WO2016101733A1 - 连翘脂素葡萄糖醛酸衍生物、制备方法及其应用 - Google Patents

连翘脂素葡萄糖醛酸衍生物、制备方法及其应用 Download PDF

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WO2016101733A1
WO2016101733A1 PCT/CN2015/094864 CN2015094864W WO2016101733A1 WO 2016101733 A1 WO2016101733 A1 WO 2016101733A1 CN 2015094864 W CN2015094864 W CN 2015094864W WO 2016101733 A1 WO2016101733 A1 WO 2016101733A1
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reaction
forsythiaside
glycosidation
virus
glucuronic acid
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French (fr)
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富力
樊宏宇
姜人武
王凯乾
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富力
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • the present invention belongs to the field of medicinal chemistry, and in particular, to the forsythiaside glucuronic acid derivative, a process for the preparation thereof, and a pharmacological action of such a derivative in antiviral action.
  • Forsythia saponin which is the glycosidic part of forsythin, also known as forsythin, is the main active component of forsythia of Forsythia genus, and its structure is shown in the following formula. Modern pharmacological studies show that even The psygoxin has anti-virus, anti-oxidation, blood lipid lowering, free radical scavenging, antibacterial, anti-tumor, anti-inflammatory and the like.
  • Forsythias molecules are unstable, easily oxidized, and the molecular configuration is susceptible to change in an acidic environment.
  • the metabolism of forsythin in rats by intestinal bacteria was found to be easily metabolized into new metabolites by the intestinal flora.
  • the drug containing phenolic hydroxyl structure is easily metabolized by glucuronidase in vivo to the glucuronic acid derivative of phenol, and most of the glucuronic acid derivatives have good activity.
  • Efficient Preparations of the ⁇ -Glucuronides of Dihydroartemisinin and Structural Confirmation of the Human Glucuronide Metabolite Efficient Preparations of the ⁇ -Glucuronides of Dihydroartemisinin and Structural Confirmation of the Human Glucuronide Metabolite.
  • the technical problem to be solved by the present invention is to prepare a forsythin glucuronic acid derivative by a chemical synthesis method.
  • the forsythiaside glucuronic acid derivative provided by the present invention provides a method for preparing a forsythiaside glucuronic acid derivative, which is suitable for industrial scale-up production.
  • the present invention provides a forsythin glucuronic acid derivative represented by the formula (I),
  • the R group selects H, Me, Na+, K+.
  • Another aspect of the present invention provides a method for preparing a forsythin glucuronic acid derivative, comprising the steps of:
  • reaction temperature of the hydroxyl group activation reaction in the step 1) is 0-20 ° C, preferably 0-10 ° C; and the reaction time is 10-20 min.
  • step 1) forsythiaside and an organic base are dissolved in an organic solvent, and the hydroxyl group activation reaction is carried out under stirring to obtain a forsythiaside-organic base complex.
  • the organic solvent is selected from one of dichloromethane, toluene or pyridine, preferably dichloromethane.
  • the organic solvent is selected from one of anhydrous dichloromethane, anhydrous toluene or anhydrous pyridine, preferably anhydrous dichloromethane.
  • the ratio (g/ml) of the mass of forsythin to the volume of the organic solvent is from 1:40 to 50, preferably 1:45.
  • organic base is selected from N,N-diisopropylethylamine or 1,8-diazabicycloundec-7-ene.
  • the molar ratio of the forsythiaside to the organic base is from 1:5 to 8, preferably 1:6.
  • the temperature of the glycosidation reaction in the step 2) is >0 ° C, preferably 0-20 ° C, preferably 5-15 ° C, further preferably 10 ° C; the reaction time is 4-15 h, preferably 8-10 h, More preferably, it is 10h.
  • the glycosidation reaction is carried out under an inert gas atmosphere.
  • the inert gas is selected from nitrogen, argon or helium, preferably nitrogen.
  • the molar ratio of the glycosyl donor to forsythiaside is from 1 to 7:1, preferably from 1.5 to 7:1, further preferably from 1.5:1.
  • the glycosyl donor in step 2) is selected from 2,3,4,-tri-O-acyl- ⁇ -D-glucopyranuronate, preferably 2,3,4-tri-O- Methyl benzoyl- ⁇ -D-bromopyranuronic acid or methyl 2,3,4-tri-O-acetyl- ⁇ -D-bromopyranuronic acid; selection of the organic solvent Dichloromethane, chloroform, 1,2-dichloroethane or toluene, preferably dichloromethane.
  • the glycosyl donor 2,3,4,-tri-O-acyl- ⁇ -D-glucopyranuronic acid ester is used in a small amount, and the yield of the glycosidation product is low, and The increase in the amount leads to an increase in by-products, and the molar ratio of the glycosyl donor 2,3,4,-tri-O-acyl- ⁇ -D-glucopyranuronic acid ester to forsythiaside is preferably 1.5 to 2.5:1. .
  • the catalyst in step 2) selects silver carbonate.
  • the molar ratio of the catalyst to the glycosyl donor is from 4 to 8:1, preferably from 4 to 6:1, further preferably 6:1.
  • the low amount of catalyst leads to the decomposition of the glycosyl donor 2,3,4,-tri-O-acyl- ⁇ -D-glucopyranuronic acid ester, which reduces the yield; the high amount of the catalyst leads to the triacyl forsythiaside Decomposition of methyl glucuronide (i.e., glycosidation reaction product) reduces the yield.
  • the glycosyl donor and the catalyst are added to an organic solvent under an inert gas atmosphere at a temperature of -20 to 0 ° C or lower, and uniformly mixed to obtain the glycosidation reagent.
  • the glycosyl donor and the catalyst are added to an organic solvent under the protection of an inert gas at a temperature of -10 to 0 ° C, and uniformly mixed to obtain the glycosidation reagent; the temperature is preferably 0 °C.
  • the forsythiaside-organic base complex is added to the glycosidation reagent under the protection of an inert gas at -20 to 0 ° C or lower, and then the glycosidation reaction is carried out.
  • the temperature is preferably 0 ° C .
  • the invention prepares the glycosidation reagent under the condition of lower temperature and inert gas protection and adds the forsythiaside-organic base complex to the glycosidation reagent to prevent the catalyst from deactivating, and the low temperature ensures that the reaction is reacted according to the SN2 route to ensure the reaction.
  • the reaction selectivity of the catalyst increases the selectivity of the reaction.
  • the inert gas is selected from nitrogen, argon or helium, preferably nitrogen.
  • the reaction temperature of the deacylation reaction in the step 3) is 0 to 10 ° C, preferably 0 to 5 ° C, and more preferably 0 ° C.
  • the basic compound described in the step 3) is selected from sodium hydroxide, potassium hydroxide or sodium methoxide; and the pH adjuster is selected from acetic acid, propionic acid or hydrochloric acid, preferably acetic acid.
  • the molar ratio of the basic compound to the glycosyl donor in step 2) is from 3-5:1, preferably to 3.5:1.
  • the prepared basic compound solution has a mass percentage concentration of 27-30%.
  • the pH of the solution mixture is adjusted to a pH of 5-8, preferably a pH of 7.
  • it also includes the step 4) after adjusting the pH of the mixture solution, separating and purifying the mixture.
  • the separation and purification treatment is performed by silica gel column chromatography on the mixture.
  • the structural formula of the compound of the present invention prepared by the above method is a forsythiaside glucuronic acid derivative as follows:
  • the forsythiaside glucuronic acid derivative is a white solid at room temperature and is soluble in chloroform and ethanol. After spraying on a TLC plate (chromic solution was chloroform/methanol 10:1, Rf was 0.4), a 10% H 2 SO 4 -ethanol reagent was sprayed to give a purple-red color.
  • the antiviral is influenza virus, anti-parainfluenza virus, anti-respiratory syncytial virus (RSV), anti-coxsackie virus B3 (CVB3), anti-coxsackie virus A16 (CoxA16), anti-enteric virus EV71, Anti-adenovirus (AdV), anti-herpes simplex virus type I (HSV-1).
  • RSV anti-parainfluenza virus
  • RSV anti-respiratory syncytial virus
  • CVB3 anti-coxsackie virus B3
  • CoxA16 anti-coxsackie virus A16
  • AdV Anti-adenovirus
  • HSV-1 anti-herpes simplex virus type I
  • Yet another aspect of the present invention provides the use of a forsythiaside glucuronic acid derivative for the preparation of an antiviral drug or a health care product.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the forsythiaside glucuronic acid derivative of the present invention, and a pharmaceutically acceptable excipient.
  • a pharmaceutically acceptable excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, carrier, pH adjuster, ionic strength modifier, sustained release or controlled release agent, encapsulating material or other formulation excipient.
  • the carrier used may be adapted to the corresponding administration form, and may be formulated into an injection, a lyophilized powder for injection, a spray, an oral solution, an oral suspension, a tablet, a capsule, or the like, which is known to those skilled in the art.
  • Formulations such as enteric-coated tablets, pills, powders, granules, sustained release or delayed release.
  • the forsythiaside sulfate derivative of the first aspect of the invention is administered by injection or by digestive tract, and therefore, the pharmaceutical composition of the present invention is preferably an injection or a preparation for administration via the digestive tract, that is, it is suitable for formulation.
  • Excipients for injection and administration by the digestive tract are particularly preferred.
  • administered by the digestive tract refers to a mode of administration of a pharmaceutical preparation through the digestive tract of a patient, including oral, intragastric, and enema administration, preferably oral, as may be known to those skilled in the art.
  • the auxiliary material is formulated into an oral solution, an oral suspension, a tablet, a capsule, an enteric-coated tablet, a pill, a powder, a granule, a sustained release or a delayed release release; wherein the preparation for injection is mainly an injection and Powder injection.
  • the advanced and practical value of the method for chemically synthesizing forsythiaglycine glucuronic acid according to the present invention is that the source of the raw material is convenient, the catalyst for glycosidation reaction is cheap and easy to obtain, the preparation cost is significantly reduced, and the chemical synthesis product forsythiaside is chemically synthesized.
  • the quality of glucuronic acid derivatives is easy to control, and the comprehensive yield of products is high, which is suitable for industrial large-scale production.
  • Figure 1 is a nuclear magnetic resonance spectrum of forsythiaside methyl glucuronide of the present invention
  • Figure 2 is a nuclear magnetic resonance carbon spectrum of the forsythiamin glucuronide methyl ester of the present invention
  • Figure 3 is a high resolution mass spectrum of forsythiaside methyl glucuronide of the present invention.
  • Figure 4 is a pathological section of lung tissue of influenza virus pneumonia model mice, wherein: A is normal mouse lung tissue; B is influenza virus pneumonia mouse lung tissue; C is influenza virus pneumonia mouse model positive drug ribavirin Lung tissue of mice after treatment; D is the lung tissue of mice treated with high dose of forsythiaside glucuronide in the treatment of influenza virus pneumonia; E is the middle dose of forsythiaside glucuronide in the treatment of influenza Mouse lung tissue after viral pneumonia; F is the lung tissue of mice treated with low dose of forsythiaside glucuronide in the treatment of influenza virus pneumonia;
  • Figure 5 is a pathological section of lung tissue of a parainfluenza virus pneumonia model mouse, wherein: A is normal mouse lung tissue; B is parainfluenza virus pneumonia mouse lung tissue; C is parainfluenza virus pneumonia mouse model positive drug Liba The lung tissue of the treated mice after Weilin; D Lung for the treatment of parainfluenza pneumonia in the high dose group of forsythiaside glucuronide; E is the lung tissue of the middle dose group of forsythiaside glucuronide in the treatment of parainfluenza virus pneumonia F is the lung tissue of mice after treatment of parainfluenza pneumonia in a low-dose group of forsythiaside glucuronide.
  • the invention is further described by the following examples, which are intended to illustrate the invention and are not to be construed as limiting.
  • the reagents and raw materials in the examples can be obtained through commercial channels. If there are any defects, the organic synthesis guide, the guidelines of the drug regulatory agency, and the manufacturer's instructions of the corresponding instruments and reagents can be referred to.
  • the present invention is applicable to the present invention under the condition that the reaction temperature is controlled at 0-20 ° C during the hydroxy activation reaction, and the embodiment of the present invention is exemplified by 10 ° C.
  • the molar ratio of forsythia sulphate to organic base is in addition to 1:6, and other molar ratios of 1:5-8 are suitable for use in the present invention.
  • the embodiment of the present invention is described by taking 1:6 as an example.
  • the hydroxyl activation reaction is to activate the hydroxyl group with a base to make the glycosidation reaction easier.
  • the organic base takes away the hydrogen of the hydroxyl group of the reaction substrate forsylin, the resulting active intermediate complex is oxidized by encountering oxygen, and the inert gas protection causes the intermediate to lose contact with oxygen to ensure the normal reaction.
  • Inert gases other than nitrogen, helium and argon are suitable for use in the present invention.
  • the glycosidation reaction mixture was dissolved in methanol under stirring, and then a sodium hydroxide solution (8.36 ml) having a mass percentage of 27% was added while stirring at 0 ° C, NaOH and 2, 3, 4 - 3 -O-acetyl ⁇ -D bromoglucopyranonic acid methyl ester in a molar ratio of 3.5:1, after deacylation reaction for 30 min, adding acidic pH adjuster acetic acid to adjust the deacylation reaction
  • the pH of the mixture is up to 7;
  • methanol as a solvent is added to dissolve the glycosidation reaction mixture; the alkalinity of the added base (such as sodium hydroxide, potassium hydroxide, sodium methoxide) does not destroy the glycosidic bond. It can be used as a base for the deacylation reaction, removes an acyl protecting group, and promotes the progress of the glycosidation reaction.
  • the deacylation reaction time is at least 30 min, preferably 30-45 min.
  • acetic acid is added to the mixture after the deacylation reaction to adjust the pH of the mixture, and the excess alkali is neutralized to terminate the reaction, while the acetic acid activity is moderate, the generated glycosidic bond is not broken, and the yield of the product is improved.
  • Compound 1 was dissolved in water or ethanol.
  • the 10% H 2 SO 4 -ethanol reagent was sprayed on a TLC plate (the chromatographic solution was chloroform/methanol 3:1, Rf was 0.4).
  • the glycosyl donor in step 2 is 2, 3, 4-tri-O-
  • the mass of benzoyl ⁇ -D bromoglucopyranonic acid methyl ester is 43.89 g (75.25 mmoL)
  • the ratio of the molar ratio of glycosyl donor to forsythiaside is 7:1
  • the catalyst silver carbonate (124.5 g, A white solid (compound 2, 3 g) was obtained in the same manner as in Example 1 except that the molar ratio of the catalyst to the glycosyl donor was 6:1, and the total yield was 79.8%.
  • Compound 2 is a white solid, soluble in water and ethanol.
  • the 10% H 2 SO 4 -ethanol reagent was sprayed on a TLC plate (the chromatographic solution was chloroform/methanol 3:1, Rf was 0.4).
  • Foragex (4g, 10.75mmol) was added to dry anhydrous dichloromethane (180ml) at a temperature of 0 ° C, stirred and dissolved, and then added to the organic base 1,8-diazabicyclo-11 9.63 mL (64.5 mmol) of carbon-7-ene, stirred for 20 minutes, and subjected to hydroxyl activation reaction to prepare a forsythiaside-organic base complex solution for use;
  • the glycosidation reaction mixture was dissolved in methanol under stirring, and then 28% potassium hydroxide solution (11.28 ml), KOH and 2,3,4-tri-O-acetyl were added while stirring at 5 °C.
  • the molar ratio of ⁇ -D-bromopyranose glucuronide methyl ester is 3.5:1, stirred, and subjected to deacylation reaction for 30 min, and then an acidic pH adjuster acetic acid is added to adjust the pH of the deacylated reaction mixture. Value to 7;
  • Compound 3 was dissolved in water and ethanol.
  • the 10% H 2 SO 4 -ethanol reagent was sprayed on a TLC plate (the chromatographic solution was chloroform/methanol 3:1, Rf was 0.4).
  • Compound 4 was dissolved in water or ethanol.
  • the 10% H 2 SO 4 -ethanol reagent was sprayed on a TLC plate (the chromatographic solution was chloroform/methanol 3:1, Rf was 0.4).
  • the compound 4 was for example, for example, potassium forsylate.
  • the glycosyl donor can also use 2,3,4-tri-O-benzoyl ⁇ - Methyl D-bromopyranose glucuronide.
  • the glycosidation reaction mixture was dissolved in methanol under stirring, and then a potassium hydroxide solution (11.7 ml) having a mass percentage of 27% was added while stirring at 0 ° C, KOH and 2, 3, 4 - 3 -O-acetyl ⁇ -D-bromopyranose glucuronide molar ratio of 3.5:1, stirring, deacylation reaction for 30 min, adding acidic pH adjuster acetic acid to adjust the pH of the deacylated mixture To 5;
  • Compound 5 was dissolved in water and ethanol.
  • the 10% H 2 SO 4 -ethanol reagent was sprayed on a TLC plate (the chromatographic solution was chloroform/methanol 3:1, Rf was 0.4).
  • Foragex (4g, 10.75mmo) was added to dry anhydrous dichloromethane (180ml) at a temperature of 10 ° C, stirred and dissolved, and then added to the organic base 1,8-diazabicyclo-11 Carbon-7-ene 9.63 mL (64.5 mmo), stirred for 10 minutes to carry out hydroxyl groups Activating the reaction to prepare a forsythiaside-organic base complex solution, which is ready for use;
  • the glycosyl donor can also use 2,3,4-tri-O-benzoyl ⁇ - Methyl D-bromopyranose glucuronide.
  • the glycosidation reaction mixture was dissolved in methanol under stirring, and then a 30% by mass sodium methoxide solution (10.16 ml), NaOCH 3 and 2, 3 , 4 - 3 were added while stirring at 0 ° C. -O-acetyl ⁇ -D-bromopyranose glucuronide molar ratio of 3.5:1, stirring, deacylation reaction for 30 min, adding acidic pH adjuster acetic acid to adjust the pH of the deacylated mixture To 7;
  • Compound 6 was dissolved in chloroform or ethanol. After spraying on a TLC plate (chromic solution was chloroform/methanol 10:1, Rf was 0.4), a 10% H 2 SO 4 -ethanol reagent was sprayed to give a purple-red color.
  • compound 6 is for example, for example, for example, for example, (2R, 3R, 4R, 5S)-6-(5- ((1R,4S)-4-(3,4-Dimethoxyphenyl)hexahydroindole[3,4-c]furan-1-yl)-2-methoxyphenyl)-3,4 , 5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid methyl ester;
  • Molecular Formula: C 28 H 34 O 12 The structural formula is:
  • forsythiagine glucuronic acid derivative forsythiaside sodium gluconate prepared in Example 1, forsythiaside glucuronide prepared in Example 3, forsythiaside glucan prepared in Example 5
  • the acid for example, forsythiaside methyl glucuronate prepared in Example 6 was produced by Dalian Fusheng Natural Medicine Development Co., Ltd., and the two areas of high performance liquid chromatography (ultraviolet detector, evaporative light scattering detector) were normalized. The purity of the method was 99.3%, 99.2%, 99.7%, and 99.6%, respectively;
  • the above drugs are all dissolved in purified water, filtered, sterilized and dispensed, and stored at 4 ° C for the test.
  • RSV respiratory syncytial virus
  • Biological safety cabinet BHC-1300IIA/B3, AIRTECH; CO2 incubator: MCO-18AIC, SANYO; inverted microscope: CKX41, OLYMPUS; electronic analytical balance: AR1140/C, DHAUS; medium: DMEM, HyClone; fetal bovine serum: HyClone; Trypsin: Gibco; MTT: Sigma; DMSO: Tianjin Beilian Fine Chemicals Development Co., Ltd.
  • Vero cells were subcultured for 1-2 days to make them into slices. The boundary was clear. When the stereoscopic effect and the refractive power were strong, trypsin digestion was performed. After the cell surface appeared a pinpoint-like pore, the digestive juice was aspirated, and several milliliters of the culture solution was blown away. The cells were counted and diluted with a culture solution (DMEM containing 10% fetal bovine serum) to about 5107 cells/L, and then seeded in a 96-well culture plate until the cells were grown into a single layer.
  • DMEM containing 10% fetal bovine serum
  • Cytotoxicity test The drug was diluted at the concentrations shown in Table 1-1 for cytotoxicity assays.
  • viruses were diluted 10-fold to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 different dilutions , and sequentially seeded in a single layer of Vero cell 96-well culture plate Above, 100 ⁇ L per well, 6 wells per dilution, and a normal cell control group. Incubate at 37 ° C, 5% CO 2 for 2 h, discard the virus solution, then add 100 ⁇ L of cell maintenance solution per well, and incubate at 37 ° C, 5% CO 2 .
  • the cytopathic effect was observed under the microscope on the third day, and the results were recorded on the 7th to 8th day, so that the highest dilution of 50% cell hole positive lesions was used as the end point, and the virus titer was calculated by the karber method.
  • TCID 50 50% tissue cell infection
  • ⁇ pi the sum of the percentage of lesions per dilution
  • the OD value was measured at 492 nm using the MTT colorimetric method to calculate the drug antiviral efficiency (ER%).
  • ANOVA was used to compare the significant differences in the antiviral efficacy of each drug in the SPSS 18.0 statistical software.
  • ER% (mean OD value of drug treatment group - average OD value of virus control group) / (average OD value of cell control group - average OD value of virus control group) ⁇ 100%
  • TC 0 maximum non-toxic concentration
  • TC 50 half toxicity concentration
  • Table 1-3 show that the inhibitory rate and effective rate of forsythiaside glucuronic acid derivatives against influenza virus, parainfluenza virus, herpes simplex virus type I (HSV-I), enterovirus EV71 are over 90%. Compared with the virus control group, the difference was statistically significant. The antiviral efficacy of forsythiaside glucuronic acid derivatives over many viruses is superior to that of forsythiaside, ribavirin and oseltamivir.
  • the forsythiaside methylglyoxylate produced by Dalian Fusheng Natural Medicine Development Co., Ltd. is produced by high performance liquid chromatography (UV detector and evaporative light scattering detector). By chemical method, the purity is 99.6%;
  • the above drugs are all dissolved in purified water, filtered, sterilized and dispensed, and stored at 4 ° C for the test.
  • influenza virus and the parainfluenza virus were diluted 10 times to a virus solution having a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , and 10 -5 .
  • 120 Kunming mice 60 influenza virus and parainfluenza virus groups were randomly divided into 6 groups. The mice were lightly anesthetized with ether and infected with different dilutions of virus solution 0.03mL/only. At the same time, a blank control was set, and the physiological suspension was used instead of the viral suspension. Death and survival were observed and observed daily until 14 days after infection. The death within 24 hours of infection was non-specific death, not counted, and the Karber method calculated the LD50 of the virus solution.
  • mice 960 Kunming mice of four weeks old were taken for two trials. 480 mice were randomly divided into 48 groups, 10 in each group, for the determination of the lung index and lung index inhibition rate of forsythiaside glucuronic acid derivatives in mice infected with influenza virus, 3 repeated tests, each Eight mice were taken. Another 480 mice were randomly divided into 48 groups, 10 in each group, for the determination of hemagglutination titer of the lung suspension virus by forsythiaside glucuronic acid derivative, three repeated tests, each taking small 80 rats.
  • mice Put a group of absorbent cotton in a beaker of 200 to 300 mL size, then pour the appropriate amount of ether (to make the cotton wool wet), pour the beaker containing the cotton wool, and place the mouse for anesthesia, see the mouse. Extremely excited, when apparently weak, the mice were placed supine, nasally infected with 15LD 50 influenza virus and parainfluenza virus 0.03ml/nostrils, and the normal control group was replaced with physiological saline instead of the virus suspension.
  • Forsythiaside glucuronic acid derivative group ribavirin and oseltamivir phosphate control group were given conventional intragastric administration on the day before infection, and forsythiaside glucuronic acid derivatives were high, medium and low.
  • the doses were 13.0, 8.0, 4.0 mg/kg, the dose of ribavirin-positive drug was 58.5 mg/kg, the dose of oseltamivir-positive drug was 19.5 mg/kg, and the forsythiaside group The dose was 13.0 mg/kg once a day for 5 days, and the virus control group was administered the same volume of physiological saline.
  • the lungs of each group of mice on the 5th day after treatment were taken and homogenized by a homogenizer at a low temperature.
  • the physiological saline was diluted to 10% of the lung tissue suspension, and the supernatant was centrifuged and diluted to 0.2 ml/
  • the holes were dropped on the titration plate, 0.2 ml of 1% chicken red blood cell suspension was added to each well, and the mixture was allowed to stand at room temperature for 30 min, and the blood coagulation titer was observed.
  • the end point of red blood cell agglutination (++) is expressed as the titer of the suspension dilution factor.
  • the Kunming mice in the experimental group were infected with different concentrations of influenza virus and parainfluenza virus in 30 ⁇ L, and the mice in the third group (virus concentration 10 -1 group, 10 -2 group, 10 -3 group) before the third day of infection. There were different degrees of symptoms: shrub, shivering, and decreased diet. On the 5th day, the mice walked and waved; on the 6th day, the mice in the highest virus concentration group began to die, and the other groups continued on the 7th day after infection. Death has occurred. After the end of 14 days, the number of deaths of each group of mice was counted. The results are shown in Tables 1-4 and 1-5 below. The LD 50 of the influenza virus was calculated to be a dilution of 10 - 2.9 and the LD 50 of the parainfluenza virus was a dilution of 10 - 2.5 .
  • the Karber method calculates the LD 50 of the virus.
  • the LogLD 50 of the influenza virus is as follows:
  • the Karber method calculates the LD 50 of the virus.
  • the LogLD 50 of the parainfluenza virus is as follows:
  • the hemagglutination titer (InX) of lung tissue in the infection model group was 32.40 and 33.11, respectively, and the lungs were treated with different concentrations of forsythiaside glucuronic acid derivative (methyl ester) for 5 days.
  • the hemagglutination titer of tissue virus decreased, and the difference was significant compared with the infection model group (P ⁇ 0.01).
  • the mid- and high-dose groups of forsythiaside glucuronic acid derivatives were influenza and parainfluenza viruses.
  • the blood coagulation titer was significantly lower than that of the model group, and the inhibition rate was higher than that of the forsythiaside group (P ⁇ 0.05, p ⁇ 0.01).
  • the test results are shown in Tables 1-8 and 1-9.
  • the alveolar septum was thinner.
  • the number of mononuclear cells infiltrated in the alveolar wall and bronchiole wall was small, and there was no exudation in the cavity, and the lesion was significantly relieved.
  • the mid- and high-dose groups of forsythiaside glucuronic acid derivatives treated the parainfluenza pneumonia were significantly thinner, the number of infiltrating mononuclear cells was less, and there was no exudation in the cavity. .
  • forsythiaside glucuronic acid derivatives had a significant inhibitory effect on influenza virus and parainfluenza virus and mouse viral pneumonia caused by the dose range of 3.25 ⁇ 13mg/kg/d. It can significantly reduce the lung index and hemagglutination titer, and the lung histopathology is also significantly improved, which is significantly different from the virus model control group; and the middle and high dose groups of forsythiaside glucuronic acid derivatives are significantly better than forsythia Lipoprotein (*P ⁇ 0.05 or **P ⁇ 0.01), while showing a trend superior to ribavirin and oseltamivir phosphate.

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Abstract

本发明提供了如式（I）所示的连翘脂素葡萄糖醛酸衍生物、制备方法及其抗病毒应用等，其中，R为H、Me、Na+、K+等。

Description

连翘脂素葡萄糖醛酸衍生物、制备方法及其应用 技术领域
本发明属于药物化学领域，具体而言，本发明涉及连翘脂素葡萄糖醛酸衍生物及其制备方法，以及这一类衍生物在抗病毒方面的药理作用。
背景技术
连翘脂素，为连翘苷的糖配基部分，又称作连翘苷元，为木犀科连翘属植物连翘的主要活性成分，其结构如下式所示，现代药理研究表明，连翘脂素有抗病毒、抗氧化、降低血脂、清除自由基、抑菌、抗肿瘤、抗炎等作用。
Figure PCTCN2015094864-appb-000001
连翘脂素分子不稳定，易被氧化，且在酸性环境下分子构型容易发生改变。通过大鼠肠内菌模拟连翘苷的代谢研究发现，极易被肠道内菌群代谢为新的代谢物。通过含酚类结构药物代谢研究发现，含酚羟基结构的药物易被体内葡萄糖醛酸酶代谢为酚的葡萄糖醛酸衍生物，大部分葡萄糖醛酸衍生物具有良好的活性。例如青蒿素葡萄糖醛酸衍生物(Efficient Preparations of theβ-Glucuronides of Dihydroartemisinin and Structural Confirmation of the Human Glucuronide Metabolite.Paul M.O'Neill,Feodor Scheinmann,Andrew V.Stachulski,James L.Maggs,and B.Kevin Park.J.Med.Chem.,2001,44(9),pp 1467–1470)、依达拉奉葡萄糖醛酸衍生物(Synthesis of the metabolites of a free radical scavenger edaravone(MCI-186,RadicutTM).Kazutoshi Watanabe,Masao Taniguchi,Masaki Shinoda.Redox Report,Vol.8,No.3,2003，157-161)、风车子素A-1葡萄糖醛酸衍生物(Regio-and Stereospecific Synthesis of Mono-β-d-Glucuronic Acid Derivatives of Combretastatin A-1.Rajendra P.Tanpure,Tracy E.Strecker,David J.Chaplin，Bronwyn G.Siim,Mary Lynn Trawick and Kevin G.Pinney.J.Nat.Prod.,2010,73(6),pp 1093–1101)、白藜芦醇葡萄糖醛酸衍生物(WANG LAIXI；Heredia,A.；Song,HJ；ZHANG ZHAOJUN；YU BIAO；Davis,C.；Redfield,R.Resveratrol glucuronides as the metabolites of resveratrol in humans:Characterization,synthesis,and anti-HIV activity.J.Pharm.Sci.2004,93(10),2448-2457)和姜黄素葡萄糖醛酸衍生物(K.S.Psrvathy,M.Sc.University of Mysore.2009)等等。因此我们设计出连翘脂素的葡萄糖醛酸衍生物，并进行了化学合成及药理研究。
发明内容
本发明的要解决的技术问题是采用化学合成方法制备连翘脂素葡萄糖醛酸衍生物。本发明提供的连翘脂素葡萄糖醛酸衍生物。另外，本发明还提供了制备连翘脂素葡萄糖醛酸衍生物的方法，适合工业化放大生产。
首先，本发明提供了如式(Ⅰ)所示的连翘脂素葡萄糖醛酸衍生物，
Figure PCTCN2015094864-appb-000002
其中，R基团选择H、Me、Na+、K+。
本发明另一方面提供一种连翘脂素葡萄糖醛酸衍生物的制备方法，包括如下顺序进行的步骤：
1)连翘脂素与有机碱进行羟基活化反应，制得连翘脂素-有机碱配合物；
2)将糖基供体、催化剂加入到有机溶剂中，混匀制得糖苷化试剂，然后加入连翘脂素-有机碱配合物，进行糖苷化反应，制得糖苷化反应混合物；
3)将糖苷化反应混合物加入到甲醇中，接着加入碱性化合物，进行脱酰基反应，然后加入pH调节剂，调节混合物溶液pH至4-8.5即得。
其中，步骤1)中所述羟基活化反应的反应温度为0-20℃，优选为0-10℃；反应时间为10-20min。
特别是，步骤1)中将连翘脂素与有机碱溶于有机溶剂，在搅拌状态下进行所述的羟基活化反应，制得连翘脂素-有机碱配合物。
尤其是，所述有机溶剂选择二氯甲烷、甲苯或吡啶中的一种，优选为二氯甲烷。
特别是，所述有机溶剂选择无水二氯甲烷、无水甲苯或无水吡啶中的一种，优选为无水二氯甲烷。
尤其是，所述连翘脂素质量与有机溶剂的体积之比(g/ml)为1：40-50，优选为1：45。
其中，所述有机碱选择N,N-二异丙基乙基胺或1,8-二氮杂二环十一碳-7-烯。
特别是，所述连翘脂素与有机碱的摩尔配比为1：5-8，优选为1：6。
其中，步骤2)中所述糖苷化反应的温度>0℃，优选为0-20℃，优选为5-15℃，进一步优选为10℃；反应时间为4-15h，优选为8-10h，进一步优选为10h。
特别是，在惰性气体气氛下进行所述的糖苷化反应。
尤其是，所述惰性气体选择氮气、氩气或氦气，优选为氮气。
其中，所述糖基供体和连翘脂素的摩尔比为1-7：1，优选为1.5～7：1，进一步优选为1.5：1。
特别是，步骤2)中所述糖基供体选择2,3,4,-三-O-酰基-α-D-吡喃葡萄糖醛酸酯，优选为2,3,4-三-O-苯甲酰基-α-D-溴代吡喃葡萄醛酸甲酯或2,3,4-三-O-乙酰基-α-D-溴代吡喃葡萄醛酸甲酯；所述有机溶剂选择二氯甲烷、三氯甲烷，1，2-二氯乙烷或甲苯，优选为二氯甲烷。
本发明进行糖苷化反应过程中，糖基供体2,3,4,-三-O-酰基-α-D-吡喃葡萄糖醛酸酯的用量少，糖苷化产物的得率低，而用量增加则导致副产物增多，糖基供体2,3,4,-三-O-酰基-α-D-吡喃葡萄糖醛酸酯和连翘脂素的摩尔比优选为1.5～2.5：1。
其中，步骤2)中所述催化剂选择碳酸银。
特别是，所述催化剂与所述糖基供体的摩尔比为4-8：1，优选为4-6：1，进一步优选为6：1。
催化剂的用量低导致糖基供体2,3,4,-三-O-酰基-α-D-吡喃葡萄糖醛酸酯的分解，降低产率；催化剂用量高，导致三酰基连翘脂素葡萄糖醛酸甲酯(即糖苷化反应产物)的分解，降低产率。
特别是，在-20～0℃以下，并且在惰性气体保护下将糖基供体、催化剂加入到有机溶剂中，混均匀，制得所述的糖苷化试剂。
尤其是，在温度为-10～0℃，并且在惰性气体保护下将糖基供体、催化剂加入到有机溶剂中，混均匀，制得所述的糖苷化试剂；温度优选为0℃。
特别是，在-20～0℃以下，并且在惰性气体保护下将连翘脂素-有机碱配合物加入到所述的糖苷化试剂中，然后再进行所述的糖苷化反应。
尤其是，在温度为-10～0℃，并且在惰性气体保护性将连翘脂素-有机碱配合物加入到所述的糖苷化试剂中，然后再进行糖苷化反应；温度优选为0℃。
本发明在较低温度和惰性气体保护的条件下配制糖苷化试剂以及将连翘脂素-有机碱配合物加入到糖苷化试剂中，防止催化剂失活，低温保证反应按照SN2途径进行反应，保证了催化剂的反应选择性，提高了反应的选择性。
特别是，所述惰性气体选择氮气、氩气或氦气，优选为氮气。
其中，步骤3)中所述脱酰基化反应的反应温度0-10℃，优选为0-5℃，进一步优选为0℃。
特别是，步骤3)中所述所述碱性化合物选择氢氧化钠、氢氧化钾或甲醇钠；所述pH调节剂选择醋酸、丙酸或盐酸，优选为醋酸。
其中，碱性化合物与步骤2)中所述糖基供体的摩尔比为3-5：1，优选为3.5：1。
特别是，将碱性化合物溶于水中，制备成碱性化合物溶液后，再加入，进行所述的脱羧基反应。
尤其是，制备的碱性化合物溶液的质量百分比浓度为27-30％。
特别是，调节混合物溶液的pH值为5-8，优选为pH值为7。
特别是，还包括步骤4)在调节混合物溶液pH值后，对混合物进行分离、纯化处理。
特别是，还包括步骤4)分离、纯化处理，对pH调节至中心的混合物溶液进行浓缩处理和硅胶柱层析处理。
其中，所述的分离、纯化处理是对混合物进行硅胶柱层析。
特别是，所述硅胶柱层析过程中选用GF254硅胶；洗脱剂选择氯仿/甲醇＝9：1。
通过上述方法制备得到的本发明化合物为连翘脂素葡萄糖醛酸衍生物的的结构式如下：
Figure PCTCN2015094864-appb-000003
连翘脂素葡萄糖醛酸衍生物在常温下为白色固体，溶于氯仿、乙醇。在TLC板上展开(层析液为氯仿/甲醇10：1，Rf为0.4)后喷雾10％H2SO4-乙醇试剂呈现紫红色。
本发明再一方面提供一种连翘脂素葡萄糖醛酸衍生物的抗病毒应用。
其中，所述抗病毒为流感病毒、抗副流感病毒、抗呼吸道合胞病毒(RSV)、抗柯萨奇病毒B3(CVB3)、抗柯萨奇病毒A16(CoxA16)、抗肠道病毒EV71、抗腺病毒(AdV)、抗单纯疱疹病毒I型(HSV-1)。
本发明又一方面提供连翘脂素葡萄糖醛酸衍生物在制备抗病毒药物或保健品中的应用。
本发明提供了药物组合物，其包括本发明连翘脂素葡萄糖醛酸衍生物，以及药学上可接受的辅料。
在本文中，药学上可接受的辅料指无毒固态、半固态或液态填充剂、稀释剂、载体、pH调节剂、离子强度调节剂、缓释或控释剂、包裹材料或其他制剂辅料。所用载体可与相应的给药形式相适应，可使用本领域技术人员所知晓的辅料配成注射剂、(注射用)冻干粉、喷雾剂、口服溶液、口服混悬液、片剂、胶囊、肠溶片、丸剂、粉剂、颗粒剂、持续释放或延迟释释放等制剂。优选本发明第一方面的连翘脂素硫酸酯衍生物通过注射或经消化道方式给药，因此，本发明的药物组合物优选为注射剂或经消化道给药的制剂，即适于配制成注射和经消化道方式给药的辅料特别优选的。其中，“经消化道给药”在本文中指药物制剂通过患者消化道的给药方式，包括口服、灌胃给药和灌肠给药等，优选是口服，如可使用本领域技术人员所知晓的辅料配成口服溶液、口服混悬液、片剂、胶囊、肠溶片、丸剂、粉剂、颗粒剂、持续释放或延迟释释放等制剂；其中，注射给药的制剂主要是针剂和 粉针剂。
本发明的连翘脂素葡萄糖醛酸衍生物的化学合成反应式如下：
Figure PCTCN2015094864-appb-000004
其中，结构式A为连翘脂素；结构B为2,3,4,-三-O-酰基-α-D-溴代吡喃葡萄糖醛酸酯；结构C为三酰基连翘脂素葡萄糖醛酸甲酯；结构D为连翘脂素葡萄糖醛酸衍生物。
本发明所述的化学合成连翘脂素葡萄糖醛酸方法的先进性和实用价值在于，原料来源方便，糖苷化反应的催化剂廉价易得，使制备成本明显下降，并且化学合成产品连翘脂素葡萄糖醛酸衍生物的质量易于控制，产品的综合得率高，适宜工业化大规模生产。
附图说明
图1为本发明连翘脂素葡萄糖醛酸甲酯的核磁共振氢谱；
图2本发明连翘脂素葡萄糖醛酸甲酯的核磁共振碳谱；
图3本发明连翘脂素葡萄糖醛酸甲酯的高分辨质谱；
图4为流感病毒肺炎模型小鼠肺组织病理切片，其中：A为正常小鼠肺组织；B为流感病毒肺炎小鼠肺组织；C为流感病毒肺炎小鼠模型经阳性药物利巴韦林的治疗后的小鼠的肺组织；D为连翘脂素葡萄糖醛酸甲酯高剂量组治疗流感病毒肺炎后的小鼠肺组织；E为连翘脂素葡萄糖醛酸甲酯中剂量组治疗流感病毒肺炎后的小鼠肺组织；F为连翘脂素葡萄糖醛酸甲酯低剂量组治疗流感病毒肺炎后的小鼠肺组织；
图5为副流感病毒肺炎模型小鼠肺组织病理切片，其中：A为正常小鼠肺组织；B为副流感病毒肺炎小鼠肺组织；C为副流感病毒肺炎小鼠模型经阳性药物利巴韦林的治疗后的小鼠的肺组织；D 为连翘脂素葡萄糖醛酸甲酯高剂量组治疗副流感病毒肺炎后的小鼠肺组织；E为连翘脂素葡萄糖醛酸甲酯中剂量组治疗副流感病毒肺炎后的小鼠肺组织；F为连翘脂素葡萄糖醛酸甲酯低剂量组治疗副流感病毒肺炎后的小鼠肺组织。
具体实施方式
以下通过实施例进一步描述本发明，但这些实施例仅是说明本发明，而不应理解为对本发明范围的任何限制。另外，实施例中的试剂、原料都可以通过商业渠道获得，如有未尽之处，可以参考有机合成指南、药品监管机构的指引以及相应仪器、试剂的厂商说明书等。
实施例1
1、制备连翘脂素-有机碱配合物
在温度为10℃的条件下将连翘脂素(4g，10.75mmol)加入到干燥的无水二氯甲烷(180ml)中，搅拌溶解后加入有机碱N,N-二异丙基乙基胺11.26mL(64.5mmoL)，搅拌10分钟，进行羟基活化反应，制得连翘脂素-有机碱配合物溶液备用，连翘脂素与有机碱的摩尔之比为1：6；
本发明进行所述羟基活化反应的过程中控制反应温度在0-20℃的条件下，均适用于本发明，本发明实施例以10℃为例进行说明。本发明中连翘脂素与有机碱的摩尔之比除了1：6之外，其他1：5-8的摩尔比均适用于本发明。本发明实施例以1：6为例进行说明。
所述羟基活化反应是利用碱活化羟基，使糖苷化反应更容易进行。
2、糖苷化反应
2-1)在-10℃以及氮气气氛下，将2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯6.4g(16.125mmoL)和碳酸银17.78g(64.5mmoL)溶于二氯甲烷中，搅拌均匀，制得糖苷化试剂，其中糖基供体与连翘脂素的摩尔之比为1.5：1，催化剂碳酸银与糖基供体的摩尔之比为4：1；
2-2)在-10℃以及氮气气氛下，边搅拌边将连翘脂素-有机碱配合物溶液加入到糖苷化试剂中，然后加热升温至10℃，并在温度保持为10℃的条件下，进行糖苷化反应10h，制得糖苷化反应混合物；
有机碱夺去反应底物连翘脂素的羟基的氢后，生成的活性中间体配合物遇到氧气会被氧化，通过惰性气体保护使中间体失去与氧接触的机会，保证反应正常进行。惰性气体除了氮气之外，氦气、氩气均适用于本发明。
3、脱酰基反应
在搅拌状态下，将糖苷化反应混合物溶于甲醇中，然后在0℃的条件下边搅拌边加入质量百分比浓度为27％的氢氧化钠溶液(8.36ml)，NaOH与2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯的摩尔比为3.5：1，进行脱酰基反应30min之后，加入酸性pH调节剂醋酸，调节脱酰基反应 的混合物的pH值至7；
本发明中进行脱酰基化反应的过程中加入作为溶剂的甲醇，将糖苷化反应混合物溶解；加入的碱(如氢氧化钠、氢氧化钾、甲醇钠)的碱性既不会破坏糖苷键又可以用作脱酰基化反应的碱，脱除酰基保护基，促进糖苷化反应的进行。脱酰基反应时间至少30min，优选为30-45min。
本发明中向脱酰基反应后的混合物中加入醋酸调节混合物的pH，中和过量的碱，终止反应，同时醋酸活性适中，不会破坏生成的糖苷键，提高产物的得率。
4、分离、纯化处理
采用旋转蒸发仪在真空状态下进行减压蒸馏、浓缩，蒸发去除溶剂，获得脱酰基反应混合物固体；将脱酰基反应混合物样品进行硅胶柱层析分离、纯化处理，其中，洗脱剂：氯仿/甲醇＝9：1；柱层析硅胶：GF254硅胶；得白色固体(化合物1、4.89g)，总产率79.8％。
化合物1溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3：1，Rf为0.4)后喷雾10％H2SO4-乙醇试剂呈现紫红色。
化合物1的1H-NMR、13C-NMR、高分辨质谱、红外光谱图如图1-4所示。
ESI-MS谱中，m/z[M-Na]-为547.18210，分子量为570.52277。
1H-NMR(400MHz，d6-DMSO)如下：δ(ppm):7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(11H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
13C-NMR(100MHz，d6-DMSO)如下：δ(ppm):169.75(C-34)，149.51(C-19)，148.95(C-14)，148.09(C-13)，145.74(C-18)，136.26(C-10)，131.67(C-9)，118.55(C-16)，118.05(C-17)，115.72(C-11)，112.03(C-12)，111.07(C-20)，109.92(C-15)，100.21(C-28)，87.11(C-4)，81.74(C-6)，76.26(C-32)，75.70(C-30)，73.41(C-31)，71.91(C-29)，70.81(C-1)，69.46(C-8)，56.15，55.99，55.94(C-25、C-26、C-27)，54.47(C-3)，49.79(C-2)。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物1为连翘脂素葡萄糖醛酸钠，分子式为：C27H31O12Na，英文名：sodium(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate；中文名：(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-二甲氧基苯基)六氢骈[3,4-c]呋喃-1-基)-2-甲氧基苯基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酸钠；结构式为：
Figure PCTCN2015094864-appb-000005
实施例2
除了步骤1中加入有机碱1,8-二氮杂二环十一碳-7-烯(9.63mL，64.5mmol)；步骤2中的糖基供体为2、3、4-三-O-苯甲酰基α-D溴代吡喃葡萄糖醛酸甲酯的质量为43.89g(75.25mmoL)，糖基供体与连翘脂素的摩尔之比为7：1；催化剂碳酸银(124.5g，0.4515mol)，催化剂与糖基供体的摩尔之比为6：1之外，其余与实施例1相同，制得白色固体(化合物2、3g)，总产率79.8％。
化合物2为白色固体，溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3：1，Rf为0.4)后喷雾10％H2SO4-乙醇试剂呈现紫红色。
化合物2的ESI-MS谱中，m/z[M-Na]-为547.18210，分子量为570.52277。
化合物2的1H-NMR、13C-NMR、IR与实施例1制得的化合物1相同。
根据ESI-MS、1H-NMR和13C-NMR、IR的测试数据，确定化合物2为连翘脂素葡萄糖醛酸钠。
实施例3
1、制备连翘脂素-有机碱配合物
在温度为0℃的条件下将连翘脂素(4g，10.75mmol)加入到干燥的无水二氯甲烷(180ml)中，搅拌溶解后加入有机碱1,8-二氮杂二环十一碳-7-烯9.63mL(64.5mmol)，搅拌20分钟，进行羟基活化反应，制得连翘脂素-有机碱配合物溶液备用；
2、糖苷化反应
2-1)在0℃以及氮气气氛下，将2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯6.4g(16.125mmoL)和碳酸银17.78g(64.5mmoL)溶于二氯甲烷中，搅拌均匀，制得糖苷化试剂；
2-2)在0℃以及氮气气氛下，边搅拌边将连翘脂素-有机碱配合物溶液加入到糖苷化试剂中，然后加热升温至20℃，并在温度保持为20℃的条件下，进行糖苷化反应10h，制得糖苷化反应混合物；
3、脱酰基反应
在搅拌状态下，将糖苷化反应混合物溶于甲醇中，然后在5℃的条件下边搅拌边加入28％的氢氧化钾溶液(11.28ml)，KOH与2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯的摩尔比为3.5：1，搅拌，进行脱酰基反应30min之后，加入酸性pH调节剂醋酸，调节脱酰基反应的混合物的pH 值至7；
4、分离、纯化处理
采用旋转蒸发仪在真空状态下进行减压蒸馏、浓缩，蒸发去除溶剂，获得脱酰基反应混合物固体；将脱酰基反应混合物样品进行硅胶在柱层析分离、纯化处理，其中，洗脱剂：氯仿/甲醇＝9：1；柱层析硅胶：GF254硅胶；得白色固体(化合物3、4.96g)，总产率81％。
化合物3溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3：1，Rf为0.4)后喷雾10％H2SO4-乙醇试剂呈现紫红色。
ESI-MS谱中，m/z[M-K]-为547.18213，分子量为586.63185。
1H-NMR(400MHz，d6-DMSO)如下：δ(ppm):7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(11H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
13C-NMR(100MHz，d6-DMSO)如下：δ(ppm):169.75(C-34)，149.51(C-19)，148.95(C-14)，148.09(C-13)，145.74(C-18)，136.26(C-10)，131.67(C-9)，118.55(C-16)，118.05(C-17)，115.72(C-11)，112.03(C-12)，111.07(C-20)，109.92(C-15)，100.21(C-28)，87.11(C-4)，81.74(C-6)，76.26(C-32)，75.70(C-30)，73.41(C-31)，71.91(C-29)，70.81(C-1)，69.46(C-8)，56.15，55.99，55.94(C-25、C-26、C-27)，54.47(C-3)，49.79(C-2)。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物3为连翘脂素葡萄糖醛酸钾，中文名：(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-二甲氧基苯基)六氢骈[3,4-c]呋喃-1-基)-2-甲氧基苯基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酸钾；英文名：potassium(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate；分子式为：C27H31O12K；结构式为：
Figure PCTCN2015094864-appb-000006
实施例4
除了步骤1中加入有机碱N,N-二异丙基乙基胺(11.26mL，64.5mmoL)，搅拌时间为10min；步骤2中的糖基供体为2、3、4-三-O-苯甲酰基α-D溴代吡喃葡萄糖醛酸甲酯的质量为43.89g(75.25mmoL)，糖基供体与连翘脂素的摩尔之比为7：1；催化剂碳酸银(124.5g，0.4515mol)，催化剂与糖基供体的摩尔之比为6：1，糖苷化反应温度为5℃，反应时间为9h；步骤3中调节pH至8之外，其余与实施例3相同，制得白色固体(化合物4、5.14g)，总产率84％。
化合物4溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3：1，Rf为0.4)后喷雾10％H2SO4-乙醇试剂呈现紫红色。
化合物4的ESI-MS谱中，m/z[M-K]-为547.18213，分子量为586.63185。
化合物4的1H-NMR、13C-NMR与实施例3制得的化合物3相同。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物4为连翘脂素葡萄糖醛酸钾。
实施例5
1、制备连翘脂素-有机碱配合物
在温度为20℃的条件下将连翘脂素(4g，10.75mmo)加入到干燥的无水二氯甲烷(180ml)中，搅拌溶解后加入有机碱N,N-二异丙基乙基胺11.26mL(64.5mmoL)，搅拌10分钟，进行羟基活化反应，制得连翘脂素-有机碱配合物溶液，备用；
2、糖苷化反应
2-1)在-10℃以及氮气气氛下，将2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯6.4g(16.125mmoL)和碳酸银17.78g(64.5mmoL)溶于二氯甲烷中，搅拌均匀，制得糖苷化试剂，其中糖基供体与连翘脂素的摩尔之比为1.5：1，催化剂碳酸银与糖基供体的摩尔之比为4：1；
糖基供体除了2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯之外，还可以使用2、3、4-三-O-苯甲酰基α-D溴代吡喃葡萄糖醛酸甲酯。
2-2)在-10℃以及氮气气氛下，边搅拌边将连翘脂素-有机碱配合物溶液滴加入到糖苷化试剂中，然后加热升温至0℃，并在温度保持为0℃的条件下，进行糖苷化反应10h，制得糖苷化反应混合物；
3、脱酰基反应
在搅拌状态下，将糖苷化反应混合物溶于甲醇中，然后在0℃的条件下边搅拌边加入质量百分比浓度为27％的氢氧化钾溶液(11.7ml)，KOH与2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯的摩尔比为3.5：1，搅拌，进行脱酰基反应30min之后，加入酸性pH调节剂醋酸，调节脱酰基反应的混合物的pH值至5；
4、分离、纯化处理
采用旋转蒸发仪在真空状态下进行减压蒸馏、浓缩，蒸发去除溶剂，获得脱酰基反应混合物固 体；将脱酰基反应混合物样品进行硅胶在柱层析分离、纯化处理，其中，洗脱剂：氯仿/甲醇＝9：1；柱层析硅胶：GF254硅胶；得白色固体(化合物5、5.08g)，总产率83％。
化合物5溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3：1，Rf为0.4)后喷雾10％H2SO4-乙醇试剂呈现紫红色。
ESI-MS谱中，m/z[M+Na]+为571.17916，分子量为548.54109。
1H-NMR(400MHz，d6-DMSO)如下：δ(ppm):12.0(1H，s，COOH)，7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(11H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
13C-NMR(100MHz，d6-DMSO)如下：δ(ppm):169.75(C-34)，149.51(C-19)，148.95(C-14)，148.09(C-13)，145.74(C-18)，136.26(C-10)，131.67(C-9)，118.55(C-16)，118.05(C-17)，115.72(C-11)，112.03(C-12)，111.07(C-20)，109.92(C-15)，100.21(C-28)，87.11(C-4)，81.74(C-6)，76.26(C-32)，75.70(C-30)，73.41(C-31)，71.91(C-29)，70.81(C-1)，69.46(C-8)，56.15，55.99，55.94(C-25、C-26、C-27)，54.47(C-3)，49.79(C-2)。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物5为连翘脂素葡萄糖醛酸，中文名：(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-二甲氧基苯基)六氢骈[3,4-c]呋喃-1-基)-2-甲氧基苯基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酸；英文名：(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid；分子式为：C27H32O12；结构式为：
Figure PCTCN2015094864-appb-000007
实施例6
1、制备连翘脂素-有机碱配合物
在温度为10℃的条件下将连翘脂素(4g，10.75mmo)加入到干燥的无水二氯甲烷(180ml)中，搅拌溶解后加入有机碱1,8-二氮杂二环十一碳-7-烯9.63mL(64.5mmo)，搅拌10分钟，进行羟基 活化反应，制得连翘脂素-有机碱配合物溶液，备用；
2、糖苷化反应
2-1)在0℃以及氮气气氛下，将2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯6.4g(16.125mmoL)和碳酸银17.78g(64.5mmoL)溶于二氯甲烷中，搅拌均匀，制得糖苷化试剂，其中糖基供体与连翘脂素的摩尔之比为1.5：1，催化剂碳酸银与糖基供体的摩尔之比为4：1；
糖基供体除了2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯之外，还可以使用2、3、4-三-O-苯甲酰基α-D溴代吡喃葡萄糖醛酸甲酯。
2-2)在0℃以及氮气气氛下，边搅拌边将连翘脂素-有机碱配合物溶液滴加入到糖苷化试剂中，然后加热升温至10℃，并在温度保持为10℃的条件下，进行糖苷化反应10h，制得糖苷化反应混合物；
3、脱酰基反应
在搅拌状态下，将糖苷化反应混合物溶于甲醇中，然后在0℃的条件下边搅拌边加入质量百分比浓度为30％的甲醇钠溶液(10.16ml)，NaOCH3与2、3、4-三-O-乙酰基α-D溴代吡喃葡萄糖醛酸甲酯的摩尔比为3.5：1，搅拌，进行脱酰基反应30min之后，加入酸性pH调节剂醋酸，调节脱酰基反应的混合物的pH值至7；
4、分离、纯化处理
采用旋转蒸发仪在真空状态下进行减压蒸馏、浓缩，蒸发去除溶剂，获得脱酰基反应混合物固体；将脱酰基反应混合物样品进行硅胶在柱层析分离、纯化处理，其中，洗脱剂：氯仿/甲醇＝9：1；柱层析硅胶：GF254硅胶；得白色固体(化合物6、5.33g)，总产率87％。
化合物6溶于氯仿、乙醇。在TLC板上展开(层析液为氯仿/甲醇10：1，Rf为0.4)后喷雾10％H2SO4-乙醇试剂呈现紫红色。
ESI-MS谱中，m/z[M+Na]+为585.19481，分子量为562.56802。
1H-NMR(400MHz，d6-DMSO)如下：δ(ppm):7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(11H，d，J＝8.4Hz，O-CH3)，3.642(3H，t，J＝8.1Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
13C-NMR(100MHz，d6-DMSO)如下：δ(ppm):169.76(C-34)，149.53(C-19)，148.96(C-14)，148.10(C-13)，145.75(C-18)，136.26(C-10)，131.68(C-9)，118.54(C-16)，118.06(C-17)，115.74(C-11)，112.04(C-12)，111.09(C-20)，109.93(C-15)，100.22(C-28)，87.11(C-4)， 81.74(C-6)，76.28(C-32)，75.69(C-30)，73.42(C-31)，71.92(C-29)，70.83(C-1)，69.47(C-8)，56.17，55.97，55.94(C-25、C-26、C-27)，54.48(C-3)，52.41(C-40)，49.80(C-2)。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物6为连翘脂素葡萄糖醛酸甲酯，中文名：(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-二甲氧基苯基)六氢骈[3,4-c]呋喃-1-基)-2-甲氧基苯基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酸甲酯；英文名：(2R,3R,4R,5S)-methyl6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetra hydro-2H-pyran-2-carboxylate；分子式为：C28H34O12；结构式为：
Figure PCTCN2015094864-appb-000008
试验例1体外抗病毒试验
1.1试验材料
(1)药物
①连翘脂素葡萄糖醛酸衍生物：实施例1制备的连翘脂素葡萄糖醛酸钠、实施例3制备的连翘脂素葡萄糖醛酸钾、实施例5制备的连翘脂素葡萄糖醛酸、实施例6制备的连翘脂素葡萄糖醛酸甲酯均由大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器(紫外检测器、蒸发光散射检测器)面积归一化法测定，其纯度分别为为99.3％、99.2％、99.7％、99.6％；
②阳性对照药：利巴韦林注射液，无色透明液体，由河南润弘股份有限公司生产，产品批号：1206261，国药准字：H19993553，100mg/ml，作为本次试验阳性对照药物；磷酸奥司他韦，中国药品生物制品检定所，产品批号：101096-200901，100mg/支作为本次试验阳性对照药物；连翘脂素，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器(紫外检测器和蒸发光散射检测器)面积归一化法测定，其纯度为99.2％。
上述药品均用纯净水溶解，滤过，除菌分装，4℃备用，为本次试验待测药物。
(2)细胞株Vero(非洲绿猴肾细胞细胞)由吉林大学基础医学院保存细胞株。
(3)病毒株①流感病毒株、副流感病毒株、呼吸道合胞病毒(RSV)株：购于中国预防医学科学院病毒研究所；②柯萨奇病毒B3(CVB3)株：购自中科院武汉病毒所；③柯萨奇病毒A16(CoxA16)株、肠道病毒EV71株：购自日本仙台国立医院；④腺病毒(AdV)：购于白求恩医科大学 一院儿科研究室；⑤单纯疱疹病毒I型(HSV-1)：购于中国药品生物制品检定所。
(4)主要设备与试剂
生物安全柜：BHC-1300ⅡA/B3，AIRTECH；CO2培养箱：MCO-18AIC，SANYO；倒置显微镜：CKX41，OLYMPUS；电子分析天平：AR1140/C，DHAUS；培养基：DMEM，HyClone；胎牛血清：HyClone；胰蛋白酶：Gibco；MTT：Sigma；DMSO：天津市北联精细化学品开发有限公司。
1.2试验方法
(1)细胞准备
Vero细胞传代培养1-2d，使之成片，界线清晰，立体感及折光度强时，用胰酶消化，待细胞面出现针尖样小孔，吸尽消化液，取数毫升培养液吹散细胞，计数，用培养液(含10％胎牛血清的DMEM)稀释至约5 107个/L后，接种于96孔培养板内，待细胞长成单层。
(2)药物毒性测定
细胞毒性试验：将药物按表1-1所示浓度进行稀释，用于细胞毒性测定。
表1-1      药物稀释参照表(单位：g/L)
Figure PCTCN2015094864-appb-000009
将上述用维持液(含2％胎牛血清的DMEM)稀释好的不同浓度的药品滴加于Vero单层细胞上，每孔0.2ml，每个浓度6个复孔，另设6孔正常对照(不加药物的正常对照组)和6孔空白对照(培养液)，置37℃，5％CO2培养箱中培养，每日置倒置显微镜观察CPE并记录。72h后，每孔加入MTT溶液20μL(5mg·mL－1)，继续孵育4h，吸弃各孔培养液，每孔加入100μL DMSO，振荡5min，492nm测定OD值，计算细胞存活率。在SPSS 18.0统计软件中，将细胞存活率进行Probit回归分析，计算药物对Vero细胞的最大无毒浓度(TC0)和半数毒性浓度(TC50)。
(3)各种病毒TCID50的测定
将各种病毒进行10倍递减稀释为10-1,10-2,10-3，10-4，10-5，10-6不同稀释度，按序接种于单层的Vero细胞96孔培养板上，每孔100μL，每个稀释度6孔，同时设正常细胞对照组。置37℃，5％CO2 中孵育2h，弃病毒液，随即每孔加细胞维持液100μL，置37℃，5％CO2中培养。第3天开始在显微镜下观察细胞病变结果，第7-8天判定结果并做好记录，以能使50％细胞孔发生阳性病变的最高稀释度作为终点，用karber法计算病毒滴度。
公式
Figure PCTCN2015094864-appb-000010
TCID50：50％组织细胞感染量
XM：病毒最高浓度稀释度的对数
d：稀释度系数(倍数)的对数
Σpi：每个稀释度病变百分数的总和
(4)药物对病毒致细胞病变作用的影响
取已长满单层细胞的培养板，吸弃培养液，以100TCID50对应的病毒攻击量接种细胞，37℃，5％CO2培养箱吸附2h，加入特定浓度(最大无毒浓度左右)的各药液，每浓度6个复孔培养，200μL/孔。设利巴韦林注射液和磷酸奥司他韦为阳性药物对照组，同时设正常对照组(不加病毒不加药)和病毒对照组(加病毒但不加药物的对照组)，观察药物对病毒致CPE的影响。72h后，用MTT比色法，在492nm波长下测定OD值，计算药物抗病毒有效率(ER％)。在SPSS 18.0统计软件中用ANOVA法比较各药物抗病毒有效率之间的显著性差异。
ER％＝(药物处理组平均OD值－病毒对照组平均OD值)/(细胞对照组平均OD值－病毒对照组平均OD值)×100％
1.3试验结果
(1)各种病毒的TCID50
副流感病毒：
Figure PCTCN2015094864-appb-000011
流感病毒：
Figure PCTCN2015094864-appb-000012
CVB3：
Figure PCTCN2015094864-appb-000013
HSV-1：
Figure PCTCN2015094864-appb-000014
AdV：
Figure PCTCN2015094864-appb-000015
RSV：
Figure PCTCN2015094864-appb-000016
CoxA16：
Figure PCTCN2015094864-appb-000017
EV71：
Figure PCTCN2015094864-appb-000018
(2)药物毒性测定
1)药物对细胞毒性的测定
各药物对Vero细胞的最大无毒浓度(TC0)、半数毒性浓度(TC50)见表1-2。
表1-2  药物细胞毒性实验结果(单位：g/L)
Figure PCTCN2015094864-appb-000019
2)药物对病毒致细胞病变保护作用结果
药物抗各种病毒的有效率及ANOVA法单因素方差分析结果，详见表1-3。
表1-3  药物抗病毒有效率(ER％)统计表
Figure PCTCN2015094864-appb-000020
注：与病毒对照组相比，*P<0.05，**P<0.01；与连翘脂素比，#P<0.05，##P<0.01。
表1-3结果显示，连翘脂素葡萄糖醛酸衍生物对流感病毒、副流感病毒、单纯疱疹病毒Ⅰ型(HSV-I)、肠道病毒EV71的抑制率及有效率均超过90％，与病毒对照组相比差异明显，具有统计学意义。连翘脂素葡萄糖醛酸衍生物对多种病毒的抗病毒疗效表现优于连翘脂素、利巴韦林和磷酸奥司他韦的优势。
试验例2体内抗病毒试验
2.1实验材料
(1)实验动物
昆明小鼠，体重18～22g，雌雄各半性，购自大连医科大学实验动物中心，质量合格证号：SCXK(13)2012-0003。
(2)药物
①连翘脂素葡萄糖醛酸衍生物：
实施例6制备的连翘脂素葡萄糖醛酸甲酯：白色固体，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器(紫外检测器和蒸发光散射检测器)面积归一化法测定，其纯度为99.6％；
②利巴韦林注射液，无色透明液体，由河南润弘股份有限公司生产，产品批号：1206261，国药准字：H19993553，100mg/ml，作为本次试验阳性对照药物；
③磷酸奥司他韦，中国药品生物制品检定所，产品批号：101096-200901，100mg/支，作为本次试验阳性对照药物；
④连翘脂素，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器(紫外检测器和蒸发光散射检测器)面积归一化法测定，其纯度为99.2％。
上述药品均用纯净水溶解，滤过，除菌分装，4℃备用，为本次试验待测药物。
(2)检测仪器、试剂
Figure PCTCN2015094864-appb-000021
2.2实验方法
(1)流感病毒和副流感病毒对小鼠半数致死量的测定
将流感病毒和副流感病毒(细胞裂解液)10倍递比稀释为10-1、10-2、10-3、10-4、10-5浓度的病毒液。取昆明种小鼠120只，流感病毒和副流感病毒组各60只，分别随机分成6组，乙醚轻度麻醉小鼠，滴鼻感染不同稀释度病毒液0.03mL/只。同时设空白对照，用生理盐水代替病毒悬液。以死亡和生存为观察指标，每天观察，直至感染后的14天。感染24h内死亡的为非特异死亡，不予统计，Karber法计算病毒液LD50。计算公式：
Figure PCTCN2015094864-appb-000022
[其中：LD50：半数致死量；XM：病毒最高浓度稀释度的对数；d：稀释度系数(倍数)的对数；Σpi：每个稀释度病变百分数的总和]。
(2)连翘脂素葡萄糖醛酸甲酯抗流感病毒和副流感病毒感染所致肺炎的研究
1)试验动物及分组
取四周龄的昆明小鼠960只，进行2项试验。取小鼠480只，随机分成48组，每组10只，用于连翘脂素葡萄糖醛酸衍生物对流感病毒感染小鼠肺指数和肺指数抑制率的测定试验,3次重复试验，每次取小鼠80只。另取小鼠480只，随机分成48组，每组10只，用于连翘脂素葡萄糖醛酸衍生物对肺悬液病毒血凝滴度的测定试验，3次重复试验，每次取小鼠80只。
2)感染方法
在200～300mL大小的烧杯内放入一团脱脂棉，然后倒入适量的乙醚(使脱脂棉变湿即可)，把装有脱脂棉的烧杯倒扣过来，把小鼠放入进行麻醉，见小鼠极度兴奋，明显呈无力样时，将小鼠仰卧，滴鼻感染15LD50流感病毒和副流感病毒0.03ml/鼻孔，正常对照组用生理盐水代替病毒悬液。
3)给药方法及给药剂量
连翘脂素葡萄糖醛酸衍生物组、利巴韦林和磷酸奥司他韦对照组，分别于感染前一天开始常规灌胃给药，连翘脂素葡萄糖醛酸衍生物高、中、低给药剂量分别为13.0、8.0、4.0mg/kg，利巴韦林阳性药给药剂量为58.5mg/kg，磷酸奥司他韦阳性药给药剂量为19.5mg/kg，连翘脂素组给药剂量为13.0mg/kg，每天一次，连续给药5d，病毒对照组灌服相同体积的生理盐水。
4)观察指标
①肺指数测定
小鼠用药后第5天，先禁食水8小时，称体重后摘眼球放血处死动物，打开胸腔摘出全肺，以生理盐水洗涤两次，用滤纸吸干表面水份，电子天平称肺重，按下列公式计算计算肺指数和肺指数抑制率：
肺指数＝(小鼠肺重/小鼠体重)×100％；肺指数抑制率＝(感染模型组平均肺指数-实验组平均肺指数)/感染模型组平均肺指数×100％。
②肺悬液病毒血凝滴度测定
分别取治疗后第5天的各组小鼠肺，低温下置匀浆器研磨成匀浆，生理盐水稀释为10％的肺组织悬液，离心取上清，倍比稀释，按0.2ml/孔滴于滴定板上,每孔加入0.2ml 1％鸡红细胞悬液,混匀,置室温30min,观察记录血凝滴度。以红细胞凝集(++)时为終点，以悬液稀释倍数表示其滴度。
③肺组织形态学观察
分别取治疗后第5天的各组小鼠肺，肉眼观察记录肺脏大体病变情况。生理盐水中漂洗干净，用滤纸吸干，取一部分10％甲醛固定，石蜡包埋，切片，HE染色，显微镜下观察并拍照，如图4所示。
2.3实验结果及分析
(1)流感病毒和副流感病毒对小鼠半数致死量的测定结果
实验组昆明种小鼠分别被滴鼻感染不同浓度流感病毒、副流感病毒液30μL，感染第3天前3组(病毒浓度为10-1组、10-2组、10-3组)小鼠均出现不同程度的发病症状：耸毛、发抖、饮食减少等；第5天小鼠出现走路摇摆不定；第6天最高病毒浓度组小鼠开始出现死亡，其余各组于感染后第7天陆续出现死亡现象。观察14天结束后，统计各组小鼠死亡数目，结果见下表1-4、1-5。计算该流感病毒的LD50为稀释度10-2.9，副流感病毒的LD50为稀释度10-2.5。
表1-4  流感病毒半数致死量试验结果统计
Figure PCTCN2015094864-appb-000023
Karber法计算病毒的LD50。流感病毒的LogLD50如下：
Figure PCTCN2015094864-appb-000024
表1-5  副流感病毒半数致死量试验结果统计
Figure PCTCN2015094864-appb-000025
Karber法计算病毒的LD50。副流感病毒的LogLD50如下：
Figure PCTCN2015094864-appb-000026
(2)连翘脂素葡萄糖醛酸甲酯抗流感病毒和副流感病毒感染所致肺炎的作用结果
①肺指数测定
流感病毒和副流感病毒感染小鼠后，平均肺指数测定结果显示：与感染模型组比较，连翘脂素 葡萄糖醛酸衍生物(甲酯)浓度在3.25～13.0mg/kg/d范围内有一定保护作用,肺指数均明显降低；连翘脂素葡萄糖醛酸衍生物高剂量组对流感病毒和副流感病毒的疗效优于连翘脂素组(P<0.05)。试验结果见表1-6、1-7。
表1-6  连翘脂素葡萄糖醛酸衍生物对流感病毒感染小鼠肺指数和肺指数的抑制率(n＝3)
Figure PCTCN2015094864-appb-000027
与病毒对照组比较,*P<0.05，**P0.01；与连翘脂素组比较，#P<0.05，##P<0.01。
表1-7  连翘脂素葡萄糖醛酸衍生物对副流感病毒感染小鼠肺指数和肺指数抑制率的影(n＝3)
Figure PCTCN2015094864-appb-000028
与病毒对照组比较,*P<0.05，**P0.01；与连翘脂素组比较，#P<0.05，##P<0.01。
②肺悬液病毒血凝滴度测定
流感病毒和副流感病毒感染小鼠后，感染模型组肺组织病毒血凝滴度(InX)分别为32.40和33.11，不同浓度连翘脂素葡萄糖醛酸衍生物(甲酯)治疗5天后，肺组织病毒血凝滴度均有所下降，与感染模型组比较，差异有显著性,(P<0.01)；其中，连翘脂素葡萄糖醛酸衍生物中、高剂量组对流感、副流感病毒血凝滴度均明显低于模型组，抑制率均高于连翘脂素组，差异显著(P<0.05，p<0.01)。试验结果见表1-8、1-9。
表1-8  连翘脂素葡萄糖醛酸衍生物对流感病毒感染小鼠肺悬液血凝滴度的影响(n＝3)
Figure PCTCN2015094864-appb-000029
与病毒对照组比较,*P<0.05，**P<0.01；与连翘脂素组比较，#P<0.05，##P<0.01。
表1-9  连翘脂素葡萄糖醛酸衍生物对副流感病毒感染小鼠肺悬液血凝滴度的影响(n＝3)
Figure PCTCN2015094864-appb-000030
与病毒对照组比较,*P<0.05，**P<0.01；与连翘脂素组比较，#P<0.05，##P<0.01。
③肺组织学检测结果
由图4、5的试验结果可以看出：流感病毒和副流感病毒肺炎模型组小鼠肺脏大多数有充血，水肿病变，有些呈暗褐色外观的实变区，严重病例呈现棕红色的出血病灶。镜下可见，支气管、细支气管壁、肺泡壁等肺间质充血、水肿以及淋巴细胞、单核细胞浸润，肺泡壁增宽，肺泡呈炎症反应。流感病毒和副流感病毒肺炎小鼠模型经连翘脂素葡萄糖醛酸衍生物治疗后，各组鼠肺大体病变明显减轻，部分肺组织形态结构正常；与感染模型组相比，肺泡间隔较薄，肺泡壁和细支气管壁单个核细胞浸润数量较少，腔内无渗出，病变明显减轻。连翘脂素葡萄糖醛酸衍生物治疗副流感病毒肺炎的中、高剂量组与感染模型组相比，肺泡间隔明显较薄，单个核细胞浸润数量较少，腔内无渗出，病变明显减轻。
2.4结论
体内抗病毒试验结果显示，连翘脂素葡萄糖醛酸衍生物在3.25～13mg/kg/d的剂量范围对流感病毒和副流感病毒及所引起的小鼠病毒性肺炎具有较明显的抑制作用，能明显降低其肺指数和血凝滴度，肺组织病理学也有明显改善，与病毒模型对照组比较差异显著；且连翘脂素葡萄糖醛酸衍生物中、高剂量组疗效明显优于连翘脂素(*P<0.05或**P<0.01)，同时表现出优于利巴韦林和磷酸奥司他韦的趋势。

Claims (10)

  1. 一种连翘脂素葡萄糖醛酸衍生物，结构通式如下：
    Figure PCTCN2015094864-appb-100001
    其中，R基团选择H、Me、Na+、K+。
  2. 一种连翘脂素葡萄糖醛酸衍生物的制备方法，其特征是，包括如下顺序进行的步骤：
    1)连翘脂素与有机碱进行羟基活化反应，制得连翘脂素-有机碱配合物；
    2)将糖基供体、催化剂加入到有机溶剂中，混均匀制得糖苷化试剂，然后加入连翘脂素-有机碱配合物，进行糖苷化反应，制得糖苷化反应混合物；
    3)将糖苷化反应混合物加入到甲醇中，接着加入碱性化合物，进行脱酰基反应，然后加入pH调节剂，调节混合物溶液pH至4-8.5即得。
  3. 如权利要求2所述的方法，其特征是，步骤1)中所述有机碱选择N,N-二异丙基乙基胺或1,8-二氮杂二环十一碳-7-烯。
  4. 如权利要求2或3所述的制备方法，其特征是，步骤1)中所述羟基活化反应的反应时间为10～20分钟。
  5. 如权利要求2或3所述的制备方法，其特征是，步骤2)中所述糖基供体选择2,3,4-三-O-酰基-α-D-溴代吡喃葡萄醛酸酯。
  6. 如权利要求2或3所述的制备方法，其特征是，步骤2)中所述催化剂选择碳酸银。
  7. 如权利要求2或3所述的制备方法，其特征是，步骤2)中所述催化剂和糖基供体的摩尔比1：6.0～8.0。
  8. 如权利要求2或3所述的制备方法，其特征是，步骤2)中所述的糖苷化反应在惰性气体保护下进行。
  9. 如权利要求2或3所述的制备方法，其特征是，还包括步骤4)分离、纯化处理，对调节pH后的混合物溶液进行浓缩处理和硅胶柱层析处理。
  10. 如权利要求1所述连翘脂素葡萄糖醛酸衍生物的抗病毒应用。
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