Synthesis method of sacubitril intermediate

Abstract

The invention discloses a chemical enzyme cascade synthesis method of a sacubitril intermediate. An enzyme catalysis reaction is used as a core basic reaction, an intermediate compound VII # imgabs2 # is obtained from a compound V # imgabs0 # and a compound VI # imgabs1 # under the action of olefin reductase by using two schemes, and meanwhile, the synthesis method of the compound V and the compound VI is optimized. The method is short in route and high in efficiency, an intermediate with high chiral purity is efficiently obtained through enzyme catalysis, the process cost is reduced, and the purity of a finished product is improved. The whole process is low in experimental condition requirement, simple to operate, high in yield and suitable for process amplification.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for synthesizing a sacubitril intermediate by catalysis of olefin reductase. Technical Background

[0002] Sacubitril-Valsartan Sodium Figure 1 ) is a new type of antihypertensive drug developed by Novartis to reduce cardiovascular death and heart failure. It can also replace angiotensin converting enzyme inhibitors (ACEI) or angiotensin II receptor antagonists (ARB) and be used in combination with other drugs to treat heart failure. Sacubitril-valsartan sodium contains sacubitril (AHU-377) and valsartan, which form a single co-crystal in a molar ratio of 1:1. The structural formula is as follows:

[0003] Compared with traditional compound preparations, the drug composition of sacubitril and valsartan is constant and stable. Compared with valsartan in other marketed tablets, the bioavailability of valsartan in sacubitril is higher. There are three synthetic routes for Scaubitril: First, in 1995, the Journal of Medicinal Chemistry, Volume 38, Pages 1689-1700 reported that chiral tyrosine methyl ester was used as raw material, trifluoromethanesulfonic anhydride was used to protect the hydroxyl group, and a biphenyl structure was formed through Suzuki reaction, followed by strong base hydrolysis, lithium aluminum tetrahydride reduction, wittig reaction, hydrogen and palladium carbon reduction, etc. Its synthetic route is as follows:

[0004]

[0005] The reaction route is too long and uses palladium metal catalysts, lithium aluminum hydroxide catalysts, etc. The production cost is too high, a lot of hazardous waste is generated, and it is not easy to scale up production.

[0006] Secondly, patent WO2014032627 discloses the synthesis of Scaubitril using 4-bromobiphenyl as substrate and S-epichlorohydrin through Grignard reaction, amino protection and deprotection, Mitsunobu reaction, oxidation reaction, and Wittig reaction. The synthesis route is as follows:

[0007]

[0008] Compared with the first route, the number of steps is reduced, but the use of chiral substrates and expensive metal catalysts greatly limits the industrial production of this method.

[0009] Thirdly, document CN 116004562 A reported that 4-benzaldehyde was used as a substrate, a transaminase mutant was obtained by directed evolution, and the transaminase mutant was used to catalyze 4,4'-biphenylpyruvic acid to synthesize D-4,4'-biphenylalanine. The synthesis route is as follows:

[0010]

[0011] Compared with the wild-type transaminase parent, the unit enzyme activity is increased by about 30 times while ensuring the chiral purity of the product. It has higher stereoselectivity and conversion rate, but only one chiral center is constructed, which limits its industrial application. Summary of the invention

[0012] In view of the problems of harsh reaction conditions and low conversion rate in the current preparation process of sacubitril, the present invention provides a chemoenzyme cascade method for synthesizing a sacubitril precursor with enzyme catalysis as the core reaction. At the same time, the preparation method of the key substrate in the synthesis route is optimized. After screening the existing olefin reductases in the laboratory, we obtained two olefin reductases that can efficiently catalyze the construction of sacubitril precursors. This method is simple to operate, has little pollution, is easy to separate and purify, is easy to scale up, and realizes industrial production.

[0013] To achieve the above object, the present invention provides the following technical solutions:

[0014]

[0015] The specific steps include:

[0016] In step 1, compound I and compound II are heated to reflux under the action of a metal catalyst. After the reaction is completed, they are extracted, concentrated, separated by column chromatography, and dried to obtain compound III.

[0017] In step 2, compound III is dissolved in dichloromethane, and Dess-Martin periodinane is added to react for 1-3 hours. After the reaction is completed, saturated sodium thiosulfate is added to quench the reaction, and the mixture is extracted, concentrated, separated by column chromatography, and dried to obtain compound IV.

[0018] In step 3, compound III is dissolved in 1,4-dioxane, phenylboronic acid, inorganic base, catalyst are added, heated to reflux for 24-48h, and after the reaction is completed, extracted, concentrated, separated by column chromatography, and dried to obtain compounds V and VI at the same time. Among them, in this synthetic route, two products (Formula V and Formula VI) can be obtained at the same time, and Formula V will isomerize to Formula VI under alkaline conditions. Therefore, two compounds can be obtained at the same time, which can be separated by column chromatography and the like, preferably by column chromatography, and more specifically, petroleum ether: ethyl acetate = 10:1 is used as the separation solvent.

[0019] In step 4, compounds V and VI are reacted with olefin reductase to obtain the target compound with high stereoselectivity.

[0020] Wherein, in step 1, the solvent selected is one of dichloromethane, methanol, ethyl acetate, tetrahydrofuran or acetonitrile.

[0021] Wherein, in step 1, the metal catalyst selected is one of Fe, Pd, Cu and Zn.

[0022] Wherein, in said step 1, the reaction temperature is 50-100°C.

[0023] Wherein, in said step 1, the reaction time is 12-24h.

[0024] Wherein, in step 2, the solvent selected is one of dichloromethane, methanol, ethyl acetate, tetrahydrofuran or acetonitrile.

[0025] Wherein, in step 2, the catalyst selected is one of hydrogen peroxide, pyridinium chlorochromate, potassium permanganate, 2-iodoacylbenzoic acid, and Dess-Martin periodinane.

[0026] Wherein, in said step 2, the reaction temperature is 20-80°C.

[0027] Wherein, in said step 2, the reaction time is 0.5-2h.

[0028] Wherein, in step 3, the solvent selected is one of dichloromethane, methanol, ethyl acetate, tetrahydrofuran or 1,4-dioxane.

[0029] Wherein, in step 3, the inorganic base selected is one of potassium carbonate, sodium carbonate and cesium fluoride.

[0030] Wherein, in step 3, the catalyst selected is one of tetrakistriphenylphosphine palladium, palladium dichloride, and tris(dibenzylideneacetone)dipalladium.

[0031] Wherein, in said step 3, the reaction temperature is 50-150°C.

[0032] Wherein, in said step 3, the reaction time is 24-48h.

[0033] In step 4, the two enzymes used in the reaction are respectively olefin reductase from Enterobacter cloacae (PDB number: 3P62_A) or olefin reductase from Gluconobacter oxydans (PDB number: 6O08_A).

[0034] The amino acid sequence of 3P62_A is as follows (SEQ ID NO: 1):

[0035] MSAEKLFTPLKVGAVTAPNRVFMAPLTRLRSIEPGDIPTPLMGEYYRQRASAGLIISEATQISAQAKGYAGAPGLHSPEQIAAWKKITAGVHAEDGRIAVQLWHTGRISHSSIQPGGQAPVSASALNANTRTSLRDENGNAIRVDTTTPRALELDEIPGIVNDFRQAVANAREAGFDLVELH SAHGYLLHQFLSPSSNQRTDQYGGSVENRARLVLEVVDAVCNEWSADRIGIRVSPIGTFQNVDNGPNEEADALYLIEELAKRGIAYLHMSETDLAGGKPYSEAFRQKVRERFHGVIIGAGAYTAEKAEDLIGKGLIDAVAFGRDYIANPDLVARLQKKAELNPQRPESFYGGGAEGYTDYPSL

[0036] The amino acid sequence of 6008_A is as follows (SEQ ID NO: 2):

[0037] MPTLFDPIDFGPIHAKNRIVMSPLTRGRADKEAVPTPIMAEYYAQRASAGLIITEATGISREGLGWPFAPGIWSDAQVEAWKPIVAGVHAKGGKIVCQLWHMGRMVHSSVTGTQPVSSSATTAPGEVHTYEGKKPFEQARAIDAADISRILNDYENAARNAIRAGFDGVQIHAANGYLIDE FLRNGTNHRTDEYGGVPENRIRFLKEVTERVIAAIGADRTGVRLSPNGDTQGCIDSAPETVFVPAAKLLQDLGVAWLELREPGPNGTFGKTDQPKLSPQIRKVFLRPLVLNQDYTFEAAQTALAEGKADAIAFGRKFISNPDLPERFARGIALQPDDMKTWYSQGPEGYTDYPSATSGPN.

[0038] Wherein, in step 4, the substrate concentration is 1-50 mM.

[0039] Wherein, in step 4, the type of buffer solution is potassium phosphate or sodium phosphate solution.

[0040] Wherein, in step 4, the concentration of the buffer solution is 10-200 mM.

[0041] Wherein, in step 4, the pH value of the buffer solution is 5-10.

[0042] Wherein, in said step 4, the reaction temperature is 20-50°C.

[0043] Wherein, in said step 4, the reaction time is 1-24h.

[0044] The present invention uses enzyme catalysis as the core basic reaction, and uses two schemes to obtain the intermediate compound VII from the compound of formula V and the compound of formula VI under the action of olefin reductase. At the same time, the synthesis method of the compound of formula V and the compound of formula VI is optimized in the steps, which is simpler than the previous synthesis operation, less polluting, easy to separate and purify, easy to scale up, and realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 NMR of the concentrated compound V separated by column chromatography.

[0046] Figure 2 NMR of the concentrated compound VI separated by column chromatography.

[0047] Figure 3 Liquid phase diagram of compound VII prepared from compound V by olefin reductase.

[0048] Figure 4 Liquid phase diagram of compound VII prepared from compound VI via olefin reductase. DETAILED DESCRIPTION

[0049] The present invention is described in detail below in conjunction with specific embodiments, but is not intended to limit the present invention.

[0050] Example 1 (Olefin reductase that efficiently catalyzes the construction of sacubitril precursor)

[0051] In order to obtain an olefin reductase that can efficiently catalyze the construction of sacubitril precursor, the present inventors screened multiple olefin reductases in order to obtain an enzyme with optimal reaction activity and selectivity.

[0052] First, formula V was selected as the substrate and the existing olefin reductase enzyme library was screened. The reaction conditions were: 1 mL system, formula V (10 mM), GDH (0.5 mg / mL), NADP + (1mM), glucose (20mM), sodium phosphate buffer solution (50mM, pH=7) 600uL, 30°C, 800rpm, crude enzyme solution (400uL). Samples were taken after 24h and detected by high performance liquid chromatography.

[0053]

[0054] The reaction results are as follows:

[0055] No. Enzyme (uniprot) source Conv. ee 1 Q02899 Saccharomyces pastorianus ＜5 ＜5 2 Q31R14 Synechococcus sp. PCC 7942 ＜5 ＜5 3 Q3M1A7 Anabaena variabilis ＜5 ＜5 4 B0C3H7 Acaryochloris marina MBIC11017 ＜5 ＜5 5 P40952 Kluyveromyces lactis ＜5 ＜5 6 Q9R9V9 Pseudomonas putida 90 95 7 A0A2G4TY30 Yersinia bercovieri 4 ＜5 8 6O08_A Gluconobacter oxydans 24 99 9 3P62_A Enterobacter cloacae 99 97

[0056] Secondly, Formula VI was selected as the substrate and the existing olefin reductase enzyme library was screened. The reaction conditions were: 1 mL system, Formula VI (10 mM), GDH (0.5 mg / mL), NADP + (1mM), glucose (20mM), sodium phosphate buffer solution (50mM, pH=7) 600uL, 30°C, 800rpm, crude enzyme solution (400uL). Samples were taken after 24h and detected by high performance liquid chromatography.

[0057]

[0058] The reaction results are as follows:

[0059]

[0060]

[0061] After screening, two olefin reductases that can efficiently catalyze the construction of sacubitril precursor were finally obtained, namely, the olefin reductase from Enterobacter cloacae (PDB number: 3P62_A), or the olefin reductase from Gluconobacteroxydans (PDB number: 6O08_A), and the amino acid sequences of the two are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0062] Example 2 (Synthesis of Compound V and Formula VI)

[0063] The synthetic route of compound V is as follows:

[0064]

[0065] In step 1, 4-bromophenylacetaldehyde (1970 mg, 10 mmol), 2-bromomethylacrylate (1930 mg, 10 mmol), and metal catalyst Zn powder (650 mg, 10 mmol) were added to a mixed solution (60 mL) of tetrahydrofuran and saturated ammonium chloride, and the mixture was heated to reflux. The reaction was monitored by TLC. After the reaction was completed, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography. The yield was 92% and the purity was 99%.

[0066] In step 2, Dess-Martin periodinane (3180 mg, 7.5 mmol) was added to a dichloromethane solution (80 mL) of the compound obtained in step 1 (1565 mg, 5 mmol), and the mixture was stirred at room temperature. The reaction was monitored by TLC. After the reaction was completed, the reaction was quenched with saturated sodium thiosulfate, extracted, dried, separated by column chromatography, and concentrated. The yield was 95% and the purity was 99%.

[0067] In step 3, phenylboric acid (1210 mg, 10 mmol), tetrakistriphenylphosphine palladium (5 mol%, 288 mg), potassium carbonate (4.8 g, 34.5 mmol) were added to compound IV (1555 mg, 5 mmol) obtained in step 2, and 1,4-dioxane (100 mL) was added. The mixture was protected by nitrogen and heated to reflux. The reaction was monitored by TLC. After the reaction was completed, the mixture was extracted, dried, and separated by silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1). The mixture was concentrated to obtain compound V (R f =0.5) and compound VI (R f =0.4), the yield of compound V was 53%, the yield of compound VI was 32%, and the total yield was 85% ( Figure 1 and Figure 2 ).

[0068] Example 3 (Preparation of Compound VII from Compound V via Olefin Reductase)

[0069] The route of preparing compound VII from compound V via olefin reductase:

[0070]

[0071] In a 100mL system, compound V (100mM), potassium phosphate buffer solution (pH=7.4, 50mM), glucose (20mM), GDH (0.5mg / mL), NADP+ (1mM), olefin reductase (3P62_A, source: Enterobactercloacae) lysate (0.1g / mL), reaction temperature 37°C, speed 1000rpm. Reaction time 24h. After the reaction, centrifugation, extraction, and HPLC monitoring. Conversion rate 99%, ee% 97% ( Figure 3 ).

[0072] Example 4 (Preparation of Compound VII from Compound VI via Olefin Reductase)

[0073] The route for preparing compound VII from compound VI via olefin reductase is as follows:

[0074]

[0075] In a 100mL system, compound VI (100mM), potassium phosphate buffer solution (pH=7.4, 50mM), glucose (20mM), GDH (0.5mg / mL), NADP+ (1mM), olefin reductase (6O08_A, source: Gluconobacteroxydans) lysate (0.1g / mL), reaction temperature 37°C, speed 1000rpm. Reaction time 24h. After the reaction, centrifugation, extraction, and HPLC monitoring. Conversion rate 99%, ee% 99% ( Figure 4 ).

Claims

1. A method for synthesizing a sacubitril intermediate, characterized in that: The specific steps include: Step 1: Compound I With raw material compound Ⅱ The reaction yielded compound III Step 2: Compound III reacts to obtain compound IV Step 3: Compound IV reacts to obtain compound V and compound VI R is H or Et; Step 4: Compound V and compound VI are catalyzed by enzyme to obtain compound VII Among them, the enzyme used is olefin reductase.

2. The method for synthesizing a sacubitril intermediate according to claim 1, wherein: In the step 1, the solvent selected is one of dichloromethane, methanol, ethyl acetate, tetrahydrofuran or acetonitrile; the metal catalyst selected is one of Fe, Pd, Cu, and Zn, the reaction temperature is 50-100°C, and after heating and reflux, the reaction is completed, extracted, concentrated, separated, and dried to obtain compound III.

3. The method for synthesizing a sacubitril intermediate according to claim 1, wherein: In the step 2, the solvent selected is one of dichloromethane, methanol, ethyl acetate, tetrahydrofuran or acetonitrile; the catalyst selected is one of hydrogen peroxide, pyridinium chlorochromate, potassium permanganate, 2-iodoacetylbenzoic acid, and Dess-Martin periodinane; the reaction temperature is 20-80°C; after the reaction is completed, saturated sodium thiosulfate is added to quench the reaction, extraction, concentration, separation, and drying are performed to obtain compound IV.

4. The method for synthesizing a sacubitril intermediate according to claim 1, wherein: In step 3, the solvent is one of dichloromethane, methanol, ethyl acetate, tetrahydrofuran or 1,4-dioxane; the inorganic base is one of potassium carbonate, sodium carbonate and cesium fluoride; the catalyst is one of tetrakistriphenylphosphine palladium, palladium dichloride and tris(dibenzylideneacetone)dipalladium; the reaction temperature is 50-150°C; Specifically, compound III is dissolved in a solvent, phenylboric acid, an inorganic base, and a catalyst are added, and the mixture is heated to reflux. After the reaction is completed, the mixture is extracted, concentrated, separated, and dried to obtain compounds V and VI at the same time. The compounds can be separated by column chromatography or the like, preferably silica gel column chromatography, and more specifically, petroleum ether: ethyl acetate = 10:1 is used as the separation solvent.

5. The method for preparing a sacubitril intermediate according to claim 1, characterized in that: In the step 4, the olefin reductase is an olefin reductase derived from Enterobacter cloacae, whose PDB number is 3P62_A; or an olefin reductase derived from Gluconobacter oxydans, whose PDB number is 6O08_A.

6. The method for preparing a sacubitril intermediate according to claim 5, characterized in that: In step 4, the substrate concentration in the reaction system is 1-50 mM, the buffer solution is potassium phosphate or sodium phosphate solution; the pH of the buffer solution is 5-10, the reaction temperature is 20-50° C.; and the reaction time is 12-24 h.

7. Olefin reductase catalyzes the reaction of compound Ⅴ or compound VI The reaction gave compound VII Application in which R is H or Et.

8. The application according to claim 7, characterized in that: The olefin reductase is an olefin reductase derived from Enterobacter cloacae, whose PDB number is 3P62_A; or an olefin reductase derived from Gluconobacter oxydans, whose PDB number is 6O08_A.

9. The application according to claim 8, characterized in that: During the reaction, the substrate concentration is 1-50 mM, the buffer solution is potassium phosphate or sodium phosphate solution; and the pH of the buffer solution is 5-10.

10. The application according to claim 9, characterized in that: The reaction temperature is 20-50°C.

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