CN106432380A - New sesquiterpene glycoside compound, preparation method and uses thereof - Google Patents
New sesquiterpene glycoside compound, preparation method and uses thereof Download PDFInfo
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Abstract
The present invention provides a new sesquiterpene glycoside compound, wherein the compound and a pharmaceutically acceptable carrier form a pharmaceutical composition. The present invention further provides applications of the compound and the pharmaceutical composition in preparation of anti-hepatitis C virus drugs.
Description
Technical field
The present invention relates to field of medicaments is and in particular to a kind of new sesquiterpene glucoside compound, Preparation Method And The Use.
Background technology
Hepatitis C viruss (HCV) are a kind of RNA viruses it is considered that mainly having 6 kinds of HCV genotype (1-6 type) and multiple hypotype；HCV genotype and its hypotype have comparative prevalence difference in each area of the world, only have 1b and 2a amphitypy in China mainland, and based on 1b type, in South China Urban, 1b type accounts for more than 90%.HCV infection may result in the necrosis of liver chronic inflammatory disease and fibrosiss, and some patientss can develop into liver cirrhosis or even hepatocarcinoma, brings great harm to the health and lives of patient, it has also become serious society and public health problem.The medicament categories being commonly used in treatment hepatitis c virus infection at present are less, predominantly broad-spectrum antiviral medicament such as interferon (IFN-α) or Pegylation IFN-α, or IFN-α, Pegylation IFN-α are combined with ribavirin.But, these medicines usually can bring serious untoward reaction, for example：IFN-α may result in influenza-like illness, bone marrow depression, psychological problem, thyroid disease, loss of appetite, lose weight, suffer from diarrhoea, erythra, alopecia and injection site aseptic inflammation etc., and ribavirin then may produce haemolysis and teratogenesis.In view of the significant damage that HCV causes to human health is relative with anti-hepatitis C medicine deficient, it is very necessary for developing new safe and effective, Small side effects anti-hepatitis C virus medicine.
Active skull cap components are because its effect is definite, safety is good, Small side effects the advantages of and the favor of extremely doctor and patient.Sesquiterpene (sesquiterpenoids) is the compound group that a class is widely present in plant kingdom and microbial world, especially with the abundantest in Compositae, Magnoliaceae, rutaceae.In recent years, the sesquiterpene finding in the Sargassum in marine organisms and coelenteron, sponge, Mollusca gets more and more, and is most classes in terpenoid.Sesquiterpenoidss often coexist in volatile oil together with monoterpenes compound, are the main constituents of high boiling range part in volatile oil, in plant how presented in alcohol, ketone, lactone or glycosides, also with the presence of with alkaloid form.Have stronger fragrance and biological activity more the containing oxygen derivative of sesquiterpene, be the important source material of medicine, food, cosmetics industry.The framework types of sesquiterpenoid are very diversified, the structural framework being currently known is more than 200 kinds, compound numbers have as many as thousands of kinds, its biological activity is also various, and such as Santonin and its derivant have drive ascarid effect, and Herba Artemisiae Annuae have stronger antimalarial active, gossypol has spermicide effect, coriamyrtin, tutin are used for treating schizophrenia, and turmerone has choleretic effect, and euparotin, gaillardin's equimultiple sesquiterpene lactone compounds have active anticancer etc..But rare report pharmacologically active in terms of anti-hepatitis C virus for such compound at present.
Content of the invention
An object of the present invention is to provide the sesquiterpene glucoside compound shown in a kind of formula I
Invention further provides the pharmaceutical composition that type I compound is formed with pharmaceutically acceptable carrier.
Present invention also offers the application in preparation treatment anti-infection of hepatitis C virus medicine of described type I compound and its pharmaceutical composition；Wherein, described hepatitis C virus are preferably HCV-1b type hepatitis C virus；
Meanwhile, present invention also offers the preparation method of described type I compound, specifically include：
(1) weigh the fresh Folium Pini of appropriate Pinus massoniana Lamb, add in 8 times amount boiling water, reflux, extract, 1h, filter, add 6 times amount water, reflux, extract, 1 hour again, filter, merging filtrate, concentrating under reduced pressure, standby；
(2) medicinal liquid being obtained in step (1) is extracted 3 times respectively with the water-saturated n-butanol of the ethyl acetate of 1/3 volume ratio, 1/3 volume ratio successively, collect water-saturated n-butanol extract and be concentrated into extractum, obtain n-butyl alcohol extract；
(3), after will be water-soluble for n-butyl alcohol extract, with macroporous resin adsorption, water elution, collect eluent, concentrating under reduced pressure is dried, standby；
(4) by above-mentioned water elution part MCI column chromatography for separation, with water-ethanol example by volume：1:0,9:1,8:2,5:5,0:1 carries out gradient elution 2-4 post journey, and collected volume is than for 8:2 water-ethanol eluent, concentrate drying, standby；
(5) eluate of step (4) is carried out polydextran gel column chromatography for separation, water elution, collect eluent, concentrate drying, standby；
(6) by silica gel column chromatography in the eluate of step (5), with volume ratio 5:1 EtOAc：Ethanol elution, collects eluent, standby；
(7) by the eluent ODS column chromatography for separation in step (6), 10% ethanol elution, then carry out silica gel column chromatography separation, be 5 with volume ratio:1 CH2Cl2：Ethanol elution, obtains type I compound.
Wherein, the filler of described MCI post is preferably MCI reverse phase filler, and particle diameter is 75～150 μm；The filler of described sephadex column is sephadex G type, preferably sephadex G25Type, particle diameter is 50 μm；The filler of described ODS post is C18 bonded silica gel, and particle diameter is 50 μm.
Specific embodiment
Experiment material
Type I compound, according to the method preparation of embodiment 1, is configured to 100mM mother solution with 100%DMSO and keeps in nitrogen cabinet；HCV-1b replicon cell, by medicine, bright Kant builds and provides.
96 hole cell culture microwell plates, manufacturer：German Greiner bio-one, model：Greiner 655090；
CO2 cell culture incubator, manufacturer：U.S. Thermo Fisher, model：HERAcell 240；
CellTiter-Fluor reagent (cell growth fluoremetry detectable), supplier：Promega, article No.：G6082；
Bright-Glo luciferase luminous substrate, supplier：Promega, article No.：E2650；
DMEM cell culture fluid, supplier：Invitrogen, article No.：11960067.
GraphPad Prism software, supplier：GraphPad Software, model：Prism 5
Multiple labeling micropore board detector, supplier：PerkinElmer, model：Envision
Aperture resin gel (MCI), supplier：Chengdu science popularization biology company limited, model：SBC MCI GEL reversed phase chromatography filler F type, 75-150 μm
Polydextran gel, supplier：GE Healthcare Life Sciences, model：sephadex G25Medium, 50 μm
C18 bonded silica gel (ODS), supplier：Sepax Technologies, Inc, model：GP-C18,50 μm
The preparation of embodiment 1 type I compound
It is prepared according to the following steps type I compound：
(1) weigh Pinus massoniana Lamb fresh Folium Pini 24kg, add in 8 times amount boiling water, reflux, extract, 1h, filter, add 6 times amount water, reflux, extract, 1 hour again, filter, merging filtrate, concentrating under reduced pressure, standby；
(2) by the medicinal liquid being obtained in step (1), with the ethyl acetate of 1/3 volume ratio, (medicinal liquid and ethyl acetate ratio are 3 successively:1), (medicinal liquid and n-butyl alcohol ratio are 3 to the water-saturated n-butanol of 1/3 volume ratio:1) extract 3 times respectively, collect water-saturated n-butanol extract and be concentrated into extractum, obtain n-butyl alcohol extract；
(3), after will be water-soluble for n-butyl alcohol extract, with macroporous resin adsorption, water elution, collect eluent, concentrating under reduced pressure is dried, standby；
(4) by above-mentioned water elution part MCI column chromatography for separation, with water-ethanol in proportion：1:0,9:1,8:2,5:5,0:1 carries out gradient elution 2-4 post journey, collects water-ethanol (8:2) eluent, concentrate drying, standby；
(5) eluate of step (4) is carried out polydextran gel column chromatography for separation, water elution, collect eluent, concentrate drying, standby；
(6) by silica gel column chromatography in the eluate of step (5), use EtOAc：Ethanol (5:1) eluting, collects eluent, standby；
(7) by the eluent ODS column chromatography for separation in step (6), 10% ethanol elution, then carry out silica gel column chromatography separation, use CH2Cl2：Ethanol (5:1) eluting, obtains type I compound.
Said method be obtained type I compound through physics and chemistry nature examination, IR, HR-MS,1H NMR、13The identification of C-NMR, HMBC collection of illustrative plates, confirms its structure, specific as follows：
The type I compound of the present invention is white powder,Ethanol can be dissolved in.IR：3427,2960,2920,2932,2877,1637,1459,1369,1304,1265,1198,1163,1077,1021.HR-MS：441.2446([M+Na]+,C21H38NaO8 +；Calc.441.2459), in conjunction with1H-,13C-NMR infers that compound molecule formula is：C21H38O8, degree of unsaturation：3.
Type I compound1The gem-dimethyl signal of HNMR spectrum two isopropyls of display：δ H 0.84 (d, J=6.8Hz, 3H-12,3H-13), angular methyl signal δ H 0.96 (s, 3H-14), and the methyl signals being connected on oxygen-containing replacement carbon：δ H 1.03 (s, 3H-15)；Three methine signals：δ H 3.37 (d, J=8.8Hz, H-1), 1.34-1.37 (m, H-5,11)；And 5 methylene signals chemical shifts are interval：δH 1.25-1.74.13C H NMR spectroscopy shows, has three oxygen-containing replacement carbon signals, and one of is methine carbon signal：δ C 87.0 (CH-1), two other is quaternary carbon signal：δ C 71.5 (C-4) and 74.7 (C-7).HMBC composes dependency relation：δ H 0.88 (3H-14) and δ C 87.0 (CH-1), 37.8 (C-10) and 44.9 (CH-5) are remotely related；δ H 1.03 (3H-15) is remotely related to δ C 71.5 (C-4) and 44.9 (CH-5)；δ H 0.84 (6H-12,13) is remotely related to δ C 74.7 (C-7) and 38.6 (CH-11), and above-mentioned dependency relation shows oxygen-containing replacement respectively on C-1, C-4 and C-7 position.The above-mentioned data of comprehensive analysis, points out this compound to contain a sesquiterpene fragment, structure is shown below：
In addition to above-mentioned NMR signal, type I compound1H H NMR spectroscopy also shows 5 oxygen-containing replacement methine signals：δ H 4.42 (d, J=7.8Hz, H-1 '), 3.17 (t, J=9.0Hz, H-2 '), 3.40-3.42 (m, H-3 '), 3.28 (t, J=9.0Hz, H-4 '), and 3.28 (ddd, J=10.2,6.0,1.8Hz, H-5 '), and an oxygen-containing substituted methylene signal：(dd, J=12.0,1.8, a), 3.61 (dd, J=12.0,6.0, H-6 ' is b) for H-6 ' for δ H 3.82.13C H NMR spectroscopy also shows 6 oxygen-containing replacement signals：100.6 (CH-1 '), 73.1 (CH-2 '), 75.9 (CH-3 '), 69.9 (CH-4 '), 75.9 (CH-5 ') and 60.9 (CH2-6’).Above-mentioned NMR data prompting type I compound contains a glucosyl group and replaces.
In the HMBC spectrum of type I compound, there is long-range dependency relation in H-1 and C-1 ' and H-1 ' and C-1, point out glucosyl group by C-O bonded on 1 of sesquiterpene.Therefore identify that type I compound is：(1R, 4S, 4aR, 6S, 8aR)-decahydro-4,6-dihydroxy-6- (1-methyethyl) -4,8a-dimethy-1-naphthalenyl- β-D-glucopyranoside, structure is as shown in formula I.
Type I compound1H and13The ownership of C NMR see table 1.
Table 1
The anti-hepatitis C activity of embodiment 2 type I compound
1st, experimental technique
Modus ponens I compound stock solutions, obtain, with 0.5% DMSO solution dilution, the test solution that concentration is respectively 500,166.7,55.56,18.52,6.173,2.058,0.686,0.229 μM；Separately take 0.5% DMSO solution as blank；Above-mentioned test solution and placebo solution are separately added into 96 orifice plates equipped with DMEM culture fluid.
Cell is processed：Above-mentioned 96 porocyte plates are planted respectively into HCV-1b replicon cell (8,000 cells/well), be subsequently placed at 37 DEG C, 5%CO2Cultivate 3 days in incubator.
Cytoactive detection：Every hole addition CellTiter-Fluor reagent, 37 DEG C, 5%CO2After incubator cultured cells 1 hour, detect fluorescence signal value with multiple labeling micropore board detector detecting system Envision.
Anti-HCV activity detects：Every hole adds Bright-Glo luciferase luminous substrate, uses multiple labeling micropore board detector chemiluminescence detection system Envision to detect values of chemiluminescence in 5 minutes.
2nd, data processing：
Using the fluorescence data in each hole in cytoactive detection test, it is calculated as follows cell viability percentage：
Using the fluorescence data in each hole in anti-HCV activity detection test, it is calculated as follows suppression HCV percent：
CPD：The signal value in type I compound hole.
HPE(Hundred percent effect):Substrate hole signal value, only has DMEM culture fluid in hole.
ZPE(Zero percent effect):Blank control wells signal value.
Cell viability percent, suppression percentage are directed respectively into GraphPad Prism software carry out data processing and draw the corresponding curve of compound and its cytotoxicity (CC50) and the inhibitory activity (EC to HCV50) numerical value.
3rd, experimental result
Type I compound anti-HCV activity result of the test of the present invention see table 2:
From data in table 2, type I compound of the present invention all shows different degrees of HCV inhibitory activity under the conditions of 0.229-500 μM, its EC50 value is 145.2 μM, under 500 μM, 97.5% is reached to the suppression ratio of HCV, and do not show obvious cytotoxicity, it can be seen that the type I compound of the present invention can effectively suppress HCV virus to infect, and no obvious toxic-side effects.
Claims (9)
1. the sesquiterpene glucoside compound shown in a kind of formula I
2. application in preparation treatment anti-infection of hepatitis C virus medicine for the compound described in claim 1.
3. application according to claim 2 is it is characterised in that described hepatitis C virus are HCV-1b type hepatitis C virus.
4. the compound described in claim 1 and pharmaceutically acceptable carrier form pharmaceutical composition.
5. pharmaceutical composition according to claim 4 is it is characterised in that described pharmaceutical composition is in preparation treatment anti-hepatitis C disease
Application in malicious infection medicine.
6. the preparation method of the compound described in claim 1 is it is characterised in that described preparation method comprises the steps：
(1) weigh the fresh Folium Pini of appropriate Pinus massoniana Lamb, add in 8 times amount boiling water, reflux, extract, 1h, filters, adds 6 times again
Amount water, reflux, extract, 1 hour, filter, merging filtrate, concentrating under reduced pressure, standby；
(2) medicinal liquid being obtained in step (1) is divided with the water-saturated n-butanol of the ethyl acetate of 1/3 volume ratio, 1/3 volume ratio successively
Not Cui Qu 3 times, collect water-saturated n-butanol extract and be concentrated into extractum, obtain n-butyl alcohol extract；
(3), after will be water-soluble for n-butyl alcohol extract, with macroporous resin adsorption, water elution, collect eluent, concentrating under reduced pressure is dried, standby；
(4) by above-mentioned water elution part MCI column chromatography for separation, with water-ethanol example by volume：1:0,9:1,8:2,5:5,
0:1 carries out gradient elution 2-4 post journey, and collected volume compares 8:2 water-ethanol eluent, concentrate drying, standby；
(5) eluate of step (4) is carried out polydextran gel column chromatography for separation, water elution, collect eluent, concentrate drying,
Standby；
(6) by silica gel column chromatography in the eluate of step (5), with volume ratio 5:1 EtOAc：Ethanol elution, collects eluent,
Standby；
(7) by the eluent ODS column chromatography for separation in step (6), 10% ethanol elution, then carry out silica gel column chromatography separation,
With volume ratio 5:1 CH2Cl2：Ethanol elution, obtains type I compound.
7. it is characterised in that the filler of described MCI post is preferably, MCI is anti-phase to be filled out preparation method according to claim 6
Material, particle diameter is 75～150 μm.
8. preparation method according to claim 7 is it is characterised in that the filler of described sephadex column is polydextran gel
G type, preferably sephadex G25Type, particle diameter is 50 μm.
9. preparation method according to claim 8 it is characterised in that described ODS post filler be C18 bonded silica gel,
Particle diameter is 50 μm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110141576A (en) * | 2018-02-12 | 2019-08-20 | 成都康弘制药有限公司 | A kind of compound is used to prepare the application in treatment oxidative damage related disease drug |
| CN115651054A (en) * | 2019-01-08 | 2023-01-31 | 成都康弘药业集团股份有限公司 | Steroid compound, use and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110141576A (en) * | 2018-02-12 | 2019-08-20 | 成都康弘制药有限公司 | A kind of compound is used to prepare the application in treatment oxidative damage related disease drug |
| CN115651054A (en) * | 2019-01-08 | 2023-01-31 | 成都康弘药业集团股份有限公司 | Steroid compound, use and preparation method thereof |
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