CN106188176A - The preparation method and applications of phillygenol glucal acid derivative - Google Patents

Abstract

The invention provides preparation method and the antiviral application thereof etc. of the phillygenol glucal acid derivative being shown below.Wherein, R is H, Me, Na⁺、K⁺Deng.

Description

The preparation method and applications of phillygenol glucal acid derivative

Technical field

The invention belongs to medicinal chemistry art, specifically, the present invention relates to phillygenol glucal acid derivative and preparation method thereof, and this analog derivative is in the pharmacological action of anti-virus aspect.

Background technology

Phillygenol, sugared aglucon part for phillyrin, also referred to as Fructus Forsythiae aglycon, main active for Oleaceae forsythia Fructus Forsythiae, its structure is shown below, modern pharmacological research shows, phillygenol has antiviral, antioxidation, reduction blood fat, removes the effect such as free radical, antibacterial, antitumor, antiinflammatory.

Phillygenol molecule is unstable, the most oxidized, and molecular configuration is susceptible to change under sour environment.Found by the metabolism research of rat intestinal flora simulation phillyrin, be easily new metabolite by intestinal flora metabolism.By finding containing phenol type structure drug metabolism study, the medicine of phenolic hydroxy group structure is easily by the glucal acid derivative that internal glucuronic acid enzymes metabolism is phenol, and major part glucal acid derivative has good activity.nullSuch as arteannuin glucal acid derivative (Efficient Preparations of the β-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 glucal acid derivative (Synthesis of the metabolites of a free radical scavenger edaravone (MCI-186,Radicut^TMnull).Kazutoshi Watanabe,Masao Taniguchi,Masaki Shinoda.Redox Report,Vol.8,No.3,2003，157-161)、Combretastatin A-1 glucal 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 glucal acid derivative (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) with curcumin glucal acid derivative (K.S.Psrvathy, M.Sc.University of Mysore.2009) etc..Therefore we design the glucal acid derivative of phillygenol, and have carried out chemosynthesis and pharmacological research.

Summary of the invention

Solving the technical problem that of the present invention is to use chemical synthesis process to prepare phillygenol glucal acid derivative.The phillygenol glucal acid derivative that the present invention provides.It addition, present invention also offers the method preparing phillygenol glucal acid derivative, it is suitable for industrial amplification production.

First, the invention provides the phillygenol glucal acid derivative as shown in formula I,

Wherein, R group selects H, Me, Na+, K+.

Another aspect of the present invention provides the preparation method of a kind of phillygenol glucal acid derivative, the step carried out including order below:

1) phillygenol and organic base carry out hydroxy activated reaction, prepare phillygenol-organic base coordination compound；

2) joining in organic solvent by glycosyl donor, catalyst, mixing prepares glycosylation reagent, is subsequently adding phillygenol-organic base coordination compound, carries out glycosylation reaction, prepares glycosylation reaction mixture；

3) glycosylation reaction mixture is joined in methanol, be subsequently added into alkali compounds, carry out deacylated tRNA base reaction, be subsequently adding pH adjusting agent, regulate mixture solution pH to 4-8.5 and get final product.

Wherein, step 1) described in the reaction temperature of hydroxy activated reaction be 0-20 DEG C, preferably 0-10 DEG C；Response time is 10-20min.

Particularly, step 1) in phillygenol and organic base are dissolved in organic solvent, under stirring, carry out described hydroxy activated reaction, prepare phillygenol-organic base coordination compound.

Especially, described organic solvent selects the one in dichloromethane, toluene or pyridine, preferably dichloromethane.

Particularly, described organic solvent selects the one in anhydrous methylene chloride, dry toluene or anhydrous pyridine, preferably anhydrous methylene chloride.

Especially, described phillygenol quality is 1:40-50, preferably 1:45 with the ratio (g/ml) of the volume of organic solvent.

Wherein, described organic base selects N, N-diisopropyl ethyl amine or 1,8-diazabicylo 11 carbon-7-alkene.

Particularly, described phillygenol is 1:5-8, preferably 1:6 with the mol ratio of organic base.

Wherein, step 2) described in the temperature of glycosylation reaction > 0 DEG C, preferably 0-20 DEG C, preferably 5-15 DEG C, more preferably 10 DEG C；Response time is 4-15h, preferably 8-10h, more preferably 10h.

Particularly, described glycosylation reaction is carried out under inert gas atmosphere.

Especially, described noble gas selects nitrogen, argon or helium, preferably nitrogen.

Wherein, the mol ratio of described glycosyl donor and phillygenol is 1-7:1, preferably 1.5～7:1, more preferably 1.5:1.

Particularly, step 2) described in glycosyl donor select 2,3,4;-three-O-acyl-alpha-D-glucopyranosiduronic acid esters, preferably 2,3; 4-tri--O-benzoyl-α-D-bromo glucopyra aldehydic acid methyl ester or 2,3,4-tri--O-acetyl group-α-D-bromo glucopyra aldehydic acid methyl ester；Described organic solvent selects dichloromethane, chloroform, 1,2-dichloroethanes or toluene, preferably dichloromethane.

During the present invention carries out glycosylation reaction; glycosyl donor 2; 3,4, the consumption of-three-O-acyl-alpha-D-glucopyranosiduronic acid esters is few; the yield of glycosylation product is low; consumption increase then causes by-product to increase, glycosyl donor 2,3; 4, the mol ratio of-three-O-acyl-alpha-D-glucopyranosiduronic acid esters and phillygenol is preferably 1.5～2.5:1.

Wherein, step 2) described in catalyst choice Disilver carbonate.

Particularly, described catalyst is 4-8:1, preferably 4-6:1, more preferably 6:1 with the mol ratio of described glycosyl donor.

The low glycosyl donor 2 that causes of the consumption of catalyst, the decomposition of 3,4 ,-three-O-acyl-alpha-D-glucopyranosiduronic acid esters, reduces productivity；Catalyst amount is high, causes the decomposition of three acyl group phillygenol glucuronic acid methyl esters (i.e. glycosylation reaction product), reduces productivity.

Particularly, below-20～0 DEG C, and under inert gas shielding, glycosyl donor, catalyst are joined in organic solvent, be mixed even, prepare described glycosylation reagent.

Especially, it is-10～0 DEG C in temperature, and under inert gas shielding, glycosyl donor, catalyst is joined in organic solvent, be mixed even, prepare described glycosylation reagent；Temperature is preferably 0 DEG C.

Particularly, below-20～0 DEG C, and under inert gas shielding, phillygenol-organic base coordination compound is joined in described glycosylation reagent, carry out described glycosylation reaction the most again.

Especially, it is-10～0 DEG C in temperature, and in inert gas shielding, phillygenol-organic base coordination compound is joined in described glycosylation reagent, carry out glycosylation reaction the most again；Temperature is preferably 0 DEG C.

The present invention prepares glycosylation reagent under conditions of lower temperature and inert gas shielding and joins in glycosylation reagent by phillygenol-organic base coordination compound; prevent catalysqt deactivation; low temperature ensures that reaction is reacted according to SN2 approach; ensure that the reaction selectivity of catalyst, improve the selectivity of reaction.

Particularly, described noble gas selects nitrogen, argon or helium, preferably nitrogen.

Wherein, step 3) described in reaction temperature 0-10 DEG C of deacylation reaction, preferably 0-5 DEG C, more preferably 0 DEG C.

Particularly, step 3) described in described alkali compounds select sodium hydroxide, potassium hydroxide or Feldalat NM；Described pH adjusting agent selects acetic acid, propanoic acid or hydrochloric acid, preferably acetic acid.

Wherein, alkali compounds and step 2) described in the mol ratio of glycosyl donor be 3-5:1, preferably 3.5:1.

Particularly, alkali compounds is soluble in water, after being prepared as alkaline compound solution, add, carry out described decarboxylation reaction.

Especially, the mass percent concentration of the alkaline compound solution of preparation is 27-30%.

Particularly, regulation mixture solution pH value be 5-8, preferably pH value be 7.

Particularly, also include step 4) after regulation mixture solution pH value, mixture is separated, purification process.

Particularly, also include step 4) separate, purification process, the mixture solution of pH regulator to center is carried out concentration and silica gel column chromatography processes.

Wherein, described separation, purification process are that mixture is carried out silica gel column chromatography.

Particularly, GF254 silica gel is selected during described silica gel column chromatography；Eluant selects chloroform/methanol=9:1.

The compounds of this invention prepared by said method be phillygenol glucal acid derivative structural formula as follows:

Phillygenol glucal acid derivative is white solid at normal temperatures, is dissolved in chloroform, ethanol.Spray 10%H afterwards to launch (chromatographic solution is chloroform/methanol 10:1, and Rf is 0.4) on the tlc plate₂SO₄-ethanol reagent presents aubergine.

Further aspect of the present invention provides the antiviral application of a kind of phillygenol glucal acid derivative.

Wherein, described antiviral is influenza virus, anti-parainfluenza virus, anti respiratory syncytial virus (RSV), anti-Coxsackie B virus 3 (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 the application in preparation antiviral drugs or health product of the phillygenol glucal acid derivative.

The invention provides pharmaceutical composition, it includes phillygenol glucal acid derivative of the present invention, and pharmaceutically acceptable adjuvant.

In this article, pharmaceutically acceptable adjuvant refers to nontoxic solid-state, semisolid or liquid filler, diluent, carrier, pH adjusting agent, ionic strength adjustor, slow release or controlled release agent, lapping or other pharmaceutical adjuncts.Used carrier can adapt with corresponding form of medication, and the adjuvant of dawn known to those skilled in the art can be used to be made into the preparations such as injection, (injection) lyophilized powder, spray, oral administration solution, oral administration mixed suspension, tablet, capsule, enteric coatel tablets, pill, powder, granule, sustained release or delay release.The preferably phillygenol sulfate derivative of first aspect present invention by injection or is administered through digestive tract mode, therefore, the pharmaceutical composition of the present invention is preferably injection or the preparation being administered through digestive tract, is i.e. suitable to be configured to injection and the adjuvant being administered through digestive tract mode is particularly preferred.Wherein,-refer to that the administering mode in people digest road is passed through in pharmaceutical preparation in this article through digestive tract administration ‖, including being administered orally, gastric infusion and coloclysis administration etc., it is the most oral, as the adjuvant of dawn known to those skilled in the art can be used to be made into the preparations such as oral administration solution, oral administration mixed suspension, tablet, capsule, enteric coatel tablets, pill, powder, granule, sustained release or delay release；Wherein, the preparation of drug administration by injection is mainly injection and injectable powder.

The chemosynthesis reaction formula of the phillygenol glucal acid derivative of the present invention is as follows:

Wherein, structural formula A is phillygenol；Structure B is 2,3,4 ,-three-O-acyl-alpha-D-bromo glucopyranosiduronic acid esters；Structure C is three acyl group phillygenol glucuronic acid methyl esters；Structure D is phillygenol glucal acid derivative.

Advance and the practical value of chemosynthesis phillygenol glucuronic acid method of the present invention are, raw material sources are convenient, the catalyst of glycosylation reaction is cheap and easy to get, preparation cost is made to be decreased obviously, and the quality of chemically synthesized product phillygenol glucal acid derivative is easily controllable, the comprehensive yield of product is high, large-scale production suitable for industrialized.

Accompanying drawing explanation

Fig. 1 is the proton nmr spectra of phillygenol glucuronic acid methyl ester of the present invention；

The carbon-13 nmr spectra of Fig. 2 phillygenol of the present invention glucuronic acid methyl ester；

The high resolution mass spectrum of Fig. 3 phillygenol of the present invention glucuronic acid methyl ester；

Fig. 4 is the section of Influenza Virus Pneumonia model mice pathologic, wherein: A is normal mouse lung tissue；B is Influenza Virus Pneumonia mouse lung tissue；C is the lung tissue of Influenza Virus Pneumonia mouse model mice after the treatment of positive drug ribavirin；D is the mouse lung tissue after phillygenol glucuronic acid methyl ester high dose group treatment Influenza Virus Pneumonia；E is the mouse lung tissue in phillygenol glucuronic acid methyl ester after dosage group treatment Influenza Virus Pneumonia；F is the mouse lung tissue after phillygenol glucuronic acid methyl ester low dose group treatment Influenza Virus Pneumonia；

Fig. 5 is the section of parainfluenza virus pneumonia model mice pathologic, wherein: A is normal mouse lung tissue；B is parainfluenza virus pneumonia mouse lung tissue；C is the lung tissue of parainfluenza virus pneumonia mouse model mice after the treatment of positive drug ribavirin；D is the mouse lung tissue after phillygenol glucuronic acid methyl ester high dose group treatment parainfluenza virus pneumonia；E is the mouse lung tissue in phillygenol glucuronic acid methyl ester after dosage group treatment parainfluenza virus pneumonia；F is the mouse lung tissue after phillygenol glucuronic acid methyl ester low dose group treatment parainfluenza virus pneumonia.

Detailed description of the invention

Further describe the present invention by the following examples, but these embodiments are illustrative of the invention, and should not be construed as any limitation on the scope of the present invention.It addition, reagent, raw material in embodiment can be obtained by commercial channel, if any not most part, it is referred to organic synthesis guide, the guide of Drug Administration mechanism and corresponding instrument, the manufacturers instruction etc. of reagent.

Embodiment 1

1, phillygenol-organic base coordination compound is prepared

By phillygenol (4g under conditions of temperature is 10 DEG C, 10.75mmol) join in dry anhydrous methylene chloride (180ml), organic base N is added after stirring and dissolving, N-diisopropyl ethyl amine 11.26mL (64.5mmoL), stir 10 minutes, carry out hydroxy activated reaction, prepare phillygenol-organic base complex solution standby, phillygenol and organic base mole ratio be 1:6；

The present invention carries out controlling during described hydroxy activated reaction reaction temperature under conditions of 0-20 DEG C, is all applicable to the present invention, and the embodiment of the present invention illustrates as a example by 10 DEG C.In the present invention phillygenol and organic base mole ratio in addition to 1:6, the mol ratio of other 1:5-8 is all applicable to the present invention.The embodiment of the present invention illustrates as a example by 1:6.

Described hydroxy activated reaction is to utilize alkali activated hydroxyl groups, makes glycosylation reaction be easier to make for.

2, glycosylation reaction

2-1) under-10 DEG C and nitrogen atmosphere; 2,3,4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester 6.4g (16.125mmoL) and Disilver carbonate 17.78g (64.5mmoL) are dissolved in dichloromethane; stir; prepare glycosylation reagent; wherein glycosyl donor and phillygenol mole ratio be 1.5:1, catalyst Disilver carbonate and glycosyl donor mole ratio be 4:1；

2-2) under-10 DEG C and nitrogen atmosphere, while stirring phillygenol-organic base complex solution is joined in glycosylation reagent, be then heated to 10 DEG C, and under conditions of temperature remains 10 DEG C, carry out glycosylation reaction 10h, prepare glycosylation reaction mixture；

After the hydrogen of the hydroxyl that organic base seizes reaction substrate phillygenol, the reactive intermediate coordination compound of generation runs into oxygen can be oxidized, makes intermediate lose the chance contacted with oxygen by inert gas shielding, it is ensured that reaction is normally carried out.In addition to nitrogen, helium, argon are all applicable to the present invention to noble gas.

3, deacylated tRNA base reaction

Under stirring; glycosylation reaction mixture is dissolved in methanol; then under conditions of 0 DEG C, add the sodium hydroxide solution (8.36ml) that mass percent concentration is 27% while stirring; NaOH and 2,3, the mol ratio of 4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester be 3.5:1; after carrying out deacylated tRNA base reaction 30min; adding acidic ph modifier acetic acid, the pH value of the mixture of regulation deacylated tRNA base reaction is to 7；

Add the methanol as solvent during the present invention carries out deacylation reaction, glycosylation reaction mixture is dissolved；The alkalescence of the alkali (such as sodium hydroxide, potassium hydroxide, Feldalat NM) added not only will not destroy glycosidic bond but also can serve as the alkali of deacylation reaction, removes acyl protecting groups, promotes the carrying out of glycosylation reaction.Deacylated tRNA base response time at least 30min, preferably 30-45min.

Adding the pH of acetic acid regulation mixture in the present invention in the reacted mixture of deacylated tRNA base, neutralize the alkali of excess, terminate reaction, acetic acid activity is moderate simultaneously, will not destroy the glycosidic bond of generation, improves the yield of product.

4, separation, purification process

Rotary Evaporators is used to carry out decompression distillation under vacuum conditions, concentrate, evaporative removal solvent, it is thus achieved that deacylated tRNA base reactant mixture solid；Deacylated tRNA base reactant mixture sample is carried out silica gel column chromatography separation, purification process, wherein, eluant: chloroform/methanol=9:1；Column chromatography silica gel: GF254 silica gel；Obtain white solid (compound 1,4.89g), gross production rate 79.8%.

Compound 1 is dissolved in water, ethanol.Spray 10%H afterwards to launch (chromatographic solution is chloroform/methanol 3:1, and Rf is 0.4) on the tlc plate₂SO₄-ethanol reagent presents aubergine.

Compound 1¹H-NMR、¹³C-NMR, high resolution mass spectrum, infrared spectrogram are as Figure 1-4.

In ESI-MS spectrum, m/z [M-Na]^-Being 547.18210, molecular weight is 570.52277.

¹H-NMR (400MHz, d₆null-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, d₆null-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, determine that compound 1 is phillygenol D-Glucuronic acid sodium salt, and molecular formula is: 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] furan-1-base)-2-methoxyphenyl)-3,4,5-trihydroxy tetrahydrochysene-2H-pyrans-2-carboxylic acid sodium；Structural formula is:

Embodiment 2

Except step 1 adds organic base 1,8-diazabicylo 11 carbon-7-alkene (9.63mL, 64.5mmol)；Glycosyl donor in step 2 is 2,3, the quality of 4-tri--O-benzoyl α-D bromo glucopyranosiduronic acid methyl ester be 43.89g (75.25mmoL), glycosyl donor and phillygenol mole ratio be 7:1；Catalyst Disilver carbonate (124.5g, 0.4515mol), catalyst and glycosyl donor mole ratio be 6:1 outside, remaining is same as in Example 1, prepares white solid (compound 2,3g), gross production rate 79.8%.

Compound 2 is white solid, is dissolved in water, ethanol.Spray 10%H afterwards to launch (chromatographic solution is chloroform/methanol 3:1, and Rf is 0.4) on the tlc plate₂SO₄-ethanol reagent presents aubergine.

In the ESI-MS spectrum of compound 2, m/z [M-Na]^-Being 547.18210, molecular weight is 570.52277.

Compound 2¹H-NMR、¹³The compound 1 that C-NMR, IR prepare with embodiment 1 is identical.

According to ESI-MS,¹H-NMR and¹³The test data of C-NMR, IR, determine that compound 2 is for phillygenol D-Glucuronic acid sodium salt.

Embodiment 3

1, phillygenol-organic base coordination compound is prepared

By phillygenol (4g under conditions of temperature is 0 DEG C, 10.75mmol) join in dry anhydrous methylene chloride (180ml), organic base 1 is added after stirring and dissolving, 8-diazabicylo 11 carbon-7-alkene 9.63mL (64.5mmol), stir 20 minutes, carry out hydroxy activated reaction, prepare phillygenol-organic base complex solution standby；

2, glycosylation reaction

2-1) under 0 DEG C and nitrogen atmosphere; 2,3,4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester 6.4g (16.125mmoL) and Disilver carbonate 17.78g (64.5mmoL) are dissolved in dichloromethane; stir, prepare glycosylation reagent；

2-2) under 0 DEG C and nitrogen atmosphere, while stirring phillygenol-organic base complex solution is joined in glycosylation reagent, be then heated to 20 DEG C, and under conditions of temperature remains 20 DEG C, carry out glycosylation reaction 10h, prepare glycosylation reaction mixture；

3, deacylated tRNA base reaction

Under stirring; glycosylation reaction mixture is dissolved in methanol; then under conditions of 5 DEG C, add the potassium hydroxide solution (11.28ml) of 28% while stirring; KOH and 2,3, the mol ratio of 4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester be 3.5:1; stirring; after carrying out deacylated tRNA base reaction 30min, adding acidic ph modifier acetic acid, the pH value of the mixture of regulation deacylated tRNA base reaction is to 7；

4, separation, purification process

Rotary Evaporators is used to carry out decompression distillation under vacuum conditions, concentrate, evaporative removal solvent, it is thus achieved that deacylated tRNA base reactant mixture solid；Deacylated tRNA base reactant mixture sample is carried out silica gel in column chromatography for separation, purification process, wherein, eluant: chloroform/methanol=9:1；Column chromatography silica gel: GF254 silica gel；Obtain white solid (compound 3,4.96g), gross production rate 81%.

Compound 3 is dissolved in water, ethanol.Spray 10%H afterwards to launch (chromatographic solution is chloroform/methanol 3:1, and Rf is 0.4) on the tlc plate₂SO₄-ethanol reagent presents aubergine.

In ESI-MS spectrum, m/z [M-K]^-Being 547.18213, molecular weight is 586.63185.

¹H-NMR (400MHz, d₆null-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, d₆null-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, determine that compound 3 is for 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] furan-1-base)-2-methoxyphenyl)-3,4,5-trihydroxy tetrahydrochysene-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 is: C₂₇H₃₁O₁₂K；Structural formula is:

Embodiment 4

Except adding organic base N in step 1, N-diisopropyl ethyl amine (11.26mL, 64.5mmoL), mixing time is 10min；Glycosyl donor in step 2 is 2,3, the quality of 4-tri--O-benzoyl α-D bromo glucopyranosiduronic acid methyl ester be 43.89g (75.25mmoL), glycosyl donor and phillygenol mole ratio be 7:1；Catalyst Disilver carbonate (124.5g, 0.4515mol), catalyst and glycosyl donor mole ratio be 6:1, glycosylation reaction temperature is 5 DEG C, and the response time is 9h；Regulating outside pH to 8 in step 3, remaining is same as in Example 3, prepares white solid (compound 4,5.14g), gross production rate 84%.

Compound 4 is dissolved in water, ethanol.Spray 10%H afterwards to launch (chromatographic solution is chloroform/methanol 3:1, and Rf is 0.4) on the tlc plate₂SO₄-ethanol reagent presents aubergine.

In the ESI-MS spectrum of compound 4, m/z [M-K]^-Being 547.18213, molecular weight is 586.63185.

Compound 4¹H-NMR、¹³The compound 3 that C-NMR prepares with embodiment 3 is identical.

According to ESI-MS,¹H-NMR and¹³The test data of C-NMR, determine that compound 4 is for phillygenol glucuronic acid potassium.

Embodiment 5

1, phillygenol-organic base coordination compound is prepared

By phillygenol (4g under conditions of temperature is 20 DEG C, 10.75mmo) join in dry anhydrous methylene chloride (180ml), organic base N is added after stirring and dissolving, N-diisopropyl ethyl amine 11.26mL (64.5mmoL), stir 10 minutes, carry out hydroxy activated reaction, prepare phillygenol-organic base complex solution, standby；

2, glycosylation reaction

2-1) under-10 DEG C and nitrogen atmosphere; 2,3,4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester 6.4g (16.125mmoL) and Disilver carbonate 17.78g (64.5mmoL) are dissolved in dichloromethane; stir; prepare glycosylation reagent; wherein glycosyl donor and phillygenol mole ratio be 1.5:1, catalyst Disilver carbonate and glycosyl donor mole ratio be 4:1；

Glycosyl donor except 2,3, in addition to 4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester, it is also possible to use 2,3,4-tri--O-benzoyl α-D bromo glucopyranosiduronic acid methyl ester.

2-2) under-10 DEG C and nitrogen atmosphere, while stirring phillygenol-organic base complex solution is added dropwise in glycosylation reagent, is then heated to 0 DEG C, and under conditions of temperature remains 0 DEG C, carry out glycosylation reaction 10h, prepare glycosylation reaction mixture；

3, deacylated tRNA base reaction

Under stirring; glycosylation reaction mixture is dissolved in methanol; then under conditions of 0 DEG C, add the potassium hydroxide solution (11.7ml) that mass percent concentration is 27% while stirring; KOH and 2,3, the mol ratio of 4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester be 3.5:1; stirring; after carrying out deacylated tRNA base reaction 30min, adding acidic ph modifier acetic acid, the pH value of the mixture of regulation deacylated tRNA base reaction is to 5；

4, separation, purification process

Rotary Evaporators is used to carry out decompression distillation under vacuum conditions, concentrate, evaporative removal solvent, it is thus achieved that deacylated tRNA base reactant mixture solid；Deacylated tRNA base reactant mixture sample is carried out silica gel in column chromatography for separation, purification process, wherein, eluant: chloroform/methanol=9:1；Column chromatography silica gel: GF254 silica gel；Obtain white solid (compound 5,5.08g), gross production rate 83%.

Compound 5 is dissolved in water, ethanol.Spray 10%H afterwards to launch (chromatographic solution is chloroform/methanol 3:1, and Rf is 0.4) on the tlc plate₂SO₄-ethanol reagent presents aubergine.

In ESI-MS spectrum, m/z [M+Na]⁺Being 571.17916, molecular weight is 548.54109.

¹H-NMR (400MHz, d₆null-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, d₆null-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, determine that compound 5 is for 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] furan-1-base)-2-methoxyphenyl)-3,4,5-trihydroxy tetrahydrochysene-2H-pyrans-2-carboxylic acids；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-carboxylic acid；Molecular formula is: C₂₇H₃₂O₁₂；Structural formula is:

Embodiment 6

1, phillygenol-organic base coordination compound is prepared

By phillygenol (4g under conditions of temperature is 10 DEG C, 10.75mmo) join in dry anhydrous methylene chloride (180ml), organic base 1 is added after stirring and dissolving, 8-diazabicylo 11 carbon-7-alkene 9.63mL (64.5mmo), stir 10 minutes, carry out hydroxy activated reaction, prepare phillygenol-organic base complex solution, standby；

2, glycosylation reaction

2-1) under 0 DEG C and nitrogen atmosphere; 2,3,4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester 6.4g (16.125mmoL) and Disilver carbonate 17.78g (64.5mmoL) are dissolved in dichloromethane; stir; prepare glycosylation reagent; wherein glycosyl donor and phillygenol mole ratio be 1.5:1, catalyst Disilver carbonate and glycosyl donor mole ratio be 4:1；

Glycosyl donor except 2,3, in addition to 4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester, it is also possible to use 2,3,4-tri--O-benzoyl α-D bromo glucopyranosiduronic acid methyl ester.

2-2) under 0 DEG C and nitrogen atmosphere, while stirring phillygenol-organic base complex solution is added dropwise in glycosylation reagent, is then heated to 10 DEG C, and under conditions of temperature remains 10 DEG C, carry out glycosylation reaction 10h, prepare glycosylation reaction mixture；

3, deacylated tRNA base reaction

Under stirring, glycosylation reaction mixture is dissolved in methanol, under conditions of 0 DEG C, then adds the sodium methoxide solution (10.16ml) that mass percent concentration is 30%, NaOCH while stirring₃With 2,3, the mol ratio of 4-tri--O-acetyl group α-D bromo glucopyranosiduronic acid methyl ester be 3.5:1, stirring, after carrying out deacylated tRNA base reaction 30min, add acidic ph modifier acetic acid, the pH value of the mixture of regulation deacylated tRNA base reaction is to 7；

4, separation, purification process

Rotary Evaporators is used to carry out decompression distillation under vacuum conditions, concentrate, evaporative removal solvent, it is thus achieved that deacylated tRNA base reactant mixture solid；Deacylated tRNA base reactant mixture sample is carried out silica gel in column chromatography for separation, purification process, wherein, eluant: chloroform/methanol=9:1；Column chromatography silica gel: GF254 silica gel；Obtain white solid (compound 6,5.33g), gross production rate 87%.

Compound 6 is dissolved in chloroform, ethanol.Spray 10%H afterwards to launch (chromatographic solution is chloroform/methanol 10:1, and Rf is 0.4) on the tlc plate₂SO₄-ethanol reagent presents aubergine.

In ESI-MS spectrum, m/z [M+Na]⁺Being 585.19481, molecular weight is 562.56802.

¹H-NMR (400MHz, d₆null-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, d₆null-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, determine that compound 6 is for phillygenol glucuronic acid methyl 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] furan-1-base)-2-methoxyphenyl)-3,4,5-trihydroxy tetrahydrochysene-2H-pyrans-2-carboxylate methyl esters；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 is: C₂₈H₃₄O₁₂；Structural formula is:

Test example 1 In vitro antibacterial test

1.1 test material

(1) medicine

1. phillygenol glucal acid derivative: the phillygenol D-Glucuronic acid sodium salt of embodiment 1 preparation, the phillygenol glucuronic acid potassium of embodiment 3 preparation, the phillygenol glucuronic acid of embodiment 5 preparation, the phillygenol glucuronic acid methyl ester of embodiment 6 preparation are produced by Dalian Fu Sheng natural drug development corporation, Ltd., measuring through high performance liquid chromatography two kinds of detectors (UV-detector, evaporative light scattering detector) area normalization method, its purity is respectively 99.3%, 99.2%, 99.7%, 99.6%；

2. positive control drug: ribavirin injection, colourless transparent liquid, effluent Nan Run expands limited company and produces, batch number: 1206261, and the quasi-word of traditional Chinese medicines: H19993553,100mg/ml tests positive control medicine as this；Oseltamivir phosphate, Nat'l Pharmaceutical & Biological Products Control Institute, batch number: 101096-200901,100mg/ prop up and test positive control medicine as this；Phillygenol, white powder, Dalian Fu Sheng natural drug development corporation, Ltd. produces, and measures through high performance liquid chromatography two kinds of detectors (UV-detector and evaporative light scattering detector) area normalization method, and its purity is 99.2%.

Above-mentioned medicine all by dissolved in purified water, filters, degerming subpackage, and 4 DEG C standby, tests medicine to be measured for this.

(2) cell strain Vero (African green monkey kidney cell cell) is preserved cell strain by preclinical medicine institute of Jilin University.

(3) Strain 1. strains of influenza viruses, parainfluenza virus strain, respiratory syncytial virus (RSV) strain: be purchased from Inst. of Viruses, China Preventive Medicine Science Academy；2. Coxsackie B virus 3 (CVB3) strain: purchased from Wuhan virus institute of the Chinese Academy of Sciences；3. coxsackie virus A 16 (CoxA16) strain, enterovirus EV 71 strain: purchased from Japan's celestial platform state hospital；4. adenovirus (AdV): be purchased from department of pediatrics research department of the First Academy of Norman Bethune Medical University；5. herpes simplex virus I-type (HSV-1): be purchased from Nat'l Pharmaceutical & Biological Products Control Institute.

(4) capital equipment and reagent

Biohazard Safety Equipment: BHC-1300 II A/B3, AIRTECH；CO2 incubator: MCO-18AIC, SANYO；Inverted microscope: CKX41, OLYMPUS；Electronic analytical balance: AR1140/C, DHAUS；Culture medium: DMEM, HyClone；Hyclone: HyClone；Trypsin: Gibco；MTT:Sigma；DMSO: Tianjin Bei Lian fine chemicals development corporation, Ltd..

1.2 test method

(1) cell prepares

Vero passage cultivates 1-2d, being allowed in blocks, boundary line is clear, when third dimension and diopter are strong, with trypsinization, treating that needle point sample aperture occurs in cell face, exhaust Digestive system, peek milliliter culture fluid dispels cell, counting, after being diluted to about 5107/L with culture fluid (containing the DMEM of 10% hyclone), it is inoculated in 96 well culture plates, treats that cell grows up to monolayer.

(2) drug toxicity measures

Cell toxicity test: medicine concentration as shown in table 1-1 is diluted, for cytotoxic assay.

Table 1-1 drug dilution reference table (unit: g/L)

The medicine of the above-mentioned variable concentrations maintaining liquid (containing the DMEM of 2% hyclone) to dilute is dripped on Vero cell monolayer, every hole 0.2ml, the multiple hole of each concentration 6, separately set 6 hole normal controls (being not added with the Normal group of medicine) and 6 hole blanks (culture fluid), put 37 DEG C, 5%CO₂Incubator is cultivated, puts inverted microscope every day and observe CPE record.After 72h, every hole adds MTT solution 20 μ L (5mg mL^{- 1}), to continue to hatch 4h, inhale and abandon each hole culture fluid, every hole adds 100 μ L DMSO, and vibration 5min, 492nm measure OD value, calculate cell survival rate.In SPSS 18.0 statistical software, cell survival rate is carried out Probit regression analysis, calculate the medicine maximal non-toxic concentration (TC to Vero cell₀) and half toxic concentration (TC₅₀)。

(3) various virus TCID₅₀Mensuration

Various viruses carry out 10 times successively decrease and be diluted to 10^-1,10^-2,10^-3, 10^-4, 10^-5, 10^-6Different dilution factors, are sequentially inoculated on Vero cell 96 well culture plate of monolayer, every hole 100 μ L, each dilution factor 6 hole, set normal cell controls group simultaneously.Put 37 DEG C, 5%CO₂In hatch 2h, abandon virus liquid, every hole adds cell maintenance medium 100 μ L immediately, puts 37 DEG C, 5%CO₂Middle cultivation.3rd day starts to examine under a microscope Cytopathic effect, and 7-8 days result of determination also make a record, and so that 50% cell hole can be made to occur the highest dilution of positive pathological changes as terminal, calculates virus titer by karber method.

Formula $\mathrm{Log}{\mathrm{TCID}}_{50}=\mathrm{XM}+\frac{1}{2}d-d\frac{\mathrm{\ΣPi}}{100}$

TCID₅₀: 50% tissue cytopathogenic dose

XM: the virus dilution logarithm of maximum concentration

D: the logarithm of dilution factor coefficient (multiple)

Σ pi: the summation of each dilution factor pathological changes percent

(4) impact of drug on viral cytopathic effect

Take the culture plate covering with cell monolayer, inhale and abandon culture fluid, with 100TCID₅₀Corresponding virus attack amount inoculating cell, 37 DEG C, 5%CO₂Incubator absorption 2h, adds each medicinal liquid of certain concentration (about maximal non-toxic concentration), and the multiple hole of every concentration 6 is cultivated, 200 μ L/ holes.If ribavirin injection and oseltamivir phosphate are positive drug control group, set Normal group (being not added with virus not dosing) and virus control group (add virus but be not added with the matched group of medicine) simultaneously, observe drug on viral and cause the impact of CPE.After 72h, use MTT colorimetry, under 492nm wavelength, measure OD value, calculate Antiviral Effect effective percentage (ER%).The significant difference between ANOVA method more each Antiviral Effect effective percentage is used in SPSS 18.0 statistical software.

ER%=(drug treating 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 result of the test

(1) TCID of various viruses₅₀

Parainfluenza virus: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+50}{100}=-4$

Influenza virus: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+50}{100}=-4$

CVB₃: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$

HSV-1: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+30}{100}=-4.8$

AdV: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+50}{100}=-4$

RSV: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$

CoxA16: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$

EV71: $\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$

(2) drug toxicity measures

1) medicine is to Cytotoxic mensuration

Each medicine maximal non-toxic concentration (TC 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 on viral cytopathogenic effect protective effect result

Medicine resists effective percentage and the ANOVA method one factor analysis of variance result of various virus, refers to table 1-3.

Table 1-3 Antiviral Effect effective percentage (ER%) statistical table

Note: compared with virus control group, * P < 0.05, * * P < 0.01；With phillygenol ratio,^#P < 0.05,^##P<0.01。

Table 1-3 result shows, phillygenol glucal acid derivative infected by influenza, parainfluenza virus, herpes simplex virus I-form (HSV-I), the suppression ratio of enterovirus EV 71 and effective percentage are all more than 90%, obvious difference compared with virus control group, has statistical significance.Phillygenol glucal acid derivative shows the advantage being better than phillygenol, ribavirin and oseltamivir phosphate to the Anti-viral Treatment of multiple virus.

Test example 2 Antiviral assay in vivo

2.1 experiment material

(1) laboratory animal

Kunming mice, body weight 18～22g, male and female half and half property, purchased from Dalian Medical Univ's Experimental Animal Center, the certification of fitness number: SCXK (13) 2012-0003.

(2) medicine

1. phillygenol glucal acid derivative:

The phillygenol glucuronic acid methyl ester of embodiment 6 preparation: white solid, Dalian Fu Sheng natural drug development corporation, Ltd. produces, measuring through high performance liquid chromatography two kinds of detectors (UV-detector and evaporative light scattering detector) area normalization method, its purity is 99.6%；

2. ribavirin injection, colourless transparent liquid, effluent Nan Run expands limited company and produces, batch number: 1206261, and the quasi-word of traditional Chinese medicines: H19993553,100mg/ml tests positive control medicine as this；

3. oseltamivir phosphate, Nat'l Pharmaceutical & Biological Products Control Institute, batch number: 101096-200901,100mg/ prop up, test positive control medicine as this；

4. phillygenol, white powder, Dalian Fu Sheng natural drug development corporation, Ltd. produces, and measures through high performance liquid chromatography two kinds of detectors (UV-detector and evaporative light scattering detector) area normalization method, and its purity is 99.2%.

Above-mentioned medicine all by dissolved in purified water, filters, degerming subpackage, and 4 DEG C standby, tests medicine to be measured for this.

(2) detecting instrument, reagent

2.2 experimental technique

(1) influenza virus and the parainfluenza virus mensuration to mice median lethal dose(LD 50)

Influenza virus and 10 times of ratios of two term of parainfluenza virus (cell pyrolysis liquid) are diluted to 10^-1、10^-2、10^-3、10^-4、10^-5The virus liquid of concentration.Taking each 60 of Kunming mouse 120, influenza virus and parainfluenza virus group, be randomly divided into 6 groups respectively, ether light anesthesia mice, collunarium only infects different dilution factor virus liquid 0.03mL/.Set blank simultaneously, replace viral suspension with normal saline.With dead and existence as observation index, every day observes, until metainfective 14 days.Infecting in 24h dead for nonspecific death, not statistics, Karber method calculates virus liquid LD50.Computing formula:[wherein: LD₅₀: median lethal dose(LD 50)；XM: the virus dilution logarithm of maximum concentration；D: the logarithm of dilution factor coefficient (multiple)；Σ pi: the summation of each dilution factor pathological changes percent].

(2) research of pneumonia caused by phillygenol glucuronic acid methyl ester resisiting influenza virus and parainfluenza virus infection

1) experimental animal and packet

Take the kunming mice 960 of four week old, carry out 2 tests.Take mice 480, be randomly divided into 48 groups, often group 10, for phillygenol glucal acid derivative influenza virus infected Lung Exponent and the determination test of lung index, repeat test for 3 times, take mice 80 every time.Separately take mice 480, be randomly divided into 48 groups, often group 10, for the phillygenol glucal acid derivative determination test to lung suspension viral hemoagglutination titre, repeat test for 3 times, take mice 80 every time.

2) method is infected

An absorbent cotton is put in the beaker of 200～300mL sizes, it is subsequently poured into appropriate ether (making absorbent cotton get wet), beaker back-off equipped with absorbent cotton is come, mice is put into and anaesthetizes, see that mice is on wires, time substantially in unable sample, being lain on the back by mice, collunarium infects 15LD₅₀Influenza virus and parainfluenza virus 0.03ml/ nostril, Normal group normal saline replaces viral suspension.

3) medication and dosage

Phillygenol glucal acid derivative group, ribavirin and oseltamivir phosphate matched group, conventional gastric infusion is started the previous day respectively at infecting, the high, medium and low dosage of phillygenol glucal acid derivative is respectively 13.0,8.0,4.0mg/kg, ribavirin positive drug dosage is 58.5mg/kg, oseltamivir phosphate positive drug dosage is 19.5mg/kg, phillygenol group dosage is 13.0mg/kg, once a day, successive administration 5d, virus control group gavages the normal saline of same volume.

4) observation index

1. Lung Exponent measures

After mice medication the 5th day, first fasting water 8 hours, pluck eyeball sacrificed by exsanguination animal after weighing, open thoracic cavity and extract full lung, with brine twice, blot surface moisture content with filter paper, electronic balance claims lung weight, by following equation calculating Lung Exponent and lung index:

Lung Exponent=(mouse lung weight/Mouse Weight) × 100%；Lung index=(the infection model group average Lung Exponent of average Lung Exponent-experimental group)/average Lung Exponent × 100% of infection model group.

2. lung suspension viral hemoagglutination titer determination

After taking treatment respectively the 5th day respectively organize mouse lung, the underlying homogenizer of low temperature grinds to form homogenate, normal saline dilution is the lung tissue suspension of 10%, centrifuging and taking supernatant, doubling dilution, drips on titer plate by 0.2ml/ hole, every hole adds 0.2ml 1% chicken erythrocyte suspension, mixing, puts room temperature 30min, observed and recorded Hemagglutination titer.With red cell agglutination (++) Shi Weidian, represent its titre with suspension extension rate.

3. lung morphology is observed

After taking treatment respectively the 5th day respectively organize mouse lung, perusal record lungs substantially pathological changes situation.Rinsed clean in normal saline, blots with filter paper, takes a part of 10% formaldehyde and fixes, paraffin embedding, section, and HE dyes, and basis of microscopic observation is also taken pictures, as shown in Figure 4.

2.3 experimental results and analysis

(1) influenza virus and the parainfluenza virus measurement result to mice median lethal dose(LD 50)

Experimental group Kunming mouse respectively by collunarium infect variable concentrations influenza virus, parainfluenza virus venom 30 μ L, before infecting the 3rd day 3 groups (virus concentration is 10^-1Group, 10^-2Group, 10^-3Group) all there is disease symptom in various degree in mice: alarm hair, shake, diet minimizing etc.；5th day mice occurs walking and plays pendulum；6th day virus concentration group mice the highest starts death occur, and remaining each group the phenomena of mortality occurs on the 7th day after infecting successively.Observing after within 14 days, terminating, add up each group of dead mouse number, result see table 1-4,1-5.Calculate the LD of this influenza virus₅₀For dilution factor 10^-2.9, the LD of parainfluenza virus₅₀For dilution factor 10^-2.5。

Table 1-4 influenza virus median lethal dose(LD 50) result of the test is added up

Karber method calculates the LD of virus₅₀.The LogLD of influenza virus₅₀As follows:

$\mathrm{Log}{\mathrm{LD}}_{50}=\mathrm{XM}+\frac{1}{2}d-d\frac{\mathrm{\&Sigma;Pi}}{100}=-1+0.5-(80\%+60\%+40\%+20\%+0\%+0\%)=-2.9$

Table 1-5 parainfluenza virus median lethal dose(LD 50) result of the test is added up

Karber Method calculates virus LD ₅₀ 。The LogLD of parainfluenza virus₅₀As follows:

$\mathrm{Log}{\mathrm{LD}}_{50}=\mathrm{XM}+\frac{1}{2}d-d\frac{\mathrm{\&Sigma;Pi}}{100}=-1+0.5-(90\%+70\%+40\%+30\%+10\%+0\%)=-2.5$

(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 mice; average Lung Exponent measurement result shows: compare with infection model group; phillygenol glucal acid derivative (methyl ester) concentration has certain protective role, Lung Exponent the most substantially to reduce in the range of 3.25～13.0mg/kg/d；The curative effect of phillygenol glucal acid derivative high dose group infected by influenza and parainfluenza virus is better than phillygenol group (P < 0.05).Result of the test is shown in Table 1-6,1-7.

Table 1-6 phillygenol glucal acid derivative influenza virus infected Lung Exponent and the suppression ratio (n=3) of Lung Exponent

Compare with virus control group,^*P < 0.05,^**P0.01；Compare with phillygenol group,^#P < 0.05,^##P<0.01。

The table 1-7 phillygenol glucal acid derivative shadow (n=3) to parainfluenza virus infection mouse lung index and lung index

Compare with virus control group,^*P < 0.05,^**P0.01；Compare with phillygenol group,^#P < 0.05,^##P<0.01。

2. lung suspension viral hemoagglutination titer determination

After influenza virus and parainfluenza virus infection mice, infection model group lung tissue viral hemoagglutination titre (InX) is respectively 32.40 and 33.11, after variable concentrations phillygenol glucal acid derivative (methyl ester) treats 5 days, lung tissue viral hemoagglutination titre has declined, compare with infection model group, difference has significance, (P < 0.01)；Wherein, phillygenol glucal acid derivative middle and high dosage group is to influenza, parainfluenza virus Hemagglutination titer all significantly lower than model group, and suppression ratio is above phillygenol group, significant difference (P < 0.05, p < 0.01).Result of the test is shown in Table 1-8,1-9.

The impact (n=3) of table 1-8 phillygenol glucal acid derivative influenza virus infected lung suspension Hemagglutination titer

Compare with virus control group,^*P < 0.05,^**P<0.01；Compare with phillygenol group,^#P < 0.05,^##P<0.01。

The impact (n=3) on parainfluenza virus infection mouse lung suspension Hemagglutination titer of the table 1-9 phillygenol glucal acid derivative

Compare with virus control group,^*P < 0.05,^**P<0.01；Compare with phillygenol group,^#P < 0.05,^##P<0.01。

3. lung tissue testing result

By Fig. 4,5 result of the test it can be seen that influenza virus and parainfluenza virus pneumonia model group mice lungs great majority have hyperemia, edema pathological changes, some is the consolidation district of crineous outward appearance, and several cases presents henna stigma.Under mirror visible, the interstitial lung such as bronchus, bronchioles wall, alveolar wall is congested, edema and lymphocyte, monocyte infiltration, and alveolar wall is broadening, and alveolar is inflammatory reaction.Influenza virus and parainfluenza virus pneumonia mouse model are after phillygenol glucuronic acid derivatives for treatment, and each group Mus lung substantially pathological changes substantially alleviates, and lung tissue segment morphosis is normal；Compared with infection model group, alveolar septum is relatively thin, and alveolar wall and bronchioles wall mononuclear cells infiltration negligible amounts, intracavity is without oozing out, and pathological changes substantially alleviates.The middle and high dosage group of phillygenol glucuronic acid derivatives for treatment parainfluenza virus pneumonia is compared with infection model group, and alveolar septum is the most relatively thin, mononuclear cells infiltration negligible amounts, and intracavity is without oozing out, and pathological changes substantially alleviates.

2.4 conclusion

Antiviral assay in vivo result shows, phillygenol glucal acid derivative has obvious inhibitory action at dosage range infected by influenza and parainfluenza virus and the caused Mouse Virus Pneumonia of 3.25～13mg/kg/d, can substantially reduce its Lung Exponent and Hemagglutination titer, lung tissue disease's Neo-Confucianism also has clear improvement, notable with virus model matched group comparing difference；And phillygenol glucal acid derivative middle and high dosage group curative effect is substantially better than phillygenol (* P < 0.05 or * * P < 0.01), show the trend being better than ribavirin and oseltamivir phosphate simultaneously.

Claims (10)

1. a phillygenol glucal acid derivative, general structure is as follows:

Wherein, R group selects H, Me, Na⁺、K⁺。

2. a preparation method for phillygenol glucal acid derivative, is characterized in that, the step carried out including order below:

1) phillygenol and organic base carry out hydroxy activated reaction, prepare phillygenol-organic base coordination compound；

2) joining in organic solvent by glycosyl donor, catalyst, being mixed even prepares glycosylation reagent, is subsequently adding phillygenol-organic Alkali coordination compound, carries out glycosylation reaction, prepares glycosylation reaction mixture；

3) glycosylation reaction mixture is joined in methanol, be subsequently added into alkali compounds, carry out deacylated tRNA base reaction, be subsequently adding pH Regulator, regulates mixture solution pH to 4-8.5 and get final product.

3. method as claimed in claim 2, is characterized in that, step 1) described in organic base select N, N-diisopropyl ethyl amine or 1,8- Diazabicylo 11 carbon-7-alkene.

4. as claimed in claim 2 or claim 3 preparation method, is characterized in that, step 1) described in response time of hydroxy activated reaction be 10～20 minutes.

5. as claimed in claim 2 or claim 3 preparation method, is characterized in that, step 2) described in glycosyl donor select 2,3,4-tri--O-acyls Base-α-D-bromo glucopyra aldehydic acid ester.

6. as claimed in claim 2 or claim 3 preparation method, is characterized in that, step 2) described in catalyst choice Disilver carbonate.

7. as claimed in claim 2 or claim 3 preparation method, is characterized in that, step 2) described in catalyst and the mol ratio 1 of glycosyl donor: 6.0～8.0.

8. as claimed in claim 2 or claim 3 preparation method, is characterized in that, step 2) described in glycosylation reaction protect at noble gas Protect down carry out.

9. as claimed in claim 2 or claim 3 preparation method, is characterized in that, also include step 4) separate, purification process, to regulation pH After mixture solution carry out concentration and silica gel column chromatography and process.

10. the antiviral of phillygenol glucal acid derivative is applied as claimed in claim 1.