CN106074527A - The compositions of Schiglautone A derivant is used for preparing antiviral drugs - Google Patents

The compositions of Schiglautone A derivant is used for preparing antiviral drugs Download PDF

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CN106074527A
CN106074527A CN201610424346.3A CN201610424346A CN106074527A CN 106074527 A CN106074527 A CN 106074527A CN 201610424346 A CN201610424346 A CN 201610424346A CN 106074527 A CN106074527 A CN 106074527A
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compositions
virus
compound
antiviral drugs
cell
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陆贤
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Nanjing Guangkangxie Biomedical Technology Co Ltd
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Nanjing Guangkangxie Biomedical Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/04—Five-membered rings

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  • Health & Medical Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The compositions of Schiglautone A derivant of the present invention is used for preparing antiviral drugs, relates to organic synthesis and medicinal chemistry art, is specifically related to compositions and the purposes in preparation antiviral drugs thereof.Disclose a kind of compositions and preparation method thereof.Pharmacological experiment shows, the compositions of the present invention has antivirus action, has the value of exploitation antiviral drugs.

Description

The compositions of Schiglautone A derivant is used for preparing antiviral drugs
Technical field
The present invention relates to organic synthesis and medicinal chemistry art, be specifically related to compositions, preparation method and its usage.
Background technology
Herpes simplex virus type 1 (Herpes simplex virus, HSV-1) and herpes simplex virus type 2 (Herpes Simplex virus, HSV-2) belong to herpes coe virus, HSV-1 and HSV-2 not only causes lip or genital mucosal herpes, with Time be also to cause encephalitis, hepatitis, the severe systemic infection even important pathogen of intrauterine infection.
Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) mainly causes respiratory system infection, Cause serious pneumonia infant, old age and immunodeficiency crowd, be one of important pathogen of infecting of human upper respiratory tract.
Influenza A virus (Influenza A virus, Flu-A) belongs to Orthomyxoviridae, at the mankind and other biological There is the popular of little scope every year, cause upper respiratory system to infect.When being very popular, serious respiratory system infection can be caused, notably Cause pneumonia, encephalitis the most dead.
There is the problem that toxicity is big, safety is low, from natural product in above-mentioned three kinds of viral current existing medicines for the treatment of Middle searching compound or lead compound also carry out structural modification and obtain its derivant, thus obtain the potential drug of high-efficiency low-toxicity Most there is important value.
The compound I that the present invention relates to be one within 2011, deliver (Fan-Yu Meng et al., 2011.Schiglautone A,a New Tricyclic Triterpenoid with a Unique 6/7/9-Fused Skeleton from the Stems of Schisandra glaucescens.Organic Letters 13(2011) 1502 1505) compound, we have carried out structural modification to compound I, it is thus achieved that two i.e. compounds of new derivant III and compound IV, and it is prepared for compositions with compound III and compound IV, and said composition antiviral activity is carried out Evaluating, it has antiviral activity.
Summary of the invention
The invention discloses a new compositions, said composition is made up of compound III and compound IV, said composition The mass percent of middle compound III and compound IV is respectively 40% and 60%.
Compositions disclosed by the invention can make pharmaceutically acceptable salt or pharmaceutically acceptable carrier.
Pharmacodynamic experiment shows, the compositions of the present invention has preferable antivirus action.The present invention pharmaceutically can connect The salt being subject to has same drug effect.
The experiment in vitro of compositions shows, compositions has the activity of the strongest anti-HSV-1, HSV-2, RSV and flu A, Therefore the compositions of the present invention is expected to be used for preparing novel antiviral medicine.
The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not by concrete real Execute any restriction of example, but be defined in the claims.
Detailed description of the invention
The preparation of embodiment 1 compound Schiglautone A
Document (the Fan-Yu that the preparation method of compound Schiglautone A (I) is delivered with reference to Fan-Yu Meng et al. Meng et al.,2011.Schiglautone A,a New Tricyclic Triterpenoid with a Unique 6/ 7/9-Fused Skeleton from the Stems of Schisandra glaucescens.Organic Letters 13 (2011) 1,502 1505) method.
The synthesis of O-bromoethyl derivant (II) of embodiment 2Schiglautone A
Compound I (502mg, 1.00mmol) is dissolved in 15mL benzene, in solution, adds tetrabutyl ammonium bromide (TBAB) (0.08g), glycol dibromide (7.520g, 40.00mmol) and 50% sodium hydroxide solution of 6mL.Mixture is Celsius 35 Degree stirring 8h.After 8h, reactant liquor is poured in frozen water, be extracted twice with dichloromethane immediately, merge organic phase solution.Then Washing organic phase solution 3 times with water and saturated aqueous common salt successively, then be dried with anhydrous sodium sulfate, last concentrating under reduced pressure is removed molten Agent obtains product crude product.Product crude product silica gel column chromatography purification (flowing is mutually: petroleum ether/acetone=100:1.0, v/v), receives Collection brown is concentrated elution band and flings to solvent and i.e. obtain the brown ceramic powder (508mg, 71%) of compound II.
1H NMR(500MHz,DMSO-d6)δ13.40(s,1H),6.10(s,1H),5.63(s,1H),5.53(s,1H), 3.85 (d, J=11.2Hz, 4H), 3.52 (d, J=10.8Hz, 4H), 2.96 (s, 1H), 2.20 (s, 1H), 2.16 (s, 2H), 2.00 (s, 1H), 1.84 (d, J=13.9Hz, 4H), 1.69 (s, 1H), 1.58 (dd, J=22.2,8.5Hz, 4H), 1.51 (s, 1H), 1.47 (s, 1H), 1.26 (dd, J=9.1,4.4Hz, 4H), 1.21 (s, 1H), 1.08 0.98 (m, 4H), 0.96-0.94 (m,9H),0.94-0.85(m,6H).
13C NMR(125MHz,DMSO-d6)δ211.46(s),209.14(s),170.06(s),161.12(s),143.51 (s),132.01(s),127.77(s),85.96(s),82.40(s),70.19(s),69.10(s),57.14(s),52.73 (s),51.90(s),45.77(s),40.67(s),38.57(s),38.32(s),35.04(s),33.55(s),33.27(s), 29.85(s),28.98(s),26.71(s),25.50(s),24.05(s),22.31(s),21.06(s),20.56(s),20.00 (s),18.69(s),18.11(s),15.07(s).
HRMS(ESI)m/z[M-H]-calcd for C34H51Br2O6:715.2032；found 715.2027.
The synthesis of O-(the 1H-tetrazole base) ethyl derivative (III) of embodiment 3Schiglautone A
Compound II (358mg, 0.5mmol) is dissolved in the middle of 15mL acetonitrile, be added thereto to Anhydrous potassium carbonate (345mg, 2.5mmol), potassium iodide (84mg, 0.5mmol) and 1H-tetrazole (1401mg, 20mmol), mixture is heated to reflux 5h.Reaction Reactant liquor is poured in 20mL frozen water after end, extract three times with equivalent dichloromethane, merge organic facies.Successively with water and saturated Organic facies after brine It merging, then be dried with anhydrous sodium sulfate, concentrating under reduced pressure is removed solvent and is obtained product crude product.Cause For tautomerization, 1H-tetrazole base and two kinds of substitution products of 2H-tetrazole base can be generated at reaction conditions.Product is thick Product silica gel column chromatography purification (flowing is mutually: petroleum ether/acetone=100:1, v/v), collects faint yellow concentration elution band, then will Faint yellow elution band concentrates, and purifies (flowing is mutually: petroleum ether/acetone=100:0.5, v/v) with silica gel column chromatography, collects successively Two flaxen elution bands, concentrate front 1 elution band and i.e. obtain the faint yellow solid (79.8mg, 23%) of compound III.
1H NMR(500MHz,DMSO-d6)δ16.29(s,1H),9.99(s,1H),9.93(s,1H),6.25(s,1H), 5.76 (s, 1H), 5.40 (s, 1H), 4.57 (s, 1H), 4.38 (s, 1H), 4.32 (s, 1H), 4.21 (s, 1H), 3.96 (d, J= 11.9Hz,4H),3.33(s,1H),2.55(s,1H),2.51(s,2H),2.39(s,1H),1.98(s,3H),1.82–1.76 (m, 3H), 1.73 (s, 1H), 1.65 (d, J=6.5Hz, 2H), 1.60 (s, 1H), 1.53 (s, 1H), 1.47 1.37 (m, 4H), 1.30 (s, 1H), 1.19 (s, 1H), 1.10 (t, J=12.5Hz, 3H), 1.08 (t, J=12.5Hz, 9H), 1.03 (s, 3H), 0.86(s,3H).
13C NMR(125MHz,DMSO-d6)δ211.72(s),209.42(s),170.36(s),161.38(s),145.37 (s),143.80(s),132.29(s),128.04(s),86.25(s),82.68(s),66.14(s),65.60(s),57.33 (s),52.91(s),52.08(s),46.81(s),45.95(s),40.84(s),38.76(s),38.50(s), 35.21(s), 33.74(s),30.01(s),29.16(s),26.91(s),25.66(s),24.23(s),22.51(s),21.22(s),20.74 (s),20.20(s),18.85(s),18.29(s),15.27(s).
HRMS(ESI):m/z[M+H]+calcd for C36H55N8O6:695.4245；found:695.4248.
The synthesis of O-(benzimidazolyl) ethyl derivative of embodiment 4Schiglautone A
Compound II (358mg, 0.5mmol) is dissolved in the middle of 15mL acetonitrile, be added thereto to Anhydrous potassium carbonate (345mg, 2.5mmol), potassium iodide (84mg, 0.5mmol) and benzimidazole (1180mg, 10mmol), mixture is heated to reflux 6h.Reaction After end, reactant liquor is poured in frozen water, extract three times with equivalent dichloromethane, merge organic facies.Successively with water and saturated common salt Organic facies after water washing merging, then be dried with anhydrous sodium sulfate, concentrating under reduced pressure is removed solvent and is obtained product crude product.Product is thick Product silica gel column chromatography purification (flowing is mutually: petroleum ether/acetone=100:1.5, v/v), collects brown and concentrates elution band, concentrate I.e. obtain the brown solid (169.9mg, 43%) of compound IV.
1H NMR (500MHz, DMSO-d6) δ 13.32 (s, 1H), 8.38 (s, 2H), 7.66 (d, J=25.0Hz, 4H), 7.39 (s, 2H), 7.32 (s, 2H), 6.25 (s, 1H), 5.64 (d, J=18.7Hz, 2H), 4.34 (s, 1H), 4.28 (s, 1H), 4.18 (d, J=13.7Hz, 2H), 4.04 (s, 2H), 3.99 (s, 2H), 3.13 (s, 1H), 2.65 (s, 2H), 2.26 (s, 1H), 2.17 (s, 1H), 2.06 (s, 1H), 1.98 (s, 3H), 1.87 (d, J=16.4Hz, 2H), 1.66 (dd, J=8.0,6.6Hz, 4H), 1.62 (s, 1H), 1.49 (s, 1H), 1.41 (dd, J=19.6,10.4Hz, 2H), 1.37 (d, 2H), 1.20 1.11 (m, 4H), 1.07 (d, J=20.0Hz, 6H), 103 (d, J=20.0Hz, 3H), 1.01 (d, J=20.0Hz, 3H), 0.95 (d, J= 20.0Hz,3H).
13C NMR(125MHz,DMSO-d6)δ211.30(s),208.98(s),169.90(s),160.96(s),146.28 (s),143.34(s),139.64(s),133.48(s),131.85(s),127.61(s),123.92(s),123.35(s), 118.55(s),110.85(s),85.79(s),82.26(s),67.74(s),67.20(s),57.00(s),52.56(s), 51.73(s),45.63(s),44.94(s),40.49(s),38.42(s),38.17(s),34.86(s),33.40(s),29.68 (s),28.81(s),26.57(s),25.33(s),23.88(s),22.17(s),20.89(s),20.39(s),19.86(s), 18.52(s),17.87(s),14.93(s).
HRMS(ESI):m/z[M+H]+calcd for C48H63N4O6:791.4748；found:791.4744.
Embodiment 5 compositions antiviral activity
(1) experimental example: the compositions Effect study to herpesvirus:
(1) cell strain and Strain:
HEKC (HEK293, for HSV-1, HSV-2 virus sensitive cells) is purchased from Clontech company of the U.S.；Merely Herpesvirus 1 type (HSV-1) Sm44 strain, simple herpes virus 2 type (HSV-2) 333 strain are quoted from China's CDC virosis institute.
(2) cytotoxic assay:
The preparation of compositions: the powder that will cross the 40mg compound III of 200 mesh nets after grinding crosses 200 after grinding The powder of the 60mg compound IV of mesh net loads in tubule with cover and i.e. obtains 100mg compositions with the mixing of turbine stirring instrument, Obtain the solution of compositions by the compositions of this 100mg of water dissolution during use.
Reference literature (Wang Shouchuan, Wang Lin, old superfine, ground by oral liquid for clearing away lung-heat Contained Serum by the experiment of viral inhibition Study carefully, Nanjing University of Traditional Chinese Medicine's journal, 2008；24 (1): 25～27) method, does serial dilution (2 by each sample with 2 multiple proportions0 ～-5), then it being inoculated on the HEK293 in 96 plate holes by dilution sequential lateral, every hole 100uL, every dilution factor longitudinally repeats 3 holes (A, B, C row), parallel 1～6 holes setting microwell plate D are as cell controls, and 7～12 hole not inoculating cells are blank, microscope Observe CPE, Continuous Observation 7d, neutral red staining lower every day, measure OD value at 540nm wavelength, by experimental group and cell controls group OD value compares calculating cell survival rate, calculates medicine half cytotoxic concentration (TD by Reed-Muench method50)。
(4) toxin inhibitory test:
Reference literature (Wang Shouchuan, Wang Lin, old superfine, ground by oral liquid for clearing away lung-heat Contained Serum by the experiment of viral inhibition Study carefully, Nanjing University of Traditional Chinese Medicine's journal, 2008；24 (1): 25～27) method, by sample by dilution order (2-3～-14) laterally connect Planting on the cell monolayer in 96 plate holes, every hole 100uL, every dilution factor longitudinally repeats 4 holes, and A, B, C row of microwell plate adds containing 100 Individual TCID50Viral 100uL as test group, D row supplements and waits capacity cell maintenance medium is variable concentrations drug control, parallel sets F row is as virus control, and the 1 of E row～12 as cell controls.37 DEG C, 5%CO2Cultivate, observe CPE, Continuous Observation every day 4d.When more than 90% CPE occurs in virus control, add 1% neutral red staining, read A value by microplate reader at wavelength 540nm wavelength, Each group A value removes virus control group A value, by each test group compared with cell controls group A value, it is thus achieved that cell survival rate, uses Reed- Muench method calculates half and presses down poison concentration (IC50), TD the most at last50And IC50Compare acquisition and press down poison index (TI).
(5) result:
1. sample TD50Mensuration: cellular control unit basis of microscopic observation adherent growth is fine and close, form is good.Compositions, change Compound III and the TD of compound IV50It is respectively 2 at HEK293-2.68、2-2.49、2-2.83。
2. poison experiment is pressed down: the adherent densification of cellular control unit, form are good.Microscopic observation finds, HSV-1, HSV-2 virus is right Occur that according to group the CPE, HSV 1 and HSV 2 of more than 90% then merges with swelling, many cells at HEK293CPE respectively at 3d, 3d (HSV 1 causes multiple cell warm one-tenth bulla, and HSV 2 causes to be fused to vesicle), become round, come off for principal character.
And compositions toxin inhibitory test respectively organizes cell, diluting gradient according to composition sample, there is CPE:HSV in gradient rule 1-HEK293 before the 10th dilution factor, HSV 2-HEK293 group cell before the 9th dilution factor protected completely, occur afterwards CPE, CPE reduce with sample concentration and gradually increase the weight of.
And compound III and compound IV process group are 2-4～-6The effect of suppression virus is just observed in concentration range.
By above-mentioned brassboard with 1% neutral red staining, surveying 450nmA value by microplate reader, each group A value cuts virus control group A After value, each experimental group A value obtains IC compared with cell controls group A value50, IC50With TD50Compare the TI obtaining compositions: suppression CPE, IC is there is in HSV-1, HSV-2 at HEK293 cell50It is respectively 2-12.36、2-12.74, TI is respectively 820.3,1067.5.Chemical combination The TI of thing III: corresponding IC50It is respectively 2-4.71、2-4.58, TI is respectively 4.7,4.3.The TI of compound IV: corresponding IC50Point It is not 2-5.36、2-4.12, TI is respectively 5.8,2.4.
Conclusion: compositions has an effect of significant herpes coe virus (including HSV-1, HEV-2), and safety Height, therefore compositions has a wide range of applications background in treatment herpesvirus infection disease.Compound III and compound IV is not There is the effect of significant herpes coe virus (including HSV-1, HEV-2), and safety be extremely low, therefore compound III and Compound IV does not has application background in treatment herpesvirus infection disease.
(2) experimental example: the compositions Effect study to respiratory syncytial virus (RSV)
(1) compositions, compound III and the configuration of compound IV: medicine is dissolved in cell maintenance medium (containing 2% new born bovine The MEM of serum, GIBICO Products) in standby.
(2) cell strain and Strain:
HEKC (HEK293, RSV virus sensitive cells) is purchased from Clontech company of the U.S.；Respiratory syncytial virus (RSV) Long strain is quoted from China's CDC virosis institute.
(3) cytotoxic assay:
Reference literature (Wang Shouchuan, Wang Lin, old superfine, made by oral liquid for clearing away lung-heat Contained Serum by respiratory syncytial virus suppression Experimentation, Nanjing University of Traditional Chinese Medicine's journal, 2008；24 (1): 25～27) method, does series by sample with 2 multiple proportions Dilution (20～-5), then it being inoculated on the HEK293 in 96 plate holes by dilution sequential lateral, every hole 100uL, every dilution factor is longitudinally Repeating 3 holes (A, B, C row), parallel 1～6 holes setting microwell plate D are as cell controls, and 7～12 hole not inoculating cells are blank, Microscope observes CPE, Continuous Observation 7d, neutral red staining lower every day, measures OD value at 540nm wavelength, by experimental group and cell Matched group OD value compares calculating cell survival rate, calculates medicine half cytotoxic concentration (TD by Reed-Muench method50)。
(4) toxin inhibitory test:
Reference literature (Wang Shouchuan, Wang Lin, old superfine, made by oral liquid for clearing away lung-heat Contained Serum by respiratory syncytial virus suppression Experimentation, Nanjing University of Traditional Chinese Medicine's journal, 2008；24 (1): 25～27) method, by sample by dilution order (2-2～-13) be laterally inoculated on the cell monolayer in 96 plate holes, every hole 100uL, every dilution factor longitudinally repeats 4 holes, the A of microwell plate, B, C row adds containing 100 TCID50Viral 100uL as test group, D row supplements and waits capacity cell maintenance medium is variable concentrations medicine Thing compares, and the parallel F row that sets is as virus control, and the 1 of E row～12 as cell controls.37 DEG C, 5%CO2Cultivating, every day observes CPE, Continuous Observation 4d.When more than 90% CPE occurs in virus control, add 1% neutral red staining, by microplate reader at wavelength 540nm Wavelength reads A value, and each group A value removes virus control group A value, by each test group compared with cell controls group A value, it is thus achieved that cell is deposited Motility rate, calculates half by Reed-Muench method and presses down poison concentration (IC50), TD the most at last50And IC50Compare acquisition and press down poison index (TI)。
(5) result:
1. sample TD50Mensuration: cellular control unit basis of microscopic observation adherent growth is fine and close, form is good.Compositions, change Compound III and the compound IV TD to HEK29350It is respectively 2-2.92、2-2.41、2-2.36。
2. poison experiment is pressed down: the adherent densification of cellular control unit, form are good.Microscopic observation finds, RSV virus control group is the 2d occurs that the CPE, RSV of more than 90% strengthen at HEK293CPE with refractivity, become round, come off, are broken for principal character.
And compositions toxin inhibitory test respectively organizes cell, according to diluted sample gradient, there is CPE in gradient rule:
RSV-HEK293 test group cell is before the 9th dilution factor, and cell is protected completely, occur afterwards CPE, CPE with Sample concentration reduces and gradually increases the weight of.
And compound III and compound IV process group are 2-4～-6The effect of suppression virus is just observed in concentration range.
By above-mentioned brassboard with 1% neutral red staining, surveying 450nmA value by microplate reader, each group A value cuts virus control group A After value, each experimental group A value obtains IC compared with cell controls group A value50, IC50With TD50Compare acquisition TI: compositions suppression RSV CPE, IC is there is at HEK293 cell50It is 2-12.17, TI is 608.9；The IC of compound III50It is 2-4.36, TI is 3.9；Compound The IC of IV50It is 2-4.51, TI is 4.4.
Conclusion: compositions has significant anti-pair and glues the effect of coe virus respiratory syncytial virus RSV, can be used to prepare Anti-pair glues coe virus respiratory syncytial virus RSV medicine.Compound III and compound IV glues coe virus without significant anti-pair and breathes The effect of road syncytial virus RSV, it is not possible to be used for preparing anti-pair and glue coe virus respiratory syncytial virus RSV medicine.
(3) experimental example: the compositions Effect study to influenza A virus:
(1) cell strain and Strain: Madin-Darby canine kidney(cell line) (MDCK) (MDCK, influenza virus sensitive cells) is ground quoted from China's CDC virosis Study carefully institute；Influenza virus A type (Flu A) 1995.AII:32094 strains is quoted from China's CDC virosis institute.
(2) cytotoxic assay:
Reference literature (Wang Shouchuan, Wang Lin, old superfine, ground by oral liquid for clearing away lung-heat Contained Serum by the experiment of viral inhibition Study carefully, Nanjing University of Traditional Chinese Medicine's journal, 2008；24 (1): 25～27) method, does serial dilution (2 by each sample with 2 multiple proportions-1～-5), then it being inoculated on the MDCK in 96 plate holes by dilution sequential lateral, every hole 100uL, every dilution factor longitudinally repeats 3 holes (A, B, C row), parallel 1～6 holes setting microwell plate D are as cell controls, and 7～12 hole not inoculating cells are blank, microscope Observe CPE, Continuous Observation 7d, neutral red staining lower every day, measure OD value at 540nm wavelength, by experimental group and cell controls group OD value compares calculating cell survival rate, calculates medicine half cytotoxic concentration (TD by Reed-Muench method50)。
(3) toxin inhibitory test:
Reference literature (Wang Shouchuan, Wang Lin, old superfine, ground by oral liquid for clearing away lung-heat Contained Serum by the experiment of viral inhibition Study carefully, Nanjing University of Traditional Chinese Medicine's journal, 2008；24 (1): 25～27) method, is inoculated in 96 plates by sample by dilution sequential lateral On cell monolayer in hole, every hole 100uL, every dilution factor longitudinally repeats 4 holes, and A, B, C row of microwell plate adds containing 100 TCID50 Viral 100uL as test group, D row supplements and waits capacity cell maintenance medium is variable concentrations drug control, parallel sets F row conduct Virus control, the 1 of E row～12 as cell controls.37 DEG C, 5%CO2Cultivate, observe CPE, Continuous Observation 4d every day.Virus is right Break forth existing more than 90% CPE time, add 1% neutral red staining, read A value by microplate reader at wavelength 540nm wavelength, respectively organize A value and go Except virus control group A value, by each test group compared with cell controls group A value, it is thus achieved that cell survival rate, use Reed-Muench side Method calculates half and presses down poison concentration (IC50), TD the most at last50And IC50Compare acquisition and press down poison index (TI).
(4) result:
1. sample TD50Mensuration: cellular control unit basis of microscopic observation adherent growth is fine and close, form is good.Compositions, change Compound III and the TD of compound IV pair50It is respectively 2 at MDCK-1.94、2-2.57、2-2.82。
2. poison experiment is pressed down: the adherent densification of cellular control unit, form are good.Microscopic observation find, Flu A MDCK CPE with Refractivity strengthens, downright bad, be broken for principal character.
And compositions toxin inhibitory test respectively organizes cell, according to diluted sample gradient, there is CPE:FluA-MDCK group in gradient rule Cell cell before the 8th dilution factor is protected completely, occurs that CPE, CPE reduce with sample concentration afterwards and gradually increases the weight of.
And compound III and compound IV process group are 2-4～-6The effect of suppression virus is just observed in concentration range.
By above-mentioned brassboard 1% neutral red staining, the A value of surveying 450nm by microplate reader, respectively organize A value and cut virus control After group A value, each experimental group A value obtains IC compared with cell controls group A value50, IC50With TD50Compare acquisition TI: compositions suppression CPE, IC is there is in Flu A at mdck cell50It is 2-12.72, TI is 1758.3；The IC of compound III50It is 2-5.14, TI is 5.4；Change The IC of compound IV50It is 2-5.63, TI is 4.6.
Conclusion: compositions has a strongest inhibitory action to influenza A virus, and safety, therefore compositions is in treatment Influenza a virus infection disease has a wide range of applications background.Influenza A virus is not had by compound III and compound IV Having significant inhibitory action, and safety is extremely low, therefore compound III and compound IV is at treatment influenza a virus infection Disease does not has application background.
The preparation of embodiment 6 composition tablet involved in the present invention
Taking 2 grams of compositionss, the customary adjuvant 18 grams of tablet is prepared in addition, and mixing, conventional tablet presses makes 100.
The preparation of embodiment 7 composition capsule involved in the present invention
Taking 2 grams of compositionss, the customary adjuvant such as starch 18 grams of capsule is prepared in addition, mixing, encapsulated makes 100.

Claims (9)

1. a compositions, is characterized by that said composition is made up of compound III and compound IV, compound in said composition The mass percent of III and compound IV is respectively 40% and 60%,
2. the preparation method of compositions as claimed in claim 1, is characterized by: by powder and the compound IV of compound III Powder be sufficiently mixed according to mass percent respectively 40% and 60%.
3. a compositions as claimed in claim 1 application in antiviral drugs.
4. compositions application in antiviral drugs as claimed in claim 3, it is characterised in that: described virus is A type stream Influenza Virus.
5. compositions application in antiviral drugs as claimed in claim 3, it is characterised in that: described virus is simple bleb Exanthema virus.
6. compositions application in antiviral drugs as claimed in claim 3, it is characterised in that: described virus is respiratory tract Syncytial virus.
7. compositions application in antiviral drugs as claimed in claim 5, it is characterised in that: described herpes simplex virus For herpes simplex virus type 1 or simple herpes virus 2 type.
8. compositions application in antiviral drugs as claimed in claim 7, it is characterised in that: described herpes simplex virus 1 type is the Sm44 strain of herpes simplex virus type 1.
9. compositions application in antiviral drugs as claimed in claim 7, it is characterised in that: described herpes simplex virus 2 types are simple herpes virus 2 type 333 strain.
CN201610424346.3A 2016-06-13 2016-06-13 The compositions of Schiglautone A derivant is used for preparing antiviral drugs Pending CN106074527A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104825467A (en) * 2015-04-29 2015-08-12 南京广康协生物医药技术有限公司 Application of cleistanone O-(benzimidazolyl) ethyl derivative in preparation of anti-inflammatory drugs
CN104825465A (en) * 2015-04-15 2015-08-12 南京广康协生物医药技术有限公司 Application of O-(1H-tetrazol)ethyl derivative of Cleistanone in preparation of antiviral drugs
CN105232510A (en) * 2015-11-09 2016-01-13 南京广康协生物医药技术有限公司 Composition and application thereof to antiviral drugs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104825465A (en) * 2015-04-15 2015-08-12 南京广康协生物医药技术有限公司 Application of O-(1H-tetrazol)ethyl derivative of Cleistanone in preparation of antiviral drugs
CN104825467A (en) * 2015-04-29 2015-08-12 南京广康协生物医药技术有限公司 Application of cleistanone O-(benzimidazolyl) ethyl derivative in preparation of anti-inflammatory drugs
CN105232510A (en) * 2015-11-09 2016-01-13 南京广康协生物医药技术有限公司 Composition and application thereof to antiviral drugs

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