WO2021121239A1 - 制备胆固醇、其衍生物及类似物的方法 - Google Patents

制备胆固醇、其衍生物及类似物的方法 Download PDF

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WO2021121239A1
WO2021121239A1 PCT/CN2020/136678 CN2020136678W WO2021121239A1 WO 2021121239 A1 WO2021121239 A1 WO 2021121239A1 CN 2020136678 W CN2020136678 W CN 2020136678W WO 2021121239 A1 WO2021121239 A1 WO 2021121239A1
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formula
alkyl
compound
group
formula iii
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PCT/CN2020/136678
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English (en)
French (fr)
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甘红星
谢来宾
羊向新
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湖南科瑞生物制药股份有限公司
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Priority to EP20902848.9A priority Critical patent/EP3967700A4/en
Priority to JP2021575975A priority patent/JP7311921B2/ja
Priority to US17/617,956 priority patent/US20220315620A1/en
Publication of WO2021121239A1 publication Critical patent/WO2021121239A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
    • C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
    • C07J7/008—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms substituted in position 21
    • C07J7/0085—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms substituted in position 21 by an halogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
    • C07J7/008—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms substituted in position 21
    • C07J7/009—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms substituted in position 21 by only one oxygen atom doubly bound
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
    • 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 present invention relates to the field of medicinal chemistry, and specifically to methods for preparing cholesterol, its derivatives and analogs.
  • Cholesterol derivatives include but are not limited to 7-dehydrocholesterol, 25-hydroxycholesterol, and 25-hydroxy-7-dehydrocholesterol , Ergosterol.
  • the steroid is a derivative of cyclopentane polyhydrophenanthrene, and its basic structure contains the A, B, C, and D rings shown in the figure below.
  • Cholesterol also known as cholesterol, is an important steroid compound that is widely present in animals. It is not only involved in the formation of cell membranes, but also the raw material for the synthesis of bile acids, 7-dehydrocholesterol and many steroid hormones in the body. 7-Dehydrocholesterol is converted into vitamin D3 by ultraviolet radiation.
  • vitamin D3 One of the metabolites of vitamin D3 in the liver is 25-hydroxyvitamin D3, which is several times more effective than vitamin D3.
  • the derivative of 7-dehydrocholesterol, 25-hydroxy-7-dehydrocholesterol, can be used in the industrial synthesis of 25-hydroxyvitamin D3.
  • the main sources of cholesterol used in the industry are the extraction of animal materials and the semi-synthetic method using plant materials.
  • the methods for extracting animal materials have low yield, high purification difficulty, limited raw material sources, and safety risks, such as mad cow disease and swine streptococcus infection.
  • the application of the semi-synthetic method using plant raw materials is limited by the supply and price of plant raw materials.
  • CN1772760A discloses a method for synthesizing cholesterol from diosgenin.
  • CN106632565A and CN105237603A disclose methods for synthesizing cholesterol from stigmasterol.
  • CN105669813A, CN102030794B, and CN100494149C disclose the following methods for synthesizing 7-dehydrocholesterol from cholesterol dehydrogenation, in which an air oxidation method is used and a hydrazone reagent is used.
  • the main sources of 25-hydroxycholesterol and 25-hydroxy-7-dehydrocholesterol are prepared from cholesterol or cholesterol analogs.
  • EP594229 and J. Chem. Research, 1999, p708 disclose the following method for preparing 25-hydroxycholesterol from cholesterol, in which trifluoroacetone peroxide or chromium trioxide/acetic anhydride is used as an oxidizing agent to introduce 25-hydroxyl.
  • CN104910231B discloses the following method for preparing 25-hydroxy-7-dehydrocholesterol from 25-hydroxycholesterol, in which a hydrazone reagent is used.
  • CN106831921A discloses the following method for preparing 25-hydroxy-7-dehydrocholesterol from 5,7,24-triencholesterol, in which a Diels-Alder protecting group is used.
  • the present invention provides a method for preparing a compound of formula III, including steps (A) and (B), and step (B) includes steps (B-1) or (B-2):
  • R a and R b are each independently selected from H, -OH, and C 1-3 alkyl;
  • PG is a hydroxy protecting group, preferably a C 1-8 silyl group, an acetyl group, a trifluoroacetyl group, or a benzoyl group optionally substituted with one or more C 1-8 alkyl groups;
  • R is L 1 -LG or L 1 -R 1 ;
  • L 1 does not exist, or is a C 1-8 alkylene group
  • LG is a leaving group
  • PG 1 is a hydroxy protecting group, preferably selected from a C 1-8 silyl group or a C 1-6 alkoxy substituted methyl group;
  • the present invention can adopt two methods to prepare the compound of formula III.
  • the first method first constructs the AB ring and then the side chain; the second method first constructs the side chain and then the AB ring.
  • the first method includes:
  • Step (1) The compound of formula ii is converted into the compound of formula a;
  • Step (2) The compound of formula a is reduced to the compound of formula b under the action of sodium borohydride, calcium chloride and pyridine;
  • Step (3) The compound of formula b is converted into the compound of formula III;
  • Ra, Rb, PG, RL1, LG, and R have the same definitions as described above, and the present invention will not be repeated here.
  • the second method includes:
  • Step (1) The compound of formula ii is converted into the compound of formula I;
  • Step (2) The compound of formula I is converted into the compound of formula II;
  • Step (3) The compound of formula II is reduced to the compound of formula III under the action of sodium borohydride and calcium chloride.
  • Ra, Rb, PG, RL1, LG, and R have the same definitions as described above, and the present invention will not be repeated here.
  • the structure of the compound of formula III is in another preferred aspect, the compound of formula III is selected from cholesterol, 7-dehydrocholesterol, 25-hydroxycholesterol, 25-hydroxy-7-dehydrocholesterol, derivatives or analogs thereof.
  • one (species) or more (species) can mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (species) Or more (species).
  • mn used herein refers to the range of m to n and the sub-range composed of each point value therein and each point value.
  • C 1 -C 8 or “C 1 - 8” encompasses the range of 1 to 8 carbon atoms, and should be understood to also encompass any subranges and values therein for each point, for example, C 2 -C 5 , C 3 -C 4 , C 1 -C 2 , C 1 -C 3 , C 1 -C 4 , C 1 -C 5 , C 1 -C 6 , C 1 -C 7, etc., and C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 and so on.
  • C 3 -C 10 or "C 3 - 10" should be understood in a similar way, for example, may encompass any sub-ranges and wherein the point values, for example C 3 -C 9, C 6 -C 9 , C 6 -C 8 , C 6 -C 7 , C 7 -C 10 , C 7 -C 9 , C 7 -C 8 , C 8 -C 9, etc. and C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 and so on.
  • the expression "three yuan to ten yuan” should be understood as covering any sub-range and each point value, such as three yuan to five yuan, three yuan to six yuan, three yuan to seven yuan, three yuan to eight yuan , Four yuan to five yuan, four yuan to six yuan, four yuan to seven yuan, four yuan to eight yuan, five yuan to seven yuan, five yuan to eight yuan, six yuan to seven yuan, six yuan to eight yuan, nine Yuan to ten Yuan, etc., and three, four, five, six, seven, eight, nine, ten Yuan, etc.
  • Other similar expressions in this article should also be understood in a similar way.
  • alkyl refers to a linear or branched saturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond.
  • Alkyl group may have 1 to 8 carbon atoms, i.e., "C 1 - 8 alkyl", for example C 1 - 4 alkyl, C 1 - 3 alkyl, C 1 - 2 alkyl, C 3 alkyl, C 4 alkyl, C 1 - 6 alkyl, C 3 - 6 alkyl.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2- Methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-di Methylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or their isomers body.
  • alkylene when used alone or in combination with other groups herein, refers to a linear or branched saturated divalent hydrocarbon group.
  • C 1-6 alkylene refers to alkylene groups having 1 to 6 carbon atoms, for example C 1 - 5 alkylene, C 1 - 4 alkylene, C 1 - 3 alkylene group, C 1 - 2 alkylene group, C 3 alkylene and C 1 alkylene group, i.e. a methylene group.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 -) CH 2 -), 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), etc.
  • alkenyl refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. Alkenyl group can have 2 to 8 carbon atoms, i.e., "C 2 - 8 alkenyl group", for example, C 2 - 4 alkenyl, C 3 - 4 alkenyl group.
  • alkenyl groups include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl , (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, etc.
  • alkynyl refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond.
  • An alkynyl group can have 2 to 8 carbon atoms, i.e., "C 2 - 8 alkynyl group", for example, C 2 - 4 alkynyl, C 3 - 4 alkynyl group.
  • Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, and the like.
  • alkoxy refers to an alkyl group as defined above connected to an oxygen atom by a single bond. The alkoxy group is connected to the rest of the molecule through an oxygen atom.
  • the alkoxy group can be represented as -O (alkyl).
  • C 1-8 alkoxy or “-O (C 1-8 alkyl)” refers to an alkoxy group containing 1-8 carbon atoms, in which the alkyl part can be linear, branched or cyclic ⁇ Like structure.
  • Alkoxy includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, n-pentoxy, cyclopentyloxy, cyclohexyloxy and the like.
  • silyl group refers to an alkyl group as defined above, in which at least one C atom is replaced by a Si atom. The silyl group is connected to the rest of the molecule through a silicon atom.
  • C 1-8 silyl group refers to a silyl group containing 1-8 carbon atoms, in which the alkyl part can be a straight chain, branched chain or cyclic structure.
  • Silyl groups include, but are not limited to, trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS, or TBDMS), dimethylisopropylsilyl (IPDMS), di-tert-butylmethylsilyl Base etc. (DTBMS).
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Conformers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to Within the scope of the present invention.
  • the purification and separation of such substances can be achieved by standard techniques known in the art.
  • hydrocarbon solvent refers to a solvent of linear, branched or cyclic hydrocarbon having 1-10 carbon atoms.
  • the hydrocarbon can be saturated or unsaturated.
  • hydrocarbon solvents include, for example, alkane solvents, including but not limited to n-pentane, n-hexane, cyclohexane, n-heptane, octane or a combination thereof, preferably hexane or heptane.
  • hydrocarbon solvents also include aromatic hydrocarbon solvents, which contain at least one aromatic ring and are optionally substituted with linear, branched, or cyclic hydrocarbons.
  • Aromatic solvents include but are not limited to benzene, toluene, xylene or a combination thereof, preferably toluene, toluene or a combination thereof.
  • halogenated alkanes solvent refers to the alkane solvents mentioned above, of which one or more (e.g. 1-6, 1-5, 1-4, 1-3, or 1-2 A) The hydrogen atom is replaced by a halogen.
  • the halogens may be the same or different, and may be located on the same or different C atoms.
  • Halogenated alkane solvents include, but are not limited to, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, hexachloroethane and 1,2,3-trichloropropane or a combination thereof, preferably two Chloroform, trichloromethane, 1,2-dichloroethane or a combination thereof, preferably dichloromethane, chloroform, dichloroethane and a combination thereof, especially dichloromethane.
  • Alcohols solvents are solvents of alcohols having 1-10 carbon atoms.
  • Alcohol solvents include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, cyclohexanol or a combination thereof, preferably methanol, ethanol and a combination thereof.
  • esteer solvent refers to solvents of esters having 3-10 carbon atoms.
  • Ester solvents include, but are not limited to, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, pentyl acetate or a combination thereof, preferably ethyl acetate, isopropyl acetate, butyl acetate or a combination thereof, especially Ethyl acetate.
  • ether-based solvent refers to an ether solvent having 2-10 carbon atoms.
  • ether solvents include, but are not limited to, diethyl ether, isopropyl ether, tetrahydrofuran, dimethyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, methyl tert-butyl ether or a combination thereof, preferably diethyl ether, tetrahydrofuran, Methyltetrahydrofuran or a combination thereof.
  • sulfoxide solvent means dimethyl sulfoxide.
  • ketone solvent refers to ketones with 3-8 carbon atoms, examples of which include but are not limited to acetone, 2-butanone, 3-methyl-2-butanone, 4-methyl-2-pentanone Or a combination.
  • room temperature refers to about 10 to 30°C, preferably about 20 to 25°C.
  • the first aspect of the present invention provides a method for preparing a compound of formula III, comprising steps (A) and (B), and step (B) includes step (B-1) or (B-2); wherein
  • Formula I, Formula II, and Formula III both represent a single bond, or "" in Formula I, Formula II, and Formula III simultaneously represent a double bond, or In Formula I, it represents a single bond or a double bond, and in Formula II and Formula III, it represents a double bond;
  • R a and R b are each independently selected from H, -OH, and C 1-3 alkyl;
  • PG is a hydroxy protecting group, preferably a C 1-8 silyl group, an acetyl group, a trifluoroacetyl group, or a benzoyl group optionally substituted with one or more C 1-8 alkyl groups;
  • R is L 1 -LG or L 1 -R 1 ;
  • L 1 does not exist, or is a C 1-8 alkylene group
  • LG is a leaving group
  • PG 1 is a hydroxy protecting group, preferably selected from a C 1-8 silyl group or a C 1-6 alkoxy substituted methyl group;
  • step (B-1) and step (B-2) both represent the process of preparing the compound of formula III in the presence of a reducing agent.
  • Step (B-1) represents chiral selective obtaining of the target product (compound of formula III).
  • Step (B-2) means that in some cases, the compound obtained by the reaction (compound of formula III') does not reach the required optical purity, so isomer separation is performed to obtain the compound of formula III.
  • PG is selected from acetyl and trifluoroacetyl. In a preferred embodiment, PG is acetyl.
  • step (A) is carried out in the presence of an acylating agent, which is selected from acetic anhydride, acetyl chloride, isopropenyl acetate and combinations thereof, or selected from trifluoroacetic anhydride, trifluoroacetyl chloride and Its combination.
  • the acylating agent is selected from acetic anhydride, acetyl chloride, and isopropenyl acetate, and combinations thereof.
  • the acylating reagent is isopropenyl acetate.
  • the acylating agent is a mixture of acetic anhydride and acetyl chloride.
  • step (A) is carried out in the presence of an acid, which is an inorganic acid or an organic acid.
  • the acid is selected from p-TsOH, MsOH, HCl, H 2 SO 4 , HClO 4 and combinations thereof.
  • the acid is p-TsOH.
  • step (A) is carried out at a temperature of 0 to 110°C, preferably room temperature to 110°C.
  • the reaction is carried out at a temperature of 40 to 110°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or a combination thereof.
  • L 1 is absent.
  • L 1 is a C 1-6 alkylene group, preferably a C 1-6 alkylene group, more preferably a C 1-4 alkylene group.
  • L 1 is C 1-2 alkylene, especially methylene.
  • LG is selected from halogen, optionally halogen-substituted C 1-6 alkyl sulfonate leaving group and optionally substituted C 1-6 alkyl tosylate Leaving group.
  • LG is OTf, OTs or OMs.
  • LG is OTs.
  • LG is selected from Cl, Br, and I.
  • R 1 is selected from H, C 1-8 alkyl, C 1-8 alkenyl, wherein said alkyl or alkenyl is optionally selected from -O( C 1-8 alkyl) and -O-PG 1 group.
  • PG 1 is TMS, TBS or MOM.
  • the chiral center represented by * is a racemic carbon atom. In one embodiment, the chiral center indicated by * is a carbon atom enriched in the (S)-configuration. In another embodiment, the chiral center indicated by * is a carbon atom enriched in the (R)-configuration.
  • the reducing agent is selected from borohydride reagents, lithium aluminum hydride.
  • the borohydride reagent is selected from Ca(BH 4 ) 2 , NaBH 4 , KBH 4 and combinations thereof.
  • the borohydride reagent is Ca(BH 4 ) 2 .
  • the reaction is carried out in the presence of a base, which is an organic base, an inorganic base, or a combination thereof.
  • the inorganic base is selected from NaOH, KOH, NaOMe, t-BuOK and combinations thereof.
  • the organic base is selected from pyridine, triethylamine, DMAP, and combinations thereof.
  • the base is pyridine.
  • the reaction is carried out in an organic solvent.
  • the organic solvent is selected from alcohol solvents, ether solvents, and combinations thereof.
  • the ether solvent is selected from diethyl ether, tetrahydrofuran and combinations thereof.
  • the alcohol solvent is selected from methanol, ethanol, isopropanol, and a combination thereof, preferably methanol, ethanol, or a combination thereof, or ethanol, isopropanol, or a combination thereof.
  • the organic solvent is a mixed solvent of tetrahydrofuran and at least one of the following alcohols: methanol, ethanol, and isopropanol.
  • the organic solvent is a mixed solvent of tetrahydrofuran and at least one of the following alcohols: methanol, ethanol.
  • the organic solvent is a mixed solvent of tetrahydrofuran, ethanol and isopropanol.
  • the ratio of tetrahydrofuran, ethanol and isopropanol in the mixed solvent of tetrahydrofuran, ethanol and isopropanol is 1:(0.1-1):(0.1-1), preferably 1:(0.3-0.8) :(0.3-0.8), especially 1:0.5:0.5.
  • step (B) is carried out at a temperature of -20 to 80°C, preferably -20 to 70°C.
  • the reaction is carried out at a temperature of -20 to 60°C.
  • the reaction is carried out at a temperature of -20 to 40°C, preferably -10 to 40°C, such as -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C , 30°C, 35°C or 40°C.
  • formula I represents a single bond or a double bond
  • the compound of formula I when it represents a single bond, can be represented as formula I-1, and the method of the present invention optionally includes the following step (a) before step (A), In order to convert the single bond into a double bond; the structure obtained after the transformation is represented as Formula I-2, and is still covered by the definition of Formula I.
  • L 1 , LG, PG and PG 1 are as defined above.
  • step (a) is carried out in the presence of a dehydrogenating agent.
  • the dehydrogenating agent is selected from DDQ and chloranil.
  • the dehydrogenating agent is selected from chloranil.
  • the dehydrogenation reaction is carried out by the method described in CN104910231B or Journal of Organic Chemistry, 2005, 70(21), 8513-8521; Tetrahedron Letters, 2004, 45(40), 7479-7482.
  • any one or more of the compounds of formula I, formula II, formula III', and formula III optionally react, and the reaction includes any one or more of the following steps and a combination of steps: step (b), step (c), step (d), step (e), step (f), combination of step (g)-step (h), step (i), step (j).
  • each The structure represented in the product is the same as the structure represented in the reactant.
  • the structure of L 1 -LG or L 1 -R 1 in the compounds of formula i, formula ii, formula iii, formula iii', formula iii", and formula iii"' is encompassed within the definition of R described above.
  • the compounds of formula i, formula ii, formula iii, formula iii', formula iii", and formula iii"' are each independently encompassed within the structural scope of formula I, formula II, formula III' or formula III.
  • the 3-position of the steroid is a carbonyl group, and the 3-position of the steroid is The carbonyl oxygen atom is not connected to R 2 through a chemical bond, and R 2 does not exist, and the structure of the AB ring is In one embodiment, the structure of the AB ring in formula i, formula ii, formula iii, formula iii′, formula iii”, formula iii”′, and formula iv corresponds to formula I.
  • At least one of formula i, formula ii, formula iii, formula iii', formula iii", formula iii"', and formula iv, the 3-position of the steroid in the formula is hydroxyl, and the steroid 3- The oxygen atom of the hydroxyl group is connected to R 2 through a chemical bond, and the structure of the AB ring is In one embodiment, the structure of the AB ring in formula i, formula ii, formula iii, formula iii', formula iii", formula iii"', and formula iv corresponds to formula II.
  • At least one of formula i, formula ii, formula iii, formula iii', formula iii", formula iii"', and formula iv, the structure of the AB ring in the formula is In one embodiment, the structure of the AB ring in formula i, formula ii, formula iii, formula iii', formula iii", formula iii"', and formula iv corresponds to formula III' or formula III.
  • At least one of formula i, formula ii, formula iii, formula iii', formula iii", formula iii"', and formula iv, the structure of the AB ring in the formula is In another embodiment, the structure of the AB ring in at least one of formula i, formula ii, formula iii, formula iii', formula iii", formula iii"', and formula iv is
  • R 2 is PG, and PG is as defined above;
  • LG is a C 1-6 alkylsulfonate leaving group optionally substituted by halogen or a benzenesulfonate leaving group optionally substituted by C 1-6 alkyl, reacting In the presence of a sulfonylation reagent; or
  • LG is a halogen, and the reaction is carried out in the presence of a halogenating reagent.
  • step (b) is performed before step (A), formula i and formula ii are both covered by the structure of formula I, and step (b) is as follows
  • LG is OTf, OMs or OTs.
  • the sulfonylating reagent is (Tf) 2 O, MsCl, or TsCl.
  • LG is Cl, Br or I.
  • the halogenating reagent is SOCl 2 , POCl 3 , chlorosilane reagent, phosphorous halide, HBr, NBS, (PhO) 3 P/CH 3 I system, or Ph 3 P/I 2 /imidazole system.
  • R 2 is PG, and PG is as defined above;
  • LG' is a C 1-6 alkylsulfonate leaving group optionally substituted by halogen or a benzenesulfonate leaving group optionally substituted by C 1-6 alkyl; LG is halogen ;
  • the reaction is carried out in the presence of a halogenating reagent.
  • step (c) is performed before step (A), formula ii' and formula ii are both covered by the structure of formula I, and step (c) is as follows
  • LG' is OTf, OMs or OTs.
  • LG is Cl, Br or I.
  • the halogenating agent is hydrogen halide, I 2 , POCl 3 , PBr 3 or metal halide.
  • the metal halide is selected from sodium chloride, lithium chloride, magnesium chloride, and combinations thereof, or selected from sodium bromide, lithium bromide, magnesium bromide, and combinations thereof, or sodium iodide.
  • R 2 is H or PG
  • LG is selected from halogen, C 1-6 alkylsulfonate leaving group optionally substituted by halogen and benzenesulfonate leaving group optionally substituted by C 1-6 alkyl;
  • LG1 is MgX or CuLi; n is 1 or 2;
  • R 1 is selected from H, C 1-8 alkyl, C 1-8 alkenyl, wherein the alkyl or alkenyl is optionally selected from -O (C 1-8 alkyl) by 1, 2 or 3 And -O-PG 1 group substitution;
  • L 1 , PG, and PG 1 are as defined above.
  • step (d) is carried out between the first step and the second step of step (B-2), formula ii and formula iii are both covered by the structure scope of formula III', step (d) Including the following steps (d-1)
  • step (d) is performed after step (B), formula ii and formula iii are both covered by the structure of formula III, step (d) includes the following steps (d-2)
  • LG is OTf, OMs or OTs. In another embodiment, LG is Cl, Br, or I.
  • LG 1 is MgX, wherein X is Cl, Br or I, and n is 1, Belongs to Grignard reagent.
  • step (d) is carried out in the presence of a catalyst, and the catalyst is selected from the group consisting of copper halide, cuprous halide, lithium chloride, lithium copper double salt, and combinations thereof.
  • the catalyst is selected from one or more of the following: CuI, CuBr, LiCl, CuCl 2 , CuCl, Li 2 CuCl 4 , LiCuCl 3 .
  • LG 1 is CuLi
  • n is 2
  • step (d) further includes a step of protecting the 3-carbonyl group and a step of deprotecting.
  • the step of protecting the 3-carbonyl group is achieved by forming the 3-carbonyl group into a ketal.
  • the protecting group protecting the 3-carbonyl group is 1,3-dioxolane.
  • R 2 is H or PG, and PG is as defined above.
  • LG is selected from halogen, a C 1-6 alkyl sulfonate leaving group optionally substituted with halogen, and a benzene sulfonate leaving group optionally substituted with C 1-6 alkyl.
  • LG is halogen.
  • the acrylic acid derivatives are selected from alkyl acrylates, wherein the alkyl groups are each independently selected from C 1-8 alkyl groups.
  • the alkyl groups are each independently selected from C 1-6 alkyl groups, more preferably C 1-4 alkyl groups.
  • the reducing agent is selected from zinc, iron, aluminum, and magnesium. In a particular embodiment, the reducing agent is zinc.
  • step (e) is carried out between the first step and the second step of step (B-2), formula ii and formula iii are both covered by the structure scope of formula III', step (e) Including the following steps (e-1)
  • step (e) is performed after step (B), formula ii and formula iii are both covered by the structure of formula III, and step (e) includes the following steps (e-2)
  • the acrylic acid derivative is a C 1-6 alkyl acrylate.
  • the alkyl acrylate is selected from methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, and isobutyl acrylate.
  • the alkyl acrylate is selected from methyl acrylate and ethyl acrylate.
  • Step (e) for the catalyst is selected from CuI, Ni (0) / ligand, Zn / NiCl 2 / ligand in the presence of a catalyst in one embodiment.
  • the ligand is selected from PPh 3 , bipyridine, o-phenanthroline, pyridine, DMAP, and combinations thereof.
  • the catalyst is Zn/NiCl 2 /pyridine.
  • step (e) is carried out at a temperature of 0 to 80°C, preferably 0 to 70°C.
  • the reaction is carried out at a temperature of 0 to 60°C.
  • the reaction is carried out at a temperature of 10 to 60°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or Its combination.
  • the reaction is carried out in an organic solvent.
  • the organic solvent is selected from ethyl acetate, tetrahydrofuran, dioxane, diethyl ether, butyl acetate, methyltetrahydrofuran, and pyridine. In one embodiment, the organic solvent is pyridine.
  • step (e) includes the following steps i) to iii):
  • step ii) is performed at a temperature of 30 to 80°C, preferably 30 to 70°C, more preferably 40 to 60°C, such as 40°C, 45°C, 50°C, 55°C or 60°C.
  • step ii) is carried out by slowly adding acrylic acid derivatives to the mixture obtained in i).
  • the reaction time of step ii) after the mixing is completed is 0.5 to 4 hours, preferably 1-2 hours.
  • step iii) is carried out at a temperature of 0 to 60°C, preferably 0 to 40°C, more preferably 0 to 30°C, such as 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C.
  • the reaction time of step iii) after the mixing is completed is 0.5 to 8 hours, preferably 1-6 hours, more preferably 3-4 hours.
  • R 2 is H or PG, and PG is as defined above;
  • R 1 is selected from C 1-8 alkyl, C 1-8 alkenyl and Wherein said alkyl or alkenyl is selected from 1 or 2 Is substituted with the group of, wherein R 3 is selected from C 1-8 alkyl;
  • the alkylating agent is R 3 MgX, or R 3 Li, where X is Cl or Br.
  • R 3 is selected from C 1-4 alkyl. In a particular embodiment, R 3 is methyl.
  • step (f) is carried out between the first step and the second step of step (B-2), formula iii' and formula iii are both covered by the structure scope of formula III', step (f ) Includes the following steps (f-1)
  • step (f) is performed after step (B), formula iii' and formula iii are both covered by the structure of formula III, and step (f) includes the following steps (f-2)
  • step (f) further includes a step of protecting 3-OH and a step of deprotecting.
  • the protecting group for protecting 3-OH is selected from C 1-8 silyl, acetyl, trifluoroacetyl, benzyl optionally substituted by methoxy or C 1-6 alkoxy substituted The methyl group.
  • R 1 is -CH 2 OH
  • R 2 is PG
  • L 1 and PG are as defined above.
  • step (g) is performed before step (A), formula iii is covered by the structure of formula I, and step (g) includes the following steps (g-1)
  • step (g) is performed between step (A) and step (B), formula iii is covered by the structure of formula II, and step (g) includes the following steps (g-2)
  • the oxidant is selected from TEMPO/NaBr/NaClO, TEMPO/NaBr/Ca(ClO) 2 , TEMPO/TCCA, DMSO/SO 3 -Py/Et 3 N, NaNO 2 /FeCl 3 /TEMPO/air, NaNO 2 /FeCl 3 /TEMPO/O 2 and combinations thereof.
  • the oxidizing agent is TEMPO/NaBr/NaClO.
  • the reaction is carried out in an organic solvent.
  • the organic solvent is selected from hydrocarbon solvents, halogenated hydrocarbon solvents, ester solvents, ketone solvents, and combinations thereof.
  • the hydrocarbon solvent is selected from benzene, toluene, and combinations thereof.
  • the halogenated hydrocarbon solvent is selected from dichloromethane, chloroform, dichloroethane and combinations thereof, preferably dichloromethane.
  • the ester solvent is ethyl acetate.
  • the ketone solvent is acetone.
  • R 2 is H or PG
  • L 1 , PG, and PG 1 are as defined above.
  • the CR 4 R 5 structure is selected from
  • step (h) is performed before step (A), formula iii is covered by the structure of formula I, and step (h) includes the following steps (h-1)
  • step (h) is performed between the first and second steps of step (B-2), formula iii is covered by the structure of formula III', and step (h) includes the following steps ( h-2)
  • step (h) is performed after step (B), formula iii is covered by the structure of formula III, and step (h) includes the following steps (h-3)
  • the Wittig reagent is prepared from a halogenated hydrocarbon and a phosphorus reagent, wherein the halogenated hydrocarbon is X-CHR 4 R 5 , and X is Cl, Br, or I.
  • the phosphorus reagent is selected from triphenylphosphine, trimethyl phosphite, triethyl phosphite, and the like. In a particular embodiment, the phosphorus reagent is triphenylphosphine.
  • the reaction of preparing the Wittig reagent from the halogenated hydrocarbon and the phosphorus reagent is carried out in the presence of a base, the base being an organic base, an inorganic base, or a combination thereof.
  • the base is selected from potassium tert-butoxide, sodium methoxide, potassium methoxide, sodium hydride, butyl lithium, lithium diisopropylamide, and combinations thereof.
  • the base is potassium tert-butoxide.
  • step (h) is carried out at a temperature of -20 to 80°C, preferably -20 to 70°C. In one embodiment, the reaction is carried out at a temperature of -20 to 60°C.
  • the reaction is carried out at a temperature of -10 to 60°C, preferably 0 to 50°C, more preferably 10 to 40°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C. °C.
  • the reaction is carried out in an organic solvent.
  • the organic solvent is selected from sulfoxide-based solvents, ether-based solvents, hydrocarbon-based solvents, and combinations thereof.
  • the sulfoxide solvent is DMSO.
  • the ether solvent is selected from diethyl ether, tetrahydrofuran, dimethyltetrahydrofuran, and combinations thereof.
  • the hydrocarbon solvent is selected from benzene, toluene, and combinations thereof.
  • R 2 is H
  • L 1 and PG 1 are as defined above.
  • step (i) is carried out between the first step and the second step of step (B-2), formula iii" and formula iii are both covered by the structure of formula III', step (i ) Includes the following steps (i-1)
  • step (i) is performed after step (B), formula iii" and formula iii are both covered by the structure of formula III, and step (i) includes the following steps (i-2)
  • step (i) is carried out by a catalytic hydrogenation reaction.
  • R 2 is H
  • L 1 and PG 1 are as defined above.
  • step (j) is carried out between the first step and the second step of step (B-2), formula iii"' and formula iii are both covered by the structure scope of formula III', step ( j) Including the following steps (j-1)
  • step (j) is performed after step (B), formula iii"' and formula iii are both covered by the structure of formula III, and step (j) includes the following steps (j-2)
  • step (A), step (B), step (a) to step (j) of the first or second aspect of the present invention are summarized in Table 1 below.
  • the second aspect of the present invention is to provide a method for preparing a compound of formula III, comprising:
  • Step (1) The compound of formula ii is converted into the compound of formula a;
  • Step (2) The compound of formula a is reduced to the compound of formula b under the action of sodium borohydride, calcium chloride and pyridine;
  • Step (3) The compound of formula b is converted into the compound of formula III;
  • Ra, Rb, PG, RL1, LG, and R have the same definitions as described above, and the present invention will not be repeated here.
  • PG is selected from acetyl and trifluoroacetyl. In a preferred embodiment, PG is acetyl.
  • step (1) is carried out in the presence of an acylating agent, which is selected from acetic anhydride, acetyl chloride, isopropenyl acetate and combinations thereof, or selected from trifluoroacetic anhydride, trifluoroacetyl chloride and Its combination.
  • the acylating agent is selected from acetic anhydride, acetyl chloride, and isopropenyl acetate, and combinations thereof.
  • the acylating reagent is isopropenyl acetate.
  • the acylating agent is a mixture of acetic anhydride and acetyl chloride.
  • step (1) is carried out in the presence of an acid, which is an inorganic acid or an organic acid.
  • the acid is selected from p-TsOH, MsOH, HCl, H2SO4, HClO4, and combinations thereof.
  • the acid is p-TsOH.
  • step (1) is carried out at a temperature of 0 to 110°C, preferably room temperature to 110°C.
  • the reaction is carried out at a temperature of 40 to 110°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or a combination thereof.
  • L 1 is absent.
  • L 1 is a C 1-6 alkylene group, preferably a C 1-6 alkylene group, more preferably a C 1-4 alkylene group.
  • L 1 is C 1-2 alkylene, especially methylene.
  • LG is selected from halogen, optionally halogen-substituted C 1-6 alkyl sulfonate leaving group and optionally substituted C 1-6 alkyl tosylate Leaving group.
  • LG is OTf, OTs or OMs.
  • LG is OTs.
  • LG is selected from Cl, Br, and I.
  • R 1 is selected from H, C 1-8 alkyl, C 1-8 alkenyl, wherein said alkyl or alkenyl is optionally selected from -O( C 1-8 alkyl) and -O-PG 1 group.
  • PG 1 is TMS, TBS or MOM.
  • step (2) the compound of formula a is reduced to the compound of formula b under the action of sodium borohydride, calcium chloride and pyridine.
  • the reaction is carried out in an organic solvent.
  • the organic solvent is selected from a mixed solvent of alcohol solvents and tetrahydrofuran.
  • the alcohol solvent is selected from methanol, ethanol, isopropanol, and a combination thereof, preferably methanol, ethanol, or a combination thereof, or ethanol, isopropanol, or a combination thereof.
  • the volume ratio of ethanol or isopropanol is 1:1.
  • the organic solvent is a mixed solvent of tetrahydrofuran and at least one of the following alcohols: methanol, ethanol, and isopropanol.
  • the organic solvent is a mixed solvent of tetrahydrofuran and at least one of the following alcohols: methanol, ethanol.
  • the organic solvent is a mixed solvent of tetrahydrofuran, ethanol and isopropanol.
  • the volume ratio of the alcohol and tetrahydrofuran is 1-2:1, especially 1:1.
  • the ratio of tetrahydrofuran, ethanol and isopropanol in the mixed solvent of tetrahydrofuran, ethanol and isopropanol is 1:(0.1-1):(0.1-1), preferably 1:(0.3-0.8) :(0.3-0.8), especially 1:0.5:0.5.
  • step (2) is carried out at a temperature of -20 to 80°C, preferably -20 to 70°C.
  • the reaction is carried out at a temperature of -20 to 60°C.
  • the reaction is carried out at a temperature of -20 to 40°C, preferably -10 to 40°C, such as -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C , 30°C, 35°C or 40°C.
  • step (3) is carried out according to the above step (d), even if the leaving group of the compound of formula b reacts with the organometallic reagent to construct the side chain to obtain the compound of formula III; the construction process is the same as that described above Similar, the present invention will not be repeated here.
  • step (3) is carried out according to the above step (e), that is, the leaving group of the compound of formula b reacts with the acrylic acid derivative under the action of a reducing agent to construct a side chain to generate the compound of formula III;
  • step (3) is carried out according to the above step (e), that is, the leaving group of the compound of formula b reacts with the acrylic acid derivative under the action of a reducing agent to construct a side chain to generate the compound of formula III;
  • step f reacting a compound containing an ester group in the side chain with an alkylating agent to prepare a compound containing a tertiary hydroxyl group in the side chain.
  • the process is similar to that described above. This will not be repeated here.
  • formula I represents a single bond or a double bond
  • the compound of formula ii can be represented as formula ii-1
  • the method of the present invention optionally includes the following step (a) before step (1), The compound undergoes a dehydrogenation reaction to convert the single bond into a double bond; the structure obtained after the conversion is represented by formula ii-2, and is still covered by the definition of formula I. This process is similar to step a described above, and the present invention will not be repeated here.
  • the method further includes step (b), reacting the primary hydroxyl group on the side chain of the compound with a sulfonylating agent or a halogenating agent to prepare a compound containing a leaving group in the side chain.
  • step (b) reacting the primary hydroxyl group on the side chain of the compound with a sulfonylating agent or a halogenating agent to prepare a compound containing a leaving group in the side chain.
  • the method further includes step (c): reacting the compound of formula ii' containing a sulfonate group with a halogenating reagent to prepare a compound of formula ii containing a halogen leaving group.
  • step (c) reacting the compound of formula ii' containing a sulfonate group with a halogenating reagent to prepare a compound of formula ii containing a halogen leaving group.
  • the third aspect of the present invention is to provide another method for preparing a compound of formula III, comprising:
  • Step (11) Convert the compound of formula ii into the compound of formula I;
  • Step (13) The compound of formula II is reduced to the compound of formula III under the action of sodium borohydride and calcium chloride;
  • R a , R b , PG, RL 1 , LG, and R have the same definitions as described above, and the present invention will not be repeated here.
  • PG is selected from acetyl and trifluoroacetyl. In a preferred embodiment, PG is acetyl.
  • step (12) is carried out in the presence of an acylating agent, which is selected from acetic anhydride, acetyl chloride, isopropenyl acetate and combinations thereof, or selected from trifluoroacetic anhydride, trifluoroacetyl chloride and Its combination.
  • the acylating agent is selected from acetic anhydride, acetyl chloride, and isopropenyl acetate, and combinations thereof.
  • the acylating reagent is isopropenyl acetate.
  • the acylating agent is a mixture of acetic anhydride and acetyl chloride.
  • step (12) is carried out in the presence of an acid, which is an inorganic acid or an organic acid.
  • the acid is selected from p-TsOH, MsOH, HCl, H 2 SO 4 , HClO 4 and combinations thereof.
  • the acid is p-TsOH.
  • step (12) is carried out at a temperature of 0 to 110°C, preferably room temperature to 110°C.
  • the reaction is carried out at a temperature of 40 to 110°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or a combination thereof.
  • L 1 is absent.
  • L 1 is a C 1-6 alkylene group, preferably a C 1-6 alkylene group, more preferably a C 1-4 alkylene group.
  • L 1 is C 1-2 alkylene, especially methylene.
  • LG is selected from halogen, optionally halogen-substituted C 1-6 alkyl sulfonate leaving group and optionally substituted C 1-6 alkyl tosylate Leaving group.
  • LG is OTf, OTs or OMs.
  • LG is OTs.
  • LG is selected from Cl, Br, and I.
  • R 1 is selected from H, C 1-8 alkyl, C 1-8 alkenyl, wherein said alkyl or alkenyl is optionally selected from -O( C 1-8 alkyl) and -O-PG 1 group.
  • PG 1 is TMS, TBS or MOM.
  • step (13) the compound of formula II is reduced to the compound of formula III under the action of sodium borohydride and calcium chloride.
  • the reaction is carried out in an organic solvent.
  • the organic solvent is selected from a mixed solvent of alcohol solvents and dichloromethane.
  • the alcohol solvent is selected from methanol, ethanol, isopropanol, and a combination thereof, preferably methanol, ethanol, or a combination thereof, or ethanol, isopropanol, or a combination thereof.
  • the volume ratio of ethanol or isopropanol is 1:1.
  • the organic solvent is a mixed solvent of dichloromethane and at least one of the following alcohols: methanol, ethanol, and isopropanol. In another particular embodiment, the organic solvent is a mixed solvent of dichloromethane and at least one of the following alcohols: methanol, ethanol. In a particular embodiment, the organic solvent is a mixed solvent of dichloromethane, ethanol and isopropanol. In one embodiment, the volume ratio of the alcohol and dichloromethane is 1-2:1, especially 1:1.
  • the ratio of dichloromethane, ethanol and isopropanol in the mixed solvent of dichloromethane, ethanol and isopropanol is 1:(0.1-1):(0.1-1), preferably 1:( 0.3-0.8): (0.3-0.8), especially 1:0.5:0.5.
  • step (13) is carried out at a temperature of -20 to 80°C, preferably -20 to 70°C.
  • the reaction is carried out at a temperature of -20 to 60°C.
  • the reaction is carried out at a temperature of -20 to 40°C, preferably -10 to 40°C, such as -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C , 30°C, 35°C or 40°C.
  • step (11) can be performed according to step d, so that the compound of formula ii containing a leaving group is converted into a compound of formula I under the action of an organometallic reagent to construct a side chain.
  • This process is similar to the process described above, and the present invention will not be repeated here.
  • step (11) can be performed according to step e, so that the compound of formula ii containing a leaving group is converted into a compound of formula I under the action of an acrylic acid derivative and a reducing agent to construct a side chain.
  • This process is similar to the process described above, and the present invention will not be repeated here.
  • step f The compound of formula ii with an ester group in its side chain is reacted with an alkylating agent to prepare a compound of formula I with a tertiary hydroxyl group in its side chain. This process is similar to the process described above, and the present invention will not be repeated here.
  • formula I represents a single bond or a double bond
  • the compound of formula ii can be represented as formula ii-1
  • the method of the present invention optionally includes step (a) before step (12), so that The compound of formula ii undergoes a dehydrogenation reaction to prepare a dehydrogenated compound of formula ii-2. This process is similar to the process described above, and the present invention will not be repeated here.
  • the method further includes step (b): reacting the compound of formula i containing a primary hydroxyl group on the side chain with a sulfonylating agent or a halogenating agent to prepare a compound of formula ii containing a leaving group.
  • the method further includes step (g): reacting the compound of formula iii containing a primary hydroxyl group on the side chain with an oxidizing agent to prepare a compound of formula iv containing an aldehyde group on the side chain.
  • the method further includes step (h), causing the compound containing an aldehyde group in the side chain to undergo a Wittig reaction to prepare a compound containing a double bond in the side chain.
  • step (h) causing the compound containing an aldehyde group in the side chain to undergo a Wittig reaction to prepare a compound containing a double bond in the side chain.
  • the present invention also provides a method for preparing the compound of formula III-1 from the compound of formula I-1:
  • step (j) can be carried out simultaneously with other reactions.
  • the compound of the following formula I can be prepared by microbial transformation using plant sterols as raw materials. Therefore, in an optional aspect of the present invention, the compound of formula I can be prepared from phytosterols through microbial transformation and chemical synthesis, and the method includes the step (A')
  • R is -CH 2 OH.
  • the present invention provides a new method for preparing cholesterol and its derivatives urgently needed in the art, especially a method for preparing 25-hydroxycholesterol and 25-hydroxy-7-dehydrocholesterol, which solves the technical problems in the prior art, such as the source of raw materials.
  • the problem and the difficulty of constructing the 25-hydroxy group in the side chain are very different from the prior art.
  • the raw materials of the method of the invention are easily available.
  • the step operation is simple, there is no special separation means, the reaction yield is high, the production cost is low, and it is suitable for industrial production.
  • precious metal catalysts highly toxic reagents such as trifluoroacetone peroxide or chromium trioxide/acetic anhydride system, and no need to use hydrazone reagents, so the introduction of nitrogen-containing wastewater that pollutes the environment can be avoided.
  • the method of the present invention can optionally use low-cost and easily available plant sterols as raw materials to prepare intermediates through microbial transformation, with high efficiency, little or no pollution to the environment, and more importantly, the present invention starts from a completely plant source. Starting from the starting material, cholesterol and its derivatives were successfully prepared, avoiding the safety risks faced by the previous synthetic process of animal starting material, such as mad cow disease, swine streptococcus infection, etc. It has high economic value and broad application prospects.
  • the system was cooled to 10° C., and 80 mL of 50% methanol aqueous solution was added dropwise to quench the reaction, and then 600 mL of water was added for liquid separation, and the organic layer was washed with water. Concentrate under reduced pressure to remove most of the solvent, add an appropriate amount of methanol, and continue to concentrate until the DCM is completely removed. Keep about 200mL methanol, lower the temperature to 0-10°C, crystallize under stirring for 1h, filter with suction, rinse with methanol, and dry at 45-50°C to obtain 2A. The weight yield is about 140%, and the purity is greater than 98%.
  • the solid was dissolved with 300 mL DCM, the aqueous layer was separated, and the organic phase was concentrated under reduced pressure to remove most of the solvent. Add methanol and continue to concentrate (this operation is performed 3 times), finally retain about 100 mL of methanol, cool to 0°C to crystallize for 1 hour, filter with suction, rinse with ice methanol, and dry at 45-50°C to obtain 4A.
  • the weight yield is about 75%, and the purity detected by HPLC is greater than 92%.
  • the organic phase was added with saturated sodium sulfite aqueous solution (containing 50g of sodium sulfite) and stirred for 1 hour, allowed to stand and separate, the organic phase was concentrated under reduced pressure to remove most of the solvent, methanol was added and the concentration was continued (this operation was performed 3 times), about 100 mL of methanol was retained, and the temperature was reduced to Crystallize at 0°C for 1h, filter with suction, rinse with methanol, and dry at 45-50°C to obtain 2a. The weight yield is about 90%, and the purity is greater than 93%.
  • intermediate 2a 100 g
  • 500 mL acetic anhydride 500 mL
  • 200 mL acetyl chloride 200 mL
  • TLC monitors that the remaining raw material is less than 5%.
  • the reaction solution was concentrated to dryness under reduced pressure at about 75°C, cooled to room temperature, 50 mL methanol was added dropwise to quench the remaining acetic anhydride, 100 mL acetone was added, and concentrated under reduced pressure to remove most of the solvent, 200 mL acetone was added and the concentration continued, leaving about 100 mL acetone , Cooling down to 0°C, crystallizing for 1h, filtering, rinsing with ice acetone, drying the solid at 45-50°C to obtain 3a, the weight yield is about 90%, and the purity is greater than 95%.
  • TLC monitoring shows that there is no raw material remaining; the reaction solution is slowly poured into 1000mL ice Stir while adding to the water, stir 20 mL after the solids are separated out, slowly drop 20 mL of glacial acetic acid into the system, filter with suction, and rinse with water.
  • the solid was dissolved in 300 mL DCM, the water layer was separated, the organic phase was concentrated under reduced pressure to remove most of the solvent, methanol was added and the concentration was continued (this operation was performed 3 times), and finally about 100 mL of methanol was retained, and the temperature was lowered to 0°C to crystallize for 1 h. Filter by suction, rinse with ice methanol, and dry at 45-50°C to obtain 4a.
  • the temperature of the reaction liquid 2 is lowered to -40 to -10°C.
  • step (5) Add 125 mL of DCM to the mixture obtained in step (5) to dissolve, add hydrochloric acid to adjust the pH to 3-4, stir at room temperature for 1 h, separate the organic phase, and wash with water (50 mL ⁇ 2). Concentrate under reduced pressure at 40 ⁇ 45°C until almost no fraction. Add 25 mL of methanol and continue to concentrate at 40-50°C. Add 25 mL methanol, cool to 10-30°C and stir for 0.5-1 hour, filter, and wash the filter cake with 10 mL methanol. The solid is dried under vacuum at 50°C to obtain 5b. The weight yield is 65%.
  • TLC monitoring shows that there is no raw material remaining; Slowly pour into 1000 mL of ice water, stir while adding, and stir 20 mL after the solid has separated out, slowly drop 20 mL of glacial acetic acid into the system, filter with suction, and rinse with water. The solid was dissolved in 300 mL DCM, the water layer was separated, the organic phase was concentrated under reduced pressure to remove most of the solvent, methanol was added and the concentration was continued (this operation was performed 3 times), and finally 9 methanol was kept about 100 mL, and the temperature was lowered to 0°C to crystallize for 1 h , Suction filtration, methanol elution, drying at 45-50°C to obtain 4A.
  • the filter cake was rinsed with ethyl acetate; 180 mL of 10% hydrochloric acid was added to wash, and the layers were separated. The organic layer was washed with a 6V 10% sodium carbonate aqueous solution and separated. The organic phase was concentrated to dryness, and methanol was added for recrystallization to obtain intermediate 5B. The weight yield is about 80%.
  • the filter cake After filtering through a celite layer, the filter cake is rinsed with ethyl acetate; the combined organic phases are concentrated under reduced pressure and concentrated to dryness.
  • the obtained oily substance is separated by column chromatography (eluent is ethyl acetate: petroleum ether 1:6), concentrated to a paste, cooled to 0°C for crystallization, filtered, and the filter cake is dried to obtain intermediate 5c.
  • the weight yield is about 65%.
  • the combined organic phases were concentrated under reduced pressure below 50°C to remove most of the solvent, added toluene and continued to concentrate until a solid precipitated out. Cool to 10°C, stir for 1h, and filter. The filter cake was transferred to a reaction flask, and 150 mL of DCM and 100 mL of methanol were added until the solution was clear. Concentrate under reduced pressure below 50°C to remove most of the solvent, add methanol and continue to concentrate to a paste. Stir at 0-10°C for 1h and filter. The filter cake was washed with a small amount of methanol and dried at 50°C to obtain 25 hydroxydehydrocholesterol with a weight yield of about 80%.
  • the 1 H NMR data are basically the same as those measured in step (6) of Example 3.
  • the filter cake was rinsed with ethyl acetate; 200L 5% hydrochloric acid was added to wash, separated, and then washed with 200mL of water, separated, the organic layer was concentrated to a small volume, and ethanol was added to continue concentration to a paste.
  • the temperature is lowered to 0-10 DEG C for crystallization for 1 hour, filtered, and the filter cake is dried to obtain the product with a weight yield of about 70% and a purity of 94%.
  • the solid was dissolved in 300 mL DCM, the water layer was separated, the organic phase was concentrated under reduced pressure to remove most of the solvent, methanol was added and the concentration was continued, and finally about 100 mL of methanol was retained.
  • the temperature was lowered to 0-10°C to crystallize for 1 h, filtered with suction, and methanol leached. Wash and dry at 45-50°C to obtain Intermediate 5B.
  • the weight yield is about 70%, and the purity is greater than 97%.
  • the 1 H NMR data are basically the same as those measured in step (5) of Example 4.
  • Compound 5 was prepared according to the step (7) of Example 4, and the 1 H NMR data were basically the same as those measured in Example 4.
  • the filter cake is rinsed with ethyl acetate; the combined filtrate is added with 100 mL of 10% hydrochloric acid to wash, and the layers are separated.
  • the organic layer was washed with 200 mL of water and separated. Concentrate to dryness, add about 100 mL of methanol, cool to 0-5°C to crystallize for one hour, filter, and dry the filter cake to obtain 3'b.
  • the weight yield is about 78%, and the purity is 92%.
  • the solid was dissolved in 300 mL DCM, the water layer was separated, the organic phase was concentrated under reduced pressure to remove most of the solvent, methanol was added and the concentration was continued, and finally about 100 mL of methanol was retained.
  • the temperature was lowered to 0-10°C to crystallize for 1 h, filtered with suction, and methanol leached. Wash and dry at 45-50°C to obtain intermediate 5'B.
  • the weight yield is about 70%, and the purity is greater than 97%.
  • Compound 4 was prepared according to the step (7) of Example 5, and the 1 H NMR data were basically the same as those measured in Example 5.
  • Example 10 The influence of using different reagents on the reaction
  • acylation reagents shown in the following table were used to perform the reaction of step (A) in the first to seventh aspects of the present invention to prepare enol ester intermediates 3A-m and intermediate 3a with different PG groups -m, which corresponds to the compound of formula II described in the first to seventh aspects of the present invention.
  • m is the serial number and PG is the hydroxyl protecting group.
  • intermediates 3A-m and intermediates 3a-m are used as raw materials, and the reagents shown in the following table are used to perform the reaction of step (B) described in the first to seventh aspects of the present invention to prepare intermediates 4A and Intermediate 4a, which corresponds to the compound of formula III described in the first to seventh aspects of the present invention.

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Abstract

本发明涉及药物化学领域，并且具体地涉及制备胆固醇、其衍生物及类似物的方法，胆固醇衍生物包括但不限于7-脱氢胆固醇、25-羟基胆固醇、25-羟基-7-脱氢胆固醇，麦角甾醇。在本发明中，可以以植物甾醇为原料，通过微生物转化制备式I的化合物，然后再进行胆固醇、其衍生物及类似物的制备。

Description

制备胆固醇、其衍生物及类似物的方法
本申请要求于2019年12月19日提交中国国家知识产权局、国际申请号为PCT/CN2019/126744、发明名称为“制备胆固醇、其衍生物及类似物的方法”的PCT专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及药物化学领域，并且具体地涉及制备胆固醇、其衍生物及类似物的方法，胆固醇衍生物包括但不限于7-脱氢胆固醇、25-羟基胆固醇、25-羟基-7-脱氢胆固醇，麦角甾醇。
背景技术
甾体为环戊烷多氢菲的衍生物，基本结构中包含下图所示的A、B、C、D环。
Figure PCTCN2020136678-appb-000001
胆固醇又称胆甾醇，其为重要甾体化合物，广泛存在于动物体内。它不仅参与形成细胞膜，而且是体内合成胆汁酸，7-脱氢胆固醇以及许多甾体激素的原料。7-脱氢胆固醇经紫外线照射转变为维生素D3。
维生素D3在肝脏内的代谢产物之一为25-羟基维生素D3，其效果是维生素D3的数倍。7-脱氢胆固醇的衍生物25-羟基-7-脱氢胆固醇可以用于工业合成25-羟基维生素D3。
目前产业上使用的胆固醇主要来源有动物材料提取和使用植物原料半合成的方法。动物材料提取的方法(例如CN201811062895.6、CN201810722994.6)收率低、纯化难度高、原料来源受限制，并且存在安全风险，如疯牛病、猪链球菌病感染等。使用植物原料半合成的方法的应用受到植物原料的供应量与价格的限制。例如CN1772760A公开了由薯蓣皂素合成胆固醇的方法。CN106632565A和CN105237603A公开了由豆甾醇合成胆固醇的方法。
7-脱氢胆固醇主要来源有从动物皮下组织提取和使用植物原料半合成的方法。CN105669813A、CN102030794B、CN100494149C公开了如下的由胆固醇脱氢合成7-脱氢胆固醇的方法，其中使用了空气氧化方法，并且使用了腙试剂。
Figure PCTCN2020136678-appb-000002
Journal of Organic Chemistry,2005,70(21),8513-8521；Tetrahedron Letters,2004,45(40),7479-7482公开了如下的由胆固醇脱氢合成7-脱氢胆固醇的方法，其中使用了溴取代-脱溴法来引入7-双键。
Figure PCTCN2020136678-appb-000003
25-羟基胆固醇和25-羟基-7-脱氢胆固醇主要来源为由胆固醇或胆固醇类似物制备。EP594229和J.Chem.Research,1999,p708公开了如下由胆固醇制备25-羟基胆固醇的方法，其中使用过氧化三氟丙酮或三氧化铬/乙酸酐作为氧化剂来引入25-羟基。
Figure PCTCN2020136678-appb-000004
Chin.Chem.Lett.,1992,vol.3,p 409公开了如下由胆固醇提取过程中的副产物24-脱氢胆固醇制备25-羟基胆固醇的方法，其中使用汞试剂向24-双键引入25-羟基。
Figure PCTCN2020136678-appb-000005
CN104910231B公开了如下由25-羟基胆固醇制备25-羟基-7-脱氢胆固醇的方法，其中使用了腙试剂。
Figure PCTCN2020136678-appb-000006
CN106831921A公开了如下由5,7,24-三烯胆固醇制备25-羟基-7-脱氢胆固醇的方法，其中使用了Diels-Alder保护基。
Figure PCTCN2020136678-appb-000007
发明内容
在一方面，本发明提供一种制备式III化合物的方法，包括步骤(A)和步骤(B)，并且步骤(B)包括步骤(B-1)或(B-2)：
步骤(A)：
Figure PCTCN2020136678-appb-000008
步骤(B)：
步骤(B-1)：
Figure PCTCN2020136678-appb-000009
步骤(B-2)：
Figure PCTCN2020136678-appb-000010
其中，
Figure PCTCN2020136678-appb-000011
在式I、式II、式III中同时表示单键，或者
Figure PCTCN2020136678-appb-000012
在式I、式II、式III中同时表示双键，或者
Figure PCTCN2020136678-appb-000013
在式I中表示单键或双键，在式II和式III中表示双键；
R a、R b各自独立地选自H、-OH、C 1-3烷基；
PG为羟基保护基，优选为C 1-8硅烷基、乙酰基、三氟乙酰基、或任选地被一个或多个C 1-8烷基取代的苯甲酰基；
R为L 1-LG或者L 1-R 1；
L 1不存在，或为C 1-8亚烷基；
LG为离去基团；
R 1选自H、C 1-8烷基、C 1-8烯基、-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
PG 1为羟基保护基，优选选自C 1-8硅烷基或C 1-6烷氧基取代的甲基；
*表示手性中心。
具体而言，本发明可以采用两种方法制备式III化合物，第一种方法首先构造AB环，然后构造侧链；第二种方法首先构造侧链，再构造AB环。
第一种方法包括：
步骤(1)：式ii化合物转化为式a化合物；
Figure PCTCN2020136678-appb-000014
步骤(2)：式a化合物在硼氢化钠、氯化钙和吡啶的作用下还原为式b化合物；
Figure PCTCN2020136678-appb-000015
步骤(3)：式b化合物转化为式III化合物；
Figure PCTCN2020136678-appb-000016
Ra、Rb、PG、RL1、LG、R具有与上文所述相同的定义，本发明在此不再赘述。
第二种方法包括：
步骤(1)：式ii化合物转化为式I化合物；
步骤(2)：式I化合物转化为式II化合物；
步骤(3)：式II化合物在硼氢化钠和氯化钙的作用下还原为式III化合物。
其中，
Figure PCTCN2020136678-appb-000017
Ra、Rb、PG、RL1、LG、R具有与上文所述相同的定义，本发明在此不再赘述。
在一优选的方面，式III化合物结构为
Figure PCTCN2020136678-appb-000018
在另一优选的方面，式III化合物选自胆固醇、7-脱氢胆固醇、25-羟基胆固醇、25-羟基-7-脱氢胆固醇、其衍生物或类似物。
具体实施方式
除非另有定义，本文所用所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。若存在矛盾，则以本申请提供的定义为准。当本文中出现商品名时，意在指代其对应的商品或其活性成分。本文引用的所有专利、已经公开的专利申请和出版物均通过引用并入到本文中。
一般术语和定义
除非另有定义，本文所用所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。若存在矛盾，则以本申请提供的定义为准。当本文中出现商品名时，意在指代其对应的商品或其活性成分。本文引用的所有专利、已经公开的专利申请和出版物均通过引用并入到本文中。
术语“一个(种)或多个(种)”或者类似的表述“至少一个(种)”可以表示例如1、2、3、4、5、6、7、8、9、10个(种)或更多个(种)。
本文所用的表述m-n指m至n的范围以及由其中的各个点值组成的亚范围以及各个点值。例如，表述“C 1-C 8”或“C 1- 8”涵盖1-8个碳原子的范围，并应理解为还涵盖其中的任意亚范围以及每个点值，例如C 2-C 5、C 3-C 4、C 1-C 2、C 1-C 3、C 1-C 4、C 1-C 5、C 1-C 6、C 1-C 7等，以及C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8等。例如，表述“C 3-C 10”或“C 3- 10”也应当以类似的方式理解，例如可以涵盖包含于其中的任意亚范围和点值，例如C 3-C 9、C 6-C 9、C 6-C 8、C 6-C 7、C 7-C 10、C 7-C 9、C 7-C 8、C 8-C 9等以及C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10等。又例如，表述“三元至十元”应理解为涵盖其中的任意亚范围以及每个点值，例如三元至五元、三元至六元、三元至七元、三元至八元、四元至五元、四元至六元、四元至七元、四元至八元、五元至七元、五元至八元、六元至七元、六元至八元、九元至十元，等，以及三、四、五、六、七、八、九、十元，等。本文中其他类似的表述也应当以类似的方式理解。
术语“烷基”是指由碳原子和氢原子组成的直链或支链的饱和的脂肪烃基团，其通过单 键与分子的其余部分连接。“烷基”可以具有1-8个碳原子，即“C 1- 8烷基”，例如C 1- 4烷基、C 1- 3烷基、C 1- 2烷基、C 3烷基、C 4烷基、C 1- 6烷基、C 3- 6烷基。烷基的非限制性实例包括但不限于甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1,2-二甲基丙基、新戊基、1,1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、2,3-二甲基丁基、1,3-二甲基丁基或1,2-二甲基丁基，或者它们的异构体。
术语“亚烷基”，在本文中单独或与其他基团组合使用时，指直链或支链的饱和的二价烃基。例如，术语“C 1-6亚烷基”指具有1-6个碳原子的亚烷基，例如C 1- 5亚烷基、C 1- 4亚烷基、C 1- 3亚烷基、C 1- 2亚烷基、C 3亚烷基，以及C 1亚烷基，即亚甲基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2-)、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)等。
术语“烯基”是指由碳原子和氢原子组成的直链或支链的具有至少一个双键的不饱和脂肪族烃基。烯基可以具有2-8个碳原子，即“C 2- 8烯基”，例如C 2- 4烯基、C 3- 4烯基。烯基的非限制性实例包括但不限于乙烯基、烯丙基、(E)-2-甲基乙烯基、(Z)-2-甲基乙烯基、(E)-丁-2-烯基、(Z)-丁-2-烯基、(E)-丁-1-烯基、(Z)-丁-1-烯基等。
术语“炔基”是指由碳原子和氢原子组成的直链或支链的具有至少一个三键的不饱和脂肪族烃基。炔基可以具有2-8个碳原子，即“C 2- 8炔基”，例如C 2- 4炔基、C 3- 4炔基。炔基的非限制性实例包括但不限于乙炔基、丙-1-炔基、丙-2-炔基、丁-1-炔基、丁-2-炔基、丁-3-炔基等。
术语“烷氧基”，是指与氧原子以单键相连的如上定义的烷基。烷氧基与分子的其他部分通过氧原子相连。烷氧基可以表示为-O(烷基)。“C 1-8烷氧基”或“-O(C 1-8烷基)”是指含有1-8个碳原子的烷氧基，其中的烷基部分可为直链、支链或环状结构。烷氧基包括但不仅限于，甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、正戊氧基、环戊基氧基、环己基氧基等。
术语“硅烷基”，是指如上定义的烷基，其中至少一个C原子被Si原子取代。硅烷基与分子的其他部分通过硅原子相连。“C 1-8硅烷基”是指含有1-8个碳原子的硅烷基，其中的烷基部分可为直链、支链或环状结构。硅烷基基包括但不仅限于，三甲基硅基(TMS)、叔丁基 二甲基硅基(TBS，或称TBDMS)、二甲基异丙基硅基(IPDMS)、二叔丁基甲基硅基等(DTBMS)。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。此类物质的纯化和分离可通过本领域已知的标准技术实现。
术语“烃类”溶剂指具有1-10个碳原子的直链、支链或环状烃的溶剂。所述烃可以是饱和或不饱和的。烃类溶剂的实例例如有烷烃类溶剂，包括但不限于正戊烷、正己烷、环己烷、正庚烷、辛烷或其组合，优选己烷或庚烷。烃类溶剂的实例还例如有芳烃类溶剂，其含有至少一个芳香环，并任选地被直链、支链或环状烃取代。芳烃类溶剂包括但不限于苯、甲苯、二甲苯或其组合，优选甲苯、甲苯或其组合。
术语“卤代烷烃类”溶剂，是指上文所述的烷烃类溶剂，其中一个或多个(例如1-6个、1-5个、1-4个、1-3个，或者1-2个)氢原子被卤素代替。本领域技术人员应当理解，当卤素取代基多于一个时，卤素可以相同也也可以不同，并且可以位于相同或不同的C原子上。卤代烷烃类溶剂包括但不限于二氯甲烷、三氯甲烷、四氯化碳、1,2-二氯乙烷、六氯乙烷和1,2,3-三氯丙烷或其组合，优选二氯甲烷、三氯甲烷、1,2-二氯乙烷或其组合，优选二氯甲烷、氯仿、二氯乙烷及其组合，特别是二氯甲烷。
术语“醇类”溶剂具有1-10个碳原子的醇的溶剂。醇类溶剂包括但不限于甲醇、乙醇、正丙醇、异丙醇、正丁醇、正戊醇、环己醇或其组合，优选甲醇、乙醇及其组合。术语“酯类”溶剂是指具有3-10个碳原子的酯的溶剂。酯类溶剂包括但不限于乙酸乙酯、乙酸丙酯、乙酸异丙酯、乙酸丁酯、乙酸戊酯或其组合，优选乙酸乙酯、乙酸异丙酯、乙酸丁酯或其组合，特别是乙酸乙酯。
术语“醚类”溶剂是指具有2-10个碳原子的醚的溶剂。醚类溶剂的实例包括但不限于乙醚、异丙醚、四氢呋喃、二甲基四氢呋喃、二氧六环、乙二醇二甲醚、甲基叔丁基醚或其组合，优选乙醚、四氢呋喃、二甲基四氢呋喃或其组合。
术语“亚砜类”溶剂意指二甲亚砜。
术语“酮类”溶剂是指具有3-8个碳原子的酮，其实例包括但不限于丙酮、2-丁酮、3-甲基-2-丁酮、4-甲基-2-戊酮或其组合。
如本文中所使用的，术语“室温”指约10至30℃，优选约20至25℃。
下述发明详述旨在举例说明非限制性实施方案，使本领域其它技术人员更充分地理解本发明的技术方案、其原理及其实际应用，以便本领域其它技术人员可以以许多形式修改和实施本发明，使其可最佳地适应特定用途的要求。
本发明的制备方法
本发明的第一方面提供一种制备式III化合物的方法，包括步骤(A)和步骤(B)，并且步骤(B)包括步骤(B-1)或(B-2)；其中
步骤(A)：
Figure PCTCN2020136678-appb-000019
步骤(B)：
步骤(B-1)：
Figure PCTCN2020136678-appb-000020
步骤(B-2)：
Figure PCTCN2020136678-appb-000021
其中，
Figure PCTCN2020136678-appb-000022
在式I、式II、式III中同时表示单键，或者“”在式I、式II、式III中同时表示双键，或者
Figure PCTCN2020136678-appb-000023
在式I中表示单键或双键，在式II和式III中表示双键；
R a、R b各自独立地选自H、-OH、C 1-3烷基；
PG为羟基保护基，优选为C 1-8硅烷基、乙酰基、三氟乙酰基、或任选地被一个或多个C 1-8烷基取代的苯甲酰基；
R为L 1-LG或者L 1-R 1；
L 1不存在，或为C 1-8亚烷基；
LG为离去基团；
R 1选自H、C 1-8烷基、C 1-8烯基、-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
PG 1为羟基保护基，优选选自C 1-8硅烷基或C 1-6烷氧基取代的甲基；
*表示手性中心。
其中，步骤(B-1)和步骤(B-2)均表示在还原剂存在下制备式III化合物的过程。步骤(B-1)表示手性选择性地得到目标产物(式III化合物)。步骤(B-2)表示在一些情况下，反应得到的化合物(式III’化合物)未达到需要的光学纯度，因此进行异构体分离以获取式III化合物。
在一实施方案中，PG选自乙酰基和三氟乙酰基。在一优选实施方案中，PG为乙酰基。
在一实施方案中，步骤(A)在酰基化试剂存在下进行，酰基化试剂选自醋酐、乙酰氯、醋酸异丙烯酯及其组合，或者选自三氟乙酸酐、三氟乙酰氯及其组合。在另一实施方案中，酰基化试剂选自醋酐、乙酰氯和醋酸异丙烯酯及其组合。在一特别的实施方案中，酰基化试剂为醋酸异丙烯酯。在又一特别的实施方案中，酰基化试剂为醋酐、乙酰氯混合物。在一实施方案中，步骤(A)在酸存在下进行，所述酸为无机酸或有机酸。在一实施方案中，酸选自p-TsOH、MsOH、HCl、H 2SO 4、HClO 4及其组合。在一特别的实施方案中，酸为p-TsOH。
在一实施方案中，步骤(A)在0至110℃温度下进行，优选室温至110℃。在一实施方案中，反应在40至110℃温度下进行，例如40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃、90℃、95℃、100℃或其组合。
在一实施方案中，L 1不存在。在另一实施方案中，L 1为C 1-6亚烷基，优选C 1-6亚烷基，更优选C 1-4亚烷基。在一特别的实施方案中，L 1为C 1-2亚烷基，尤其是亚甲基。
在一实施方案中，LG选自卤素、任选地被卤素取代的C 1-6烷基磺酸酯基离去基团和任选地被C 1-6烷基取代的苯磺酸酯基离去基团。在一实施方案中，LG为OTf、OTs或OMs。在一实施方案中，LG为OTs。在一实施方案中，LG选自Cl、Br和I。
在一实施方案中，R 1选自H、C 1-8烷基、C 1-8烯基，其中所述烷基或烯基任选地被1、2或3个个选自-O(C 1-8烷基)和-O-PG 1的基团取代。
在一实施方案中，R 1为-CH＝CR 4R 5，其中R 4和R 5各自独立地选自H、C 1-6烷基、C 1-6烯基、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自 -OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代。
在一实施方案中，R 1选自-(CH 2) 2-C(＝O)O(C 1-8烷基)、-(CH 2) 2-C(＝O)N(C 1-8烷基) 2。优选地，R 1选自-(CH 2) 2-C(＝O)O(C 1-6烷基)、-(CH 2) 2-C(＝O)N(C 1-6烷基) 2。
在一特别的实施方案中，PG 1为TMS、TBS或MOM。
在一实施方案中，*表示的手性中心为消旋碳原子。在一实施方案中，*表示的手性中心为(S)-构型富集的碳原子。在另一实施方案中，*表示的手性中心为(R)-构型富集的碳原子。
在一实施方案中，还原剂选自硼氢化试剂、氢化锂铝。在另一实施方案中，硼氢化试剂选自Ca(BH 4) 2、NaBH 4、KBH 4及其组合。在一优选的实施方案中，硼氢化试剂为Ca(BH 4) 2。在一实施方案中，反应在碱的存在下进行，所述碱为有机碱、无机碱或其组合。在一优选的实施方案中，所述无机碱选自NaOH、KOH、NaOMe、t-BuOK及其组合。在另一优选的实施方案中，所述有机碱选自吡啶、三乙胺、DMAP及其组合。在另一特别的实施方案中，所述碱为吡啶。在一实施方案中，反应在有机溶剂中进行。在一实施方案中，有机溶剂选自醇类溶剂、醚类溶剂及其组合。在一优选的实施方案中，醚类溶剂选自乙醚、四氢呋喃及其组合。在另一优选的实施方案中，醇类溶剂选自甲醇、乙醇、异丙醇及其组合，优选为甲醇、乙醇或其组合，或者乙醇、异丙醇或其组合。在一特别的实施方案中，有机溶剂为四氢呋喃与以下醇类中的至少一个的混合溶剂：甲醇、乙醇和异丙醇。在另一特别的实施方案中，有机溶剂为四氢呋喃与以下醇类中的至少一个的混合溶剂：甲醇、乙醇。在一特别的实施方案中，有机溶剂为四氢呋喃、乙醇与异丙醇的混合溶剂。在一特别的实施方案中，四氢呋喃、乙醇与异丙醇混合溶剂中四氢呋喃、乙醇与异丙醇的比例为1:(0.1-1):(0.1-1)，优选1:(0.3-0.8):(0.3-0.8)，尤其是1:0.5:0.5。
在一实施方案中，步骤(B)在-20至80℃温度下进行，优选-20至70℃。在一实施方案中，反应在-20至60℃温度下进行。在一实施方案中，反应在-20至40℃温度下进行，优选-10至40℃，例如-10℃、-5℃、0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃或40℃。在一实施方案中，
Figure PCTCN2020136678-appb-000024
在式I中表示单键或双键，当其表示单键时，式I化合物可以表示为式I-1，并且本发明的方法在步骤(A)之前任选地包括以下步骤(a)，以使该单键转化为双键；转化后得到的结构表示为式I-2，并且仍然涵盖在式I的定义内。
步骤(a)：使式I-1化合物发生脱氢反应，以制备式I-2化合物
Figure PCTCN2020136678-appb-000025
其中：
R为L 1-LG或者L 1-R 1，R 1选自H、C 1-8烷基、C 1-8烯基、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
L 1、LG、PG和PG 1如上文所定义。
在一实施方案中，步骤(a)在脱氢试剂存在下进行。在一实施方案中，脱氢试剂选自DDQ和四氯苯醌。在一优选的实施方案中，脱氢试剂选自四氯苯醌。在另一实施方案中，脱氢反应通过CN104910231B或Journal of Organic Chemistry,2005,70(21),8513-8521；Tetrahedron Letters,2004,45(40),7479-7482记载的方法进行。
在一实施方案中，式I、式II、式III’和式III化合物中的任意一个或多个任选地发生反应，所述反应包括以下步骤和步骤组合中的任意一个或多个：步骤(b)、步骤(c)、步骤(d)、步骤(e)、步骤(f)、步骤(g)-步骤(h)的组合、步骤(i)、步骤(j)。通过上述反应，式I、式II、式III’或式III化合物中的R发生基团转换，转换后得到的基团仍然涵盖在R的定义内。以下式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv化合物每次出现时，其中的
Figure PCTCN2020136678-appb-000026
各自独立地表示单键或双键，以使式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv的甾体AB环结构各自独立地对应式I、式II、式III’或式III的甾体AB环结构。在步骤(b)、(c)、(d)、(e)、(f)、(g)、(h)、(i)和(j)中的任一步骤之内，每个
Figure PCTCN2020136678-appb-000027
在产物中表示的结构与其在反应物中表示的结构相同。式i、式ii、式iii、式iii’、式iii”、式iii”'化合物中的L 1-LG或L 1-R 1结构涵盖在上文所述R的定义内。每次出现时，式i、式ii、式iii、式iii’、式iii”、式iii”'化合物各自独立地涵盖在式I、式II、式III’或式III的结构范围之内。
在一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中的至少一个式中的甾体3-位为羰基，甾体3-位羰基氧原子不通过化学键连接R 2，并且R 2不存在，并且AB环的结构为
Figure PCTCN2020136678-appb-000028
在一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中AB环的结构与式I相对应。
在另一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中的至少一个式中的甾体3-位为羟基，甾体3-位羟基氧原子通过化学键与R 2连接，并且AB环的结构为
Figure PCTCN2020136678-appb-000029
在一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中AB环的结构与式II相对应。
在一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中的至少一个式中的AB环的结构为
Figure PCTCN2020136678-appb-000030
在一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中AB环的结构与式III’或式III相对应。在另一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中的至少一个式中的AB环的结构为
Figure PCTCN2020136678-appb-000031
在又一实施方案中，式i、式ii、式iii、式iii’、式iii”、式iii”'、式iv中的至少一个式中的AB环的结构为
Figure PCTCN2020136678-appb-000032
当R为L 1-OH时，式I、式II、式III’或式III化合物的结构如下式i所示。当R为L 1-LG时，式I、式II、式III’或式III化合物的结构如下式ii所示。
步骤(b)：使式i化合物与磺酰化试剂或卤化试剂反应，以制备式ii化合物
Figure PCTCN2020136678-appb-000033
其中：
R 2为PG，PG如上文所定义；
(i)LG为任选地被卤素取代的C 1-6烷基磺酸酯基离去基团或任选地被C 1-6烷基取代的苯磺酸酯基离去基团，反应在磺酰化试剂存在下进行；或者
(ii)LG为卤素，反应在卤化试剂存在下进行。
在一实施方案中，步骤(b)在步骤(A)之前进行，式i和式ii同时涵盖在式I的结构范围之内，步骤(b)如下
Figure PCTCN2020136678-appb-000034
在一实施方案中，LG为OTf、OMs或OTs。在一实施方案中，磺酰化试剂为(Tf) 2O、MsCl或TsCl。
在一实施方案中，LG为Cl、Br或I。在一实施方案中，卤化试剂为SOCl 2、POCl 3、氯硅烷试剂、卤化磷、HBr、NBS、(PhO) 3P/CH 3I体系或Ph 3P/I 2/咪唑体系。
当R为L 1-LG’时，式I、式II、式III’或式III化合物的结构如下式ii’所示。
步骤(c)：使式ii’化合物与卤化试剂反应，以制备式ii化合物
Figure PCTCN2020136678-appb-000035
其中：
R 2为PG，PG如上文所定义；
LG’为任选地被卤素取代的C 1-6烷基磺酸酯基离去基团或任选地被C 1-6烷基取代的苯磺酸酯基离去基团；LG为卤素；反应在卤化试剂存在下进行。
在一实施方案中，步骤(c)在步骤(A)之前进行，式ii’和式ii同时涵盖在式I的结构范围之内，步骤(c)如下
Figure PCTCN2020136678-appb-000036
在一实施方案中，LG’为OTf、OMs或OTs。在一实施方案中，LG为Cl、Br或I。在一实施方案中，卤化试剂为卤化氢、I 2，POCl 3、PBr 3或金属卤化物。在一实施方案中，金属卤化物选自氯化钠、氯化锂、氯化镁及其组合，或者选自溴化钠、溴化锂、溴化镁及其组合，或者为碘化钠。
当R为L 1-R 1时，式I、式II、式III’或式III化合物的结构如下式iii所示。
步骤(d)：使式ii化合物与有机金属试剂反应，以制备式iii化合物
Figure PCTCN2020136678-appb-000037
其中：
R 2为H或PG；
LG选自卤素、任选地被卤素取代的C 1-6烷基磺酸酯基离去基团和任选地被C 1-6烷基取代的苯磺酸酯基离去基团；
Figure PCTCN2020136678-appb-000038
为有机金属试剂；LG1为MgX或CuLi；n为1或2；
R 1选自H、C 1-8烷基、C 1-8烯基，其中所述烷基或烯基任选地被1、2或3个选自-O(C 1-8烷基)和-O-PG 1的基团取代；
L 1、PG、PG 1如上文所定义。
在一实施方案中，步骤(d)在步骤(B-2)的第一步与第二步之间进行，式ii和式iii同时涵盖在式III’的结构范围之内，步骤(d)包括以下步骤(d-1)
Figure PCTCN2020136678-appb-000039
在一实施方案中，步骤(d)在步骤(B)之后进行，式ii和式iii同时涵盖在式III的结构范围之内，步骤(d)包括以下步骤(d-2)
Figure PCTCN2020136678-appb-000040
在一实施方案中，LG为OTf、OMs或OTs。在另一实施方案中，LG为Cl、Br或I。
在一实施方案中，LG 1为MgX，其中X为Cl、Br或I，n为1，
Figure PCTCN2020136678-appb-000041
属于格氏试剂。在一实施方案中，步骤(d)在催化剂存在下进行，催化剂选自卤代铜、卤代亚铜、氯化锂、锂铜复盐及其组合。在一实施方案中，催化剂选自以下的一种或多种：CuI、CuBr、LiCl、CuCl 2、CuCl、Li 2CuCl 4、LiCuCl 3。
在一实施方案中，LG 1为CuLi，n为2，
Figure PCTCN2020136678-appb-000042
属于烃基铜锂试剂。
在一实施方案中，步骤(d)还包括保护3-羰基的步骤以及脱保护的步骤。在一实施方案中，保护3-羰基的步骤通过使3-羰基形成缩酮来实现。在一实施方案中，保护3-羰基的保护基为1,3-二氧戊环。
步骤(e)：使式ii化合物与丙烯酸衍生物在还原剂存在下反应，以制备式iii化合物
Figure PCTCN2020136678-appb-000043
其中，R 2为H或PG，PG如上文所定义。
LG选自卤素、任选地被卤素取代的C 1-6烷基磺酸酯基离去基团和任选地被C 1-6烷基取代的苯磺酸酯基离去基团。优选地，LG为卤素。
丙烯酸衍生物选自丙烯酸烷基酯，其中所述烷基各自独立地选自C 1-8烷基。优选地，所述烷基各自独立地选自C 1-6烷基，更优选C 1-4烷基。
还原剂选自锌、铁、铝、镁。在一特别的实施方案中，还原剂为锌。
R 1选自-(CH 2) 2-C(＝O)O(C 1-8烷基)。优选地，R 1选自-(CH 2) 2-C(＝O)O(C 1-6烷基)。更优选地，R 1选自-(CH 2) 2-C(＝O)O(C 1-4烷基)。
在一实施方案中，步骤(e)在步骤(B-2)的第一步与第二步之间进行，式ii和式iii同时涵盖在式III’的结构范围之内，步骤(e)包括以下步骤(e-1)
Figure PCTCN2020136678-appb-000044
在一实施方案中，步骤(e)在步骤(B)之后进行，式ii和式iii同时涵盖在式III的结构范围之内，步骤(e)包括以下步骤(e-2)
Figure PCTCN2020136678-appb-000045
在一实施方案中，丙烯酸衍生物为丙烯酸C 1-6烷基酯。在另一实施方案中，丙烯酸烷基酯选自丙烯酸甲酯、丙烯酸乙酯、丙烯酸丙酯、丙烯酸正丁酯和丙烯酸异丁酯。在一优选的实施方案中，丙烯酸烷基酯选自丙烯酸甲酯和丙烯酸乙酯。
在一实施方案中，步骤(e)在催化剂存在下进行，催化剂选自CuI，Ni(0)/配体，Zn/NiCl 2/配体。在一实施方案中，配体选自PPh 3、联吡啶、邻菲罗啉、吡啶、DMAP及其组合。在一特别的实施方案中，催化剂为Zn/NiCl 2/吡啶。
在一实施方案中，步骤(e)在0至80℃温度下进行，优选0至70℃。在一实施方案中，反应在0至60℃温度下进行。在一实施方案中，反应在10至60℃温度下进行，例如10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃或其组合。在一实施方案中，反应在有机溶剂中进行。在一实施方案中，有机溶剂选自乙酸乙酯，四氢呋喃，二氧六环，乙醚，醋酸丁酯，甲基四氢呋喃，吡啶。在一实施方案中，有机溶剂为吡啶。
在一实施方案中，步骤(e)包括以下步骤i)至步骤iii)：
i)将还原剂与催化剂与溶剂混合得到混合物；
ii)将i)中得到的混合物与丙烯酸衍生物混合，反应得到活化的丙烯酸衍生物；
iii)将ii)中得到的活化的丙烯酸衍生物与式ii化合物混合，反应得到式iii化合物。
在一实施方案中，步骤ii)在30至80℃温度下进行，优选30至70℃，更优选40至60℃，例如40℃、45℃、50℃、55℃或60℃。
在一实施方案中，步骤ii)通过向i)中得到的混合物缓慢加入丙烯酸衍生物来进行。在一实施方案中，步骤ii)在混合完毕之后的反应时间为0.5至4小时，优选1-2小时。
在一实施方案中，步骤iii)在0至60℃温度下进行，优选0至40℃，更优选0至30℃，例如0℃、5℃、10℃、15℃、20℃、25℃、30℃。在一实施方案中，步骤iii)在混合完毕之后的反应时间为0.5至8小时，优选1-6小时，更优选3-4小时。
当R为L 1-R 1’时，式I、式II、式III’或式III化合物的结构如下式iii’所示。
步骤(f)：使式iii’化合物与烷基化试剂反应，以制备式iii化合物
Figure PCTCN2020136678-appb-000046
其中R 2为H或PG，PG如上文所定义；
R 1’选自C 1-8烷基、C 1-8烯基和-C(＝O)O(C 1-8烷基)，其中所述烷基或烯基被1或2个选自-C(＝O)O(C 1-8烷基)的基团取代；
R 1选自C 1-8烷基、C 1-8烯基和
Figure PCTCN2020136678-appb-000047
其中所述烷基或烯基被1或2个选自
Figure PCTCN2020136678-appb-000048
的基团取代，其中R 3选自C 1-8烷基；
烷基化试剂为R 3MgX，或R 3Li，其中X为Cl或Br。
在一实施方案中，R 3选自C 1-4烷基。在一特别的实施方案中，R 3为甲基。
在一实施方案中，步骤(f)在步骤(B-2)的第一步与第二步之间进行，式iii’和式iii同时涵盖在式III’的结构范围之内，步骤(f)包括以下步骤(f-1)
Figure PCTCN2020136678-appb-000049
在一实施方案中，步骤(f)在步骤(B)之后进行，式iii’和式iii同时涵盖在式III的结构范围之内，步骤(f)包括以下步骤(f-2)
。
在一实施方案中，步骤(f)还包括保护3-OH的步骤以及脱保护的步骤。在一实施方案中，保护3-OH的保护基选自C 1-8硅烷基、乙酰基、三氟乙酰基、任选地被甲氧基取代的苄基或C 1-6烷氧基取代的甲基。
当R为L 1-CHO时，式I、式II、式III’或式III化合物的结构如下式iv所示。
步骤(g)：使式iii化合物与氧化剂反应，以制备式iv化合物
Figure PCTCN2020136678-appb-000050
其中R 1为-CH 2OH；R 2为PG；
L 1、PG如上文所定义。
在一实施方案中，步骤(g)在步骤(A)之前进行，式iii涵盖在式I的结构范围之内，步骤(g)包括以下步骤(g-1)
Figure PCTCN2020136678-appb-000051
在一实施方案中，步骤(g)在步骤(A)和步骤(B)之间进行，式iii涵盖在式II的结构范围之内，步骤(g)包括以下步骤(g-2)
Figure PCTCN2020136678-appb-000052
在一实施方案中，氧化剂选自TEMPO/NaBr/NaClO、TEMPO/NaBr/Ca(ClO) 2、TEMPO/TCCA、DMSO/SO 3-Py/Et 3N、NaNO 2/FeCl 3/TEMPO/空气、NaNO 2/FeCl 3/TEMPO/O 2及其组合。在一特别的实施方案中，氧化剂为TEMPO/NaBr/NaClO。
在一实施方案中，反应在有机溶剂中进行。在一实施方案中，有机溶剂选自烃类溶剂、卤代烃类溶剂、酯类溶剂、酮类溶剂及其组合。在一优选的实施方案中，烃类溶剂选自苯、甲苯及其组合。在又一优选的实施方案中，卤代烃类溶剂选自二氯甲烷、氯仿、二氯乙烷及其组合，优选二氯甲烷。在一优选的实施方案中，酯类溶剂为乙酸乙酯。在另一优选的实施方案中，酮类溶剂为丙酮。
步骤(h)：使式iv化合物与Wittig试剂反应，以制备式iii化合物
Figure PCTCN2020136678-appb-000053
R 1为-CH＝CR 4R 5，其中R 4和R 5各自独立地选自H、C 1-6烷基、C 1-6烯基、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1或2个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
R 2为H或PG；
L 1、PG、PG 1如上文所定义。
在一实施方案中，CR 4R 5结构选自
Figure PCTCN2020136678-appb-000054
在一实施方案中，步骤(h)在步骤(A)之前进行，式iii涵盖在式I的结构范围之内，步骤(h)包括以下步骤(h-1)
Figure PCTCN2020136678-appb-000055
在一实施方案中，步骤(h)在步骤(B-2)的第一步与第二步之间进行，式iii涵盖在式III’的结构范围之内，步骤(h)包括以下步骤(h-2)
Figure PCTCN2020136678-appb-000056
在一实施方案中，步骤(h)在步骤(B)之后进行，式iii涵盖在式III的结构范围之内，步骤(h)包括以下步骤(h-3)
Figure PCTCN2020136678-appb-000057
在一实施方案中，Wittig试剂由卤代烃与磷试剂制备，其中卤代烃为X-CHR 4R 5，X为Cl、Br或I。
在一实施方案中，磷试剂选自三苯基膦，亚磷酸三甲酯，亚磷酸三乙酯等。在一特别的实施方案中，磷试剂为三苯基膦。
在一实施方案中，由卤代烃与磷试剂制备Wittig试剂的反应在碱的存在下进行，所述碱为有机碱、无机碱或其组合。在一实施方案中，碱选自叔丁醇钾、甲醇钠、甲醇钾、氢化钠、丁基锂、二异丙氨基锂及其组合。在一特别的实施方案中，碱为叔丁醇钾。在一实施方案中，步骤(h)在-20至80℃温度下进行，优选-20至70℃。在一实施方案中，反应在-20至60℃温度下进行。在一实施方案中，反应在-10至60℃温度下进行，优选0至50℃，更优选10至40℃，例如10℃、15℃、20℃、25℃、30℃、35℃或40℃。在一实施方案中，反应在有机溶剂中进行。在一实施方案中，有机溶剂选自亚砜类溶剂、醚类溶剂、烃类溶剂及其组合。在一优选的实施方案中，亚砜类溶剂为DMSO。在另一优选的实施方案中，醚类溶剂选自乙醚、四氢呋喃、二甲基四氢呋喃及其组合。在又一优选的实施方案中，烃类溶剂选自苯、甲苯及其组合。
当R为L 1-R 1”时，式I、式II、式III’或式III化合物的结构如下式iii”所示。
步骤(i)：使式iii”化合物还原，以制备式iii化合物
Figure PCTCN2020136678-appb-000058
R 1”为C 1-8烯基，其中所述烯基任选地被1或2个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)的基团取代；
R 1为C 1-8烷基，其中所述烷基任选地被1或2个选自-OH、-O(C 1-8烷基)、-O-PG 1、-CH 2OH、-C(＝O)O(C 1-8烷基)的基团取代；
R 2为H；
L 1、PG 1如上文所定义。
在一实施方案中，步骤(i)在步骤(B-2)的第一步与第二步之间进行，式iii”和式iii同时涵盖在式III’的结构范围之内，步骤(i)包括以下步骤(i-1)
Figure PCTCN2020136678-appb-000059
在一实施方案中，步骤(i)在步骤(B)之后进行，式iii”和式iii同时涵盖在式III的结构范围之内，步骤(i)包括以下步骤(i-2)
Figure PCTCN2020136678-appb-000060
当R为L 1-R 1”'时，式I、式II、式III’或式III化合物的结构如下式iii”'所示。
在一实施方案中，步骤(i)通过催化氢化反应进行。
步骤(j)：使式iii”'化合物脱保护基，以制备式iii化合物
Figure PCTCN2020136678-appb-000061
R 1”'选自H、C 1-8烷基、C 1-8烯基、-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1或2个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
R 1选自H、C 1-8烷基、C 1-8烯基、-OH、-O(C 1-8烷基)、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-OH、-O(C 1-8烷基)、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
R 2为H；
L 1、PG 1如上文所定义。
在一实施方案中，步骤(j)在步骤(B-2)的第一步与第二步之间进行，式iii”'和式iii同时涵盖在式III’的结构范围之内，步骤(j)包括以下步骤(j-1)
Figure PCTCN2020136678-appb-000062
在一实施方案中，步骤(j)在步骤(B)之后进行，式iii”'和式iii同时涵盖在式III的结构范围之内，步骤(j)包括以下步骤(j-2)
Figure PCTCN2020136678-appb-000063
上述式i、式ii、式iii、式iii”、式iii”'化合物涵盖在式I、式II、式III’或式III的结构范围之内，式iv中AB环的结构与式iii相同，因此式i、式ii、式iii、式iii”、式iii”'、式iv中AB环存在多种结构时，多种结构之间可以根据步骤(A)和步骤(B)所述的方法进行转化。
本发明第一或第二方面的步骤(A)、步骤(B)、步骤(a)至步骤(j)的反应总结于下表1中。
表1
Figure PCTCN2020136678-appb-000064
本发明的第二方面在于提供一种制备式III化合物的方法，包括：
步骤(1)：式ii化合物转化为式a化合物；
Figure PCTCN2020136678-appb-000065
步骤(2)：式a化合物在硼氢化钠、氯化钙和吡啶的作用下还原为式b化合物；
Figure PCTCN2020136678-appb-000066
步骤(3)：式b化合物转化为式III化合物；
Figure PCTCN2020136678-appb-000067
其中，
Figure PCTCN2020136678-appb-000068
Ra、Rb、PG、RL1、LG、R具有与上文所述相同的定义，本发明在此不再赘述。
在一实施方案中，PG选自乙酰基和三氟乙酰基。在一优选实施方案中，PG为乙酰基。在一实施方案中，步骤(1)在酰基化试剂存在下进行，酰基化试剂选自醋酐、乙酰氯、醋酸异丙烯酯及其组合，或者选自三氟乙酸酐、三氟乙酰氯及其组合。在另一实施方案中，酰基化试剂选自醋酐、乙酰氯和醋酸异丙烯酯及其组合。在一特别的实施方案中，酰基化试剂为醋酸异丙烯酯。在又一特别的实施方案中，酰基化试剂为醋酐、乙酰氯混合物。在一实施方案中，步骤(1)在酸存在下进行，所述酸为无机酸或有机酸。在一实施方案中，酸选自p-TsOH、MsOH、HCl、H2SO4、HClO4及其组合。在一特别的实施方案中，酸为p-TsOH。在一实施方案中，步骤(1)在0至110℃温度下进行，优选室温至110℃。在一实施方案中，反应在40至110℃温度下进行，例如40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃、90℃、95℃、100℃或其组合。
在一实施方案中，L 1不存在。在另一实施方案中，L 1为C 1-6亚烷基，优选C 1-6亚烷基，更优选C 1-4亚烷基。在一特别的实施方案中，L 1为C 1-2亚烷基，尤其是亚甲基。
在一实施方案中，LG选自卤素、任选地被卤素取代的C 1-6烷基磺酸酯基离去基团和任选地被C 1-6烷基取代的苯磺酸酯基离去基团。在一实施方案中，LG为OTf、OTs或OMs。在一实施方案中，LG为OTs。在一实施方案中，LG选自Cl、Br和I。
在一实施方案中，R 1选自H、C 1-8烷基、C 1-8烯基，其中所述烷基或烯基任选地被1、2或3个个选自-O(C 1-8烷基)和-O-PG 1的基团取代。
在一实施方案中，R 1为-CH＝CR 4R 5，其中R 4和R 5各自独立地选自H、C 1-6烷基、C 1-6烯基、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代。
在一实施方案中，R 1选自-(CH 2) 2-C(＝O)O(C 1-8烷基)、-(CH 2) 2-C(＝O)N(C 1-8烷基) 2。优选地，R 1选自-(CH 2) 2-C(＝O)O(C 1-6烷基)、-(CH 2) 2-C(＝O)N(C 1-6烷基) 2。
在一特别的实施方案中，PG 1为TMS、TBS或MOM。
在一实施方案中，步骤(2)中式a化合物在硼氢化钠、氯化钙和吡啶的作用下还原为式b化合物。在一实施方案中，反应在有机溶剂中进行。在一实施方案中，有机溶剂选自醇类溶剂和四氢呋喃的混合溶剂。在另一优选的实施方案中，醇类溶剂选自甲醇、乙醇、异丙醇及其组合，优选为甲醇、乙醇或其组合，或者乙醇、异丙醇或其组合。在一个实施方案中，乙醇或异丙醇的体积比为1:1。在一特别的实施方案中，有机溶剂为四氢呋喃与以下醇类中的至少一个的混合溶剂：甲醇、乙醇和异丙醇。在另一特别的实施方案中，有机溶剂为四氢呋喃与以下醇类中的至少一个的混合溶剂：甲醇、乙醇。在一特别的实施方案中，有机溶剂为四氢呋喃、乙醇与异丙醇的混合溶剂。在一个实施方案中，所述醇和四氢呋喃的体积比为1～2:1，尤其是1:1。在一特别的实施方案中，四氢呋喃、乙醇与异丙醇混合溶剂中四氢呋喃、乙醇与异丙醇的比例为1:(0.1-1):(0.1-1)，优选1:(0.3-0.8):(0.3-0.8)，尤其是1:0.5:0.5。
在一实施方案中，步骤(2)在-20至80℃温度下进行，优选-20至70℃。在一实施方案中，反应在-20至60℃温度下进行。在一实施方案中，反应在-20至40℃温度下进行，优选-10至40℃，例如-10℃、-5℃、0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃或40℃。
在一实施方案中，步骤(3)按照上述步骤(d)进行，即使式b化合物的离去基团与有机金属试剂反应，以构建侧链得到式III化合物；其构建过程与上文所述相似，本发明在此不再赘述。
在一实施方案中，步骤(3)按照上述步骤(e)进行，即式b化合物的离去基团与丙烯酸衍生物在还原剂的作用下发生反应，以构建侧链生成式III化合物；其构建过程与上文相似，本发明在此不再赘述。
在一实施方案中，还包括步骤f，使侧链含有酯基的化合物与烷基化试剂发生反应，以制备侧链含有三级羟基的化合物，其过程与上文所述类似，本发明在此不再赘述。
在一实施方案中，
Figure PCTCN2020136678-appb-000069
在式I中表示单键或双键，当其表示单键时，式ii化合物可以表示为式ii-1，并且本发明的方法在步骤(1)之前任选地包括以下步骤(a)，使化合物发生脱氢反应，使该单键转化为双键；转化后得到的结构表示为式ii-2，并且仍然涵盖在式I的定义内。该过程与上文所述步骤a相似，本发明在此不再赘述。
在一些实施方案中，还包括步骤(b)，使化合物侧链上的一级羟基与磺酰化试剂或卤化试剂反应，以制备侧链含有离去基团的化合物。该过程与上文所述过程相似，本发明在此不再赘述。
在一些实施方案中，还包括步骤(c)：使含有磺酸酯基的式ii’化合物与卤化试剂反应，以制备含有卤素离去基团的式ii化合物，该过程与上文所述过程相似，本发明在此不再赘述。
在一些实施例中，还包括步骤(i)：使侧链含有双键的式iii”化合物还原，以制备具有侧链的式iii化合物。该过程与上文所述过程相似，本发明在此不再赘述。
在一个实施例中，还包括步骤(j)：使侧链含有保护剂的式iii”'化合物脱保护基，以构建式iii化合物的侧链。该过程与上文所述过程相似，本发明在此不再赘述。
本发明的第三方面在于提供另一种制备式III化合物的方法，包括：
步骤(11)：式ii化合物转化为式I化合物；
Figure PCTCN2020136678-appb-000070
步骤(12)：式I化合物转化为式II化合物；
步骤(13)：式II化合物在硼氢化钠和氯化钙的作用下还原为式III化合物；
Figure PCTCN2020136678-appb-000071
其中，其中，
Figure PCTCN2020136678-appb-000072
R a、R b、PG、RL 1、LG、R具有与上文所述相同的定义，本发明在此不再赘述。
在一实施方案中，PG选自乙酰基和三氟乙酰基。在一优选实施方案中，PG为乙酰基。
在一实施方案中，步骤(12)在酰基化试剂存在下进行，酰基化试剂选自醋酐、乙酰氯、醋酸异丙烯酯及其组合，或者选自三氟乙酸酐、三氟乙酰氯及其组合。在另一实施方案中，酰基化试剂选自醋酐、乙酰氯和醋酸异丙烯酯及其组合。在一特别的实施方案中，酰基化试剂为醋酸异丙烯酯。在又一特别的实施方案中，酰基化试剂为醋酐、乙酰氯混合物。在一实施方案中，步骤(12)在酸存在下进行，所述酸为无机酸或有机酸。在一实施方案中，酸选自p-TsOH、MsOH、HCl、H 2SO 4、HClO 4及其组合。在一特别的实施方案中，酸为p-TsOH。在一实施方案中，步骤(12)在0至110℃温度下进行，优选室温至110℃。在一实施方案中，反应在40至110℃温度下进行，例如40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃、90℃、95℃、100℃或其组合。
在一实施方案中，L 1不存在。在另一实施方案中，L 1为C 1-6亚烷基，优选C 1-6亚烷基，更优选C 1-4亚烷基。在一特别的实施方案中，L 1为C 1-2亚烷基，尤其是亚甲基。
在一实施方案中，LG选自卤素、任选地被卤素取代的C 1-6烷基磺酸酯基离去基团和任选地被C 1-6烷基取代的苯磺酸酯基离去基团。在一实施方案中，LG为OTf、OTs或OMs。在一实施方案中，LG为OTs。在一实施方案中，LG选自Cl、Br和I。
在一实施方案中，R 1选自H、C 1-8烷基、C 1-8烯基，其中所述烷基或烯基任选地被1、2或3个个选自-O(C 1-8烷基)和-O-PG 1的基团取代。
在一实施方案中，R 1为-CH＝CR 4R 5，其中R 4和R 5各自独立地选自H、C 1-6烷基、C 1-6烯基、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代。
在一实施方案中，R 1选自-(CH 2) 2-C(＝O)O(C 1-8烷基)、-(CH 2) 2-C(＝O)N(C 1-8烷基) 2。优选地，R 1选自-(CH 2) 2-C(＝O)O(C 1-6烷基)、-(CH 2) 2-C(＝O)N(C 1-6烷基) 2。
在一特别的实施方案中，PG 1为TMS、TBS或MOM。
在一实施方案中，步骤(13)中式II化合物在硼氢化钠和氯化钙的作用下还原为式III化合物。在一实施方案中，反应在有机溶剂中进行。在一实施方案中，有机溶剂选自醇类溶剂和二氯甲烷的混合溶剂。在另一优选的实施方案中，醇类溶剂选自甲醇、乙醇、异丙醇及其组合，优选为甲醇、乙醇或其组合，或者乙醇、异丙醇或其组合。在一个实施方案中，乙醇或异丙醇的体积比为1:1。在一特别的实施方案中，有机溶剂为二氯甲烷与以下醇类中的至少一个的混合溶剂：甲醇、乙醇和异丙醇。在另一特别的实施方案中，有机溶剂为二氯甲烷与以下醇类中的至少一个的混合溶剂：甲醇、乙醇。在一特别的实施方案中，有机溶剂为二氯甲烷、乙醇与异丙醇的混合溶剂。在一个实施方案中，所述醇和二氯甲烷的体积比为1～2:1，尤其是1:1。在一特别的实施方案中，二氯甲烷、乙醇与异丙醇混合溶剂中二氯甲烷、乙醇与异丙醇的比例为1:(0.1-1):(0.1-1)，优选1:(0.3-0.8):(0.3-0.8)，尤其是1:0.5:0.5。
在一实施方案中，步骤(13)在-20至80℃温度下进行，优选-20至70℃。在一实施方案中，反应在-20至60℃温度下进行。在一实施方案中，反应在-20至40℃温度下进行，优选-10至40℃，例如-10℃、-5℃、0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃或40℃。
在一实施方案中，步骤(11)可以按照步骤d进行，使含有离去基团的式ii化合物在有机金属试剂的作用下转化为式I化合物，以构建侧链。该过程与上文所述过程相似，本发明在此不再赘述。
在一些实施方案中，步骤(11)可以按照步骤e进行，使含有离去基团的式ii化合物在丙烯酸衍生物和还原剂的作用下转化为式I化合物，以构建侧链。该过程与上文所述过程相似，本发明在此不再赘述。
在一实施方案中，还包括步骤f，侧链含有酯基的式ii化合物与烷基化试剂发生反应，制备侧链含有三级羟基的式I化合物。该过程与上文所述过程相似，本发明在此不再赘述。
在一实施方案中，
Figure PCTCN2020136678-appb-000073
在式I中表示单键或双键，当其表示单键时，式ii化合物可以表示为式ii-1，并且本发明的方法在步骤(12)之前任选地包括步骤(a)，使式ii化合物发生脱氢反应，制备脱氢的式ii-2化合物。该过程与上文所述过程相似，本发明在此不再赘述。
在一些实施方案中，还包括步骤(b)：使侧链上含有一级羟基的式i化合物与磺酰化试剂或卤化试剂反应，以制备含有离去基团的式ii化合物。该过程与上文所述过程相似，本发明在此不再赘述。
在一些实施方案中，还包括步骤(c)：使含有磺酸酯基离去基团的式ii’化合物与卤化试剂反应，以制备含有卤素离去基团的式ii化合物。该过程与上文所述过程相似，本发明在此不再赘述。
在一个实施例中，还包括步骤(g)：使侧链上含有一级羟基的式iii化合物与氧化剂反应，以制备侧链上含有醛基的式iv化合物。该过程与上文所述过程相似，本发明在此不再赘述。
在一实施方案中，还包括步骤(h)，使侧链含有醛基的化合物发生Wittig反应，制备侧链含有双键的化合物。该过程与上文所述过程相似，本发明在此不再赘述。
本发明还提供了由式I-1化合物制备式III-1化合物的方法：
Figure PCTCN2020136678-appb-000074
制备过程与上文所述相同，区别在于取代基和手性，本发明在此不再赘述。
应当理解，本领域技术人员也可根据需要对本发明的合成方法进行适当调整，例如调整反应步骤的顺序，以及增加或省略保护/脱保护反应步骤。此外，根据需要以及实际情况，本发明的合成方法的各个步骤之间可以分开进行，也可以同时进行，不做特别的限定。例如以上所述各个方面中，步骤(j)可以与其他反应同时进行。
根据本领域中已有的知识，可以以植物甾醇为原料，通过微生物转化制备以下式I的化合物。因此，在本发明的一个任选的方面，可以由植物甾醇经过微生物转化和化学合成制备式I化合物，所述方法包括步骤(A’)
步骤(A’)：使式I’化合物与分枝杆菌(Mycobacterium)属的微生物或其培养物接触，以制备式I化合物
Figure PCTCN2020136678-appb-000075
其中式I’化合物为植物甾醇，R’选自
Figure PCTCN2020136678-appb-000076
R为-CH 2OH。
有益效果
本发明提供本领域亟需的制备胆固醇及其衍生物的新方法，尤其是制备25-羟基胆固醇、25-羟基-7-脱氢胆固醇的方法，解决了现有技术中的技术难题例如原料来源问题及侧链中25-羟基构建难度大的问题。
本发明的方法原料易得。步骤操作简单，无特殊分离手段，反应收率高，生产成本低，适合工业化生产。并且无需使用贵金属催化剂、剧毒试剂如过氧化三氟丙酮或三氧化铬/乙酸酐体系，无需使用腙试剂，因此可以避免引入污染环境的含氮废水。
本发明的方法可以任选地以价格低廉且易于获得的植物甾醇为原料，通过微生物转化制备中间体，效率高，对环境污染小或无污染，更重要的是本发明从完全植物源的起始物出发，成功制备了胆固醇及其衍生物，避免了以往动物源起始物合成工艺面临的安全风险，如疯牛病，猪链球菌病感染等，具有较高的经济价值和广阔应用前景。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：由化合物1制备胆固醇
Figure PCTCN2020136678-appb-000077
步骤(1)中间体2A的制备
室温下向反应瓶中加入200g化合物1、10g DMAP、200mL三乙胺及1000mL DCM，氮气置换，搅拌至溶液澄清。升温回流。向体系中滴加150g对甲苯磺酰氯的DCM(400mL)溶液，约30min滴加完毕，继续回流反应1-2h。TLC监测反应至原料消失。反应完毕，将体系降温至10℃，滴加50％甲醇水溶液80mL淬灭反应，然后加入600mL水，分液，有机层用水洗涤。减压浓缩以除去大部分溶剂，加入适量甲醇，继续浓缩至DCM被完全除去。保留约200mL甲醇，降温至0-10℃，搅拌下析晶1h，抽滤，甲醇淋洗，45-50℃烘干得2A。重量收率约140％，纯度大于98％。 1H NMR(400MHz,CDCl 3)δ7.75(d,J＝8.1Hz,2H),7.32(d,J＝8.0Hz,2H),5.69(s,1H),3.93(dd,J＝9.2,2.9Hz,1H),3.75(dd,J＝9.1,6.5Hz,1H),2.52–2.16(m,7H),2.06–1.88(m,2H),1.86–1.32(m,8H),1.18–0.78(m,13H),0.65(s,3H).
步骤(2)中间体3A的制备
室温下向反应瓶中加入200g中间体2A、20g p-TsOH、400mL醋酸异丙烯酯、600L二氯甲烷，搅拌下缓慢升温至回流反应，大约3-4h反应完毕，TLC监测无原料剩余，降温至30℃，减压浓缩以除去大部分溶剂。加入200mL甲醇并继续浓缩(该操作进行3次)，最后浓缩至稠状，降温至0℃度，搅拌析晶1h，抽滤，甲醇淋洗，45-50℃烘干得3A。重量收率约104％，纯度大于98％。 1H NMR(400MHz,CDCl 3)δ7.76(d,J＝7.9Hz,2H),7.33(d,J＝7.9Hz,2H),5.66(s,1H),5.36(s,1H),4.06–3.87(m,1H),3.85–3.67(m,1H),2.41(d,J＝16.4Hz,4H),2.19–2.04(m,5H),1.92(d,J＝12.5Hz,1H),1.81(dd,J＝12.5,5.2Hz,1H),1.74–1.47(m,6H),1.44–0.89(m,14H),0.65(s,3H).
步骤(3)中间体4A的制备
室温下向反应瓶中加入7g无水氯化钙、40g吡啶、400mL甲醇、400mL THF，搅拌溶解；而后降温至-10～-15℃，分4批加入硼氢化钠，每批4g，投料间隔10min，保持反应体系温度低于-10℃；全部加完后，加入中间体3A(100g)，自然升至室温反应，大约8-10h反应完毕；搅拌下将反应液缓慢倒入1000mL冰水中，待固体析出完毕。继续搅拌20mL，向体系中缓慢滴加20mL冰醋酸，抽滤，水淋洗。将固体用300mL DCM溶解，分去水层，有机相减压浓缩以除去大部分溶剂。加入甲醇并继续浓缩(该操作进行3次)，最后保留甲醇约100mL，降温至0℃析晶1h，抽滤，冰甲醇淋洗，45-50℃烘干得4A。重量收率约75％，HPLC检测纯度大于92％。 1H NMR(400MHz,CDCl 3)δ7.76(t,J＝7.6Hz,2H),7.33(t,J＝7.4Hz,2H),5.30(d,J＝3.9Hz,1H),3.93(dd,J＝8.9,2.3Hz,1H),3.83–3.66(m,1H),3.58–3.36(m,1H),2.51–2.36(m,3H),2.34–2.10(m,2H),2.06–1.84(m,3H),1.80(d,J＝10.6Hz,2H),1.51(dddd,J＝25.8,21.7,12.1,8.1Hz,8H),1.23–0.78(m,13H),0.67–0.55(m,3H). 13C NMR(101MHz,CDCl 3)δ144.52,140.73,133.00,129.68,127.78,121.32,75.59,71.55,56.23,51.68,49.89,42.33,42.11,39.31,37.14,36.36,36.08,31.79,31.71,31.47,27.33,24.15,21.54,20.90,19.28,16.76,11.7.(2个碳信号与其他信号重叠)
步骤(4)胆固醇(化合物2)的制备
室温下向250mL干燥的反应瓶加入2.33g氯化铜，1.47g氯化锂，80mL THF，氮气置换三次。控制温度在10-30℃反应1～2小时至溶液澄清，得到四氯铜酸锂溶液1。室温下向1L干燥的反应瓶加入9.37g镁屑，58.9g溴代异戊烷，400mL THF，氮气置换三次。缓慢升温至37～42℃反应约3～4小时，得到格氏试剂反应液2。将反应液2降温至-40℃。滴加四氯铜酸锂溶液，约1小时滴完。然后滴加中间体4A(25g)的THF(250mL)溶液。滴加完毕，缓慢升温至室温反应3h，取样点板，原料基本消失，控制温度在0-20℃，加入200mL 5％盐酸淬灭，搅拌10～30分钟。减压浓缩至基本无馏分。加入100mL水和200mL DCM，搅拌溶解0.5～1小时。分液。有机层于40～45℃减压浓缩至基本无馏分。加入25mL甲醇并于40～50℃继续浓缩。加入25mL甲醇，降温至10～30℃下搅拌0.5～1小时，过滤，得粗品。固体用DCM和甲醇重结晶，得到的固体于50℃以下烘干得5A。重量收率52％，纯 度约99％。 1H NMR(400MHz,CDCl 3)δ5.35(t,J＝2.8Hz,1H),3.02-3.43(m,1H),2.36-2.27(m,2H),2.00-1.82(m,5H),1.56-0.87(m,28H),0.86(d,J＝1.6Hz,6H),0.68(s,3H).
13C NMR(101MHz,CDCl 3)δ140.78,121.71,71.80,56.78,56.18,50.15,42.33,39.80,39.53,37.27,36.51,36.21,35.80,31.92,31.92,31.68,28.24,28.02,24.31,23.85,22.83,22.72,22.57,21.10,19.41,19.22,12.35.(1个碳信号与其他信号重叠)
实施例2：由化合物1制备7-脱氢胆固醇
Figure PCTCN2020136678-appb-000078
步骤(1)参见实施例1。
步骤(2)中间体2a
室温下，搅拌下向反应瓶中加入中间体2A(100g)、无水甲醇500mL、PTS(5g)、原乙酸三甲酯(80mL)，保持30℃，大约3h反应完毕，TLC监测，原料反应完毕后，加入400mL丙酮，70mL水，80g四氯苯醌，搅拌下缓慢升温至40℃左右反应，TLC监测，大约3-4h反应完毕，将反应体系倒入1000mL水中以析出固体，过滤，固体用400mL氯仿加热至50℃溶解，趁热过滤，滤饼用100mL氯仿加热溶解，过滤，合并有机相。有机相加入饱和亚硫酸钠水溶液(含50g亚硫酸钠)搅拌1h，静置分层，有机相减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩(该操作进行3次)，保留约100mL甲醇，降温至0℃析晶1h，抽滤，甲醇淋洗，45-50℃烘干得2a。重量收率约90％，纯度大于93％。 1H NMR(400MHz,CDCl 3)δ7.74(t,J＝7.1Hz,2H),7.32(d,J＝7.7Hz,2H),6.06(d,J＝6.6Hz,2H),5.62(d,J＝6.5Hz,1H),3.94(dd,J＝9.0,2.3Hz,1H),3.84–3.61(m,1H),2.64–2.46(m,1H),2.45–2.25(m,4H),2.13(t,J＝10.1Hz,1H),2.04–1.84(m,2H),1.82–1.31(m,6H),1.31–1.02(m,9H),1.04–0.89(m,3H),0.73–0.58(m,3H).
步骤(3)中间体3a
室温下，向反应瓶中加入中间体2a(100g)、500mL醋酐、200mL乙酰氯，避光升温至回流反应，约6-8h后TLC监测，原料剩余小于5％。将反应液在75℃左右减压浓缩至干，降温至室温，滴加50mL甲醇淬灭剩余醋酐加入100mL丙酮，减压浓缩以除去大部分溶剂，加入200mL丙酮并继续浓缩，保留约100mL丙酮，降温至0℃析晶1h，过滤，冰丙酮淋洗，固体45-50℃烘干得3a，重量收率约90％，纯度大于95％。 1H NMR(400MHz,CDCl 3)δ7.76(d,J＝7.8Hz,2H),7.32(d,J＝7.9Hz,2H),5.73(s,1H),5.55(d,J＝5.7Hz,1H),5.47(s,1H),3.95(dd,J＝9.2,2.3Hz,1H),3.85–3.69(m,1H),2.54(dd,J＝21.0,8.4Hz,1H),2.42(s, 3H),2.19–1.96(m,6H),1.87(dd,J＝12.4,5.3Hz,2H),1.77–1.49(m,6H),1.48–1.14(m,4H),1.03–0.91(m,6H),0.56(s,3H).
步骤(4)中间体4a
室温下，向反应瓶中加入7g无水氯化钙、40g吡啶、400mL甲醇、400mL THF，搅拌溶解；而后降温至-10～-15℃，分4批加入硼氢化钠，每批4g间隔10min，全部加完后，加入中间体3a(100g)，加完后保持体系温度-5～-10℃反应，大约8-10h反应完毕，TLC监测，无原料剩余；将反应液缓慢倒入1000mL冰水中，边加边搅拌，待固体析出完毕搅拌20mL，向体系中缓慢滴加20mL冰醋酸，抽滤，水淋洗。将固体用300mL DCM溶解，分去水层，有机相减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩(该操作进行3次)，最后保留甲醇约100mL，降温至0℃析晶1h，抽滤，冰甲醇淋洗，45-50℃烘干得4a。重量收率约70％，纯度大于95％。 1H NMR(400MHz,CDCl 3)δ7.77(d,J＝8.1Hz,2H),7.33(d,J＝8.0Hz,2H),5.54(d,J＝3.7Hz,1H),5.44–5.20(m,1H),3.95(dd,J＝9.2,2.7Hz,1H),3.80(dd,J＝9.1,6.4Hz,1H),3.67–3.52(m,1H),2.58–2.37(m,4H),2.26(t,J＝12.8Hz,1H),2.08–1.78(m,6H),1.74–1.14(m,11H),0.98(dd,J＝11.3,6.1Hz,3H),0.87(d,J＝31.0Hz,3H),0.59(d,J＝23.9Hz,3H). 13C NMR(101MHz,CDCl 3)δ144.59,140.44,140.05,133.01,129.72,127.84,119.39,116.57,75.49,70.26,53.97,51.43,46.03,42.94,40.67,38.77,38.27,36.94,36.44,31.84,27.26,22.93,21.57,20.94,16.92,16.19,11.70.(2个碳信号与其他信号重叠)
步骤(5)中间体4c
室温条件下，反应瓶中加入500mL DMF、100g中间体4a、39g溴化锂、10g碳酸锂，搅拌均匀，氮气置换三次，避光保护。体系缓慢升温至80-85℃反应，TLC监控反应，反应完毕。将反应液降温至10-30℃，控制温度在10-30℃，将反应液转移至10V水中，搅拌1-2小时。过滤，适量水淋洗，约50℃下烘箱避光干燥，重量收率约80％。 1H NMR(400MHz,CDCl 3)δ5.57(d,J＝3.8Hz,1H),5.45–5.30(m,1H),3.63(dd,J＝13.3,9.3Hz,1H),3.51(dd,J＝9.7,2.0Hz,1H),3.37(dd,J＝9.7,5.9Hz,1H),2.47(dd,J＝14.1,2.3Hz,1H),2.28(t,J＝12.8Hz,1H),2.13–1.83(m,6H),1.68(ddd,J＝26.3,16.9,7.6Hz,5H),1.51–1.22(m,6H),1.10(t,J＝8.6Hz,3H),0.95(d,J＝11.5Hz,3H),0.67(d,J＝23.9Hz,3H).
步骤(6)7-脱氢胆固醇(化合物3)
室温下，向250mL干燥的反应瓶加入0.80g氯化铜、0.51g氯化锂、30mL THF，氮气置换三次。控制温度在10-30℃反应1～2小时至溶液澄清，得到四氯铜酸锂溶液1。室温下，向1L干燥的反应瓶加入4.16g镁屑，100mL THF，氮气置换三次。滴加26.2g溴代异戊烷，缓慢升温至37～42℃反应约3～4小时，得到格氏试剂反应液2。将反应液2降温至-40～-10℃。滴加四氯铜酸锂溶液，约1小时滴完。控制温度在-20～-10℃，加入20g中间体4c固体。加完毕，缓慢升温至室温反应4小时，取样点板，控制原料基本消失，控制温度在0-20℃，加入225mL氯化铵溶液淬灭，控制温度在40～45℃减压浓缩至基本无馏分。加入100mL DCM萃取。分液，再次水洗一次。有机层于40～45℃减压浓缩至基本无馏分。加入25mL 甲醇并于40～50℃继续浓缩。加入25mL甲醇，降温至10℃搅拌0.5～1小时，过滤，滤饼用10mL甲醇洗涤。固体于50℃以下真空烘干得化合物3。重量收率65％。 1H NMR(400MHz,CDCl 3)δ5.57-5.40(m,2H),3.64-3.58(m,1H),2.49-2.23(m,2H),2.12-1.83(m,7H),1.72-0.83(m,29H),0.62(s,3H).
实施例3：由化合物1制备25-羟基-7-脱氢胆固醇
Figure PCTCN2020136678-appb-000079
步骤(1)-步骤(4)参见实施例2
步骤(5)中间体5a
室温下向250mL干燥的反应瓶加入2.41g氯化铜、1.52g氯化锂、80mL THF，氮气置换三次。控制温度在10-30℃反应1～2小时至溶液澄清，得到四氯铜酸锂溶液1。室温下向1L干燥的反应瓶加入12.5g镁屑，350mL THF，氮气置换三次。滴加138.5g 4-溴-2-甲基-2-三甲基硅氧基丁烷，缓慢升温至37～42℃反应约3～4小时，得到格氏试剂反应液2。将反应液2降温至-40℃。滴加四氯铜酸锂溶液，约1小时滴完。控制温度在-20～-10℃，加中间体4a固体25g。加完毕，缓慢升温至室温反应4小时，取样点板，控制原料基本消失，加入225mL氯化铵溶液淬灭，控制温度在40～45℃减压浓缩至基本无馏分，得到的混合物直接用于下一步反应。
步骤(6)25-羟基-7-脱氢胆固醇(化合物4)
向步骤(5)得到的混合物加入125mL DCM溶解，加入盐酸调节pH至3-4，室温搅拌1h，分出有机相，用水洗涤(50mL×2)。于40～45℃减压浓缩至基本无馏分。加入25mL甲醇并于40～50℃继续浓缩。加入25mL甲醇，降温至10～30℃下搅拌0.5～1小时，过滤，滤饼用10mL甲醇洗涤。固体于50℃以下真空烘干得5b。重量收率65％。 1H NMR(400MHz,CDCl 3)δ5.57(t,J＝2.8Hz,1H),5.39(t,J＝2.8Hz,1H),3.72-3.56(m,1H),2.48-2.08(m,3H),1.98-1.82(m,5H),1.67-1.18(m,23H),1.10-1.06(m,1H),0.95-0.94(m,6H),0.62(s,3H).
实施例4：由化合物1制备25-羟基胆固醇
Figure PCTCN2020136678-appb-000080
步骤(1)参见实施例1。
步骤(2)中间体2C
室温下，反应瓶中加入500mL THF、100g中间体2A、39g溴化锂、10g碳酸锂，搅拌均匀，氮气置换三次，避光保护。体系缓慢升温至55-60℃反应，TLC监控反应反应完毕。浓缩至小体积回收THF，将反应液降温至10-30℃，控制温度在10-30℃将反应液转移至1L水中，搅拌1-2小时。过滤，适量水淋洗，约50℃下烘箱避光干燥，重量收率约80％，纯度96％。
步骤(3)中间体3C
室温下，向反应瓶中加入200g中间体2A、20g PTS、600mL醋酐，搅拌下缓慢升温至30-35反应℃，大约4-6h反应完毕，TLC监测无原料剩余，将降温至20℃以下，缓慢倒入4L冰水中，边倒边剧烈搅拌，继续搅拌2h，抽滤，用大量水淋洗至中性，45-50℃烘干得3C。重量收率约98％，纯度大于96％。
步骤(4)中间体4C
室温下，向反应瓶中加入7g无水氯化钙、40g吡啶、400mL甲醇、400mL THF，搅拌溶解；而后降温至-10～-15℃，分4批加入硼氢化钠，每批4g间隔10min，保持反应体系温度低于-10℃；全部加完后，加入中间体3C(100g)，加完后自然升至室温反应，大约8-10h反应完毕，TLC监测，无原料剩余；将反应液缓慢倒入1000mL冰水中，边加边搅拌，待固体析出完毕搅拌20mL，向体系中缓慢滴加20mL冰醋酸，抽滤，水淋洗。将固体用300mL DCM溶解，分去水层，有机相减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩(该操作进行3次)，最后保留9甲醇约100mL，降温至0℃析晶1h，抽滤，甲醇淋洗，45-50℃烘干得4A。重量收率约80％，纯度大于95％。 1H NMR(400MHz,CDCl 3)δ5.35(d,J＝5.2Hz,1H),3.50(dd,J＝9.8,2.6Hz,2H),3.35(dd,J＝9.7,5.9Hz,1H),2.26(dt,J＝24.0,9.9Hz,2H),2.04–1.92(m,2H),1.84(d,J＝10.3Hz,3H),1.73–1.60(m,3H),1.49(td,J＝14.6,9.6Hz,6H),1.31–1.18(m,3H),1.08(t,J＝7.8Hz,5H),1.01(s,3H),0.97–0.89(m,1H),0.70(s,3H).
步骤(5)中间体5B
反应瓶中加入180mL吡啶、30g锌粉、21g六水氯化镍。氮气置换三次，滴加45g丙烯酸甲酯，10min滴加完毕，升温至60℃剧烈搅拌1h，溶液会变黑，体系基本成均相；将 体系降温至15-20℃，加入30g中间体4C固体，加完后，在20-30℃左右反应3h，TLC检测反应完后加150mL乙酸乙酯搅拌20min，体系大量悬浊物产生。
经过硅藻土层过滤，滤饼用乙酸乙酯淋洗；加入180mL 10％盐酸洗涤，分层。有机层加入6V 10％碳酸钠水溶液洗涤，分液。有机相浓缩至干，加入甲醇重结晶得中间体5B。重量收率约80％。 1H NMR(400MHz,CDCl 3)δ5.32(d,J＝4.1Hz,1H),3.64(s,3H),3.49(td,J＝10.7,5.4Hz,1H),2.41–2.08(m,4H),2.01–1.91(m,3H),1.79(t,J＝15.3Hz,3H),1.68(dd,J＝11.2,6.9Hz,1H),1.59–1.26(m,9H),1.23–0.87(m,14H),0.65(s,3H). 13C NMR(101MHz,CDCl 3)δ174.32,140.74,121.56,71.67,56.69,55.76,51.39,50.06,42.28,42.21,39.70,37.21,36.45,35.44,35.36,34.47,31.84,31.86,31.56,28.10,24.21,21.48,21.02,19.34,18.55,11.79.
步骤(6)25-羟基胆固醇(化合物5)
在反应瓶加入50g中间体5B，500mL THF，氮气置换三次。降温至0-10℃，缓慢滴加300mL 2M的甲基氯化镁的THF溶液，0.5h滴加完毕，滴完升至室温反应1h。TLC检测反应，确认反应完全，降温至0-10℃，缓慢滴加至氯化铵水溶液淬灭反应。分层，水层用150mL DCM萃取。合并有机层，在50℃以下减压浓缩以除去大部分溶剂，加入甲苯并继续浓缩至糊状。10-30℃搅拌1h，过滤。滤饼转移至反应瓶，加入DCM和3甲醇溶清，50℃以下减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩至约固体析出，10-30℃搅拌1h，过滤。滤饼用少量甲醇洗涤，50℃下烘干得化合物5，重量收率约70％。 1H NMR(400MHz,CDCl 3)δ5.27(s,1H),4.59(s,1H),4.03(s,1H),2.13-2.03(m,2H),1.96-1.91(m,2H),1.78-1.68(m,3H),1.67-0.95(m,33H),0.66(s,3H).
实施例5：由化合物1制备25-羟基-7-脱氢胆固醇
Figure PCTCN2020136678-appb-000081
步骤(1)-步骤(4)参见实施例2
步骤(5)中间体4d
室温下，反应瓶中加入500mL丙酮、100g中间体4a、77.5g碘化钠，搅拌均匀，氮气置换三次，避光保护。体系缓慢升温至55-60℃反应，TLC监控反应反应完毕。减压浓缩大部分丙酮，将反应液降温至10-30℃，加入1L冰水，搅拌1-2小时。过滤，适量水淋洗，约50℃下烘箱避光干燥得中间体4d，重量收率约90％。
步骤(6)中间体5c
反应瓶中加入160mL吡啶、26.8g锌粉、18.8g六水氯化镍。氮气置换三次，滴加40.2g丙烯酸甲酯，10min滴加完毕，升温至60℃剧烈搅拌1h，溶液会变黑，体系基本成均相；将体系降温至15-20℃，加入30g中间体4d固体，加完20-30℃左右反应3h，TLC检测反应完后加150mL乙酸乙酯搅拌20min，体系大量悬浊物产生。经过硅藻土层过滤，滤饼用乙酸乙酯淋洗；合并有机相减压浓缩浓至干。得到的油状物柱层析分离(淋洗剂为乙酸乙酯：石油醚1:6)，浓缩至糊状，降温至0℃结晶，过滤，滤饼烘干得中间体5c。重量收率约65％。 1H NMR(400MHz,CDCl 3)δ5.54(d,J＝3.9Hz,1H),5.41–5.27(m,1H),3.71–3.51(m,4H),2.44(dd,J＝14.0,2.8Hz,1H),2.33–2.17(m,3H),2.13–1.80(m,8H),1.74–1.18(m,12H),1.15–1.01(m,1H),0.96–0.86(m,6H),0.59(s,3H). 13C NMR(101MHz,CDCl 3)δ174.31,141.14,139.81,119.51,116.31,70.33,55.48,54.41,51.40,46.18,42.87,40.72,39.13,38.34,36.97,35.78,35.29,34.46,31.90,27.97,22.95,21.49,21.05,18.69,16.23,11.74.
步骤(7)25-羟基-7-脱氢胆固醇(化合物4)
在反应瓶加入50g中间体5c，500mL，氮气置换三次。降温至0-10℃，缓慢滴加2M的甲基氯化镁300mL，0.5h滴加完毕，滴完升至室温反应1h。TLC检测反应，确认反应完全，降温至0-10℃，缓慢滴加至氯化铵水溶液淬灭反应。分液，水层用150mL DCM萃取。合并有机相50℃以下减压浓缩以除去大部分溶剂，加入甲苯并继续浓缩至固体析出。降温至10℃搅拌1h，过滤。滤饼转移至反应瓶，加入150mL DCM和100mL甲醇至溶液澄清。50℃以下减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩至糊状。0-10℃搅拌1h，过滤。滤饼用少量甲醇洗涤，50℃下烘干得25羟脱氢胆固醇，重量收率约80％。其 1H NMR数据与实施例3步骤(6)测定的基本相同。
实施例6：由化合物1制备25-羟基胆固醇
Figure PCTCN2020136678-appb-000082
步骤(1)参见实施例1
步骤(2)中间体2’D
室温下，反应瓶中加入500mL丙酮、100g中间体2A、80g碘化钠，搅拌均匀，氮气置换三次，避光保护。体系缓慢升温至55-60℃反应2-3h，TLC监控反应,反应完毕。减压浓缩大部分丙酮，将反应液降温至10-30℃，加入1L冰水，搅拌1-2小时。过滤，适量水淋洗，约50℃下烘箱避光干燥，重量收率约90％，纯度大于97％。
步骤(3)中间体3’B
反应瓶中加入160mL吡啶、26.8g锌粉、18.8g六水氯化镍。氮气置换三次，滴加40.2g丙烯酸甲酯，10min滴加完毕，升温至60℃剧烈搅拌1h，溶液变黑，体系基本成均相；将体系降温至15-20℃，加入30g中间体2’D，加完25-30℃左右反应3h，TLC检测反应完后加150mL乙酸乙酯搅拌20min，体系大量悬浊物产生。经过硅藻土层过滤，滤饼用乙酸乙酯淋洗；加入200L 5％盐酸洗涤，分液，再用200mL水洗涤，分液，有机层浓缩至小体积，加入乙醇继续浓缩至糊状，降温至0-10℃析晶1h，过滤，滤饼烘干得产品，重量收率约70％，纯度94％。 1H NMR(400MHz,CDCl 3)δ5.71(s,1H),3.65(s,3H),2.49–2.14(m,6H),2.01(dd,J＝13.3,3.3Hz,2H),1.92–1.31(m,10H),1.31–0.83(m,15H),0.69(s,3H).
步骤(4)中间体4’B
反应瓶中加入100mL醋酸酐、50g中间体3’B、氮气保护下、加入5g对甲苯磺酸，加热至25-30℃反应4-6h，TLC监测反应，原料基本反应完全。剧烈搅拌下将反应体系缓慢滴入1000mL水中，固体析出，搅拌2h后，过滤，滤饼用大量水洗涤至中性。45-50℃烘干，重量收率104％，纯度95％。
步骤(5)中间体5B
室温下，向反应瓶中加入10g无水氯化钙，400mL无水乙醇，搅拌至溶液澄清，加入200mL DCM，然后一次性加入100g中间体4’B，搅拌均匀，分4批加入硼氢化钠，每批4g间隔5min，全部加完后，继续保持室温反应2-4h TLC监测，无原料剩余；向体系中缓慢滴加20mL冰醋酸淬灭反应，浓缩除去有机溶剂，加入1000mL冰水以析出固体，抽滤，水淋洗。将固体用300mL DCM溶解，分去水层，有机相减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩，最后保留甲醇约100mL，降温至0-10℃析晶1h，抽滤，甲醇淋洗，45-50℃烘干得中间体5B。重量收率约70％，纯度大于97％。其 1H NMR数据与实施例4步骤(5)测定的基本相同。
步骤(7)25-羟基胆固醇(化合物5)
按照实施例4步骤(7)制备化合物5， 1H NMR数据与实施例4测定的基本相同。
实施例7：由化合物1制备25-羟基-7-脱氢胆固醇
Figure PCTCN2020136678-appb-000083
步骤(1)-步骤(2)参见实施例2
步骤(3)中间体2’d
室温下，反应瓶中加入500mL DMF、100g中间体2a、39g溴化锂，搅拌均匀，氮气置换三次。体系缓慢升温至80-85℃，反应3-5h，TLC监控反应。反应完毕后将反应液降温至10-30℃，控制温度在10-30℃将反应液转移至1L水中，搅拌1-2小时。过滤，适量水淋洗，约50℃下烘箱干燥，重量收率约78％，纯度96％。
步骤(4)中间体3’b
反应瓶中加入180mL吡啶、30g锌粉、21g六水氯化镍。氮气置换三次，滴加45g丙烯酸甲酯，10min滴加完毕，升温至60℃剧烈搅拌1h，溶液会变黑，体系基本成均相；将体系降温至15-20℃，加入30g中间体2’d固体，加完20-30℃左右反应3h，TLC检测反应完后加150mL乙酸乙酯搅拌20min，体系大量悬浊物产生。
经过硅藻土层过滤，滤饼用乙酸乙酯淋洗；合并滤液加入100mL 10％盐酸洗涤，分液。有机层加入200mL水洗涤，分液。浓缩至干，加入约100mL甲醇，降温至0-5℃析晶一小时，过滤，滤饼烘干得3’b。重量收率约78％,纯度92％。
步骤(5)中间体4’b
室温下，向反应瓶中加入中间体3’b(100g)、300mL醋酐、200mL乙酰氯，避光升温至75℃-85℃反应，TLC监测，原料剩余小于5％(大约6-8h)，75℃左右减压浓缩至干，降温至室温，滴加50mL甲醇淬灭剩余醋酐；加入100mL丙酮，减压浓缩至小体积，加入200mL丙酮并继续浓缩，保留约50mL丙酮，降温至0℃析晶1h，过滤，冰丙酮淋洗，固体45-50℃烘干得4’b，重量收率约95％，纯度大于95％。
步骤(6)中间体5c
室温下，向反应瓶中加入10g无水氯化钙、400mL无水甲醇，搅拌至溶液澄清，加入200mL DCM，然后一次性加入100g中间体4’b，搅拌均匀，加入硼氢化钠10g，继续保持室温反应2-4h。TLC监测监测反应完毕后，向体系中缓慢滴加20mL冰醋酸淬灭反应，浓缩以除去有机溶剂，加入1000mL冰水以析出固体，抽滤，水淋洗。将固体用300mL DCM溶解，分去水层，有机相减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩，最后保留甲醇约100mL，降温至0-10℃析晶1h，抽滤，甲醇淋洗，45-50℃烘干得中间体5’B。重量收率约70％，纯度大于97％。 1H NMR(400MHz,CDCl 3)δ5.54(d,J＝3.9Hz,1H),5.41–5.27(m,1H),3.71–3.51(m,4H),2.44(dd,J＝14.0,2.8Hz,1H),2.33–2.17(m,3H),2.13–1.80(m,8H),1.74–1.18(m,12H),1.15–1.01(m,1H),0.96–0.86(m,6H),0.59(s,3H). 13C NMR(101MHz,CDCl 3)δ174.31,141.14,139.81,119.51,116.31,70.33,55.48,54.41,51.40,46.18,42.87,40.72,39.13,38.34,36.97,35.78,35.29,34.46,31.90,27.97,22.95,21.49,21.05,18.69,16.23,11.74.
步骤(7)25-羟基-7-脱氢胆固醇(化合物4)
按照实施例5步骤(7)制备化合物4， 1H NMR数据与实施例5测定的基本相同。
实施例8：由化合物1制备麦角甾醇
Figure PCTCN2020136678-appb-000084
步骤(1)中间体2O
在3000mL三口瓶中加入200g化合物1、1g TEMPO，加入700mL DCM，搅拌溶解，加入溴化钠/碳酸氢钠的水溶液(含溴化钠7.2g和碳酸氢钠9.88g，水50mL)，降温至5℃以下，控制温度在-5～5℃，滴加500mL 8％的次氯酸钠的溶液，TLC检测反应完全(乙酸乙酯:甲苯＝1:1)，加入硫代硫酸钠水溶液(硫代硫酸钠17.2g，水178mL)终止反应，升温至30～35℃搅拌10min，分液，水相用DCM(400mL*2)萃取，合并有机相，用水洗涤，有机层加80mL水，继续浓缩除去溶剂，加1200mL水，搅拌降温至0～5℃，析晶1小时，过滤，50℃下干燥，得中间体2O，HPLC检测纯度>98.5％，收率97％。 1H NMR(400MHz,CDCl 3)δ9.53(d,J＝3.1Hz,1H),5.69(s,1H),2.50–2.15(m,5H),2.01–1.89(m,2H),1.88–1.73(m,2H),1.72–1.57(m,2H),1.56–1.28(m,5H),1.25–0.83(m,11H),0.72(s,3H).
步骤(2)中间体3O
三口反应瓶中加入100g中间体2O、原乙酸三甲酯100mL，室温搅拌10min，氮气保护下，加入1g对甲苯磺酸，缓慢升温至40～45℃，保温反应4～5h。TLC检测反应(展开剂为PE:EA＝5:1)。反应完成后，降温至室温(20～25℃)，加入三乙胺调节体系pH值为8左右，然后向反应瓶中加入500mL丙酮、80g水，搅拌10min，加入80g四氯苯醌，然后升温至35～40℃反应4h。加入200g水，然后滴加80mL浓盐酸，40℃下搅拌2h水解，TLC确认水解完毕，反应液倒入1000mL水中，常温搅拌2h，过滤收集析出的固体，滤饼用水洗至中性，然后与400mL氯仿混合，加热使固体溶解后，趁热过滤。向滤液中加入300mL亚硫酸钠水溶液(含50g亚硫酸钠)搅拌1h，静置后分出有机相，减压浓缩以除去大部分溶剂，加入甲醇并继续浓缩(该操作进行3次)，保留约100mL甲醇，降温至0℃析晶1h，抽滤，甲醇淋洗，45-50℃烘干得中间体3O。重量收率93％，纯度大于95％。
1H NMR(400MHz,CDCl 3)δ9.54(d,J＝2.9Hz,1H),6.17–5.95(m,2H),5.63(s,1H),2.58–2.47(m,1H),2.45–2.29(m,2H),2.17(t,J＝10.5Hz,1H),2.03–1.74(m,4H),1.67(td,J＝13.8,5.0Hz,1H),1.58–1.12(m,9H),1.08(d,J＝5.0Hz,5H),0.74(d,J＝18.5Hz,3H). 13C NMR(101MHz,CDCl 3)δ204.43,199.29,163.44,140.73,127.91,123.57,52.68,50.70,50.54,49.25,43.83,39.09,37.52,35.93,33.81,33.79,26.89,23.96,20.50,16.19,13.33,12.17.
步骤(3)中间体4O
在1000mL三口瓶中加入500mL乙腈，然后加入100g溴代异戊烷和100g三苯基膦。氮气保护，回流反应24h。TLC检测基本反应完全，浓缩除去溶剂，加入适量石油醚搅拌1h后过滤除去反应剩余的三苯基膦。得到滤饼，为叶立德试剂。
在1000mL三口瓶中加入1000mL无水THF，加入制备得到的叶立德试剂，氮气保护下，0℃下滴加正丁基锂300mL，控制温度在10℃以下，然后加入100g中间体3O，缓慢升至常温反应1h。TLC监测反应。反应完毕后，滴加50mL水淬灭反应。浓缩除去THF，加500mL水，水相用石油醚(400mL*2)萃取，合并有机相，浓缩，加入甲醇并继续浓缩至小体积，搅拌降温至0～5℃，析晶1小时，过滤，50℃下干燥得到中间体4O，收率70％。
步骤(4)中间体5O
向洁净干燥避光的反应瓶中依次加入280mL醋酐、140mL乙酰氯和70g中间体4O，氮气置换三次。搅拌下缓慢升温至75-85℃避光反应(剧烈回流)，TLC监测(甲苯：丙酮＝4:1)，约6-8h反应完毕；反应完毕后，70-80℃避光减压浓缩至小体积，滴加甲醇，保持温度小于10℃，滴加完甲醇后搅拌15-20min，继续加入200mL甲醇浓缩至糊状，降温至0-5℃析晶，过滤，45-50℃下避光烘干，得到中间体5O，收率约90％，纯度94％。
步骤(5)麦角甾醇(化合物6)
往反应瓶中加入180g无水乙醇，180g DCM，6g无水氯化钙，搅拌无水氯化钙溶解；加入60g中间体5O，搅拌。控制釜内温度为20℃左右，开始分批加入6g硼氢化钠，20min内加完。30-35度反应2-3小时。TLC跟踪至无原料，未反应完全延长时间直至反应完全，展开剂PE:EA:DCM 5:1:2。反应结束后，控制温度，缓慢滴加备用液。60mL 3％盐酸。浓缩掉DCM和乙醇，加200mL DCM，用3％盐酸将调节至弱酸性。分出有机相，用碳酸氢钠水溶液洗涤，有机相减压浓缩，加入乙醇并继续浓缩至无DCM，继续浓缩至稠状，0℃下搅拌析晶1-2小时。过滤收集析出的固体，45-50℃避光烘干，得固体45g。收率约75％，纯度96％。 1H NMR(400MHz,CDCl 3)δ5.56(dd,J＝10.6,7.2Hz,1H),5.37(dd,J＝14.1,11.3Hz,1H),5.26–5.04(m,2H),3.71–3.50(m,1H),2.45(dt,J＝30.1,15.1Hz,1H),2.26(dd,J＝25.2,12.7Hz,1H),2.11–1.18(m,19H),1.02(t,J＝7.4Hz,3H),0.96–0.88(m,6H),0.82(dd,J＝13.1,6.7Hz,6H),0.63(s,3H).
13C NMR(101MHz,CDCl 3)δ141.35,139.77,135.56,131.98,119.59,116.28,70.47,55.74,54.56,46.26,42.83,40.79,40.41,39.09,38.38,37.04,33.09,31.99,28.28,23.00,21.11,19.95,19.64,17.60,16.28,12.05
实施例9：按照wittig路线合成胆固醇
Figure PCTCN2020136678-appb-000085
步骤(1)参见实施例8。
步骤(2)中间体3P
在1000mL三口瓶中加入500mL甲苯，再加入110g溴代异戊烷和100g三苯基膦，氮气保护，100℃反应24h。过滤，滤饼用石油醚冲洗，挤压除去大部分溶剂，得叶立德试剂。在1000mL三口瓶中加入300mL DMSO，加入制备好的叶立德试剂，氮气保护下，加入叔丁醇钾40g，控制温度在10℃以下，然后加入80g中间体2O，室温反应1-2h。取样TLC，观察原料是否反应完全。如果未反应完全，延长反应至反应完毕。滴加50mL水淬灭反应。倒入至500mL冰水，水相用石油醚(400mL*2)萃取，合并有机相浓缩，加入甲醇至小体积，搅拌降温至0～5℃，析晶1小时，过滤，50℃下干燥，收率70％。 1H NMR(400MHz,CDCl 3)δ5.62(s,1H),5.28–5.16(m,2H),2.50–2.36(m,3H),2.24–1.75(m,6H),1.61–1.30(m,8H),1.26–0.97(m,22H).
步骤(3)中间体4P
室温下，反应瓶中加入150mL叔丁醇，氮气置换3次，加入20g叔丁醇钾加热至40℃搅拌溶解清澈，严格氮气保护下加入中间体3P固体，加入后继续保温反应2h，TLC检测反应原料基本消失，则停止反应降温至室温。准备冰水200mL，加入冰乙酸10g，抗坏血酸钠5g，搅拌均匀制备成缓冲液1，将叔丁醇反应溶液快速倒入缓冲液1中，固体析出，继续搅拌20min，过滤。得到中间体4P湿固体。
步骤(4)中间体5P
反应瓶中加入上述湿品，加入200mL乙醇搅拌均匀，加入将2.5g硼氢化钠搅拌反应2h，反应完毕加入2mL冰乙酸淬灭反应，浓缩乙醇至小体积，加入100mL水水析，过滤得中间体5P粗品，甲醇重结晶得中间体5P，收率80％。纯度96％。 1H NMR(400MHz,CDCl 3)δ5.35(s,1H),5.17(d,J＝6.8Hz,2H),3.63–3.38(m,1H),2.48–2.09(m,2H),1.93(m,6H),1.70–1.36(m,10H),1.22–0.82(m,19H),0.70(d,J＝11.2Hz,3H).
步骤(5)胆固醇(化合物2)
反应瓶中加入10g中间体5P，加入25mL THF和25mL甲醇溶解，加入1g钯碳(5％)，氮气置换，然后氢气置换，连接一个氢气球，保温15-20℃反应24h，过滤，滤饼加入甲醇洗涤，合并滤液，减压浓缩，加入甲醇并继续浓缩，至除去全部THF，甲醇中析晶，室温 搅拌1h，过滤，干燥得胆固醇，重量收率85％，纯度99％。其 1H NMR和 13C NMR数据与实施例1测定的基本相同。
实施例10：使用不同试剂对反应的影响
分别使用以下表格所示的酰基化试剂进行本发明的第一至第七方面所述步骤(A)的反应，以制备带有不同PG基团的烯醇酯中间体3A-m和中间体3a-m，其对应本发明的第一至第七方面所述的式II化合物。其中m为序号，PG为羟基保护基。进一步地，以中间体3A-m、中间体3a-m为原料，分别使用以下表格所示的试剂进行本发明的第一至第七方面所述步骤(B)的反应，制备中间体4A和中间体4a，其对应本发明的第一至第七方面所述的式III化合物。
1.中间体4A
Figure PCTCN2020136678-appb-000086
(1)向反应容器中加入中间体2A(20g)、氯仿40mL，然后加入酰基化试剂(8eq.)、催化剂p-TsOH(1g)，加热回至回流，反应4-8h。反应完毕降温至0-20℃，缓慢滴加甲醇淬灭反应。加入40mL水，分出有机相，浓缩后加入甲醇，继续浓缩至糊状，降温至0-5℃搅拌1h，过滤，烘干得中间体3A-m。向反应瓶中加入中间体3A-m(1g)、10mL二氯甲烷、10mL甲醇。体系降温至0-5℃，分三批(每批次间隔20min)加入0.2g硼氢化钠，加入完毕后，保持温度，反应12h。滴加1mL冰醋酸淬灭反应，浓缩除去溶剂，加入10mL水，搅拌得到混合物，然后过滤，烘干。硅胶柱层析分离得到中间体4A，计算摩尔收率。结果如表2所示。
表2使用不同酰基化试剂对反应的影响
Figure PCTCN2020136678-appb-000087
由表2可知使用使用其中的酰基化试剂时，烯醇酯化反应的转化率都较高。PG为乙酰基、任选被烷基取代的苯甲酰基，特别是乙酰基时，可以达到较高的终产品收率。
(2)向反应瓶中加入中间体3A-m(1g)、10mL二氯甲烷、10mL甲醇。体系降温至0-5℃，根据情况在需要时加入CaCl 2或ZnCl 2(0.3eq.)，分三批(每批次间隔10min)加入0.2g硼氢化钠，加入完毕后，马上加入对应的碱(2eq.)。保持温度，反应12h。滴加1mL冰醋酸淬灭反应，浓缩除去溶剂，加入10mL水，搅拌得到混合物，然后过滤，烘干。硅胶柱层析分离得到中间体4A，计算摩尔收率。结果如表3所示。
表3使用不同试剂制备中间体4A
序号 还原剂 碱(2eq.) 溶剂(1:1) 转化率％ 4A摩尔收率％
1 NaBH4 - CH 2Cl 2/MeOH >99 66
2 KBH4 - CH 2Cl 2/MeOH 42 <10
3 NaBH 4/CaCl 2 - CH 2Cl 2/MeOH 80 40
4 NaBH 4/ZnCl 2 - CH 2Cl 2/MeOH 0 0
5 NaBH 4/CaCl 2 - THF/MeOH >99 71
6 NaBH 4/CaCl 2 - MeOH 72 43
7 NaBH 4/CaCl 2 - THF/EtOH >99 74
8 NaBH 4/CaCl 2 吡啶 THF/MeOH >99 81
9 NaBH 4/CaCl 2 DMAP THF/MeOH >99 65
10 NaBH 4/CaCl 2 NaOH THF/MeOH >99 60
由表3可知使用NaBH 4/CaCl 2进行反应，总体上能够达到较好的效果。当使用THF作为含醇(甲醇)的混合溶剂中的组分时，效果优于使用CH 2Cl 2。对于反应中使用的碱，当加入DMAP或NaOH时，与未加入碱时相比产率降低(分别降低约8.5％和15.5％)；而当加入吡啶时，与未加入碱时相比产率提高约14.1％。
2.中间体4a
Figure PCTCN2020136678-appb-000088
步骤(1)制备中间体3a-m
向反应容器中加入中间体2a(20g)、1,2-二氯乙烷40mL，然后加入酰基化试剂(10eq.)、催化剂p-TsOH(2g)，加热至80℃，反应6-12h。反应完毕降温至0-20℃，缓慢滴加甲醇淬灭反应。加入60mL水，分出有机相，用水洗涤，浓缩除去溶剂，然后加入乙酸乙酯，继续浓缩至糊状，降温至0-5℃搅拌1h，过滤，烘干得酯化物。然后进行还原反应，结果如表4所示。
表4使用不同酰基化试剂制备烯醇酯
Figure PCTCN2020136678-appb-000089
a：加压反应
由表4可知使用乙酸酐/乙酰氯混合物、三氟乙酸酐作为酰基化试剂时，酰基化反应收率较高。
步骤(2)制备中间体4a
向反应瓶中加入中间体3a-m(1g)、10mL溶剂、根据情况在需要时加入CaCl 2或ZnCl 2(0.1g)。体系降温至0-5℃，分三批(每批次间隔10min)加入0.2g硼氢化钠，然后马上加入 对应碱(2eq.)，加入完毕后，保持温度，反应12h。滴加1mL冰醋酸淬灭反应，浓缩除去溶剂，加入10mL水，搅拌得到混合物，然后过滤，烘干。硅胶柱层析分离得到中间体4a，计算摩尔收率。结果见表5。
表5使用不同试剂制备中间体4a
序号 还原剂 碱(2eq.) 溶剂 转化率％ 4a收率％
1 NaBH4 - THF/MeOH a >99 54
2 NaBH 4/ZnCl 2 - THF/MeOH a - -
3 NaBH 4/CaCl 2 - THF/MeOH a >99 55
4 NaBH 4/CaCl 2 吡啶 THF/MeOH a >99 70
5 NaBH 4/CaCl 2 吡啶 MeOH a 91 65
6 NaBH 4/CaCl 2 吡啶 THF/EtOH a >99 74
7 NaBH 4/CaCl 2 吡啶 THF/i-PrOH a 87 58
8 NaBH 4/CaCl 2 吡啶 THF/EtOH/i-PrOH b >99 86
9 NaBH 4/CaCl 2 吡啶 THF/EtOH/MeOH c >99 71
a:(1:1)；b:THF/EtOH/i-PrOH＝(1:0.5:0.5)；c:THF/EtOH/MeOH＝(1:0.5:0.5)
由表5可知使用NaBH 4/CaCl 2进行反应，能够达到较好的效果，优于单独使用NaBH 4，或与其相当。含醇的混合溶剂中所含的醇的种类对于反应有一定影响，在测试的混合溶剂中，四氢呋喃/乙醇/异丙醇实现了最好的效果。使用混合的乙醇/异丙醇(例如以1:1混合的乙醇/异丙醇)作为含醇的混合溶剂中所含的醇组分，与单独使用乙醇或异丙醇相比，产率分别提高了10.8％和48.3％。发明人出人意料地发现，使用混合的乙醇/异丙醇作为混合溶剂中的醇组分，能大幅提高反应的产率。

Claims (10)

  1. 一种制备式III化合物的方法，包括：
    步骤(1)：式ii化合物转化为式a化合物；
    Figure PCTCN2020136678-appb-100001
    步骤(2)：式a化合物在硼氢化钠、氯化钙和吡啶的作用下还原为式b化合物；
    Figure PCTCN2020136678-appb-100002
    步骤(3)：式b化合物转化为式III化合物；
    Figure PCTCN2020136678-appb-100003
    其中，
    Figure PCTCN2020136678-appb-100004
    在式ii、式a、式b、式III中同时表示单键，或者
    Figure PCTCN2020136678-appb-100005
    在式ii、式a、式b、式III中同时表示双键，或者
    Figure PCTCN2020136678-appb-100006
    在式ii中表示单键或双键，在式a、式b和式III中表示双键；
    R a、R b各自独立地选自H、-OH、C 1-3烷基；
    PG为羟基保护基，优选为C 1-8硅烷基、乙酰基、三氟乙酰基、或任选地被一个或多个C 1-8烷基取代的苯甲酰基；
    R为L 1-R 1；
    L 1不存在，或为C 1-8亚烷基；
    LG为离去基团；
    R 1选自H、C 1-8烷基、C 1-8烯基、-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
    PG 1为羟基保护基，优选选自C 1-8硅烷基或C 1-6烷氧基取代的甲基。
  2. 根据权利要求1所述的方法，其特征在于，所述步骤(2)中，式a化合物在硼氢化钠、氯化钙和吡啶的作用下，在醇和四氢呋喃的混合溶剂中还原为式b化合物。
  3. 根据权利要求2所述的方法，其特征在于，所述醇选自甲醇、乙醇和异丙醇中的一种或多种。
  4. 根据权利要求2所述的方法，其特征在于，所述醇和四氢呋喃的体积比为1～2:1。
  5. 根据权利要求3或4所述的方法，其特征在于，所述醇选自乙醇和异丙醇，所述乙醇和异丙醇的体积比为1:1。
  6. 根据权利要求1所述的方法，其特征在于，PG选自乙酰基、对苯甲酰基或三氟乙酰基；
    LG选自被卤素取代的C 1-6烷基磺酸酯基离去基团、任选地被C 1-6烷基取代的苯磺酸酯基离去基团或者卤素。
  7. 一种制备式III化合物的方法，包括：
    步骤(11)：式ii化合物转化为式I化合物；
    Figure PCTCN2020136678-appb-100007
    步骤(12)：式I化合物转化为式II化合物；
    Figure PCTCN2020136678-appb-100008
    步骤(13)：式II化合物在硼氢化钠和氯化钙的作用下还原为式III化合物；
    Figure PCTCN2020136678-appb-100009
    其中，
    Figure PCTCN2020136678-appb-100010
    在式ii、式a、式b、式III中同时表示单键，或者
    Figure PCTCN2020136678-appb-100011
    在式ii、式a、式b、式III中同时表示双键，或者
    Figure PCTCN2020136678-appb-100012
    在式ii中表示单键或双键，在式a、式b和式III中表示双键；
    R a、R b各自独立地选自H、-OH、C 1-3烷基；
    PG为羟基保护基，优选为C 1-8硅烷基、乙酰基、三氟乙酰基、或任选地被一个或多个C 1-8烷基取代的苯甲酰基；
    R为L 1-R 1；
    L 1不存在，或为C 1-8亚烷基；
    LG为离去基团；
    R 1选自H、C 1-8烷基、C 1-8烯基、-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)、-C(＝O)N(C 1-8烷基) 2，其中所述烷基或烯基任选地被1、2或3个选自-OH、-O(C 1-8烷基)、-O-PG 1、-C(＝O)O(C 1-8烷基)和-C(＝O)N(C 1-8烷基) 2的基团取代；
    PG 1为羟基保护基，优选选自C 1-8硅烷基或C 1-6烷氧基取代的甲基。
  8. 根据权利要求7所述的方法，其特征在于，式II化合物在硼氢化钠和 氯化钙的作用下、在二氯甲烷和醇的混合溶剂中还原为式III化合物。
  9. 根据权利要求8所述的方法，其特征在于，所述醇选自甲醇、乙醇和异丙醇中的一种或多种；
    所述醇和四氢呋喃的体积比为1～2:1。
  10. 根据权利要求7所述的方法，其特征在于，PG选自乙酰基、对苯甲酰基或三氟乙酰基；
    LG选自被卤素取代的C 1-6烷基磺酸酯基离去基团、任选地被C 1-6烷基取代的苯磺酸酯基离去基团或者卤素。
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