CN106188176B - Phillygenol glucuronic acid derivative, preparation method and applications - Google Patents

Phillygenol glucuronic acid derivative, preparation method and applications Download PDF

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CN106188176B
CN106188176B CN201510320579.4A CN201510320579A CN106188176B CN 106188176 B CN106188176 B CN 106188176B CN 201510320579 A CN201510320579 A CN 201510320579A CN 106188176 B CN106188176 B CN 106188176B
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phillygenol
glucuronic acid
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樊宏宇
富力
姜人武
王凯乾
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Abstract

The present invention provides phillygenol glucuronic acid derivative, preparation method and its antiviral applications for being shown below etc.:

Description

Phillygenol glucuronic acid derivative, preparation method and applications
Technical field
The invention belongs to field of medicinal chemistry, specifically, the present invention relates to phillygenol glucuronic acid derivative and The pharmacological action of preparation method and this analog derivative in anti-virus aspect.
Background technique
Phillygenol is the sugared aglucon part of forsythin, also referred to as Fructus Forsythiae aglycon, is Oleaceae forsythia Fructus Forsythiae Main active, structure are shown below, modern pharmacological studies have shown that phillygenol has antiviral, anti-oxidant, reduction blood Rouge, remove free radical, it is antibacterial, antitumor, anti-inflammatory the effects of.
Phillygenol molecule is unstable, is easily oxidized, and molecular configuration is easy to happen change under acidic environment.By big Mouse enteral bacterium simulates the metabolism of forsythin the study found that being easily metabolized as new metabolin by intestinal flora.By containing phenols Structure medicament is metabolized the study found that the drug of phenolic hydroxy group structure is easily metabolized as the grape alditol of phenol by internal glycuronidase Acid derivative, most of glucuronic acid derivative have good activity.Such as qinghaosu glucuronic acid derivative (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), Edaravone glucuronic acid derivative (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), combretastatin A-1 glucuronic acid derivative (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), resveratrol glucuronic acid spreads out Biology (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) and curcumin glucuronic acid derivative (K.S.Psrvathy, M.Sc.University of Mysore.2009) etc..Therefore we design the glucuronic acid derivative of phillygenol, and carried out chemical synthesis and Pharmacological research.
Summary of the invention
It is derivative the technical problem to be solved by the invention is phillygenol glucuronic acid is prepared using chemical synthesis process Object.Phillygenol glucuronic acid derivative provided by the invention.In addition, the present invention also provides prepare phillygenol glucose The method of aldehydic acid derivative is suitble to industrial amplification production.
Firstly, the present invention provides the phillygenol glucuronic acid derivative as shown in formula (I),
Wherein, R group selects H, Me, Na, K.
Another aspect of the present invention provides a kind of preparation method of phillygenol glucuronic acid derivative, including following sequence Progress the step of:
1) phillygenol and organic base carry out it is hydroxy activated react, phillygenol-organic base complex is made；
2) glycosyl donor, catalyst are added in organic solvent, glycosylation reagent is made in mixing, and Fructus Forsythiae rouge is then added Element-organic base complex carries out glycosylation reaction, and glycosylation reaction mixture is made；
3) glycosylation reaction mixture is added in methanol, is subsequently added into alkali compounds, carry out the reaction of deacylation base, so After be added pH adjusting agent, adjust mixture solution pH to 4-8.5 to obtain the final product.
Wherein, the reaction temperature of hydroxy activated reaction described in step 1) is 0-20 DEG C, preferably 0-10 DEG C；Reaction time For 10-20min.
In particular, phillygenol and organic base are dissolved in organic solvent in step 1), under stirring described in progress Phillygenol-organic base complex is made in hydroxy activated reaction.
Especially, one of the organic solvent selection methylene chloride, toluene or pyridine, preferably methylene chloride.
In particular, one of the organic solvent selection anhydrous methylene chloride, dry toluene or anhydrous pyridine, preferably Anhydrous methylene chloride.
Especially, the ratio between the phillygenol quality and the volume of organic solvent (g/ml) be 1:40-50, preferably 1: 45。
Wherein, the organic base selects N, N- diisopropyl ethyl amine or 1,11 carbon -7- alkene of 8- diazabicylo.
In particular, the mol ratio of the phillygenol and organic base is 1:5-8, preferably 1:6.
Wherein, temperature > 0 DEG C of glycosylation reaction described in step 2), preferably 0-20 DEG C, preferably 5-15 DEG C, into one Preferably 10 DEG C of step；Reaction time is 4-15h, preferably 8-10h, further preferably 10h.
In particular, carrying out the glycosylation reaction under inert gas atmosphere.
Especially, the inert gas selection nitrogen, argon gas or helium, preferably nitrogen.
Wherein, the molar ratio of the glycosyl donor and phillygenol be 1-7:1, preferably 1.5~7:1, further preferably For 1.5:1.
In particular, the selection of glycosyl donor described in step 2) 2,3,4 ,-three-O- acyl-alpha-D- glucopyranosiduronic acid esters, Preferably tri--O- acetyl group-α-D- bromine of tri--O- benzoyl-α-D- bromo glucopyra aldehydic acid methyl esters of 2,3,4- or 2,3,4- For glucopyra aldehydic acid methyl esters；The organic solvent selects methylene chloride, chloroform, 1,2- dichloroethanes or toluene, preferably For methylene chloride.
During the present invention carries out glycosylation reaction, glycosyl donor 2,3,4 ,-three-O- acyl-alpha-D- glucopyra alditols The dosage of acid esters is few, and the yield of glycosylation product is low, and dosage increase then causes by-product to increase, glycosyl donor 2,3, and 4 ,-three- The molar ratio of O- acyl-alpha-D- glucopyranosiduronic acid ester and phillygenol is preferably 1.5~2.5:1.
Wherein, catalyst choice silver carbonate described in step 2).
In particular, the molar ratio of the catalyst and the glycosyl donor is 4-8:1, preferably 4-6:1, further preferably For 6:1.
The dosage of catalyst is low to lead to glycosyl donor 2,3,4, point of-three-O- acyl-alpha-D- glucopyranosiduronic acid esters Solution reduces yield；Catalyst amount is high, leads to three acyl group phillygenol glucuronic acid methyl esters (i.e. glycosylation reaction product) It decomposes, reduces yield.
In particular, at -20~0 DEG C hereinafter, and being under inert gas protection added to glycosyl donor, catalyst organic In solvent, it is mixed even, the obtained glycosylation reagent.
Especially, it is -10~0 DEG C in temperature, and has under inert gas protection been added to glycosyl donor, catalyst In solvent, it is mixed even, the obtained glycosylation reagent；Temperature is preferably 0 DEG C.
In particular, at -20~0 DEG C hereinafter, and phillygenol-organic base complex is added under inert gas protection Into the glycosylation reagent, the glycosylation reaction is then carried out again.
Especially, it is -10~0 DEG C in temperature, and adds phillygenol-organic base complex in inert gas shielding Enter into the glycosylation reagent, then carries out glycosylation reaction again；Temperature is preferably 0 DEG C.
The present invention prepares glycosylation reagent under conditions of lower temperature and inert gas shielding and has phillygenol- Machine alkali complex is added in glycosylation reagent, prevents catalyst inactivation, and low temperature guarantees that reaction is reacted according to SN2 approach, The reaction selectivity that ensure that catalyst improves the selectivity of reaction.
In particular, the inert gas selection nitrogen, argon gas or helium, preferably nitrogen.
Wherein, 0-10 DEG C of reaction temperature of the reaction of deacylation described in step 3), preferably 0-5 DEG C, further preferably It is 0 DEG C.
In particular, the alkali compounds selection sodium hydroxide, potassium hydroxide or sodium methoxide described in step 3)；The pH Regulator selects acetic acid, propionic acid or hydrochloric acid, preferably acetic acid.
Wherein, the molar ratio of alkali compounds and glycosyl donor described in step 2) is 3-5:1, preferably 3.5:1.
In particular, alkali compounds is soluble in water, after being prepared into alkaline compound solution, add, carries out described Decarboxylation reaction.
Especially, the mass percent concentration of the alkaline compound solution of preparation is 27-30%.
In particular, the pH value for adjusting mixture solution is 5-8, preferably pH value is 7.
In particular, further including step 4) after adjusting mixture solution pH value, mixture is separated, purification process.
In particular, further including step 4) separation, purification process, the pH mixture solution for being adjusted to center is carried out at concentration Reason and silica gel column chromatography processing.
Wherein, the separation, purification process are to carry out silica gel column chromatography to mixture.
In particular, selecting GF254 silica gel during the silica gel column chromatography；Eluant, eluent selects chloroform/methanol=9:1.
It is the structure of phillygenol glucuronic acid derivative by the compounds of this invention that the above method is prepared Formula is as follows:
Phillygenol glucuronic acid derivative is white solid at normal temperature, is dissolved in chloroform, ethyl alcohol.It opens up on the tlc plate It opens (chromatographic solution is chloroform/methanol 10:1, Rf 0.4) and is sprayed 10%H afterwards2SO4Aubergine is presented in ethanol reagent.
Further aspect of the present invention provides a kind of antiviral application of phillygenol glucuronic acid derivative.
Wherein, described antiviral for influenza virus, anti-parainfluenza virus, anti respiratory syncytial virus (RSV), anti-Coxsack Viral B3 (CVB3), anti-coxsackie virus A 16 (CoxA16), anti-enterovirus EV 71, anti-adenovirus (AdV), anti-herpes simplex Virus I-type (HSV-1).
Another aspect of the invention provides phillygenol glucuronic acid derivative in preparation antiviral drugs or health care product Application.
The present invention provides pharmaceutical compositions comprising phillygenol glucuronic acid derivative of the present invention and pharmacy Upper acceptable auxiliary material.
Herein, pharmaceutically acceptable auxiliary material refer to nontoxic solid-state, semisolid or liquid filler, diluent, carrier, PH adjusting agent, ionic strength adjustor, sustained release or controlled release agent, lapping or other pharmaceutical adjuncts.Used carrier can with it is corresponding Form of medication be adapted, auxiliary material known to a person skilled in the art can be used to be made into injection, (injection) freeze-dried powder, spray Mist agent, oral administration solution, oral administration mixed suspension, tablet, capsule, enteric coatel tablets, pill, pulvis, granule, sustained release or delay are released Put equal preparations.It is preferred that the phillygenol sulfate derivative of first aspect present invention is administered by injection or through alimentary canal mode, Therefore, pharmaceutical composition of the invention is preferably injection or the preparation through digesting canal drug administration, that is, is suitable for being configured to inject and passing through The auxiliary material that alimentary canal mode is administered is particularly preferred.Wherein, refer to that pharmaceutical preparation passes through patient herein " through digesting canal drug administration " Gastral administration mode, including oral, gastric infusion and bowel lavage administration etc., it is preferably oral, art technology such as can be used The auxiliary material that personnel are known is made into oral administration solution, oral administration mixed suspension, tablet, capsule, enteric coatel tablets, pill, pulvis, granule, holds The preparations such as continuous release or delay release；Wherein, the preparation of drug administration by injection is mainly injection and powder-injection.
The chemosynthesis reaction formula of phillygenol glucuronic acid derivative of the invention is as follows:
Wherein, structural formula A is phillygenol；Structure B is 2,3,4 ,-three-O- acyl-alpha-D- bromo glucopyranosiduronic acids Ester；Structure C is three acyl group phillygenol glucuronic acid methyl esters；Structure D is phillygenol glucuronic acid derivative.
Chemical synthesis phillygenol glucuronic acid method of the present invention advance and practical value be, raw material The catalyst of convenient sources, glycosylation reaction is cheap and easy to get, is decreased obviously preparation cost, and chemically synthesized product Fructus Forsythiae rouge The quality of plain glucuronic acid derivative is easily controllable, and the synthesis yield of product is high, suitable for large-scale industrialized production.
Detailed description of the invention
Fig. 1 is the nuclear magnetic resonance spectroscopy of phillygenol glucuronic acid methyl ester of the present invention；
The carbon-13 nmr spectra of Fig. 2 phillygenol glucuronic acid methyl ester of the present invention；
The high resolution mass spectrum of Fig. 3 phillygenol glucuronic acid methyl ester of the present invention；
Fig. 4 is Influenza Virus Pneumonia model mice pathologic slice, in which: A is normal mouse lung tissue；B is stream Influenza Virus Pneumonia Mice lung tissue；C is mouse of the Influenza Virus Pneumonia mouse model after the treatment of positive drug Ribavirin Lung tissue；D is that phillygenol glucuronic acid methyl ester high dose group treats the mouse lung tissue after Influenza Virus Pneumonia；E is Phillygenol glucuronic acid methyl ester middle dose group treats the mouse lung tissue after Influenza Virus Pneumonia；F is phillygenol grape Uronic acid methyl esters low dose group treats the mouse lung tissue after Influenza Virus Pneumonia；
Fig. 5 is parainfluenza virus pneumonia model mice pathologic slice, in which: A is normal mouse lung tissue；B is Parainfluenza virus pneumonia mouse lung tissue；C is parainfluenza virus pneumonia mouse model after the treatment of positive drug Ribavirin Mouse lung tissue；D is that phillygenol glucuronic acid methyl ester high dose group treats the mouse lung after parainfluenza virus pneumonia Tissue；E is that phillygenol glucuronic acid methyl ester middle dose group treats the mouse lung tissue after parainfluenza virus pneumonia；F is to connect Mouse lung tissue after sticking up rouge element glucuronic acid methyl ester low dose group treatment parainfluenza virus pneumonia.
Specific embodiment
The present invention is further described by the following examples, but these embodiments are illustrative of the invention, and should not be understood For any limitation on the scope of the present invention.In addition, the reagent, raw material in embodiment can be obtained by commercial channel, if any Not most place, can refer to the manufacturers instruction of organic synthesis guide, the guide of Drug Administration mechanism and corresponding instrument, reagent Deng.
Embodiment 1
1, phillygenol-organic base complex is prepared
Phillygenol (4g, 10.75mmol) is added to dry anhydrous methylene chloride under conditions of temperature is 10 DEG C In (180ml), organic base N, N- diisopropyl ethyl amine 11.26mL (64.5mmoL) are added after stirring and dissolving, stirs 10 minutes, Hydroxy activated reaction is carried out, obtained phillygenol-organic base complex solution is spare, the ratio between mole of phillygenol and organic base For 1:6；
The present invention control during the hydroxy activated reaction reaction temperature under conditions of 0-20 DEG C, is applicable in In the present invention, the embodiment of the present invention is illustrated for 10 DEG C.The ratio between mole of phillygenol and organic base removes in the present invention Except 1:6, the molar ratio of other 1:5-8 is suitable for the present invention.The embodiment of the present invention is illustrated by taking 1:6 as an example.
The hydroxy activated reaction is to be easier to carry out glycosylation reaction using alkali activated hydroxyl groups.
2, glycosylation reaction
2-1) under -10 DEG C and nitrogen atmosphere, by 2,3, tri--O- acetyl group α-D bromo glucopyranosiduronic acid first of 4- Ester 6.4g (16.125mmoL) and silver carbonate 17.78g (64.5mmoL) are dissolved in methylene chloride, are stirred evenly, are made glycosylation Reagent, wherein the ratio between mole of glycosyl donor and phillygenol is 1.5:1, the ratio between mole of catalyst silver carbonate and glycosyl donor For 4:1；
2-2) under -10 DEG C and nitrogen atmosphere, phillygenol-organic base complex solution is added to sugar while stirring In glycosidation reagent, 10 DEG C are then heated to, and under conditions of temperature remains 10 DEG C, carry out glycosylation reaction 10h, system Obtain glycosylation reaction mixture；
After organic base seizes the hydrogen of the hydroxyl of reaction substrate phillygenol, the reactive intermediate complex of generation encounters oxygen It can be oxidized, so that intermediate is lost the chance contacted with oxygen by inert gas shielding, guarantee that reaction is normally carried out.Inert gas In addition to nitrogen, helium, argon gas are suitable for the present invention.
3, deacylation base reacts
Under stirring, glycosylation reaction mixture is dissolved in methanol, is then added while stirring under conditions of 0 DEG C Enter the sodium hydroxide solution (8.36ml) that mass percent concentration is 27%, NaOH and 2,3, tri--O- acetyl group α-D bromo pyrrole of 4- The molar ratio of glucopyranoside aldehydic acid methyl esters is 3.5:1, and after carrying out deacylation base reaction 30min, acidic ph modifier acetic acid is added, The pH value of the mixture of deacylation base reaction is adjusted to 7；
It carries out that the methanol as solvent is added during deacylation reaction in the present invention, by glycosylation reaction mixture Dissolution；The alkalinity of the alkali (such as sodium hydroxide, potassium hydroxide, sodium methoxide) of addition will not only destroy glycosidic bond but also may be used as deacylation The alkali of glycosylation reaction removes acyl protecting groups, promotes the progress of glycosylation reaction.Deacylation base reaction time at least 30min, preferably For 30-45min.
The pH that acetic acid adjusts mixture is added in the present invention into the mixture after the reaction of deacylation base, neutralizes excessive alkali, Reaction is terminated, while acetic acid activity is moderate, will not destroy the glycosidic bond of generation, improves the yield of product.
4, separation, purification process
It is evaporated under reduced pressure, is concentrated under vacuum conditions using Rotary Evaporators, it is anti-to obtain deacylation base for evaporative removal solvent Answer mixture solid；Deacylation base reaction mixture sample is subjected to silica gel column chromatography separation, purification process, wherein eluant, eluent: chlorine Imitative/methanol=9:1；Column chromatography silica gel: GF254 silica gel；It obtains white solid (compound 1,4.89g), gross production rate 79.8%.
Compound 1 is dissolved in water, ethyl alcohol.Expansion (chromatographic solution is chloroform/methanol 3:1, Rf 0.4) is spraying afterwards on the tlc plate 10%H2SO4Aubergine is presented in ethanol reagent.
Compound 11H-NMR、13C-NMR, high resolution mass spectrum figure are as shown in Figs. 1-3.
In ESI-MS spectrum, m/z [M-Na]-It is 547.18210, molecular weight 570.52277.
1H-NMR (400MHz, d6- DMSO) as follows: 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) as follows: 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).
According to ESI-MS,1H-NMR and13The test data of C-NMR determines that compound 1 is phillygenol glucuronic acid Sodium, molecular formula are as follows: C27H31O12Na, English name: 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；Chinese name: (2R, 3R, 4R, 5S) -6- (5- ((1R, 4S) -4- (3,4- Dimethoxyphenyl) hexahydro a pair of horses going side by side [3,4-c] furans -1- base) -2- methoxyphenyl) -3,4,5- trihydroxy four Hydrogen -2H- pyrans -2- carboxylic acid sodium；Structural formula are as follows:
Embodiment 2
In addition to organic base 1,11 carbon -7- alkene (9.63mL, 64.5mmol) of 8- diazabicylo are added in step 1；Step 2 In glycosyl donor be 2,3, the quality of tri--O- benzoyl α-D bromo glucopyranosiduronic acid methyl esters of 4- be 43.89g The ratio between mole of (75.25mmoL), glycosyl donor and phillygenol is 7:1；Catalyst silver carbonate (124.5g, 0.4515mol), The ratio between mole of catalyst and glycosyl donor be 6:1 except, remaining is same as Example 1, be made white solid (compound 2, 3g), gross production rate 79.8%.
Compound 2 is white solid, is dissolved in water, ethyl alcohol.(chromatographic solution is chloroform/methanol 3:1, and Rf is for expansion on the tlc plate 0.4) it is sprayed 10%H afterwards2SO4Aubergine is presented in ethanol reagent.
In the ESI-MS spectrum of compound 2, m/z [M-Na]-It is 547.18210, molecular weight 570.52277.
Compound 21H-NMR、13C-NMR, IR are identical as compound 1 made from embodiment 1.
According to ESI-MS,1H-NMR and13The test data of C-NMR, IR determine that compound 2 is phillygenol grape alditol Sour sodium.
Embodiment 3
1, phillygenol-organic base complex is prepared
Phillygenol (4g, 10.75mmol) is added to dry anhydrous methylene chloride under conditions of temperature is 0 DEG C In (180ml), organic base 1,11 carbon -7- alkene 9.63mL (64.5mmol) of 8- diazabicylo, stirring are added after stirring and dissolving 20 minutes, hydroxy activated reaction is carried out, it is spare that phillygenol-organic base complex solution is made；
2, glycosylation reaction
2-1) under 0 DEG C and nitrogen atmosphere, by 2,3, tri--O- acetyl group α-D bromo glucopyranosiduronic acid methyl esters of 4- 6.4g (16.125mmoL) and silver carbonate 17.78g (64.5mmoL) are dissolved in methylene chloride, stir evenly, glycosylation examination is made Agent；
2-2) under 0 DEG C and nitrogen atmosphere, phillygenol-organic base complex solution is added to glucosides while stirring Change in reagent, be then heated to 20 DEG C, and under conditions of temperature remains 20 DEG C, carry out glycosylation reaction 10h, is made Glycosylation reaction mixture；
3, deacylation base reacts
Under stirring, glycosylation reaction mixture is dissolved in methanol, is then added while stirring under conditions of 5 DEG C Enter 28% potassium hydroxide solution (11.28ml), KOH and 2,3, tri--O- acetyl group α-D bromo glucopyranosiduronic acid methyl esters of 4- Molar ratio be 3.5:1, stirring, carry out deacylation base reaction 30min after, be added acidic ph modifier acetic acid, adjust deacylation base The pH value of the mixture of reaction is to 7；
4, separation, purification process
It is evaporated under reduced pressure, is concentrated under vacuum conditions using Rotary Evaporators, it is anti-to obtain deacylation base for evaporative removal solvent Answer mixture solid；Deacylation base reaction mixture sample is subjected to silica gel in column chromatography for separation, purification process, wherein eluant, eluent: Chloroform/methanol=9:1；Column chromatography silica gel: GF254 silica gel；It obtains white solid (compound 3,4.96g), gross production rate 81%.
Compound 3 is dissolved in water, ethyl alcohol.Expansion (chromatographic solution is chloroform/methanol 3:1, Rf 0.4) is spraying afterwards on the tlc plate 10%H2SO4Aubergine is presented in ethanol reagent.
In ESI-MS spectrum, m/z [M-K]-It is 547.18213, molecular weight 586.63185.
1H-NMR (400MHz, d6- DMSO) as follows: 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) as follows: 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).
According to ESI-MS,1H-NMR and13The test data of C-NMR determines that compound 3 is phillygenol glucuronic acid Potassium, Chinese name: (2R, 3R, 4R, 5S) -6- (5- ((1R, 4S) -4- (3,4- Dimethoxyphenyl) hexahydro a pair of horses going side by side [3,4-c] furans - 1- yl) -2- methoxyphenyl) -3,4,5- trihydroxy tetrahydro -2H- pyrans -2- carboxylic acid potassium；English name: 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；Molecular formula are as follows: C27H31O12K；Structural formula are as follows:
Embodiment 4
In addition to organic base N, N- diisopropyl ethyl amine (11.26mL, 64.5mmoL) are added in step 1, mixing time is 10min；Glycosyl donor in step 2 is 2,3, the quality of tri--O- benzoyl α-D bromo glucopyranosiduronic acid methyl esters of 4- be The ratio between mole of 43.89g (75.25mmoL), glycosyl donor and phillygenol is 7:1；Catalyst silver carbonate (124.5g, 0.4515mol), the ratio between mole of catalyst and glycosyl donor is 6:1, and glycosylation reaction temperature is 5 DEG C, reaction time 9h； It is adjusted except pH to 8 in step 3, remaining is same as Example 3, is made white solid (compound 4,5.14g), gross production rate 84%.
Compound 4 is dissolved in water, ethyl alcohol.Expansion (chromatographic solution is chloroform/methanol 3:1, Rf 0.4) is spraying afterwards on the tlc plate 10%H2SO4Aubergine is presented in ethanol reagent.
In the ESI-MS spectrum of compound 4, m/z [M-K]-It is 547.18213, molecular weight 586.63185.
Compound 41H-NMR、13C-NMR is identical as compound 3 made from embodiment 3.
According to ESI-MS,1H-NMR and13The test data of C-NMR determines that compound 4 is phillygenol glucuronic acid Potassium.
Embodiment 5
1, phillygenol-organic base complex is prepared
Phillygenol (4g, 10.75mmo) is added to dry anhydrous methylene chloride under conditions of temperature is 20 DEG C In (180ml), organic base N, N- diisopropyl ethyl amine 11.26mL (64.5mmoL) are added after stirring and dissolving, stirs 10 minutes, Hydroxy activated reaction is carried out, phillygenol-organic base complex solution is made, it is spare；
2, glycosylation reaction
2-1) under -10 DEG C and nitrogen atmosphere, by 2,3, tri--O- acetyl group α-D bromo glucopyranosiduronic acid first of 4- Ester 6.4g (16.125mmoL) and silver carbonate 17.78g (64.5mmoL) are dissolved in methylene chloride, are stirred evenly, are made glycosylation Reagent, wherein the ratio between mole of glycosyl donor and phillygenol is 1.5:1, the ratio between mole of catalyst silver carbonate and glycosyl donor For 4:1；
Glycosyl donor can also use other than 2,3, tri--O- acetyl group α-D bromo glucopyranosiduronic acid methyl esters of 4- 2,3, tri--O- benzoyl α-D bromo glucopyranosiduronic acid methyl esters of 4-.
2-2) under -10 DEG C and nitrogen atmosphere, phillygenol-organic base complex solution is added dropwise to while stirring In glycosylation reagent, it is then heated to 0 DEG C, and under conditions of temperature remains 0 DEG C, carries out glycosylation reaction 10h, system Obtain glycosylation reaction mixture；
3, deacylation base reacts
Under stirring, glycosylation reaction mixture is dissolved in methanol, is then added while stirring under conditions of 0 DEG C Enter the potassium hydroxide solution (11.7ml) that mass percent concentration is 27%, KOH and 2,3, tri--O- acetyl group α-D bromo pyrrole of 4- The molar ratio of glucopyranoside aldehydic acid methyl esters is 3.5:1, and acidic ph modifier is added after carrying out deacylation base reaction 30min in stirring Acetic acid adjusts the pH value of the mixture of deacylation base reaction to 5；
4, separation, purification process
It is evaporated under reduced pressure, is concentrated under vacuum conditions using Rotary Evaporators, it is anti-to obtain deacylation base for evaporative removal solvent Answer mixture solid；Deacylation base reaction mixture sample is subjected to silica gel in column chromatography for separation, purification process, wherein eluant, eluent: Chloroform/methanol=9:1；Column chromatography silica gel: GF254 silica gel；It obtains white solid (compound 5,5.08g), gross production rate 83%.
Compound 5 is dissolved in water, ethyl alcohol.Expansion (chromatographic solution is chloroform/methanol 3:1, Rf 0.4) is spraying afterwards on the tlc plate 10%H2SO4Aubergine is presented in ethanol reagent.
In ESI-MS spectrum, m/z [M+Na]+It is 571.17916, molecular weight 548.54109.
1H-NMR (400MHz, d6- DMSO) as follows: 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) as follows: 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).
According to ESI-MS,1H-NMR and13The test data of C-NMR determines that compound 5 is phillygenol glucuronic acid, Chinese name: (2R, 3R, 4R, 5S) -6- (5- ((1R, 4S) -4- (3,4- Dimethoxyphenyl) hexahydro a pair of horses going side by side [3,4-c] furans -1- Base) -2- methoxyphenyl) -3,4,5- trihydroxy tetrahydro -2H- pyrans -2- carboxylic acid；English name: (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-carboxylicacid；Molecular formula Are as follows: C27H32O12；Structural formula are as follows:
Embodiment 6
1, phillygenol-organic base complex is prepared
Phillygenol (4g, 10.75mmo) is added to dry anhydrous methylene chloride under conditions of temperature is 10 DEG C In (180ml), organic base 1,11 carbon -7- alkene 9.63mL (64.5mmo) of 8- diazabicylo, stirring 10 are added after stirring and dissolving Minute, hydroxy activated reaction is carried out, phillygenol-organic base complex solution is made, it is spare；
2, glycosylation reaction
2-1) under 0 DEG C and nitrogen atmosphere, by 2,3, tri--O- acetyl group α-D bromo glucopyranosiduronic acid methyl esters of 4- 6.4g (16.125mmoL) and silver carbonate 17.78g (64.5mmoL) are dissolved in methylene chloride, stir evenly, glycosylation examination is made Agent, wherein the ratio between mole of glycosyl donor and phillygenol is 1.5:1, and the ratio between mole of catalyst silver carbonate and glycosyl donor is 4:1；
Glycosyl donor can also use other than 2,3, tri--O- acetyl group α-D bromo glucopyranosiduronic acid methyl esters of 4- 2,3, tri--O- benzoyl α-D bromo glucopyranosiduronic acid methyl esters of 4-.
2-2) under 0 DEG C and nitrogen atmosphere, phillygenol-organic base complex solution is added dropwise to sugar while stirring In glycosidation reagent, 10 DEG C are then heated to, and under conditions of temperature remains 10 DEG C, carry out glycosylation reaction 10h, system Obtain glycosylation reaction mixture；
3, deacylation base reacts
Under stirring, glycosylation reaction mixture is dissolved in methanol, is then added while stirring under conditions of 0 DEG C Enter the sodium methoxide solution (10.16ml) that mass percent concentration is 30%, NaOCH3With 2,3, tri--O- acetyl group α-D bromo of 4- The molar ratio of glucopyranosiduronic acid methyl esters is 3.5:1, and stirring is added acid pH and adjusts after carrying out deacylation base reaction 30min Agent acetic acid adjusts the pH value of the mixture of deacylation base reaction to 7；
4, separation, purification process
It is evaporated under reduced pressure, is concentrated under vacuum conditions using Rotary Evaporators, it is anti-to obtain deacylation base for evaporative removal solvent Answer mixture solid；Deacylation base reaction mixture sample is subjected to silica gel in column chromatography for separation, purification process, wherein eluant, eluent: Chloroform/methanol=9:1；Column chromatography silica gel: GF254 silica gel；It obtains white solid (compound 6,5.33g), gross production rate 87%.
Compound 6 is dissolved in chloroform, ethyl alcohol.Expansion (chromatographic solution is chloroform/methanol 10:1, Rf 0.4) is sprayed afterwards on the tlc plate Mist 10%H2SO4Aubergine is presented in ethanol reagent.
In ESI-MS spectrum, m/z [M+Na]+It is 585.19481, molecular weight 562.56802.
1H-NMR (400MHz, d6- DMSO) as follows: 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) as follows: 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)。
According to ESI-MS,1H-NMR and13The test data of C-NMR determines that compound 6 is phillygenol glucuronic acid first Ester, Chinese name: (2R, 3R, 4R, 5S) -6- (5- ((1R, 4S) -4- (3,4- Dimethoxyphenyl) hexahydro a pair of horses going side by side [3,4-c] furans - 1- yl) -2- methoxyphenyl) -3,4,5- trihydroxy tetrahydro -2H- pyrans -2- carboxylate methyl ester；English name: (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；Molecular formula are as follows: C28H34O12；Structural formula are as follows:
1 In vitro antibacterial test of test example
1.1 test material
(1) drug
1. phillygenol glucuronic acid derivative: the phillygenol Sodium Glucuronate of the preparation of embodiment 1, embodiment 3 The Fructus Forsythiae of phillygenol glucuronic acid, the preparation of embodiment 6 prepared by the phillygenol glucuronic acid potassium of preparation, embodiment 5 Rouge element glucuronic acid methyl ester is produced by Dalian Fu Sheng natural drug development corporation, Ltd., is detected through two kinds of high performance liquid chromatography The measurement of device (UV detector, evaporative light scattering detector) area normalization method, purity is 99.3%, 99.2%, 99.7%, 99.6%；
2. positive control drug: ribavirin injection, colourless transparent liquid are produced by Henan Run Hong limited liability company, Batch number: 1206261, national drug standard: H19993553,100mg/ml test positive control medicine as this；Phosphoric acid is difficult to understand Si Tawei, Nat'l Pharmaceutical & Biological Products Control Institute, batch number: 101096-200901,100mg/ branch are positive as this test Control drug；Phillygenol, white powder, Dalian Fu Sheng natural drug development corporation, Ltd. production, through high performance liquid chromatography two Kind detector (UV detector and evaporative light scattering detector) area normalization method measurement, purity 99.2%.
Above-mentioned drug uses dissolved in purified water, filtration, degerming packing, and 4 DEG C spare, tests drug to be measured for this.
(2) cell strain Vero (African green monkey kidney cell cell) saves cell strain by preclinical medicine institute of Jilin University.
(3) Strain 1. strains of influenza viruses, parainfluenza virus strain, Respiratory Syncytial Virus(RSV) (RSV) strain: it is purchased from Chinese prevention Institute of Viruses of Academy of Medical Sciences；Coxsackie virus B 3 2. (CVB3) strain: being purchased from Chinese Academy of Sciences Wuhan virus institute；3. Coxsackie virus A16 (CoxA16) strain, enterovirus EV 71 strain: purchased from Japanese celestial platform state hospital；4. adenovirus (AdV): being purchased from Bethune doctor Paediatrics research department of the First Academy of university of section；5. herpes simplex virus I-type (HSV-1): being purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
(4) capital equipment and reagent
Biohazard Safety Equipment: BHC-1300 IIA/B3, AIRTECH；CO2 incubator: MCO-18AIC, SANYO；It is inverted micro- Mirror: CKX41, OLYMPUS；Electronic analytical balance: AR1140/C, DHAUS；Culture medium: DMEM, HyClone；Fetal calf serum: HyClone；Trypsase: Gibco；MTT:Sigma；DMSO: Tianjin Bei Lian fine chemicals development corporation, Ltd..
1.2 test method
(1) cell prepares
Vero cell secondary culture 1-2d is allowed in flakes, and boundary line is clear, when three-dimensional sense and strong diopter, is digested with pancreatin, There is needle point sample aperture to cell face, exhaust digestive juice, access milliliter culture solution dispels cell, counts, and (contains 10% with culture solution The DMEM of fetal calf serum) be diluted to about 5 107/L after, be inoculated in 96 well culture plates, grow up to single layer to cell.
(2) drug toxicity measures
Cell toxicity test: drug is diluted by concentration shown in table 1-1, is used for cytotoxicity assay.
Table 1-1 drug dilution is referring to table (unit: g/L)
The drug of the above-mentioned various concentration diluted with maintaining liquid (DMEM containing 2% fetal calf serum) is added dropwise mono- in Vero On confluent monolayer cells, every hole 0.2ml, 6 multiple holes of each concentration separately set 6 hole normal controls (Normal group that drug is not added) and 6 holes Blank control (culture solution), sets 37 DEG C, 5%CO2It cultivates, set inverted microscope observation CPE daily and records in incubator.72h Afterwards, 20 μ L (5mgmL of MTT solution is added in every hole- 1), continue to be incubated for 4h, inhale and abandon each hole culture solution, 100 μ L are added in every hole DMSO vibrates 5min, and 492nm measures OD value, calculates cell survival rate.In 18.0 statistical software of SPSS, by cell survival rate Probit regression analysis is carried out, calculates drug to the maximal non-toxic concentration (TC of Vero cell0) and half toxic concentration (TC50)。
(3) various virus TCID50Measurement
Various 10 times of virus progress are successively decreased and is diluted to 10-1,10-2,10-3, 10-4, 10-5, 10-6Different dilutions, sequentially It is inoculated on 96 well culture plate of Vero cell of single layer, every 100 μ L of hole, each 6 hole of dilution, while setting normal cell controls Group.37 DEG C are set, 5%CO2Middle incubation 2h abandons virus liquid, and every hole adds 100 μ L of cell maintenance medium immediately, sets 37 DEG C, 5%CO2Middle training It supports.Cytopathic effect is observed in beginning in 3rd day under the microscope, and the 7-8 days judgement results simultaneously make a record, 50% thin can make The highest dilution of positive lesion occurs for hilum as terminal, calculates virus titer with karber method.
Formula
TCID50: 50% tissue cytopathogenic dose
XM: the logarithm of viral maximum concentration dilution
D: the logarithm of dilution coefficient (multiple)
Σ pi: the summation of each dilution lesion percentage
(4) influence that drug acts on pathological changes caused by virus
The culture plate for having covered with cell monolayer is taken, inhales and abandons culture solution, with 100TCID50Corresponding virus attack amount inoculation is thin Born of the same parents, 37 DEG C, 5%CO2Incubator adsorbs 2h, and each medical fluid of certain concentration (maximal non-toxic concentration or so) is added, and every concentration 6 multiple Hole culture, 200 holes μ L/.If ribavirin injection and Oseltamivir phosphate are positive drug control group, while setting normal control Group (viral not dosing is not added) and virus control group (add virus but the control group of drug is not added), observation drug cause CPE to virus Influence.After 72h, with MTT colorimetric method, OD value is measured under 492nm wavelength, it is efficient (ER%) to calculate Antiviral Effect.? With the significant difference between more each Antiviral Effect effective percentage of ANOVA method in 18.0 statistical software of SPSS.
ER%=(drug-treated group mean OD value-virus control group mean OD value)/(cell controls group mean OD value- Virus control group mean OD value) × 100%
1.3 test result
(1) TCID of various viruses50
Parainfluenza virus:
Influenza virus:
CVB3:
HSV-1:
AdV:
RSV:
CoxA16:
EV71:
(2) drug toxicity measures
1) measurement of the drug to cytotoxicity
Maximal non-toxic concentration (TC of each drug to Vero cell0), half toxic concentration (TC50) it is shown in Table 1-2.
Table 1-2 drug cytotoxicity experimental result (unit: g/L)
2) drug is to pathological changes caused by virus protective effect result
The effective percentage and ANOVA method one-way analysis of variance that drug resists various viruses are as a result, see Table 1 for details -3.
Efficient (ER%) statistical form of table 1-3 Antiviral Effect
Note: compared with virus control group, P < 0.01 * P < 0.05, * *；With phillygenol ratio,#P < 0.05,##P<0.01。
Table 1-3 is the results show that phillygenol glucuronic acid derivative infected by influenza, parainfluenza virus, herpe simplex Virus I-form (HSV-I), the inhibiting rate of enterovirus EV 71 and effective percentage are more than 90%, and difference is bright compared with virus control group It is aobvious, there is statistical significance.Phillygenol glucuronic acid derivative is better than Fructus Forsythiae to the Anti-viral Treatment performance of a variety of viruses The advantage of rouge element, Ribavirin and Oseltamivir phosphate.
2 Antiviral assay in vivo of test example
2.1 experimental material
(1) experimental animal
Kunming mice, 18~22g of weight, half male and half female are purchased from Dalian Medical Univ's Experimental Animal Center, up-to-standard Card number: SCXK (13) 2012-0003.
(2) drug
1. phillygenol glucuronic acid derivative:
Phillygenol glucuronic acid methyl ester prepared by embodiment 6: white solid, the raw natural drug exploitation of Dalian richness are limited Company's production, is surveyed through high performance liquid chromatography two kinds of detectors (UV detector and evaporative light scattering detector) area normalization method It is fixed, purity 99.6%；
2. ribavirin injection, colourless transparent liquid are produced, batch number by Henan Run Hong limited liability company: 1206261, national drug standard: H19993553,100mg/ml test positive control medicine as this；
3. Oseltamivir phosphate, Nat'l Pharmaceutical & Biological Products Control Institute, batch number: 101096-200901,100mg/ Branch tests positive control medicine as this；
4. phillygenol, white powder, Dalian Fu Sheng natural drug development corporation, Ltd. production, through high performance liquid chromatography two Kind detector (UV detector and evaporative light scattering detector) area normalization method measurement, purity 99.2%.
Above-mentioned drug uses dissolved in purified water, filtration, degerming packing, and 4 DEG C spare, tests drug to be measured for this.
(2) detecting instrument, reagent
2.2 experimental method
(1) measurement of influenza virus and parainfluenza virus to mouse median lethal dose
10 times of ratios of two term of influenza virus and parainfluenza virus (cell pyrolysis liquid) are diluted to 10-1、10-2、10-3、10-4、10-5 The virus liquid of concentration.Taking Kunming mouse 120, influenza virus and parainfluenza virus group each 60 are randomly divided into 6 groups respectively, Ether light anesthesia mouse, collunarium infect different dilution virus liquid 0.03mL/ only.Blank control is set simultaneously, uses physiological saline Instead of viral suspension.It with dead and existence for observation index, observes daily, until metainfective 14 days.Death in infection for 24 hours It for nonspecific death, not counts, Karber method calculates virus liquid LD50.Calculation formula:[wherein: LD50: median lethal dose；XM: the logarithm of viral maximum concentration dilution；D: the logarithm of dilution coefficient (multiple)；Σ pi: each The summation of dilution lesion percentage].
(2) research of pneumonia caused by phillygenol glucuronic acid methyl ester resisiting influenza virus and parainfluenza virus infection
1) experimental animal and grouping
Kunming mice 960 of four week old are taken, 2 tests are carried out.Mouse 480 are taken, is randomly divided into 48 groups, every group 10 Only, the measurement for phillygenol glucuronic acid derivative influenza virus infected Lung Exponent and lung index tries It tests, 3 repetitions are tested, and take mouse 80 every time.Mouse 480 separately are taken, 48 groups is randomly divided into, every group 10, is used for Fructus Forsythiae rouge Plain glucuronic acid derivative tests the measurement of lung suspension viral hemoagglutination titre, and 3 repetitions are tested, and takes mouse 80 every time.
2) method is infected
An absorbent cotton is put into the beaker of 200~300mL size, being subsequently poured into suitable ether (becomes absorbent cotton It is wet), the beaker back-off equipped with absorbent cotton is come, mouse is put into and is anaesthetized, sees that mouse is on wires, hence it is evident that is in nothing When power sample, mouse is lain on the back, collunarium infects 15LD50Influenza virus and the nostril parainfluenza virus 0.03ml/, Normal group life It manages salt water and replaces viral suspension.
3) medication and dosage
Phillygenol glucuronic acid derivative group, Ribavirin and Oseltamivir phosphate control group, before infection Daystart routine gastric infusion, the high, medium and low dosage of phillygenol glucuronic acid derivative is respectively 13.0,8.0, 4.0mg/kg, Ribavirin positive drug dosage are 58.5mg/kg, and Oseltamivir phosphate positive drug dosage is 19.5mg/kg, phillygenol group dosage are 13.0mg/kg, and once a day, successive administration 5d, virus control group gavages phase The physiological saline of same volume.
4) observation index
1. Lung Exponent measures
The 5th day after mouse medication, first fasting water 8 hours plucks eyeball sacrificed by exsanguination animal after weighing, and opens thoracic cavity extraction Full lung twice with brine blots surface moisture content with filter paper, and electronic balance claims lung weight, and calculating is calculated according to the following formula Lung Exponent and lung index:
Lung Exponent=(mouse lung weight/mouse weight) × 100%；Lung index=(the infection model group lung that is averaged refers to Number-experimental group is averaged Lung Exponent)/infection model group is averaged Lung Exponent × 100%.
2. lung suspension viral hemoagglutination titer determination
The 5th day after treating each group mouse lung is taken respectively, and the underlying homogenizer of low temperature is ground into homogenate, and normal saline dilution is 10% lung tissue suspension, centrifuging and taking supernatant, doubling dilution, by the hole 0.2ml/ drop on titer plate, 0.2ml 1% is added in every hole Chicken erythrocyte suspension mixes, sets room temperature 30min, observe and record Hemagglutination titer.Using when erythrocyte agglutination (++) as terminal, with outstanding Liquid extension rate indicates its titre.
3. lung morphology is observed
The each group mouse lung for taking the 5th day after treating respectively visually observes record lungs substantially lesion situation.In physiological saline Rinsed clean is blotted with filter paper, and a part of 10% formaldehyde is taken to fix, and paraffin embedding, slice, HE dyeing, microscopically observation is simultaneously It takes pictures, as shown in Figure 4.
2.3 experimental results and analysis
(1) measurement result of influenza virus and parainfluenza virus to mouse median lethal dose
Experimental group Kunming mouse infects various concentration influenza virus, 30 μ L of parainfluenza virus venom, infection by collunarium respectively 3 groups of (virus concentrations 10 before 3rd day-1Group, 10-2Group, 10-3Group) there is different degrees of disease symptom in mouse: alarm hair, hair Tremble, diet reduce etc.；Walking, which occurs, in 5th day mouse to be played pendulum；6th day highest virus concentration group mouse starts death occur, There are the phenomena of mortality on the 7th day after infection successively in remaining each group.After observation 14 days, each group dead mouse number, knot are counted Fruit see the table below 1-4,1-5.Calculate the LD of the influenza virus50For dilution 10-2.9, the LD of parainfluenza virus50For dilution 10-2.5。
Table 1-4 influenza virus median lethal dose test result statistics
Karber method calculates the LD of virus50.The LogLD of influenza virus50It is as follows:
Table 1-5 parainfluenza virus median lethal dose test result statistics
Karber method calculates the LD of virus50。The LogLD of parainfluenza virus50It is as follows:
(2) exercising result of pneumonia caused by phillygenol glucuronic acid methyl ester resisiting influenza virus and parainfluenza virus infection
1. Lung Exponent measures
After influenza virus and parainfluenza virus infection mouse, average Lung Exponent measurement result is shown: with infection model group ratio Compared with, phillygenol glucuronic acid derivative (methyl esters) concentration has certain protective role within the scope of 3.25~13.0mg/kg/d, Lung Exponent is substantially reduced；The curative effect of phillygenol glucuronic acid derivative high dose group infected by influenza and parainfluenza virus Better than phillygenol group (P < 0.05).Test result is shown in Table 1-6,1-7.
The inhibiting rate of table 1-6 phillygenol glucuronic acid derivative influenza virus infected Lung Exponent and Lung Exponent (n=3)
Compared with virus control group, * P < 0.05, * * P0.01；Compared with phillygenol group,#P < 0.05,##P<0.01。
Table 1-7 phillygenol glucuronic acid derivative is to parainfluenza virus infection mouse Lung Exponent and lung index Shadow (n=3)
Compared with virus control group, * P < 0.05, * * P0.01；Compared with phillygenol group,#P < 0.05,##P<0.01。
2. lung suspension viral hemoagglutination titer determination
After influenza virus and parainfluenza virus infection mouse, infection model group lung tissue virus Hemagglutination titer (InX) is respectively For 32.40 and 33.11, after various concentration phillygenol glucuronic acid derivative (methyl esters) is treated 5 days, lung tissue viral hemoagglutination Titre is declined, and compared with infection model group, difference has conspicuousness, (P < 0.01)；Wherein, phillygenol glucuronic acid The middle and high dosage group of derivative is significantly lower than model group to influenza, parainfluenza virus Hemagglutination titer, and inhibiting rate is above Fructus Forsythiae rouge Plain group, significant difference (P < 0.05, p < 0.01).Test result is shown in Table 1-8,1-9.
Influence (the n of table 1-8 phillygenol glucuronic acid derivative influenza virus infected lung suspension Hemagglutination titer =3)
Compared with virus control group, P < 0.01 * P < 0.05, * *；Compared with phillygenol group,#P < 0.05,##P<0.01。
Influence of the table 1-9 phillygenol glucuronic acid derivative to parainfluenza virus infection mouse lung suspension Hemagglutination titer (n=3)
Compared with virus control group, P < 0.01 * P < 0.05, * *；Compared with phillygenol group,#P < 0.05,##P<0.01。
3. lung tissue testing result
By Fig. 4,5 test result it can be seen that influenza virus and parainfluenza virus pneumonia model group mouse lung are most Number has hyperemia, and oedema lesion, some are in the consolidation area of dun colored appearance, and the stigma of brownish red is presented in several cases.Under mirror As it can be seen that the hyperemia of the interstitial lungs such as bronchus, bronchiole wall, alveolar wall, oedema and lymphocyte, monocyte infiltration, alveolar Wall is broadening, and alveolar is in inflammatory reaction.Influenza virus and parainfluenza virus pneumonia mouse model spread out through phillygenol glucuronic acid After biological therapy, each group mouse lung substantially lesion is substantially reduced, and lung tissue segment morphosis is normal；Compared with infection model group, Alveolar septum is relatively thin, and alveolar wall and bronchiole wall mononuclear cells infiltration negligible amounts, intracavitary no exudation, lesion obviously subtract Gently.The middle and high dosage group of phillygenol glucuronic acid derivatives for treatment parainfluenza virus pneumonia is compared with infection model group, lung It is substantially reduced that bubble is spaced obviously relatively thin, mononuclear cells infiltration negligible amounts, intracavitary no exudation, lesion.
2.4 conclusion
Antiviral assay in vivo the results show that phillygenol glucuronic acid derivative 3.25~13mg/kg/d agent Measuring range infected by influenza and parainfluenza virus and caused Mouse Virus Pneumonia has obvious inhibiting effect, can be bright Aobvious its Lung Exponent of reduction and Hemagglutination titer, pathologic state colony also have clear improvement, aobvious with virus model control group comparing difference It writes；And the middle and high dosage group curative effect of phillygenol glucuronic acid derivative be substantially better than phillygenol (P < 0.05 * or * * P < 0.01), while the trend for being better than Ribavirin and Oseltamivir phosphate is shown.

Claims (8)

1. a kind of phillygenol glucuronic acid derivative, general structure are as follows:
Wherein, R group selects Me, Na, K.
2. a kind of preparation method of phillygenol glucuronic acid derivative as described in claim 1, characterized in that including as follows The step of sequence carries out:
1) phillygenol and organic base carry out it is hydroxy activated react, phillygenol-organic base complex is made, wherein described to have Machine alkali selects N, 11 carbon -7- alkene of N- diisopropyl ethyl amine or 1,8- diazabicylo；
2) glycosyl donor, catalyst are added in organic solvent, be mixed it is even glycosylation reagent is made, then be added Fructus Forsythiae rouge Element-organic base complex carries out glycosylation reaction, and glycosylation reaction mixture is made, wherein the glycosyl donor is 2,3,4- Three-O- acyl-alpha-D- bromo glucopyra aldehydic acid esters；
3) glycosylation reaction mixture is added in methanol, is subsequently added into alkali compounds, carry out the reaction of deacylation base, then plus Enter pH adjusting agent, adjust mixture solution pH to 4-8.5 to obtain the final product.
3. preparation method as claimed in claim 2, characterized in that the reaction time of hydroxy activated reaction described in step 1) is 10~20 minutes.
4. preparation method as claimed in claim 2 or claim 3, characterized in that catalyst choice silver carbonate described in step 2).
5. preparation method as claimed in claim 2 or claim 3, characterized in that catalyst described in step 2) and glycosyl donor rub You are than 1:6.0~8.0.
6. preparation method as claimed in claim 2 or claim 3, characterized in that glycosylation reaction described in step 2) is in indifferent gas Body protection is lower to be carried out.
7. preparation method as claimed in claim 2 or claim 3, characterized in that further include step 4) separation, purification process, to adjusting Mixture solution after pH carries out concentration and silica gel column chromatography processing.
8. application of the phillygenol glucuronic acid derivative as described in claim 1 in preparation antiviral drugs.
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