A process for the preparation of chiral isoquinoline carboxylic acids

Abstract

The application discloses a method for preparing chiral isoquinoline carboxylic acid (I), which comprises the following steps: reacting a racemate of compound I in an aqueous buffer solution under catalysis of Candida antarctica lipase B to generate a compound shown in formula (I), and performing the reaction at a preset pH value of 7.8-8.2. The method has the characteristics of mild reaction condition, high stereoselectivity, high reaction efficiency, relatively simple process and the like, and has an industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a method for preparing chiral isoquinoline carboxylic acid. Background Technology

[0002] The tetrahydroisoquinoline (THIQ) skeleton is a crucial structural module for many drugs, including trabectedin, noscapine, quinapril, and praziquantel. Novel THIQ therapeutics have attracted increasing attention due to their specific anticancer, anti-inflammatory, and immunomodulatory activities. For example, (R)-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1-methyl-1,2,3,4-tetrahydroisoquinoline are important intermediates in the synthesis of novel Parkinson's disease therapeutics. As a non-natural bicyclic amino acid, 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (1-TIC) can be used to develop bioactive peptides, such as antimicrobial peptides (AMPs) for innate immune protection. Furthermore, 1-TIC can replace structurally similar phenylalanine in the synthesis of farnesyltransferase inhibitors, potentially leading to new approaches to cancer treatment. Currently, there are no reports on the synthesis of (R)-1-TIC. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide an improved method for preparing (R)-1-TIC. This method has the characteristics of mild reaction conditions, strong stereoselectivity, high reaction efficiency, and relatively simple process, and has the prospect of industrial application.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for preparing (R)-1-TIC(Ⅰ),

[0006]

[0007] The method includes: reacting the racemic mixture of 1-TIC ester in an aqueous buffer solution under the catalysis of Candida antarctica lipase B to generate the compound shown in formula (Ⅰ), wherein the reaction is always carried out at a preset pH value of 7.8-8.2.

[0008] According to some preferred aspects of the present invention, the method further includes the step of detecting the pH value of the reaction solution before and during the reaction, and when the detected pH value is lower than the preset pH value, adjusting the pH value of the reaction solution to always be the preset pH value by adding a pH adjuster.

[0009] In some specific embodiments of the present invention, the pH adjuster is ammonia, alkali metal hydroxide, or an aqueous solution thereof.

[0010] According to a specific and preferred aspect of the invention, the pH adjuster is 20wt% to 35wt% ammonia.

[0011] According to another specific aspect of the present invention, the pH adjuster is an aqueous solution of sodium hydroxide or potassium hydroxide.

[0012] According to some preferred aspects of the invention, the reaction is carried out at a temperature of 20-35°C. Preferably, the reaction is carried out at a temperature of 25-32°C. More preferably, the reaction is carried out at a temperature of 28-32°C. Setting the reaction temperature as described above can significantly shorten the reaction time, avoid the decrease in catalytic performance of the enzyme due to soaking and swelling, improve the space-time yield of the reaction, and avoid the energy consumption problems caused by low-temperature reactions in the prior art.

[0013] According to some preferred aspects of the present invention, the aqueous buffer solution is an aqueous ammonium acetate buffer solution.

[0014] According to some preferred aspects of the present invention, the Candida antarcticis lipase B is in the form of an immobilized enzyme preparation.

[0015] According to some preferred aspects of the present invention, the *Candida antarcticis* lipase B is one or more of the following: lipase QLlip-9 from Suzhou Tongli Biopharmaceutical Co., Ltd., Novozyme 435 from Suzhou Tongli Biopharmaceutical Co., Ltd., Immo 8285 from Purolite Co., Ltd., Immo plus from Purolite Co., Ltd., D5544 from Purolite Co., Ltd., and SZ-PLE-100(CAL-B)-IMMO from Shangke Biopharmaceutical (Shanghai) Co., Ltd. The selection of the above-mentioned lipases not only achieves better catalytic effects (manifested in catalytic efficiency and selectivity), but also allows for recycling and reuse. The recovered enzyme still exhibits excellent catalytic effects, with its catalytic capacity remaining essentially unchanged, significantly reducing the amount of enzyme used and greatly saving costs.

[0016] More preferably, the Candida antarcticis lipase B is lipase QLlip-9 selected from Suzhou Tongli Biomedical Co., Ltd.

[0017] According to some preferred aspects of the invention, the mass ratio of the Antarctic Candida lipase B to the racemic mixture of the 1-TIC ester is 1:1.8-2.2.

[0018] According to some preferred aspects of the present invention, the specific implementation of the method is as follows: weigh the racemic mixture of 1-TIC ester and Candida antarcticis lipase B, then add the racemic mixture of 1-TIC ester to an aqueous ammonium acetate buffer to obtain a substrate solution, adjust the pH of the substrate solution to a preset pH value using a pH adjuster, then add the weighed Candida antarcticis lipase B to the substrate solution to obtain a reaction solution, and allow the reaction solution to react at a preset temperature to generate the compound shown in formula (Ⅰ). During the reaction process, the pH value of the reaction solution is always controlled to be the preset pH value by adding a pH adjuster.

[0019] The Antarctic Candida lipase B is lipase QLlip-9 from Suzhou Tongli Biopharmaceutical Co., Ltd., Novozyme 435 from Suzhou Tongli Biopharmaceutical Co., Ltd., Immo 8285 from Purolite Co., Ltd., Immo plus from Purolite Co., Ltd., D5544 from Purolite Co., Ltd., or SZ-PLE-100(CAL-B)-IMMO from Shangke Biopharmaceutical (Shanghai) Co., Ltd., and the preset temperature is 28-32℃.

[0020] Due to the implementation of the above technical solutions, the present invention has the following beneficial effects compared with the prior art:

[0021] This invention reveals that by controlling the reaction under a essentially constant specific pH environment, (R)-1-TIC can be prepared efficiently with good selectivity, high yield, and a significantly shortened time for complete conversion. The reaction conditions are mild, and the production rate is high (reaching 0.24 g·(h·g)). 酶 ) -1 (Above), the product obtained contains ee p ≥99% [ee] p = (Amount of R acid product - Amount of S acid product) ÷ (Amount of R acid product + Amount of S acid product) × 100%], and the process is relatively simple. Detailed Implementation

[0022] The above-mentioned solution will be further described below with reference to specific embodiments; it should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments; the implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0023] Unless otherwise specified, all raw materials described below are either commercially available or prepared using conventional methods in the field; QLlip-9 was purchased from Suzhou Tongli Biomedical Co., Ltd., Novozyme 435 was purchased from Suzhou Tongli Biomedical Co., Ltd., and SZ-PLE-100(CAL-B)-IMMO was purchased from Shangke Biomedical (Shanghai) Co., Ltd.

[0024] Example 1: Preparation and Separation of (R)-1-TIC

[0025] Preparation of (R)-1-TIC:

[0026] Preparation of substrate solution: Prepare a 10 g / L racemic solution of 1-TIC ester using 0.1 M aqueous ammonium acetate buffer (pH = 8.0) and adjust the initial pH of the solution to 8.0 with 30% ammonia.

[0027] 300 mL of the prepared substrate solution was transferred to a round-bottom flask and placed in an external constant-temperature water bath. QLlip-9 was added to the substrate solution until the concentration of QLlip-9 reached 5 g / L. The reaction was carried out at 30°C for 6 hours. During the reaction, the pH of the reaction solution was adjusted to approximately 8.0 by adding 30% ammonia. After the reaction was completed, a certain amount of hydrochloric acid was used to adjust the reaction solution to a strongly acidic state to terminate the reaction. The entire reaction solution was transferred to a volumetric flask, diluted to volume with the mobile phase, and then filtered through a microporous organic filter membrane. Finally, the solution was analyzed using high-performance liquid chromatography (HPLC). The conversion rate [(1 - residual substrate at the end of the reaction ÷ substrate at the beginning of the reaction) × 100%] was greater than 99%. p The percentage of (R acid product amount - S acid product amount) ÷ (R acid product amount + S acid product amount) × 100% is 96.20%.

[0028] Separation of (R)-1-TIC:

[0029] After the reaction in the aforementioned preparation process was completed, the QLlip-9 particles were separated from the reaction solution by vacuum filtration. The reaction solution was concentrated by rotary evaporation at 60°C, and the crystals precipitated during the process were dried at 50°C to finally obtain dry (R)-1-TIC crystals. The separation yield [separation yield = mass of weighed product ÷ theoretical product mass × 100%, theoretical product mass = substrate mass ÷ 241.5 × 177] was 81.06%, and the optical purity was greater than 99%.

[0030] Example 2(R)-1 Preparation and Separation of TIC

[0031] Preparation of (R)-1-TIC:

[0032] Preparation of substrate solution: Prepare a 10 g / L racemic solution of 1-TIC ester using 0.1 M aqueous ammonium acetate buffer (pH = 8.0) and adjust the initial pH of the solution to 8.0 with 30% ammonia.

[0033] 1000 mL of the prepared substrate solution was transferred to a reaction vessel and placed in an external constant-temperature water bath. QLlip-9 was added to the substrate solution until the concentration of QLlip-9 reached 5 g / L. The reaction was carried out at 30°C for 6 hours. During the reaction, the pH of the reaction solution was adjusted to approximately 8.0 by adding 30% ammonia. After the reaction was completed, a certain amount of hydrochloric acid was used to adjust the reaction solution to a strongly acidic state to terminate the reaction. The entire reaction solution was transferred to a volumetric flask, diluted to volume with the mobile phase, and then filtered through a microporous organic filter membrane. Finally, the solution was analyzed using high-performance liquid chromatography (HPLC). The conversion rate [(1 - residual substrate amount at the end of the reaction ÷ substrate amount at the beginning of the reaction) × 100%] was greater than 99%. p The percentage of (R acid product amount - S acid product amount) ÷ (R acid product amount + S acid product amount) × 100% is 96.21%.

[0034] Separation of (R)-1-TIC:

[0035] After the reaction in the aforementioned preparation process was completed, the QLlip-9 particles were separated from the reaction solution by vacuum filtration. The reaction solution was concentrated by rotary evaporation at 60°C, and the crystals precipitated during the process were dried at 50°C to finally obtain dry (R)-1-TIC crystals. The separation yield [separation yield = mass of weighed product ÷ theoretical product mass × 100%, theoretical product mass = substrate mass ÷ 241.5 × 177] was 80.27%, and the optical purity was greater than 99%.

[0036] Example 3(R)-1-TIC Preparation and Separation

[0037] Preparation of (R)-1-TIC:

[0038] Preparation of substrate solution: Prepare a 10 g / L racemic solution of 1-TIC ester using 0.1 M aqueous ammonium acetate buffer (pH = 8.0) and adjust the initial pH of the solution to 8.0 with 30% ammonia.

[0039] 20,000 mL of the prepared substrate solution was transferred to a reaction vessel. The stirred reactor was fitted with an external insulation jacket. QLlip-9 was added to the substrate solution until the concentration reached 5 g / L. The reaction was carried out at 30°C for 6 hours. During the reaction, the pH of the reaction solution was adjusted to approximately 8.0 by adding 30% ammonia. After the reaction was complete, a certain amount of hydrochloric acid was used to adjust the reaction solution to a strongly acidic state to terminate the reaction. The entire reaction solution was transferred to a volumetric flask, diluted to volume with the mobile phase, and then filtered through a microporous organic filter membrane. Finally, the solution was analyzed using high-performance liquid chromatography (HPLC). The conversion rate [(1 - residual substrate at the end of the reaction ÷ substrate at the beginning of the reaction) × 100%] was greater than 99%. p The percentage of (R acid product amount - S acid product amount) ÷ (R acid product amount + S acid product amount) × 100% is 96.16%.

[0040] Separation of (R)-1-TIC:

[0041] After the reaction in the aforementioned preparation process was completed, the QLlip-9 particles were separated from the reaction solution by vacuum filtration. The reaction solution was concentrated by rotary evaporation at 60°C, and the crystals precipitated during the process were dried at 50°C to finally obtain dry (R)-1-TIC crystals. The separation yield [separation yield = mass of weighed product ÷ theoretical product mass × 100%, theoretical product mass = substrate mass ÷ 241.5 × 177] was 80.18%, and the optical purity was greater than 99%.

[0042] Example 4(R)-1-TIC Preparation and Separation

[0043] Preparation of (R)-1-TIC:

[0044] Preparation of substrate solution: Prepare a racemic solution of 1-TIC ester at a concentration of 9.5 g / L using 0.1 M aqueous ammonium acetate buffer (pH = 8.0) and adjust the initial pH of the solution to 7.9 with 30% ammonia.

[0045] 300 mL of the prepared substrate solution was transferred to a reaction vessel, and an external constant-temperature water bath was placed on top. Novozyme 435 was added to the substrate solution until the concentration of Novozyme 435 reached 5 g / L. The reaction was carried out at 32°C for 6 hours. During the reaction, the pH of the reaction solution was adjusted to approximately 7.9 by adding 30% ammonia. After the reaction was completed, a certain amount of hydrochloric acid was used to adjust the reaction solution to a strongly acidic state to terminate the reaction. The entire reaction solution was transferred to a volumetric flask, diluted to volume with the mobile phase, and then filtered through a microporous organic filter membrane. Finally, the solution was analyzed using high-performance liquid chromatography (HPLC). The conversion rate [(1 - residual substrate amount at the end of the reaction ÷ substrate amount at the beginning of the reaction) × 100%] was greater than 99%. pThe percentage of (R acid product amount - S acid product amount) ÷ (R acid product amount + S acid product amount) × 100% is 96.04%.

[0046] Separation of (R)-1-TIC:

[0047] After the reaction in the aforementioned preparation process was completed, the Novozyme 435 particles were separated from the reaction solution by vacuum filtration. The reaction solution was concentrated by rotary evaporation at 60°C, and the crystals precipitated during the process were dried at 50°C to finally obtain dry (R)-1-TIC crystals. The separation yield [separation yield = mass of weighed product ÷ theoretical product mass × 100%, theoretical product mass = substrate mass ÷ 241.5 × 177] was 80.14%, and the optical purity was greater than 99%.

[0048] Example 5(R)-1-TIC Preparation and Separation

[0049] Preparation of (R)-1-TIC:

[0050] Preparation of substrate solution: Prepare a 10.5 g / L racemic solution of 1-TIC ester using 0.1 M aqueous ammonium acetate buffer (pH = 8.0) and adjust the initial pH of the solution to 8.1 with 30% ammonia.

[0051] 20,000 mL of the prepared substrate solution was transferred to a reaction vessel and placed in an external constant-temperature water bath. SZ-PLE-100(CAL-B)-IMMO was added to the substrate solution until the concentration of SZ-PLE-100(CAL-B)-IMMO reached 5 g / L. The reaction was carried out at 28°C for 6 hours. During the reaction, the pH of the reaction solution was adjusted to approximately 8.1 by adding 30% ammonia. After the reaction was completed, a certain amount of hydrochloric acid was used to adjust the reaction solution to a strongly acidic state to terminate the reaction. The entire reaction solution was transferred to a volumetric flask, diluted to volume with the mobile phase, and then filtered through a microporous organic filter membrane. Finally, the solution was analyzed using high-performance liquid chromatography (HPLC). The conversion rate [(1 - residual substrate at the end of the reaction ÷ substrate at the beginning of the reaction) × 100%] was greater than 99%. p The percentage of (R acid product amount - S acid product amount) ÷ (R acid product amount + S acid product amount) × 100% is 95.89%.

[0052] Separation of (R)-1-TIC:

[0053] After the reaction in the aforementioned preparation process was completed, the SZ-PLE-100(CAL-B)-IMMO particles were separated from the reaction solution by vacuum filtration. The reaction solution was concentrated by rotary evaporation at 60°C, and the crystals precipitated during the process were dried at 50°C to finally obtain dry (R)-1-TIC crystals. The separation yield [separation yield = mass of weighed product ÷ theoretical product mass × 100%, theoretical product mass = substrate mass ÷ 241.5 × 177] was 79.85%, and the optical purity was greater than 99%.

[0054] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a chiral isoquinoline carboxylic acid (I), （Ⅰ）， The method comprises: The racemate of isoquinoline carboxylate is reacted in a water-phase buffer solution to generate a compound shown in formula (I) under the catalysis of Candida antarctica lipase B, characterized in that the specific implementation of the method for preparing the chiral isoquinoline carboxylic acid (I) is as follows: the racemate of isoquinoline carboxylate and Candida antarctica lipase B are weighed respectively, then the racemate of isoquinoline carboxylate is added into a water-phase ammonium acetate buffer solution to obtain a substrate solution, a pH adjuster is used to adjust the pH value of the substrate solution to a preset pH value, then the weighed Candida antarctica lipase B is added into the substrate solution to obtain a reaction solution, and the reaction solution is reacted at a preset temperature to generate a compound shown in formula (I) ; wherein the pH value of the reaction solution is controlled to be always the preset pH value by adding a pH adjuster during the reaction, the preset pH value is 7.8-8.2, the pH adjuster is ammonia, an alkali metal hydroxide or an aqueous solution thereof; the Candida antarctica lipase B is lipase QLlip-9 of Suzhou Tongli Biological Medicine Co., Ltd., Novozyme 435 of Suzhou Tongli Biological Medicine Co., Ltd. or SZ-PLE-100 (CAL-B) -IMMO of Shangke Biological Medicine (Shanghai) Co., Ltd., and the preset temperature is 28-32 °C.

2. The method of claim 1, wherein, The pH adjuster is 20wt%-35wt% ammonia.

3. The method of claim 1, wherein, The mass ratio of the Candida antarctica lipase B to the racemate of isoquinoline carboxylate is 1:1.8-2.2.

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