-aminobutyronitrile loading is 3-50 g / L, the R-type aminotransferase wet cell concentration is 0-100 g / L, the final PLP concentration is 10 mM, the final amine receptor concentration is 4.14-69 g / L, the reaction system pH is 8.5, and the reaction temperature is 30°C.

8. The method according to claims 5-7, characterized in that R-aminotransferases include but are not limited to those from the genera Cormierella and Pseudofluorescens, and amine acceptors include but are not limited to acetone, acetophenone, and pyruvate.

9. An enzymatic synthesis route for (S)-3-aminobutyronitrile, which uses cyanoacetone or 3-aminobutyronitrile as starting materials, converts cyanoacetone into (S)-3-aminobutyronitrile by S-transaminase, or converts racemic 3-aminobutyronitrile into (S)-3-aminobutyronitrile by R-transaminase.

10. The method according to claim 9, characterized in that The S-aminotransferase sequence is shown in SEQ ID No. 1-2, and the R-aminotransferase sequence is shown in SEQ ID No. 3-4.

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