WO2018050099A1 - 二醇型人参皂苷衍生物及其制备方法和应用 - Google Patents

二醇型人参皂苷衍生物及其制备方法和应用 Download PDF

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Publication number
WO2018050099A1
WO2018050099A1 PCT/CN2017/101880 CN2017101880W WO2018050099A1 WO 2018050099 A1 WO2018050099 A1 WO 2018050099A1 CN 2017101880 W CN2017101880 W CN 2017101880W WO 2018050099 A1 WO2018050099 A1 WO 2018050099A1
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Prior art keywords
compound
ethyl acetate
present
formula
type ginsenoside
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PCT/CN2017/101880
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English (en)
French (fr)
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李晓辉
贾乙
黄琰
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李晓辉
重庆天百康生物科技有限公司
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Application filed by 李晓辉, 重庆天百康生物科技有限公司 filed Critical 李晓辉
Priority to US16/333,803 priority Critical patent/US11091511B2/en
Priority to EP17850301.7A priority patent/EP3502120B1/en
Priority to JP2019536633A priority patent/JP6867577B2/ja
Priority to RU2019107605A priority patent/RU2730495C1/ru
Publication of WO2018050099A1 publication Critical patent/WO2018050099A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of only two carbon atoms, e.g. pregnane derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a glycol type ginsenoside derivative and a preparation method and application thereof.
  • Atherosclerosis is one of the diseases with high morbidity in today's society. It is the common pathological basis of many serious cardiovascular diseases (such as coronary heart disease, angina pectoris, cerebrovascular embolism, etc.), which is a serious threat to human health.
  • cardiovascular diseases such as coronary heart disease, angina pectoris, cerebrovascular embolism, etc.
  • the drugs for treating atherosclerosis mainly mainly regulate blood lipids, including statins, fibrates and niacin drugs.
  • statins mainly regulate blood lipids, including statins, fibrates and niacin drugs.
  • Atherosclerosis has been recognized as a chronic inflammatory disease of blood vessels. Therefore, it is important to develop anti-atherosclerotic drugs that simultaneously regulate blood lipids and anti-inflammatory activities.
  • Ginsenoside is the main active ingredient of ginseng and has the main pharmacological activity of ginseng. More than 40 kinds of ginsenoside monomers have been identified. It has been found that diol-type ginsenosides have certain atherosclerosis control effects. Further mechanism studies have shown that its pharmacological effects include regulating blood lipids, reducing inflammation, and reducing smooth muscle. Proliferation, etc., but its activity is low, and it has certain cytotoxicity and lacks practical application value.
  • the object of the present invention is to provide a diol type ginsenoside derivative and a preparation method and application thereof, and aim to provide a diol type ginsenoside derivative having high activity and low toxicity, which can be applied to prepare a medicament for preventing and treating atherosclerosis. in.
  • the present invention provides a glycol type ginsenoside derivative having the structure of Formula I or Formula II:
  • R 1 is Wherein R 3 is a C1 to C4 alkyl group
  • the R 2 has the structure shown in Formula III:
  • the n 0, 1 or 2
  • the R 4 is a methyl group or an ethyl group
  • the R 5 is a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted One of a benzyl group and a C4 to C9 heterocycloalkyl group.
  • the R 5 includes a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group or a C3-C5 ester alkyl group.
  • the R 5 comprises -CH 2 CH(CH 3 ) 2 , -CH(CH 3 ) 2 , -CH(OH)CH 3 , -CH 2 OH, -CH 2 CH 2 COOCH 2 CH 3 or - CH 2 COOCH 2 CH 3 .
  • the R 5 includes
  • the R 2 comprises
  • the R 1 includes
  • the invention provides a preparation method of the diol type ginsenoside derivative according to the above technical solution, which comprises the following steps:
  • parent compound in the step (1) has the structure represented by the formula IV or V:
  • the oxidation reaction in the step (2) is specifically:
  • the second intermediate precursor in the step (21) is subjected to a secondary oxidation reaction in the presence of a second oxidizing agent and an organic solvent to obtain a second intermediate product.
  • the first oxidizing agent in the step (21) comprises hydrogen peroxide, hypochlorous acid, calcium hypochlorite, peroxyacetone or m-chloroperoxybenzoic acid; and the second oxidizing agent in the step (22) comprises permanganic acid. Potassium, manganese dioxide, periodic acid or Sharit reagent.
  • the present invention provides the diol type ginsenoside derivative described in the above technical solution or the preparation method described in the above technical solution.
  • the present invention provides a diol type ginsenoside derivative having the structure represented by Formula I or Formula II, and the diol type ginsenoside derivative provided by the present invention has low cytotoxicity and can significantly reduce atherosclerosis of apoE-/- mice.
  • the percentage of plaque area at the same time, it can effectively reduce the level of low-density lipoprotein cholesterol in serum and increase the level of high-density lipoprotein cholesterol; it can significantly reduce the level of TNF- ⁇ in the arteries of apoE-/- mice, and has better resistance.
  • Inflammatory effect significantly reduced the formation of RAW264.7 cell-derived foam cells at a dose of 30 ⁇ mol/L. This indicates that the diol type ginsenoside derivative provided by the present invention can be used as an active ingredient in the preparation of a medicament for controlling atherosclerosis.
  • the present invention provides a preparation method of the diol type ginsenoside derivative, which is simple in operation and high in yield.
  • the present invention provides a glycol type ginsenoside derivative having the structure of Formula I or Formula II:
  • R 1 is Wherein R 3 is a C1 to C4 alkyl group
  • the R 2 has the structure shown in Formula III:
  • the n 0, 1 or 2
  • the R 4 is a methyl group or an ethyl group
  • the R 5 is a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted One of a benzyl group and a C4 to C9 heterocycloalkyl group.
  • the R 1 is Wherein R 3 is a C1-C4 alkyl group, preferably a methyl group, an ethyl group, a n-propyl group, an isopropyl group or an isobutyl group.
  • R 1 preferably includes
  • R 2 has the structure shown in Formula III:
  • the n 0, 1 or 2
  • the R 4 is a methyl group or an ethyl group
  • the R 5 is a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted One of a benzyl group and a C4 to C9 heterocycloalkyl group.
  • the substituted or unsubstituted C1-C5 alkyl group preferably includes a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group or a C3-C5 ester alkyl group.
  • the C1 ⁇ C5 alkyl group preferably comprises -CH 2 CH (CH 3) 2 or -CH (CH 3) 2; said C1 ⁇ C5 hydroxyalkyl groups preferably comprise -CH (OH) CH 3 or -CH 2 OH; the C3 to C5 ester alkyl group preferably includes -CH 2 CH 2 COOCH 2 CH 3 or -CH 2 COOCH 2 CH 3 .
  • the substituted benzyl group preferably includes a hydroxybenzyl group, and specifically
  • the C4 to C9 heterocycloalkyl group preferably includes
  • the R 2 preferably includes
  • the diol type ginsenoside derivative having the structure represented by Formula I preferably includes
  • the diol type ginsenoside derivative having the structure represented by Formula II preferably includes
  • the invention provides a preparation method of the diol type ginsenoside derivative according to the above technical solution, which comprises the following steps:
  • parent compound in the step (1) has the structure represented by the formula IV or V:
  • a parent compound having a structure represented by Formula IV or V and an acid anhydride are subjected to a nucleophilic substitution reaction in the presence of an alkaline reagent to obtain a first intermediate product.
  • the mass ratio of the parent compound to the acid anhydride and the alkaline agent is preferably 1: (1 to 20): (1 to 20), more preferably 1: (4 to 15): (4 to 15) , most preferably 1: (8 ⁇ 12): (8 ⁇ 12).
  • the type of the acid anhydride of the present invention is not particularly limited, and an acid anhydride having the structure of R 1 which is well known to those skilled in the art can be obtained.
  • the acid anhydride preferably includes acetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride or isovaleric anhydride.
  • the type of the alkaline agent to be used in the present invention is not particularly limited, and an organic basic compound, an inorganic basic compound, and/or an alkali metal element which are well known to those skilled in the art may be used.
  • the type of the organic basic compound to be used in the present invention is not particularly limited, and an organic basic compound well known to those skilled in the art may be used.
  • the organic basic compound preferably includes triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, pyridine, N-methylmorpholine, tetramethylethylenediamine. , potassium t-butoxide, sodium methoxide, potassium ethoxide or sodium ethoxide.
  • the type of the inorganic basic compound to be used in the present invention is not particularly limited, and an inorganic basic compound well known to those skilled in the art may be used.
  • the inorganic basic compound preferably includes sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate or sodium hydride.
  • the type of the alkali metal element is not particularly limited in the present invention, and an alkali metal element known to those skilled in the art may be used, such as sodium metal.
  • the temperature of the nucleophilic substitution reaction is preferably 0 to 100 ° C, more preferably 20 to 75 ° C, most preferably 35 to 60 ° C; and the time of the nucleophilic substitution reaction is preferably 1 to 48 h. It is more preferably 5 to 38 hours, and most preferably 15 to 25 hours.
  • the nucleophilic substitution reaction is preferably carried out under stirring.
  • the stirring rate is preferably 800 to 1200 rpm, more preferably 900 to 1100 rpm.
  • the manner of the agitation of the present invention is not particularly limited, and a stirring method well known to those skilled in the art may be employed.
  • the invention preferably employs magnetic stirring.
  • the obtained product is post-treated to obtain a first intermediate product.
  • the post-processing preferably includes the following steps:
  • the specific operation steps and reagents used in the silica gel column chromatography of the present invention are not particularly limited, and silica gel column chromatography well known to those skilled in the art can be used.
  • the crude product is preferably mixed with ethyl acetate, and the mixture is sampled by silica gel and used for column chromatography.
  • the mass ratio of the crude product to ethyl acetate is preferably 1: (1 to 10), more preferably 1: (3 to 8), and most preferably 1: (4 to 6).
  • ethyl acetate and n-hexane are preferably used as an eluent, and the volume ratio of ethyl acetate to n-hexane in the eluent is preferably 1: (1 to 10), more preferably 1: (3 to 8). Most preferably 1: (4 to 6).
  • the present invention oxidizes the first intermediate product in the presence of an oxidizing agent and an organic solvent to obtain a second intermediate product.
  • the oxidation reaction is specifically:
  • the second intermediate precursor is subjected to a secondary oxidation reaction in the presence of a second oxidizing agent and an organic solvent to obtain a second intermediate product.
  • the first intermediate product is subjected to a primary oxidation reaction in the presence of a first oxidizing agent and an organic solvent to obtain a second intermediate product precursor.
  • the mass ratio of the first intermediate product, the first oxidizing agent and the organic solvent is preferably 1: (0.1 to 0.5): (5 to 10), more preferably 1: (0.2 to 0.4): (6) ⁇ 8).
  • the first oxidizing agent preferably includes hydrogen peroxide, hypochlorous acid, calcium hypochlorite, peroxyacetone or m-chloroperoxybenzoic acid.
  • the type of the organic solvent required for carrying out the primary oxidation reaction of the present invention is not particularly limited, and a polar organic solvent or nonpolar which is well known to those skilled in the art capable of matching the first intermediate product and the first oxidant is used.
  • the organic solvent may be, for example, pyridine, N-methylpyrrolidone, chloroform, dichloromethane, carbon tetrachloride, tetrahydrofuran or 1,4-dioxane.
  • the temperature of the primary oxidation reaction is preferably 10 to 80 ° C, more preferably 15 to 55 ° C, and most preferably 20 to 35 ° C; specifically, in the embodiment of the present invention, the primary oxidation The reaction is carried out at room temperature without heating the reaction system or cooling.
  • the time of the primary oxidation reaction is preferably from 1 to 10 hours, more preferably 3 to 8 h, most preferably 4 to 6 h.
  • the obtained product is post-treated to obtain a second intermediate product precursor.
  • the post-processing preferably includes the following steps:
  • the material obtained after the completion of the initial oxidation reaction was stirred with water, filtered, and the solvent was removed to obtain a crude product; the crude product was purified by silica gel column chromatography to give a second intermediate product.
  • the mass ratio of the material to water obtained after the completion of the primary oxidation reaction is preferably 1: (1 to 100), more preferably (8 to 70), and most preferably (20 to 40).
  • the stirring rate is preferably 800 to 1200 rpm, more preferably 900 to 1100 rpm; and the stirring time is preferably 1 to 10 h, more preferably 3 to 8 h, and most preferably 4 to 6 h.
  • the manner of the agitation of the present invention is not particularly limited, and a stirring method well known to those skilled in the art may be employed.
  • the invention preferably employs magnetic stirring.
  • the present invention is not particularly limited to the filtration, and a filtration scheme well known to those skilled in the art may be employed.
  • the method for removing the solvent in the present invention is not particularly limited, and a technical solution for removing the solvent well known to those skilled in the art may be employed. In the present invention, it is preferred to remove the solvent by distillation under reduced pressure.
  • the specific operation steps and reagents used in the silica gel column chromatography of the present invention are not particularly limited, and silica gel column chromatography well known to those skilled in the art can be used.
  • the crude product is preferably mixed with ethyl acetate, and the mixture is sampled by silica gel and used for column chromatography.
  • the mass ratio of the crude product to ethyl acetate is preferably 1: (1 to 10), more preferably 1: (3 to 8), and most preferably 1: (4 to 6).
  • ethyl acetate and n-hexane are preferably used as an eluent, and the volume ratio of ethyl acetate to n-hexane in the eluent is preferably 1: (1 to 10), more preferably 1: (3 to 8). Most preferably 1: (4 to 6).
  • the present invention After obtaining the second intermediate product precursor, the present invention performs the second oxidation reaction of the second intermediate product precursor in the presence of the second oxidizing agent and the organic solvent to obtain a second intermediate product.
  • the mass ratio of the second intermediate precursor, the second oxidizing agent and the organic solvent is preferably 1: (0.1 to 0.3): (5 to 10), more preferably 1: (0.15 to 0.25): (6-8).
  • the second oxidizing agent preferably includes potassium permanganate, manganese dioxide, periodic acid or a Sharit reagent.
  • the type of the organic solvent required for carrying out the secondary oxidation reaction of the present invention is not particularly limited, and a polar organic solvent which is compatible with the second intermediate precursor and the second oxidant, which is well known to those skilled in the art, or A non-polar organic solvent such as chloroform, dichloromethane, carbon tetrachloride, acetonitrile, tetrahydrofuran or 1,4-dioxane may be used.
  • the temperature of the secondary oxidation reaction is preferably 10 to 100 ° C, more preferably 15 to 65 ° C, and most preferably 20 to 45 ° C; specifically, in the embodiment of the present invention, the second The sub-oxidation reaction is carried out at room temperature without heating or cooling the reaction system.
  • the time of the secondary oxidation reaction is preferably from 1 to 10 h, more preferably from 3 to 8 h, and most preferably from 4 to 6 h.
  • the obtained product is subjected to post-treatment to obtain a second intermediate product.
  • the post-processing preferably includes the following steps:
  • the material obtained after the completion of the second oxidation reaction is extracted with an organic solvent, washed, and the solvent is removed to obtain a crude product; the crude product is purified by silica gel column chromatography to obtain a second intermediate product.
  • the mass ratio of the material obtained after the completion of the secondary oxidation reaction to the extracting agent is preferably 1: (10 to 30), more preferably 1: (15 to 25), and most preferably 1: (18). ⁇ 22).
  • the extractant preferably includes ethyl acetate, chloroform, dichloromethane or carbon tetrachloride.
  • the number of extractions is preferably 2 to 4 times. In the present invention, specifically, the organic phase obtained after the extraction is washed.
  • the present invention is not particularly limited to the washing, and a washing technique known to those skilled in the art may be employed.
  • the material obtained after the extraction is preferably washed successively with an alkali solution and a sodium chloride solution.
  • the volume ratio of the material obtained after the extraction to the alkali solution or the sodium chloride solution is preferably 1: (1 to 10), more preferably 1: (3 to 8), most preferably 1 :(4 ⁇ 6).
  • the type of the lye according to the present invention is not particularly limited, and a lye well known to those skilled in the art, such as a sodium hydrogencarbonate solution, may be used.
  • the concentration of the alkali solution and the sodium chloride solution in the present invention is not particularly limited, and the concentration of the solution suitable for washing can be used by those skilled in the art.
  • the washing is carried out specifically using a saturated lye and a saturated sodium chloride solution.
  • the material obtained after the extraction is preferably washed 3 to 5 times with an alkali solution, and then washed 3 to 5 times with a sodium chloride solution.
  • the invention has no special limitation on the method for removing the solvent. It is sufficient to use a technical solution for removing the solvent well known to those skilled in the art. In the present invention, it is preferred to remove the solvent by distillation under reduced pressure.
  • the specific operation steps and reagents used in the silica gel column chromatography of the present invention are not particularly limited, and silica gel column chromatography well known to those skilled in the art can be used.
  • the crude product is preferably mixed with ethyl acetate, and the mixture is sampled by silica gel and used for column chromatography.
  • the mass ratio of the crude product to ethyl acetate is preferably 1: (1 to 10), more preferably 1: (3 to 8), and most preferably 1: (4 to 6).
  • ethyl acetate and n-hexane are preferably used as an eluent, and the volume ratio of ethyl acetate to n-hexane in the eluent is preferably 1: (1 to 10), more preferably 1: (3 to 8). Most preferably 1: (4 to 6).
  • the present invention reductively reacts the second intermediate product with the amino compound in the presence of an organic solvent and a reducing agent to obtain a diol type ginseng having the structure represented by Formula I or Formula II.
  • a saponin derivative in the present invention, the mass ratio of the second intermediate product to the amino compound, the reducing agent and the organic solvent is preferably 1: (1 to 100): (5 to 10), more preferably 1: (8) ⁇ 70): (6 to 9), more preferably 1: (20 to 50): (7 to 8).
  • the amino compound preferably includes L-leucine methyl ester hydrochloride, L-valine methyl ester hydrochloride, L-threonine methyl ester hydrochloride, L-serine methyl ester salt Acid salt, L-glutamic acid dimethyl ester hydrochloride, glycine methyl ester hydrochloride, ⁇ -alanine ethyl ester hydrochloride, aminomalonic acid diethyl ester hydrochloride, L-phenylalanine Methyl ester hydrochloride, L-tyrosine methyl ester, L-histidine methyl ester hydrochloride or L-tryptophan methyl ester hydrochloride.
  • the reducing agent preferably includes sodium borohydride, sodium cyanoborohydride or sodium triacetylborohydride.
  • the type of the organic solvent required for carrying out the reductive amination reaction of the present invention is not particularly limited, and a polar organic solvent which is well known to those skilled in the art and which is compatible with the second intermediate product and the amino compound and the reducing agent or A non-polar organic solvent such as methanol, ethyl acetate, chloroform, dichloromethane, carbon tetrachloride.
  • the temperature at which the reducing agent is mixed with the second intermediate product, the amino compound and the organic solvent is preferably -2 to 2 °C.
  • the method for controlling the temperature at which the reducing agent is mixed with the second intermediate product, the amino compound and the organic solvent is not particularly limited, and a method of controlling the temperature well known to those skilled in the art may be employed.
  • the reducing agent is specifically mixed with the second intermediate product, the amino compound and the organic solvent under ice water bath conditions.
  • the mixing of the reducing agent with the second intermediate product, the amino compound and the organic solvent is preferably carried out under stirring.
  • the stirring rate is preferably 800 to 1200 rpm, more preferably 900 to 1100 rpm.
  • the manner of the agitation of the present invention is not particularly limited, and a stirring method well known to those skilled in the art may be employed.
  • the invention preferably employs magnetic stirring.
  • the temperature of the reductive amination reaction is preferably -10 to 80 ° C, more preferably 0 to 55 ° C, and most preferably 20 to 35 ° C; specifically, in the embodiment of the present invention, The reductive amination reaction is carried out at room temperature without heating the reaction system or cooling.
  • the time of the reductive amination reaction is preferably from 1 to 48 h, more preferably from 8 to 32 h, and most preferably from 13 to 20 h.
  • the obtained product is post-treated to obtain a diol type ginsenoside derivative having a structure represented by Formula I or Formula II.
  • the post-processing preferably includes the following steps:
  • the material obtained after the reductive amination reaction is extracted with an organic solvent, washed, and the solvent is removed to obtain a crude product; the crude product is purified by silica gel column chromatography to obtain the formula I or formula II.
  • a structured glycol-type ginsenoside derivative is obtained after the reductive amination reaction.
  • the mass ratio of the material obtained after the reductive amination reaction to the extractant is preferably 1: (1 to 50), more preferably 1: (8 to 40), and most preferably 1: (15). ⁇ 25).
  • the extractant preferably includes ethyl acetate, chloroform, dichloromethane or carbon tetrachloride.
  • the number of extractions is preferably 2 to 4 times. In the present invention, specifically, the organic phase obtained after the extraction is washed.
  • the present invention is not particularly limited to the washing, and a washing technique known to those skilled in the art may be employed.
  • it is preferred to wash the material obtained after the extraction with a sodium chloride solution.
  • the concentration of the sodium chloride solution in the present invention is not particularly limited, and the concentration of the solution suitable for washing can be used by those skilled in the art.
  • the washing is carried out specifically using a saturated sodium chloride solution.
  • the volume ratio of the material obtained after the extraction to the sodium chloride solution is preferably 1: (1 to 30), more preferably 1: (5 to 23), and most preferably 1: (10) ⁇ 15).
  • the number of washings is preferably from 3 to 5 times.
  • the method for removing the solvent in the present invention is not particularly limited, and a technical solution for removing the solvent well known to those skilled in the art may be employed. In the present invention, it is preferred to remove the solvent by distillation under reduced pressure.
  • the specific operation steps and reagents used in the silica gel column chromatography of the present invention are not particularly limited, and silica gel column chromatography well known to those skilled in the art can be used.
  • the crude product is preferably mixed with ethyl acetate, and the mixture is sampled by silica gel and used for column chromatography.
  • the mass ratio of the crude product to ethyl acetate is preferably 1: (1 to 10), more preferably 1: (3 to 8), and most preferably 1: (4 to 6).
  • ethyl acetate and n-hexane are preferably used as an eluent, and the volume ratio of ethyl acetate to n-hexane in the eluent is preferably 1: (1 to 10), more preferably 1: (3 to 8). Most preferably 1: (4 to 6).
  • the invention provides the use of the glycol type ginsenoside derivative of the above technical solution or the diol type ginsenoside derivative obtained by the preparation method of the above technical solution for preparing a medicament for preventing and treating atherosclerosis.
  • the atherogenic drug preferably comprises an active ingredient and an adjuvant.
  • the active ingredient for controlling the atherosclerotic drug is the diol type ginsenoside derivative described in the above technical solution of the present invention or the diol type ginsenoside derivative obtained by the above-mentioned preparation method.
  • the type of the auxiliary material is not particularly limited in the present invention, and an auxiliary material well known to those skilled in the art may be used.
  • the ratio of the active ingredient to the auxiliary material in the present invention is not particularly limited, and the ratio of the active ingredient to the auxiliary material can be determined according to actual conditions.
  • the dosage form and dosage of the anti-atherosclerotic drug are not particularly limited, and the dosage form and dosage of the atherosclerotic drug can be determined according to actual needs.
  • the mixture was used for column chromatography, and a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:6) to obtain 0.03 g of the title compound 5 as a white solid. 60%.
  • the structure of the target compound 5 is:
  • the ester was mixed, and the mixture was applied to a column of silica gel, and the mixture was applied to a column chromatography, and a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:3) to obtain 0.03 g of a target compound as a white solid. 8, the yield of 55%.
  • the structure of the target compound 8 is:
  • a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:3) to obtain 0.03 g of a target compound 12 as a white solid.
  • the rate is 50%.
  • the structure of the target compound 12 is:
  • the structure of the target compound 14 is:
  • a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:10) to obtain 0.03 g of a target compound 19 as a white solid.
  • the rate is 72%.
  • the structure of the target compound 19 is:
  • Example 18 0.033 g (0.6 mmol) of the compound 18 prepared in Example 18 was mixed with 0.012 g (0.7 mmol) of L-valine methyl ester hydrochloride, 2 mL of ethyl acetate, and 0.033 g was added to the ice bath under magnetic stirring at 1000 rpm.
  • a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:9) to obtain 0.03 g of a target compound 20 as a white solid.
  • the rate is 75%.
  • the structure of the target compound 20 is:
  • the yield was 50%.
  • the structure of the target compound 21 is:
  • the structure of the target compound 22 is:
  • the mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:6) to obtain a target of 0.028 g of a white solid.
  • the structure of the target compound 23 is:
  • Example 18 0.033 g (0.6 mmol) of the compound 18 prepared in Example 18 was mixed with 0.011 g (0.7 mmol) of ⁇ -alanine ethyl ester hydrochloride, 2 mL of methanol, and 0.033 g (5.3 g) was added in an ice bath under magnetic stirring at 1000 rpm.
  • Example 18 0.033 g (0.6 mmol) of the compound 18 prepared in Example 18 was mixed with 0.015 g (0.7 mmol) of diethylaminomalonate hydrochloride, 2 mL of methanol, and 0.033 g (5.3 g) was added in an ice bath under a magnetic stirring at 900 rpm.
  • a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:3) to obtain 0.026 g of a target compound 26 as a white solid.
  • the rate is 60%.
  • the structure of the target compound 26 is:
  • the structure of the target compound 27 is:
  • Example 18 0.033 g (0.6 mmol) of the compound 18 prepared in Example 18 was mixed with 0.017 g (0.7 mmol) of L-histidine methyl ester hydrochloride, 2 mL of methanol, and 0.033 g (5.3 g) was added to the ice bath under magnetic stirring at 1000 rpm.
  • a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:5) to obtain 0.025 g of a target compound 29 as a white solid.
  • the rate is 50%.
  • the structure of the target compound 29 is:
  • a mixture of ethyl acetate and n-hexane was used as an eluent (ethyl acetate and n-hexane in a volume ratio of 1:5) to obtain 0.02 g of a target compound 30 as a white solid.
  • the rate is 45%.
  • the structure of the target compound 30 is:
  • Kunming mice (18-22 g, male, purchased from the Experimental Animal Center of the Third Military Medical University) were randomly divided into 31 groups, and the compounds prepared in Examples 1 to 30 of the present invention were applied to the Kunming mice ( A single oral gavage (500 mg/kg) was administered, and a saline control group (1 mL) was set up for 14 days.
  • the mouse macrophage cell line RAW264.7 was cultured, and the compounds prepared in Inventive Examples 1 to 30 were used in a medium to prepare a concentration of 10 ⁇ mol/L.
  • the solvent was physiological saline
  • the co-solvent was dimethyl sulfoxide (DMSO, the final concentration was 0.1%)
  • a group of 0.1% DMSO physiological saline control group was set up.
  • Cell viability was determined by CCK8 method, and the data was expressed as x ⁇ s, and SPSS 10.0 was used between groups.
  • the factor analysis of variance was significant with p ⁇ 0.05, and the results are shown in Table 1. As can be seen from Table 1, Compounds 1, 2 and 3 have significant cytotoxicity, and other compounds have no significant cytotoxicity at this concentration.
  • Animals 160 healthy, clean-grade apoE-/- mice (purchased from Peking University Experimental Animal Center), male, weighing 22-25 g, kept in a sterile laminar feeding room, freely drinking water. .
  • Saline control group saline administration
  • TC total cholesterol
  • LDL-C low-density cholesterol lipoprotein
  • HDL-C high-density lipoprotein cholesterol
  • TNF- ⁇ level measurement Mcytomag-70K-3, Mouse Cytokine/Chemokine Magnetic Bead Panel was determined by Millipore liquid phase chip method.
  • the compounds prepared in Examples 1 to 30 of the present invention all significantly reduced the percentage of atherosclerotic plaque area in apoE-/- mice, which was statistically different from the control group.
  • the compounds prepared in Examples 1 to 30 of the present invention all significantly increased HDL-C levels, and some compounds significantly decreased the level of LDL-C in the serum of mice, which was statistically different from the control group.
  • the compounds prepared in Examples 1 to 30 of the present invention can significantly reduce the local TNF- ⁇ level in the apoE-/- mouse arteries, which is statistically different from the control group, indicating that the present invention is in the range of 1 to 30.
  • the prepared compound has a good anti-inflammatory effect.
  • RAW264.7 cells were cultured in high glucose DMEM medium (GIBCO) to a cell concentration of 1 ⁇ 10 6 , planted in 6-well plates, and 60 mg/L ox-LDL (oxidized modified low-density lipoprotein, Beijing) Coordination Medical University) obtained foam cells after 48 hours of incubation.
  • GEBCO high glucose DMEM medium
  • the obtained foam cells were divided into 1 group per 3 well plates, and were divided into 61 groups. After adding the drug (100 ⁇ L/well) for 48 hours, the medium was discarded, and after staining with oil red O, the excess oil red O was washed away. The absorbance values were measured at 490 nm after the cells were dissolved in isopropanol. The OD value of the model group was 100%, and the OD value of each group corrected by the number of cells was compared with that of the model group to obtain the ratio of intracellular lipid content. The larger the value, the more intracellular lipids, foam cells The higher the degree of formation.
  • Saline control group normal saline
  • the compounds prepared in Examples 1 to 30 of the present invention can significantly reduce the degree of foam cell formation at a dose of 30 ⁇ mol/L, and some compounds can significantly reduce the degree of foam cell formation at a dose of 10 ⁇ mol/L.
  • the diol-type ginsenoside derivative provided by the present invention has low cytotoxicity, can significantly reduce the percentage of atherosclerotic plaque area in apoE-/- mice, and can effectively reduce the serum in mice.
  • Low-density lipoprotein cholesterol level, high-density lipoprotein cholesterol level can significantly reduce the local TNF- ⁇ level in apoE-/- mouse arteries, has a good anti-inflammatory effect; can significantly reduce RAW264 at 30 ⁇ mol/L dose. 7

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Abstract

提供具有式I或式II所示结构的二醇型人参皂苷衍生物，并提供其在制备防治动脉粥样硬化药物中的应用。所述的二醇型人参皂苷衍生物细胞毒性小，能够显著减少apoE-/-小鼠动脉粥样硬化斑块面积的百分比；同时能够有效降低小鼠血清中低密度脂蛋白胆固醇水平，提高高密度脂蛋白胆固醇水平；能够显著降低apoE-/-小鼠动脉局部TNF-α水平，具有较好的抗炎作用；在30μM剂量下能够显著降低RAW264.7细胞源泡沫细胞形成的程度。此外，所述二醇型人参皂苷衍生物的制备方法，操作简便，产率高。

Description

二醇型人参皂苷衍生物及其制备方法和应用 技术领域
本发明属于医药技术领域，具体涉及二醇型人参皂苷衍生物及其制备方法和应用。
背景技术
动脉粥样硬化是当今社会高发病率的疾病之一，是多种严重心血管疾病(如冠心病、心绞痛、脑血管栓塞等)的共同病理基础，严重威胁人类健康。目前治疗动脉粥样硬化的药物主要以调节血脂为主，包括他汀类、贝特类和烟酸类药物等。尽管以上类别的药物能够达到很好的降低血脂的效果，但动脉粥样硬化的发病率仍居高不下且逐年上升，这说明仅仅具有调节血脂功能的药物不能满足对动脉粥样硬化防治的需求。研究表明，炎症免疫因素在动脉粥样硬化的发病过程中具有重要意义，而且动脉粥样硬化已经被公认为是一种血管的慢性炎症性疾病。因此，研发同时具有调节血脂和抗炎活性的抗动脉粥样硬化药物具有重要意义。
人参自古被誉为“百草之王”，具有大补元气、复脉固脱、补脾益肺、生津养血、安神益智的功效。人参皂苷是人参的主要有效成分，具有人参的主要药理活性。现已明确结构的人参皂苷单体约有40余种，研究发现二醇型人参皂苷具有一定的动脉粥样硬化防治作用，进一步的机制研究表明其药理作用包括调节血脂、减低炎症反应、减少平滑肌增殖等，但其活性偏低，而且具有一定的细胞毒性，缺乏实际应用价值。
发明内容
本发明的目的在于提供二醇型人参皂苷衍生物及其制备方法和应用，旨在提供具有较高活性和较低毒性的二醇型人参皂苷衍生物，能够应用于制备防治动脉粥样硬化药物中。
本发明提供了二醇型人参皂苷衍生物，具有式I或式II所示结构：
Figure PCTCN2017101880-appb-000001
式I和式II中，所述R1为
Figure PCTCN2017101880-appb-000002
其中，R3为C1～C4的烷基；
所述R2具有式III所示结构：
Figure PCTCN2017101880-appb-000003
式III中，所述n＝0、1或2，所述R4为甲基或乙基，所述R5为氢原子、取代或未取代的C1～C5的烷基、取代或未取代的苄基和C4～C9的杂环烷基中的一种。
优选的，所述R5包括C1～C5的烷基、C1～C5的羟烷基或C3～C5的酯烷基。
优选的，所述R5包括-CH2CH(CH3)2、-CH(CH3)2、-CH(OH)CH3、-CH2OH、-CH2CH2COOCH2CH3或-CH2COOCH2CH3。
优选的，所述R5包括
Figure PCTCN2017101880-appb-000004
优选的，所述R2包括
Figure PCTCN2017101880-appb-000005
Figure PCTCN2017101880-appb-000006
优选的，所述R1包括
Figure PCTCN2017101880-appb-000007
本发明提供了上述技术方案所述二醇型人参皂苷衍生物的制备方法，包括以下步骤：
(1)将母体化合物与酸酐在碱性试剂存在的条件下进行亲核取代反应，得到第一中间产物；
(2)将所述步骤(1)中第一中间产物在氧化剂和有机溶剂存在的条件下进行氧化反应，得到第二中间产物；
(3)将所述步骤(2)中第二中间产物与氨基化合物在有机溶剂和还原剂存在的条件下进行还原胺化反应，得到具有式I或式II所示结构的二醇型人参皂苷衍生物；
其中，所述步骤(1)中母体化合物具有式IV或V所示结构：
Figure PCTCN2017101880-appb-000008
优选的，步骤(2)所述氧化反应具体为：
(21)将所述步骤(1)中第一中间产物在第一氧化剂和有机溶剂存在的条件下进行初次氧化反应，得到第二中间产物前体；
(22)将所述步骤(21)中第二中间产物前体在第二氧化剂和有机溶剂存在的条件下进行二次氧化反应，得到第二中间产物。
优选的，步骤(21)所述第一氧化剂包括过氧化氢、次氯酸、次氯化钙、过氧丙酮或间氯过氧苯甲酸；步骤(22)所述第二氧化剂包括高锰酸钾、二氧化锰、高碘酸或沙瑞特试剂。
本发明提供了上述技术方案所述二醇型人参皂苷衍生物或上述技术方案所述制备方法得 到的二醇型人参皂苷衍生物在制备防治动脉粥样硬化药物中的应用。
本发明提供了具有式I或式II所示结构的二醇型人参皂苷衍生物，本发明提供的二醇型人参皂苷衍生物细胞毒性小，能够显著减少apoE-/-小鼠动脉粥样硬化斑块面积的百分比；同时能够有效降低小鼠血清中低密度脂蛋白胆固醇水平，提高高密度脂蛋白胆固醇水平；能够显著降低apoE-/-小鼠动脉局部TNF-α水平，具有较好的抗炎作用；在30μmol/L剂量下能够显著降低RAW264.7细胞源泡沫细胞形成的程度。这说明本发明提供的二醇型人参皂苷衍生物能够作为活性组分应用于制备防治动脉粥样硬化药物中。
此外，本发明提供了所述二醇型人参皂苷衍生物的制备方法，操作简便，产率高。
具体实施方式
本发明提供了二醇型人参皂苷衍生物，具有式I或式II所示结构：
Figure PCTCN2017101880-appb-000009
式I和式II中，所述R1为
Figure PCTCN2017101880-appb-000010
其中，R3为C1～C4的烷基；
所述R2具有式III所示结构：
Figure PCTCN2017101880-appb-000011
式III中，所述n＝0、1或2，所述R4为甲基或乙基，所述R5为氢原子、取代或未取代的C1～C5的烷基、取代或未取代的苄基和C4～C9的杂环烷基中的一种。
在本发明中，所述R1为
Figure PCTCN2017101880-appb-000012
其中，R3为C1～C4的烷基，优选为甲基、乙基、正丙基、异丙基或异丁基。相应的，在本发明中，所述R1优选包括
Figure PCTCN2017101880-appb-000013
Figure PCTCN2017101880-appb-000014
在本发明中，所述R2具有式III所示结构：
Figure PCTCN2017101880-appb-000015
式III中，所述n＝0、1或2，所述R4为甲基或乙基，所述R5为氢原子、取代或未取代的C1～C5的烷基、取代或未取代的苄基和C4～C9的杂环烷基中的一种。
在本发明中，所述取代或未取代的C1～C5的烷基优选包括C1～C5的烷基、C1～C5的羟烷基或C3～C5的酯烷基。在本发明中，所述C1～C5的烷基优选包括-CH2CH(CH3)2或-CH(CH3)2；所述C1～C5的羟烷基优选包括-CH(OH)CH3或-CH2OH；所述C3～C5的酯烷基优 选包括-CH2CH2COOCH2CH3或-CH2COOCH2CH3。
在本发明中，所述取代的苄基优选包括羟基苄基，具体可为
Figure PCTCN2017101880-appb-000016
在本发明中，所述C4～C9的杂环烷基优选包括
Figure PCTCN2017101880-appb-000017
相应的，在本发明中，所述R2优选包括
Figure PCTCN2017101880-appb-000018
Figure PCTCN2017101880-appb-000019
在本发明中，所述具有式I所示结构的二醇型人参皂苷衍生物优选包括
Figure PCTCN2017101880-appb-000020
在本发明中，所述具有式II所示结构的二醇型人参皂苷衍生物优选包括
Figure PCTCN2017101880-appb-000021
本发明提供了上述技术方案所述二醇型人参皂苷衍生物的制备方法，包括以下步骤：
(1)将母体化合物与酸酐在碱性试剂存在的条件下进行亲核取代反应，得到第一中间产物；
(2)将所述步骤(1)中第一中间产物在氧化剂和有机溶剂存在的条件下进行氧化反应，得到第二中间产物；
(3)将所述步骤(2)中第二中间产物与氨基化合物在有机溶剂和还原剂存在的条件下进行还原胺化反应，得到具有式I或式II所示结构的二醇型人参皂苷衍生物；
其中，所述步骤(1)中母体化合物具有式IV或V所示结构：
Figure PCTCN2017101880-appb-000022
本发明将具有式IV或V所示结构的母体化合物与酸酐在碱性试剂存在的条件下进行亲核取代反应，得到第一中间产物。在本发明中，所述母体化合物与酸酐和碱性试剂的质量比优选为1：(1～20)：(1～20)，更优选为1：(4～15)：(4～15)，最优选为1：(8～12)： (8～12)。
本发明对于所述酸酐的种类没有特殊的限定，采用本领域技术人员熟知的能够得到具有所述R1结构的酸酐即可。在本发明中，所述酸酐优选包括乙酸酐、丙酸酐、丁酸酐、异丁酸酐或异戊酸酐。
本发明对于所述碱性试剂的种类没有特殊的限定，采用本领域技术人员熟知的有机碱性化合物、无机碱性化合物和/或碱金属单质即可。
本发明对于所述有机碱性化合物的种类没有特殊的限定，采用本领域技术人员熟知的有机碱性化合物即可。在本发明中，所述有机碱性化合物优选包括三乙胺、N,N-二异丙基乙胺、4-二甲氨基吡啶、吡啶、N-甲基吗啉、四甲基乙二胺、叔丁醇钾、甲醇钠、乙醇钾或乙醇钠。
本发明对于所述无机碱性化合物的种类没有特殊的限定，采用本领域技术人员熟知的无机碱性化合物即可。在本发明中，所述无机碱性化合物优选包括氢氧化钠，氢氧化钾、碳酸钾、碳酸钠或氢化钠。
本发明对于所述碱金属单质的种类没有特殊的限定，采用本领域技术人员熟知的碱金属单质即可，具体如金属钠。
在本发明中，所述亲核取代反应的温度优选为0～100℃，更优选为20～75℃，最优选为35～60℃；所述亲核取代反应的时间优选为1～48h，更优选为5～38h，最优选为15～25h。在本发明中，所述亲核取代反应优选在搅拌条件下进行。在本发明中，所述搅拌的速率优选为800～1200rpm，更优选为900～1100rpm。本发明对于所述搅拌的方式没有特殊的限定，采用本领域技术人员熟知的搅拌方式即可。本发明优选采用磁力搅拌。
本发明优选在所述亲核取代反应完成后，对得到的产物进行后处理，得到第一中间产物。在本发明中，所述后处理优选包括以下步骤：
亲核取代反应结束后，减压蒸馏，得到粗产品；采用硅胶柱层析法对所述粗产品进行提纯，得到第一中间产物。
本发明对于所述硅胶柱层析法的具体操作步骤及所采用的试剂没有特殊的限定，采用本领域技术人员熟知的硅胶柱层析法即可。本发明优选将粗产品与乙酸乙酯混合，硅胶拌样后用于柱层析。在本发明中，所述粗产品与乙酸乙酯的质量比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。本发明优选采用乙酸乙酯和正己烷为洗脱剂，所述洗脱剂中乙酸乙酯和正己烷的体积比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。
得到第一中间产物后，本发明将所述第一中间产物在氧化剂和有机溶剂存在的条件下进行氧化反应，得到第二中间产物。在本发明中，所述氧化反应具体为：
将所述第一中间产物在第一氧化剂和有机溶剂存在的条件下进行初次氧化反应，得到第二中间产物前体；
将所述第二中间产物前体在第二氧化剂和有机溶剂存在的条件下进行二次氧化反应，得到第二中间产物。
本发明将所述第一中间产物在第一氧化剂和有机溶剂存在的条件下进行初次氧化反应，得到第二中间产物前体。在本发明中，所述第一中间产物、第一氧化剂和有机溶剂的质量比优选为1：(0.1～0.5)：(5～10)，更优选为1：(0.2～0.4)：(6～8)。
在本发明中，所述第一氧化剂优选包括过氧化氢、次氯酸、次氯化钙、过氧丙酮或间氯过氧苯甲酸。
本发明对于进行所述初次氧化反应所需的有机溶剂种类没有特殊的限定，采用本领域技术人员熟知的能够与所述第一中间产物和第一氧化剂相匹配的极性有机溶剂或非极性有机溶剂即可，如吡啶、N-甲基吡咯烷酮、氯仿、二氯甲烷、四氯化碳、四氢呋喃或1,4-二氧六环。
在本发明中，所述初次氧化反应的温度优选为10～80℃，更优选为15～55℃，最优选为20～35℃；具体的，在本发明的实施例中，所述初次氧化反应在室温下进行，无需对反应体系进行加热也无需进行冷却。在本发明中，所述初次氧化反应的时间优选为1～10h，更优选为 3～8h，最优选为4～6h。
本发明优选在所述初次氧化反应完成后，对得到的产物进行后处理，得到第二中间产物前体。在本发明中，所述后处理优选包括以下步骤：
向所述初次氧化反应结束后得到的物料中加水搅拌、过滤，除去溶剂，得到粗产品；采用硅胶柱层析法对所述粗产品进行提纯，得到第二中间产物前体。
在本发明中，所述初次氧化反应结束后得到的物料与水的质量比优选为1：(1～100)，更优选为(8～70)，最优选为(20～40)。在本发明中，所述搅拌的速率优选为800～1200rpm，更优选为900～1100rpm；所述搅拌的时间优选为1～10h，更优选为3～8h，最优选为4～6h。本发明对于所述搅拌的方式没有特殊的限定，采用本领域技术人员熟知的搅拌方式即可。本发明优选采用磁力搅拌。本发明对于所述过滤没有特殊的限定，采用本领域技术人员熟知的过滤的技术方案即可。本发明对于所述除去溶剂的方法没有特殊的限定，采用本领域技术人员熟知的除去溶剂的技术方案即可。本发明优选采用减压蒸馏除去溶剂。
本发明对于所述硅胶柱层析法的具体操作步骤及所采用的试剂没有特殊的限定，采用本领域技术人员熟知的硅胶柱层析法即可。本发明优选将粗产品与乙酸乙酯混合，硅胶拌样后用于柱层析。在本发明中，所述粗产品与乙酸乙酯的质量比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。本发明优选采用乙酸乙酯和正己烷为洗脱剂，所述洗脱剂中乙酸乙酯和正己烷的体积比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。
得到第二中间产物前体后，本发明将所述第二中间产物前体在第二氧化剂和有机溶剂存在的条件下进行二次氧化反应，得到第二中间产物。在本发明中，所述第二中间产物前体、第二氧化剂和有机溶剂的质量比优选为1：(0.1～0.3)：(5～10)，更优选为1：(0.15～0.25)：(6～8)。
在本发明中，所述第二氧化剂优选包括高锰酸钾、二氧化锰、高碘酸或沙瑞特试剂。
本发明对于进行所述二次氧化反应所需的有机溶剂种类没有特殊的限定，采用本领域技术人员熟知的能够与所述第二中间产物前体和第二氧化剂相匹配的极性有机溶剂或非极性有机溶剂即可，如氯仿、二氯甲烷、四氯化碳、乙腈、四氢呋喃或1,4-二氧六环。
在本发明中，所述二次氧化反应的温度优选为10～100℃，更优选为15～65℃，最优选为20～45℃；具体的，在本发明的实施例中，所述二次氧化反应在室温下进行，无需对反应体系进行加热也无需进行冷却。在本发明中，所述二次氧化反应的时间优选为1～10h，更优选为3～8h，最优选为4～6h。
本发明优选在所述二次氧化反应完成后，对得到的产物进行后处理，得到第二中间产物。在本发明中，所述后处理优选包括以下步骤：
采用有机溶剂对所述二次氧化反应结束后得到的物料进行萃取，洗涤，除去溶剂，得到粗产品；采用硅胶柱层析法对所述粗产品进行提纯，得到第二中间产物。
在本发明中，所述二次氧化反应结束后得到的物料与萃取剂的质量比优选为1：(10～30)，更优选为1：(15～25)，最优选为1：(18～22)。在本发明中，所述萃取剂优选包括乙酸乙酯、氯仿、二氯甲烷或四氯化碳。在本发明中，所述萃取的次数优选为2～4次。在本发明中，具体是将萃取后得到的有机相进行洗涤。
本发明对于所述洗涤没有特殊的限定，采用本领域技术人员熟知的洗涤的技术方案即可。本发明优选将萃取后得到的物料依次采用碱液和氯化钠溶液洗涤。在本发明中，所述萃取后得到的物料与所述碱液或氯化钠溶液的体积比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。本发明对于所述碱液的种类没有特殊的限定，采用本领域技术人员熟知的碱液即可，如碳酸氢钠溶液。本发明对于所述碱液和氯化钠溶液的浓度没有特殊的限定，采用本领域技术人员熟知的适用于进行洗涤的溶液的浓度即可。在本发明的实施例中，具体采用饱和碱液和饱和氯化钠溶液进行所述洗涤。本发明优选将萃取后得到的物料采用碱液洗涤3～5次，然后采用氯化钠溶液洗涤3～5次。本发明对于所述除去溶剂的方法没有特殊的限 定，采用本领域技术人员熟知的除去溶剂的技术方案即可。本发明优选采用减压蒸馏除去溶剂。
本发明对于所述硅胶柱层析法的具体操作步骤及所采用的试剂没有特殊的限定，采用本领域技术人员熟知的硅胶柱层析法即可。本发明优选将粗产品与乙酸乙酯混合，硅胶拌样后用于柱层析。在本发明中，所述粗产品与乙酸乙酯的质量比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。本发明优选采用乙酸乙酯和正己烷为洗脱剂，所述洗脱剂中乙酸乙酯和正己烷的体积比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。
得到第二中间产物后，本发明将所述第二中间产物与氨基化合物在有机溶剂和还原剂存在的条件下进行还原胺化反应，得到具有式I或式II所示结构的二醇型人参皂苷衍生物；在本发明中，所述第二中间产物与氨基化合物、还原剂和有机溶剂的质量比优选为1：(1～100)：(5～10)，更优选为1：(8～70)：(6～9)，更优选为1：(20～50)：(7～8)。
在本发明中，所述氨基化合物优选包括L-亮氨酸甲酯盐酸盐、L-缬氨酸甲酯盐酸盐、L-苏氨酸甲酯盐酸盐、L-丝氨酸甲酯盐酸盐、L-谷氨酸二甲酯盐酸盐、甘氨酸甲酯盐酸盐、β-丙氨酸乙酯盐酸盐、氨基丙二酸二乙酯盐酸盐、L-苯丙氨酸甲酯盐酸盐、L-酪氨酸甲酯、L-组氨酸甲酯盐酸盐或L-色氨酸甲酯盐酸盐。
在本发明中，所述还原剂优选包括硼氢化钠、氰基硼氢化钠或三乙酰基硼氢化钠。
本发明对于进行所述还原胺化反应所需的有机溶剂种类没有特殊的限定，采用本领域技术人员熟知的能够与所述第二中间产物和氨基化合物、还原剂相匹配的极性有机溶剂或非极性有机溶剂即可，如甲醇、乙酸乙酯、氯仿、二氯甲烷、四氯化碳。
在本发明中，所述还原剂与所述第二中间产物、氨基化合物和有机溶剂混合时的温度优选为-2～2℃。本发明对于控制所述还原剂与所述第二中间产物、氨基化合物和有机溶剂混合温度的方式没有特殊的限定，采用本领域技术人员熟知的控制温度的方式即可。在本发明的实施例中，具体在冰水浴条件下将所述还原剂与所述第二中间产物、氨基化合物和有机溶剂混合。在本发明中，将所述还原剂与所述第二中间产物、氨基化合物和有机溶剂混合优选在搅拌条件下进行。在本发明中，所述搅拌的速率优选为800～1200rpm，更优选为900～1100rpm。本发明对于所述搅拌的方式没有特殊的限定，采用本领域技术人员熟知的搅拌方式即可。本发明优选采用磁力搅拌。
在本发明中，所述还原胺化反应的温度优选为-10～80℃，更优选为0～55℃，最优选为20～35℃；具体的，在本发明的实施例中，所述还原胺化反应在室温下进行，无需对反应体系进行加热也无需进行冷却。在本发明中，所述还原胺化反应的时间优选为1～48h，更优选为8～32h，最优选为13～20h。
本发明优选在所述还原胺化反应完成后，对得到的产物进行后处理，得到具有式I或式II所示结构的二醇型人参皂苷衍生物。在本发明中，所述后处理优选包括以下步骤：
采用有机溶剂对所述还原胺化反应结束后得到的物料进行萃取，洗涤，除去溶剂，得到粗产品；采用硅胶柱层析法对所述粗产品进行提纯，得到具有式I或式II所示结构的二醇型人参皂苷衍生物。
在本发明中，所述还原胺化反应结束后得到的物料与萃取剂的质量比优选为1：(1～50)，更优选为1：(8～40)，最优选为1：(15～25)。在本发明中，所述萃取剂优选包括乙酸乙酯、氯仿、二氯甲烷或四氯化碳。在本发明中，所述萃取的次数优选为2～4次。在本发明中，具体是将萃取后得到的有机相进行洗涤。
本发明对于所述洗涤没有特殊的限定，采用本领域技术人员熟知的洗涤的技术方案即可。本发明优选采用氯化钠溶液洗涤所述萃取后得到的物料。本发明对于所述氯化钠溶液的浓度没有特殊的限定，采用本领域技术人员熟知的适用于进行洗涤的溶液的浓度即可。在本发明的实施例中，具体采用饱和氯化钠溶液进行所述洗涤。在本发明中，所述萃取后得到的物料与所述氯化钠溶液的体积比优选为1：(1～30)，更优选为1：(5～23)，最优选为1：(10～15)。 在本发明中，所述洗涤次数优选为3～5次。本发明对于所述除去溶剂的方法没有特殊的限定，采用本领域技术人员熟知的除去溶剂的技术方案即可。本发明优选采用减压蒸馏除去溶剂。
本发明对于所述硅胶柱层析法的具体操作步骤及所采用的试剂没有特殊的限定，采用本领域技术人员熟知的硅胶柱层析法即可。本发明优选将粗产品与乙酸乙酯混合，硅胶拌样后用于柱层析。在本发明中，所述粗产品与乙酸乙酯的质量比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。本发明优选采用乙酸乙酯和正己烷为洗脱剂，所述洗脱剂中乙酸乙酯和正己烷的体积比优选为1：(1～10)，更优选为1：(3～8)，最优选为1：(4～6)。
本发明提供了上述技术方案所述二醇型人参皂苷衍生物或上述技术方案所述制备方法得到的二醇型人参皂苷衍生物在制备防治动脉粥样硬化药物中的应用。在本发明中，所述防治动脉粥样硬化药物优选包括活性成分和辅料。在本发明中，所述防治动脉粥样硬化药物的活性成分为本发明上述技术方案所述二醇型人参皂苷衍生物或上述技术方案所述制备方法得到的二醇型人参皂苷衍生物。本发明对于所述辅料的种类没有特殊的限定，采用本领域技术人员熟知的辅料即可。本发明对于所述活性成分与辅料的配比没有特殊的限定，可根据实际情况确定所述活性成分与辅料的配比。本发明对于所述防治动脉粥样硬化药物的剂型和用量没有特殊的限定，可根据实际需要确定所述防治动脉粥样硬化药物的剂型和用量。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
将1g(16mmol)具有式IV所示结构的化合物与10mL乙酸酐、10mL吡啶混合，在40℃、900rpm磁力搅拌的条件下进行亲核取代反应3h；减压蒸馏，将得到的粗产品与6mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到1.3g白色固体的化合物1，收率90％。所述化合物1的结构为：
Figure PCTCN2017101880-appb-000023
实施例2
将1g(11mmol)实施例1制备的化合物1，5mL二氯甲烷，0.2g(11mmol)间氯过氧苯甲酸混合，在室温下进行初次氧化反应1h；向所述初次氧化反应结束后得到的物料中加25mL水，于1100rpm磁力搅拌1h，有白色固体析出，过滤得到澄清溶液，减压蒸馏除去溶剂，将得到的粗产品与8mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：8)，得到0.87g白色固体的化合物2，产率90％。所述化合物2的结构为：
Figure PCTCN2017101880-appb-000024
实施例3
将0.8g(9mmol)实施例2制备的化合物2，5mL四氢呋喃、0.32g(14mmol)高碘酸混合，在室温下进行二次氧化反应2h；采用乙酸乙酯对所述二次氧化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为40mL)，再依次用饱和碳酸氢钠溶液和饱和氯化钠溶液分别洗涤4次得到澄清溶液(每次洗涤所需饱和碳酸氢钠溶液的体积为50mL，饱和氯化钠溶液的体积为60mL)，减压蒸馏除去溶剂，将得到的粗产品与4mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：4)，得到0.73g白色固体的化合物3，产率92％。所述化合物3的结构为：
Figure PCTCN2017101880-appb-000025
实施例4
将0.05g(0.6mmol)实施例3制备的化合物3与0.013g(0.7mmol)L-亮氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、1000rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为10mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为30mL)，减压蒸馏除去溶剂，将得到的粗产品与6mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：7)，得到0.03g白色固体的目标化合物4，产率52％。所述目标化合物4的结构为：
Figure PCTCN2017101880-appb-000026
1H NMR(600MHz,cdcl3)δ5.90(t,1H),5.70(dd,1H),5.11(t,1H),4.87(m,2H),4.55(m,1H),4.41(m,1H),4.29(m,1H),4.19(m,1H),3.62(d,2H),3.05(m,1H),2.55(m,2H),2.283(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例5
将0.05g(0.6mmol)实施例3制备的化合物3与0.012g(0.7mmol)L-缬氨酸甲酯盐酸盐、2mL乙酸乙酯混合，在冰浴、800rpm磁力搅拌条件下加入0.05g(8mmol)硼氢化钠，在室温下进行还原胺化反应3h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为100mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为50mL)，减压蒸馏除去溶剂，将得到的粗产品与10mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：6)，得到0.03g白色固体的目标化合物5，产率60％。所述目标化合物5的结构为：
Figure PCTCN2017101880-appb-000027
1H NMR(600MHz,cdcl3)δ6.98(t,1H),5.22(m,3H),4.86(m,1H),4.69(dd,1H),4.50(m,1H),4.42(m,2H),4.27(m,1H),4.01(m,1H),3.66(s,3H),2.67(m,1H),2.58(m,2H),2.33(m,1H),2.283(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.98(s,3H),0.94(s,3H),0.96(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例6
将0.05g(0.6mmol)实施例3制备的化合物3与0.012g(0.7mmol)L-苏氨酸甲酯盐酸盐、2mL氯仿混合，在冰浴、900rpm磁力搅拌条件下加入0.05g(8mmol)三乙酰基硼氢化钠，在室温下进行还原胺化反应6h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为90mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为10mL)，减压蒸馏除去溶剂，将得到的粗产品与1mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：1)，得到0.02g白色固体的目标化合物6，产率40％。所述目标化合物6的结构为：
Figure PCTCN2017101880-appb-000028
1H NMR(600MHz,cdcl3)δ5.66(t,1H),5.37(m,2H),5.03(m,2H),4.42(s,2H),4.28(m,1H),4.09(m,1H),3.77(m,1H),3.66(s,3H),2.74(m,1H),2.57(m,2H),2.283(m,1H),2.062(m,2H),12.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.18(s,3H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例7
将0.05g(0.6mmol)实施例3制备的化合物3与0.011g(0.7mmol)L-丝氨酸甲酯盐酸盐、2mL二氯甲烷混合，在冰浴、1000rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应9h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为20mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为90mL)，减压蒸馏除去溶剂，将得到的粗产品与2mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：2)，得到0.02g白色固体的目标化合物7，产率40％。所述目标化合物7的结构为：
Figure PCTCN2017101880-appb-000029
1H NMR(600MHz,cdcl3)δ6.26(t,1H),5.37(t,1H),5.29(m,1H),4.95(m,2H),4.45(m,1H),4.28(m,2H),4.15(m,2H),3.77(m,1H),3.66(s,3H),3.25(t,1H),2.57(m,2H),2.283(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例8
将0.05g(0.6mmol)实施例3制备的化合物3与0.017g(0.7mmol)L-谷氨酸二甲酯盐酸盐、2mL四氯化碳混合，在冰浴、1100rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为30mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为80mL)，减压蒸馏除去溶剂，将得到的粗产品与3mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：3)，得到0.03g白色固体的目标化合物8，产率55％。所述目标化合物8的结构为：
Figure PCTCN2017101880-appb-000030
1H NMR(600MHz,cdcl3)δ6.68(t,1H),5.63(t,1H),5.06(m,1H),4.95(t,2H),4.82(m,1H),4.55(m,2H),4.26(t,1H),3.99(m,1H),3.66(s,3H),3.60(s,3H),3.55(t,1H),2.57(m,2H),2.35(m,2H),2.28(m,1H),2.22(m,1H),2.09(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例9
将0.05g(0.6mmol)实施例3制备的化合物3与0.009g(0.7mmol)甘氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、1200rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为40mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为70mL)，减压蒸馏除去溶剂，将得到的粗产品与4mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：4)，得到0.03g白色固体的目标化合物9，产率60％。所述目标化合物9的结构为：
Figure PCTCN2017101880-appb-000031
1H NMR(600MHz,cdcl3)δ6.66(t,1H),4.95-4.82(m,5H),4.55(m,1H),4.44(m,1H),4.28(t,1H),4.01(m,1H),3.72(s,3H),3.62(d,2H),2.57(m,2H),2.44(m,1H),2.283(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例10
将0.05g(0.6mmol)实施例3制备的化合物3与0.011g(0.7mmol)β-丙氨酸乙酯盐酸盐、2mL甲醇混合，在冰浴、1200rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为50mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为60mL)，减压蒸馏除去溶剂，将得到的粗产品与5mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到0.03g白色固体的目标化合物10，产率60％。所述目标化合物10的结构为：
Figure PCTCN2017101880-appb-000032
1H NMR(600MHz,cdcl3)δ5.16(t,1H),4.97(t,Hz,1H),4.89(dd,1H),4.66(d,1H),4.10–4.06(m,1H),4.04(dd,J＝12.3,5.3Hz,1H),3.60(ddd,1H),2.70(ddd,1H),2.37–2.29(m,1H),2.283(m,1H),2.14–2.08(m,2H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.07(m,3H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例11
将0.05g(0.6mmol)实施例3制备的化合物3与0.015g(0.7mmol)氨基丙二酸二乙酯盐酸盐、2mL甲醇混合，在冰浴、1100rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为40mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为70mL)，减压蒸馏除去溶剂，将得到的粗产品与4mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：4)，得到0.03g白色固体的目标化合物11，产率50％。所述目标化合物11的结构为：
Figure PCTCN2017101880-appb-000033
1H NMR(600MHz,cdcl3)δ5.16(t,1H),4.97(t,1H),4.89(dd,8.0Hz,1H),4.78(ddd,1H),4.66(d,1H),4.46(d,1H),4.11(m,5.3Hz,4H),4.09(m,2H),3.60(ddd,1H),2.77(ddd,1H),2.540(m,1H),2.283(m,1H),2.062(m,2H),2.00(m,18H),1.82(dd,1H),1.81(m,1H),1.74(t,1H),1.67(s,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.55(m,2H),1.49(m,2H),1.39(m,2H),1.37(m,1H),1.33(s,3H),1.25(m,2H),1.220(m,1H),1.07(m,2H),1.01(s,3H),0.96(s,3H),0.92(s,3H),0.91(s,3H),0.77(s,3H)。
实施例12
将0.05g(0.6mmol)实施例3制备的化合物3与0.01g(0.7mmol)L-苯丙氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、1100rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为30mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为80mL)，减压蒸馏除去溶剂，将得到的粗产品与3mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：3)，得到0.03g白色固体的目标化合物12，产率50％。所述目标化合物12的结构为：
Figure PCTCN2017101880-appb-000034
1H NMR(600MHz,cdcl3)δ7.31(t,1H),7.11(m,5H),5.32(m,1H),4.96(m,1H),4.79(m,2H),4.61(t,1H),4.42(m,1H),4.31(m,1H),4.16(dd,1H),4.06(t,1H),3.70(s,3H),3.32(m,1H),2.94(m,2H),2.55(m,2H),2.283(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例13
将0.05g(0.6mmol)实施例3制备的化合物3与0.014g(0.7mmol)L-酪氨酸甲酯、2mL甲醇混合，在冰浴、1000rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为20mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为90mL)，减压蒸馏除去溶剂，将得到的粗产品与2mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：2)，得到0.02g白色固体的目标化合物13，产率35％。所述目标化合物13的结构为：
Figure PCTCN2017101880-appb-000035
1H NMR(600MHz,cdcl3)δ7.01(d,2H),6.75(d,2H),6.04(m,1H),5.49(dd,1H),5.18(m,2H),4.56(m,1H),4.50(s,2H),4.37(m,1H),4.28(m,1H),4.18(m,1H),3.63(m,3H),3.25(s,1H),2.97(m,1H),2.57(t,2H),2.28(m,1H),2.06(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例14
将0.05g(0.6mmol)实施例3制备的化合物3与0.017g(0.7mmol)L-组氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、900rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为10mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为100mL)，减压蒸馏除去溶剂，将得到的粗产品与1mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：1)，得到0.03g白色固体的目标化合物14，产率51％。所述目标化合物14的结构为：
Figure PCTCN2017101880-appb-000036
1H NMR(600MHz,cdcl3)δ8.67(d,1H),7.63(d,1H),6.02(t,1H),5.90(dd,1H),4.99(m,1H),4.75(t,1H),4.55(m,1H),4.42(m,2H),4.26(m,1H),4.16(m,1H),3.66(s,3H),3.48(t,1H),3.14(m,1H),2.77(m,1H),2.55(m,2H),2.283(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例15
将0.05g(0.6mmol)实施例3制备的化合物3与0.015g(0.7mmol)L-色氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、800rpm磁力搅拌条件下加入0.05g(8mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为50mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为50mL)，减压蒸馏除去溶剂，将得到的粗产品与5mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到0.02g白色固体的目标化合物15，产率33％。所述目标化合物15的结构为：
Figure PCTCN2017101880-appb-000037
1H NMR(600MHz,cdcl3)δ7.26(dd,1H),6.85(ddd,1H),6.76(ddd,1H),6.67(ddd,1H),6.26(t,1H),5.15(t,1H),4.92(m,1H),4.55(dd,1H),4.44(m,1H),4.28(t,1H),4.11(m,2H),3.68(s,3H),3.52(m,2H),3.45(m,1H),3.37(m,1H),2.54(m,2H),2.48(m,1H),2.20(m,1H),2.283(m,1H),2.062(m,2H),2.00(ddd,18H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例16
将1g(21mmol)具有式V所示结构的化合物与10mL乙酸酐、10mL吡啶混合，在60℃、1000rpm磁力搅拌的条件下进行亲核取代反应3h；减压蒸馏，将得到的粗产品与5mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到1.1g白色固体的化合物16，收率90％。所述化合物16的结构为：
Figure PCTCN2017101880-appb-000038
实施例17
将1g(17mmol)实施例16制备的化合物16，5mL二氯甲烷，0.31g(11mmol)间氯过氧苯甲酸混合，在室温下进行初次氧化反应1h；向所述初次氧化反应结束后得到的物料中加40mL水，于1000rpm磁力搅拌2h，有白色固体析出，过滤得到澄清溶液，减压蒸馏除去溶剂，将得到的粗产品与5mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到0.92g白色固体的化合物17，产率90％。所述化合物17的结构为：
Figure PCTCN2017101880-appb-000039
实施例18
将0.9g(15mmol)实施例17制备的化合物17，5mL四氢呋喃、0.83g(32mmol)高碘酸混合，在室温下进行二次氧化反应2h；采用乙酸乙酯对所述二次氧化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为40mL)，再依次用饱和碳酸氢钠溶液和饱和氯化钠溶液分别洗涤4次得到澄清溶液(每次洗涤所需饱和碳酸氢钠溶液的体积为50mL，饱和氯化钠溶液的体积为50mL)，减压蒸馏除去溶剂，将得到的粗产品与5mL乙酸乙酯混合， 硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到0.71g白色固体的化合物18，产率85％。所述化合物18的结构为：
Figure PCTCN2017101880-appb-000040
实施例19
将0.033g(0.6mmol)实施例18制备的化合物18与0.013g(0.7mmol)L-亮氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、800rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为10mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为100mL)，减压蒸馏除去溶剂，将得到的粗产品与10mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：10)，得到0.03g白色固体的目标化合物19，产率72％。所述目标化合物19的结构为：
Figure PCTCN2017101880-appb-000041
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),3.65(s,3H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例20
将0.033g(0.6mmol)实施例18制备的化合物18与0.012g(0.7mmol)L-缬氨酸甲酯盐酸盐、2mL乙酸乙酯混合，在冰浴、1000rpm磁力搅拌条件下加入0.033g(5.3mmol)硼氢化钠，在室温下进行还原胺化反应3h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为20mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为90mL)，减压蒸馏除去溶剂，将得到的粗产品与9mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：9)，得到0.03g白色固体的目标化合物20，产率75％。所述目标化合物20的结构为：
Figure PCTCN2017101880-appb-000042
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),3.65(s,3H),2.88(m,1H),2.56(t,2H),2.00 (ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.98(s,3H),0.94(s,3H),0.96(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例21
将0.033g(0.6mmol)实施例18制备的化合物18与0.012g(0.7mmol)L-苏氨酸甲酯盐酸盐、2mL氯仿混合，在冰浴、1100rpm磁力搅拌条件下加入0.033g(5.3mmol)三乙酰基硼氢化钠，在室温下进行还原胺化反应6h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为30mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为80mL)，减压蒸馏除去溶剂，将得到的粗产品与8mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：8)，得到0.04g白色固体的目标化合物21，产率50％。所述目标化合物21的结构为：
Figure PCTCN2017101880-appb-000043
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),3.65(s,3H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例22
将0.033g(0.6mmol)实施例18制备的化合物18与0.011g(0.7mmol)L-丝氨酸甲酯盐酸盐、2mL二氯甲烷混合，在冰浴、1200rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应9h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为40mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为70mL)，减压蒸馏除去溶剂，将得到的粗产品与7mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：7)，得到0.028g白色固体的目标化合物22，产率73％。所述目标化合物22的结构为：
Figure PCTCN2017101880-appb-000044
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),3.65(s,3H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例23
将0.033g(0.6mmol)实施例18制备的化合物18与0.017g(0.7mmol)L-谷氨酸二甲酯盐酸盐、2mL四氯化碳混合，在冰浴、1200rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为50mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为60mL)，减压蒸馏除去溶剂，将得到的粗产品与6mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：6)，得到0.028g白色固体的目标化合物23，产率67％。所述目标化合物23的结构为：
Figure PCTCN2017101880-appb-000045
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),3.65(s,3H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例24
将0.033g(0.6mmol)实施例18制备的化合物18与0.009g(0.7mmol)甘氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、1100rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为60mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为50mL)，减压蒸馏除去溶剂，将得到的粗产品与5mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到0.026g白色固体的目标化合物24，产率70％。所述目标化合物24的结构为：
Figure PCTCN2017101880-appb-000046
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),3.65(s,3H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例25
将0.033g(0.6mmol)实施例18制备的化合物18与0.011g(0.7mmol)β-丙氨酸乙酯盐酸盐、2mL甲醇混合，在冰浴、1000rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为70mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶 液(每次洗涤所需饱和氯化钠溶液的体积为40mL)，减压蒸馏除去溶剂，将得到的粗产品与4mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：4)，得到0.025g白色固体的目标化合物25，产率65％。所述目标化合物25的结构为：
Figure PCTCN2017101880-appb-000047
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),3.92(t,2H),2.93(t,2H),2.56(t,2H),2.46(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例26
将0.033g(0.6mmol)实施例18制备的化合物18与0.015g(0.7mmol)氨基丙二酸二乙酯盐酸盐、2mL甲醇混合，在冰浴、900rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为80mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为30mL)，减压蒸馏除去溶剂，将得到的粗产品与3mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：3)，得到0.026g白色固体的目标化合物26，产率60％。所述目标化合物26的结构为：
Figure PCTCN2017101880-appb-000048
1H NMR(600MHz,cdcl3)δ4.95(m,1H),4.25(t,1H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.07(m,3H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例27
将0.033g(0.6mmol)实施例18制备的化合物18与0.01g(0.7mmol)L-苯丙氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、800rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为90mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为20mL)，减压蒸馏除去溶剂，将得到的粗产品与2mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：2)，得到0.029g白色固体的目标化合物27，产率70％。所述 目标化合物27的结构为：
Figure PCTCN2017101880-appb-000049
1H NMR(600MHz,cdcl3)δ7.31(t,1H),7.11(m,5H),δ4.95(m,1H),4.25(t,1H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例28
将0.033g(0.6mmol)实施例18制备的化合物18与0.014g(0.7mmol)L-酪氨酸甲酯、2mL甲醇混合，在冰浴、1000rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为100mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为10mL)，减压蒸馏除去溶剂，将得到的粗产品与1：1mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：1)，得到0.024g白色固体的目标化合物28，产率55％。所述目标化合物28的结构为：
Figure PCTCN2017101880-appb-000050
1H NMR(600MHz,cdcl3)δ7.01(d,2H),6.75(d,2H),δ4.95(m,1H),4.25(t,1H),3.66(s,3H),3.50(t,1H),3.25(m,1H),3.05(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例29
将0.033g(0.6mmol)实施例18制备的化合物18与0.017g(0.7mmol)L-组氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、1000rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为50mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为50mL)，减压蒸馏除去溶剂，将得到的粗产品与5mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到0.025g白色固体的目标化合物29，产率50％。所述目标化合物29的结构为：
Figure PCTCN2017101880-appb-000051
1H NMR(600MHz,cdcl3)δ8.73(d,1H),7.65(d,1H),4.95(m,1H),4.25(t,1H),3.45(t,2H),3.98(dd,1H),2.82(dd,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例30
将0.033g(0.6mmol)实施例18制备的化合物18与0.015g(0.7mmol)L-色氨酸甲酯盐酸盐、2mL甲醇混合，在冰浴、1000rpm磁力搅拌条件下加入0.033g(5.3mmol)氰基硼氢化钠，在室温下进行还原胺化反应1h；采用乙酸乙酯对所述还原胺化反应结束后得到的物料萃取3次(每次萃取所需乙酸乙酯的体积为50mL)，再用饱和氯化钠溶液洗涤4次得到澄清溶液(每次洗涤所需饱和氯化钠溶液的体积为50mL)，减压蒸馏除去溶剂，将得到的粗产品与5mL乙酸乙酯混合，硅胶拌样后用于柱层析，以乙酸乙酯和正己烷的混合物为洗脱剂(乙酸乙酯和正己烷的体积比为1：5)，得到0.02g白色固体的目标化合物30，产率45％。所述目标化合物30的结构为：
Figure PCTCN2017101880-appb-000052
1H NMR(600MHz,cdcl3)δ7.26(dd,1H),6.85(ddd,1H),6.76(ddd,1H),6.67(ddd,1H),δ4.95(t,1H),4.25(m,1H),2.88(m,1H),2.56(t,2H),2.00(ddd,9H),1.92(m,2H),1.85(m,2H),1.82(m,1H),1.81(m,1H),1.74(m,1H),1.67(m,3H),1.65(m,3H),1.63(m,3H),1.53(m,2H),1.49(m,2H),1.39(m,1H),1.33(m,3H),1.26(m,1H),1.21(m,1H),1.20(m,2H),1.11(s,3H),1.09(m,1H),1.03(m,1H),0.94(s,3H),0.92(s,3H),0.85(m,1H),0.84(s,3H),0.82(s,3H)。
实施例31
对本发明实施例1～30制备的化合物的毒性进行测试，具体步骤如下：
取昆明小鼠(18-22g，雄性，购自第三军医大学实验动物中心)186只，随机平均分为31组，将本发明实施例1～30制备的化合物作用于所述昆明小鼠(一次性灌胃500mg/kg)，并设置生理盐水对照组(1mL)，连续观察14天。
结果：各实验组昆明小鼠未见死亡且生存状态良好，食量和毛色正常，体重与生理盐水对照组相比较无显著差异。
为了进一步验证本发明实施例1～30制备的化合物的毒性，取小鼠巨噬细胞系RAW264.7细胞培养，将发明实施例1～30制备的化合物使用培养基配制成浓度为10μmol/L，其中溶剂为生理盐水，助溶剂为二甲基亚砜(DMSO，终浓度为0.1％)，并设置一组0.1％DMSO的生理盐水对照组。采用CCK8法测定细胞生存率，数据以x±s表示，组间用SPSS 10.0行单 因素方差分析，以p<0.05为相差显著，结果如表1所示。由表1可知，化合物1、2和3具有显著的细胞毒性，其它化合物在该浓度无显著细胞毒性。
表1.实施例1～30制备的化合物对RAW264.7细胞存活率的影响(
Figure PCTCN2017101880-appb-000053
n＝3)
Figure PCTCN2017101880-appb-000054
注：a与生理盐水对照组相比p<0.05
实施例32
本发明实施例1～30制备的化合物对高脂饮食apoE-/-小鼠动脉粥样硬化斑块形成面积占整条动脉面积百分比以及低密度脂蛋白胆固醇、高密度脂蛋白水平的影响，具体步骤如下：
1.动物：10周龄健康清洁级apoE-/-小鼠(购自北京大学实验动物中心)160只，雄性，体重22～25g，于无菌层流饲养室中分笼饲养，自由饮水摄食。以高脂饮食(常规饲料+0.15％ 胆固醇+21％猪油)饲养共25周，保持室温24℃，相对湿度50％，光照时间7:30～19:30。
2.分组、给药方法：
饲养10周后按体重以均衡随机法将动物分为以下32组(n＝5)，给药15周后处死。
生理盐水对照组：生理盐水灌胃
阳性对照组：辛伐他汀10mg/(kg·d)灌胃
本发明实施例1～30制备的化合物组(共30组)：各30mg/(kg·d)灌胃
3.检测指标与方法：
①动脉粥样硬化斑块面积及其占总面积百分比分析：处死动物后分离主动脉，位置从主动脉根部至腹主动脉末端分叉处。经福尔马林固定后用苏丹Ⅳ染色，红染部分即为动脉粥样硬化病变区域。利用Image Pro Plus 5.0软件计算病变部分面积占总面积的百分比。
②总胆固醇(TC)、低密度胆固醇脂蛋白(LDL-C)及高密度脂蛋白胆固醇(HDL-C)水平测定：利用ROCHE 7060全自动生化分析仪进行检测。
③TNF-α水平测定：采用Millipore公司液相芯片法测定(Mcytomag-70K-3,Mouse Cytokine/Chemokine Magnetic Bead Panel)。
4.统计学分析方法：
数据以
Figure PCTCN2017101880-appb-000055
表示，组间用SPSS 10.0行单因素方差分析，以p<0.05为相差显著。
5.结果：
①如表2所示，本发明实施例1～30制备的化合物均能显著减少apoE-/-小鼠动脉粥样硬化斑块面积的百分比，与对照组比较有统计学差异。
表2.实施例1～30制备的化合物对apoE-/-小鼠动脉粥样硬化斑块面积占主动脉总面积百分比的影响(
Figure PCTCN2017101880-appb-000056
n＝5)
分组 斑块面积/主动脉总面积(％)
生理盐水对照组 14.67±3.58
辛伐他汀 7.95±2.03a
化合物1 10.65±2.62a
化合物2 8.27±2.54a
化合物3 9.74±3.19a
化合物4 9.52±4.21a
化合物5 10.29±2.67a
化合物6 8.16±2.83a
化合物7 11.02±2.71a
化合物8 9.31±3.58a
化合物9 10.55±2.19a
化合物10 8.52±3.25a
化合物11 9.68±3.52a
化合物12 10.23±3.54a
化合物13 9.44±3.24a
化合物14 9.34±3.01a
化合物15 10.22±2.19a
化合物16 10.41±2.09a
化合物17 8.53±2.57a
化合物18 9.15±2.85a
化合物19 8.34±2.41a
化合物20 9.67±2.98a
化合物21 8.28±3.29a
化合物22 10.32±2.74a
化合物23 8.64±2.46a
化合物24 9.54±2.65a
化合物25 8.33±2.44a
化合物26 9.78±3.17a
化合物27 10.65±3.82a
化合物28 8.18±2.89a
化合物29 9.67±2.51a
化合物30 9.99±3.68a
注：a与对照组相比p<0.05
②如表3所示，本发明实施例1～30制备的化合物均能显著升高HDL-C水平，部分化合物能显著降低小鼠血清中LDL-C水平，与对照组比较有统计学差异。
表3.实施例1～30制备的化合物对apoE-/-小鼠血脂的影响(mmol/L，
Figure PCTCN2017101880-appb-000057
n＝5)
分组 LDL-C HDL-C
生理盐水对照组 7.33±1.03 3.74±0.62
辛伐他汀 2.75±1.89a 3.69±0.72a
化合物1 6.39±1.09 4.55±0.86a
化合物2 5.56±1.24a 5.47±0.91a
化合物3 6.12±1.28 4.97±0.95a
化合物4 4.62±1.58a 5.48±0.61a
化合物5 5.58±1.89a 5.21±0.73a
化合物6 6.04±1.07 5.51±0.94a
化合物7 5.50±1.82a 4.52±0.46a
化合物8 4.34±1.19a 5.63±0.84a
化合物9 6.84±0.85 4.41±0.67a
化合物10 6.54±1.06 4.50±0.94a
化合物11 6.59±1.56 4.93±0.58a
化合物12 5.27±1.74a 4.44±0.83a
化合物13 5.07±0.99a 5.29±1.18a
化合物14 5.58±1.11a 4.39±0.59a
化合物15 6.54±1.36 4.12±0.82a
化合物16 5.18±1.26a 4.12±0.90a
化合物17 4.83±1.51a 5.55±0.56a
化合物18 6.15±1.28 4.72±0.91a
化合物19 4.62±1.34a 5.13±0.83a
化合物20 6.62±0.89 4.72±0.52a
化合物21 4.31±1.84a 5.82±0.95a
化合物22 6.31±1.17 4.29±0.84a
化合物23 4.28±1.46a 5.39±0.61a
化合物24 5.65±1.04a 4.68±0.67a
化合物25 4.82±1.54a 5.14±0.87a
化合物26 6.28±0.84 4.45±0.62a
化合物27 5.55±1.06a 4.37±0.56a
化合物28 4.59±1.62a 5.55±0.63a
化合物29 6.27±1.59 4.10±0.78a
化合物30 6.37±0.75 4.42±0.57a
注：a与对照组相比p<0.05
③如表4所示，本发明实施例1～30制备的化合物能够显著降低apoE-/-小鼠动脉局部TNF-α水平，与对照组比较有统计学差异，说明本发明实施例1～30制备的化合物具有较好的抗炎作用。
表4.实施例1～30制备的化合物对apoE-/-小鼠动脉局部TNF-α水平的影响(
Figure PCTCN2017101880-appb-000058
n＝5)
分组 TNF-α水平(ng/g)
生理盐水对照组 25.58±6.44
辛伐他汀 14.84±3.22a
化合物1 19.41±3.81a
化合物2 13.24±3.71a
化合物3 16.68±4.98a
化合物4 11.04±3.68a
化合物5 17.73±4.87a
化合物6 11.35±4.97a
化合物7 17.77±4.63a
化合物8 12.67±3.11a
化合物9 12.85±4.17a
化合物10 11.66±2.65a
化合物11 12.33±5.81a
化合物12 18.27±5.24a
化合物13 15.65±6.74a
化合物14 16.92±5.04a
化合物15 16.07±3.67a
化合物16 15.94±4.29a
化合物17 15.27±4.65a
化合物18 16.65±4.57a
化合物19 11.23±3.69a
化合物20 15.98±5.33a
化合物21 10.76±3.54a
化合物22 18.14±6.82a
化合物23 12.61±3.74a
化合物24 15.47±2.88a
化合物25 11.65±5.45a
化合物26 16.02±3.11a
化合物27 17.95±2.15a
化合物28 11.84±2.91a
化合物29 14.93±3.67a
化合物30 16.11±4.19a
注：a与对照组相比p<0.05
实施例33
本发明实施例1～30制备的化合物对RAW264.7细胞源泡沫细胞形成的影响，具体步骤如下：
1.细胞培养及泡沫细胞模型建立：
将RAW264.7细胞于高糖DMEM培养基(GIBCO)中进行培养至细胞浓度1×106，种植于6孔板中，加入60mg/L的ox-LDL(氧化修饰型低密度脂蛋白，北京协和医科大学)孵育48h后获得泡沫细胞。
2.分组及指标检测：
将获得的泡沫细胞每3个孔板为1组，分为61组，加入药物(100μL/孔)处理48h后，弃培养基，用油红O染色后，洗去多余的油红O，将细胞用异丙醇溶解后在490nm处测定吸光度值。以模型组OD值为100％，将用细胞数矫正过后的各组OD值与模型组相比，得到细胞内脂质含量的比值，该值越大则说明细胞内脂质越多，泡沫细胞形成程度越高。
生理盐水对照组：生理盐水
本发明实施例1～30制备的化合物的低剂量组：10μmol/L(溶剂为生理盐水)
本发明实施例1～30制备的化合物的高剂量组：30μmol/L(溶剂为生理盐水)
3.统计学分析方法：
数据以
Figure PCTCN2017101880-appb-000059
表示，组间用SPSS10.0统计学软件行单因素方差分析，以p<0.05为相差显著。
4.结果：本发明实施例1～30制备的化合物在30μmol/L剂量下均能够显著降低泡沫细胞形成的程度，部分化合物在10μmol/L剂量下能够显著降低泡沫细胞形成的程度。
表5.本发明实施例1～30制备的化合物对RAW264.7源性泡沫细胞形成程度的影响(％，
Figure PCTCN2017101880-appb-000060
n＝3)
Figure PCTCN2017101880-appb-000061
Figure PCTCN2017101880-appb-000062
Figure PCTCN2017101880-appb-000063
注：a与对照组相比p<0.05
由以上实施例可以看出，本发明提供的二醇型人参皂苷衍生物细胞毒性小，能够显著减少apoE-/-小鼠动脉粥样硬化斑块面积的百分比；同时能够有效降低小鼠血清中低密度脂蛋白胆固醇水平，提高高密度脂蛋白胆固醇水平；能够显著降低apoE-/-小鼠动脉局部TNF-α水平，具有较好的抗炎作用；在30μmol/L剂量下能够显著降低RAW264.7细胞源泡沫细胞形成的程度。这说明本发明提供的二醇型人参皂苷衍生物能够作为活性组分，应用于制备防治动脉粥样硬化药物。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 二醇型人参皂苷衍生物，具有式I或式II所示结构：
    Figure PCTCN2017101880-appb-100001
    式I和式II中，所述R1为
    Figure PCTCN2017101880-appb-100002
    其中，R3为C1～C4的烷基；
    所述R2具有式III所示结构：
    Figure PCTCN2017101880-appb-100003
    式III中，所述n＝0、1或2，所述R4为甲基或乙基，所述R5为氢原子、取代或未取代的C1～C5的烷基、取代或未取代的苄基和C4～C9的杂环烷基中的一种。
  2. 根据权利要求1所述的二醇型人参皂苷衍生物，其特征在于，所述R5包括C1～C5的烷基、C1～C5的羟烷基或C3～C5的酯烷基。
  3. 根据权利要求2所述的二醇型人参皂苷衍生物，其特征在于，所述R5包括-CH2CH(CH3)2、-CH(CH3)2、-CH(OH)CH3、-CH2OH、-CH2CH2COOCH2CH3或-CH2COOCH2CH3。
  4. 根据权利要求1所述的二醇型人参皂苷衍生物，其特征在于，所述R5包括
    Figure PCTCN2017101880-appb-100004
  5. 根据权利要求1～4任一项所述的二醇型人参皂苷衍生物，其特征在于，所述R2包括
    Figure PCTCN2017101880-appb-100005
  6. 根据权利要求1所述的二醇型人参皂苷衍生物，其特征在于，所述R1包括
    Figure PCTCN2017101880-appb-100006
    Figure PCTCN2017101880-appb-100007
  7. 权利要求1～6任一项所述二醇型人参皂苷衍生物的制备方法，包括以下步骤：
    (1)将母体化合物与酸酐在碱性试剂存在的条件下进行亲核取代反应，得到第一中间产物；
    (2)将所述步骤(1)中第一中间产物在氧化剂和有机溶剂存在的条件下进行氧化反应，得到第二中间产物；
    (3)将所述步骤(2)中第二中间产物与氨基化合物在有机溶剂和还原剂存在的条件下进行还原胺化反应，得到具有式I或式II所示结构的二醇型人参皂苷衍生物；
    其中，所述步骤(1)中母体化合物具有式IV或V所示结构：
    Figure PCTCN2017101880-appb-100008
  8. 根据权利要求7所述的制备方法，其特征在于，步骤(2)所述氧化反应具体为：
    (21)将所述步骤(1)中第一中间产物在第一氧化剂和有机溶剂存在的条件下进行初次氧化反应，得到第二中间产物前体；
    (22)将所述步骤(21)中第二中间产物前体在第二氧化剂和有机溶剂存在的条件下进行二次氧化反应，得到第二中间产物。
  9. 根据权利要求8所述的制备方法，其特征在于，步骤(21)所述第一氧化剂包括过氧化氢、次氯酸、次氯化钙、过氧丙酮或间氯过氧苯甲酸；步骤(22)所述第二氧化剂包括高锰酸钾、二氧化锰、高碘酸或沙瑞特试剂。
  10. 权利要求1～6任一项所述二醇型人参皂苷衍生物或权利要求7～9任意一项所述制备方法得到的二醇型人参皂苷衍生物在制备防治动脉粥样硬化药物中的应用。
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