CN105250290A - Composition and application thereof to antiviral medicines - Google Patents

Abstract

The invention relates to the field of organic synthesis and medicinal chemistry and in particular relates to a composition and an application thereof to preparation of antiviral medicines. The invention discloses the composition and the preparation method thereof. Pharmacological experiments show that the composition has the antiviral effect and has the value in developing the antiviral medicines.

Description

A kind of compositions and the application in antiviral drugs thereof

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 (Herpessimplexvirus, and herpes simplex virus type 2 (Herpessimplexvirus HSV-1), HSV-2) herpes coe virus is belonged to, HSV-1 and HSV-2 not only causes lip or genital mucosal herpes, is also cause encephalitis, hepatitis, the severe systemic infection even important pathogen of intrauterine infection simultaneously.

Respiratory syncytial virus (RespiratorySyncytialVirus, RSV) mainly causes respiratory system infection, causes serious pneumonia infant, old age and immunodeficiency crowd, is one of important pathogen of infecting of human upper respiratory tract.

Influenza A virus (InfluenzaAvirus, Flu-A) belongs to Orthomyxoviridae, has among a small circle every year popular, cause upper respiratory system to infect the mankind and other biological.When being very popular, serious respiratory system infection can be caused, notably cause pneumonia, encephalitis even dead.

There is the problem that toxicity is large, safety is low in above-mentioned three kinds of viral current existing medicines for the treatment of, from natural product, find compound or lead compound and carry out structural modification and obtain its derivant, thus the potential drug obtaining high-efficiency low-toxicity there is important value most.

The Compound I that the present invention relates to is one and delivers (AyumiOhsakietal. in 2011,2011.SalviskinoneA, aditerpenewithanewskeletonfromSalviaprzewalskii.Tetrahed ronLetters52 (2011) 1375 – 1377) compound, we have carried out structural modification to Compound I, obtain two new derivants and compound III and compound IV, and prepared compositions by compound III and compound IV, and said composition antiviral activity is evaluated, 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, and in said composition, the mass percent of compound III and compound IV is respectively 25% and 75%.

Compositions disclosed by the invention can make pharmaceutically acceptable salt or pharmaceutically acceptable carrier.

Pharmacodynamic experiment shows, compositions of the present invention has good antivirus action.Pharmaceutically acceptable salt of the present invention has same drug effect.

The experiment in vitro of compositions shows, compositions has the activity of very strong anti-HSV-1, HSV-2, RSV and fluA, and therefore compositions of the present invention is expected to be used to prepare novel antiviral medicine.

The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not by any restriction of specific embodiment, but be limited by claim.

Detailed description of the invention

The preparation of embodiment 1 compound S alviskinoneA

Document (the AyumiOhsakietal. that the people such as the preparation method reference AyumiOhsaki of compound S alviskinoneA (I) deliver, 2011.SalviskinoneA, aditerpenewithanewskeletonfromSalviaprzewalskii.Tetrahed ronLetters52 (2011) 1375 – 1377) method.

The synthesis of the O-bromoethyl derivant (II) of embodiment 2SalviskinoneA

By Compound I (312mg, 1.00mmol) be dissolved in 15mL benzene, add in solution tetrabutyl ammonium bromide (TBAB) (0.08g), 1,50% sodium hydroxide solution of 2-Bromofume (3.760g, 20.00mmol) and 6mL.Mixture stirs 12h at 35 degrees Celsius.After 12h, reactant liquor is poured in frozen water, use dichloromethane extraction twice immediately, merge organic phase solution.Then use water and saturated common salt water washing 4 times successively to organic phase solution, then use anhydrous sodium sulfate drying, last concentrating under reduced pressure is removed solvent and is obtained product crude product.Product crude product purification by silica gel column chromatography (mobile phase is: petroleum ether/acetone=100:1.5, v/v), collects brown concentrated elution band and flings to the brown ceramic powder (327mg, 78%) that namely solvent obtains Compound II per.

¹HNMR(500MHz,DMSO-d ₆)δ6.63(s,1H),6.37(s,1H),5.81(s,1H),4.51(s,2H),3.84(s,1H),3.79(s,2H),2.15(s,1H),2.04(s,1H),1.91(s,1H),1.65(s,1H),1.39(s,3H),1.08(s,6H),0.99(s,6H).

¹³CNMR(125MHz,DMSO-d6)δ188.07(s),183.70(s),154.38(s),147.57(s),140.21(s),136.40(s),134.71(s),131.25(s),128.40(s),118.83(s),72.12(s),45.49(s),37.54(s),33.58(s),31.78(s),26.17(s),25.12(s),24.66(s),23.51(s),23.17(s),22.65(s).

HRMS(ESI)m/z[M+H] ⁺calcdforC ₂₂H ₂₈BrO ₃:419.1222；found419.1220.

The synthesis of the O-(nafoxidine base) ethyl derivative (III) of embodiment 3SalviskinoneA

Compound II per (209mg, 0.5mmol) is dissolved in the middle of 20mL acetonitrile, adds Anhydrous potassium carbonate (345mg wherein, 2.5mmol), potassium iodide (84mg, 0.5mmol) and pyrrolidine (1420mg, 20mmol), mixture reflux 8h.After reaction terminates, reactant liquor is poured in frozen water, with equivalent dichloromethane extraction 3 times, merge organic facies.Organic facies after merging with water and saturated common salt water washing successively, then use anhydrous sodium sulfate drying, concentrating under reduced pressure is removed solvent and is obtained product crude product.Product crude product purification by silica gel column chromatography (mobile phase is: petroleum ether/acetone=100:1.0, v/v), collects brown concentrated elution band and flings to the yellow powder (131.6mg, 65%) that namely solvent obtains compound III.

¹HNMR(500MHz,DMSO-d6)δ6.57(s,1H),6.29(s,1H),5.82(s,1H),4.25(s,2H),3.86(s,1H),3.10(s,2H),2.55(s,4H),2.16(s,1H),2.07(s,1H),1.893(s,1H),1.73(s,4H),1.70(s,1H),1.38(s,3H),1.09(s,6H),1.02(s,6H).

¹³CNMR(125MHz,DMSO-d6)δ188.10(s),183.71(s),154.37(s),147.57(s),140.19(s),136.41(s),134.73(s),131.25(s),128.38(s),118.82(s),69.23(s),54.54(d,J＝16.6Hz),45.51(s),37.54(s),33.56(s),26.16(s),25.19(d,J＝10.3Hz),24.73(s),23.58(s),23.24(s),22.69(s).

HRMS(ESI):m/z[M+H] ⁺calcdforC ₂₆H ₃₆NO ₃:410.2695；found:410.2693。

The synthesis of O-(piperidyl) ethyl derivative (IV) of embodiment 4SalviskinoneA

Compound II per (209mg, 0.5mmol) is dissolved in the middle of 20mL acetonitrile, adds Anhydrous potassium carbonate (345mg wherein, 2.5mmol), potassium iodide (84mg, 0.5mmol) and piperidines (852mg, 10mmol), mixture reflux 10h.After reaction terminates, reactant liquor is poured in 25mL frozen water, with equivalent dichloromethane extraction 2 times, merge organic facies.Organic facies after merging with water and saturated common salt water washing successively, then use anhydrous sodium sulfate drying, concentrating under reduced pressure is removed solvent and is obtained product crude product.Product crude product purification by silica gel column chromatography (mobile phase is: petroleum ether/acetone=100:0.5, v/v), collects yellow concentrated elution band and flings to the yellow powder (116.3mg, 55%) that namely solvent obtains compound IV.

¹HNMR(500MHz,Chloroform-d1)δ6.44(d,J＝95.3Hz,1H),6.31(s,1H),5.73(s,1H),4.20(s,2H),3.74(s,1H),3.01(s,2H),2.39(s,4H),2.07(s,1H),1.96(s,1H),1.86(s,1H),1.60(s,1H),1.44(s,4H),1.33(d,J＝15.0Hz,5H),1.00(s,6H),0.91(s,6H).

¹³CNMR(125MHz,DMSO-d6)δ188.16(s),183.79(s),154.48(s),147.63(s),140.27(s),136.51(s),134.79(s),131.33(s),128.47(s),118.88(s),69.31(s),54.69(d,J＝16.2Hz),45.56(s),37.61(s),33.62(s),26.21(s),25.21(s),24.65(d,J＝19.1Hz),23.50(d,J＝17.2Hz),23.23(s),22.68(s).

HRMS(ESI):m/z[M+H] ⁺calcdforC ₂₇H ₃₈NO ₃:424.2852；found:424.2856。

Embodiment 5 compositions antiviral activity

(1) experimental example: compositions is to the Effect study of herpesvirus:

(1) cell strain and Strain:

HEKC (HEK293 is HSV-1, HSV-2 virus sensitive cells) purchased from American Clontech company; Herpes simplex virus type 1 (HSV-1) Sm44 strain, simple herpes virus 2 type (HSV-2) 333 strain are drawn from Chinese CDC virosis institute.

(2) cytotoxic assay:

The preparation of compositions: loaded by the powder of 75mg compound IV crossing 200 order nets after crossing the powder of the 25mg compound III of 200 order nets and grinding after grinding to mix in tubule with cover and with turbine stirring instrument and namely obtain 100mg compositions, obtains the solution of compositions by the compositions of this 100mg of water dissolution during use.

Reference literature (Wang Shouchuan, Wang Lin, old superfine, oral liquid for clearing away lung-heat Contained Serum to the experimentation of viral inhibition, 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 ^{0 ~-5}), then be 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 are capable), parallel 1 ~ 6 hole of microwell plate D of setting is as cell controls, 7 ~ 12 holes not inoculating cell are blank, microscope observes CPE lower every day, Continuous Observation 7d, neutral red staining, measure OD value at 540nm wavelength, by experimental group calculating cell survival rate compared with cell controls group OD value, calculate medicine half cytotoxic concentration (TD by Reed-Muench method ₅₀).

(4) toxin inhibitory test:

Reference literature (Wang Shouchuan, Wang Lin, old superfine, oral liquid for clearing away lung-heat Contained Serum to the experimentation of viral inhibition, Nanjing University of Traditional Chinese Medicine's journal, 2008; 24 (1): 25 ~ 27) method, by sample by dilution order (2 ^{-3 ~-14}) be laterally inoculated in 96 plate holes cell monolayer on, every hole 100uL, every dilution factor longitudinally repeats 4 holes, and A, B, C of microwell plate are capable to be added containing 100 TCID ₅₀viral 100uL as test group, capable the supplementing of D waits capacity cell maintenance medium to be variable concentrations drug control, and the parallel F that establishes is capable of virus control, E capable 1 ~ 12 as cell controls.37 DEG C, 5%CO ₂cultivate, every day observes CPE, Continuous Observation 4d.When there is more than 90% CPE in virus control, add 1% neutral red staining, A value is read at wavelength 540nm wavelength by microplate reader, each group of A value removes virus control group A value, by each test group compared with cell controls group A value, obtain cell survival rate, calculate half by Reed-Muench method and press down malicious concentration (IC ₅₀), TD the most at last ₅₀and IC ₅₀compare to obtain and press down malicious index (TI).

(5) result:

1. sample TD ₅₀mensuration: cellular control unit basis of microscopic observation adherent growth is fine and close, form is good.The TD of compositions, compound III and compound IV ₅₀2 are respectively at HEK293 ^-2.15, 2 ^-2.26, 2 ^-2.38.

2. poison experiment is pressed down: the adherent densification of cellular control unit, form are good.Microscopic observation finds, the CPE of more than 90% is there is respectively in HSV-1, HSV-2 virus control group at 3d, 3d, HSV1 with HSV2 HEK293CPE then with swelling, many cells merge (HSV1 cause multiple cell warm become bulla, and HSV2 cause be fused to vesicle), become circle, come off into principal character.

And compositions toxin inhibitory test respectively organizes cell; according to composition sample dilution gradient; gradient rule occur CPE:HSV1-HEK293 before the 10th dilution factor, HSV2-HEK293 group cell before the 9th dilution factor is completely protected, occurs CPE afterwards, CPE reduces with sample concentration and increases the weight of gradually.

And compound III and compound IV processed group are 2 ^{-4 ~-6}the effect suppressing virus is just observed in concentration range.

By above-mentioned brassboard 1% neutral red staining, survey 450nmA value by microplate reader, after each group A value cuts virus control group A value, each experimental group A value obtains IC compared with cell controls group A value ₅₀, IC ₅₀with TD ₅₀compare the TI obtaining compositions: suppress HSV-1, HSV-2, at HEK293 cell, CPE, IC occur ₅₀be respectively 2 ^-11.87, 2 ^-11.98, TI is respectively 843.4,910.2.The TI of compound III: corresponding IC ₅₀be respectively 2 ^-3.17, 2 ^-5.01, TI is respectively 1.9,6.7.The TI of compound IV: corresponding IC ₅₀be respectively 2 ^-5.18, 2 ^-4.63, TI is respectively 7.0,4.8.

Conclusion: compositions has the effect of significant herpes coe virus (comprising HSV-1, HEV-2), and safety is high, therefore compositions has a wide range of applications background in treatment herpesvirus infection disease.Compound III and compound IV do not have the effect of significant herpes coe virus (comprising HSV-1, HEV-2), and safety is extremely low, and therefore compound III and compound IV do not have application background in treatment herpesvirus infection disease.

(2) experimental example: compositions is to the Effect study of respiratory syncytial virus (RSV)

(1) configuration of compositions, compound III and compound IV: medicine is dissolved in cell maintenance medium (containing the MEM of 2% new-born calf serum, GIBICO Products) for subsequent use.

(2) cell strain and Strain:

HEKC (HEK293, RSV virus sensitive cells) purchased from American Clontech company; Respiratory syncytial virus (RSV) Long strain is drawn from Chinese CDC virosis institute.

(3) cytotoxic assay:

Reference literature (Wang Shouchuan, Wang Lin, old superfine, oral liquid for clearing away lung-heat Contained Serum to the inhibiting experimentation of respiratory syncytial virus, Nanjing University of Traditional Chinese Medicine's journal, 2008; 24 (1): 25 ~ 27) method, does serial dilution (2 by sample with 2 multiple proportions ^{0 ~-5}), then be 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 are capable), parallel 1 ~ 6 hole of microwell plate D of setting is as cell controls, 7 ~ 12 holes not inoculating cell are blank, microscope observes CPE lower every day, Continuous Observation 7d, neutral red staining, measure OD value at 540nm wavelength, by experimental group calculating cell survival rate compared with cell controls group OD value, calculate medicine half cytotoxic concentration (TD by Reed-Muench method ₅₀).

(4) toxin inhibitory test:

Reference literature (Wang Shouchuan, Wang Lin, old superfine, oral liquid for clearing away lung-heat Contained Serum to the inhibiting experimentation of respiratory syncytial virus, 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 in 96 plate holes cell monolayer on, every hole 100uL, every dilution factor longitudinally repeats 4 holes, and A, B, C of microwell plate are capable to be added containing 100 TCID ₅₀viral 100uL as test group, capable the supplementing of D waits capacity cell maintenance medium to be variable concentrations drug control, and the parallel F that establishes is capable of virus control, E capable 1 ~ 12 as cell controls.37 DEG C, 5%CO ₂cultivate, every day observes CPE, Continuous Observation 4d.When there is more than 90% CPE in virus control, add 1% neutral red staining, A value is read at wavelength 540nm wavelength by microplate reader, each group of A value removes virus control group A value, by each test group compared with cell controls group A value, obtain cell survival rate, calculate half by Reed-Muench method and press down malicious concentration (IC ₅₀), TD the most at last ₅₀and IC ₅₀compare to obtain and press down malicious index (TI).

(5) result:

1. sample TD ₅₀mensuration: cellular control unit basis of microscopic observation adherent growth is fine and close, form is good.Compositions, compound III and compound IV are to the TD of HEK293 ₅₀be respectively 2 ^-2.11, 2 ^-2.35, 2 ^-2.74.

2. poison experiment is pressed down: the adherent densification of cellular control unit, form are good.Microscopic observation finds, RSV virus control group occurs the CPE of more than 90% at 2d, and RSV strengthens with refractivity at HEK293CPE, change is justified, comes off, is 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 completely protected, occurs CPE afterwards, and CPE reduces with sample concentration and increases the weight of gradually.

And compound III and compound IV processed group are 2 ^{-4 ~-6}the effect suppressing virus is just observed in concentration range.

By above-mentioned brassboard 1% neutral red staining, survey 450nmA value by microplate reader, after each group A value cuts virus control group A value, each experimental group A value obtains IC compared with cell controls group A value ₅₀, IC ₅₀with TD ₅₀compare and obtain TI: compositions suppresses RSV, at HEK293 cell, CPE, IC occur ₅₀be 2 ^-12.12, TI is 1031.2; The IC of compound III ₅₀be 2 ^-5.04, TI is 6.5; The IC of compound IV ₅₀be 2 ^-4.68, TI is 3.8; .

Conclusion: compositions has the effect that significant anti-pair glues coe virus respiratory syncytial virus RSV, can be used for preparing anti-pair and glue coe virus respiratory syncytial virus RSV medicine.Compound III and compound IV glue the effect of coe virus respiratory syncytial virus RSV without significant anti-pair, are not available to prepare anti-pair and glue coe virus respiratory syncytial virus RSV medicine.

(3) experimental example: compositions is to the Effect study of influenza A virus:

(1) cell strain and Strain: Madin-Darby canine kidney(cell line) (MDCK) (MDCK, influenza virus sensitive cells) is drawn from Chinese CDC virosis institute; Influenza virus A type (FluA) 1995.AII:32094 strains draws from Chinese CDC virosis institute.

(2) cytotoxic assay:

Reference literature (Wang Shouchuan, Wang Lin, old superfine, oral liquid for clearing away lung-heat Contained Serum to the experimentation of viral inhibition, 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 be 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 are capable), parallel 1 ~ 6 hole of microwell plate D of setting is as cell controls, 7 ~ 12 holes not inoculating cell are blank, microscope observes CPE lower every day, Continuous Observation 7d, neutral red staining, measure OD value at 540nm wavelength, by experimental group calculating cell survival rate compared with cell controls group OD value, calculate medicine half cytotoxic concentration (TD by Reed-Muench method ₅₀).

(3) toxin inhibitory test:

Reference literature (Wang Shouchuan, Wang Lin, old superfine, oral liquid for clearing away lung-heat Contained Serum to the experimentation of viral inhibition, Nanjing University of Traditional Chinese Medicine's journal, 2008; 24 (1): 25 ~ 27) method, be inoculated on the cell monolayer in 96 plate holes by sample by dilution sequential lateral, every hole 100uL, every dilution factor longitudinally repeats 4 holes, and A, B, C of microwell plate are capable to be added containing 100 TCID ₅₀viral 100uL as test group, capable the supplementing of D waits capacity cell maintenance medium to be variable concentrations drug control, and the parallel F that establishes is capable of virus control, E capable 1 ~ 12 as cell controls.37 DEG C, 5%CO ₂cultivate, every day observes CPE, Continuous Observation 4d.When there is more than 90% CPE in virus control, add 1% neutral red staining, A value is read at wavelength 540nm wavelength by microplate reader, each group of A value removes virus control group A value, by each test group compared with cell controls group A value, obtain cell survival rate, calculate half by Reed-Muench method and press down malicious concentration (IC ₅₀), TD the most at last ₅₀and IC ₅₀compare to obtain and press down malicious index (TI).

(4) result:

1. sample TD ₅₀mensuration: cellular control unit basis of microscopic observation adherent growth is fine and close, form is good.The TD that compositions, compound III and compound IV are right ₅₀2 are respectively at MDCK ^-2.29, 2 ^-2.57, 2 ^-2.94.

2. poison experiment is pressed down: the adherent densification of cellular control unit, form are good.Microscopic observation finds, FluA strengthens with refractivity at MDCKCPE, downright bad, be broken for principal character.

And compositions toxin inhibitory test respectively organizes cell, according to diluted sample gradient, gradient rule occurs that CPE:FluA-MDCK group cell cell before the 8th dilution factor is completely protected, occurs CPE afterwards, and CPE reduces with sample concentration and increases the weight of gradually.

And compound III and compound IV processed group are 2 ^{-4 ~-6}the effect suppressing virus is just observed in concentration range.

By above-mentioned brassboard 1% neutral red staining, the A value surveying 450nm by microplate reader, after each group A value cuts virus control group A value, each experimental group A value obtains IC compared with cell controls group A value ₅₀, IC ₅₀with TD ₅₀compare and obtain TI: compositions suppresses FluA at mdck cell, CPE to occur, IC ₅₀be 2 ^-11.82, TI is 739.3; The IC of compound III ₅₀be 2 ^-4.51, TI is 3.8; The IC of compound IV ₅₀be 2 ^-3.27, TI is 1.3.

Conclusion: compositions has very strong inhibitory action to influenza A virus, and safety, therefore compositions has a wide range of applications background in treatment influenza a virus infection disease.Compound III and compound IV do not have significant inhibitory action to influenza A virus, and safety is extremely low, and therefore compound III and compound IV do not have application background in treatment influenza a virus infection disease.

The preparation of embodiment 6 composition tablet involved in the present invention

Get 2 grams of compositionss, add the customary adjuvant 18 grams preparing tablet, mixing, conventional tablet presses makes 100.

The preparation of embodiment 7 composition capsule involved in the present invention

Get 2 grams of compositionss, add prepare capsule customary adjuvant as starch 18 grams, mixing, encapsulatedly makes 100.

Claims (9)

1. a compositions, it is characterized by said composition and be made up of compound III and compound IV, in said composition, the mass percent of compound III and compound IV is respectively 25% and 75%,

2. the preparation method of compositions as claimed in claim 1, is characterized by: the powder of compound III and the powder of compound IV are respectively 25% and 75% according to mass percent and fully mix.

3. the application of compositions as claimed in claim 1 in antiviral drugs.

4. the application of compositions in antiviral drugs as claimed in claim 3, is characterized in that: described virus is influenza A virus.

5. the application of compositions in antiviral drugs as claimed in claim 3, is characterized in that: described virus is herpes simplex virus.

6. the application of compositions in antiviral drugs as claimed in claim 3, is characterized in that: described virus is respiratory syncytial virus.

7. the application of compositions in antiviral drugs as claimed in claim 5, is characterized in that: described herpes simplex virus is herpes simplex virus type 1 or simple herpes virus 2 type.

8. the application of compositions in antiviral drugs as claimed in claim 7, is characterized in that: described herpes simplex virus type 1 is the Sm44 strain of herpes simplex virus type 1.

9. the application of compositions in antiviral drugs as claimed in claim 7, is characterized in that: described herpes simplex virus type 2 is the strain of simple herpes virus 2 type 333.