Synthesis process of travoprost intermediate

Abstract

The invention relates to the technical field of medicines, in particular to a synthesis process of a travoprost intermediate. According to the technical scheme, the method comprises the following steps: S1, enzymatic esterification: adding 2-(4-trifluoromethylphenoxy)-propionic acid into isopropenyl acetate, then adding lipase A, adjusting the temperature to 37-45 DEG C, stirring for 12-18 hours, and then filtering, cleaning and concentrating through a diatomite layer to obtain an intermediate mixture which comprises a (R) compound F and an (S) compound G; s2, performing configuration overturning; S3, performing enzyme catalysis to obtain a target compound. According to the method, the problems of tedious process steps and low resolution efficiency are effectively improved, the original five-step reaction is simplified into three steps, the reaction is mild and easy to operate, the safety is high, the enzyme can be reused, the yield is increased, the resolution purity is ensured, and the economic benefit is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a synthesis process of travoprost intermediate. BACKGROUND

[0002] Bimatoprost and travoprost are a class of highly effective eye pressure-lowering drugs developed by Allergan Company in the United States. They are unique in pharmacology and have a strong effect on reducing eye pressure. Their pharmacological activity is highly dependent on the absolute configuration (R configuration) of intermediate A. According to the requirements of the pharmacopoeia, the enantiomeric excess value (e.e. value) of the chiral intermediate in the raw material drug needs to reach more than 99.8% to ensure the safety and effectiveness of the drug. If the chiral control is insufficient, the S configuration impurity may cause unpredictable side effects (such as fluctuations in intraocular pressure or inflammatory reactions), and even lead to a decrease in clinical efficacy.

[0003] Currently, the synthesis and resolution of travoprost intermediate A mainly rely on the following two methods. The Organic Letters (2022), 24(48), 8836-8889 document reports that compound 1 is used as a raw material, amide is formed with dimethylamine hydrochloride under the catalysis of 4-(dimethylamino) pyridine, then combined with ethynyl triisopropylsilane under the action of n-butyllithium to form compound 3, then selectively reduced with a specific catalyst to obtain compound 4, and finally the triisopropylsilyl group is removed to obtain the target product A. The method disclosed in patent US20090259058 first obtains racemic intermediate B, and then performs selective acetylation on the R configuration and photo extension reaction on the unreacted S configuration to flip the configuration to obtain the R configuration formylated product. Then, guanidine is used to deacylate the acetylated and formylated mixture to obtain compound A. However, the former has four reaction steps and needs to involve rare and valuable metals, and the obtained ee value is only 99%, which does not meet the requirement of the pharmacopoeia of ee value of more than 99.8%. The ee value of the obtained compound A in the latter is about 90%. In order to meet the requirement of chiral control of the raw material drug, the enzyme resolution reaction and guanidine hydrolysis reaction need to be performed again, resulting in a long process, low efficiency and high cost.

[0004] Scheme 1 Scheme 2 SUMMARY

[0005] In order to effectively improve the problems of long process steps and low resolution efficiency in the above process, the present application provides a synthesis process of travoprost intermediate, which has the advantages of high yield, shortening of reaction steps, high selectivity, improvement of production efficiency, reduction of production cost, and suitability for industrial production.

[0006] The synthesis process of the fluvastatin intermediate provided by the application adopts the following technical scheme: The synthesis process of the fluvastatin intermediate comprises the following steps: S1: Enzymatic esterification: 2-(4-trifluoromethyl phenoxy)-propionic acid is added into isopropenyl acetate, then lipase A is added, the temperature is adjusted to 37-45 DEG C, stirring is carried out for 12-18 h, then filtration is carried out through diatomite layer, washing and concentration are carried out to obtain an intermediate mixture, wherein the intermediate mixture contains (R) compound F and (S) compound G; S2: Configuration inversion: under the cold bath of 0-10 DEG C, the intermediate mixture obtained in the step S1 is added into a polar solvent, then triphenylphosphine and formic acid are added, then di-tert-butyl azodicarboxylate is slowly added dropwise, after the dropwise addition is completed, the temperature is increased to 10-70 DEG C and stirring is carried out for 28-26 h, the raw material is detected to be reacted completely through the middle control TLC, concentration is carried out under reduced pressure, then washing is carried out through filtration to obtain a mixture containing (R) compound F and (R) compound H; S3: Enzymatic catalysis: under the protection of nitrogen, the mixture containing (R) compound F and (R) compound H obtained in the step S2 is taken, lipase B and a solvent are added, stirring is carried out at 15-20 DEG C for 6-30 h, the raw material is detected to be reacted completely through the middle control TLC, then filtration is carried out, and the mother liquor is concentrated to obtain compound A. The specific synthesis route is shown in the following: Through the above technical scheme, firstly, the intermediate raw material 2-(4-trifluoromethyl phenoxy)-propionic acid is added into isopropenyl acetate as an acyl donor to react with the active center of lipase to generate an acyl-enzyme intermediate, so that the specific binding of (R)-hydroxyl is induced and the exclusion mechanism of (S)-hydroxyl is induced, so that the (S)-configuration cannot enter the active center of the enzyme, the hydroxyl remains as -OH, the yield of the intermediate mixture is improved, through further separation and purification, the synergistic effect of triphenylphosphine and di-tert-butyl azodicarboxylate (DBAD), formic acid as a hydrogen donor, the hydroxyl of S-ester is oxidized to formate, then configuration inversion generates R-ester, and racemization is inhibited, side reactions are reduced, then immobilized lipase B is used as a biological catalyst, the active site thereof has a specific spatial structure, only R-configuration isomers can be recognized and combined, the hydrolysis of the isomers is catalyzed through a nucleophilic substitution reaction, and S-configuration isomers cannot match the active site of the enzyme due to steric hindrance, so no reaction occurs, and high selective catalysis is realized. Compared with the traditional enzyme process, the purification steps are reduced and the e.e. value is improved, the selectivity is improved through the double chiral recognition mechanism, the (R)-substrate can be enriched, after the reaction is completed, high-purity and high-e.e. value (more than 99.8%) compound A is obtained through post-treatment and purification, the generation of difficult-to-remove impurities is avoided, the reaction steps are shortened, the production operation is simplified, the production efficiency is improved, the production cost is reduced, and the process is suitable for industrial production.

[0007] In one specific embodiment, in S1, the lipase A is a modified silica immobilized lipase, and the preparation method thereof comprises: (1) dispersing silica in toluene, adding trimethylchlorosilane, refluxing at 75-80°C under nitrogen protection for 8-12h, adding water after centrifugation, adding 0.1M HCl dropwise to adjust pH to 5-6, and uniformly dispersing by ultrasonic to obtain modified silica; (2) dissolving hexadecyl-D-phenylalanine in water at 60-70°C, adjusting pH to 12-13, stirring for 1-2h to obtain a mixed solution; adding the modified silica obtained in step (1), and adding TEOS and APTES ethanol mixture dropwise under stirring at 500-800rpm; stirring at 30-35°C for 20-24h, standing, filtering, washing and drying, and low-temperature calcination to obtain chiral R-type silica carrier; (3) dissolving lipase PS in PBS buffer, adding the chiral R-type silica carrier, oscillating at 4-5°C for 10-12h, centrifuging, and freeze-drying to obtain the modified silica immobilized lipase.

[0008] In S1, the mass ratio of the hexadecyl-D-phenylalanine and the lipase PS is 1:(0.06-0.1).

[0009] By adopting the above technical solution, a mesoporous framework is provided by modifying mesoporous silica, and N-hexadecyl-R-phenylalanine is added during the synthesis stage of constructing a silica layer on its surface. Through self-assembly, the chiral arrangement of the pores is induced, generating chiral pores. This allows enzyme molecules to connect with the chiral R-type silica layer at multiple points, which can enhance the stereoselective adsorption of the substrate. The reaction between the R-conformation and the active site of the lipase in the dual-inducible reactant of the synthesized specific enzyme and R-conformation further improves the specificity and selectivity of the enzyme reaction. First, mesoporous silica is amino-modified to provide active sites for hexadecyl-D-phenylalanine anchoring and silicon source condensation. As a support carrier, hexadecyl-D-phenylalanine with long hydrophobic chains self-assembles into helical micelles under alkaline conditions. Through electrostatic adsorption, the silicon source is directionally induced to extend outward along the R-configuration micelles, constructing a chiral R-type silica layer. Then, through the electrostatic interaction between enzyme molecules and silica, and the interaction between enzyme molecules and the hexadecyl hydrophobic chain, a conformational change is triggered and the binding force is enhanced, thus enhancing the enzyme immobilization effect. In this way, the modified silica immobilized lipase has a dual effect of enhancing R-configuration-induced adsorption, which in turn enables the R-configuration to specifically bind to the acyl-enzyme intermediate and dually excludes the (S)-hydroxyl configuration, thereby enhancing stereoselectivity and minimizing the loss of side reactions caused by configuration inversion in step S2, thereby enhancing yield and selectivity. Furthermore, through the interaction between the modified silica support in (1) and the amino group of the outer dynamic silica layer, the pore blockage caused by a single amino active group is avoided, the overall stability is improved, and the fixation of the enzyme is enhanced. While reducing enzyme leakage, it can prevent the enzyme molecules from stretching and deforming, thus reducing enzyme activity. It can also play the role of the enzyme and the chiral R-type silica layer, preventing the self-degradation of enzyme molecules due to intermolecular interactions, thereby reducing activity. The applicant found that within the mass range of this application, hexadecyl-D-phenylalanine and lipase PS interact. The alkyl chain of hexadecyl-D-phenylalanine can further enhance the binding with the enzyme active site, reduce non-specific adsorption, and the applicant noted that lipase PS has a higher affinity for D-type substrates, which can promote the loading and reactivity of lipase, thereby controlling the appropriate reaction rate, improving yield and purity. If the content of hexadecyl-D-phenylalanine is too low, it will lead to a decrease in generation efficiency, the generation of by-products, and a decrease in chiral purity.

[0010] In one specific implementation, in S1, the mass ratio of 2-(4-trifluoromethylphenoxy)-propionic acid, isopropyl acetate, and lipase A is (5-7):1:(0.25-0.45).

[0011] By adopting the technical scheme, the conversion rate and efficiency of the intermediate mixture are improved by using 2-(4-trifluoromethylphenoxy)-propionic acid and isopropenyl acetate in a certain mass ratio and lipase A, which is conducive to the subsequent step and optimizes the entire synthesis process and improves the economic benefit. Too much isopropenyl acetate can occupy the active site of lipase A, hinder the effective combination of 2-(4-trifluoromethylphenoxy)-propionic acid and lipase A, and generate impurities that are difficult to separate, thereby reducing the yield. Moreover, it interferes with the recognition of the substrate configuration by lipase A, resulting in a decrease in stereoselectivity. If there is too little lipase A, the selective catalysis of the specific configuration substrate will be reduced, resulting in poor stereoselectivity.

[0012] In S3, the lipase B is one of immobilized BCA lipase and immobilized BL-IM lipase.

[0013] In S3, the mass ratio of the mixture containing (R) compound F and (R) compound H to lipase B is (50-100):1.

[0014] Preferably, the mass ratio of the mixture containing (R) compound F and (R) compound H to lipase B is (50-60):1.

[0015] By adopting the technical scheme, the stereoselectivity stability can be improved by selecting lipase B according to the application, so that the reaction process is stable, no obvious impurities are generated and increased, the conversion rate is ensured, the high-purity compound A can be finally obtained, the process can be simplified, and the economic benefit can be improved. In addition, too much lipase B can increase the collision probability between molecules, which can increase the possibility of side reactions. On the one hand, the occurrence of side reactions can consume part of the reactants, resulting in a decrease in the raw materials for generating the target product compound A, thereby reducing the yield.

[0016] In S3, the solvent includes one or both of an alcohol solvent and a non-polar solvent.

[0017] The alcohol solvent includes one or more of methanol, ethanol, isopropyl alcohol, n-pentanol, n-propanol and tert-butanol.

[0018] The non-polar solvent includes one or more of n-pentane, n-hexane, n-heptane and n-octane.

[0019] Preferably, the solvent is ethanol and n-heptane in a mass ratio of 2:1.

[0020] By employing the above technical solution, ethanol and n-heptane are selected as solvents. Ethanol, containing polar hydroxyl groups, promotes the dissolution and diffusion of reactants, facilitating sufficient contact between the enzyme and substrate and accelerating the reaction rate. n-Heptane, being a nonpolar solvent, provides a suitable hydrophobic environment for the reaction. This hydrophobic environment helps maintain the active conformation of the lipase and improves its catalytic efficiency. The combined use of ethanol and n-heptane allows for the selective dissolution of specific substances based on the polarity differences between reactants and products, thus facilitating the separation of small amounts of unreacted S-chiral isomers and the generated compound A. Furthermore, the combined use of these two solvents makes the reaction system more stable, avoiding side reactions that may arise from using a single solvent, ensuring the high efficiency and stability of the entire synthesis process, and ultimately leading to the acquisition of compound A with high purity and a high ee value.

[0021] S3 also includes a purification step using silica gel column chromatography.

[0022] In summary, this application includes at least one of the following beneficial technical effects: 1. This application enhances selectivity through a dual chiral recognition mechanism, enabling pre-enrichment of (R)-substrate and further increasing the ee value of compound A. After the reaction, post-processing purification yields compound A with high purity and a high ee value (above 99.8%). The entire process avoids the generation of difficult-to-remove impurities, shortens reaction steps, simplifies production operations, improves production efficiency, and reduces production costs. It features mild reaction conditions, high safety and good stability, and significantly improved separation yield and chiral purity, making it suitable for industrial production.

[0023] 2. By modifying mesoporous silica to provide a mesoporous framework, and adding N-hexadecyl-R-phenylalanine during the synthesis stage of constructing the silica layer, chiral channels are generated through self-assembly-induced chiral arrangement of pores. This allows enzyme molecules to connect to the chiral R-type silica layer at multiple points, which can enhance the stereoselective adsorption of substrates. The synthesis of specific enzymes and the reaction between the R-conformation and the active site of lipase in the dual-inducing reactants of R-configuration further improve the specificity and selectivity of enzyme reactions. Attached Figure Description

[0024] Figure 1 This is the HNMR spectrum of the travoprost intermediate prepared in Example 1 of this application.

[0025] Figure 2 This is a CNMR image of the travoprost intermediate prepared in Example 1 of this application. Detailed Implementation

[0026] Lipase PS (purity≥500 U / g) of the examples: L299010, Aldrin; immobilized BCA lipase and immobilized BL-IM lipase were purchased from Shanghai Banglin Biological Co., Ltd.; mesoporous silica, SBA-15, pore size 6-10 nm, other raw materials were commercially available.

[0027] Preparation Example 1 The lipase A is a modified silica immobilized lipase, and its preparation method is as follows: (1) 1.0 g of silica is dispersed in 50 ml of toluene, 0.15 g of trimethylchlorosilane is added, and refluxing is carried out at 80°C under nitrogen protection for 12 h. After centrifugation, it is dispersed in 50 mL of water, 0.1 M HCl is added dropwise to adjust the pH to 5, ultrasonic dispersion is carried out for 30 min, centrifugal washing is carried out, and vacuum drying is carried out at 60°C to obtain modified silica; (2) 1 g of hexadecyl-D-phenylalanine is dissolved in 50 ml of water at 70°C, the pH is adjusted to 12, and stirring is carried out for 1 h to obtain a mixed solution; 1 g of the modified silica obtained in step (1) is added, and 30 ml of an ethanol mixture containing 1.46 g of TEOS and 0.23 g of APTES is added dropwise under stirring at 800 rpm; stirring is carried out at 30°C for 24 h, and after standing for 24 h, filtration, water washing, and drying at 60°C are carried out, and the temperature is increased to 250°C at a rate of 1°C / min, and calcination is carried out for 6 h to obtain a chiral R type silica carrier; (3) 0.08 g of lipase PS is dissolved in 20 ml of PBS buffer with pH of 7.0, and the above chiral R type silica carrier is added, and oscillation is carried out at 4°C for 12 h, and centrifugal separation is carried out at 800 rpm, and freeze-drying is carried out to obtain a modified silica immobilized lipase.

[0028] Preparation Example 2 The lipase A is a modified silica immobilized lipase, and its preparation method is as follows: (1) 1.0 g of silica is dispersed in 50 ml of toluene, 0.15 g of trimethylchlorosilane is added, and refluxing is carried out at 80°C under nitrogen protection for 12 h. After centrifugation, it is dispersed in 50 mL of water, 0.1 M HCl is added dropwise to adjust the pH to 5, ultrasonic dispersion is carried out for 30 min, centrifugal washing is carried out, and vacuum drying is carried out at 60°C to obtain modified silica; (2) 1.5 g of hexadecyl-D-phenylalanine is dissolved in 50 ml of water at 70°C, the pH is adjusted to 12, and stirring is carried out for 1 h to obtain a mixed solution; 0.5 g of the modified silica obtained in step (1) is added, and 30 ml of an ethanol mixture containing 1.46 g of TEOS and 0.23 g of APTES is added dropwise under stirring at 800 rpm; stirring is carried out at 30°C for 24 h, and after standing for 24 h, filtration, water washing, and drying at 60°C are carried out, and the temperature is increased to 250°C at a rate of 1°C / min, and calcination is carried out for 6 h to obtain a chiral R type silica carrier; (3) 0.08 g of lipase PS is dissolved in 20 ml of PBS buffer with pH of 7.0, and the above chiral R type silica carrier is added, and oscillation is carried out at 4°C for 12 h, and centrifugal separation is carried out at 800 rpm, and freeze-drying is carried out to obtain a modified silica immobilized lipase.

[0029] Preparation Example 3 The lipase A is a modified silica immobilized lipase, which is prepared by the following method: (1) 1.0 g of silica is dispersed in 50 ml of toluene, 0.15 g of trimethylchlorosilane is added, and refluxing is carried out at 80°C for 12 h under nitrogen protection. After centrifugation, it is dispersed in 50 ml of water, 0.1 M HCl is added dropwise to adjust the pH to 5, ultrasonic dispersion is carried out for 30 min, centrifugal washing is carried out, and vacuum drying is carried out at 60°C to obtain modified silica; (2) 0.5 g of hexadecyl-D-phenylalanine is dissolved in 50 ml of water at 70°C, the pH is adjusted to 12, and stirring is carried out for 1 h to obtain a mixed solution; 1.5 g of the modified silica obtained in step (1) is added, and 30 ml of an ethanol mixture containing 1.46 g of TEOS and 0.23 g of APTES is added dropwise under stirring at 800 rpm; stirring is carried out at 30°C for 24 h, and after standing for 24 h, filtration, water washing, drying at 60°C, and heating at 1°C / min to 250°C are carried out, and calcination is carried out for 6 h to obtain a chiral R-type silica carrier; (3) 0.08 g of lipase PS is dissolved in 20 ml of PBS buffer with a pH of 7.0, and the above chiral R-type silica carrier is added, and oscillation is carried out at 4°C for 12 h, centrifugal separation is carried out at 800 rpm, and freeze-drying is carried out to obtain a modified silica immobilized lipase.

[0030] Preparation Example 4 (2) 1 g of hexadecyl-D-phenylalanine is dissolved in 50 ml of water at 70°C, the pH is adjusted to 12, and stirring is carried out for 1 h to obtain a mixed solution; 1 g of silica is added, and 30 ml of an ethanol mixture containing 1.46 g of TEOS and 0.23 g of APTES is added dropwise under stirring at 800 rpm; stirring is carried out at 30°C for 24 h, and after standing for 24 h, filtration, water washing, drying at 60°C, heating at 1°C / min to 250°C, and calcination are carried out for 6 h to obtain a chiral R-type silica carrier; (3) 0.08 g of lipase PS is dissolved in 20 ml of PBS buffer with a pH of 7.0, and the above chiral R-type silica carrier is added, and oscillation is carried out at 4°C for 12 h, centrifugal separation is carried out at 800 rpm, and freeze-drying is carried out to obtain a modified silica immobilized lipase.

[0031] Preparation Example 5 The lipase A is a modified silica immobilized lipase, and the preparation method is as follows: (1) 1.0 g of silica is dispersed in 50 ml of toluene, 0.15 g of trimethylchlorosilane is added, and refluxing is carried out at 80°C for 12 h under nitrogen protection. After centrifugation, it is dispersed in 50 ml of water, 0.1 M HCl is added dropwise to adjust the pH to 5, ultrasonic dispersion is carried out for 30 min, centrifugal washing is carried out, and vacuum drying is carried out at 60°C to obtain modified silica; (2) 2 g of the modified silica obtained in step (1) is added, and 30 ml of an ethanol mixture containing 1.46 g of TEOS and 0.23 g of APTES is added dropwise under stirring at 800 rpm; stirring is carried out at 30°C for 24 h, and after standing for 24 h, filtration, water washing and drying at 60°C are carried out, and the temperature is increased to 250°C at 1°C / min, and calcination is carried out for 6 h to obtain a silica carrier; (3) 0.08 g of lipase PS is dissolved in 20 ml of PBS buffer with pH of 7.0, and the above silica carrier is added, and oscillation is carried out at 4°C for 12 h, and centrifugal separation is carried out at 800 rpm, and freeze-drying is carried out to obtain a modified silica immobilized lipase.

[0032] Preparation Example 6 The lipase A is a modified silica immobilized lipase, and the preparation method is as follows: (1) 1.0 g of silica is dispersed in 50 ml of toluene, 0.15 g of trimethylchlorosilane is added, and refluxing is carried out at 80°C for 12 h under nitrogen protection. After centrifugation, it is dispersed in 50 ml of water, 0.1 M HCl is added dropwise to adjust the pH to 5, ultrasonic dispersion is carried out for 30 min, centrifugal washing is carried out, and vacuum drying is carried out at 60°C to obtain modified silica; (2) 1 g of D-phenylalanine is dissolved in 50 ml of water at 70°C, the pH is adjusted to 12, and stirring is carried out for 1 h to obtain a mixed solution; 1 g of the modified silica obtained in step (1) is added, and 30 ml of an ethanol mixture containing 1.46 g of TEOS and 0.23 g of APTES is added dropwise under stirring at 800 rpm; stirring is carried out at 30°C for 24 h, and after standing for 24 h, filtration, water washing and drying at 60°C are carried out, the temperature is increased to 250°C at 1°C / min, and calcination is carried out for 6 h to obtain a chiral R-type silica carrier; (3) 0.08 g of lipase PS is dissolved in 20 ml of PBS buffer with pH of 7.0, and the above chiral R-type silica carrier is added, and oscillation is carried out at 4°C for 12 h, and centrifugal separation is carried out at 800 rpm, and freeze-drying is carried out to obtain a modified silica immobilized lipase.

[0033] Example 1 The synthesis process of the intermediate of the compound of the present application comprises the following steps: S1: enzymatic esterification: 5 g of 2-(4-trifluoromethyl phenoxy)-propionic acid is added into 1 g of isopropenyl acetate, then 0.4 g of the lipase A prepared in the preparation example 1 is added, the temperature is adjusted to 37℃, and stirring is carried out for 18 h, then filtration is carried out through a diatomite layer, washing is carried out three times with ethyl acetate, and concentration is carried out, so as to obtain an intermediate mixture containing (R) compound F and (S) compound G; (R) compound F (substrate yield 61.1%, chiral purity 97.2%); S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100 ml of THF under a cold bath with a temperature of 5℃, then 3.8 g of triphenylphosphine and 1 g of formic acid are added, 1.8 g of di-tert-butyl azodicarboxylate is slowly added dropwise, after the dropwise addition is completed, the temperature is increased to 10℃, and stirring is carried out for 28 h, the raw material is detected to be reacted completely through a TLC control, concentration is carried out under reduced pressure, washing is carried out through filtration, so as to obtain a mixture containing (R) compound F and (R) compound H; S3: enzyme catalysis: 0.7 g of the mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7 mg of BL-IM lipase and 14 ml of ethanol under nitrogen protection, stirring is carried out at 20℃ for 24 h, the raw material is detected to be reacted completely through a TLC control, filtration is carried out, and the mother liquor is concentrated, so as to obtain compound A; the chiral purity of compound A is 99.8%, and the crude product yield is 90%; Example 2 The synthesis process of the intermediate of the compound of the present application comprises the following steps: S1: enzymatic esterification: 5 g of 2-(4-trifluoromethyl phenoxy)-propionic acid is added into 1 g of isopropenyl acetate, then 0.4 g of the lipase A prepared in the preparation example 1 is added, the temperature is adjusted to 37℃, and stirring is carried out for 18 h, then filtration is carried out through a diatomite layer, washing is carried out three times with ethyl acetate, and concentration is carried out, so as to obtain an intermediate mixture containing (R) compound F and (S) compound G; (R) compound F (substrate yield 61.1%, chiral purity 97.2%); S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100 ml of THF under a cold bath with a temperature of 5℃, then 3.8 g of triphenylphosphine and 1 g of formic acid are added, 1.8 g of di-tert-butyl azodicarboxylate is slowly added dropwise, after the dropwise addition is completed, the temperature is increased to 10℃, and stirring is carried out for 28 h, the raw material is detected to be reacted completely through a TLC control, concentration is carried out under reduced pressure, washing is carried out through filtration, so as to obtain a mixture containing (R) compound F and (R) compound H; S3: enzyme catalysis: 0.7 g of the mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7 mg of BL-IM lipase and 14 ml of ethanol under nitrogen protection, stirring is carried out at 20℃ for 24 h, the raw material is detected to be reacted completely through a TLC control, filtration is carried out, and the mother liquor is concentrated, so as to obtain compound A.

[0034] Example 3 The synthesis process of a treprostinil intermediate comprises the following steps: S1: enzymatic esterification: 5 g of 2-(4-trifluoromethylphenoxy)-propionic acid is added into 1 g of isopropenyl acetate, then 0.4 g of lipase A prepared in Preparation Example 2 is added, the temperature is adjusted to 37 DEG C, and stirring is conducted for 18 h, then filtration is conducted through a diatomite layer, washing is conducted with ethyl acetate for three times, and concentration is conducted to obtain an intermediate mixture containing (R) compound F and (S) compound G; (R) compound F (substrate yield 58.7%, chiral purity 96.7%); S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100 ml of THF under a cold bath at 5 DEG C, then 3.8 g of triphenylphosphine and 1 g of formic acid are added, 1.8 g of di-tert-butyl azodicarboxylate is slowly added dropwise, after the dropwise addition is completed, the temperature is increased to 10 DEG C, and stirring is conducted for 28 h, the raw material is detected to be reacted completely through TLC, concentration is conducted under reduced pressure, washing is conducted through filtration, and an intermediate mixture containing (R) compound F and (R) compound H is obtained; S3: enzyme catalysis: 0.7 g of the intermediate mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7 mg of BL-IM lipase and 14 ml of ethanol under nitrogen protection, stirring is conducted at 20 DEG C for 24 h, the raw material is detected to be reacted completely through TLC, filtration is conducted, and the mother liquor is concentrated to obtain compound A.

[0035] Example 4 The synthesis process of a treprostinil intermediate comprises the following steps: S1: enzymatic esterification: 5 g of 2-(4-trifluoromethylphenoxy)-propionic acid is added into 1 g of isopropenyl acetate, then 0.4 g of lipase A prepared in Preparation Example 2 is added, the temperature is adjusted to 37 DEG C, and stirring is conducted for 18 h, then filtration is conducted through a diatomite layer, washing is conducted with ethyl acetate for three times, and concentration is conducted to obtain an intermediate mixture containing (R) compound F and (S) compound G; (R) compound F (substrate yield 58.7%, chiral purity 96.7%); S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100 ml of THF under a cold bath at 5 DEG C, then 3.8 g of triphenylphosphine and 1 g of formic acid are added, 1.8 g of di-tert-butyl azodicarboxylate is slowly added dropwise, after the dropwise addition is completed, the temperature is increased to 10 DEG C, and stirring is conducted for 28 h, the raw material is detected to be reacted completely through TLC, concentration is conducted under reduced pressure, washing is conducted through filtration, and an intermediate mixture containing (R) compound F and (R) compound H is obtained; S3: enzyme catalysis: 0.7 g of the intermediate mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7 mg of BL-IM lipase and 14 ml of ethanol under nitrogen protection, stirring is conducted at 20 DEG C for 24 h, the raw material is detected to be reacted completely through TLC, filtration is conducted, and the mother liquor is concentrated to obtain compound A.

[0036] Example 5 The synthesis process of travoprost intermediate includes the following steps: S1: Enzymatic esterification: 5g of 2-(4-trifluoromethylphenoxy)-propionic acid was added to 1g of isopropyl acetate, followed by 0.4g of lipase A prepared in Example 4. The temperature was adjusted and stirred at 37°C for 18h. The mixture was then filtered through a diatomaceous earth layer, washed three times with ethyl acetate, and concentrated to obtain an intermediate mixture containing compound F (R) and compound G (S); compound F (substrate yield 58.2%, chiral purity 96.9%). S2: Configuration Inversion: Under a cold bath at 5°C, the intermediate mixture obtained in step S1 was added to 100 ml of THF, followed by the addition of 3.8 g of triphenylphosphine and 1 g of formic acid. Then, 1.8 g of di-tert-butyl azodicarboxylate was slowly added dropwise. After the addition was complete, the temperature was raised to 10°C and the mixture was stirred for 28 h. The reaction was confirmed by TLC under central control. The mixture was concentrated under reduced pressure, filtered, and washed to obtain a mixture containing compounds F and H. S3: Enzyme Catalysis: Under nitrogen protection, 0.7 g of the mixture containing compounds F and H obtained in step S2 was added to 7 mg of BL-IM lipase and 14 ml of ethanol. The mixture was stirred at 20°C for 24 h. The reaction was confirmed by TLC under central control. The mixture was filtered and the mother liquor was concentrated to obtain compound A.

[0037] Example 6 The synthesis process of travoprost intermediate includes the following steps: S1: Enzymatic esterification: 5g of 2-(4-trifluoromethylphenoxy)-propionic acid was added to 1g of isopropyl acetate, followed by 0.4g of lipase A prepared in Preparation Example 5. The temperature was adjusted and stirred at 37°C for 18h. The mixture was then filtered through a diatomaceous earth layer, washed three times with ethyl acetate, and concentrated to obtain an intermediate mixture containing compound F (R) and compound G (S); compound F (substrate yield 57.5%, chiral purity 96.1%). S2: Configuration Inversion: Under a cold bath at 5°C, the intermediate mixture obtained in step S1 was added to 100 ml of THF, followed by the addition of 3.8 g of triphenylphosphine and 1 g of formic acid. Then, 1.8 g of di-tert-butyl azodicarboxylate was slowly added dropwise. After the addition was complete, the temperature was raised to 10°C and the mixture was stirred for 28 h. The reaction was confirmed by TLC under central control. The mixture was concentrated under reduced pressure, filtered, and washed to obtain a mixture containing compounds F and H. S3: Enzyme Catalysis: Under nitrogen protection, 0.7 g of the mixture containing compounds F and H obtained in step S2 was added to 7 mg of BL-IM lipase and 14 ml of ethanol. The mixture was stirred at 20°C for 24 h. The reaction was confirmed by TLC under central control. The mixture was filtered and the mother liquor was concentrated to obtain compound A.

[0038] Example 7 The synthesis process of a treprostinil intermediate comprises the following steps: S1: enzymatic esterification: 5 g of 2-(4-trifluoromethylphenoxy)-propionic acid is added into 1 g of isopropenyl acetate, then 0.4 g of lipase A prepared in Preparation Example 6 is added, the temperature is adjusted to 37 ℃, and stirring is performed for 18 h, then filtration is performed through a diatomite layer, washing is performed three times with ethyl acetate, and concentration is performed to obtain an intermediate mixture containing (R) compound F and (S) compound G; (R) compound F (substrate yield 59.7%, chiral purity 97.0%); S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100 ml of THF under a cold bath at a temperature of 5 ℃, then 3.8 g of triphenylphosphine and 1 g of formic acid are added, 1.8 g of di-tert-butyl azodicarboxylate is slowly added dropwise, after the dropwise addition is completed, the temperature is increased to 10 ℃, and stirring is performed for 28 h, the raw material is detected to be completely reacted by TLC, concentration is performed under reduced pressure, washing is performed through filtration, and an intermediate mixture containing (R) compound F and (R) compound H is obtained; S3: enzyme catalysis: 0.7 g of the intermediate mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7 mg of BL-IM lipase and 14 ml of ethanol under nitrogen protection, stirring is performed at 20 ℃ for 24 h, the raw material is detected to be completely reacted by TLC, filtration is performed, the mother liquor is concentrated, and compound A is obtained; the chiral purity of the compound A is 99.8%, and the crude product yield is 90%; Example 8 The synthesis process of a treprostinil intermediate comprises the following steps: S1: enzymatic esterification: 5 g of 2-(4-trifluoromethylphenoxy)-propionic acid is added into 1 g of isopropenyl acetate, then 0.4 g of lipase A prepared in Preparation Example 6 is added, the temperature is adjusted to 37 ℃, and stirring is performed for 18 h, then filtration is performed through a diatomite layer, washing is performed three times with ethyl acetate, and concentration is performed to obtain an intermediate mixture containing (R) compound F and (S) compound G; S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100 ml of THF under a cold bath at a temperature of 5 ℃, then 3.8 g of triphenylphosphine and 1 g of formic acid are added, 1.8 g of di-tert-butyl azodicarboxylate is slowly added dropwise, after the dropwise addition is completed, the temperature is increased to 10 ℃, and stirring is performed for 28 h, the raw material is detected to be completely reacted by TLC, concentration is performed under reduced pressure, washing is performed through filtration, and an intermediate mixture containing (R) compound F and (R) compound H is obtained; S3: enzyme catalysis: 0.7 g of the intermediate mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7 mg of BL-IM lipase and 14 ml of ethanol under nitrogen protection, stirring is performed at 20 ℃ for 24 h, the raw material is detected to be completely reacted by TLC, filtration is performed, the mother liquor is concentrated, and compound A is obtained; the chiral purity of the compound A is 99.8%, and the crude product yield is 90%; Example 9 The synthesis process of the intermediate of the compound of the application comprises the following steps: S1: enzymatic esterification: 5g of 2-(4-trifluoromethylphenoxy)-propionic acid is added into 1g of isopropenyl acetate, and then 0.4g of the lipase A prepared in the preparation example 1 is added, and then the temperature is adjusted to 37℃, and stirred for 18h, and then filtered through diatomite layer, washed with ethyl acetate for three times, and concentrated to obtain an intermediate mixture containing (R) compound F and (S) compound G; S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100ml of THF under the temperature of 5℃, and then 3.8g of triphenylphosphine and 1g of formic acid are added, and then 1.8g of di-tert-butyl azodicarboxylate is slowly added dropwise, and after the dropwise addition is completed, the temperature is increased to 10℃, and stirred for 28h, and then the raw material is detected to be reacted completely by TLC, and then concentrated under reduced pressure, filtered and washed to obtain a mixture containing (R) compound F and (R) compound H; S3: enzyme catalysis: 0.7g of the mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7mg of BL-IM lipase and 14ml of a mixture of ethanol and n-heptane with a mass ratio of 2:1 under the protection of nitrogen, and then stirred for 24h at 20℃, and then the raw material is detected to be reacted completely by TLC, and then filtered, and the mother liquor is concentrated to obtain compound A; the chiral purity of the compound A is 99.8%, and the yield of the crude product is 93% Example 10 The synthesis process of the intermediate of the compound of the application comprises the following steps: S1: enzymatic esterification: 5g of 2-(4-trifluoromethylphenoxy)-propionic acid is added into 1g of isopropenyl acetate, and then 0.4g of the lipase A prepared in the preparation example 1 is added, and then the temperature is adjusted to 37℃, and stirred for 18h, and then filtered through diatomite layer, washed with ethyl acetate for three times, and concentrated to obtain an intermediate mixture containing (R) compound F and (S) compound G; S2: configuration inversion: the intermediate mixture obtained in the step S1 is added into 100ml of THF under the temperature of 5℃, and then 3.8g of triphenylphosphine and 1g of formic acid are added, and then 1.8g of di-tert-butyl azodicarboxylate is slowly added dropwise, and after the dropwise addition is completed, the temperature is increased to 10℃, and stirred for 28h, and then the raw material is detected to be reacted completely by TLC, and then concentrated under reduced pressure, filtered and washed to obtain a mixture containing (R) compound F and (R) compound H; S3: enzyme catalysis: 0.7g of the mixture containing (R) compound F and (R) compound H obtained in the step S2 is added into 7mg of BL-IM lipase and 14ml of a mixture of ethanol and n-heptane with a mass ratio of 2:1 under the protection of nitrogen, and then stirred for 24h at 20℃, and then the raw material is detected to be reacted completely by TLC, and then filtered, and the mother liquor is concentrated to obtain compound A; the chiral purity of the compound A is 99.8%, and the yield of the crude product is 93%

[0039] ByFigure 1 and Figure 2 The hydrogen spectrum and carbon spectrum of compound A in the above-mentioned compounds A, Figure 1 and Figure 2 can determine the conformation and purity of the synthesized compound A. The synthesis process of the example of fluvastatin intermediate can improve the selectivity by double chiral recognition mechanism, can enrich (R)-substrate, and after the reaction is completed, high purity and high e.e. value (more than 99.8%) of compound A can be obtained by post-treatment and purification. The whole process avoids the generation of difficult-to-remove impurities, shortens the reaction steps, simplifies the production operation, improves the production efficiency, reduces the production cost, and is suitable for industrial production. Compared with examples 1 and 2-6, by preparing modified silica fixed lipase, mesoporous silica modification provides a mesoporous skeleton, and N-hexadecyl-R-phenylalanine is added in the synthesis stage of the silica layer on the surface thereof, the chiral channel is arranged by self-assembly induction, the chiral channel is generated, the enzyme molecules are connected with the chiral R-type silica layer at multiple points, the stereoselective adsorption of the substrate is enhanced, the specific enzyme and the R-conformation double-induction reactant are synthesized, the (S)-hydroxyl configuration is excluded, and the stereoselectivity is further enhanced. If the content of hexadecyl-D-phenylalanine is too low or too high, by-products will be generated, and the chiral purity will decrease.

[0040] Compared with examples 1 and 8, in S3, the mixture of (R) compound F and (R) compound H and lipase B are within the preferred range of the present application, which helps to finally obtain high-purity compound A. Too much lipase B will increase the collision probability between molecules, increase the possibility of side reactions, and too much raw material will cause subsequent separation difficulties and affect the yield.

[0041] Compared with examples 1 and 9-10, the present application discloses that the ratio of ethanol and n-heptane as a solvent helps to maintain the active conformation of the lipase, improve the catalytic efficiency of the enzyme, and make the yield of the crude product higher. Through the gradient elution of column chromatography in the present application, the yield of the pure product in S3 step reaches 82%, the present application simplifies the original five-step reaction to three steps, and the operation is mild, easy and safe, the enzyme can be reused, the yield is improved, the purity of the separation is ensured, and the economic benefit is improved.

[0042] The above are preferred embodiments of the present application, and do not limit the protection scope of the present application, so: any equivalent changes made according to the structure, shape, principle of the present application should be covered within the protection scope of the present application.

Claims

1. A synthetic process for travoprost intermediates, characterized in that: The process includes the following steps: S1: Enzymatic esterification: 2-(4-trifluoromethylphenoxy)-propionic acid is added to isopropyl acetate, followed by lipase A. The temperature is adjusted to 37-45℃ and stirred for 12-18 hours. The mixture is then filtered through a diatomaceous earth layer, washed, and concentrated to obtain an intermediate mixture containing (R) compound F and (S) compound G. S2: Configuration inversion: Under a cold bath at 0-10℃, the intermediate mixture obtained in step S1 was added to a polar solvent, followed by the addition of triphenylphosphine and formic acid. Then, di-tert-butyl azodicarboxylate was slowly added dropwise. After the addition was complete, the temperature was raised to 10-70℃ and the mixture was stirred for 28-26 hours. The reaction of the raw materials was detected by TLC under central control. The mixture was concentrated under reduced pressure, filtered and washed to obtain a mixture containing compound (R) F and compound (R) H. S3: Enzyme catalysis: Under nitrogen protection, take the mixture containing (R) compound F and (R) compound H obtained in step S2, add lipase B and solvent, stir at 15-20℃ for 6-30h, and monitor the reaction of the raw materials by central TLC until the reaction is complete. Filter and concentrate the mother liquor to obtain compound A.

2. The synthetic process for the travoprost intermediate according to claim 1, characterized in that: In S1, the lipase A is a modified silica-immobilized lipase, and its preparation method includes: (1) Disperse silica in toluene, add trimethylchlorosilane, reflux at 75-80℃ for 8-12h under nitrogen protection, centrifuge, add water, add 0.1M HCl to adjust pH to 5-6, and sonicate to obtain modified silica; (2) Dissolve hexadecyl-D-phenylalanine in water at 60-70℃, adjust pH to 12-13, stir for 1-2h to obtain a mixed solution; add the modified silica obtained in step (1), add TEOS and APTES ethanol mixture dropwise under stirring at 500-800rpm; stir at 30-35℃ for 20-24h, stand, filter, wash and dry, and calcine at low temperature to obtain chiral R-type silica carrier; (3) Dissolve lipase PS in PBS buffer, add chiral R-type silica carrier, shake at 4-5℃ for 10-12h, centrifuge, freeze dry to obtain modified silica immobilized lipase.

3. The synthesis process of the travoprost intermediate according to claim 2, characterized in that: In S1, the mass ratio of hexadecyl-D-phenylalanine to lipase PS is 1:(0.06-0.1).

4. The synthetic process for the travoprost intermediate according to claim 1, characterized in that: In S1, the mass ratio of 2-(4-trifluoromethylphenoxy)-propionic acid, isopropyl acetate, and lipase A is (5-7):1:(0.25-0.45).

5. The synthesis process of the travoprost intermediate according to claim 1, characterized in that: In S3, the lipase B is one of immobilized BCA lipase and immobilized BL-IM lipase.

6. The synthetic process for the travoprost intermediate according to claim 1, characterized in that: In S3, the mass ratio of the mixture containing (R) compound F and (R) compound H to lipase B is (50-100):

1.

7. The synthetic process for the travoprost intermediate according to claim 1, characterized in that: In S3, the solvent includes one or both of alcohol solvents and nonpolar solvents.

8. The synthetic process for the travoprost intermediate according to claim 1, characterized in that: S3 also includes a purification step using silica gel column chromatography.

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