WO2019196262A1 - Preparation method for hexahydrofuro-furan-ol derivative, and intermediate of derivative and preparation method therefor - Google Patents

Preparation method for hexahydrofuro-furan-ol derivative, and intermediate of derivative and preparation method therefor Download PDF

Info

Publication number
WO2019196262A1
WO2019196262A1 PCT/CN2018/097736 CN2018097736W WO2019196262A1 WO 2019196262 A1 WO2019196262 A1 WO 2019196262A1 CN 2018097736 W CN2018097736 W CN 2018097736W WO 2019196262 A1 WO2019196262 A1 WO 2019196262A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
reaction
formula
preparation
hexahydrofuro
Prior art date
Application number
PCT/CN2018/097736
Other languages
French (fr)
Chinese (zh)
Inventor
高照波
陈建华
万志东
何大伟
周增乐
马晓东
向韦
林荆鑫
梅义将
Original Assignee
江苏瑞科医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏瑞科医药科技有限公司 filed Critical 江苏瑞科医药科技有限公司
Publication of WO2019196262A1 publication Critical patent/WO2019196262A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C235/16—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/80—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms having carbon atoms of carboxamide groups and keto groups bound to the same carbon atom, e.g. acetoacetamides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C07D213/803—Processes of preparation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/89—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
    • C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D237/10—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D237/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D241/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D309/28—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/30—Oxygen atoms, e.g. delta-lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/32—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the field of pharmaceutical synthesis, in particular to a preparation method of a hexahydrofuranfuranol derivative, an intermediate thereof and a preparation method thereof.
  • a compound having the structure of the following formula Z is a chemical name of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol:
  • One of the derivatives of hexahydrofuranfuranol is an intermediate of the anti-AIDS drug darunavir.
  • the compound of the formula (3) is prepared from the starting compound of the formula (1).
  • European Patent Application EP 2 634 180 A1 (Application Date: 2012-1-3), which is incorporated herein by reference.
  • the chirality of the Nave intermediate the specification lists a number of commercially available enzymes such as Saccharomyces cerevisiae YNL331C, etc., and mentions that the compound represented by the following formula Ia is a suitable configuration.
  • the preparation method of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention starts from the selection of starting materials and the construction of chiral configuration, and researches and develops different
  • the preparation method of the key intermediate of darunavir is prepared from the starting materials in the above prior patent application.
  • the preparation method of the invention adopts the enzymatic method to construct chirality, has low cost and mild reaction conditions, and provides another suitable industrialization route for the preparation of the key intermediate of darunavir.
  • 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, which has the following structural formula:
  • R Z is alkoxy, -NR 4 R 5 ;
  • R P is hydrogen, -CCO-Bn, -CC-OH, -C-CO 2 R 6 , -C-CO 2 Ar;
  • R L is -OH Or a hydroxy protecting group.
  • the hydroxy protecting group may be -O-Bn, -O-Bz, -OCOR 6 , -OR 6 , -OAr, -OCOAr, etc.
  • Ar may be an aryl group, a heteroaryl group, a substituted aryl group or a substituted heteroaryl group.
  • the heteroaryl group may be furan, pyridine, thiophene, anthracene or naphthalene;
  • R 4 , R 5 are the same or different phenyl, alkyl or substituted phenyl;
  • 6 is an alkyl group; * means chirality.
  • R P is hydrogen, -CCO-Bn, -CC-OH, -C-CO 2 R 6 , -C-CO 2 Ar;
  • R L is -OH, -O-Bn, -O-Bz, -OCOR6 , -OR 6 , -OAr, -OCOAr, etc.;
  • R 4 , R 5 are the same or different phenyl, alkyl or substituted phenyl;
  • R 6 is alkyl; * represents chirality.
  • the second aspect of the present invention provides a preparation method of the above intermediate compound, which is prepared by enzymatic reduction reaction of a compound of formula B to form a chiral preparation.
  • R Z , R P , and R L are the same as defined above.
  • the enzyme is a biological enzyme such as an aldosterone reductase, wherein the aldosterone reductase is derived from the Saccharomyces kudriavzevii strain.
  • the protein having the amino acid sequence of SEQ ID NO: 1, or the protein having aldehyde ketoreductase activity after substitution, deletion or addition of one or several amino acid residues of SEQ ID NO: 1, or SEQ ID NO: 1
  • the amino acid sequence shown has a protein having 80% or more homology and having aldosterone reductase activity; the nucleotide sequence thereof is shown in SEQ ID NO: 2 in the Sequence Listing.
  • the aldosterone reductase may be derived from genetically engineered whole cells, disrupted enzyme solution, lyophilized powder or immobilized enzyme or immobilized cells.
  • the enzyme may be fed in an amount of from 50 to 100 g/L, and the reaction temperature may be from 25 to 37 °C.
  • the enzymatic enzyme may be selectively added to the enzymatic reduction reaction, and the coenzyme is NADP+ or NADPH.
  • 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 water or a mixed solvent of a buffer solution and an organic solvent.
  • the buffer solution is selected from one or more of a phosphate buffer solution, a carbonate buffer solution, a Tri-HCl buffer solution, a citrate buffer solution, or a 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.
  • the biotransformation process of the enzymatic reduction reaction of the present invention is monitored by HPLC-MS and HPLC until the substrate is fully utilized.
  • the chiral preparation is carried out by enzymatic reduction reaction of the compound of formula B2,
  • R P and R L are the same as described above.
  • the method of constructing the chirality by the reduction reaction is an enzymatic method, and the definition of the enzyme is the same as described above.
  • the above intermediate compound of the present invention is used for the preparation of an intermediate compound of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol.
  • the third aspect of the present invention provides a process for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which is prepared by a reduction reaction of a compound of formula C,
  • R Z , R P and R L are the same as described above.
  • the compound of formula C2 is subjected to a reduction reaction for the preparation of a compound of formula C-i1,
  • the reducing agent in the reduction reaction may be a boron-based reducing agent, an aluminum-based reducing agent or a silicon-lithium-based reducing agent.
  • a boron-based reducing agent such as sodium borohydride, sodium cyanoborohydride, lithium tetrahydrogenate, red aluminum, lithium aluminum hydride, diisobutylaluminum hydride, lithium diisopropylamide, lithium hexamethyldisilazide.
  • R P and R L are the same as defined above.
  • the reagent for the ring closure reaction is an acid or a base which is common in the art.
  • the compound of the formula C-i1 prepared by the reduction reaction can be subjected to a ring closure reaction to prepare (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol without isolation.
  • a process for the preparation of a compound of the formula B2 which is prepared by reacting a dicarbonyl compound with an amine compound,
  • X 1 is a halogen, an alkoxy group or a leaving group
  • R P and R L have the same meanings as defined above.
  • X 1 or X 2 is the same or differently a halogen, an alkoxy group, a leaving group or a ring, and R P , R L have the same meanings as defined above.
  • reaction formulas are:
  • X is a halogen and R 6 is an alkyl group
  • the fifth aspect of the present invention provides another preparation method of the compound of the formula B2, which is prepared by reacting a dicarbonyl compound with a R L H compound and further reacting with a halogenated compound.
  • X is a halogen and R L is -O-Ar, -O-CO-Ar, -O-CO-Ar, -O-Bn, -O-Bz, -OR 6 or -OCOR 6 .
  • the dicarbonyl compound is reacted with benzyl alcohol, it is further prepared by reacting with a brominated compound,
  • the sixth aspect of the present invention provides a different preparation method of the compound of the formula B2, which is prepared by reacting a compound of the formula D with a halogenated compound such as a chloro compound, and further reacting with N-methylaniline,
  • the seventh aspect of the present invention provides a preparation method of the compound of the formula B2, which is prepared by using a compound of the formula D0, which is prepared by reacting a compound of the formula D0 with N-methylaniline under the action of a halogen such as Br 2 , and further subjecting the reaction,
  • R L is -O-Ar, -O-CO-Ar, -O-Bz, -OR 6 , -OCOR 6 or -OR 6
  • X is a halogen, more preferably bromine.
  • the preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is more preferably carried out by the compound of the formula D0 under the action of halogen and N-A. After the reaction of the aniline, the two-step substitution reaction, the cyclization reaction, the enzymatic reduction reaction and the reduction and ring-closing reaction are prepared.
  • R L is -O-Ar, -O-CO-Ar, -O-Bz, -OR 6 , -OCOR 6 or -OR 6
  • X is a halogen, more preferably bromine
  • R 6 is an alkyl group. More preferably, it is an ethyl group.
  • the preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is more preferably another embodiment: the compound of the formula D0 is reacted with a halogen After N-methylaniline is reacted, it is prepared by a two-step substitution reaction, an enzymatic reduction reaction and a reduction ring-closing reaction.
  • R L is -O-Ar, -O-CO-Ar, -O-Bz, -OCOR 6 or -OR 6 .
  • R L is acetoxy (-OCOCH 3 ) or t-butoxy
  • X is halogen, more preferably bromine
  • R 6 is alkyl, more preferably ethyl.
  • the preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is: after reacting the dicarbonyl compound with benzyl alcohol Further reacting with N-methylaniline, further reacting with a halogenate compound such as ethyl bromoacetate, followed by enzymatic reduction, reduction ring closure reaction,
  • X is a halogen, more preferably bromine
  • R 6 is an alkyl group, and more preferably an ethyl group.
  • the preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is: reacting a compound of the formula D with a halogenated compound After reacting with N-methylaniline, reacting with a halogen acid ester such as ethyl bromoacetate, followed by enzymatic reduction, reduction ring closure reaction,
  • X is a halogen, more preferably bromine
  • R 6 is an alkyl group, and more preferably an ethyl group.
  • the preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is: after reacting the dicarbonyl compound with benzyl alcohol Further reacting with a halogenated compound, reacting with an N-methylaniline compound, followed by an enzymatic reduction reaction, a reduction ring closure reaction,
  • X is a halogen
  • the preparation method of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol provided by the invention adopts enzymatic method to construct chirality, and can obtain product with high yield and high optical purity.
  • the prior art as disclosed in the above-mentioned European application discloses that a carbonyl reductase polypeptide or a microorganism containing a carbonyl reductase polypeptide achieves a reduction of a carbonyl group to a hydroxyl group, the enzyme used in the present invention has a more significant advantage, which is manifested in higher optical purity. Product 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.
  • the compound of formula D0 (30.00 g, 1.0 eq), dichloromethane (150 ml) was added to a 500 ml four-necked flask, and after cooling to -20 ° C at room temperature, bromine (57.00 g in 90 ml of DCM) was added dropwise, and the mixture was dropped.
  • N-methylaniline 38.20 g, 1.0 eq
  • dichloromethane 120 ml
  • triethylamine 36.10 g, 1.0 eq
  • N-methylaniline (7.01 g, 1.1 eq), toluene (60 ml), compound of formula D0 (5.00 g) were added to the reaction flask under nitrogen atmosphere, and the internal temperature was raised to 100 ° C by heating, and the reaction was stopped while stirring. After cooling to room temperature, 8% dilute hydrochloric acid (60.00 ml) was added and stirred for washing. After layering, the aqueous layer was added with toluene, the organic layer was combined, washed with water, and the toluene layer was added with an appropriate amount of anhydrous sodium sulfate and dried at 45 ° C. The organic layer was concentrated under reduced pressure to give 9.80 g,yield: 86.19%.
  • the dicarbonyl compound prepared in Example 1 (2.00 g, 1.0 eq), DMF (16.00 ml), potassium acetate (0.73 g, 1.0 eq) was sequentially added to the reaction mixture and stirred. After the reaction is completed, ethyl acetate and water are added in this order, and the layers are separated by stirring. The EA layer is taken, the aqueous layer is added with EA, and the EA layer is combined, washed with water, washed with saturated brine, and then dried over anhydrous sodium sulfate. After concentration and concentration under reduced pressure at 40 ° C, 1.58 g of brown oil was obtained, yield: 85.59%.
  • Recombinant aldosterone reductase genetically engineered bacteria the specific preparation method is: selecting the gene sequence of aldehyde ketone reductase derived from Saccharomyces kudriavzevii, artificially designing, and artificially designing the sequence through whole gene synthesis (trusted by Jinsi Rui Biotechnology Co., Ltd.) The company synthesized), cloned into the Nde I and Xho I restriction sites of the expression vector pET28a, transformed the host E.coli BL21 (DE3) competent cells; picked the positive transformants and sequenced them to obtain the recombinant expression vector; The recombinant expression vector was transformed into E. coli BL21 (DE3) strain to obtain a recombinant aldosterone reductase genetically engineered bacteria which can induce expression of recombinant aldosterone reductase.
  • the recombinant aldosterone reductase genetically engineered bacteria were inoculated into LB medium containing kanamycin and cultured overnight at 37 ° C to obtain a seed culture solution; the seed culture solution was inoculated into a TB medium containing kanamycin.
  • the inoculum was 1% of the volume of TB medium containing kanamycin; then cultured at 37 ° C for 2-5 h, induced by sterile IPTG, the final concentration of IPTG reached 0.1 mM, and the culture was continued at 25 ° C. 20h.
  • aldehyde ketone reductase genetically engineered bacteria derived from Saccharomyces kudriavzevii were obtained by high speed centrifugation.
  • the whole genetically engineered bacteria obtained by ultrasonication were ultrasonically disrupted to obtain a whole enzyme shattering enzyme solution of the aldehyde ketone reductase genetic engineering bacteria derived from Saccharomyces kudriavzevii.
  • the aldosterone reductase is a protein having the amino acid sequence of SEQ ID NO: 1, and the nucleotide sequence of the aldosterone reductase gene is shown in SEQ ID NO: 2 in the Sequence Listing.
  • the enzyme activity of the pure aldehyde ketoreductase protein was determined, and the reaction system was 0.25 ml, including Tris-HCl, pH 8.0, 2 mmol/L NADPH, 0.1 mmol/L substrate. As well as an appropriate amount of enzyme, the decrease in absorbance at 340 nm was determined, and the enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the oxidation of 1 umol of NADPH per minute under the above conditions.
  • the whole cells of the aldehyde ketone reductase genetic engineering bacteria used in the examples of the present invention were prepared by this method.
  • glucose dehydrogenases used in the examples and comparative examples of the present invention are all commercial enzymes purchased from sigma-aldrich.
  • Step 1 The reaction was carried out in a 1 L shake flask, and the reaction system was controlled to 300 mL.
  • the whole cell of the aldehyde ketone reductase genetically engineered bacteria was suspended in 250 mL of sterilized deionized water in a shake flask, and glucose dehydrogenase was added thereto, and 2.5 mol was added.
  • Step 2 Purify the conversion solution of the target compound obtained in the step 1. Add an equal volume of ethyl acetate to the reaction system, extract at 37 ° C for 15 min, repeat 3 times, and collect the ethyl acetate layer by centrifugation to collect the acetic acid. After adding 5% anhydrous magnesium sulfate to the ester layer for 15 minutes, the magnesium sulfate layer was removed by filtration, and the ethyl acetate layer was subjected to high temperature under reduced pressure to give the title compound 9.32 g. 97.1%, ee (anti) value is 99.5%.
  • the compound of formula B2 is added in sequence to the reaction flask (R P is R L is an acetate group (4.40 g, 1.0 eq), THF (44.00 ml), methanol (4.40 ml), cooled at room temperature, and sodium borohydride (0.4 g, 0.8 eq) was slowly added portionwise, and stirred while stirring.
  • 10% dilute sulfuric acid is slowly added dropwise to adjust the pH, and the mixture is slowly added to room temperature and stirred.
  • the mixture is successively added with ethyl acetate and water, and the layers are separated.
  • the EA layer is taken, and the aqueous layer is added with EA to extract, and the EA layer is combined and washed with water. After washing with saturated brine, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness.
  • Example 9 The product of Example 9 (2.00 g, 1.0 eq), toluene (16.00 ml) N-methylaniline (1.05 eq) was added to the reaction flask, and the mixture was heated to reflux under nitrogen atmosphere, stirred and cooled. The 5% dilute hydrochloric acid (40.00 ml) was stirred and washed, and the toluene layer was taken. Water was added thereto, and the mixture was washed with saturated brine, and then dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure at 45 ° C, 1.83 g, yield 90.15%. .
  • Step 1 The reaction was carried out in a 1 L shake flask, the reaction system was controlled to 300 mL, and 260 mL of the sterilized potassium phosphate buffer solution was used to suspend the aldosterone reductase genetically engineered whole cell disruption enzyme solution in a shake flask, and the glucose dehydrogenase was introduced.
  • the cells were disrupted by ultrasonication for 50 min, and then 25 g of glucose, 0.42 g of NADP+ was added to the shake flask, and 8 g of the reactant was weighed, dissolved in 40 mL of DMSO, and the DMSO solution in which the substrate was dissolved was slowly poured into a shake flask.
  • Step 2 Purify the conversion product of the target product obtained in the 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, and collect the acetic acid. After adding 5% anhydrous magnesium sulfate to the ester layer for 15 minutes, the magnesium sulfate layer was removed by filtration, and the ethyl acetate layer after water removal was concentrated under high pressure under reduced pressure to give the desired product 7.39 g, with a value of 96.2%, ee (anti) The value is 99.5%.
  • the compound of formula B2 is sequentially added to the hydrogenation vessel (R P is R L is -OBn) (2.00g), ethanol (20.00ml), 10% Pd/C (0.2g), three times of hydrogen replacement, maintaining hydrogen pressure (1.0MPa), stirring at room temperature (20-25 ° C) for 8.0h After suction filtration, the filter layer was washed with about 5 ml of ethanol, and the washing liquid and the filtrate were combined, and concentrated under reduced pressure at 40 ° C to obtain 1.41 g of a product. The yield was 92.00%.
  • Step 1 The reaction was carried out in a 1 L shake flask, and the reaction system was controlled to 300 mL.
  • the whole cell of the aldehyde ketone reductase genetically engineered bacteria was suspended in 250 mL of sterilized deionized water in a shake flask, and glucose dehydrogenase was added thereto, and 2.5 mol was added.
  • Step 2 Purify the conversion solution of the target compound obtained in the step 1. Add an equal volume of ethyl acetate to the reaction system, extract at 37 ° C for 15 min, repeat 3 times, and collect the ethyl acetate layer by centrifugation to collect the acetic acid. After adding 5% anhydrous magnesium sulfate to the ester layer for 15 minutes, the magnesium sulfate layer was removed by filtration, and the ethyl acetate layer was subjected to high temperature under reduced pressure to give the title compound 9.51 g. 99.1%, the ee (anti) value of the enantiomer is 99.7%.
  • Step 1 The reaction was carried out in a 1 L shake flask, the reaction system was controlled to 300 mL, and 250 mL of sterilized deionized water was used to suspend the whole cell of the aldehyde ketone reductase genetically engineered bacteria in the shake flask, and the aldehyde ketone reductase genetically engineered bacteria were used.
  • the coding sequence of the whole cell aldosterone reductase gene is as disclosed in European Patent No. EP 2634180 (i.e., SEQ ID NO 12 in Patent EP 2634180), and the sequence is prepared by whole gene synthesis (commissioned by Kingsray Biotech Co., Ltd.).
  • the method is as in Example 3, further adding glucose dehydrogenase, adding 2.5 mol/L glucose 10 mL, 0.26 g of NADP+, and weighing 10 g of the substrate, dissolved in 30 mL of butyl acetate, and the substrate is dissolved in acetic acid.
  • the butyl ester solution was slowly poured into a shake flask.
  • Step 1 The reaction was carried out in a 1 L shake flask, the reaction system was controlled to 300 mL, and 250 mL of sterilized deionized water was used to suspend the whole cell of the aldehyde ketone reductase genetically engineered bacteria of Saccharomyces kudriavzevii in a shake flask, and the aldehyde ketone was used for reduction.
  • the coding sequence of the aldehyde ketone reductase gene of the whole cell of the enzyme genetic engineering bacteria is shown in SEQ ID NO 3, (the coding sequence of the aldosterone reductase gene is not artificially designed), and the sequence is synthesized by whole genes (trusted by Kingsray Biotechnology) Co., Ltd. synthesis, preparation method as in Example 3, adding glucose dehydrogenase, adding 2.5mol / L glucose 10mL, 0.26g of NADP +, weigh 10g of substrate, dissolved in 30mL of butyl acetate, will dissolve The butyl acetate solution with substrate was slowly poured into the shake flask.
  • sequence number of SEQ ID NO 1 is:
  • the SEQ ID NO 2 sequence number is:
  • the SEQ ID NO 3 sequence number is:

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to the field of pharmaceutical synthesis, and in particularly relates to a preparation method for a hexahydrofuro-furan-ol derivative, and an intermediate of the derivative and a preparation method therefor. A dicarbonyl compound or a compound represented by formula DO is used as a starting material in the preparation method, wherein X1 and X2 are halogen or alkoxy identically or differently, or are constructed into a ring. In the preparation process for the hexahydrofuro-furan-ol derivative, an enzymic method is used to construct chirality; and such a technical means can prepare products with very high optical purity. The preparation method can commercialize and prepare a key intermediate (3R, 3aS, 6aR)-hexahydrofuro[2, 3-b]-3-ol of darunavir, which is a very economic way suitable for industrial production.

Description

六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法Preparation method of hexahydrofuranfuranol derivative, intermediate thereof and preparation method thereof
本申请要求于2018年4月12日提交中国专利局、申请号为201810326250.2、发明名称为“六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims priority to Chinese patent application filed on April 12, 2018, the Chinese Patent Office, application number 201810326250.2, the invention titled "Preparation method of hexahydrofuranfuranol derivative, its intermediate and its preparation method" The entire contents are hereby incorporated by reference.
技术领域Technical field
本发明涉及医药合成领域，具体涉及六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法。The invention relates to the field of pharmaceutical synthesis, in particular to a preparation method of a hexahydrofuranfuranol derivative, an intermediate thereof and a preparation method thereof.
背景技术Background technique
具有下列式Z结构的化合物为化学名称为(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇：A compound having the structure of the following formula Z is a chemical name of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol:
Figure PCTCN2018097736-appb-000001
Figure PCTCN2018097736-appb-000001
属于六氢呋喃并呋喃醇衍生物的一种，是抗艾滋病药物达芦那韦的中间体。One of the derivatives of hexahydrofuranfuranol is an intermediate of the anti-AIDS drug darunavir.
达芦那韦原研厂家泰博特克药品有限公司的中国专利申请号分别为02817639.1(申请日：2002-9-6)和200580010400.X提供了上述(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其中原料为如下的式(3)化合物，The above-mentioned (3R, 3aS, 6aR)-hexahydrofuran is provided in the Chinese patent application No. 02817639.1 (application date: 2002-9-6) and 200580010400.X of the original manufacturer of Dalu Nawei. A method for producing a 2,3-b]-3-ol, wherein the starting material is a compound of the formula (3):
Figure PCTCN2018097736-appb-000002
Figure PCTCN2018097736-appb-000002
式(3)化合物由起始原料式(1)化合物制备。The compound of the formula (3) is prepared from the starting compound of the formula (1).
Figure PCTCN2018097736-appb-000003
Figure PCTCN2018097736-appb-000003
达芦那韦仿制药厂家日本住友化学株式会社的中国专利申请号为200380109926.4提供了上述(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其中起始原料为如下的式VIII化合物，The preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol is provided in the Chinese patent application No. 200380109926.4 of Suvarnavir Imitation Pharmaceutical Co., Ltd., Sumitomo Chemical Co., Ltd. The starting material is a compound of formula VIII as follows
Figure PCTCN2018097736-appb-000004
Figure PCTCN2018097736-appb-000004
日本住友化学株式会社在手性构型的产生上，选择了使用手性配体催化剂，虽然这的确是构建手性构型的方法，但是，不太适合产业化。Sumitomo Chemical Co., Ltd. chose to use a chiral ligand catalyst in the generation of a chiral configuration. Although this is indeed a method for constructing a chiral configuration, it is not suitable for industrialization.
达芦那韦仿制药厂家瑞士Lonza Ltd公司的欧洲专利申请EP2634180A1(申请日：2012-1-3)涉及使用羰基还原酶多肽或含有羰基还原酶多肽的微生物实现羰基还原为羟基，来构建达芦那韦中间体的手性，其说明书中列举了许多种可以商业化购买的酶如酿酒酵母YNL331C等，并且提及如下式Ia所示的化合物是比较合适的构型，European Patent Application EP 2 634 180 A1 (Application Date: 2012-1-3), which is incorporated herein by reference. The use of a carbonyl reductase polypeptide or a microorganism containing a carbonyl reductase polypeptide to reduce the carbonyl group to a hydroxyl group to construct a ruthenium The chirality of the Nave intermediate, the specification lists a number of commercially available enzymes such as Saccharomyces cerevisiae YNL331C, etc., and mentions that the compound represented by the following formula Ia is a suitable configuration.
Figure PCTCN2018097736-appb-000005
Figure PCTCN2018097736-appb-000005
考虑到(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇是制备达芦那韦药物的关键中间体，有必要开发出更多的该关键中间体的制备方法，该方法不仅能获得高收率和高DE值的该关键中间体，而且成本低，反应条件温和，适合产业化。Considering that (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol is a key intermediate in the preparation of darunavir, it is necessary to develop more preparations of this key intermediate. The method not only can obtain the key intermediates with high yield and high DE value, but also has low cost, mild reaction conditions and is suitable for industrialization.
发明内容Summary of the invention
本发明的制备(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的方法从起始原料的选择和手性构型的构建上着手，研究开发出了不同于上述已有专利申请中的起始原料制备达芦那韦关键中间体的制备方法。本发明的制备方法采用酶法来构建手性，成本低，反应条件温和，为该达芦那韦关键中间体的制备提供了另一条适合产业化的路线。The preparation method of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention starts from the selection of starting materials and the construction of chiral configuration, and researches and develops different The preparation method of the key intermediate of darunavir is prepared from the starting materials in the above prior patent application. The preparation method of the invention adopts the enzymatic method to construct chirality, has low cost and mild reaction conditions, and provides another suitable industrialization route for the preparation of the key intermediate of darunavir.
为实现本发明的技术目的，本发明提供了如下的技术方案：To achieve the technical object of the present invention, the present invention provides the following technical solutions:
本发明第一方面提供了制备(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的中间体化合物，结构式如下：The first aspect of the present invention provides an intermediate compound for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which has the following structural formula:
Figure PCTCN2018097736-appb-000006
Figure PCTCN2018097736-appb-000006
其中，R Z为烷氧基，-NR 4R 5；R P为氢，-C-C-O-Bn，-C-C-OH，-C-CO 2R 6，-C-CO 2Ar；R L为-OH或羟基保护基。所述羟基保护基可以为-O-Bn，-O-Bz，-OCOR 6，-OR 6，-OAr，-OCOAr等，Ar可以为芳基，杂芳基，取代芳基或取代杂芳基，所述杂芳基可以为呋喃、吡啶、噻吩、吲哚或萘等；R P，R L并构成环；R 4，R 5相同或不同地为苯基，烷基或取代苯基；R 6为烷基；*表示手性。 Wherein R Z is alkoxy, -NR 4 R 5 ; R P is hydrogen, -CCO-Bn, -CC-OH, -C-CO 2 R 6 , -C-CO 2 Ar; R L is -OH Or a hydroxy protecting group. The hydroxy protecting group may be -O-Bn, -O-Bz, -OCOR 6 , -OR 6 , -OAr, -OCOAr, etc., Ar may be an aryl group, a heteroaryl group, a substituted aryl group or a substituted heteroaryl group. , the heteroaryl group may be furan, pyridine, thiophene, anthracene or naphthalene; R P , R L and form a ring; R 4 , R 5 are the same or different phenyl, alkyl or substituted phenyl; 6 is an alkyl group; * means chirality.
较优选地，结构式如下：More preferably, the structural formula is as follows:
Figure PCTCN2018097736-appb-000007
Figure PCTCN2018097736-appb-000007
其中，R P为氢，-C-C-O-Bn，-C-C-OH，-C-CO 2R 6，-C-CO 2Ar；R L为-OH，-O-Bn，-O-Bz，-OCOR6，-OR 6，-OAr，-OCOAr等；R 4，R 5相同或不同地为苯基，烷基或取代苯基；R 6为烷基；*表示手性。 Wherein R P is hydrogen, -CCO-Bn, -CC-OH, -C-CO 2 R 6 , -C-CO 2 Ar; R L is -OH, -O-Bn, -O-Bz, -OCOR6 , -OR 6 , -OAr, -OCOAr, etc.; R 4 , R 5 are the same or different phenyl, alkyl or substituted phenyl; R 6 is alkyl; * represents chirality.
更优选地，结构式如下：More preferably, the structural formula is as follows:
Figure PCTCN2018097736-appb-000008
Figure PCTCN2018097736-appb-000008
Figure PCTCN2018097736-appb-000009
Figure PCTCN2018097736-appb-000009
Figure PCTCN2018097736-appb-000010
Figure PCTCN2018097736-appb-000010
本发明第二方面提供了上述中间体化合物的制备方法，由式B化合物经酶法还原反应构建手性制备，The second aspect of the present invention provides a preparation method of the above intermediate compound, which is prepared by enzymatic reduction reaction of a compound of formula B to form a chiral preparation.
Figure PCTCN2018097736-appb-000011
Figure PCTCN2018097736-appb-000011
其中，R Z，R P，R L的定义与上述定义相同。 Wherein, the definitions of R Z , R P , and R L are the same as defined above.
所述酶是一种生物酶，如醛酮还原酶，其中，醛酮还原酶来源于Saccharomyces kudriavzevii菌株。其氨基酸序列为SEQ ID NO：1所示的蛋白质，或SEQ ID NO：1经过一个或几个氨基酸残基取代、缺失或添加后具有醛酮还原酶活性的蛋白质，或与SEQ ID NO：1所示的氨基酸序列具有80％以上同源性且具有醛酮还原酶活性的蛋白质；其核苷酸序列如序列表中SEQ ID NO：2所示。醛酮还原酶可以来自于基因工程菌全细胞、破碎酶液、冻干粉或固定化酶或固定化细胞。The enzyme is a biological enzyme such as an aldosterone reductase, wherein the aldosterone reductase is derived from the Saccharomyces kudriavzevii strain. The protein having the amino acid sequence of SEQ ID NO: 1, or the protein having aldehyde ketoreductase activity after substitution, deletion or addition of one or several amino acid residues of SEQ ID NO: 1, or SEQ ID NO: 1 The amino acid sequence shown has a protein having 80% or more homology and having aldosterone reductase activity; the nucleotide sequence thereof is shown in SEQ ID NO: 2 in the Sequence Listing. The aldosterone reductase may be derived from genetically engineered whole cells, disrupted enzyme solution, lyophilized powder or immobilized enzyme or immobilized cells.
所述酶的投料量可以为50-100g/L，所述反应温度可以为25-37℃。The enzyme may be fed in an amount of from 50 to 100 g/L, and the reaction temperature may be from 25 to 37 °C.
所述酶法还原反应中可以选择性加入辅酶，所述辅酶为NADP+或NADPH。The enzymatic enzyme may be selectively added to the enzymatic reduction reaction, and the coenzyme is NADP+ or NADPH.
所述酶法还原反应中可以选择性加入葡萄糖脱氢酶。Glucose dehydrogenase may be selectively added to the enzymatic reduction reaction.
所述酶法还原反应在溶剂存在的条件下进行，所述溶剂为水或缓冲溶液与有机溶剂组成 的混合溶剂。The enzymatic reduction reaction is carried out in the presence of a solvent which is water or a mixed solvent of a buffer solution and an organic solvent.
所述缓冲溶液选自磷酸盐缓冲溶液、碳酸盐缓冲溶液、Tri-HCl缓冲溶液、柠檬酸盐缓冲溶液或MOPS缓冲溶液中的一种或多种。The buffer solution is selected from one or more of a phosphate buffer solution, a carbonate buffer solution, a Tri-HCl buffer solution, a citrate buffer solution, or a MOPS buffer solution.
所述有机溶剂选自DMSO、乙酸乙酯、乙酸丁酯、异丙醇、DMF、TBME、二氯甲烷、乙酸乙烯酯中的一种或几种。The organic solvent is selected from one or more of DMSO, ethyl acetate, butyl acetate, isopropanol, DMF, TBME, dichloromethane, and vinyl acetate.
本发明酶法还原反应的生物转化过程中利用HPLC-MS和HPLC进行监控，直至底物完全利用。The biotransformation process of the enzymatic reduction reaction of the present invention is monitored by HPLC-MS and HPLC until the substrate is fully utilized.
较优选地，由式B2化合物经酶法还原反应构建手性制备，More preferably, the chiral preparation is carried out by enzymatic reduction reaction of the compound of formula B2,
Figure PCTCN2018097736-appb-000012
Figure PCTCN2018097736-appb-000012
其中，R P，R L的定义与上述相同。 Wherein, the definitions of R P and R L are the same as described above.
所述还原反应构建手性的方法为酶法，所述酶的定义与上述相同。The method of constructing the chirality by the reduction reaction is an enzymatic method, and the definition of the enzyme is the same as described above.
本发明上述中间体化合物用于制备(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的中间体化合物。The above intermediate compound of the present invention is used for the preparation of an intermediate compound of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol.
本发明第三方面提供了制备(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的方法，由式C化合物经还原反应制备式C-i化合物，The third aspect of the present invention provides a process for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which is prepared by a reduction reaction of a compound of formula C,
Figure PCTCN2018097736-appb-000013
Figure PCTCN2018097736-appb-000013
然后，式C-i化合物经关环反应制备(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇，Then, a compound of formula C-i is subjected to a ring closure reaction to prepare (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol,
Figure PCTCN2018097736-appb-000014
Figure PCTCN2018097736-appb-000014
其中，R Z，R P，R L的定义与上述相同。 Wherein, the definitions of R Z , R P and R L are the same as described above.
较优选地，由式C2化合物经还原反应用于制备式C-i1化合物，More preferably, the compound of formula C2 is subjected to a reduction reaction for the preparation of a compound of formula C-i1,
Figure PCTCN2018097736-appb-000015
Figure PCTCN2018097736-appb-000015
所述还原反应中还原剂可以为硼类还原剂，铝类还原剂或硅锂类还原剂。如硼氢化钠，氰基硼氢化钠，四氢铝锂，红铝，氢化铝锂，二异丁基氢化铝，二异丙基氨基锂，六甲基二硅基氨基锂。The reducing agent in the reduction reaction may be a boron-based reducing agent, an aluminum-based reducing agent or a silicon-lithium-based reducing agent. Such as sodium borohydride, sodium cyanoborohydride, lithium tetrahydrogenate, red aluminum, lithium aluminum hydride, diisobutylaluminum hydride, lithium diisopropylamide, lithium hexamethyldisilazide.
进一步，式C-i1化合物经关环反应制备(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇，Further, a compound of the formula C-i1 is subjected to a ring closure reaction to prepare (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol,
Figure PCTCN2018097736-appb-000016
Figure PCTCN2018097736-appb-000016
其中，R P，R L的定义与上述定义相同。 Wherein, the definitions of R P and R L are the same as defined above.
所述关环反应的试剂为本领域常见的酸或碱。The reagent for the ring closure reaction is an acid or a base which is common in the art.
经还原反应制备得到的式C-i1化合物可不经分离，经关环反应制备(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇。The compound of the formula C-i1 prepared by the reduction reaction can be subjected to a ring closure reaction to prepare (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol without isolation.
本发明第四方面提供了式B2化合物的制备方法，由二羰基化合物与胺类化合物反应制备，According to a fourth aspect of the present invention, there is provided a process for the preparation of a compound of the formula B2, which is prepared by reacting a dicarbonyl compound with an amine compound,
Figure PCTCN2018097736-appb-000017
Figure PCTCN2018097736-appb-000017
其中，X 1为卤素，烷氧基或离去基，R P，R L的定义与上述相同。 Wherein X 1 is a halogen, an alkoxy group or a leaving group, and R P and R L have the same meanings as defined above.
较优选地，由二羰基化合物与N-甲基苯胺反应后经进一步地取代反应制备，More preferably, it is prepared by further substituting a reaction of a dicarbonyl compound with N-methylaniline,
Figure PCTCN2018097736-appb-000018
Figure PCTCN2018097736-appb-000018
其中，X 1或X 2相同或不同地为卤素，烷氧基，离去基或并构为环，R P，R L的定义与上述相同。 Wherein X 1 or X 2 is the same or differently a halogen, an alkoxy group, a leaving group or a ring, and R P , R L have the same meanings as defined above.
更优选地，由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸酯化合物如溴乙酸乙酯反应制备，反应式分别为：More preferably, after the reaction of the dicarbonyl compound with benzyl alcohol, further reacting with N-methylaniline, further reacting with a halogenate compound such as ethyl bromoacetate, the reaction formulas are:
Figure PCTCN2018097736-appb-000019
Figure PCTCN2018097736-appb-000019
X为卤素，R 6为烷基； X is a halogen and R 6 is an alkyl group;
Figure PCTCN2018097736-appb-000020
Figure PCTCN2018097736-appb-000020
本发明第五方面提供了式B2化合物的另一种制备方法，由二羰基化合物与R LH化合物反应后，进一步与卤代化合物反应制备， The fifth aspect of the present invention provides another preparation method of the compound of the formula B2, which is prepared by reacting a dicarbonyl compound with a R L H compound and further reacting with a halogenated compound.
Figure PCTCN2018097736-appb-000021
Figure PCTCN2018097736-appb-000021
其中，X为卤素，R L为-O-Ar，-O-CO-Ar，-O-CO-Ar，-O-Bn，-O-Bz，-OR 6或-OCOR 6。 Wherein X is a halogen and R L is -O-Ar, -O-CO-Ar, -O-CO-Ar, -O-Bn, -O-Bz, -OR 6 or -OCOR 6 .
较优选地，由二羰基化合物与苯甲醇反应后，进一步与溴代化合物反应制备，More preferably, after the dicarbonyl compound is reacted with benzyl alcohol, it is further prepared by reacting with a brominated compound,
Figure PCTCN2018097736-appb-000022
Figure PCTCN2018097736-appb-000022
本发明第六方面提供了式B2化合物的不同的另一种制备方法，由式D化合物与卤代化合物如氯代化合物反应后，进一步与N-甲基苯胺反应制备，The sixth aspect of the present invention provides a different preparation method of the compound of the formula B2, which is prepared by reacting a compound of the formula D with a halogenated compound such as a chloro compound, and further reacting with N-methylaniline,
Figure PCTCN2018097736-appb-000023
当Ar为苄基时，反应式如下：
Figure PCTCN2018097736-appb-000023
When Ar is a benzyl group, the reaction formula is as follows:
Figure PCTCN2018097736-appb-000024
Figure PCTCN2018097736-appb-000024
本发明第七方面提供了式B2化合物的以式D0化合物为原料的另一种制备方法，由式D0 化合物在卤素如Br 2的作用下与N-甲基苯胺反应后经进一步取代反应制备， The seventh aspect of the present invention provides a preparation method of the compound of the formula B2, which is prepared by using a compound of the formula D0, which is prepared by reacting a compound of the formula D0 with N-methylaniline under the action of a halogen such as Br 2 , and further subjecting the reaction,
Figure PCTCN2018097736-appb-000025
Figure PCTCN2018097736-appb-000025
其中，R L为-O-Ar，-O-CO-Ar，-O-Bz，-OR 6，-OCOR 6或-OR 6，X为卤素，较优选地，为溴。 Wherein R L is -O-Ar, -O-CO-Ar, -O-Bz, -OR 6 , -OCOR 6 or -OR 6 , and X is a halogen, more preferably bromine.
本发明上述(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，较优选地实施方式为：由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，环合反应，酶法还原反应和还原关环反应制备，The preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is more preferably carried out by the compound of the formula D0 under the action of halogen and N-A. After the reaction of the aniline, the two-step substitution reaction, the cyclization reaction, the enzymatic reduction reaction and the reduction and ring-closing reaction are prepared.
Figure PCTCN2018097736-appb-000026
Figure PCTCN2018097736-appb-000026
其中，R L为-O-Ar，-O-CO-Ar，-O-Bz，-OR 6，-OCOR 6或-OR 6，X为卤素，较优选地，为溴，R 6为烷基，较优选地，为乙基。 Wherein R L is -O-Ar, -O-CO-Ar, -O-Bz, -OR 6 , -OCOR 6 or -OR 6 , X is a halogen, more preferably bromine, and R 6 is an alkyl group. More preferably, it is an ethyl group.
本发明上述(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，较优选地另一种实施方式为：由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，酶法还原反应和还原关环反应制备，The preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention is more preferably another embodiment: the compound of the formula D0 is reacted with a halogen After N-methylaniline is reacted, it is prepared by a two-step substitution reaction, an enzymatic reduction reaction and a reduction ring-closing reaction.
Figure PCTCN2018097736-appb-000027
Figure PCTCN2018097736-appb-000027
其中，R L为-O-Ar，-O-CO-Ar，-O-Bz，-OCOR 6或-OR 6。优选地，R L为乙酸基(-OCOCH 3)或叔丁氧基，X为卤素，较优选地，为溴，R 6为烷基，较优选地，为乙基。 Wherein R L is -O-Ar, -O-CO-Ar, -O-Bz, -OCOR 6 or -OR 6 . Preferably, R L is acetoxy (-OCOCH 3 ) or t-butoxy, X is halogen, more preferably bromine, R 6 is alkyl, more preferably ethyl.
本发明上述(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，较优选地其他另一种实施方式为：由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸 酯化合物如卤乙酸乙酯反应，后经酶法还原反应，还原关环反应制备，The preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention, more preferably another embodiment is: after reacting the dicarbonyl compound with benzyl alcohol Further reacting with N-methylaniline, further reacting with a halogenate compound such as ethyl bromoacetate, followed by enzymatic reduction, reduction ring closure reaction,
Figure PCTCN2018097736-appb-000028
Figure PCTCN2018097736-appb-000028
其中，X为卤素，较优选地，为溴，R 6为烷基，较优选地，为乙基。 Wherein X is a halogen, more preferably bromine, R 6 is an alkyl group, and more preferably an ethyl group.
本发明上述(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，较优选地其他另一种实施方式为：由式D化合物与卤代化合物反应后，与N-甲基苯胺反应，与卤酸酯如卤乙酸乙酯反应，后经酶法还原反应，还原关环反应制备，The preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention, more preferably another embodiment is: reacting a compound of the formula D with a halogenated compound After reacting with N-methylaniline, reacting with a halogen acid ester such as ethyl bromoacetate, followed by enzymatic reduction, reduction ring closure reaction,
Figure PCTCN2018097736-appb-000029
Figure PCTCN2018097736-appb-000029
其中，X为卤素，较优选地，为溴，R 6为烷基，较优选地，为乙基。 Wherein X is a halogen, more preferably bromine, R 6 is an alkyl group, and more preferably an ethyl group.
本发明上述(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，较优选地其他另一种实施方式为：由二羰基化合物与苯甲醇反应后，进一步与卤代化合物反应，与N-甲基苯胺化合物反应，后经酶法还原反应，还原关环反应制备，The preparation method of the above (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol of the present invention, more preferably another embodiment is: after reacting the dicarbonyl compound with benzyl alcohol Further reacting with a halogenated compound, reacting with an N-methylaniline compound, followed by an enzymatic reduction reaction, a reduction ring closure reaction,
Figure PCTCN2018097736-appb-000030
Figure PCTCN2018097736-appb-000030
其中，X为卤素。Wherein X is a halogen.
本发明提供的(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，采用酶法构建手性，能够高收率，高光学纯度的制备得到产物。虽然现有技术如上述欧洲申请中已公开羰基还原酶多肽或含有羰基还原酶多肽的微生物实现羰基还原为羟基，然而，本发明使用的酶具备更显著地优势，体现在更高光学纯度的得到产物和更适宜的反应条件上。本发明的(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法适宜于工业化生产。The preparation method of (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol provided by the invention adopts enzymatic method to construct chirality, and can obtain product with high yield and high optical purity. . Although the prior art as disclosed in the above-mentioned European application discloses that a carbonyl reductase polypeptide or a microorganism containing a carbonyl reductase polypeptide achieves a reduction of a carbonyl group to a hydroxyl group, the enzyme used in the present invention has a more significant advantage, which is manifested in higher optical purity. Product 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.
具体实施方式detailed description
为了进一步理解本发明，下面结合实施例对本发明提供的六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法进行详细说明。需要理解的是，这些实施例描述只是为进一步详细说明本发明的特征，而不是对本发明范围或本发明权利要求范围的限制。In order to further understand the present invention, the preparation method of the hexahydrofuranfuranol derivative provided by the present invention, the intermediate thereof and the preparation method thereof will be described in detail below with reference to the examples. It is to be understood that the description of the embodiments of the present invention is not intended to limit the scope of the invention or the scope of the invention.
实施例1：Example 1:
Figure PCTCN2018097736-appb-000031
Figure PCTCN2018097736-appb-000031
于500ml四口瓶中依次加入式D0化合物(30.00g，1.0eq)，二氯甲烷(150ml)，内温冷却至-20℃后，滴加溴素(57.00g溶于90mlDCM中)，滴毕；于另一500ml四口瓶中依次加入N-甲基苯胺(38.20g，1.0eq)，二氯甲烷(120ml)，三乙胺(36.10g，1.0eq)，内温冷却至-20℃后，将前一反应瓶中的反应液滴加至该反应液中，滴毕，缓慢升温至室温并保温搅拌，反应完成后，依次采用水，饱和食盐水洗涤，无水硫酸钠干燥，40℃减压浓缩干后得红棕色油状物81.29g，收率84.33％。The compound of formula D0 (30.00 g, 1.0 eq), dichloromethane (150 ml) was added to a 500 ml four-necked flask, and after cooling to -20 ° C at room temperature, bromine (57.00 g in 90 ml of DCM) was added dropwise, and the mixture was dropped. N-methylaniline (38.20 g, 1.0 eq), dichloromethane (120 ml), triethylamine (36.10 g, 1.0 eq) were added to another 500 ml four-necked flask, and cooled to -20 ° C at room temperature. The reaction liquid in the previous reaction flask is added to the reaction liquid, and the mixture is slowly heated to room temperature and stirred while stirring. After the reaction is completed, the mixture is washed with water, saturated brine, dried over anhydrous sodium sulfate, 40 ° C. The organic layer was concentrated under reduced pressure to give a brown brown oil (yield: 81.29 g).
实施例2：Example 2:
Figure PCTCN2018097736-appb-000032
Figure PCTCN2018097736-appb-000032
于氮气氛围中，反应瓶内依次加入N-甲基苯胺(7.01g，1.1eq)，甲苯(60ml)，式D0化合物(5.00g)，加热使内温升至100℃，保温搅拌，停止反应，冷却至室温后，加入8％稀盐酸(60.00ml)搅拌洗涤，分层后，水层加入甲苯反萃，合并有机层，加入水洗涤，取甲苯层加入适量无水硫酸钠干燥，45℃减压浓缩干后得油状物9.80g，收率86.19％。N-methylaniline (7.01 g, 1.1 eq), toluene (60 ml), compound of formula D0 (5.00 g) were added to the reaction flask under nitrogen atmosphere, and the internal temperature was raised to 100 ° C by heating, and the reaction was stopped while stirring. After cooling to room temperature, 8% dilute hydrochloric acid (60.00 ml) was added and stirred for washing. After layering, the aqueous layer was added with toluene, the organic layer was combined, washed with water, and the toluene layer was added with an appropriate amount of anhydrous sodium sulfate and dried at 45 ° C. The organic layer was concentrated under reduced pressure to give 9.80 g,yield: 86.19%.
实施例3：Example 3:
Figure PCTCN2018097736-appb-000033
Figure PCTCN2018097736-appb-000033
于反应瓶中依次加入实施例1制备得到的二羰基化合物(2.00g，1.0eq)，DMF(16.00ml)，乙酸钾(0.73g，1.0eq)，搅拌。反应完成后，依次加入乙酸乙酯和水，搅拌分层后，取EA层，水层加入EA反萃，合并EA层，用水洗涤，饱和食盐水洗涤后，有机相加入适量无水硫酸钠干燥，40℃减压浓缩干后得1.58g棕色油状物，收率85.59％。The dicarbonyl compound prepared in Example 1 (2.00 g, 1.0 eq), DMF (16.00 ml), potassium acetate (0.73 g, 1.0 eq) was sequentially added to the reaction mixture and stirred. After the reaction is completed, ethyl acetate and water are added in this order, and the layers are separated by stirring. The EA layer is taken, the aqueous layer is added with EA, and the EA layer is combined, washed with water, washed with saturated brine, and then dried over anhydrous sodium sulfate. After concentration and concentration under reduced pressure at 40 ° C, 1.58 g of brown oil was obtained, yield: 85.59%.
实施例4：Example 4:
Figure PCTCN2018097736-appb-000034
Figure PCTCN2018097736-appb-000034
于反应瓶中依次加入实施例2制备得到的棕色油状物(6.00g，1.0eq)，DCM(24.00ml)，内温冷却，缓慢滴加溴素(5.01g溶于12ml DCM，1.0eq)，滴毕，缓慢升至室温(20-25℃)保温搅拌。停止反应，30℃减压浓缩干得产物粗品6.96g，收率82.05％。The brown oil obtained in Example 2 (6.00 g, 1.0 eq), DCM (24.00 ml) was added to the reaction mixture, and the mixture was cooled at room temperature, and bromine (5.01 g dissolved in 12 ml DCM, 1.0 eq) was slowly added dropwise. After the dropwise addition, slowly increase to room temperature (20-25 ° C) and stir. The reaction was stopped, and concentrated under reduced pressure at 30 ° C to give a crude product (yield: 6.06 g).
实施例5：Example 5:
Figure PCTCN2018097736-appb-000035
Figure PCTCN2018097736-appb-000035
于反应瓶中依次加入式B2化合物(R P为氢，R L为乙酸基)(1.30g，1.0eq)，DMF(13.00ml)，溴乙酸乙酯(0.91g，1.05eq)，碳酸钾(0.79g，1.1eq)，室温搅拌。反应完成后，依次加入乙酸乙酯和水搅拌分层后，取EA层，水层加入EA反萃，合并EA层，水洗，饱和食盐水洗涤后，有机相加入适量无水硫酸钠干燥，40℃减压浓缩干后得1.40g棕色油状物，收率94.05％。 1HNMR(CDCl 3)：1.24(t，J＝6.8，3H)，2.10(s，3H)，2.68(dd，J 1＝17.2，J 2＝5.6，1H)，2.97(dd，J 1＝17.2，J 2＝8.8，1H)，3.31(s，3H)，3.99～4.03(m，1H)，4.09(dd，J 1＝14.4，J 2＝7.2，1H)，4.17(d，J＝17.2，1H)，4.56(d，J＝17.2，1H)，7.29～7.48(m，5H). The compound of formula B2 (R P is hydrogen, R L is acetate) (1.30 g, 1.0 eq), DMF (13.00 ml), ethyl bromoacetate (0.91 g, 1.05 eq), potassium carbonate ( 0.79 g, 1.1 eq), stirred at room temperature. After the reaction is completed, ethyl acetate and water are added successively and the layers are separated, and the EA layer is taken. The aqueous layer is added with EA, and the EA layer is combined, washed with water, washed with saturated brine, and then dried over a After concentration and concentration under reduced pressure <RTI ID=0.0></RTI></RTI><RTIgt; 1 H NMR (CDCl 3 ): 1.24 (t, J = 6.8, 3H), 2.10 (s, 3H), 2.68 (dd, J 1 = 17.2, J 2 = 5.6, 1H), 2.97 (dd, J 1 = 17.2) , J 2 = 8.8, 1H), 3.31 (s, 3H), 3.99 to 4.03 (m, 1H), 4.09 (dd, J 1 = 14.4, J 2 = 7.2, 1H), 4.17 (d, J = 17.2, 1H), 4.56 (d, J = 17.2, 1H), 7.29 to 7.48 (m, 5H).
实施例6：Example 6
醛酮还原酶基因工程菌全细胞的制备Preparation of whole cells of aldehyde ketone reductase genetic engineering bacteria
重组醛酮还原酶基因工程菌，具体制备方法是：选择来源于Saccharomyces kudriavzevii的醛酮还原酶的基因序列，进行人工设计，将人工设计后的序列通过全基因合成(委托金斯瑞生物科技有限公司合成)，克隆入表达载体pET28a的Nde I和Xho I酶切位点，转化宿主菌E.coli BL21(DE3)感受态细胞；挑取阳性转化子并测序鉴定后，得到重组表达载体；将重组表达载体转入E.coli BL21(DE3)菌株中，获得可以诱导表达重组醛酮还原酶的重组醛酮还原酶基因工程菌。Recombinant aldosterone reductase genetically engineered bacteria, the specific preparation method is: selecting the gene sequence of aldehyde ketone reductase derived from Saccharomyces kudriavzevii, artificially designing, and artificially designing the sequence through whole gene synthesis (trusted by Jinsi Rui Biotechnology Co., Ltd.) The company synthesized), cloned into the Nde I and Xho I restriction sites of the expression vector pET28a, transformed the host E.coli BL21 (DE3) competent cells; picked the positive transformants and sequenced them to obtain the recombinant expression vector; The recombinant expression vector was transformed into E. coli BL21 (DE3) strain to obtain a recombinant aldosterone reductase genetically engineered bacteria which can induce expression of recombinant aldosterone reductase.
将重组醛酮还原酶基因工程菌接种到含有卡那霉素的LB培养基中，于37℃过夜培养，得到种子培养液；将种子培养液接种到含卡那霉素的TB培养基中，接种量为含卡那霉素的TB培养基体积的1％；然后置于37℃下培养2-5h，加入无菌的IPTG诱导，使IPTG终浓度达到0.1mM，置于25℃下继续培养20h。最后通过高速离心得到来源于Saccharomyces kudriavzevii的醛酮还原酶基因工程菌全细胞。采用超声破碎方法对得到的基因工程菌全细胞进行超声破碎，得到来源于Saccharomyces kudriavzevii的醛酮还原酶基因工程菌全细胞的破碎酶液。醛酮还原酶为氨基酸序列为SEQ ID NO：1所示的蛋白质，醛酮还原酶基因的核苷酸序列如序列表中SEQ ID NO：2所示。The recombinant aldosterone reductase genetically engineered bacteria were inoculated into LB medium containing kanamycin and cultured overnight at 37 ° C to obtain a seed culture solution; the seed culture solution was inoculated into a TB medium containing kanamycin. The inoculum was 1% of the volume of TB medium containing kanamycin; then cultured at 37 ° C for 2-5 h, induced by sterile IPTG, the final concentration of IPTG reached 0.1 mM, and the culture was continued at 25 ° C. 20h. Finally, whole cells of the aldehyde ketone reductase genetically engineered bacteria derived from Saccharomyces kudriavzevii were obtained by high speed centrifugation. The whole genetically engineered bacteria obtained by ultrasonication were ultrasonically disrupted to obtain a whole enzyme shattering enzyme solution of the aldehyde ketone reductase genetic engineering bacteria derived from Saccharomyces kudriavzevii. The aldosterone reductase is a protein having the amino acid sequence of SEQ ID NO: 1, and the nucleotide sequence of the aldosterone reductase gene is shown in SEQ ID NO: 2 in the Sequence Listing.
经诱导表达后在45kDa处有明显的蛋白条带，表明醛酮还原酶在重组菌中得到了高效表达。After induction, there was a distinct protein band at 45kDa, indicating that aldosterone reductase was highly expressed in recombinant bacteria.
测定醛酮还原酶纯蛋白的酶活性，反应体系为0.25ml，包括Tris-HCl，pH为8.0，2mmol/L NADPH，0.1mmol/L底物
Figure PCTCN2018097736-appb-000036
以及适量酶，测定340nm处吸光度的减少，酶活力单位(U)定义为：在上述条件下，每分钟催化1umol NADPH氧化所需的酶量。 The enzyme activity of the pure aldehyde ketoreductase protein was determined, and the reaction system was 0.25 ml, including Tris-HCl, pH 8.0, 2 mmol/L NADPH, 0.1 mmol/L substrate.
Figure PCTCN2018097736-appb-000036
As well as an appropriate amount of enzyme, the decrease in absorbance at 340 nm was determined, and the enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the oxidation of 1 umol of NADPH per minute under the above conditions.
结果表明，重组的基因工程醛酮还原酶较欧洲专利(EP2634180A1)序列的醛酮还原酶活性提高了20％以上，较未突变的醛酮还原酶活性提高了50％以上。The results showed that the recombinant genetically engineered aldosterone reductase increased the aldehyde ketone reductase activity of the sequence of the European patent (EP2634180A1) by more than 20%, and the activity of the unmutated aldosterone reductase was increased by more than 50%.
本发明实施例中所使用的醛酮还原酶基因工程菌全细胞均采用此方法制备。The whole cells of the aldehyde ketone reductase genetic engineering bacteria used in the examples of the present invention were prepared by this method.
本发明实施例及对比例中所使用的葡萄糖脱氢酶均为购自于sigma-aldrich的商品酶。The glucose dehydrogenases used in the examples and comparative examples of the present invention are all commercial enzymes purchased from sigma-aldrich.
对映异构体过量表达的ee值的算法为：The algorithm for the ee value of the enantiomeric overexpression is:
ee(syn)＝([R，R]-[S，S])/([R，R]+[S，S])Ee(syn)=([R,R]-[S,S])/([R,R]+[S,S])
ee(anti)＝([R，S]-[S，R])/([R，S]+[S，R])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])}。De={([R,S]+[S,R])-([R,R]+[S,S])}/{([R,S]+[S,R])+([R , R]+[S,S])}.
酶法还原反应，反应式：Enzymatic reduction reaction, reaction formula:
Figure PCTCN2018097736-appb-000037
Figure PCTCN2018097736-appb-000037
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮醛酮还原酶基因工程菌全细胞，投入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL和0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h，得到含目标化合物的转化液，转化率为97.8％。Step 1: The reaction was carried out in a 1 L shake flask, and the reaction system was controlled to 300 mL. The whole cell of the aldehyde ketone reductase genetically engineered bacteria was suspended in 250 mL of sterilized deionized water in a shake flask, and glucose dehydrogenase was added thereto, and 2.5 mol was added. /L glucose 10mL and 0.26g NADP+, weigh 10g of the substrate, dissolved in 30mL of butyl acetate, slowly pour the solution of the substrate in butyl acetate into the shake flask, add 2.5 after 1h reaction 10 mL of mol/L glucose, 75 g/L of whole cells of aldehyde-ketone reductase genetically engineered bacteria, 25 mg/L of glucose dehydrogenase, and 37 °C of control conversion system; In the middle, the rotation speed of the shaker was controlled to 200 r/min, and the conversion time was 12 h, and a conversion liquid containing the target compound was obtained, and the conversion rate was 97.8%.
步骤2：对步骤1得到的目标化合物的转化液进行纯化，向反应体系中加入等体积的乙酸乙酯，37℃下萃取15min，重复3次，离心收集乙酸乙酯层，向收集的乙酸乙酯层中加入5％的无水硫酸镁振荡15min后过滤除去硫酸镁，再将除水后的乙酸乙酯层进行高温减压浓缩，得到目标化合物9.32g，所制备的目标化合物的de值为97.1％，ee(anti)值为99.5％。Step 2: Purify the conversion solution of the target compound obtained in the step 1. Add an equal volume of ethyl acetate to the reaction system, extract at 37 ° C for 15 min, repeat 3 times, and collect the ethyl acetate layer by centrifugation to collect the acetic acid. After adding 5% anhydrous magnesium sulfate to the ester layer for 15 minutes, the magnesium sulfate layer was removed by filtration, and the ethyl acetate layer was subjected to high temperature under reduced pressure to give the title compound 9.32 g. 97.1%, ee (anti) value is 99.5%.
实施例7：Example 7
Figure PCTCN2018097736-appb-000038
Figure PCTCN2018097736-appb-000038
于冰浴中，反应瓶内依次加入式B2化合物(R P为
Figure PCTCN2018097736-appb-000039
R L为乙酸基)(4.40g，1.0eq)，THF(44.00ml)，甲醇(4.40ml)，内温冷却，分批缓慢加入硼氢化钠(0.4g，0.8eq)，加毕保温搅拌。反应完成后，缓慢滴加10％稀硫酸调节pH，滴毕缓慢升至室温搅拌，依次加入乙酸乙酯和水搅拌分层后，取EA层，水层加入EA反萃，合并EA层，水洗，饱和食盐水洗涤后，有机相加入适量无水硫酸钠干燥，40℃减压浓缩干后得3.98g无色油状物，收率86.43％。 In the ice bath, the compound of formula B2 is added in sequence to the reaction flask (R P is
Figure PCTCN2018097736-appb-000039
R L is an acetate group (4.40 g, 1.0 eq), THF (44.00 ml), methanol (4.40 ml), cooled at room temperature, and sodium borohydride (0.4 g, 0.8 eq) was slowly added portionwise, and stirred while stirring. After the reaction is completed, 10% dilute sulfuric acid is slowly added dropwise to adjust the pH, and the mixture is slowly added to room temperature and stirred. The mixture is successively added with ethyl acetate and water, and the layers are separated. The EA layer is taken, and the aqueous layer is added with EA to extract, and the EA layer is combined and washed with water. After washing with saturated brine, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness.
实施例8：Example 8
Figure PCTCN2018097736-appb-000040
Figure PCTCN2018097736-appb-000040
于反应瓶中依次加入式B2化合物(R P为
Figure PCTCN2018097736-appb-000041
R L为乙酸基)(1.50g，1.0eq)，THF(20.00ml)，内温冷却，分批缓慢加入LiAlH 4(0.68g，4.0eq)，加毕缓慢升温至室温(20-25℃)保温搅拌。反应完成后，内温冷却，缓慢滴加10％稀硫酸调节pH，滴毕缓慢升至室温搅拌。内温冷却，缓慢滴加4％NaOH水 溶液调节反应液pH，调毕，加入甲苯搅拌萃取，合并甲苯层，取水层，加入DCM搅拌萃取，合并DCM层，取DCM层加入适量无水硫酸钠干燥，40℃减压浓缩干后得无色油状物0.43g，收率74.55％。 Add the compound of formula B2 in sequence to the reaction flask (R P is
Figure PCTCN2018097736-appb-000041
R L is an acetate group (1.50 g, 1.0 eq), THF (20.00 ml), cooled at room temperature, and slowly added LiAlH 4 (0.68 g, 4.0 eq) in portions, and slowly warmed to room temperature (20-25 ° C). Stir in heat. After the reaction was completed, the internal temperature was cooled, and 10% dilute sulfuric acid was slowly added dropwise to adjust the pH, and the mixture was slowly stirred to room temperature and stirred. After cooling at internal temperature, the pH of the reaction solution was adjusted slowly by adding 4% NaOH aqueous solution. After the adjustment, the toluene layer was added and stirred, and the toluene layer was combined. The aqueous layer was taken, and the mixture was extracted with DCM. The DCM layer was combined, and the DCM layer was added and dried over anhydrous sodium sulfate. After concentration and concentration under reduced pressure at 40 ° C, 0.43 g of colorless oil was obtained, yield 74.55%.
实施例9：Example 9
Figure PCTCN2018097736-appb-000042
Figure PCTCN2018097736-appb-000042
于反应瓶中依次加入式D化合物(25.55g，1.0eq)，DCM(350.00ml)，内温冷却，缓慢滴入吡啶(29.33g，2.15eq)，滴毕，保温搅拌，缓慢滴加氯代化合物(35.00g，1.07eq)，滴毕后缓慢升至室温(20-25℃)保温搅拌，加入稀盐酸溶液，搅拌静置分层后，取有机层，水层加入DCM反萃，合并DCM层，依次加入水，饱和食盐水洗涤，加入适量无水硫酸钠干燥，40℃减压浓缩干后得产物53.56g，收率96.73％。The compound of formula D (25.55 g, 1.0 eq), DCM (350.00 ml) was added to the reaction flask, and cooled at room temperature. pyridine (29.33 g, 2.15 eq) was slowly added dropwise, and the mixture was stirred, and the mixture was slowly stirred. The compound (35.00 g, 1.07 eq) was slowly warmed to room temperature (20-25 ° C) and stirred while stirring. After adding a dilute hydrochloric acid solution, the mixture was stirred and allowed to stand for separation. The organic layer was taken and the aqueous layer was added to DCM to extract and combine DCM. The layers were successively added with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 40 ° C to give the product 53.56 g.
实施例10：Example 10:
Figure PCTCN2018097736-appb-000043
Figure PCTCN2018097736-appb-000043
于反应瓶中依次加入实施例9中的产物(2.00g，1.0eq)，甲苯(16.00ml)N-甲基苯胺(1.05eq)，氮气保护下升温至回流保温搅拌，冷却至室温后，加入5％稀盐酸(40.00ml)搅拌洗涤，取甲苯层，依次加入水，饱和食盐水洗涤分液后，加入适量无水硫酸钠干燥，45℃减压浓缩的产物粗品1.83g，收率90.15％。The product of Example 9 (2.00 g, 1.0 eq), toluene (16.00 ml) N-methylaniline (1.05 eq) was added to the reaction flask, and the mixture was heated to reflux under nitrogen atmosphere, stirred and cooled. The 5% dilute hydrochloric acid (40.00 ml) was stirred and washed, and the toluene layer was taken. Water was added thereto, and the mixture was washed with saturated brine, and then dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure at 45 ° C, 1.83 g, yield 90.15%. .
实施例11：Example 11
Figure PCTCN2018097736-appb-000044
Figure PCTCN2018097736-appb-000044
于500ml四口瓶中依次加入苯甲醇(20.76g，1.0eq)，THF(180.00ml)，内温冷却，加入NaH(16.11g，2.1eq)，加毕保温搅拌，缓慢滴加二羰基化合物(30.00g，0.95eq)，加毕于室温下自然升温搅拌反应。降温，滴加5％的盐酸溶液调节pH，调毕，加入乙酸乙酯萃取分液，其中水层用乙酸乙酯萃取分液，合并有机相后用水洗涤分液，有机层继续用饱和食盐水洗涤分液，有机相加入适量无水硫酸钠干燥，45℃减压旋蒸后得油状物40.65g，收率89.62％。To a 500 ml four-necked flask, benzyl alcohol (20.76 g, 1.0 eq), THF (180.00 ml) was added, and the mixture was cooled at room temperature. NaH (16.11 g, 2.1 eq) was added, and the mixture was stirred while stirring, and the dicarbonyl compound was slowly added dropwise. 30.00 g, 0.95 eq), the reaction was stirred at room temperature under natural temperature. The temperature was lowered, the pH was adjusted by adding 5% hydrochloric acid solution, and the mixture was adjusted. The mixture was extracted with ethyl acetate. The aqueous layer was extracted with ethyl acetate. The organic phase was combined and washed with water, and the organic layer was washed with saturated brine. The organic layer was dried and dried over anhydrous sodium sulfate at 45 ° C to give 40.65 g of oil, yield 89.62%.
实施例12：Example 12
Figure PCTCN2018097736-appb-000045
Figure PCTCN2018097736-appb-000045
于反应瓶中依次加入二羰基化合物(5.00g，1.0eq)，甲苯(40.00ml)，N-甲基苯胺(1.2eq)，氮气保护下升温至回流保温搅拌，冷却至室温后，加入5％稀盐酸搅拌洗涤，取甲苯层，依次加入水，饱和食盐水洗涤 分液后，加入适量无水硫酸钠干燥，45℃减压浓缩的产物粗品3.27g，收率52.02％。Dicarbonyl compound (5.00 g, 1.0 eq), toluene (40.00 ml), N-methylaniline (1.2 eq) were added successively to the reaction flask, and the mixture was heated to reflux under nitrogen atmosphere, stirred, and cooled to room temperature. The mixture was stirred and washed with dilute hydrochloric acid, and the toluene layer was taken, and then water was added thereto, and the mixture was washed with a saturated aqueous solution of sodium chloride, and then dried over anhydrous sodium sulfate, and then, the crude product was concentrated under reduced pressure at 4 ° C, 3.27 g, yield 52.02%.
实施例13：Example 13
Figure PCTCN2018097736-appb-000046
Figure PCTCN2018097736-appb-000046
于反应瓶中依次加入式B2化合物(R P为氢，
Figure PCTCN2018097736-appb-000047
R L为-OBn)(2.00g，1.0eq)，DMF(20.00ml)，溴乙酸乙酯(1.24g，1.1eq)，碳酸钾(1.12g，1.2eq)，室温(20-25℃)搅拌，停止反应，加入EA和水搅拌静置分层，取EA层，水层加入EA反萃，合并EA层，将EA层以水洗涤后，加入适量无水硫酸钠干燥除水，40℃减压干燥后得产物粗品2.21g，收率85.59％。 Adding the compound of formula B2 in sequence to the reaction flask (R P is hydrogen,
Figure PCTCN2018097736-appb-000047
R L is -OBn) (2.00 g, 1.0 eq), DMF (20.00 ml), ethyl bromoacetate (1.24 g, 1.1 eq), potassium carbonate (1.12 g, 1.2 eq), stirred at room temperature (20-25 ° C) Stop the reaction, add EA and water, stir and disperse the layer, take the EA layer, add the EA layer to the water layer, combine the EA layer, wash the EA layer with water, add the appropriate amount of anhydrous sodium sulfate and remove the water, 40 ° C minus The product obtained by pressure drying was 2.21 g of crude product, and the yield was 85.59%.
1HNMR(CDCl 3)：1.18(t，J＝7.2，3H)，2.62(dd，J 1＝16.8，J 2＝6.4，1H)，2.78(dd，J 1＝17.2，J 2＝7.6，1H)，3.24(s，3H)，3.81(dd，J 1＝48.0，J 2＝16.8，2H)，4.01～4.06(m，2H)，4.13(t，J＝6.8，1H)，7.09～7.32(m，10H). 1 H NMR (CDCl 3 ): 1.18 (t, J = 7.2, 3H), 2.62 (dd, J 1 = 16.8, J 2 = 6.4, 1H), 2.78 (dd, J 1 = 17.2, J 2 = 7.6, 1H) ), 3.24 (s, 3H), 3.81 (dd, J 1 = 48.0, J 2 = 16.8, 2H), 4.01 to 4.06 (m, 2H), 4.13 (t, J = 6.8, 1H), 7.09 to 7.32 ( m, 10H).
实施例14：Example 14
Figure PCTCN2018097736-appb-000048
Figure PCTCN2018097736-appb-000048
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用260mL灭菌后的磷酸钾缓冲溶液悬浮醛酮还原酶基因工程菌全细胞破碎酶液，投入葡萄糖脱氢酶，超声50min破碎细胞，再向摇瓶中加入25g的葡萄糖，0.42g的NADP+，再称取8g的反应物，溶于40mL的DMSO中，将溶有底物的DMSO溶液缓慢倒入摇瓶中，反应2h后补加12g的葡萄糖，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h，得到目标产物的转化液，转化率为97.8％。Step 1: The reaction was carried out in a 1 L shake flask, the reaction system was controlled to 300 mL, and 260 mL of the sterilized potassium phosphate buffer solution was used to suspend the aldosterone reductase genetically engineered whole cell disruption enzyme solution in a shake flask, and the glucose dehydrogenase was introduced. The cells were disrupted by ultrasonication for 50 min, and then 25 g of glucose, 0.42 g of NADP+ was added to the shake flask, and 8 g of the reactant was weighed, dissolved in 40 mL of DMSO, and the DMSO solution in which the substrate was dissolved was slowly poured into a shake flask. After 2 hours of reaction, 12 g of glucose was added, and the amount of whole cells of the aldehyde-ketone reductase genetically engineered bacteria was 75 g/L, the amount of glucose dehydrogenase was 25 mg/L, and the temperature of the control system was 37 ° C; In the shaker, the rotation speed of the shaker was controlled to 200 r/min, and the conversion time was 12 h, and the conversion product of the target product was obtained, and the conversion rate was 97.8%.
步骤2：对步骤1得到的目标产物的转化液进行纯化，向反应体系中加入等体积的乙酸乙酯，37℃下萃取15min，重复3次，离心收集乙酸乙酯层，向收集的乙酸乙酯层中加入5％的无水硫酸镁振荡15min后过滤除去硫酸镁，再将除水后的乙酸乙酯层进行高温减压浓缩，得到目标产物7.39g，de值为96.2％，ee(anti)值为99.5％。Step 2: Purify the conversion product of the target product obtained in the 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, and collect the acetic acid. After adding 5% anhydrous magnesium sulfate to the ester layer for 15 minutes, the magnesium sulfate layer was removed by filtration, and the ethyl acetate layer after water removal was concentrated under high pressure under reduced pressure to give the desired product 7.39 g, with a value of 96.2%, ee (anti) The value is 99.5%.
实施例15：Example 15
Figure PCTCN2018097736-appb-000049
Figure PCTCN2018097736-appb-000049
于氢化釜中依次加入式B2化合物(R P为
Figure PCTCN2018097736-appb-000050
R L为-OBn)(2.00g)，乙醇(20.00ml)，10％Pd/C (0.2g)，氢气置换三次，保持氢气压力(1.0MPa)，室温(20-25℃)搅拌8.0h后，抽滤，滤层采用约5ml乙醇洗涤后，合并洗涤液与滤液，40℃减压浓缩得产物1.41g，收率92.00％。 The compound of formula B2 is sequentially added to the hydrogenation vessel (R P is
Figure PCTCN2018097736-appb-000050
R L is -OBn) (2.00g), ethanol (20.00ml), 10% Pd/C (0.2g), three times of hydrogen replacement, maintaining hydrogen pressure (1.0MPa), stirring at room temperature (20-25 ° C) for 8.0h After suction filtration, the filter layer was washed with about 5 ml of ethanol, and the washing liquid and the filtrate were combined, and concentrated under reduced pressure at 40 ° C to obtain 1.41 g of a product. The yield was 92.00%.
实施例16：Example 16:
Figure PCTCN2018097736-appb-000051
Figure PCTCN2018097736-appb-000051
于反应瓶中依次加入式B2化合物(R P为
Figure PCTCN2018097736-appb-000052
R L为-OH)(1.50g，1.0eq)，THF(20.00ml)，内温冷却，分批缓慢加入LiAlH 4(0.77g，4.0eq)，加毕缓慢升温至室温(20-25℃)保温搅拌。反应完成后，内温冷却，缓慢滴加10％稀盐酸调节pH，滴毕缓慢升至室温(20-25℃)搅拌。内温冷却，缓慢滴加4％NaOH水溶液调节反应液pH，调毕，加入甲苯搅拌萃取，合并甲苯层，取水层，加入DCM搅拌萃取，合并DCM层，取DCM层加入适量无水硫酸钠干燥，40℃减压浓缩干后得无色油状物0.48g，收率72.40％。 Add the compound of formula B2 in sequence to the reaction flask (R P is
Figure PCTCN2018097736-appb-000052
R L is -OH) (1.50 g, 1.0 eq), THF (20.00 ml), cooled at room temperature, and slowly added LiAlH 4 (0.77 g, 4.0 eq) in portions, and slowly warmed to room temperature (20-25 ° C). Stir in heat. After the reaction was completed, the internal temperature was cooled, and 10% dilute hydrochloric acid was slowly added dropwise to adjust the pH, and the mixture was slowly stirred to room temperature (20-25 ° C) and stirred. After cooling at internal temperature, the pH of the reaction solution was adjusted slowly by adding 4% NaOH aqueous solution. After the adjustment, the toluene layer was added and stirred, and the toluene layer was combined. The aqueous layer was taken, and the mixture was extracted with DCM. The DCM layer was combined, and the DCM layer was added and dried over anhydrous sodium sulfate. After concentration and concentration under reduced pressure at 40 ° C, 0.48 g of colorless oil was obtained, yield 72.40%.
实施例17：Example 17
Figure PCTCN2018097736-appb-000053
Figure PCTCN2018097736-appb-000053
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮醛酮还原酶基因工程菌全细胞，投入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL，0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h，得到目标化合物的转化液，转化率为97.8％。Step 1: The reaction was carried out in a 1 L shake flask, and the reaction system was controlled to 300 mL. The whole cell of the aldehyde ketone reductase genetically engineered bacteria was suspended in 250 mL of sterilized deionized water in a shake flask, and glucose dehydrogenase was added thereto, and 2.5 mol was added. /L glucose 10mL, 0.26g of NADP+, weigh 10g of substrate, dissolved in 30mL of butyl acetate, slowly pour the solution of the substrate in butyl acetate into the shake flask, add 2.5 after 1h reaction 10 mL of mol/L glucose, 75 g/L of whole cells of aldehyde-ketone reductase genetically engineered bacteria, 25 mg/L of glucose dehydrogenase, and 37 °C of control conversion system; In the middle, the rotation speed of the shaker was controlled to 200 r/min, and the conversion time was 12 h, and the conversion liquid of the target compound was obtained, and the conversion rate was 97.8%.
步骤2：对步骤1得到的目标化合物的转化液进行纯化，向反应体系中加入等体积的乙酸乙酯，37℃下萃取15min，重复3次，离心收集乙酸乙酯层，向收集的乙酸乙酯层中加入5％的无水硫酸镁振荡15min后过滤除去硫酸镁，再将除水后的乙酸乙酯层进行高温减压浓缩，得到目标化合物9.51g，所制备的目标化合物的de值为99.1％，对映异构体的ee(anti)值为99.7％。Step 2: Purify the conversion solution of the target compound obtained in the step 1. Add an equal volume of ethyl acetate to the reaction system, extract at 37 ° C for 15 min, repeat 3 times, and collect the ethyl acetate layer by centrifugation to collect the acetic acid. After adding 5% anhydrous magnesium sulfate to the ester layer for 15 minutes, the magnesium sulfate layer was removed by filtration, and the ethyl acetate layer was subjected to high temperature under reduced pressure to give the title compound 9.51 g. 99.1%, the ee (anti) value of the enantiomer is 99.7%.
实施例18：对照实施例Example 18: Comparative Example
Figure PCTCN2018097736-appb-000054
Figure PCTCN2018097736-appb-000054
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮 醛酮还原酶基因工程菌全细胞，所用的醛酮还原酶基因工程菌全细胞的醛酮还原酶基因的编码序列如欧洲专利EP2634180中公布序列(即专利EP2634180中的SEQ ID NO 12所示)，序列通过全基因合成(委托金斯瑞生物科技有限公司合成)，制备方法如实施例3，再加入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL，0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h。Step 1: The reaction was carried out in a 1 L shake flask, the reaction system was controlled to 300 mL, and 250 mL of sterilized deionized water was used to suspend the whole cell of the aldehyde ketone reductase genetically engineered bacteria in the shake flask, and the aldehyde ketone reductase genetically engineered bacteria were used. The coding sequence of the whole cell aldosterone reductase gene is as disclosed in European Patent No. EP 2634180 (i.e., SEQ ID NO 12 in Patent EP 2634180), and the sequence is prepared by whole gene synthesis (commissioned by Kingsray Biotech Co., Ltd.). The method is as in Example 3, further adding glucose dehydrogenase, adding 2.5 mol/L glucose 10 mL, 0.26 g of NADP+, and weighing 10 g of the substrate, dissolved in 30 mL of butyl acetate, and the substrate is dissolved in acetic acid. The butyl ester solution was slowly poured into a shake flask. After 1 h of reaction, 10 mL of 2.5 mol/L glucose was added, and the amount of whole cells of the aldehyde ketone reductase genetically engineered bacteria was 75 g/L, and the amount of glucose dehydrogenase was 25 mg/ L, the temperature of the control conversion system was 37 ° C; the conversion reaction was carried out in a shaker, the rotation speed of the shaker was controlled to 200 r / min, and the conversion time was 12 h.
纯化步骤如实施例3，得到目标化合物8.07g，所制备的目标化合物的de值为85.1％，对映异构体的ee(anti)值为93.3％。Purification step As in Example 3, 8.07 g of the title compound was obtained. The desired compound had a value of 85.1%, and an ee (anti) value of the enantiomer was 93.3%.
实施例19：对照实施例Example 19: Comparative Example
Figure PCTCN2018097736-appb-000055
Figure PCTCN2018097736-appb-000055
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮以Saccharomyces kudriavzevii的醛酮还原酶基因工程菌全细胞，所用的醛酮还原酶基因工程菌全细胞的醛酮还原酶基因的编码序列如SEQ ID NO 3所示，(醛酮还原酶基因的编码序列未经人工设计)，序列通过全基因合成(委托金斯瑞生物科技有限公司合成)，制备方法如实施例3，加入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL，0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h。Step 1: The reaction was carried out in a 1 L shake flask, the reaction system was controlled to 300 mL, and 250 mL of sterilized deionized water was used to suspend the whole cell of the aldehyde ketone reductase genetically engineered bacteria of Saccharomyces kudriavzevii in a shake flask, and the aldehyde ketone was used for reduction. The coding sequence of the aldehyde ketone reductase gene of the whole cell of the enzyme genetic engineering bacteria is shown in SEQ ID NO 3, (the coding sequence of the aldosterone reductase gene is not artificially designed), and the sequence is synthesized by whole genes (trusted by Kingsray Biotechnology) Co., Ltd. synthesis, preparation method as in Example 3, adding glucose dehydrogenase, adding 2.5mol / L glucose 10mL, 0.26g of NADP +, weigh 10g of substrate, dissolved in 30mL of butyl acetate, will dissolve The butyl acetate solution with substrate was slowly poured into the shake flask. After 1 h of reaction, 10 mL of 2.5 mol/L glucose was added, and the amount of whole cells of the aldehyde ketone reductase genetically engineered bacteria was 75 g/L, and glucose dehydrogenase was added. 25 mg / L, the temperature of the control conversion system was 37 ° C; the conversion reaction was carried out in a shaker, the rotation speed of the shaker was controlled to 200 r / min, and the conversion time was 12 h.
纯化步骤如实施例3，得到目标化合物7.70g，所制备的目标化合物的de值为79.6％，对映异构体的ee(anti)值为88.7。Purification step As in Example 3, 7.70 g of the title compound was obtained. The desired compound had a value of 79.6%, and the enantiomer had an ee (anti) value of 88.7.
SEQ ID NO 1序列号为：The sequence number of SEQ ID NO 1 is:
Figure PCTCN2018097736-appb-000056
Figure PCTCN2018097736-appb-000056
Figure PCTCN2018097736-appb-000057
Figure PCTCN2018097736-appb-000057
SEQ ID NO 2序列号为：The SEQ ID NO 2 sequence number is:
Figure PCTCN2018097736-appb-000058
Figure PCTCN2018097736-appb-000058
Figure PCTCN2018097736-appb-000059
Figure PCTCN2018097736-appb-000059
SEQ ID NO 3序列号为：The SEQ ID NO 3 sequence number is:
Figure PCTCN2018097736-appb-000060
Figure PCTCN2018097736-appb-000060

Claims (29)

  1. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式B，C或C-i化合物，其特征在于，结构式如下：A (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol intermediate of the formula B, C or C-i, characterized in that the structural formula is as follows:
    Figure PCTCN2018097736-appb-100001
    Figure PCTCN2018097736-appb-100001
    其中，R Z为烷氧基，-NR 4R 5；R P为氢，-C-C-O-Bn，-C-C-OH，-C-CO 2R 6，-C-CO 2Ar；R L为-OH或羟基保护基；R P，R L并构成环；R 4，R 5相同或不同地为苯基，烷基或取代苯基；R 6为烷基；*表示手性。 Wherein R Z is alkoxy, -NR 4 R 5 ; R P is hydrogen, -CCO-Bn, -CC-OH, -C-CO 2 R 6 , -C-CO 2 Ar; R L is -OH Or a hydroxy protecting group; R P , R L and forming a ring; R 4 , R 5 are the same or different phenyl, alkyl or substituted phenyl; R 6 is alkyl; * represents chirality.
  2. 根据权利要求1中的化合物，其特征在于，结构式如下：The compound according to claim 1, wherein the structural formula is as follows:
    Figure PCTCN2018097736-appb-100002
    Figure PCTCN2018097736-appb-100002
    其中，R P为氢，-C-C-O-Bn，-C-C-OH，-C-CO 2R 6，-C-CO 2Ar；R L为羟基或羟基保护基；R 4，R 5相同或不同地为苯基，烷基或取代苯基；R 6为烷基；*表示手性。 Wherein R P is hydrogen, -CCO-Bn, -CC-OH, -C-CO 2 R 6 , -C-CO 2 Ar; R L is a hydroxyl or hydroxy protecting group; and R 4 , R 5 are the same or different Is a phenyl group, an alkyl group or a substituted phenyl group; R 6 is an alkyl group; * represents a chirality.
  3. 根据权利要求1中的化合物，其特征在于，结构式如下：The compound according to claim 1, wherein the structural formula is as follows:
    Figure PCTCN2018097736-appb-100003
    Figure PCTCN2018097736-appb-100003
    Figure PCTCN2018097736-appb-100004
    Figure PCTCN2018097736-appb-100004
    Figure PCTCN2018097736-appb-100005
    Figure PCTCN2018097736-appb-100005
  4. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式C化合物的制备方法，其特征在于，由式B化合物经酶法还原反应构建手性制备，Method for preparing (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol intermediate compound C, characterized in that chiral preparation is carried out by enzymatic reduction reaction of compound of formula B ,
    Figure PCTCN2018097736-appb-100006
    Figure PCTCN2018097736-appb-100006
    所述酶为醛酮还原酶，氨基酸序列为SEQ ID NO：1的蛋白质或SEQ ID NO：1经过一个或几个氨基酸残基取代、缺失或添加后具有醛酮还原酶活性的蛋白质，或与SEQ ID NO：1所示的氨基酸序列具有80％以上同源性且具有醛酮还原酶活性的蛋白质。The enzyme is an aldehyde ketone reductase, and the amino acid sequence is a protein of SEQ ID NO: 1 or a protein having aldosterone reductase activity after substitution, deletion or addition of one or several amino acid residues of SEQ ID NO: 1, or The amino acid sequence represented by SEQ ID NO: 1 has a protein having 80% or more homology and having aldosterone reductase activity.
    其中，R Z，R P，R L的定义与权利要求1中的相同。 Wherein, the definitions of R Z , R P , and R L are the same as those in claim 1.
  5. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式C2化合物的制备方法，其特征在于，由式B2化合物经酶法还原反应构建手性制备，Method for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol intermediate compound C2, characterized in that chiral preparation is carried out by enzymatic reduction reaction of compound of formula B2 ,
    Figure PCTCN2018097736-appb-100007
    Figure PCTCN2018097736-appb-100007
    其中，R P，R L的定义与权利要求2中的相同， Wherein, the definitions of R P and R L are the same as those in claim 2,
    所述还原反应构建手性的方法为酶法，所述酶与权利要求4中的相同。The method for the chirality of the reduction reaction is enzymatic, and the enzyme is the same as in claim 4.
  6. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式C-i化合物的制备方法，其特征在于，由式C化合物经还原反应制备，Process for preparing (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol intermediate formula C-i, which is prepared by reduction reaction of compound of formula C,
    Figure PCTCN2018097736-appb-100008
    Figure PCTCN2018097736-appb-100008
    其中，R Z，R P，R L的定义与权利要求1中的相同。 Wherein, the definitions of R Z , R P , and R L are the same as those in claim 1.
  7. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其特征在于，由式C-i化合物经关环反应制备，A method for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which is prepared by a ring-closing reaction of a compound of formula C-i,
    Figure PCTCN2018097736-appb-100009
    Figure PCTCN2018097736-appb-100009
    其中，R Z，R P，R L的定义与权利要求1中的相同。 Wherein, the definitions of R Z , R P , and R L are the same as those in claim 1.
  8. 根据权利要求7所述的制备方法，其特征在于，由式C2化合物经还原反应用于制备式C-i1化合物，The method according to claim 7, wherein the compound of the formula C2 is subjected to a reduction reaction for preparing a compound of the formula C-i1,
    Figure PCTCN2018097736-appb-100010
    Figure PCTCN2018097736-appb-100010
    其中，R P，R L的定义与权利要求1中的相同。 Wherein, the definitions of R P and R L are the same as those in claim 1.
  9. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其特征在于，由式C-i1化合物经关环反应制备，A method for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which is prepared by a ring-closing reaction of a compound of formula C-i1,
    Figure PCTCN2018097736-appb-100011
    Figure PCTCN2018097736-appb-100011
    其中，R P，R L的定义与权利要求1中的相同。 Wherein, the definitions of R P and R L are the same as those in claim 1.
  10. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由二羰基化合物与胺类化合物反应制备，Process for preparing (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol intermediate compound B2, which is prepared by reacting a dicarbonyl compound with an amine compound,
    Figure PCTCN2018097736-appb-100012
    Figure PCTCN2018097736-appb-100012
    其中，X 1为卤素，烷氧基或离去基，R P，R L的定义与权利要求1中的相同。 Wherein X 1 is a halogen, an alkoxy group or a leaving group, and R P , R L have the same definitions as in claim 1.
  11. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由二羰基化合物与N-甲基苯胺反应后进一步地取代反应制备，Process for preparing (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol intermediate formula B2, which is further characterized by reacting a dicarbonyl compound with N-methylaniline Substitution reaction preparation,
    Figure PCTCN2018097736-appb-100013
    Figure PCTCN2018097736-appb-100013
    其中，X 1或X 2相同或不同地为卤素，烷氧基，离去基或并构为环，R P，R L的定义与权利要求1中的相同。 Wherein X 1 or X 2 is the same or different, halogen, alkoxy, leaving group or accommodating ring, and R P , R L have the same definitions as in claim 1.
  12. 根据权利要求11所述的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸酯化合物反应制备，The preparation method according to claim 11, wherein after the reaction of the dicarbonyl compound with benzyl alcohol, further reacting with N-methylaniline, further reacting with a halogenate compound,
    Figure PCTCN2018097736-appb-100014
    Figure PCTCN2018097736-appb-100014
    其中，X为卤素，R 6为烷基。 Wherein X is a halogen and R 6 is an alkyl group.
  13. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由二羰基化合物与R LH化合物反应后，进一步与卤代化合物反应制备， A method for preparing a compound of the formula (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, wherein the dicarbonyl compound is reacted with the R L H compound, further Prepared by reacting with a halogenated compound,
    Figure PCTCN2018097736-appb-100015
    Figure PCTCN2018097736-appb-100015
    其中，X为卤素，R L为羟基保护基。 Wherein X is a halogen and R L is a hydroxy protecting group.
  14. 根据权利要求13所述的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与溴代化合物反应制备，The preparation method according to claim 13, wherein after the dicarbonyl compound is reacted with benzyl alcohol, it is further reacted with a brominated compound,
    Figure PCTCN2018097736-appb-100016
    Figure PCTCN2018097736-appb-100016
  15. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由式D化合物与卤代化合物反应后，进一步与N-甲基苯胺反应制备，A method for preparing a compound of the formula (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, wherein the compound of the formula D is reacted with a halogenated compound, further Prepared by N-methylaniline reaction,
    Figure PCTCN2018097736-appb-100017
    Figure PCTCN2018097736-appb-100017
    其中，X为卤素，R 6与权利要求1中定义相同。 Wherein X is a halogen and R 6 is the same as defined in claim 1.
  16. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由式D0化合物在卤素的作用下与N-甲基苯胺反应后经进一步取代反应制备，Process for the preparation of a compound of the formula (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, wherein the compound of the formula D0 is bonded to the N- After the aniline reaction, it is prepared by further substitution reaction,
    Figure PCTCN2018097736-appb-100018
    Figure PCTCN2018097736-appb-100018
    其中，R L与权利要求13中定义相同。 Wherein R L is the same as defined in claim 13.
  17. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其特征在于，由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，环合反应，酶法还原反应和还原关环反应制备，Process for the preparation of (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol, characterized in that the compound of formula D0 is reacted with N-methylaniline under the action of halogen After two steps of substitution reaction, cyclization reaction, enzymatic reduction reaction and reduction ring closure reaction,
    Figure PCTCN2018097736-appb-100019
    Figure PCTCN2018097736-appb-100019
    其中，R L与权利要求13中的定义相同，X为卤素，R 6为烷基。 Wherein R L is the same as defined in claim 13, X is a halogen, and R 6 is an alkyl group.
  18. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其特征在于，由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，酶法还原反应和还原关环反应 制备，Process for the preparation of (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol, characterized in that the compound of formula D0 is reacted with N-methylaniline under the action of halogen After two steps of substitution reaction, enzymatic reduction reaction and reduction ring closure reaction,
    Figure PCTCN2018097736-appb-100020
    Figure PCTCN2018097736-appb-100020
    其中，R L与权利要求13中的定义相同，X为卤素，R 6为烷基。 Wherein R L is the same as defined in claim 13, X is a halogen, and R 6 is an alkyl group.
  19. 根据权利要求13所述的制备方法，其特征在于，所述R L为叔丁氧基，-OCOCH 3，-OBn，-OBz，-OCOR 6，-OAr，-OCOAr，Ar为芳基，杂芳基，取代芳基或取代杂芳基，所述杂芳基为呋喃、吡啶、噻吩、吲哚或萘。 The preparation method according to claim 13, wherein the R L is a tert-butoxy group, -OCOCH 3 , -OBn, -OBz, -OCOR 6 , -OAr, -OCOAr, Ar is an aryl group, and is heterozygous. Aryl, substituted aryl or substituted heteroaryl, which is furan, pyridine, thiophene, anthracene or naphthalene.
  20. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸酯化合物反应，后经酶法还原反应，还原关环反应制备，Process for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which is further reacted with N-methylaniline after reacting a dicarbonyl compound with benzyl alcohol After the reaction, it is further reacted with a halogenate compound, and then subjected to an enzymatic reduction reaction to reduce the ring-closing reaction.
    Figure PCTCN2018097736-appb-100021
    Figure PCTCN2018097736-appb-100021
    其中，X为卤素，R 6为烷基。 Wherein X is a halogen and R 6 is an alkyl group.
  21. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其特征在于，由式D化合物与卤代化合物反应后，与N-甲基苯胺反应，与卤酸酯化合物反应，后经酶法还原反应，还原关环反应制备，Process for the preparation of (3R,3aS,6aR)-hexahydrofuro[2,3-b]-3-ol, characterized by reacting a compound of formula D with a halogenated compound, and N-methylaniline The reaction is reacted with a halogenate compound, and then subjected to an enzymatic reduction reaction, a reduction ring closure reaction,
    Figure PCTCN2018097736-appb-100022
    Figure PCTCN2018097736-appb-100022
    其中，X为卤素，R 6为烷基。 Wherein X is a halogen and R 6 is an alkyl group.
  22. 一种(3R，3aS，6aR)-六氢呋喃并[2，3-b]-3-醇的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与卤代化合物反应，与N-甲基苯胺化合物反应，后经酶法还原反应，还原关环反应制备，A method for preparing (3R, 3aS, 6aR)-hexahydrofuro[2,3-b]-3-ol, which is further reacted with a halogenated compound after reacting a dicarbonyl compound with benzyl alcohol. Reacting with an N-methylaniline compound, followed by an enzymatic reduction reaction, a reduction ring closure reaction,
    Figure PCTCN2018097736-appb-100023
    Figure PCTCN2018097736-appb-100023
    其中，X为卤素。Wherein X is a halogen.
  23. 根据权利要求4所述的制备方法，其特征在于，所述醛酮还原酶基因的核苷酸序列为SEQ ID NO：2。The preparation method according to claim 4, wherein the nucleotide sequence of the aldosterone reductase gene is SEQ ID NO: 2.
  24. 根据权利要求4所述的制备方法，其特征在于，所述醛酮还原酶为基因工程菌全细胞、破碎酶液、冻干粉或固定化酶或固定化细胞。The preparation method according to claim 4, wherein the aldosterone reductase is a genetically engineered whole cell, a disrupted enzyme solution, a lyophilized powder or an immobilized enzyme or an immobilized cell.
  25. 根据权利要求4所述的制备方法，其特征在于，所述反应体系中所述醛酮还原酶基因工程菌全细胞的投入量为10-100g/L，转化温度为25-37℃。The preparation method according to claim 4, wherein the total amount of the aldehyde-ketal reductase genetically engineered bacteria in the reaction system is 10-100 g/L, and the transformation temperature is 25-37 °C.
  26. 根据权利要求4所述的制备方法，其特征在于，所述反应在溶剂存在的条件下进行。The production method according to claim 4, wherein the reaction is carried out in the presence of a solvent.
  27. 根据权利要求26所述的制备方法，其特征在于，所述溶剂为水或缓冲溶液与有机溶剂组成的混合溶剂。The production method according to claim 26, wherein the solvent is water or a mixed solvent of a buffer solution and an organic solvent.
  28. 根据权利要求27所述的制备方法，其特征在于，所述缓冲溶液选自磷酸盐缓冲溶液、碳酸盐缓冲溶液、Tri-HCl缓冲溶液、柠檬酸盐缓冲溶液或MOPS缓冲溶液中的一种或多种。The preparation method according to claim 27, wherein the buffer solution is selected from the group consisting of a phosphate buffer solution, a carbonate buffer solution, a Tri-HCl buffer solution, a citrate buffer solution or a MOPS buffer solution. Or a variety.
  29. 根据权利要求27所述的制备方法，其特征在于，所述有机溶剂选自DMSO、乙酸乙酯、乙酸丁酯、异丙醇、DMF、TBME、二氯甲烷、乙酸乙烯酯中的一种或几种。The preparation method according to claim 27, wherein the organic solvent is one selected from the group consisting of DMSO, ethyl acetate, butyl acetate, isopropanol, DMF, TBME, dichloromethane, and vinyl acetate. Several.
PCT/CN2018/097736 2018-04-12 2018-07-30 Preparation method for hexahydrofuro-furan-ol derivative, and intermediate of derivative and preparation method therefor WO2019196262A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810326250.2A CN110372532B (en) 2018-04-12 2018-04-12 Process for producing hexahydrofurofuranol derivative, intermediate therefor, and process for producing the intermediate
CN201810326250.2 2018-04-12

Publications (1)

Publication Number Publication Date
WO2019196262A1 true WO2019196262A1 (en) 2019-10-17

Family

ID=68163856

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/097736 WO2019196262A1 (en) 2018-04-12 2018-07-30 Preparation method for hexahydrofuro-furan-ol derivative, and intermediate of derivative and preparation method therefor

Country Status (2)

Country Link
CN (1) CN110372532B (en)
WO (1) WO2019196262A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114634957A (en) * 2020-12-15 2022-06-17 苏州引航生物科技有限公司 Method for synthesizing 4AA intermediate through biocatalysis

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4720572A (en) * 1984-11-30 1988-01-19 Bayer Aktiengesellschaft Process for the preparation of 4-acyloxy-3-oxo-butyric acid esters
CN1753898A (en) * 2002-12-27 2006-03-29 住友化学株式会社 Production method of hexahydrofurofuranol derivative, intermediate therefor and production method thereof
CN101941941A (en) * 2009-07-06 2011-01-12 中国中化股份有限公司 Carboxylic ester compound and applications thereof
US20110251289A1 (en) * 2008-06-05 2011-10-13 Csir New compounds for the treatment of cancer
CN104520262A (en) * 2012-08-09 2015-04-15 住友化学株式会社 Method for producing hexahydrofurofuranol derivative

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2634180A1 (en) * 2012-03-01 2013-09-04 Lonza Ltd. Enzymatic process for the preparation of butyrolactones

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4720572A (en) * 1984-11-30 1988-01-19 Bayer Aktiengesellschaft Process for the preparation of 4-acyloxy-3-oxo-butyric acid esters
CN1753898A (en) * 2002-12-27 2006-03-29 住友化学株式会社 Production method of hexahydrofurofuranol derivative, intermediate therefor and production method thereof
US20110251289A1 (en) * 2008-06-05 2011-10-13 Csir New compounds for the treatment of cancer
CN101941941A (en) * 2009-07-06 2011-01-12 中国中化股份有限公司 Carboxylic ester compound and applications thereof
CN104520262A (en) * 2012-08-09 2015-04-15 住友化学株式会社 Method for producing hexahydrofurofuranol derivative

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SADAGOPAN RAGHAVAN ET AL: "A Stereoselective Synthesis of the Carbon Backbone of Phoslactomycin B", EUR. J. ORG. CHEM., vol. 2017, no. 20, 26 May 2017 (2017-05-26), XP055642628, ISSN: 1434-193X, DOI: 10.1002/ejoc.201700379 *
YANKE LIANG ET AL: "Access to Oxetane-Containing psico-Nucleosides from 2-Me- thyleneoxetanes: A Role for Neighboring Group Participation", J. ORG. CHEM., vol. 76, no. 24, 26 October 2011 (2011-10-26), pages 9962 - 9974, XP055642644, ISSN: 0022-3263, DOI: 10.1021/jo201565h *
YUMI HAYASHI: "Research and Development of an Efficient Synthesis of a Key Building Block for Anti-AIDS Drugs by Diphenylprolinol-Catalyzed Enantio- and Diastereoselective Direct Cross Aldol Reaction", ORG. PROCESS RES. DEV., vol. 20, no. 9, 15 August 2016 (2016-08-15), pages 1615 - 1620, XP055642647, ISSN: 1083-6160, DOI: 10.1021/acs.oprd.6b00178 *

Also Published As

Publication number Publication date
CN110372532B (en) 2022-11-29
CN110372532A (en) 2019-10-25

Similar Documents

Publication Publication Date Title
WO2019007146A1 (en) Aminotransferase, mutant, and applications of aminotransferase and mutant in preparation of sitagliptin
US10526622B2 (en) Preparation method for (R)-3-hydroxyl-5-hexenoate
WO2021139689A1 (en) Use of biological enzyme for preparing orlistat intermediate, and preparation method
WO2019174176A1 (en) Preparation method for hexahydrofuro-furan-ol derivative and intermediate of derivative and preparation method therefor
CN112424171B (en) Method for synthesizing sitagliptin and intermediate thereof through biocatalysis
WO2019196263A1 (en) Method for preparing hexahydrofuro-furanol derivative, intermediate thereof and preparation method therefor
JP2020109102A (en) Method of producing pitavastatin calcium
CN113816836B (en) Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol
WO2019196262A1 (en) Preparation method for hexahydrofuro-furan-ol derivative, and intermediate of derivative and preparation method therefor
CN113930404B (en) Method for synthesizing chiral tofacitinib citrate intermediate by enzymatic method
WO2023184791A1 (en) Method for enzymatic synthesis of brivaracetam chiral intermediate
WO2022253111A1 (en) Method for using reduction to prepare (s)-nicotine
WO2015085827A1 (en) Method for preparing silodosin and intermediate thereof
CN111808893A (en) Novel biological preparation method of amino alcohol drug intermediate
WO2019120000A1 (en) METHOD FOR PREPARING β-AMINO ACID DERIVATIVE RICH IN ENANTIOMERS
CN114644636B (en) Method for preparing tofacitinib key intermediate
WO2019072199A1 (en) Deuterated azole compounds and preparation method therefor and uses thereof
CN117305258A (en) Synthesis method of chiral lactone compound, carbonyl reductase ChKRED20 mutant and application
CN114105961A (en) Preparation method of IDO1 inhibitor (LY-3381916)
CN115820576A (en) Diels-Alder reaction enzyme mutant and preparation method and application thereof
CN117210426A (en) Carbonyl reductase mutant and application thereof
CN116179503A (en) Recombinant aminotransferase and application thereof in preparation of chiral intermediate of nilaparib
CN117965476A (en) Reductive amination enzyme mutant and application thereof in rotigotine molecular synthesis
CN112300059A (en) Preparation method of PF-06651600 intermediate
CN115927227A (en) 2-valeryl benzonitrile reductase mutant and application thereof in preparation of optically pure butylphthalide

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18914824

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18914824

Country of ref document: EP

Kind code of ref document: A1