Patent Publication Number: US-2021171485-A1

Title: Method For Preparing Hexahydrofuro-Furanol Derivative, Intermediate Thereof And Preparation Method Thereof

Description:
This application claims the priority of the Chinese patent application with the application number 201810325842.2 and the invention title of “METHOD FOR PREPARING HEXAHYDROFURO-FURANOL DERIVATIVE, INTERMEDIATE THEREOF AND PREPARATION METHOD THEREOF”, filed to the National Intellectual Property Administration, PRC on Apr. 12, 2018, which is hereby incorporated herein by reference in its entirety. 
     TECHNICAL FIELD 
     The invention relates to the field of pharmaceutical synthesis, in particular to the method for preparing hexahydrofuro-furanol derivative, the intermediate thereof and preparation method thereof. 
     TECHNICAL BACKGROUND 
     The chemical name of the compound with the following formula Z structure is (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol: 
     
       
         
         
             
             
         
       
     
     which belongs to one of the hexahydrofuro-furanol derivatives and is an intermediate of the anti-AIDS drug Darunavir. 
     The Chinese patent applications No. 02817639.1 (application date: 2002-9-6) and 200580010400.X of Tibotec Pharmaceutical Co., Ltd., the original manufacturer of Darunavir, provide the preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, in which the raw material is the following compound of formula (3), 
     
       
         
         
             
             
         
       
     
     the compound of formula (3) is prepared from the starting material, the compound of formula (1). 
     
       
         
         
             
             
         
       
     
     The Chinese patent application No. 2003080109926.4 of Sumitomo Chemical Co., Ltd., a generic drug manufacturer of Darunavir, provides the preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, in which the starting material is the following compound of formula VIII 
     
       
         
         
             
             
         
       
     
     Sumitomo Chemical Co., Ltd. of Japan chose to use chiral ligand catalysts for the generation of chiral conformation. Although this is indeed a method for constructing chiral conformation, it is not suitable for industrialization. 
     European patent application EP2634180A1 (application date: 2012-1-3) of Lonza Ltd. of Switzerland, a generic drug manufacturer of Darunavir, involves using carbonyl reductase polypeptides or microorganisms containing carbonyl reductase polypeptides to achieve the reduction of carbonyl groups to hydroxyl groups so as to construct the chirality of intermediate of Darunavir, and many kinds of commercially available enzymes such as  Saccharomyces cerevisiae  YNL331C are listed in the specification, and it is mentioned that the compound represented by the following formula Ia is a more suitable configuration, 
     
       
         
         
             
             
         
       
     
     Considering that (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol is a key intermediate for the preparation of Darunavir drugs, it is necessary to develop more preparation methods for this key intermediate, which can not only obtain the key intermediate with high yield and high DE value, but also has low cost and mild reaction conditions, and is suitable for industrialization. 
     SUMMARY 
     The method for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the invention starts from the choice of starting materials and the construction of chiral configuration, and a method for preparing the key intermediate of Darunavir with the starting materials different from that in the above-mentioned prior patent applications has been developed. The preparation method of the invention adopts an enzymatic method to construct chirality, which has low cost and mild reaction conditions, and provides an alternative route suitable for industrialization for the preparation of the key intermediate of Darunavir. 
     To achieve the technical objective of the present invention, the present invention provides the following technical solutions: 
     The first aspect of the present invention provides an intermediate compound for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, having the following structural formula: 
     
       
         
         
             
             
         
       
     
     wherein, R 1 , R 2 , R 3  are hydrogen or hydroxy protecting groups; R 4 , R 5  are the same or different, are phenyl, alkyl or substituted phenyl. The hydroxy protecting group is alkyl, silyl, C 2-11  acyl, C 4-9  cycloalkenyl, aryl, aralkyl, aroyl, phenyl, substituted phenyl; the silyl is tetramethylsilyl, trimethylsilyl, triethylsilyl, tri-n-butylsilyl, tert-butyldimethylsilyl; the alkyl is a C 1 -C 8  alkyl; the aryl group is phenyl, furanyl, thienyl or indolyl; the substituted phenyl is alkyl-substituted phenyl, alkoxyalkyl-substituted phenyl, nitroalkyl-substituted phenyl or halogen-substituted phenyl; the alkyl-substituted phenyl is benzyl, benzhydryl, trityl; the alkoxyalkyl-substituted phenyl is p-methoxybenzyl; the nitroalkyl-substituted phenyl is p-nitrobenzyl; the halogen-substituted phenyl is p-chlorophenyl. 
     The second aspect of the present invention provides a method for preparing the above intermediate compound, which is prepared by reacting a compound of formula Cp with an amine compound, 
     
       
         
         
             
             
         
       
     
     wherein, the definitions of R 1 , R 2 , R 4  and R 5  are the same as above. 
     More preferably, it is prepared by reacting a compound of formula Cp with an N-methylaniline compound, 
     
       
         
         
             
             
         
       
     
     wherein, the definitions of R 1 , R 2  are the same as above. 
     It is prepared by enzymatic reduction reaction of compound of formula B to construct chirality, 
     
       
         
         
             
             
         
       
     
     wherein, the definition of R 1  is the same as above. 
     The enzyme is a biological enzyme, such as aldo-keto reductase, wherein the aldo-keto reductase is derived from the  Saccharomyces kudriavzevii  strain. Its amino acid sequence is the protein shown in SEQ ID NO: 1, or the protein having aldo-keto reductase activity obtained from SEQ ID NO: 1 after substitution, deletion or addition of one or more amino acid residues, or the protein having 80% homology with the amino acid sequence shown in SEQ ID NO:1 and having aldo-keto reductase activity; its nucleotide sequence is shown as SEQ ID NO: 2 in the sequence listing. The aldo-keto reductase may be derived from whole cells of genetically engineered bacteria, broken enzyme liquid, lyophilized powder or immobilized enzymes or immobilized cells. 
     The amount of the enzyme fed may be in the range of 50-100 g/L, and the reaction temperature may be in the range of 25−37° C. 
     A coenzyme may be selectively added to the enzymatic reduction reaction, and the coenzyme is NADP+ or NADPH. 
     A glucose dehydrogenase may be selectively added to the enzymatic reduction reaction. 
     The enzymatic reduction reaction is carried out in the presence of a solvent which is a mixed solvent that is composed of water or buffer solution and an organic solvent. 
     The buffer solution is selected from one or more of phosphate buffer solution, carbonate buffer solution, Tri-HCl buffer solution, citrate buffer solution or MOPS buffer solution. 
     The organic solvent is selected from one or more of DMSO, ethyl acetate, butyl acetate, isopropanol, DMF, TBME, dichloromethane, and vinyl acetate. 
     In the biological conversion process of the enzymatic reduction reaction of the present invention, HPLC-MS and HPLC are used for monitoring until the substrate is fully utilized. 
     After the above enzymatic reduction reaction, a protecting group can be further introduced to prepare the compound of formula Cp, 
     
       
         
         
             
             
         
       
     
     wherein, the definition of R 1  is the same as above, R 2  is a hydroxy protecting group, and the enzyme is the same as above. 
     Wherein, the compound of formula B is prepared from the compound of formula A2 by acylation, the reaction formula is as follows: 
     
       
         
         
             
             
         
       
     
     wherein, X is halogen, and the definition of R 1  is the same as above. 
     The above compound of formula A2 is prepared from the compound of formula A1 by halogenation reaction, the reaction formula is as follows: 
     
       
         
         
             
             
         
       
     
     wherein, X is halogen 
     The invention further provides a method for preparing the intermediate of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, compound of formula C-i, which is prepared from the compound of formula C-1 by deprotection reaction, 
     
       
         
         
             
             
         
       
     
     wherein, the definitions of R 1 , R 2  are the same as above. 
     The third aspect of the present invention provides a method for preparing the intermediate of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, compound of formula Cf, which is prepared from a compound of formula C L  by reduction reaction, 
     
       
         
         
             
             
         
       
     
     wherein, the definitions of R 1 , R 2 , R 4  and R 5  are the same as above. 
     More preferably, it is prepared from a compound of formula C-i by reduction reaction, 
     
       
         
         
             
             
         
       
     
     In the reduction reaction, the reducing agent may be a boron reducing agent, an aluminum reducing agent or a silicon-lithium reducing agent. Such as sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, Red-Al, lithium aluminum hydride, diisobutyl aluminum hydride, lithium diisopropylamide, lithium hexamethyldisilazide may be used. 
     Before the above reduction reaction, a deprotection reaction can be carried out first, the reaction formula is as follows: 
     
       
         
         
             
             
         
       
     
     Alternatively, a cyclization reaction can be carried out first, and the reaction formula is as follows: 
     
       
         
         
             
             
         
       
     
     The prepared compound of formula Cp3 and compound of formula Cp4 are subjected to the carbonyl reduction reaction according to the above method, and the reducing reagent is the same as above. 
     That is, the present invention provides an alternative method for preparing the intermediate of (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol, compound of formula Cp, which is prepared from a compound of formula C L  by ring closure reaction, 
     
       
         
         
             
             
         
       
     
     wherein, the definitions of R 1 , R 2 , R 4 , R 5  are the same as above. 
     The fourth aspect of the present invention provides a method for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which is prepared from a compound of formula Cf by ring closure reaction, 
     
       
         
         
             
             
         
       
     
     wherein, the definitions of R 1 , R 2 , R 4 , R 5  are the same as above. 
     More preferably, it is prepared from a compound of formula Cf-1 by ring closure reaction, 
     
       
         
         
             
             
         
       
     
     The reagent for the ring closure reaction is an acid or a base common in the art. 
     The above method for preparing (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention includes the steps of halogenation reaction, acylation reaction, enzymatic reduction reaction, reaction with amine compounds, reductive ring closure reaction, and the reaction formula is: 
     
       
         
         
             
             
         
       
     
     wherein, X is halogen; the definitions of R 1 , R 2 , R 4 , R 5  are the same as above. 
     More preferably, it is prepared by halogenation reaction, acylation reaction, enzymatic reduction reaction, reaction with amine compounds, deprotection reaction, reductive ring closure reaction 
     
       
         
         
             
             
         
       
     
     Wherein, the method for constructing chirality by the reduction reaction is an enzymatic method, and the definition of the enzyme is the same as the above. 
     The preparation method of (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol provided by the present invention adopts enzymatic method to construct chirality, and the product can be prepared with high yield and high optical purity. Although the prior art such as the above European application has disclosed that the reduction of carbonyl to hydroxyl is achieved by carbonyl reductase polypeptides or microorganisms containing carbonyl reductase polypeptides, the enzyme used in the present invention has more significant advantages, shown in the higher optical purity of the obtained products and more suitable reaction conditions. The preparation method of (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is suitable for industrial production. 
    
    
     EMBODIMENTS 
     In order to further understand the present invention, the preparation method of the hexahydrofuro-furanol derivatives provided by the present invention, the intermediates thereof and the preparation method thereof will be described in detail in combination with embodiments below. It should be understood that the description of these embodiments is only for further describing the features of the present invention in details, rather than limiting the scope of the present invention or the claims of the present invention. 
     Example 1 
     
       
         
         
             
             
         
       
     
     The compound of formula A1 and dichloromethane were added to a reaction flask, cooled down, bromine was weighed, and dichloromethane was added for dilution, the diluted bromine was transferred to a funnel and slowly added dropwise, and the internal temperature was controlled. After dropwise addition, the solution was continued reacting with the temperature kept and the internal temperature was controlled. Water was added, the temperature was controlled, and the solution was allowed to stand, the liquid separation was performed. The lower organic phase was put into another reaction flask, water was added for extraction, and the liquid separation was performed. The lower organic phase was put into another reaction flask, 5% NaHCO 3  aqueous solution was added for extraction, and the liquid separation was performed. The lower organic phase was put into another reaction flask, and the upper aqueous phases were combined and extracted by adding dichloromethane, and the liquid separation was performed. The water phase was discard, and the organic phases were combined, water was added for extraction, and the liquid separation was performed, the water phase was discard, and the lower organic phase was placed in a rotary evaporator to concentrate until no solvent flows out, the yield was 90-95%. 
     Example 2 
     
       
         
         
             
             
         
       
     
     Acetone, compound of formula A2 (X is bromine), benzoic acid were put into a reaction flask, the mixture was stirred and cooled down. Triethylamine was added to a tank and started to drop slowly, and the internal temperature was controlled. After dropwise addition, the mixture was raised to room temperature, and stirred for reaction. After the reaction was completed, suction filtration was performed, after that, the filtrate was transferred to a distillation flask to distill under reduced pressure and the temperature was controlled at 50-60° C. until a pasty solid appeared in the distilling flask, then ethyl acetate was added. Ethyl acetate was added to the distilling flask and stirred until the pasty solid was dissolved, the material liquid in the distilling flask was transferred to the reaction flask. Saturated salt water was added to the reaction flask to wash, the solution was allowed to stand and the liquid separation was performed, water layers were combined, ethyl acetate was added for extraction, the solution was allowed to stand to separate into layers, the water layer was discarded, the organic layers were combined, anhydrous sodium sulfate was added to the organic layer and stirred to dryness, and suction filtration was performed. The filtrate was transferred to a reaction flask to distill under reduced pressure and the temperature was controlled at 50-60° C. until a pasty solid appeared in the reaction solution, part of ethyl acetate was added, stirred and refluxed until dissolved to clear, the temperature was controlled at 50-60° C., n-heptane was added to the tank, and was added slowly dropwise into the reaction bottle until completely added into the bottle. After slowly cooling down, the reaction solution was stirred with the temperature kept, suction filtration was performed and the target solid crude product was dried, the yield was 70-75%. 
     Example 3 
     Preparation of Whole Cells of Genetically Engineered Bacteria of Aldo-Keto Reductase 
     The specific preparation method of genetically engineered bacteria of recombinant aldo-keto reductase was: selecting the gene sequence of aldo-keto reductase derived from  Saccharomyces kudriavzevii , designing it artificially, and synthesizing the artificially designed sequence by whole genes (synthesized by Genscript Biotechnology Co., Ltd.), cloning it into the Nde I and Xho I enzyme digestion sites of the expression vector pET28a, and transforming host  E. coli  BL21 (DE3) competent cells; picking the positive transformants and sequencing and identifying to obtain the recombinant expression vector; transforming the recombinant expression vector into  E. coli  BL21 (DE3) strain to obtain the genetically engineered bacteria of recombinant aldo-keto reductase which can induce the expression of recombinant aldo-keto reductase. 
     The genetically engineered bacteria of recombinant aldo-keto reductase were inoculated into LB medium containing kanamycin, and cultured at 37° C. overnight to obtain seed culture solution; the seed culture solution was inoculated into TB medium containing kanamycin, and the inoculation volume was 1% of the volume of TB medium containing kanamycin; then it was incubated at 37° C. for 2-5 h. Sterile IPTG was added for inducing and the final concentration of IPTG was 0.1 mM, and the inoculum was continued to be incubated at 25° C. for 20 h. Finally, whole cells of the genetically engineered bacteria of aldo-keto reductase derived from  Saccharomyces kudriavzevii  were obtained by high-speed centrifugation. The whole cells of the genetically engineered bacteria were ultrasonically broken by ultrasonic method to obtain the broken enzyme solution of the whole cells of the genetically engineered bacteria of aldo-keto reductase derived from  Saccharomyces kudriavzevii . The aldo-keto reductase is a protein whose amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the aldo-keto reductase gene is shown as SEQ ID NO: 2 in the sequence table. 
     After induced expression, there was a clear protein band at 45 kDa, indicating that the aldo-keto reductase was highly expressed in the recombinant bacteria. 
     The enzyme activity of the pure protein of aldo-keto reductase was measured, the reaction system was at a volume of 0.25 ml, including Tris-HCl, pH of 8.0, 2 mmol/L NADPH, 0.1 mmol/L substrate 
     
       
         
         
             
             
         
       
     
     and appropriate amount of enzyme, the decrease of absorbance at 340 nm was measured. The enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the oxidation of lumol NADPH per minute under the above conditions. 
     The results showed that the recombinant genetically engineered aldo-keto reductase has an activity of more than 20% higher than that of the sequence in European patent (EP2634180A1), and more than 50% higher than that of the non-mutated aldo-keto reductase. 
     The whole cells of genetically engineered bacteria of aldo-keto reductase used in the Examples of the present invention were all prepared by this method. 
     The glucose dehydrogenases used in the Examples and Comparative Examples of the present invention were all commercial enzymes purchased from sigma-aldrich. 
     The algorithm for the ee value of enantiomer overexpression was: 
         ee (syn)=([R,R]−[S,S])/([R,R]+[S,S])
 
         ee (anti)=([R,S]−[S,R])/([R,S]+[S,R])
 
         de ={([R,S]+[S,R])−([R,R]+[S,S])}/{([R,S]+[S,R])+([R,R]+[S,S])}.
 
     Enzymatic reduction reaction, with the reaction formula as follows: 
     
       
         
         
             
             
         
       
     
     Step 1: The reaction was carried out in a 1 L shake flask, and the reaction system was controlled at the volume of 300 mL. In the shake flask, the broken enzyme solution of the whole cells of the genetically engineered bacteria of aldo-keto reductase was suspended in 260 mL of sterilized potassium phosphate buffer solution. Glucose dehydrogenase was added to the shake flask, the cells were broken by ultrasound for 50 min, then 25 g glucose, 0.42 g NADP+ were added to the shake flask. Then 8 g of the reactant was weighed and dissolved in 40 mL of DMSO, the DMSO solution in which the substrate dissolved was slowly poured into the shake flask. After 2 hours of reaction, 12 g of glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 75 g/L, the amount of glucose dehydrogenase fed was 25 mg/L, and the temperature of the conversion system was controlled at 37° C. The conversion reaction was carried out in a shaker, the rotation speed of the shaker was controlled at 200 r/min, the conversion time was 12 h, and the conversion liquid of the target product was obtained, with a conversion rate of 97.8%. 
     Step 2: The conversion solution of the target product obtained in step 1 was purified, an equal volume of ethyl acetate was added to the reaction system, the extraction was carried out at 37° C. for 15 min, repeated for 3 times, the ethyl acetate layer was collected by centrifugation, and 5% anhydrous magnesium sulfate was added to the collected ethyl acetate layer and shaken for 15 min, after that, the magnesium sulfate was removed by filtration. Then the ethyl acetate layer after the removal of water was concentrated under reduced pressure at elevated temperature, 7.41 g of the target product was obtained, with a de value of 96.2% and an ee (anti) value of 99.5%. 
     Example 4 
     
       
         
         
             
             
         
       
     
     DCM (80 ml) and AlCl 3  (5.6 g) were added to a 250 mL four-necked flask successively, N-methylaniline (17.1 g) was added dropwise, after dropwise addition, then the solution of the compound of formula Cp1 (8.0 g Cp1 dissolved in 80 mL of DCM) was added dropwise. After the dropwise addition, the temperature was kept. The reaction solution was dropped into dilute sulfuric acid (10.1 g sulfuric acid dissolved in 200 mL water) and stirred while dropping. After the dropwise addition, the pH was measured. The reaction mixture was stirred and separated into layers, then liquid separation was performed. The acid water layer was extracted with 100 mL DCM. The mixture was separated into layers, the organic layers were combined, and washed with 5% NaHCO 3  solution. The liquid separation was performed, and the organic layer was washed with 100 mL of water. The liquid separation was performed, and the organic layer was dried with Na 2 SO 4 . The organic layer was subjected to suction filtration, the filtrate was concentrated under reduced pressure at 50° C. to obtain a yellow oily substance, and 27 mL EA and 27 mL heptane were added and stirred at 60° C. until dissolved to clear. The temperature was slowly lowered to 0° C. to crystallize. The obtained crystal was subjected to suction filtration, and rinsed with 20 mL of mixture (EA:heptane=1:1), and dried to obtain 10 g of white-like solid, compound of formula Cp2 (purity&gt;98%). 
     Example 5 
     
       
         
         
             
             
         
       
     
     The compound of formula Cp2 (3.57 g) and methanol (15.00 mL) were added to a reaction flask successively, and 0.15 g caustic soda liquid was added dropwise under stirring, after the temperature was kept at −20˜30° C., 5% sulfuric acid was added dropwise to adjust the pH, 0.1 g sodium bicarbonate was added, the mixture was concentrated under reduced pressure in a water bath at 30-35° C. to remove methanol. After decompression, water was added, then DCM was added for extraction, the organic phases were combined, and concentrated under reduced pressure at 40° C. to obtain 2.50 g of product with a yield of about 99%. 
     Example 6 
     
       
         
         
             
             
         
       
     
     The compound of formula Cp3 (2.50 g) and THF (20.00 mL) were added to a reaction flask successively, the inner temperature was cooled down, LiAlH 4  (1.50 g) was slowly added in batches, after addition the mixture was slowly warmed to room temperature (20-25° C.) and stirred with the temperature kept. After the reaction, the inner temperature was cooled down, 10% dilute hydrochloric acid was slowly added dropwise to adjust the pH, and after the addition, the solution was slowly warmed to room temperature (20-25° C.) and stirred. The inner temperature was cooled down, and 4% NaOH aqueous solution was slowly added to adjust the pH of the reaction solution. After adjusting the pH, toluene was added and stirred for extraction, the toluene layers were combined, the water layer was taken, DCM was added to the water layer under stirring for extraction, and the DCM layers were combined, the combined DCM layer was taken and an appropriate amount of anhydrous sodium sulfate was added to the DCM layer to dry. 1.0 g of colorless oily substance was obtained after concentration under reduced pressure at 40° C. to dryness, with a yield of 80.0%. 
     Example 7 
     
       
         
         
             
             
         
       
     
     The compound of formula Cp2 (3.57 g), THF (25.00 mL), tetramethyldisiloxane (1.34 g), and titanium tetraisopropoxide (2.84 g) were added to a reaction flask successively, and stirred at 20-30° C. for 6-24 h, then EA (50 mL) was added, and 5% sulfuric acid was added dropwise to adjust the pH, the mixture was allowed to stand to separate into layers. The water layer was further extracted once with EA (20 mL), the organic layers were combined and 5% sodium bicarbonate was added thereto, and the EA was removed by concentration under reduced pressure in a water bath at 30-45° C. After decompression, EA (5 mL) was added for making slurry, then the solution was filtrated and dried to obtain 2.20 g of white or white-like solid compound of formula Cp4, with a yield of about 90%. 
     Example 8 
     
       
         
         
             
             
         
       
     
     The solid compound of formula B (R 1  is benzoyl) (20.00 g) was added to a 500 mL dry and clean four-necked flask, and toluene was added and stirred, vacuum replacement was performed under nitrogen protection, the mixture was cooled down under nitrogen protection, and toluene was added to a constant-pressure funnel under the protection of nitrogen, and 70% Red-Al solution (26.50 g, 26.00 mL) was added to the constant-pressure funnel under the protection of nitrogen. When the preparation was completed, after the reaction solution was cooled to −15˜−10° C., the Red-Al was added, the temperature was controlled, after the dropwise addition, the temperature was kept. Pure water and ethyl acetate were added to a 1000 mL clean four-necked flask successively, sulfuric acid was added to the 1000 mL clean four-necked flask under stirring, then the solution was cooled down, after the temperature was kept constant, the reaction solution was dropped into the sulfuric acid solution, the temperature of the reaction liquid was controlled, the sulfuric acid solution was controlled at a temperature of 0-10° C., after the dropwise addition, ethyl acetate was added and then stirred; 10% sodium bicarbonate aqueous solution was added to a clean 1000 mL four-necked flask, the solution was cooled down, stop stirring, and the reaction solution was allowed to stand to separate into layers, the upper organic layer was transferred into the 10% sodium bicarbonate aqueous solution and stirred; the lower acid water layer was extracted by adding ethyl acetate, then allowed to stand to separate into layers, and the acid water layer was discarded, and the second ethyl acetate layer was transferred to 10% sodium bicarbonate aqueous solution and stirred, the temperature was controlled, and the solution was allowed to stand to separate into layers, ethyl acetate was added to the alkaline water layer for extraction and stirred, the temperature was controlled, pure water was added to the alkali-washed organic layer for washing, stirred, the temperature was controlled, the solution was allowed to separate into layers, the water-washed organic layer was obtained for further use, and the water-washed water layer was further extracted with ethyl acetate, and allowed to stand to separate into layers, and the water layer was discarded, the organic layers were combined, dried over sodium sulfate, subjected to suction filtration, and rinsed with ethyl acetate. The filtrate was concentrated under reduced pressure at 40-70° C. to obtain about 17 g of an oily substance with a yield of 84.33%. 
     Example 9 
     
       
         
         
             
             
         
       
     
     Methanol (10.00 mL) and the compound of formula C (R 1  is benzoyl) (1.00 g) were added to a reaction flask successively, cooled to −15˜−5° C., sodium hydroxide aqueous solution (10.00 mL) was added dropwise, and after the dropwise addition, the solution was stirred with the temperature kept until the raw material has been reacted completely. 10% sulfuric acid aqueous solution (4.00 g) was added dropwise, after the dropwise addition, the solution was continued reacting with the temperature kept, after the temperature start to change, saturated sodium carbonate aqueous solution was added to adjust the pH. After the methanol was removed by distillation under reduced pressure at elevated temperature, methyl tert-butyl ether was added for extraction, the organic layer was separated, sodium chloride was added to the water layer until saturation and dichloromethane was further added for extraction, the water layer was separated for recovery, and the organic layer was distilled under reduced pressure to remove dichloromethane, 0.38 g of product was obtained with a yield of 73.77%. 
     Example 10 
     
       
         
         
             
             
         
       
     
     The compound of formula Cp2 (3.50 g) and THF (20.00 mL) were added to a reaction flask successively, and the inner temperature was cooled down, LiAlH 4  (1.50 g) was slowly added in batches, after addition the mixture was slowly warmed to room temperature (20-25° C.) and stirred with the temperature kept. After the reaction was completed, the inner temperature was cooled down, 10% dilute hydrochloric acid was slowly added dropwise to adjust the pH, and after the addition, the solution was slowly warmed to room temperature (20-25° C.) and stirred. The inner temperature was cooled down, and 4% NaOH aqueous solution was slowly added to adjust the pH of the reaction solution. After adjusting the pH, toluene was added and stirred for extraction, the toluene layers were combined, the water layer was taken, DCM was added to the water layer under stirring for extraction, and DCM layers were combined, the combined DCM layer was taken and an appropriate amount of anhydrous sodium sulfate was added to the DCM layer to dry. 1.0 g of colorless oily substance was obtained after concentration under reduced pressure at 40° C. to dryness, with a yield of 80.0%. 
     Example 11 
     
       
         
         
             
             
         
       
     
     The preparation of the enzyme was the same as in Example 3. 
     Step 1: The reaction was carried out in a 5 L beaker, and the reaction system was controlled at the volume of 2 L. In the shake flask, the whole cells of the genetically engineered bacteria of aldo-keto reductase was suspended in 1.7 L of sterilized potassium phosphate buffer solution. Glucose dehydrogenase was added to the shake flask, the cells were broken by ultrasound for 50 min, then 25 g glucose, 0.42 g NADP+ were added to the beaker. Then 80 g of the substrate was weighed and dissolved in 300 mL of DMSO, the DMSO solution in which the substrate dissolved was slowly poured into the shake flask. After 2 hours of reaction, 12 g of glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 75 g/L, the amount of glucose dehydrogenase fed was 25 mg/L, and the temperature of the conversion system was controlled to 37° C. The conversion reaction was carried out in a beaker, the rotation speed of the magneton was controlled at 200 r/min, the conversion time was 12 h, and the conversion liquid of the target product was obtained with a conversion rate of 97.8%. 
     Step 2: The conversion solution containing the intermediate of Darunavir, the compound of formula VIII obtained in step 1, was purified, and the purification steps was as in Example 3, 77.10 g of the target compound was obtained. The prepared target compound had a de value of 95.3%, an ee (anti) value of 99.6%. 
     Example 12 
     
       
         
         
             
             
         
       
     
     Step 1: The reaction was carried out in a 1 L shake flask, and the reaction system was controlled at the volume of 300 mL. In the shake flask, the whole cells of the genetically engineered bacteria of aldo-keto reductase was suspended in 250 mL of sterilized deionized water. Glucose dehydrogenase was added to the shake flask, and 10 mL of 2.5 mol/L glucose, 0.26 g NADP+ were added. Then 10 g of the substrate was weighed and dissolved in 30 mL of butyl acetate, the butyl acetate solution in which the substrate was dissolved was slowly poured into the shake flask. After 1 hour of reaction, 10 mL of 2.5 mol/L glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 75 g/L, the amount of glucose dehydrogenase was 25 mg/L, and the temperature of the conversion system was controlled at 37° C. The conversion reaction was carried out in a shaker, the rotation speed of the shaker was controlled at 200 r/min, the conversion time was 12 h, and the conversion liquid of the target product was obtained with a conversion rate of 97.8%. 
     Step 2: The conversion solution of the target compound obtained in step 1 was purified, an equal volume of ethyl acetate was added to the reaction system, the extraction was carried out at 37° C. for 15 min, repeated for 3 times, the ethyl acetate layer was collected by centrifugation, and 5% anhydrous magnesium sulfate was added to the collected ethyl acetate layer and shaken for 15 min, after that, the magnesium sulfate was removed by filtration. Then the ethyl acetate layer after the removal of water was concentrated under reduced pressure at elevated temperature, 9.55 g of the target compound was obtained, the prepared target compound had a de value of 99.1%, an ee (anti) value of the enantiomer is 99.7%.  1 H NMR (600 MHz, CDCl 3 ) δ 2.269˜2.301 (m, 1H, J=6 Hz), 2.367˜2.404 (m, 1H), 2.954˜2.993 (m, 1H, J=6 Hz), 3.438˜3.466 (m, 1H), 3.520˜3.549 (m, 1H), 4.227˜4.269 (m, 1H), 4.298˜4.326 (m, 1H), 4.391˜4.420 (m, 1H). MS(ESI): m/z 210.03 [M+H] + . 
     Example 13 
     
       
         
         
             
             
         
       
     
     Step 1: The reaction was carried out in a 5 L beaker, and the reaction system was controlled at the volume of 2 L. In the beaker, the whole cells of the genetically engineered bacteria of aldo-keto reductase was suspended in 1.5 L of sterilized deionized water. Glucose dehydrogenase was added, and 100 mL of 2.5 mol/L glucose, 3 g NADP+ were added. Then 100 g of substrate was weighed and dissolved in 300 mL of butyl acetate, the butyl acetate solution in which the substrate was dissolved was slowly poured into the beaker. After 1 hour of reaction, 100 mL of 2.5 mol/L glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 100 g/L, the amount of glucose dehydrogenase fed was 25 mg/L, and the temperature of the conversion system was controlled at 28° C. The conversion reaction was carried out in a beaker, the rotation speed of the magneton was controlled at 200 r/min, the conversion time was 12 h, and the conversion liquid of the target was obtained with a conversion rate of 97.8%. 
     Step 2: The conversion solution of the target obtained in step 1 was purified, and the purification steps was as in Example 3, 9.42 g of the target was obtained. The the prepared target had a de value of 96.9%, an ee (anti) value of the enantiomer is 99.4%. 
     Example 14 
     
       
         
         
             
             
         
       
     
     Step 1: The reaction was carried out in a 1 L shake flask, and the reaction system was controlled at the volume of 300 mL. In the shake flask, the whole cells of the genetically engineered bacteria of aldo-keto reductase was suspended in 250 mL of sterilized deionized water. Glucose dehydrogenase was added, and 10 mL of 2.5 mol/L glucose, 0.26 g NADP+ were added. Then 10 g of substrate was weighed and dissolved in 30 mL of butyl acetate, the butyl acetate solution in which the substrate was dissolved was slowly poured into the shake flask. After 1 hour of reaction, 10 mL of 2.5 mol/L glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 75 g/L, the amount of glucose dehydrogenase fed was 25 mg/L, and the temperature of the conversion system was controlled at 37° C. The conversion reaction was carried out in a shaker, the rotation speed of the shaker was controlled at 200 r/min, the conversion time was 12 h, and the conversion liquid of the target was obtained with a conversion rate of 97.8%. 
     Step 2: The conversion solution of the target compound obtained in step 1 was purified, and the purification steps was as in Example 3, 9.37 g of the target was obtained. The prepared target compound had a de value of 97.1%, an ee (anti) value of 99.5%. 
     Example 15 
     
       
         
         
             
             
         
       
     
     Step 1: The reaction was carried out in a 5 L beaker, and the reaction system was controlled at the volume of 2 L. In the beaker, the whole cells of the genetically engineered bacteria of aldo-keto reductase was suspended in 1.6 L of sterilized deionized water. Glucose dehydrogenase was added, and 100 mL of 2.5 mol/L glucose, 2.5 g NADP+ were added. Then 100 g of substrate was weighed and dissolved in 200 mL of butyl acetate, the butyl acetate solution in which the substrate was dissolved was slowly poured into the beaker. After 1 hour of reaction, 100 mL of 2.5 mol/L glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 50 g/L, the amount of glucose dehydrogenase fed was 25 mg/L, and the temperature of the conversion system was controlled at 25° C. The conversion reaction was carried out in a beaker, the rotation speed of the magneton was controlled at 200 r/min, the conversion time was 12 h, and the conversion liquid of the target compound was obtained with a conversion rate of 97.8%. 
     Step 2: The conversion solution of the target compound obtained in step 1 was purified, and the purification step was as in Example 3, 93.1 g of the target compound was obtained. The prepared target compound had a de value of 95.6%, and an ee (anti) value of 99.6%. 
     Example 16: Comparative Example 
     
       
         
         
             
             
         
       
     
     Step 1: The reaction is carried out in a 1 L shake flask, and the reaction system was controlled at the volume of 300 mL. In the shake flask, the whole cells of the genetically engineered bacteria of aldo-keto reductase was suspended in 250 mL of sterilized deionized water. The coding sequence of the aldo-keto reductase gene of the whole cell of the genetically engineered bacteria of the aldo-keto reductase used was as the sequence published in the European patent EP2634180 (ie SEQ ID NO 12 shown in the patent EP2634180), and the sequence was synthesized by whole gene (synthesized by Genscript Biotechnology Co., Ltd.), the preparation method was as in Example 3. The glucose dehydrogenase was added, and 10 mL of 2.5 mol/L glucose, 0.26 g NADP+ were added. Then 10 g of substrate was weighed and dissolved in 30 mL of butyl acetate, the butyl acetate solution in which the substrate was dissolved was slowly poured into the beaker. After 1 hour of reaction, 10 mL of 2.5 mol/L glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 75 g/L, the amount of glucose dehydrogenase fed was 25 mg/L, and the temperature of the conversion system was controlled at 37° C. The conversion reaction was carried out in a shaker, the rotation speed of the shaker was controlled at 200 r/min, the conversion time was 12 h. 
     The purification step was as in Example 3, 8.11 g of the target compound was obtained. The prepared target compound had a de value of 85.1%, and an ee (anti) value of the enantiomer of 93.3%. 
     Example 17: Comparative Example 
     
       
         
         
             
             
         
       
     
     Step 1: The reaction is carried out in a 1 L shake flask, and the reaction system was controlled at the volume of 300 mL. In the shake flask, the whole cells of the genetically engineered bacteria of aldo-keto reductase of  Saccharomyces kudriavzevii  was suspended in 250 mL of sterilized deionized water. The coding sequence of the aldo-keto reductase gene of the whole cell of the genetically engineered bacteria of the aldo-keto reductase used was shown in SEQ ID NO 3 (the coding sequence of aldo-keto reductase gene has not been artificially designed), and the sequence was synthesized by whole gene (synthesized by Genscript Biotechnology Co., Ltd.), the preparation method was as in Example 3. The glucose dehydrogenase was added, and 10 mL of 2.5 mol/L glucose, 0.26 g NADP+ were added. Then 10 g of substrate was weighed and dissolved in 30 mL of butyl acetate, the butyl acetate solution in which the substrate was dissolved was slowly poured into the beaker. After 1 hour of reaction, 10 mL of 2.5 mol/L glucose was further added, the amount of the whole cells of the genetically engineered bacteria of aldo-keto reductase fed was 75 g/L, the amount of glucose dehydrogenase fed was 25 mg/L, and the temperature of the conversion system was controlled at 37° C. The conversion reaction was carried out in a shaker, the rotation speed of the shaker was controlled at 200 r/min, the conversion time was 12 h. 
     The purification step was as in Example 3, 7.73 g of the target compound was obtained. The prepared target compound had a de value of 79.6%, and a ee (anti) value of the enantiomer of 88.7. 
     
       
         
           
               
               
            
               
                 Serial number of SEQ ID NO 1: 
                   
               
               
                 Met Ser Asp Leu Phe Lys Pro Ala Pro Glu Pro Pro Thr Glu Leu Gly 
               
               
                   
               
               
                 Arg Leu Arg Val Leu Ser Lys Thr Ala Gly Ile Arg Val Ser Pro Leu 
               
               
                   
               
               
                 Ile Leu Gly Gly Ala Ser Ile Gly Asp Ala Trp Ser Gly Phe Met Gly 
               
               
                   
               
               
                 Ser Met Asn Lys Glu Gln Ala Phe Glu Leu Leu Asp Ala Phe Tyr Glu 
               
               
                   
               
               
                 Ala Gly Gly Asn Cys Val Asp Thr Ala Asn Ser Tyr Gln Asn Glu Glu 
               
               
                   
               
               
                 Ser Glu Ile Trp Ile Gly Glu Trp Met Lys Ser Arg Lys Leu Arg Asp 
               
               
                   
               
               
                 Gln Ile Val Ile Ala Thr Lys Phe Thr Gly Asp Tyr Lys Lys Tyr Glu 
               
               
                   
               
               
                 Val Gly Gly Gly Lys Ser Ala Asn Tyr Cys Gly Asn His Lys His Ser 
               
               
                   
               
               
                 Leu His Val Ser Val Arg Asp Ser Leu Arg Lys Leu Gln Thr Asp Trp 
               
               
                   
               
               
                 Ile Asp Ile Leu Tyr Val His Trp Trp Asp Tyr Met Ser Ser Ile Glu 
               
               
                   
               
               
                 Glu Val Met Asp Ser Leu His Ile Leu Ile Gln Gln Gly Lys Val Leu 
               
               
                   
               
               
                 Tyr Leu Gly Val Ser Asp Thr Pro Ala Trp Val Val Ser Ala Ala Asn 
               
               
                   
               
               
                 Asn Tyr Ala Thr Ser His Gly Lys Thr Pro Phe Ser Ile Tyr Gln Gly 
               
               
                   
               
               
                 Lys Trp Asn Val Leu Asn Arg Asp Phe Glu Arg Asp Ile Ile Pro Met 
               
               
                   
               
               
                 Ala Arg His Phe Gly Met Ala Leu Ala Pro Trp Asp Val Met Gly Gly 
               
               
                   
               
               
                 Gly Lys Phe Gln Ser Lys Lys Ala Met Glu Glu Trp Lys Lys Asn Gly 
               
               
                   
               
               
                 Glu Gly Leu Arg Thr Ala Val Gly Gly Pro Glu Gln Thr Glu Leu Glu 
               
               
                   
               
               
                 Val Lys Ile Ser Glu Ala Leu Asn Lys Ile Ala Glu Glu His Gly Thr 
               
               
                   
               
               
                 Glu Ser Val Thr Ala Ile Ala Ile Ala Tyr Val Arg Ser Lys Ala Lys 
               
               
                   
               
               
                 Asn Val Phe Pro Leu Val Gly Gly Arg Lys Ile Glu His Leu Lys Gln 
               
               
                   
               
               
                 Asn Ile Glu Ala Leu Ser Ile Lys Leu Thr Pro Glu Gln Ile Glu Tyr 
               
               
                   
               
               
                 Leu Glu Ser Ile Val Thr Phe Asp Val Gly Phe Pro Lys Ser Asn Ile 
               
               
                   
               
               
                 Gly Asp Asp Pro Ala Val Thr Lys Lys Leu Ser Pro Leu Thr Ser Met 
               
               
                   
               
               
                 Ser Ala Arg Ile Ser Phe Asp Asn 
               
               
                   
               
               
                 Serial number of SEQ ID NO 2: 
               
            
           
           
               
               
               
            
               
                 atgagcgatc tgtttaaacc ggcgccggaa ccgccgaccg aactgggccg cctgcgcgtg 
                 60 
                   
               
               
                   
               
               
                 ctgagcaaaa ccgcgggcat tcgcgtgagc ccgctgattc tgggcggcgc gagcattggc 
                 120 
               
               
                   
               
               
                 gatgcgtgga gcggctttat gggcagcatg aacaaagaac aggcgtttga actgctggat 
                 180 
               
               
                   
               
               
                 gcgttttatg aagcgggcgg caactgcgtg gataccgcga acagctatca gaacgaagaa 
                 240 
               
               
                   
               
               
                 agcgaaattt ggattggcga atggatgaaa agccgcaaac tgcgcgatca gattgtgatt 
                 300 
               
               
                   
               
               
                 gcgaccaaat ttaccggcga ttataaaaaa tatgaagtgg gcggcggcaa aagcgcgaac 
                 360 
               
               
                   
               
               
                 tattgcggca accataaaca tagcctgcat gtgagcgtgc gcgatagcct gcgcaaactg 
                 420 
               
               
                   
               
               
                 cagaccgatt ggattgatat tctgtatgtg cattggtggg attatatgag cagcattgaa 
                 480 
               
               
                   
               
               
                 gaagtgatgg atagcctgca tattctgatt cagcagggca aagtgctgta tctgggcgtg 
                 540 
               
               
                   
               
               
                 agcgataccc cggcgtgggt ggtgagcgcg gcgaacaact atgcgaccag ccatggcaaa 
                 600 
               
               
                   
               
               
                 accccgttta gcatttatca gggcaaatgg aacgtgctga accgcgattt tgaacgcgat 
                 660 
               
               
                   
               
               
                 attattccga tggcgcgcca ttttggcatg gcgctggcgc cgtgggatgt gatgggcggc 
                 720 
               
               
                   
               
               
                 ggcaaatttc agagcaaaaa agcgatggaa gaatggaaaa aaaacggcga aggcctgcgc 
                 780 
               
               
                   
               
               
                 accgcggtgg gcggcccgga acagaccgaa ctggaagtga aaattagcga agcgctgaac 
                 840 
               
               
                   
               
               
                 aaaattgcgg aagaacatgg caccgaaagc gtgaccgcga ttgcgattgc gtatgtgcgc 
                 900 
               
               
                   
               
               
                 agcaaagcga aaaacgtgtt tccgctggtg ggcggccgca aaattgaaca tctgaaacag 
                 960 
               
               
                   
               
               
                 aacattgaag cgctgagcat taaactgacc ccggaacaga ttgaatatct ggaaagcatt 
                 1020 
               
               
                   
               
               
                 gtgacctttg atgtgggctt tccgaaaagc aacattggcg atgatccggc ggtgaccaaa 
                 1080 
               
               
                   
               
               
                 aaactgagcc cgctgaccag catgagcgcg cgcattagct ttgataacta a 
                 1131 
               
               
                   
               
            
           
           
               
               
            
               
                 Serial number of SEQ ID NO 3: 
                   
               
            
           
           
               
               
               
            
               
                 atgtctgatg tatttggacc tgcacctgaa ccacctaccg agttaggacg tctaagagtt 
                 60 
                   
               
               
                   
               
               
                 ctctctaaaa cagctggtat aagagtctct ccgctaatat tgggaggtat gtcgattggt 
                 120 
               
               
                   
               
               
                 gacgcctggt caggattcat ggggtcaatg aacaaggagc gggcttttga gctgcttgat 
                 180 
               
               
                   
               
               
                 gcctttttcg aggcaggtgg aaacttcatt gatactgcaa ataattacca aaatgaacag 
                 240 
               
               
                   
               
               
                 tcagaggcat ggataggtga atggatggtt tcaagaaaat tgcgtgacca aattgttatt 
                 300 
               
               
                   
               
               
                 gccaccaaat tcaccacaga ctataagaag tatgaagtgg gcaagggcag aagtgccaac 
                 360 
               
               
                   
               
               
                 ttctgtggta atcacaagca tagtttacac gtaagtgtga gagattctct tcgcaaattg 
                 420 
               
               
                   
               
               
                 cagactgatt ggattgacat tctctatgtt cactggtggg attatatgag ttcgatcgag 
                 480 
               
               
                   
               
               
                 gaagttatgg atagtctgca tattcttgtg cagcagggca aggtcctcta cctgggagta 
                 540 
               
               
                   
               
               
                 tctgatacac ctgcatgggt cgtgtctgct gcaaattact acgctacctc tcacgggaaa 
                 600 
               
               
                   
               
               
                 actcccttca gcatctatca aggtaaatgg aatctgttga atagggactt tgagcgtgaa 
                 660 
               
               
                   
               
               
                 attattccaa tggctaggca ttttggtatg gctctcgctc catgggatgt catgggaggg 
                 720 
               
               
                   
               
               
                 ggaagatttc agagcaaaaa agctttagaa gaacggaaga agagtggaga gggcctgcgt 
                 780 
               
               
                   
               
               
                 agctttgttg gtacatctga acagacggat gcagaggtta agatcagcga ggcattgtcg 
                 840 
               
               
                   
               
               
                 aaggttgctg aggaacatgg cattgagtct gtcacagcta ttgccattgc ctatgtccgc 
                 900 
               
               
                   
               
               
                 tccaaagcga agcatgtttt cccattggtt ggaggaagga aaattgagca cctcaaacaa 
                 960 
               
               
                   
               
               
                 aatattgagg cgttgagtat taaattgaca ccaggacaga tagaatatct agaaagcatt 
                 1020 
               
               
                   
               
               
                 gtcccattcg atgttgggtt ccctagcaat ttcatcggag atgatcctgc agttactaag 
                 1080 
               
               
                   
               
               
                 aaacttgcat tccttccagc aatgtctgcc aagattgctt ttgacgatta g 
                 1131