Synthesis method and application of treprostinil chiral intermediate and synthesis method of treprostinil

Abstract

The invention provides a synthesis method and application of a treprostinil chiral intermediate and a synthesis method of treprostinil, and relates to the technical field of pharmaceutical chemical synthesis. According to the synthesis method of the treprostinil chiral intermediate, provided by the invention, efficient synthesis of a key intermediate is realized by chemical synthesis methods of constructing a cyclopentanaphthalene skeleton through a Dieckmann reaction catalyzed by LiHMDS and the like in combination with a chiral alcohol preparation technology through asymmetric reduction of carbonyl reductase. The method is efficient, low in cost and environmentally friendly, the total yield is larger than or equal to 75%, the chiral purity ee is larger than or equal to 99%, the method is suitable for large-scale production, and core technical support is provided for large-scale patent medicine of treprostinil.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemical synthesis, in particular to a synthesis method of a treprostinil chiral intermediate and an application thereof, and a synthesis method of treprostinil. Background Art

[0002] Treprostinil (Remodulin) is a core drug for the treatment of pulmonary arterial hypertension. The synthesis of its chiral intermediate is a key step in the preparation of Treprostinil (the active ingredient of Treprostinil). Currently, existing synthesis technologies are mainly faced with the following bottlenecks: 1. Traditional chemical method: The traditional chemical method uses a single chemical reaction, with a low yield of 45%-55% and poor chiral selectivity, with enantiomeric excess (ee value) less than 85%. It faces the dual challenges of yield and chirality control in the full chemical synthesis route.

[0003] 2. Single enzyme catalysis: The single enzyme catalysis method relies on a precious metal coenzyme regeneration system, with a reaction time exceeding 48 hours and a limited substrate concentration, usually below 5 g / L. The industrialization cost is high, making it a difficult problem for the industrial application of total biosynthesis.

[0004] 3. Advantages of the chemo-enzymatic method: The chemo-enzymatic method is expected to break through the bottlenecks of the above-mentioned traditional chemical method and single enzyme catalysis method, combining the advantages of chemical synthesis and biocatalysis, and showing unique potential in terms of yield, chiral selectivity, reaction time, substrate concentration and cost control.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The first object of the present invention is to provide a method for synthesizing a chiral intermediate of treprostinil, which has low cost, high efficiency and is environmentally friendly, so as to solve the above technical problems.

[0007] The second object of the present invention is to provide the application of the synthesis method of the above-mentioned treprostinil chiral intermediate in the preparation of treprostinil.

[0008] The third object of the present invention is to provide a method for synthesizing treprostinil.

[0009] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides a method for synthesizing a chiral intermediate of treprostinil, comprising the following steps: LiHMDS catalysis Dieckmann reaction to generate ; Then the hydrolysis reaction produces ; then catalyze The carbon-carbon double bond is reduced to generate ; then using carbonyl reductase mutants to catalyze Generate chiral intermediate of treprostinil ; The carbonyl reductase mutant is HMC-04, and the amino acid sequence is shown in SEQ ID NO.1: MAQYDVAGRSAIVTGGGSGIGRAIALTLAASGAAVLVTDLNEENANAVVAEISAAGGTARALAGDVTDPAFAEASVAAANELAPLRIAVNNAGIGGAAAPVGDYPLDSWRKVIEVNLNAVFYGMQA QLDAIGANGGGAIVNMASILGSVGFANYSAYVTAKHALLGLTQNAALEYAGKNVRVVAVGPGGIGGASNMDADTLAFLEGKHALGRLGEPEEVASLVAFLASDAASFITGSYHLVDGGYTAQ (SEQ ID NO.1).

[0010] As a further technical solution, the temperature of the Dieckmann reaction is 0-25°C, and the reaction time is 2-6 hours; The medium for the Dieckmann reaction includes tetrahydrofuran.

[0011] As a further technical solution, the hydrolysis reaction adopts a base catalytic system or an acid catalytic system, wherein the base catalytic system is a NaOH or KOH aqueous solution, and the acid catalytic system is a dilute H2SO4 or HCl solution; In the base catalytic system, the molar ratio of substrate to NaOH is 1:1.0-3.0, the reaction temperature is 0-50°C, and the reaction time is 2-12 hours; in the acid catalytic system, the molar ratio of substrate to H2SO4 is 1:1.0-5.0, the reaction temperature is 50-80°C, and the reaction time is 6-24 hours.

[0012] As a further technical solution, olefin reductase is used to catalyze the reduction of carbon-carbon double bonds; The temperature for catalyzing the reduction of carbon-carbon double bonds by olefin reductase is 25-35° C. and the pH is 7.5-8.5.

[0013] As a further technical solution, the reaction system catalyzed by the carbonyl reductase mutant includes: 15-25mM , 25-85 g / L of bacteria expressing the carbonyl reductase mutant, 10-20% (V / V) isopropanol, and 0.05-0.15 M phosphate buffer; The carbonyl reductase mutant catalyzes the process at a temperature of 25-35° C., a pH of 7-8, and a time of 12-36 hours.

[0014] In a second aspect, the present invention provides the application of the synthesis method of the above-mentioned treprostinil chiral intermediate in the preparation of treprostinil.

[0015] In a third aspect, the present invention provides a method for synthesizing treprostinil, comprising the following steps: Synthesized by the above-mentioned synthesis method of treprostinil chiral intermediate ; then Condensation reaction to obtain ; then Nucleophilic substitution reaction to obtain carbonyl reductase catalytic precursor ; The precursor is then catalyzed by carbonyl reductase to prepare treprostinil.

[0016] As a further technical solution, the preparation of the treprostinil intermediate using a carbonyl reductase catalytic precursor comprises the following steps: catalytic Restore Generation ; then Demethylation gives ; Afterwards Substitution reaction to obtain ; then The tert-butyl ester of treprostinil was hydrolyzed and deprotected to prepare treprostinil. .

[0017] As a further technical solution, the carbonyl reductase mutant is used to catalyze Restore Generation ; The carbonyl reductase mutant is HMC-04, and the amino acid sequence is shown in SEQ ID NO.1.

[0018] As a further technical solution, the substitution reaction comprises Substitution reaction with tert-butyl bromoacetate.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for synthesizing a chiral intermediate of treprostinil. This method uses a chemical reaction to construct an enzyme-catalyzed precursor, combined with carbonyl reductase asymmetric reduction technology, to achieve efficient synthesis of key intermediates. This method is highly efficient, low-cost, and environmentally friendly, with an overall yield of ≥75% and a chiral purity (ee) of ≥99%. It is suitable for large-scale production and provides core technical support for the large-scale development of treprostinil as a pharmaceutical. DETAILED DESCRIPTION

[0020] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0021] Example 1 A method for synthesizing a chiral intermediate of treprostinil comprises the following steps: 1. Diethyl 2,2'-(8-methoxy-1,2-dihydronaphthalene-2,3-diyl) diacetate is subjected to the Dieckmann reaction to obtain ethyl 5-methoxy-2-oxo-2,3,3a,4-tetrahydro-1H-cyclopenta[b]naphthalene-1-carboxylate. The reaction formula is as follows:

[0022] The steps include: dissolving LiHMDS (0.3 mol) in 500 mL of anhydrous THF under argon protection and cooling to 0°C; adding a substrate solution (100 g of diethyl 2,2'-(8-methoxy-1,2-dihydronaphthalene-2,3-diyl) diacetate dissolved in 200 mL of THF) dropwise within 30 minutes, heating to 25°C and reacting for 4 hours; Quenching: adding saturated NH4Cl solution, extracting with ethyl acetate (3×200 mL), and drying over anhydrous sodium sulfate; Column chromatography (silica gel, petroleum ether:ethyl acetate = 10:1) gave 92.5 g of the product with a yield of 92% and a purity of 98.2% (HPLC).

[0023] Characterization data: 1 HNMR (400 MHz, DMSO -d 6) δ 7.17 (t, J =7.9Hz, 1H), 6.80 (t, J =6.9Hz, 2H),6.03 (s,1H), 4.06–3.94 (m, 3H), 3.77 (s,3H), 3.40 (dd, J =16.6, 7.0 Hz, 1H),2.99 (m, J =13.2, 6.8 Hz, 1H), 2.60 (dd, J=18.6, 6.4 Hz, 1H), 2.18–2.06 (m, 2H), 1.36–1.21 (m, 3H). 13 CNMR (101 MHz, DMSO- d 6) δ 207.67, 180.89, 156.53, 134.39,127.23, 126.74, 123.49, 120.84, 107.89, 59.77, 55.31, 41.66, 37.70, 32.24,30.48, 20.77, 14.09.

[0024] 2. 5-methoxy-2-oxo-2,3,3a,4-tetrahydro-1H-cyclopenta[b]naphthalene-1-carboxylic acid ethyl ester is hydrolyzed to obtain 5-methoxy-2-oxo-2,3,3a,4-tetrahydro-1H-cyclopenta[b]naphthalene-1-carboxylic acid according to the following reaction formula:

[0025] The process involves slowly adding 2 mol / L NaOH solution (2.4 mL, 0.0048 mol, 1.5 eq) to a mixture of ethyl 5-methoxy-2-oxo-2,3,3a,4-tetrahydro-1H-cyclopenta[b]naphthalene-1-carboxylate (1 g, 98.5% purity, 0.0032 mol) and deionized water (10 mL) with stirring. The reaction temperature is controlled at 25°C-30°C and the reaction is allowed to proceed for 8 hours. HPLC monitoring indicates <3% residual substrate.

[0026] The reaction mixture was adjusted to pH 2.5 with 2 mol / L dilute hydrochloric acid, resulting in the precipitation of a large amount of white solid. The filter cake was then filtered and washed with deionized water (50 mL x 3). The filter cake was then dried under vacuum at 45°C for 10 hours to afford 0.82 g of a white solid (88.7% yield).

[0027] 3. Using olefin reductase, the double bond of 5-methoxy-2-oxo-2,3,3a,4-tetrahydro-1H-cyclopenta[b]naphthalene-1-carboxylic acid is eliminated to yield 5-methoxy-2-oxo-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-1-carboxylic acid. The reaction formula is as follows:

[0028] The steps include: 5-methoxy-2-oxo-2,3,3a,4-tetrahydro-1H-cyclopenta[b]naphthalene-1-carboxylic acid (1.0 eq) is reacted with olefin reductase (0.5 g / L) in a pH 8.0 Tris buffer for 24 hours to obtain 5-methoxy-2-oxo-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-1-carboxylic acid.

[0029] Reaction parameters: temperature 30°C, rotation speed 600 rpm, reaction time 24 h; Post-treatment: extraction with ethyl acetate three times, and GC detection (column temperature: 70°C → 300°C, heating rate 8°C / min).

[0030] A white powder was obtained with a yield of 80%.

[0031] 4. Carbonyl reductase catalyzes (2 R )-2-hydroxy-5-methoxy-2,3,3a,4,9,9a-hexahydro-1 H -cyclopenta[b]naphthalene-1-carboxylic acid, the reaction chemical formula is as follows:

[0032] The steps include: Enzyme solution: recombinant Escherichia coli wet cells (expressing carbonyl reductase mutant HMC-04, amino acid sequence as SEQ ID NO. 1), 50 g / L; Reaction system: 20 mM 5-methoxy-2-oxo-2,3,3a,4,9,9a-hexahydro-1 H -Cyclopenta[b]naphthalene-1-carboxylic acid, 10% isopropanol (cosolvent), 0.1 M phosphate buffer (pH 7.5); Parameters: temperature 30°C, rotation speed 600 rpm, reaction time 24 h; Post-treatment: Extract with ethyl acetate three times, combine the organic phases, and rotary evaporate to obtain the crude product; HPLC detection: conversion rate 75%, ee value 99% (Chiralpak AD-H column, mobile phase: n-hexane:IPA=66:34).

[0033] Comparative experiment: Replacing the mutant enzyme HMC-04 with the wild-type enzyme: conversion rate 7.42%, of value 17.28%; Example 2 A method for preparing treprostinil (the preparation process is a conventional process in the art, so only the synthetic route is provided), comprising the following steps: The (2R)-2-hydroxy-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-1-carboxylic acid prepared in Example 1 was condensed to obtain 2-((((2R)-2-hydroxy-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-1-formyl)oxy)carbonyl)benzoic acid, and the reaction formula is as follows:

[0034] 2-((((2R)-2-hydroxy-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-1-yl)oxy)carbonyl)benzoic acid is nucleophilically substituted to obtain 1-((1R,2R,3aS,9aS)-2-hydroxy-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-1-yl)octan-3-one, as shown in the following reaction formula:

[0035] Carbonyl reductase catalyzes the reaction of 1-((1R,2R,3aS,9aS)-2-hydroxy-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalen-1-yl)octan-3-one to produce (1R,2R,3aS,9aS)-1-((S)-3-hydroxyoctyl)-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalen-2-ol. The reaction formula is as follows:

[0036] Here are the steps: Enzyme source: carbonyl reductase mutant HMC-04 expressed in recombinant Escherichia coli, amino acid sequence as SEQ ID NO.1; Reaction system: 1-((1R,2R,3aS,9aS)-2-hydroxy-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalen-1-yl)octan-3-one (20 mM), 10% isopropanol, 0.1 M phosphate buffer (pH 7.5); Parameters: temperature 30°C, rotation speed 600 rpm, reaction time 24 h; Post-treatment: extraction with ethyl acetate three times, and GC detection (column temperature: 70°C → 300°C, heating rate 8°C / min).

[0037] (1R,2R,3aS,9aS)-1-((S)-3-hydroxyoctyl)-5-methoxy-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-2-ol is demethylated to obtain (1R,2R,3aS,9aS)-1-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-2,5-diol. The reaction formula is as follows:

[0038] (1R,2R,3aS,9aS)-1-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-2,5-diol is substituted with tert-butyl bromoacetate to obtain tert-butyl 2-(((1R,2R,3aS,9aS)-2-hydroxy-1-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-5-yl)oxy)acetate; tert-butyl 2-(((1R,2R,3aS,9aS)-2-hydroxy-1-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalene-5-yl)oxy)acetate is deBOCed to obtain treprostinil, and the reaction formula is as follows:

[0039] Treprostinil NMR data are as follows: 1 H NMR (400 MHz, DMSO -d 6) δ 12.92 (s, 1H), 7.03(t, J = 7.8 Hz, 1H), 6.72 (m, 2H), 4.62 (s, 2H), 4.55 – 4.14 (m, 2H), 3.47(q, J = 8.7 Hz, 5H), 2.70 (m, 2H), 2.42 (m, 2H), 2.20 – 1.89 (m, 2H), 1.68(m, 2H), 1.44 – 1.26 (m, 8H), 1.14 – 0.94 (m, 2H), 0.87 (t, J = 6.7 Hz, 3H). 13 CNMR (101 MHz, DMSO -d 6) δ 170.37, 154.63, 140.57, 126.76, 125.86, 120.66,109.37, 75.44, 70.14, 64.97, 51.50, 41.15, 40.48, 37.07, 35.03, 33.38, 32.43,31.54, 28.36, 25.63, 24.96, 22.19, 13.97.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for synthesizing a chiral intermediate of treprostinil, characterized in that: The steps include: LiHMDS catalysis Dieckmann reaction to generate ; Then the hydrolysis reaction produces ; then catalyze The carbon-carbon double bond is reduced to generate ; then using carbonyl reductase mutants to catalyze Generate treprostinil chiral intermediate ; The carbonyl reductase mutant is HMC-04, and the amino acid sequence is shown in SEQ ID NO.

1.

2. The method for synthesizing a chiral intermediate of treprostinil according to claim 1, wherein The temperature of the Dieckmann reaction is 0-25°C, and the reaction time is 2-6 hours; The medium for the Dieckmann reaction includes tetrahydrofuran.

3. The method for synthesizing a chiral intermediate of treprostinil according to claim 1, wherein: The hydrolysis reaction adopts a base catalytic system or an acid catalytic system, wherein the base catalytic system is a NaOH or KOH aqueous solution, and the acid catalytic system is a dilute H2SO4 or HCl solution; In the base catalytic system, the molar ratio of substrate to NaOH is 1:1.0-3.0, the reaction temperature is 0-50°C, and the reaction time is 2-12 hours; in the acid catalytic system, the molar ratio of substrate to H2SO4 is 1:1.0-5.0, the reaction temperature is 50-80°C, and the reaction time is 6-24 hours.

4. The method for synthesizing a chiral intermediate of treprostinil according to claim 1, wherein: Utilize olefin reductase to catalyze the reduction of carbon-carbon double bonds; The temperature for catalyzing the reduction of carbon-carbon double bonds by olefin reductase is 25-35° C. and the pH is 7.5-8.

5.

5. The method for synthesizing a chiral intermediate of treprostinil according to claim 1, wherein The reaction system catalyzed by the carbonyl reductase mutant includes: 15-25mM , 25-85 g / L of bacteria expressing the carbonyl reductase mutant, 10-20% (V / V) isopropanol, and 0.05-0.15 M phosphate buffer; The carbonyl reductase mutant catalyzes the process at a temperature of 25-35° C., a pH of 7-8, and a time of 12-36 hours.

6. Use of the synthesis method of the chiral intermediate of treprostinil according to any one of claims 1 to 5 in the preparation of treprostinil.

7. A method for synthesizing treprostinil, characterized in that: The steps include: The product is synthesized by the method for synthesizing the chiral intermediate of treprostinil according to any one of claims 1 to 5. ; then Condensation reaction to obtain ; then Nucleophilic substitution reaction to obtain carbonyl reductase catalytic precursor ; The precursor is then catalyzed by carbonyl reductase to prepare treprostinil.

8. The method for synthesizing treprostinil according to claim 7, wherein The preparation of the treprostinil intermediate using a carbonyl reductase catalytic precursor comprises the following steps: catalytic Restore Generation ; then Demethylation gives ; Afterwards Substitution reaction to obtain ; then The tert-butyl ester of treprostinil was hydrolyzed and deprotected to prepare treprostinil. .

9. The method for synthesizing treprostinil according to claim 8, wherein Catalysis using carbonyl reductase mutants Restore Generation ; The carbonyl reductase mutant is HMC-04, and the amino acid sequence is shown in SEQ ID NO.

1.

10. The method for synthesizing treprostinil according to claim 8, wherein The substitution reaction includes Substitution reaction with tert-butyl bromoacetate.

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