Preparation methods of hexahydrofuranofuranol derivatives, their intermediates and preparation methods

Abstract

This invention relates to the field of pharmaceutical synthesis, specifically to a method for preparing hexahydrofurano[2,3-b]-3-ol derivatives, their intermediates, and the same method. The method uses a compound of formula A1 as a starting material. During the preparation of the hexahydrofurano[2,3-b]-3-ol derivative, chirality is constructed via enzymatic methods. This technique allows for the preparation of the product with very high optical purity. This method enables the commercial production of the key intermediate (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol, a very economical route suitable for industrial production.

Description

Technical Field

[0001] This application is a divisional application of Chinese invention patent application No. 201810220506.1, filed on March 16, 2018, entitled "Preparation method of hexahydrofuranofuranol derivative, intermediate thereof and preparation method thereof". This invention relates to the field of pharmaceutical synthesis, specifically to the preparation method of hexahydrofuranofuranol derivative, intermediate thereof and preparation method thereof. Background Technology

[0002] Compounds having the following Z-structure are chemically named (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ols:

[0003]

[0004] It belongs to the category of hexahydrofuranofuranol derivatives and is an intermediate of the anti-AIDS drug darunavir.

[0005] The original manufacturer of darunavir, Tybot Pharmaceuticals Co., Ltd., has Chinese patent applications numbered 02817639.1 (application date: 2002-9-6) and 200580010400.X, which provide a method for preparing the above-mentioned (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol, wherein the raw material is the following compound of formula (3).

[0006]

[0007] Compound (3) is prepared from starting material compound (1).

[0008]

[0009] The Chinese patent application No. 200380109926.4 of Sumitomo Chemical Co., Ltd., a manufacturer of generic darunavir, provides a method for preparing the above-mentioned (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol, wherein the starting material is a compound of formula VⅢ as follows.

[0010]

[0011] Sumitomo Chemicals of Japan chose to use chiral ligand catalysts to generate chiral configurations. While this is indeed a method for constructing chiral configurations, it is not very suitable for industrialization.

[0012] The European patent application EP2634180A1 (filed on 2012-01-03) by Lonza Ltd., a Swiss manufacturer of generic darunavir, relates to the chirality of constructing darunavir intermediates by using carbonyl reductase peptides or microorganisms containing carbonyl reductase peptides to reduce carbonyl groups to hydroxyl groups. The specification lists many commercially available enzymes, such as Saccharomyces cerevisiae YNL331C, and mentions that the compound shown in formula Ia is a suitable configuration.

[0013]

[0014] Considering that (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol is a key intermediate in the preparation of darunavir, it is necessary to develop more methods for preparing this key intermediate. Such methods should not only yield the key intermediate with high yield and high DE value, but also have low cost, mild reaction conditions, and be suitable for industrialization. Summary of the Invention

[0015] The method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol of this invention starts with the selection of starting materials and the construction of chiral configurations, and develops a method for preparing darunavir key intermediates from starting materials that differs from those in the aforementioned patent applications. The preparation method of this invention uses an enzymatic method to construct the chiral configuration, which is low-cost and has mild reaction conditions, providing another suitable route for the industrialization of this darunavir key intermediate.

[0016] To achieve the technical objective of this invention, the following technical solution is provided:

[0017] Firstly, the first aspect of this invention provides a method for preparing a (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol intermediate compound, which is prepared by enzymatic reduction of compound B or compound B-2 to construct a chiral compound.

[0018]

[0019] Wherein, R1 is a hydrogen or hydroxyl protecting group;

[0020] The enzyme is a biological enzyme, such as aldehyde-ketone reductase, which is derived from the strain *Saccharomyces kudriavzevii*. Its amino acid sequence is that of the protein shown in SEQ ID NO: 1, or a protein exhibiting aldehyde-ketone reductase activity after substitution, deletion, or addition of one or more amino acid residues as shown in SEQ ID NO: 1, or a protein exhibiting aldehyde-ketone reductase activity with more than 80% homology to the amino acid sequence shown in SEQ ID NO: 1; its nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing. Aldehyde-ketone reductase can be derived from whole cells of genetically engineered bacteria, lysed enzyme solutions, lyophilized powder, immobilized enzymes, or immobilized cells.

[0021] The amount of enzyme added can be 50-100 g / L, and the reaction temperature can be 25-37℃.

[0022] In the enzymatic reduction reaction, a coenzyme can be selectively added, and the coenzyme is NADP+ or NADPH.

[0023] Glucose dehydrogenase can be selectively added to the enzymatic reduction reaction.

[0024] The enzymatic reduction reaction is carried out in the presence of a solvent, which is a mixture of water or a buffer solution and an organic solvent.

[0025] 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.

[0026] The organic solvent is selected from one or more of DMSO, ethyl acetate, butyl acetate, isopropanol, DMF, TBME, dichloromethane, and vinyl acetate.

[0027] In this invention, the enzymatic reduction reaction biotransformation process is monitored using HPLC-MS and HPLC until the substrate is completely utilized.

[0028] Furthermore, the method for preparing the (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol intermediate provided by the present invention involves constructing a chiral compound of formula B via enzymatic reduction, followed by a protecting group addition reaction.

[0029]

[0030] In this context, R1 is defined as described above, R2 is a hydroxyl protecting group, and the enzyme is the same as described above.

[0031] In the above preparation method, R1 is preferably a straight-chain or branched acyl group of C2-11, a benzoyl group or a mono- or poly-substituted benzoyl group on a benzene ring, wherein the mono- or poly-substituted group is an alkyl group, an alkoxy group, a nitro group or a cyano group.

[0032] A second aspect of the present invention provides a method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol, which is prepared from the above intermediate compound of formula C by further reduction and cyclization reaction.

[0033]

[0034] The definition of R1 is the same as above.

[0035] The present invention provides a method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol, or a method for preparing the intermediate Cp compound obtained above through further reduction and cyclization reactions.

[0036]

[0037] The definitions of R1 and R2 are the same as those described above.

[0038] The method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol provided by the present invention can be prepared by selectively separating the obtained C-2 or Cp-2 compound and then further preparing it by a cyclization reaction.

[0039] Compound B of the present invention is prepared by acylation of compound A2, as shown in the following reaction formula:

[0040]

[0041] Wherein, R1 is a C2-11 straight-chain or branched acyl benzoyl group or a mono- or poly-substituted benzoyl group on a benzene ring, wherein the mono- or poly-substituted group is an alkyl, alkoxy, nitro, or cyano group.

[0042] The compound of formula A2 of this invention is prepared from the compound of formula A1 by a halogenation reaction, as shown in the following reaction formula:

[0043]

[0044] Where X is a halogen.

[0045] This invention discloses a method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol. A preferred embodiment is as follows: preparation via halogenation, acylation, enzymatic reduction, further reduction, and cyclization.

[0046]

[0047] Wherein, X is a halogen, R1 is defined as in claim 1, the enzyme is an aldehyde-ketone reductase, the amino acid sequence is the protein of SEQ ID NO: 1 or the protein of SEQ ID NO: 1 after substitution, deletion or addition of one or more amino acid residues has aldehyde-ketone reductase activity, or the protein has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 1 and has aldehyde-ketone reductase activity.

[0048] The present invention discloses a method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol. A preferred embodiment comprises a halogenation reaction, an acylation reaction, an enzymatic reduction reaction, a protecting group addition reaction, further reduction, and a cyclization reaction.

[0049]

[0050] Wherein, X is a halogen, R1 is defined as in claim 1, R2 is defined as in claim 2, and the enzyme is defined as described above.

[0051] Following the enzymatic construction of the chiral center described above, the hydroxyl group is protected to provide an intermediate compound of (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol, with the following structural formula:

[0052]

[0053] Wherein, R1 and R3 are hydrogen or hydroxyl protecting groups, and R2 is a hydroxyl protecting group. The hydroxyl protecting group is alkyl, silyl, C2-11 acyl, C4-9 cycloalkenyl, aryl, aralkyl, arylacyl, phenyl, or substituted phenyl. The silyl group is tetramethylsilyl, trimethylsilyl, triethylsilyl, tri-n-butylsilyl, or tert-butyldimethylsilyl. The alkyl group is C1-C8. The aryl group is phenyl, furanyl, thiophene, or indole. The substituted phenyl group is alkyl-substituted phenyl, alkoxyalkyl-substituted phenyl, nitroalkyl-substituted phenyl, or halogen-substituted phenyl. The alkyl-substituted phenyl group is benzyl, diphenylmethyl, or triphenylmethyl; the alkoxyalkyl-substituted phenyl group is p-methoxybenzyl; the nitroalkyl-substituted phenyl group is p-nitrobenzyl; and the halogen-substituted phenyl group is p-chlorophenyl.

[0054] The hydroxyl protecting group is preferably benzoyl, mono- or poly-substituted benzoyl, tert-butyl, or benzyl on the benzene ring.

[0055] The reduction reaction described above relates to a method for reducing another carbonyl group to a hemiketal product, and the reaction formula is as follows:

[0056]

[0057] The two reactions mentioned above can be directly represented by the general formula:

[0058]

[0059] R3 is R2 or hydrogen. The definitions of R1 and R2 are the same as above.

[0060] The above-mentioned hemiketal products can be separated or used in the next reaction without separation. For example, a method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol via a cyclization reaction...

[0061]

[0062] R3 is R2 or hydrogen. The definitions of R1 and R2 are the same as above.

[0063] The reducing agent used in the reduction reaction to hemiacetal can be a boron-based reducing agent, an aluminum-based reducing agent, or a silicon-lithium-based reducing agent. Examples include sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, red aluminum, lithium aluminum hydride, diisobutylaluminum hydride, diisopropylaminolithium, and hexamethyldisilaminolithium.

[0064] The reagents for the ring-closing reaction are common acids or bases in the art.

[0065] A preferred embodiment of the present invention includes: first constructing a chiral center, selectively attaching a hydroxyl protecting group, then reducing it to a hemiketal product, and performing a cyclization reaction to prepare (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol.

[0066]

[0067] R3 is R2 or hydrogen. The definitions of R1 and R2 are the same as above.

[0068] However, an embodiment of the present invention can also involve first reducing the product to a hemiketal, then constructing a chiral center, selectively separating the components, and finally performing a cyclization reaction to prepare (3R, 3aS, 6aR)-hexahydrofurano[2, 3-b]-3-ol.

[0069] R3 is R2 or hydrogen. The definitions of R1 and R2 are the same as above.

[0070] In the above-described acylation reaction of the present invention,

[0071]

[0072] Preferably, R1 is a C2-11 straight-chain or branched acyl benzoyl group or a mono- or poly-substituted benzoyl group on a benzene ring.

[0073] The reagent for the acylation reaction is a substituted benzoic acid compound or its salt. The substituents of the substituted benzoic acid compound can be alkyl, alkoxy, nitro, cyano, etc., and the substitution can be monosubstituted or polysubstituted.

[0074] An acid-coating agent may be added to the acylation reaction. The acid-coating agent is a base, either an organic or inorganic base, such as triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, etc.

[0075] R1 can be any other substituent, such as tert-butyl, which can be obtained through conversion of compound A3 or by reaction of compound A2 with tert-butyric acid. Compound A3 is prepared by reacting compound A2.

[0076]

[0077] The above-mentioned compound A2 is prepared from compound A1 via a halogenation reaction, as shown in the following reaction formula:

[0078]

[0079] Where X is a halogen.

[0080] The reagents used in the halogenation reaction of this invention are hydrohalic acids, etc.

[0081] The method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol of this invention can also be achieved through halogenation, enzymatic reduction, acylation, and reductive cyclization, as shown in the following reaction formulas:

[0082]

[0083] Where X is a halogen, and R1 is defined in the same way as above.

[0084] The method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol provided by this invention employs an enzymatic method to construct the chiral form, enabling the product to be prepared in high yield and with high optical purity. Although existing technologies, such as the aforementioned European application, disclose carbonyl reductase peptides or microorganisms containing carbonyl reductase peptides to reduce carbonyl groups to hydroxyl groups, the enzyme used in this invention has more significant advantages, namely, the product obtained with higher optical purity and more suitable reaction conditions. The method for preparing (3R,3aS,6aR)-hexahydrofurano[2,3-b]-3-ol of this invention is suitable for industrial production. Detailed Implementation

[0085] To further understand the present invention, the preparation method of the hexahydrofuranofuranol derivative, its intermediate, and the preparation method thereof are described in detail below with reference to embodiments. It should be understood that these embodiments are described only to further illustrate the features of the present invention and are not intended to limit the scope of the invention or the scope of the claims.

[0086] Example 1:

[0087]

[0088] Add compound A1 and dichloromethane to a reaction flask, cool, weigh bromine, dilute with dichloromethane, transfer the diluted bromine to a dropping funnel, and slowly add it dropwise, controlling the internal temperature. After addition, maintain the reaction temperature, controlling the internal temperature. Add water, control the temperature, let stand, and separate the layers. Transfer the lower organic phase to another reaction flask, add water, extract, and separate. Transfer the lower organic phase to another reaction flask, add 5% NaHCO3 aqueous solution, extract, and separate. Transfer the lower organic phase to another reaction flask, combine the upper aqueous phase, add dichloromethane, extract, and separate. Discard the aqueous phase, combine the organic phases, add water, extract, separate, discard the aqueous phase, and concentrate the lower organic phase in a rotary evaporator until no solvent flows out, yielding 90-95%.

[0089] Example 2:

[0090]

[0091] Add acetone, compound A2 (where X is bromine), and benzoic acid to the reaction flask, stir, and cool. Add triethylamine to the dropping vessel, starting with slow dropwise addition while controlling the internal temperature. After the addition is complete, raise the temperature to room temperature and stir the reaction. After the reaction is complete, filter under vacuum. Transfer the filtrate to a distillation flask for vacuum distillation, controlling the temperature at 50-60°C, until a solid paste appears in the distillation flask. Add ethyl acetate and continue distillation. Add ethyl acetate to the distillation flask and stir until dissolved. Transfer the liquid from the distillation flask to the reaction flask, wash with saturated brine, allow to stand, separate the layers, combine the aqueous layers, extract with ethyl acetate, allow to stand, separate the layers, discard the aqueous layer, combine the organic layers, add anhydrous sodium sulfate to the organic layer, stir and dry, and filter under vacuum. The filtrate was transferred to a reaction flask for vacuum distillation, maintaining the temperature at 50-60°C, until a solid paste formed in the reaction solution. A portion of ethyl acetate was added, and the mixture was stirred and refluxed until dissolved. The temperature was maintained at 50-60°C. Heptane was then added slowly dropwise to the reaction flask from a dropping vessel until the addition was complete. After slow cooling, the mixture was kept at this temperature with stirring, filtered, and the crude target solid was dried, yielding 70-75%.

[0092] Example 3:

[0093] Preparation of whole cells of genetically engineered bacteria containing aldehyde-ketone reductase

[0094] The specific preparation method for the recombinant aldehyde-ketone reductase genetically engineered bacteria is as follows: The gene sequence of aldehyde-ketone reductase from *Saccharomyces kudriavzevii* is selected and artificially designed. The artificially designed sequence is synthesized in its entirety (contracted to GenScript Biotech Co., Ltd.), cloned into the Nde I and Xho I restriction sites of the expression vector pET28a, and transformed into competent E. coli BL21(DE3) cells. Positive transformants are selected and sequenced to obtain the recombinant expression vector. This recombinant expression vector is then transformed into *E. coli BL21(DE3)* strain to obtain a recombinant aldehyde-ketone reductase genetically engineered bacteria capable of inducing recombinant aldehyde-ketone reductase expression.

[0095] The recombinant aldehyde-ketone reductase-producing genetically engineered bacteria were inoculated into LB medium containing kanamycin and cultured overnight at 37°C to obtain a seed culture. The seed culture was then inoculated into TB medium containing kanamycin at a volume of 1% of the TB medium volume. The culture was then incubated at 37°C for 2–5 h, followed by induction with sterile IPTG to a final IPTG concentration of 0.1 mM. The culture was then continued at 25°C for 20 h. Finally, whole cells of the aldehyde-ketone reductase-producing genetically engineered bacteria from *Saccharomyces kudriavzevii* were obtained by high-speed centrifugation. The obtained whole cells were then sonicated to obtain the lysed enzyme solution of the aldehyde-ketone reductase-producing genetically engineered bacteria from *Saccharomyces kudriavzevii*. The aldehyde-ketone reductase is a protein with the amino acid sequence shown in SEQ ID NO: 1, and the nucleotide sequence of the aldehyde-ketone reductase gene is shown in SEQ ID NO: 2 of the sequence listing.

[0096] After induction, a distinct protein band was observed at 45 kDa, indicating that aldehyde reductase was efficiently expressed in the recombinant bacteria.

[0097] The enzyme activity of purified aldehyde-ketone reductase was determined using a 0.25 mL reaction system containing Tris-HCl, pH 8.0, 2 mmol / L NADPH, and 0.1 mmol / L substrate. In addition to an appropriate amount of enzyme, the decrease in absorbance at 340 nm was measured. Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the oxidation of 1 μmol NADPH per minute under the above conditions.

[0098] The results showed that the recombinant genetically engineered aldehyde-ketone reductase had an activity that was more than 20% higher than that of the aldehyde-ketone reductase sequence in the European patent (EP2634180A1) and more than 50% higher than that of the unmutated aldehyde-ketone reductase.

[0099] The whole cells of the aldehyde-ketone reductase genetically engineered bacteria used in the embodiments of this invention were all prepared using this method.

[0100] The glucose dehydrogenases used in the embodiments and comparative examples of this invention are all commercial enzymes purchased from Sigma-Aldrich.

[0101] The algorithm for calculating the ee value of enantiomer overexpression is as follows:

[0102] ee(syn)=([R ,R]-[S ,S]) / ([R ,R]+[S ,S])

[0103] 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])}.

[0104] Enzymatic reduction reaction, reaction formula:

[0105]

[0106] Step 1: The reaction was carried out in a 1L shake flask with a reaction volume of 300mL. The whole-cell lysate of the aldehyde-ketone reductase genetically engineered bacteria was suspended in 260mL of sterile potassium phosphate buffer solution. Glucose dehydrogenase was added, and the cells were sonicated for 50min to lyse. Then, 25g of glucose and 0.42g of NADP+ were added to the shake flask. 8g of the reactant was weighed and dissolved in 40mL of DMSO. The DMSO solution containing the substrate was slowly poured into the shake flask. After 2 hours of reaction, 12g of glucose was added. The total amount of whole-cell aldehyde-ketone reductase genetically engineered bacteria added was 75g / L, and the amount of glucose dehydrogenase added was 25mg / L. The temperature of the transformation system was controlled at 37℃. The transformation reaction was carried out in a shaker at 200r / min for 12 hours, yielding the target product with a conversion rate of 97.8%.

[0107] Step 2: Purify the conversion solution of the target product obtained in Step 1. Add an equal volume of ethyl acetate to the reaction system, extract at 37°C for 15 min, repeat 3 times, collect the ethyl acetate layer by centrifugation, add 5% anhydrous magnesium sulfate to the collected ethyl acetate layer, shake for 15 min, filter to remove magnesium sulfate, and then concentrate the dehydrated ethyl acetate layer under high temperature and reduced pressure to obtain 7.41 g of the target product, with a de value of 96.2% and an ee (anti) value of 99.5%.

[0108] Example 4:

[0109]

[0110] Add 20.00 g of solid compound B (R1 is benzoyl) to a clean, dry four-necked flask. Add toluene, stir, and purge under nitrogen. Cool under nitrogen protection. Add toluene to a constant-pressure dropping funnel under nitrogen protection. Add 26.50 g (26.00 ml) of 70% red aluminum solution to the constant-pressure dropping funnel under nitrogen protection. After preparation, once the reaction solution has cooled to -15 to -10°C, begin adding red aluminum dropwise, controlling the temperature. After the addition is complete, maintain the temperature. Add pure water and ethyl acetate sequentially to a 1000ml clean four-necked flask. While stirring, add sulfuric acid to the 1000ml clean four-necked flask, cool, and maintain the temperature. Then, drop the reaction solution into the sulfuric acid solution, controlling the temperature of both the reaction solution and the sulfuric acid solution (0-10°C). After the addition is complete, add ethyl acetate and stir. Add a 10% sodium bicarbonate aqueous solution to a clean 1000ml four-necked flask, cool, stop stirring, and allow the reaction solution to stand and separate into layers. Transfer the upper organic layer to a 10% sodium bicarbonate solution. After stirring the sodium aqueous solution, extract the lower acidic aqueous layer with ethyl acetate, allow it to stand and separate into layers, discard the acidic aqueous layer, and transfer the secondary ethyl acetate layer to a 10% sodium bicarbonate aqueous solution with stirring and temperature control. Allow it to stand and separate into layers, add ethyl acetate to the alkaline aqueous layer for extraction, stir, and control the temperature. Add pure water to the alkaline-washed organic layer for washing, stir, control the temperature, and allow it to separate into layers. Wash the organic layer with water and set aside. Continue extraction with ethyl acetate after washing the water layer, allow it to stand and separate into layers, discard the aqueous layer, combine the organic layers, add sodium sulfate to dry, filter, wash with ethyl acetate, and concentrate the filtrate under reduced pressure at 40-70℃ to obtain approximately 17g of oil, with a yield of 84.33%.

[0111] Example 5:

[0112]

[0113] Methanol (10.00 ml) and compound C (R1 is benzoyl) (1.00 g) were added sequentially to a reaction flask. The temperature was lowered to -15 to -5 °C, and sodium hydroxide aqueous solution (10.00 ml) was added dropwise. After the addition was complete, the mixture was kept warm and stirred until the reactants reacted completely. 10% sulfuric acid aqueous solution (4.00 g) was added dropwise. After the addition was complete, the reaction was kept warm. After the reaction was completed, saturated sodium carbonate aqueous solution was added dropwise to adjust the pH. Methanol was removed by distillation under reduced pressure. Methyl tert-butyl ether was added for extraction, and the organic layer was separated. Sodium chloride was added to the aqueous layer until saturated, and then dichloromethane was added for extraction. The aqueous layer was separated for recovery, and dichloromethane was removed from the organic layer by distillation under reduced pressure, yielding 0.38 g of product, with a yield of 73.77%.

[0114] Example 6:

[0115]

[0116] The enzyme was prepared using the same method as in Example 3.

[0117] Step 1: The reaction was carried out in a 5L beaker, with the reaction system controlled at 2L. Whole cells of the aldehyde-ketone reductase genetically engineered bacteria were suspended in 1.7L of sterile potassium phosphate buffer solution in a shake flask. Glucose dehydrogenase was added, and the cells were sonicated for 50 minutes to disrupt the cells. Then, 25g of glucose and 0.42g of NADP+ were added to the beaker. 80g of substrate was weighed and dissolved in 300mL of DMSO. The DMSO solution containing the substrate was slowly poured into the shake flask. After reacting for 2 hours, 12g of glucose was added. The total amount of whole cells of the aldehyde-ketone reductase genetically engineered bacteria added was 75g / L, and the amount of glucose dehydrogenase added was 25mg / L. The temperature of the transformation system was controlled at 37℃. The transformation reaction was carried out in a beaker with a magnetic stirrer speed controlled at 200r / min for 12 hours, yielding the transformation solution of the target compound with a conversion rate of 97.8%.

[0118] Step 2: The conversion solution containing the Darunavir intermediate compound of formula VIII obtained in Step 1 was purified as in Example 3, yielding 77.10 g of the target compound. The de value of the prepared target compound was 95.3%, and the ee (anti) value was 99.6%.

[0119] Example 7:

[0120]

[0121] Step 1: The reaction was carried out in a 1L shake flask with a reaction volume of 300mL. Whole cells of the aldehyde-ketone reductase genetically engineered bacteria were suspended in 250mL of sterile deionized water in the shake flask. Glucose dehydrogenase was added, along with 10mL of 2.5mol / L glucose and 0.26g of NADP+. 10g of substrate was weighed and dissolved in 30mL of butyl acetate. The butyl acetate solution containing the substrate was slowly poured into the shake flask. After reacting for 1 hour, 10mL of 2.5mol / L glucose was added. The total amount of whole cells of the aldehyde-ketone reductase genetically engineered bacteria added was 75g / L, and the amount of glucose dehydrogenase added was 25mg / L. The temperature of the transformation system was controlled at 37℃. The transformation reaction was carried out in a shaker at a speed of 200r / min for 12 hours, yielding the transformation solution of the target compound with a conversion rate of 97.8%.

[0122] Step 2: The conversion solution of the target compound obtained in Step 1 was purified by adding an equal volume of ethyl acetate to the reaction system and extracting at 37°C for 15 min. This process was repeated three times. The ethyl acetate layer was collected by centrifugation. 5% anhydrous magnesium sulfate was added to the collected ethyl acetate layer, and the mixture was shaken for 15 min. The magnesium sulfate was then removed by filtration. The dehydrated ethyl acetate layer was then concentrated under high temperature and reduced pressure to obtain 9.55 g of the target compound. The de value of the prepared target compound was 99.1%, and the ee (anti) value of the enantiomer was 99.7%. 1H NMR (600MHz, CDCl3) δ2 .269~2.301 (m, 1H, J=6Hz), 2.367~2.404 (m, 1H), 2.954~2.993 (m, 1H, J=6Hz), 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]+.

[0123] Example 8:

[0124]

[0125] Step 1: The reaction was carried out in a 5L beaker, with the reaction system controlled at 2L. Whole cells of the aldehyde-ketone reductase genetically engineered bacteria were suspended in 1.5L of sterile deionized water in the beaker. Glucose dehydrogenase was added, along with 100mL of 2.5mol / L glucose and 3g of NADP+. 100g of the substrate was weighed and dissolved in 300mL of butyl acetate. The ethyl acetate solution containing the substrate was slowly poured into the beaker. After reacting for 1 hour, 100mL of 2.5mol / L glucose was added. The total amount of whole cells of the aldehyde-ketone reductase genetically engineered bacteria added was 100g / L, and the amount of glucose dehydrogenase added was 25mg / L. The temperature of the transformation system was controlled at 28℃. The transformation reaction was carried out in the beaker with the magnetic stirrer rotated at 200r / min for 12 hours, yielding the target compound with a conversion rate of 97.8%.

[0126] Step 2: The conversion solution of the target compound obtained in Step 1 was purified as in Example 3, yielding 9.42 g of the target compound. The de value of the prepared target compound was 96.9%, and the ee (anti) value of the enantiomer was 99.4%.

[0127] Example 9:

[0128]

[0129] Step 1: The reaction was carried out in a 1L shake flask with a reaction volume of 300mL. Whole cells of the aldehyde-ketone reductase genetically engineered bacteria were suspended in 250mL of sterile deionized water in the shake flask. Glucose dehydrogenase was added, along with 10mL of 2.5mol / L glucose and 0.26g of NADP+. 10g of substrate was weighed and dissolved in 30mL of butyl acetate. The butyl acetate solution containing the substrate was slowly poured into the shake flask. After reacting for 1 hour, 10mL of 2.5mol / L glucose was added. The total amount of whole cells of the aldehyde-ketone reductase genetically engineered bacteria added was 75g / L, and the amount of glucose dehydrogenase added was 25mg / L. The temperature of the transformation system was controlled at 37℃. The transformation reaction was carried out in a shaker at a speed of 200r / min for 12 hours, yielding a transformation solution containing the target compound with a conversion rate of 97.8%.

[0130] Step 2: The conversion solution of the target compound obtained in Step 1 was purified as in Example 3, and 9.37 g of the target compound was obtained. The de value of the prepared target compound was 97.1%, and the ee (anti) value was 99.5%.

[0131] Example 10:

[0132]

[0133] Step 1: The reaction was carried out in a 5L beaker, with the reaction system controlled at 2L. Whole cells of the aldehyde-ketone reductase genetically engineered bacteria were suspended in 1.6L of sterile deionized water in the beaker. Glucose dehydrogenase was added, along with 100mL of 2.5mol / L glucose and 2.5g of NADP+. 100g of substrate was weighed and dissolved in 200mL of butyl acetate. The butyl acetate solution containing the substrate was slowly poured into the beaker. After reacting for 1 hour, 100mL of 2.5mol / L glucose was added. The total amount of whole cells of the aldehyde-ketone reductase genetically engineered bacteria added was 50g / L, and the amount of glucose dehydrogenase added was 25mg / L. The temperature of the transformation system was controlled at 25℃. The transformation reaction was carried out in the beaker with the magnetic stirrer rotated at 200r / min for 12 hours, yielding a transformation solution containing the target compound with a conversion rate of 97.8%.

[0134] Step 2: The conversion solution of the target compound obtained in Step 1 was purified as in Example 3. 93.1 g of the target compound was used. The de value of the prepared target compound was 95.6%, and the ee (anti) value was 99.6%.

[0135] Example 11: Comparative Example

[0136]

[0137] Step 1: The reaction was carried out in a 1L shake flask, with the reaction system controlled at 300mL. Whole cells of the aldehyde-ketone reductase genetically engineered bacteria were suspended in 250mL of sterile deionized water in the shake flask. The coding sequence of the aldehyde-ketone reductase gene from the whole cells of the genetically engineered bacteria used is as disclosed in European Patent EP2634180 (i.e., SEQ ID NO 12 in Patent EP2634180). The sequence was synthesized as a whole gene (commissioned by GenScript Biotech Co., Ltd.), and the preparation method is as described in Example 3. Glucose dehydrogenase was then added, along with 10mL of 2.5mol / L glucose and 0.26g of NADP+. 10g of the substrate was weighed and dissolved in 30mL of butyl acetate. The butyl acetate solution containing the substrate was slowly poured into the shake flask. After reacting for 1 hour, 2.5mol / L glucose was added again. 10 mL of glucose was added, along with 75 g / L of whole cells of the aldehyde-ketone reductase genetically engineered bacteria and 25 mg / L of glucose dehydrogenase. The temperature of the transformation system was controlled at 37°C. The transformation reaction was carried out in a shaker at a speed of 200 r / min for 12 h.

[0138] The purification steps were as described in Example 3, yielding 8.11 g of the target compound. The de value of the prepared target compound was 85.1%, and the ee (anti) value of the enantiomer was 93.3%.

[0139] Example 12: Comparative Example

[0140]

[0141] Step 1: The reaction was carried out in a 1L shake flask, with the reaction volume controlled at 300mL. Whole cells of the engineered aldehyde-ketone reductase strain *Saccharomyces kudriavzevii* were suspended in 250mL of sterile deionized water in the shake flask. The coding sequence of the aldehyde-ketone reductase gene from the engineered aldehyde-ketone reductase strain used is shown in SEQ ID NO 3 (the coding sequence of the aldehyde-ketone reductase gene was not artificially designed). The sequence was synthesized by whole-gene synthesis (commissioned by GenScript Biotech Co., Ltd.). The preparation method was as described in Example 3: glucose dehydrogenase was added, along with 10mL of 2.5mol / L glucose and 0.26g of NADP+. 10g of the substrate was then weighed and dissolved in 30mL of butyl acetate. The butyl acetate solution containing the substrate was slowly poured into the shake flask. After reacting for 1 hour, 2... 10 mL of 0.5 mol / L glucose was added, along with 75 g / L of whole cells of the aldehyde-ketone reductase genetically engineered bacteria and 25 mg / L of glucose dehydrogenase. The temperature of the transformation system was controlled at 37℃. The transformation reaction was carried out in a shaker at a speed of 200 r / min for 12 h.

[0142] The purification steps were as described in Example 3, yielding 7.73 g of the target compound. The de value of the prepared target compound was 79.6%, and the ee (anti) value of the enantiomer was 88.7.

[0143]

[0144]

Claims

1. A method for preparing a (3R,3aS,6aR)-hexahydrofurano[2,3-b]furan-3-ol intermediate compound, characterized in that, The steps for preparing compound C-2 via enzymatic reduction include the following: Wherein, R1 is a hydrogen or hydroxyl protecting group; The enzyme is an aldehyde-ketone reductase, a protein with the amino acid sequence SEQ ID NO:

1.

2. A method for preparing a (3R,3aS,6aR)-hexahydrofurano[2,3-b]furan-3-ol intermediate compound, characterized in that, The steps include the following: preparing compound C-2 via enzymatic reduction and further adding a protecting group: Wherein, R1 is defined as in claim 1, R2 is a hydroxyl protecting group, and the enzyme is the same as in claim 1.

3. The preparation method according to claim 1 or 2, characterized in that, R1 is C 2-11 The benzoyl group is a straight-chain or branched acyl group, a benzoyl group, or a mono- or poly-substituted benzoyl group on a benzene ring, wherein the mono- or poly-substituted group is an alkyl group, an alkoxy group, a nitro group, or a cyano group.

4. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the aldehyde-ketone reductase gene is SEQ ID NO:

2.

5. The preparation method according to claim 1, characterized in that, The aldehyde-ketone reductase is a genetically engineered bacterial whole cell, a broken enzyme solution, a lyophilized powder, or an immobilized enzyme or immobilized cell.

6. The preparation method according to claim 1, characterized in that, The amount of whole cells of the aldehyde-ketone reductase genetically engineered bacteria used in the reaction system is 10-100 g / L, and the conversion temperature is 25-37℃.

7. The preparation method according to claim 1, characterized in that, The reaction is carried out in the presence of a solvent.

8. The preparation method according to claim 7, characterized in that, The solvent is a mixture of water or a buffer solution and an organic solvent.

9. The preparation method according to claim 8, characterized in that, 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.

10. The preparation method according to claim 8, characterized in that, The organic solvent is selected from one or more of DMSO, ethyl acetate, butyl acetate, isopropanol, DMF, TBME, dichloromethane, and vinyl acetate.

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