Method for preparing eldoxaban intermediate through catalysis of oxidase

Abstract

The invention provides a preparation method of an eldoxaban intermediate (compound 2). The preparation method is characterized in that a compound 1 is oxidized into the compound 2 by using oxidase. The oxidase is screened laccase from Bacillus pumilus, the amino acid sequence of the oxidase is shown as SEQ ID NO. 1, and the nucleotide sequence for coding the oxidase is shown as SEQ ID NO. 2. The oxidase has good catalytic activity on a raw material (a compound 1), the enzyme-substrate ratio of the oxidase reaction is 2: 1 (enzyme liquid / raw material), the substrate concentration is 50g / L, the reaction conversion rate reaches 75% or above, the reaction conditions are mild, and the method has good industrial application value. Wherein R1 is hydrogen, alkyl and other groups, and R2 is hydrogen, alkyl and other groups.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of enzyme catalysis technology, and relates to enzyme catalysis synthesis of a drug intermediate, in particular to application of an oxidase in preparation of an edoxaban intermediate. BACKGROUND

[0002] Edoxaban is also called edoxaban, and its chemical name is N-(5-chloroindol-2-yl)-N'-[(1S,2R,4S)-4-(N,N-dimethylformamido)-2-(5-methyl-4,5,6,7-tetrahydro[1,3]thiazolo[5,4-c]pyridine-2-carboxamide)cyclohexyl]oxamide, and a structural formula is as shown in the following formula:

[0003]

[0004] Edoxaban

[0005] Edoxaban is an anti-platelet aggregation drug, can inhibit thrombin activity, prevent thrombosis, and can be used for treating or preventing thrombotic diseases. It is used for preventing stroke and systemic embolism in non-valvular atrial fibrillation adult patients with one or more risk factors (such as congestive heart failure, hypertension, age of 75 years old or older, diabetes, history of previous stroke or transient ischemic attack). It is used for treating adult deep vein thrombosis (DVT) and pulmonary embolism (PE), and preventing recurrence of adult deep vein thrombosis and pulmonary embolism.

[0006] 4,5,6,7-Tetrahydro-5-methylthiazolo[5,4-c]pyridine-2-carboxylic acid (CAS NO: 758685-72-2) is a key intermediate (compound 1) for synthesizing edoxaban, and a structural formula is as shown in the following formula.

[0007] 1

[0009] At present, all the synthesis methods of edoxaban intermediate 4,5,6,7-tetrahydro-5-methylthiazolo[5,4-c]pyridine-2-carboxylic acid and a derivative compound (compound 2, a structural formula is as shown in the following formula) are chemical synthesis methods, and there is no related report on enzyme catalysis synthesis.

[0010] 2

[0012] The chemical synthesis methods for compound 2 include: 1) Patent CN102002059A, which discloses the main synthesis method currently on the market. This method uses n-butyllithium or tert-butyllithium as an organometallic reagent to react with the key raw material, a brominated derivative, to form a salt. The reaction temperature is below -60℃. Industrially, this requires low-temperature equipment, and the cooling system needs to use liquid nitrogen and ethanol for cooling. Moreover, the yield of this method is not high, and industrial-scale production requires high-end equipment. 2) Patent WO2015125710A discloses a preparation method using palladium acetate or its ligand catalyst coupling. Although this method avoids low-temperature reactions, it uses expensive palladium catalysts and ligands, resulting in very high costs. Furthermore, it is a pressure reaction, requiring an autoclave, which is not suitable for industrial-scale production. The production process also involves the use of carbon monoxide gas, posing significant safety hazards.

[0013] Chemical synthesis methods all require heavy metal catalysts and harsh reaction conditions, resulting in high costs, significant environmental impact, and the generation of waste. Compared to chemical synthesis, bio-enzyme catalytic synthesis offers advantages such as high selectivity, fewer byproducts, and milder reaction conditions, making it an environmentally friendly and green synthesis method.

[0014] There are currently no literature reports on the enzymatic synthesis of intermediate compound 2, but there are similar related reports. For example, the literature ACSCatalysis (2017), 7(2), 1295-1300 uses monoamine oxidase (MAO-N) and 6-hydroxy-D-nicotine oxidase (6-HDNO) for oxidative dehydrogenation; the literature ACSCatalysis (2019), 9(8), 7264-7269 reports the use of laccase for oxidative dehydrogenation.

[0015] This invention uses compound 1 as a transformation substrate (raw material) and an oxidase as a catalyst. By developing and screening an effective oxidase, it aims to catalyze the synthesis of a key intermediate (compound 2) for the antithrombotic drug edoxaban, providing technical support for the industrial production of edoxaban. Summary of the Invention

[0016] The purpose of this invention is to provide a method for preparing compound 2 by dehydrogenation of compound 1 catalyzed by an oxidase, wherein the oxidase is derived from Bacillus pumilus. This invention is simple to operate, safe to react, environmentally friendly, and has good prospects for industrial application.

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] In a first aspect, the present invention provides a method for preparing compound 2, which utilizes an oxidase to catalyze the reaction of a raw material (compound 1) to generate an edoxaban intermediate (compound 2), the reaction formula of which is as follows:

[0019]

[0020] Wherein, R1 is hydrogen or alkyl, and R2 is hydrogen or alkyl.

[0021] Secondly, the enzyme-catalyzed preparation method described in this invention specifically involves adding a mediator TEMPO or ABTS, compound 1, and hydrogen peroxide to a reaction medium, followed by adding an oxidase to generate edoxaban intermediate compound 2. The oxidase is a laccase derived from Bacillus pumilus.

[0022] Thirdly, in one embodiment of the present invention, the method specifically involves adding raw material compound 1, oxidase, hydrogen peroxide, metal ions, and buffer solution into a reaction vessel to carry out a catalytic reaction. The substrate concentration is 50 g / L, the buffer solution is 0.1 M pH 5.0 phosphate buffer, the reaction temperature is 40°C, the final concentration of hydrogen peroxide is 1.2 g / L, and the oxidase is added at an enzyme-to-substrate ratio of 2:1 with shaking.

[0023] The oxidase is derived from Bacillus pumilus.

[0024] The amino acid sequence of the oxidase is shown in SEQ ID NO.1.

[0025] The oxidase was prepared using the following method:

[0026] (1) Synthesize the nucleotide sequence encoding the oxidase and construct engineered bacteria;

[0027] (2) Induce culture of engineered bacteria, collect bacterial cells by centrifugation, and then prepare the required enzyme preparation.

[0028] The nucleotide sequence is shown in SEQ ID NO.2.

[0029] The reaction medium is selected from phosphate buffer, triethanolamine buffer, Tris-HCl buffer, citrate buffer, sodium acetate buffer, borate-borax buffer, and preferably sodium phosphate buffer.

[0030] The reaction is carried out at a pH of 4.0-9.0, preferably at a pH of 5.

[0031] The reaction temperature is 25-45℃, and more preferably, the temperature is 40℃.

[0032] The substrate concentration is 10-100 g / L, and more preferably, the concentration is 50 g / L.

[0033] The mediator is ABTS or TEMPO, with ABTS added at a rate of 0-0.3 mM and TEMPO added at a ratio of 0-1 / 2.5 to the raw material mass, preferably 1 / 5.

[0034] Hydrogen peroxide is added to the system, with the amount of hydrogen peroxide added being 0-6 g / L, preferably 1.2 g / L.

[0035] Fourthly, the present invention provides a method for producing the oxidase, namely, expressing the oxidase gene using pET21a as a vector and E. coli BL21 as a host, then centrifuging the induced fermentation broth to remove the supernatant, collecting the bacterial cells, and then preparing the desired enzyme preparation such as enzyme solution.

[0036] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0037] (1) In this invention, genetically engineered Escherichia coli derived from the BpLaccase gene of Bacillus pumilus was screened and constructed. The oxidase was obtained through induced culture and applied to the asymmetric reduction of compound 1 to prepare compound 2. Under optimized enzyme reaction conditions, the raw material concentration was 50 g / L and the reaction conversion rate reached more than 75%.

[0038] (2) There are no reports in the literature on the use of enzyme to catalyze the reaction of compound 1 to generate compound 2. The oxidase BpLaccase from Bacillus pumilus has high enzyme activity.

[0039] (3) Compared with existing chemical methods that use heavy metal catalysts and harsh reaction conditions, the enzyme-catalyzed preparation method of the present invention has the advantages of simple reaction operation, mild reaction conditions, fewer by-products and less waste, and has good potential for industrial application. Detailed Implementation

[0040] The invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only and do not limit the scope of the claims.

[0041] Technical solution: The present invention first screens out the effective oxidase BpLaccase from Bacillus pumilus, constructs enzyme-producing recombinant genetically engineered Escherichia coli, and prepares oxidase solution from recombinant Escherichia coli expressing oxidase. Under optimized conditions, the catalytic substrate is compound 1, which is oxidized to generate compound 2.

[0042] Experimental materials and reagents: 1. All materials used in this invention are commercially available products. 2. Culture medium: LB liquid medium: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, pH 6.8-7.0;

[0043] Detection and analysis methods: 1) HPLC quantitative detection method for products: YMC-Triart C18 column 150*4.6mm*3um, column temperature 35℃, flow rate 1mL / min, detection wavelength 290nm, mobile phase A: 30mMk2HPO4 aqueous solution; mobile phase B: acetonitrile, the mobile phase gradient is shown in Table 1.

[0044] Table 1. Mobile phase gradient for HPLC analysis

[0045]

[0046] Example 1: Construction of recombinant engineered bacteria for the oxidase BpLaccase

[0047] The gene for the oxidase BpLaccase derived from Bacillus pumilus was synthesized, and NdeI and XhoI sites were introduced at both ends of the gene, respectively. The gene was then cloned into the pET21a vector to obtain the recombinant vector pET21a-BpLaccase; its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence encoding this amino acid is shown in SEQ ID NO.2. The constructed recombinant plasmid was chemically transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL ampicillin, and cultured at 37°C for 8–12 h. Single clones were picked to obtain the recombinant genetically engineered bacterium BL21(DE3) / pET21a-BpLaccase, which can be induced to express the oxidase.

[0048] Example 2: Induction and expression of recombinant engineered bacteria and preparation of enzyme solution

[0049] Seed culture: The engineered BpLaccase oxidase bacteria were inoculated into LB liquid medium containing 50 μg / mL ampicillin and cultured at 37°C for 12–16 h.

[0050] Induction of expression: The recombinant engineered bacterial seed culture was inoculated at a volume fraction of 1% (v / v) into fresh fermentation medium containing a final concentration of 50 μg / mL ampicillin, and cultured at 37℃ and 200 rpm for 2–3 h. OD 600 Add the inducing agent IPTG and copper ions when the concentration is between 0.6 and 0.8, with a final concentration of IPTG of 0.2 mM and a final concentration of copper ions of 2.5 mM, and incubate at 25°C for 20 h.

[0051] Enzyme solution preparation: After induction culture, the fermentation broth was centrifuged at high speed to obtain whole cells of the genetically engineered bacteria. The cells were suspended in 0.1M pH 7.0 Tris-HCl buffer (cell concentration 20%), and then the suspension was disrupted by ultrasonication or homogenization to obtain the enzyme solution.

[0052] Example 3: Laccase BpLaccase catalyzes reactions of different raw materials

[0053] Take six 10 mL centrifuge tubes and add the raw material aqueous solution to a final concentration of 4 g / L, 0.8 mg TEMPO (mass ratio of 1:5 to raw material), and 100 μL laccase solution. Finally, add 0.1 M pH 7.0 phosphate buffer to each tube to a final concentration of 1 mL and react at 30 °C for 24 h. The reaction results are shown in Table 2.

[0054] Table 2. Results of BpLaccase-catalyzed reactions with different raw materials

[0055]

[0056] Example 4: Laccase-catalyzed reaction in different reaction media

[0057] Take six 10 mL centrifuge tubes and add the raw material (compound 1-II) aqueous solution to a final concentration of 4 g / L, 0.8 mg TEMPO (mass ratio of 1:5 to raw material), and 100 μL laccase solution. Finally, add 0.1 M pH 7.0 phosphate, Tris-HCl, citrate, sodium acetate, triethanolamine, and borate-borax buffer to a final concentration of 1 mL, respectively. Incubate at 30 °C for 24 h. The reaction results are shown in Table 3.

[0058] Table 3 Results of BpLaccase-catalyzed reactions in different reaction media

[0059]

[0060] Example 5: Laccase-catalyzed reaction at different reaction pH

[0061] Take six 10 mL centrifuge tubes and add the raw material (compound 1-II) aqueous solution to a final concentration of 4 g / L, 0.8 mg TEMPO (mass ratio of 1:5 to raw material), and 100 μL laccase solution. Finally, add 0.1 M pH 4-9 phosphate buffer to 1 mL and react at 30 °C for 24 h. The reaction results are shown in Table 4.

[0062] Table 4 Results of BpLaccase-catalyzed reactions at different reaction pH

[0063]

[0064] Example 6: BpLaccase-catalyzed reaction at different temperatures

[0065] Take five 10 mL centrifuge tubes and add the raw material (compound 1-II) aqueous solution to a final concentration of 4 g / L, 0.8 mg TEMPO (mass ratio of 1:5 to raw material), 100 μL laccase solution, and 0.1 M pH 5.0 phosphate buffer to 1 mL. Incubate at 25-45 °C for 24 h. The reaction results are shown in Table 5.

[0066] Table 5 Results of BpLaccase-catalyzed reactions at different temperatures

[0067]

[0068] Example 7: Laccase-catalyzed reaction at different feed concentrations

[0069] Take four 50 mL centrifuge tubes and add aqueous solutions of the raw material (compound 1-II) to them to final concentrations of 10, 20, 50, and 100 g / L, respectively. Then add 10, 20, 50, and 100 mg TEMPO (at a mass ratio of 1:5 with the raw material), 500 μL of laccase enzyme solution, and 0.1 M pH 5 phosphate buffer to a final volume of 5 mL. Incubate at 40 °C for 24 h. The reaction results are shown in Table 6.

[0070] Table 6 Results of BpLaccase-catalyzed reactions at different feed concentrations

[0071]

[0072] Example 8 Effect of mediators and different addition amounts on oxidase-catalyzed reactions

[0073] Eleven 10 mL centrifuge tubes were taken. In five of these tubes, an aqueous solution of the raw material (compound 1-II) was added to a final concentration of 4 g / L and 100 μL of laccase solution. ABTS was added to a final concentration of 0-0.3 mM, and 0.1 M pH 5.0 phosphate buffer was added to a final volume of 1 mL. The tubes were then incubated at 40 °C for 24 h. In the other five tubes, an aqueous solution of the raw material (compound 1-II) was added to a final concentration of 4 g / L and 100 μL of laccase solution. TEMPO was added at a mass ratio (to raw material ratio) of 1:20-1:2.5, and 0.1 M pH 5.0 phosphate buffer was added to a final volume of 1 mL. The tubes were then incubated at 40 °C for 24 h. The reaction results are shown in Table 5.

[0074] Table 7 Results of oxidase-catalyzed reactions with different amounts of mediators.

[0075]

[0076] Example 9 Effect of hydrogen peroxide addition on oxidase-catalyzed reaction

[0077] Add 300 mg TEMPO to 3 mL DMSO. Take seven 50 mL centrifuge tubes. In one of the centrifuge tubes, add the raw material (compound 1-II) aqueous solution to a final concentration of 50 g / L, 50 mg TEMPO, 500 μL laccase solution, and hydrogen peroxide to a final concentration of 2.4 g / L. Finally, add 0.1 M pH 5.0 phosphate buffer to 5 mL and adjust to the corresponding pH. Incubate at 40 °C for 24 h. In the remaining six centrifuge tubes, add the raw material (compound 1-II) aqueous solution to a final concentration of 50 g / L, 500 μL DMSO solution containing TEMPO, 500 μL laccase solution, and hydrogen peroxide to final concentrations of 0-6 g / L. Finally, add 0.1 M pH 5.0 phosphate buffer to 5 mL and adjust to the corresponding pH. Incubate at 40 °C for 24 h.

[0078] Table 8 Results of oxidase-catalyzed reactions with different amounts of hydrogen peroxide added.

[0079]

[0080] Example 10: Preparation of edoxaban intermediate compound 2 by oxidase BpLaccase catalyzing the oxidation of compound 1

[0081] The catalytic system composition and reaction conditions are as follows: In a 100 mL reaction flask, add 10 mL of BpLaccase oxidase solution, 5 g of raw material, hydrogen peroxide to a final concentration of 1.2 g / L, 10 mL of DMSO containing 1 g TEMPO, and then add 0.1 M pH 5 sodium phosphate buffer to a final volume of 100 mL, adjusting the pH to 5.0. The reaction is carried out at 40 °C, with the pH maintained at 5.0 and the rotation speed at 200 rpm. After 24 h of reaction, samples are taken for HPLC analysis, showing a conversion rate of 77.3% for substrate compound 1.

[0082] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the enzymatic production of compound 2, characterized in that: The raw material (compound 1) is reacted by using an oxidase to generate an intermediate (compound 2) of idoxaban, and the reaction formula is as follows: wherein R1is hydrogen, alkyl, R2is hydrogen, alkyl, The oxidase is an oxidase capable of catalyzing the oxidative dehydrogenation of compound 1 to generate compound 2.

2. The enzyme-catalyzed preparation method of compound 2 according to claim 1, characterized in that: The oxidase is a laccase derived from Bacillus pumilus, and the amino acid sequence is shown as SEQ ID NO.

1.

3. The method according to claim 1 or 2, wherein the compound 2 is produced by an enzyme catalysis.

3. The method according to claim 1 or 2, wherein the compound 2 is produced by an enzyme catalysis. The oxidase is prepared by the following method: (1) synthesizing a nucleotide sequence encoding the oxidase to construct an engineering bacterium; (2) inducing the culture of the engineering bacterium, centrifuging to collect the bacterial cells, and then preparing the required enzyme preparation; The nucleotide sequence is shown as SEQ ID NO.

2.

4. The method for the enzymatic preparation of compound 2 according to claim 1 or 2, characterized in that: In a buffer reaction medium, the raw material (compound 1), a mediator, hydrogen peroxide, and the oxidase are added to perform an oxidation reaction to obtain an intermediate (compound 2) of idoxaban.

5. The method of claim 4, wherein the compound 2 is produced by the enzyme catalyzed reaction of: ###0002### 1 2 The reaction medium is selected from phosphate buffer, triethanolamine buffer, Tris-HCl buffer, citrate buffer, sodium acetate buffer, and boric acid-borax buffer.

6. The enzyme-catalyzed preparation method of compound 2 according to claim 4, characterized in that: The pH of the reaction is 4-9.

7. The method of claim 4, wherein the compound 2 is prepared by an enzymatic catalysis. The reaction temperature is 25-45℃.

8. The method of claim 4, wherein the compound 2 is produced by an enzyme catalyzed reaction. The feeding concentration of the raw material (compound 1) is 10-100 g / L.

9. The method of claim 4, wherein the compound 2 is produced by an enzyme catalyzed reaction. The mediator is ABTS or TEMPO, the addition amount of ABTS is 0-0.3 mM, and the addition amount of TEMPO is 0-1 / 2.5 relative to the mass of the raw material.

10. The method of claim 4, wherein the compound 2 is produced enzymatically. Hydrogen peroxide is added to the system, and the final concentration of hydrogen peroxide is 0-6 g / L.

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