Fluorine-containing aromatic alcohol compound as well as preparation method and application thereof

Abstract

The invention provides a fluorine-containing aromatic alcohol compound as well as a preparation method and application thereof, and belongs to the field of organic chemistry and biochemical engineering. Specifically, the method comprises the following steps: catalyzing the reduction reaction of an aromatic ketone substrate 1-(5-fluoro-2-iodophenyl) ethanone through bifunctional ketoreductase, and assisting a coenzyme NADPH or NADH and isopropanol with the effects of a cosolvent and a hydrogen donor to efficiently generate the target product (S)-1-(2-iodo-5-fluorophenyl) ethanol under mild conditions. According to the method, extra dehydrogenase and hydrogen source body requirements are not needed, cyclic regeneration of the coenzyme is achieved through oxidation of isopropanol, the reaction system is simple in design, the substrate concentration reaches up to 250 g / L, the conversion rate reaches 99.7%, and the chiral purity reaches 99.9%. The method is safe and environment-friendly in process, simple and convenient to operate and suitable for industrial production, and the prepared target compound is a key intermediate of loratinib and can be widely applied to synthesis of anti-cancer drugs.

Description

Technical Field

[0001] The invention belongs to the field of organic chemistry and biochemical engineering, and specifically relates to a process for preparing fluorine-containing aromatic alcohol compounds by using an enzyme catalysis method, in particular to a method for generating high-purity and high-chirality-selective (S)-1-(2-iodo-5-fluorophenyl)ethanol by catalyzing the reduction reaction of an aromatic ketone substrate 1-(5-fluoro-2-iodophenyl)ethanone with a bifunctional ketoreductase. Background Art

[0002] Fluorinated aromatic alcohol compounds are important intermediates in the field of pharmaceutical synthesis, and their derivatives are widely used in the preparation of anticancer drugs, antiviral drugs and other targeted therapeutic drugs. Among them, (S)-1-(2-iodo-5-fluorophenyl)ethanol is a key intermediate in the synthesis of lorlatinib. Lorlatinib (Formula I) is a new, reversible, and potent ALK and ROS1 inhibitor, mainly used to treat ALK-positive and ROS1-positive advanced non-small cell lung cancer (NSCLC). The drug has a significant inhibitory effect on a variety of ALK resistance mutations, and its efficacy in the first treatment of NSCLC patients is as high as 90%, and its intracranial efficacy is 75%. Therefore, the preparation technology of lorlatinib and its intermediates is of great significance for clinical application.

[0003]

[0004] At present, (S)-1-(2-iodo-5-fluorophenyl)ethanol (CAS No.: 1454847-96-1), with a structural formula as shown in Formula II, has preparation methods mainly including chemical synthesis and bio-enzyme catalysis. The chemical synthesis method usually uses DIPCl (diisopropylchlorosilane p-chlorophenyl) as a catalyst and 1-(5-fluoro-2-iodophenyl)ethanone as a raw material, but such methods need to be carried out under low temperature conditions, the operation is cumbersome, the reaction efficiency is low, and the product yield and purity are not ideal. In addition, some methods involve highly dangerous chemicals, which increases the safety risks of industrial production.

[0005]

[0006] The bio-enzyme catalysis method has gradually attracted attention due to its advantages such as green, environmental protection and high selectivity, but the existing technology still has the following shortcomings:

[0007] Low enzyme catalytic efficiency: Some biological enzymes have low catalytic activity on substrates, resulting in long reaction times and insufficient conversion rates;

[0008] Complex coenzyme cycle: The regeneration of coenzymes (such as NADPH or NADH) usually requires the addition of dehydrogenases and hydrogen sources (such as glucose or formic acid), which increases the complexity and cost of the reaction system;

[0009] Low substrate concentration: In existing biocatalytic processes, the substrate concentration is usually low (for example, the substrate concentration disclosed in CN109468346A is only 211.2 g / L), which is difficult to meet the needs of industrial scale.

[0010] Therefore, developing a green process with mild reaction conditions, high enzyme catalytic efficiency, simple coenzyme cycle and applicability to high substrate concentration is a key technical issue in the current technical field. Summary of the invention

[0011] In order to solve the problems of low enzyme catalytic efficiency, complex coenzyme circulation and low substrate concentration in the prior art, the present invention provides a green preparation method for catalytic reduction of aromatic ketone substrates by bifunctional ketoreductase. The method uses 1-(5-fluoro-2-iodophenyl) ethyl ketone as a substrate, a coenzyme as NADPH or NADH, and isopropanol having both the role of a cosolvent and a hydrogen donor as an important component of the reaction system, and realizes efficient reduction of the substrate by bifunctional ketoreductase, while realizing the recycling of coenzymes by isopropanol oxidation, thereby preparing high-purity, highly selective (S)-1-(2-iodo-5-fluorophenyl) ethanol. The reaction conditions of the present invention are mild, and there is no need to add dehydrogenase and complex coenzyme circulation devices, and it has the advantages of simplified process, low cost, safety and environmental protection.

[0012] According to a first aspect of the present invention, there is provided a method for preparing a fluorine-containing aromatic alcohol compound, comprising the following steps:

[0013] In a reaction system comprising a bifunctional ketoreductase, a coenzyme and a compound having both the functions of a cosolvent and a hydrogen donor,

[0014] The reduction of aromatic ketone substrates is catalyzed by a bifunctional ketoreductase.

[0015] The bifunctional ketoreductase simultaneously catalyzes the oxidation of a compound that acts as both a cosolvent and a hydrogen donor in the reaction system to achieve the cyclic regeneration of the coenzyme.

[0016] Under controlled temperature and pH conditions, target fluorinated aromatic alcohol compounds are generated.

[0017] In some technical solutions, the bifunctional ketoreductase is selected from any one or a combination of at least two of the ketoreductases SY-K01 to SY-K10.

[0018] In some technical solutions, the compound that acts as both a co-solvent and a hydrogen donor is isopropanol.

[0019] In some technical solutions, the coenzyme is any one of NADP salt, NADPH, NADH or NAD salt, or a combination thereof.

[0020] In some technical solutions, the reaction system further includes a buffer, which is any one of potassium phosphate buffer, tris(hydroxymethylaminomethane) buffer or sodium phosphate buffer, and has a pH range of 5.5 to 8.0.

[0021] In some technical solutions, the substrate concentration in the reaction system is 10 g / L to 250 g / L, and the volume fraction of the co-solvent is 10% to 20%.

[0022] In some technical solutions, the reduction reaction temperature is 25°C to 35°C, and the duration is 10h to 24h.

[0023] In some technical solutions, the aromatic ketone substrate is 1-(5-fluoro-2-iodophenyl)ethanone, and the specific preparation steps are as follows:

[0024] Adding a buffer, 1-(5-fluoro-2-iodophenyl)ethanone, a coenzyme and isopropanol into a reaction container, wherein the isopropanol serves as a cosolvent and a hydrogen donor;

[0025] Adjust the pH of the reaction system to 5.5 to 8.0, and set the reaction temperature to 25° C. to 35° C.;

[0026] Adding bifunctional ketoreductase and stirring to form a uniform reaction system;

[0027] Maintain the reaction for 10 to 24 hours and monitor the conversion rate of the substrate until the target is reached;

[0028] After the reaction is completed, the target product (S)-1-(2-iodo-5-fluorophenyl)ethanol is obtained by separation and purification methods.

[0029] According to the second aspect of the present invention, there is further provided a fluorine-containing aromatic alcohol compound, which is prepared by the above-mentioned preparation method.

[0030] According to the third aspect of the present invention, there is further provided an application of a fluorinated aromatic alcohol compound, which is the application of (S)-1-(2-iodo-5-fluorophenyl)ethanol in the preparation of lorlatinib.

[0031] The present invention adopts the above technical solution to have at least the following beneficial effects:

[0032] 1. The present invention realizes the cyclic regeneration of coenzyme by catalyzing the oxidation of isopropanol through bifunctional ketoreductase, without the need to add dehydrogenase and independent hydrogen source (such as glucose or formic acid). This design significantly simplifies the reaction system, reduces the cost and complexity of coenzyme regeneration, and reduces the dependence of the reaction system on external conditions.

[0033] 2. The bifunctional ketoreductase of the present invention has dual catalytic activities of substrate reduction and coenzyme regeneration, which can effectively reduce the reduction activation energy of the substrate and shorten the reaction time. After optimization, the present invention can achieve a substrate conversion rate of 99.7% and a chiral purity of 99.9% under the conditions of a substrate concentration of 250 g / L and a reaction temperature of 30°C, which is significantly better than the prior art.

[0034] 3. The enzyme catalysis system of the present invention can adapt to a high substrate concentration of 250 g / L. Especially under industrial production conditions, the increase in substrate concentration can significantly reduce solvent usage and separation costs, thereby improving production efficiency and economic benefits.

[0035] 4. The process of the present invention does not require high-pressure hydrogenation operation, does not require the use of hazardous chemical reagents, and only needs to be operated within the range of 25°C to 35°C. Isopropanol is used as a co-solvent and hydrogen donor, which not only reduces the use of complex solvents in traditional methods, but also reduces potential environmental and safety risks.

[0036] 5. The (S)-1-(2-iodo-5-fluorophenyl)ethanol prepared by the present invention has a purity of ≥99% and a chiral purity of ≥99%, and can be directly used in the subsequent synthesis of lorlatinib, meeting the strict requirements of the pharmaceutical industry for the quality of intermediates.

[0037] 6. The reaction system of the present invention is simple in design, does not rely on special equipment (such as a high-pressure reactor or a complex coenzyme regeneration device), has strong industrial adaptability and operability, and provides reliable technical support for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a conventional HPLC chromatogram of the (S)-1-(2-iodo-5-fluorophenyl)ethanol product, used to show the purity of the target product;

[0039] Figure 2 is a conventional HPLC chromatogram of 1-(5-fluoro-2-iodophenyl)ethanone substrate, used to show the purity of the substrate;

[0040] Figure 3 This is a conventional HPLC analysis spectrum of the reaction product in Example 2, used to show the conversion rate of the target product in the reaction system;

[0041] Figure 4 This is a chiral HPLC analysis spectrum of the reaction product in Example 2, used to show the chiral purity of the product;

[0042] Figure 5 This is the chiral HPLC analysis spectrum of the product standard racemate, which is used to compare the chirality difference between the reaction product and the racemate;

[0043] Figure 6This is the chiral HPLC analysis spectrum of the product standard R configuration, which is used to compare the chiral characteristics of the reaction products;

[0044] Figure 7 This is the chiral HPLC analysis spectrum of the S configuration of the product standard, which is used to confirm that the reaction product is of S configuration;

[0045] Figure 8 The hydrogen nuclear magnetic resonance (HNMR) spectrum of the S-configuration product is used to further verify the structure of the target product;

[0046] Fig. 9 This is a chiral HPLC analysis spectrum of the reaction product in Example 3, used to show the chiral purity and consistency in the 100-gram scale-up experiment. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with specific embodiments, but these embodiments are only exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that several improvements and modifications may be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

[0048] Example 1 (Screening example of ketoreductase)

[0049] The reaction system components are as follows: 0.1M pH 7.0 potassium phosphate buffer, substrate 1-(5-fluoro-2-iodophenyl)ethanone 10g / L, cosolvent isopropanol 5% (V / V), coenzyme NADP 0.5g / L, ketoreductase (SY-K01-110 enzyme powder) 1g / L each. Reaction at 30°C for 18h. The ketoreductases used are shown in Table 1:

[0050] Table 1

[0051] Screening Number Ketoreductase Reaction conversion rate Chiral purity (S type) 1-1 SY-K01 100.0％ 96.7％ 1-2 SY-K02 11.4％ 82.8％ 1-3 SY-K03 46.7％ 95.6％ 1-4 SY-K04 20.5％ 95.3％ 1-5 SY-K05 97.3％ 80.7％ 1-6 SY-K06 99.8％ 99.9％ 1-7 SY-K07 99.9％ 96.6％ 1-8 SY-K08 99.9％ 94.2％ 1-9 SY-K09 100.0％ 95.9％ 1-10 SY-K10 18.0％ 97.3％

[0052] The results showed that the enzyme with the best conversion effect was the ketoreductase No. 6 enzyme SY-K06, and the HPLC conversion rate was more than 99.8%.

[0053] Example 2 (Chiral Analysis of Ketoreductase Reaction Product)

[0054] The ketoreductase SY-K06 enzyme powder with good reaction results was used to prepare (S)-1-(2-iodo-5-fluorophenyl)ethanol, specifically:

[0055] Add 0.05M pH-7.0 potassium phosphate buffer, 2.5g substrate (250g / L), 2.0ml isopropanol, 2mg NADP, 10mg ketoreductase to 10ml reaction system, and make up the rest with water. React at 30℃ for 24h, and monitor the reaction with HPLC.

[0056] The peak position of the product in HPLC analysis is as follows Figure 1 As shown, the peak position of the substrate in conventional liquid phase analysis is as follows Figure 2 As shown. The reaction transformation is as follows Figure 3 , conversion rate 99.7%, chiral purity 99.9%, such as Figure 4 After the product was purified, 2.27 g of the product was obtained, with a yield of 90.2% and a content of 98.0%. The HNMR analysis chart is as follows: Figure 8 .in addition, Figure 5-7 Spectral analysis of purchased standards was performed for comparison of results.

[0057] Example 3 (100-gram scale-up of ketoreductase reaction)

[0058] Add 0.05M pH-7.0 potassium phosphate buffer, 0.5kg substrate (250g / L), 400ml isopropanol, 0.8g NADP, 4.0g ketoreductase to 2L reaction system, and make up the rest with water. React at 30℃ for 24h. Chiral purity 99.8%, such as Fig. 9 After the product was purified, 459.0 g of the product was obtained with a yield of 91.1%.

[0059] Comparative Example 1

[0060] The only difference between this example and Example 2 is that dehydrogenase GDH is added to the reaction, and the matching hydrogen source is glucose. Because gluconic acid is produced during the reaction, 10% sodium carbonate is also needed to control the pH during the reaction to maintain it at around pH 7.0.

[0061] Add 0.05M pH-7.0 potassium phosphate buffer, 2.5g substrate (250g / L), 2.0ml isopropanol, 2mg NADP, 10mg ketoreductase, 1mg GDH, 2.56g glucose to 10ml reaction system, and make up the rest with water. React at 30℃ for 18h, and monitor the reaction with HPLC. The reaction is complete, and the chiral purity of S-type product is 99.8%.

[0062] Results: The chirality of the product remained unchanged, the conversion rate remained unchanged, and the chiral purity of the S-type product was also 99.8%. However, the reaction increased the reaction components and required pH monitoring, making the operation relatively complicated.

[0063] Comparative Example 2

[0064] The only difference between this example and Example 2 is that the concentration of the substrate is increased to a certain extent.

[0065] Add 0.05M pH-7.0 potassium phosphate buffer, 2.55g substrate (255g / L), 2.0ml isopropanol, 2mg NADP, 10mg ketoreductase to 10ml reaction system, and make up the rest with water. React at 30℃ for 24h, and monitor the reaction with HPLC.

[0066] The reaction was not completed, and the conversion rate was about 95%; when the reaction time was extended to 48h, the conversion rate was 97%, and the reaction did not change. The chiral purity of the S-type product was 99.8%.

[0067] Results: With increasing substrate amount, the substrate conversion rate was 95%, and after prolonging the reaction, the conversion rate changed slightly to 97%; the chiral purity of the S-type product remained unchanged.

[0068] Comparative Example 3

[0069] The only difference between this example and Example 2 is that the amount of hydrogen source is increased.

[0070] Add 0.05M pH-7.0 potassium phosphate buffer, 2.5g substrate (250g / L), 2.5ml isopropanol, 2mg NADP, 10mg ketoreductase to 10ml reaction system, and make up the rest with water. React at 30℃ for 24h, and monitor the reaction with HPLC.

[0071] The reaction was not completed, with a conversion rate of about 96%; the reaction time was extended to 48h, with a conversion rate of 97% and no further changes. The chiral purity of the S-type product was 99.8%.

[0072] Results: With the increase of hydrogen source, the conversion rate of substrate was 96%. After the reaction was prolonged, the conversion rate changed slightly to 97%. The chiral purity of S-type product remained unchanged.

[0073] Comparative Example 4

[0074] The only difference between this example and Example 2 is that the amount of hydrogen source is reduced.

[0075] Add 0.05M pH-7.0 potassium phosphate buffer, 2.5g substrate (250g / L), 10ml isopropanol (10%), 2mg NADP, 10mg ketoreductase to 10ml reaction system, and make up the rest with water. React at 30℃ for 24h, and monitor the reaction with HPLC.

[0076] The reaction was not completed, and the conversion rate was about 97%; when the reaction time was extended to 48h, the conversion rate was 98%, and the reaction did not change. The chiral purity of the S-type product was 99.8%.

[0077] Results: By reducing the amount of hydrogen source, the substrate conversion rate was 97%, and after extending the reaction time, the conversion rate changed to 98%, and the chiral purity of the S-type product remained unchanged.

[0078] Comparative Example 5

[0079] The only difference between this example and Example 2 is that the amount of ketoreductase is reduced.

[0080] Add 0.05M pH-7.0 potassium phosphate buffer, 2.5g substrate (250g / L), 2.0ml isopropanol, 2mg NADP, 8mg ketoreductase to 10ml reaction system, and make up the rest with water. React at 30℃ for 24h, and monitor the reaction with HPLC.

[0081] The reaction was not completed, and the conversion rate was about 94%; the reaction time was extended to 48h, the conversion rate was 96%, and the reaction time was further extended to 72h, the conversion rate was 98%. The chiral purity of the S-type product was 99.8%.

[0082] Results: After reducing the enzyme dosage, the substrate conversion rate was 94%, and after extending the reaction, the conversion rate changed slightly to 98%; the chiral purity of the S-type product remained unchanged.

[0083] Comparative Example 6

[0084] The only difference between this example and Example 2 is that the amount of NADP is reduced.

[0085] Add 0.05M pH-7.0 potassium phosphate buffer, 2.5g substrate (250g / L), 2.0ml isopropanol, 1.5mg NADP, 10mg ketoreductase to 10ml reaction system, and make up the rest with water. React at 30℃ for 24h, and monitor the reaction with HPLC.

[0086] The reaction was not completed, and the conversion rate was about 96%; the reaction time was extended to 48h, the conversion rate was 98%, and the reaction time was further extended to 72h, the conversion rate was 99.5%. The chiral purity of the S-type product was 99.8%.

[0087] Results: After reducing the amount of NADP, the substrate conversion rate was 96%, and after extending the reaction for 72 hours, the conversion rate was 99.5%, which was basically completed, but the time was long and the reaction energy consumption increased; the chiral purity of the S-type product remained unchanged.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a fluorine-containing aromatic alcohol compound, characterized in that: The following steps are involved: In a reaction system comprising a bifunctional ketoreductase, a coenzyme and a compound having both the functions of a cosolvent and a hydrogen donor, The reduction of aromatic ketone substrates is catalyzed by a bifunctional ketoreductase. The bifunctional ketoreductase simultaneously catalyzes the oxidation of a compound that acts as both a cosolvent and a hydrogen donor in the reaction system to achieve the cyclic regeneration of the coenzyme. Under controlled temperature and pH conditions, target fluorinated aromatic alcohol compounds are generated.

2. The preparation method according to claim 1, characterized in that: The bifunctional ketoreductase is selected from any one or a combination of at least two of the ketoreductases SY-K01 to SY-K10.

3. The preparation method according to claim 1, characterized in that: The compound having both the functions of a co-solvent and a hydrogen donor is isopropanol.

4. The preparation method according to claim 1, characterized in that: The coenzyme is any one of NADP salt, NADPH, NADH or NAD salt or a combination thereof.

5. The preparation method according to claim 1, characterized in that: The reaction system also includes a buffer, which is any one of potassium phosphate buffer, tris(hydroxymethylaminomethane) buffer or sodium phosphate buffer, and has a pH range of 5.5 to 8.

0.

6. The preparation method according to claim 1, characterized in that: The substrate concentration in the reaction system is 10 g / L to 250 g / L, and the volume fraction of the co-solvent is 10% to 20%.

7. The preparation method according to claim 1, characterized in that: The reduction reaction is carried out at a temperature of 25° C. to 35° C. and for a time of 10 h to 24 h.

8. The preparation method according to claim 1, characterized in that: The aromatic ketone substrate is 1-(5-fluoro-2-iodophenyl)ethanone, and the specific preparation steps are as follows: Adding a buffer, 1-(5-fluoro-2-iodophenyl)ethanone, a coenzyme and isopropanol into a reaction container, wherein the isopropanol serves as a cosolvent and a hydrogen donor; Adjust the pH of the reaction system to 5.5 to 8.0, and set the reaction temperature to 25° C. to 35° C.; Adding bifunctional ketoreductase and stirring to form a uniform reaction system; Maintain the reaction for 10 to 24 hours and monitor the conversion rate of the substrate until the target is reached; After the reaction is completed, the target product (S)-1-(2-iodo-5-fluorophenyl)ethanol is obtained by separation and purification methods.

9. A fluorine-containing aromatic alcohol compound, characterized in that: The preparation method is adopted according to any one of claims 1 to 8.

10. An application of a fluorine-containing aromatic alcohol compound, characterized in that: The invention discloses an application of (S)-1-(2-iodo-5-fluorophenyl)ethanol in the preparation of lorlatinib.

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