CN101829105A - Application of benzoly-substituted silybin in preparing drugs for treating virus hepatitis - Google Patents
Application of benzoly-substituted silybin in preparing drugs for treating virus hepatitis Download PDFInfo
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Abstract
The invention relates to application of benzoly-substituted silybin in preparing drugs for treating virus hepatitis, in particular to application of benzoly-substituted silybin ester flavanolignan and pharmaceutically acceptable salts thereof in preparing drugs for inhibiting HBV DNA reproduction and treating hepatitis B virus infected diseases. The flavanolignan has the specific activity for inhibiting the hepatitis virus HBV DNA; compared with alpha-interferon in the highest concentration of 10000 unit per milliliter, the flavanolignan has twice inhibition activity in inhibiting the reproduction of the hepatitis virus HBV DNA when the high dosage is 100 microgram per milliliter, and belongs to the non-nucleoside inhibition hepatitis virus natural product, and the pharmacodynamics indicate that the flavanolignan and the pharmaceutically acceptable salts thereof can be anticipated to preparing the drugs for inhibiting HBV DNA reproduction and treating hepatitis B virus infected diseases.
Description
Technical field
The present invention relates to medical technical field; particularly; the silybin ester type flavanolignan or its officinal salt that the present invention relates to a benzoyl replacement shown in the formula (1) are used for preparation inhibition hepatitis B virus DNA (deoxyribonucleic acid) HBV dna replication dna; the purposes of treatment hepatitis B medicine; this flavanolignan has definite inhibition HBV DNA activity; it suppresses the inhibition activity of specific activity alpha-interferon when the maximum concentration 10000 units per ml concentration to duplicating of HBV DNA and exceeds twice nearly when high dose (100 mcg/ml); belong to potent non-nucleoside and suppress the hepatitis B virus natural product, above pharmacodynamic result shows that this flavanolignan or its officinal salt can be expected and is used to prepare the purposes that suppresses the HBV dna replication dna or treat hepatitis b virus infected disease medicament.
Technical background
Hepatitis B (hepatitis B) is the infectious disease that is caused by hepatitis b virus hbv, and HBV is a member of Hepadnaviridae hepadnaviridae, and it is shaped as the spheroidal particle of diameter 42 nanometers.HBV is peculiar virus, and less being infectious in other animal has only in human body or primate chimpanzee body and just can duplicate.This virus is propagated by hepatitis B virus carriers and hepatitis B patients'blood, saliva, seminal fluid, vaginal secretions, has chronic carrier state.Hepatitis B is widely current in China, and because of it is divided in vertical transmission, horizontal transmission, the family propagation, iatrogenic infection and multiple mode such as spread through sex intercourse, to crowd infection rate's height, infection rate reaches more than 35% in certain areas.According to interrelated data, hepatitis detects male patient and has reached 1.89 hundred million, and the number (carrier) nearly 400,000,000 of should not going to a doctor is one of the most serious infectious disease of current harm people ' s health.Hepatitis B clinical manifestation variation easily develops into chronic hepatitis B (CHB) and liver cirrhosis, and a few patients can change primary hepatocarcinoma into.
Along with the research of hepatopathy, developed the analysis of standardized HBV DNA in recent years, advanced understanding greatly the hepatitis B patient state of an illness.The quantitative analysis of HBV DNA can be predicted the seriousness and the prognosis thereof of hepatitis B, because HBV DNA lasting masculin (promptly continuing viremia) makes the hepatitis B disease progression easily and increases the weight of; High hepatitis B virus (HBV DNA) content promotes the formation of liver cirrhosis easily; The lasting existence of HBV DNA is that the high risk factor, particularly viral level of hepatocarcinoma (HCC) generation is higher, the course of disease is long, older or merge other liver patient; The HBV DNA that continues high concentration exists, and the mortality rate that can cause losing compensatory liver cirrhosis and constitutional liver serious symptom obviously increases.Simultaneously must recognize that the relation of HBV dna level and liver histological is extremely close: bibliographical information is through antiviral therapy, and the improvement of hepatic fibrosis and elimination are obviously; Recent international hepatopathy meeting report, potent and low chemical sproof antiviral therapy along with the reduction of HBV DNA with turn out cloudy, in various degree reverse can occur and observe liver cirrhosis, and therefore present opinion liver cirrhosis also should carry out antiviral therapy.
Therefore, the application of HBV DNA index in antiviral therapy also plays a part very important: the level of HBV DNA is the important indicator whether the decision chronic hepatitis B needs antiviral therapy; Make the difference treatment standard and the requirement of the HBeAg positive or HBeAg feminine gender respectively according to the different situations of HBV DNA; In antiviral therapy, according to the therapeutic response of HBVDNA, judge whether that virusology replys in early days and then determine the strategy of long-term prescription to reply with the virusology that obtains persistence, reach and continue the purpose that virus suppresses; According to the virological situation of replying of HBVDNA, create serological switching foundation of HBeAg and condition, to reach its good middle therapeutic goal; Continue the inhibition situation according to HBV DNA and strive for that virus continues feminine gender, to strive for reaching the final therapeutic goal of antiviral; Continue to be suppressed fully according to HBV DNA, also demonstrated improvement in various degree and the disappearance of cccDNA; In antiviral therapy, assess and prevent the risk of caused virus variation of antiviral drugs and drug resistance generation with the variation of HBV DNA; In case when virus variation or drug resistance took place, the variation of HBV DNA was unique signal at first and diagnosis basis, also be treatment drug resistance and guidance and the foundation that changes therapeutic strategy.In sum, the inhibition degree of HBV DNA there is new important meaning in the further diagnosis of hepatitis B and treatment,, assessment hepatitis B prognosis and drug resistance danger is all had bigger directive function the observation of curative effect.
By above-mentioned factor as can be known: the basic link that the inhibition hepatitis B virus is bred in vivo is to suppress duplicating of HBV DNA.The reduction of HBV dna level or be lower than detection level be the check antiviral drugs one JINYAOSHI.So, Asia-Pacific liver EASD and European liver EASD all with the HBV DNA detection less than as one of hepatitis B virus patient treatment terminal point.Also tested compounds is considered as one of the test event that must finish for the inhibition strength of HBV DNA in China's new drug development guide.Why lamivudine can become first-selected nucleoside medicine is because it has the activity of potent inhibition HBV dna replication dna.
Mandatory declaration be: the present antiviral drugs of the using inhibitor of virus replication just in fact, directly kill virus and break virus body, otherwise will damage host cell.These antiviral drugs (mostly being nucleoside medicine) also exist above-mentioned toxic and side effects greatly, easily to cause after viral gene sudden change, the drug withdrawal shortcomings such as easily knock-on, so the development of new antiviral drugs is the task of top priority in current medicament research and development field.It all has extremely important social meaning and economic implications for a large amount of hepatitis B patient and virus carrier, the control sources of infection etc. of treatment China.So, find that from the natural drug of national folk life-time service new non-nucleoside hepatitis B virus inhibitor and this type of lead compound that can suppress the HBV dna replication dna has very big guiding significance, and vast development prospect arranged.
Based on this purpose, the inventor once finished the patent and the article of multinomial anti-hepatitis virus natural product and structure of modification derivant thereof in the past, multiple inhibition hepatitis B virus surface antigen HBsAg or hepatitis B virus e antigen HBeAg activity have been found, the chemical compound that suppresses the HBV dna replication dna, thus explanation filters out from natural product and synthesis of derivatives thereof and can suppress HBVDNA and duplicate, the novelty medicine of control hepatitis B virus infection is feasible [referring to " medical usage of a class mapping eucalyptus globulus alkanol type sesquiterpene for inhibiting hepatitis virus " (Zhao Yu, Liu Guangming, Yu Rongmin, Li Haibo etc.; ZL 200610053827.4); " medical usages of 2 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Lee school Kun, Zhao Yu, Huang Kexin, Li Haibo etc.; ZL200610053749.8); " 2 α, the medical usage of 3 beta-dihydroxies-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Zhang Li and, Dong Sun Han, Li Haibo etc.; ZL200610053601.4); The purposes and the pharmaceutical composition thereof of hepatitis B virus " the eremophilane lactone suppress " (Zhao Yu, Li Haibo, Yang Leixiang, girth are new etc.; ZL 03153691.3); " a kind of Eremophilone lactones acid natural product and application thereof " (Zhao Yu, girth new, Shi Shuyun, Wang Xiaoyu etc.; ZL 200610053575.5); " a kind of eudesmane type sesquiterpenes acid and uses thereof " (Zhao Yu, Liu Guangming, Li Haibo, Wu Xiumei etc.; ZL 200610053579.3); " purposes of laggera plant abstract in inhibiting herpes simplex virus and hepatitis B virus " (Zhao Yu, girth new, Yu Rongmin, Bai Hua; CN 1989989A); " medical usage of 1 β-oxo-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Li Haibo etc.; CN 1927197A); " medical usage of 1 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Wu Xiumei etc.; CN 1935131A); " medical usage of acid of mapping eremophilane and inhibition hepatitis B surface antigen thereof " (Huang Kexin, Lee school Kun, Wang Xiaoyu, Zhao Yu etc.; CN 101239054); " 1-oxygen-substituted benzene formyl quinic acid chemical compound and inhibition hepatitis B virus purposes thereof " (Lee school Kun, Hu Lihong, Wu Xiumei, Zhao Yu etc.; CN101293836); Inhibition HBsAg/HBeAg that the inventor has delivered and HBV DNA isoreactivity article are referring to " In Vitro Antiviral Activity of Three EnantiomericSesquiterpene Lactones from Senecio Species Against Hepatitis B Virus ", Haibo Li (Li Haibo), Changxin Zhou (girth is new), Xiumei Wu (Wu Xiumei), Yu Zhao* (Zhao Yu) etc., Antiviral Chemistry; Chemotherapy, 2005,16,277-282; " Evaluation of Antiviral Activity of Compounds Isolated fromRanunculus sieboldii Miq.and Ranunculus sceleratus L ", Haibo Li (Li Haibo), Changxin Zhou (girth is new), Xiumei Wu (Wu Xiumei), Yu Zhao* (Zhao Yu) etc., Planta Medica, 2005,71 (12), 1128-1133; " Application ofhigh-speed counter-current chromatography for the isolation of antiviraleremophilenolides from Ligularia atroviolacea ", Shi, Shu-Yun (Shi Shuyun), Hai-Bo (Li Haibo), Zhao, Yu (Zhao Yu) etc., Biomedical Chromatography, 2008,22 (9), 985-991; " Purification and identification of antiviralcomponents from Laggera pterodonta by high-speed counter-currentchromatography ", Shuyun Shi (Shi Shuyun), Yu Zhao (Zhao Yu) etc., Journalof Chromatography B, 2007,859,119-124].Undoubtedly, continuing to seek the lead compound that can suppress the HBV dna replication dna from natural product and structure of modification derivant thereof is that very therefore being necessary property is also classified as one of great special project of new drug development by the Ministry of Science and Technology with urgent.
In above-mentioned treatment CHB medicine, also having a class is to protect the liver the class medicine, its clinical a large amount of uses be typically silymarin in the seed that is present in the feverfew Herba Silybi mariani.Herba Silybi mariani is extensive use clinically, its commodity Legalon by name on market
TMGrand or the Flavobion of sharp liver
TM, its representative compounds surely belongs to flavanolignan's silibinin.Flavone lignin compound belongs to weedtree quality class, is a class natural product of and a part flavone be combined into plain by a part phenylpropyl alcohol.Silibinin content is maximum in the Herba Silybi mariani, and activity is also the highest.This medicine effect mainly contain following some: (one) free radical resisting activity: silymarin has protective effect for the hepatic injury that is caused by carbon tetrachloride, galactosamine, alcohols and other hepatotoxin.People such as nineteen ninety Lotteron have reported in the Mouse Liver microsome, silymarin can reduce external lipid peroxidation that is caused by the carbon tetrachloride metabolism and the peroxidation that is caused separately by reduced coenzyme, and these show that all silymarin is the chain interruption antioxidant or is free radical scavenger.(2) protection liver plasma membrane: keep flowability of cell membranes by the anti peroxidation of lipid reaction, the protection liver plasma membrane.Can also block combining of special receptor on mycotoxin phalloidine and α-amanitin etc. and the hepatocyte, suppress it, interrupt its liver sausage circulation, thereby the enhance hepatocyte film be for the resistance of multiple damage factor hepatocellular attack and transmembrane transport.(3) promote hepatocellular reparation and regeneration: silibinin can combine with estradiol receptor after entering cell, and make it to activate, activated receptors can enhance hepatocyte nuclear RNA polymerase 1 activity, rna transcription is strengthened, promote enzyme and proteinic synthetic, and promote the synthetic of DNA indirectly, help hepatocellular reparation and regeneration.(4) antitumor action: various active oxygens can form 8-hydroxyl guanine by the oxidation guanine, cause DNA damage, and then cause tumor, and silibinin has also shown the effect of prevention and treatment tumor as an effective free radical resisting material.This medical instrument of the clinical trial certificate of three more than ten years has definite curative effect and hypotoxicity (to consult Flora, K. etc., Am.J.Gastroenterol, 1998,93,139-143; Saller, R. etc., Drugs, 2001,61 (14), 2035-2063;
R. etc., Curr.Med.Chem.2007,14,315-338;
Z. etc., Phytother.Res.2003,17,524-530;
R. etc., Bioorg.Med.Chem.2004,12,5677-5687; Varga, Z. etc.; Phytothe.Res.2001,15,608-612.Singh, R.P. etc., Curr.Cancer Drug Tar.2004,4,1-11).Therefore, the flavone lignin chemical compound that with the silibinin is representative has caused increasing concern, in the period of 2006-2009, prepare and a plurality of serial silibinin analog derivative reported also has obvious antioxidation activity (Yang Leixiang, Zhao Yu etc. as the inventor, " Design; synthesis andexamination of neuron protective properties of alkenylated and amidateddehydro-silybin derivatives ", Journal of Medicinal Chemistry, 2009,52 (23), 7732-7752; Wang Feng, Zhao Yu etc., " Preparation of C-23 esterified silybinderivatives and evaluation of their lipid peroxidation inhibitory and DNAprotective properties ", Bioorganic ﹠amp; Medicinal Chemistry, 2009,17 (17), 6380-6389; Yang Leixiang, Zhao Yu etc., " Synthesis and Antioxidant PropertiesEvaluation of Novel Silybin Analogues ", Journal of Enzyme Inhibitionand Medicinal Chemistry, 2006,21 (4), 399-404; Or the like).In the above-mentioned article of inventor report, flavanolignan's compounds of A ring, B ring, E ring and 23 replacements of a plurality of series that design and synthesize out through the inventor all demonstrates activity, antioxidant activity and the protection PC12 cell activity of the potent DPPH of catching free radical and ultra-oxygen anion free radical.But apparent: above-mentioned research only concentrates on antioxidation and the cytoprotection of studying the silibinin flavonolignan.
(2006) in the recent period, Xie Jun has reported that use in conjunction has the interferon of antiviral and immunomodulating dual function and the silibinin phosphatide complexes of hepatoprotective is treated the result of chronic viral hepatitis B, it is more obvious than using the interferon effect separately to find that drug combination reduces ALT, AST value in patient's body, illustrates that the silibinin phosphatide complexes can strengthen the effect of the treatment chronic viral hepatitis B of interferon.But similarly therapeutic scheme is still flavanolignans such as silibinin as adjuvant drug, and mainly is to be used to protect liver plasma membrane but not directly to implement antivirus action with it.
(left side is state-run for the state-run grade in a left side, Liu Shuling, Xu Guili, world Chinese digests magazine, 2006 14 13 phases of volume, the 1241-1246 page or leaf) summarized the active progress of the external anti-HBV of medicinal plants composition over nearly 20 years, the multiple natural product of touching upon in the literary composition, do not report that wherein any flavanolignan compounds has the active relative recording of anti-HBV, especially silibinin class natural product and derivant thereof, domestic almost nobody relates to, and only by inventor team it has been carried out structure of modification and the antioxidant activity research that forefathers do not add attention.
Though be the antioxidation curative effect that flavanolignan's chemical compound of representative has the above with the silibinin, yet it appears in the newspapers less relatively in the document of antiviral therapy aspect, the compounds for treating DNA of flavanolignan viroid infection especially its new purposes that is used for anti-hepatitis virus aspect (especially suppressing the HBV dna replication dna) is effectively developed as yet, so seek the reactive compound in anti-hepatitis virus field from flavanolignan, also being about to flavanolignan's structure of modification, to make it have anti-DNA viroid activity be a brand-new field.From wherein finding to suppress the lead compound challenge that forefathers did not attempt especially of HBV dna replication dna.In order to explore this field; our design has also prepared and silibinin structure a kind of new flavanolignan's derivant of difference to some extent; also i.e. benzoyl in 23 connections of former silibinin; whole molecular conjugation scope and conjugation intensity have been prolonged; so design can generate the plain flavanone alcohol compound (also being the novel flavanolignan of class chemical compound) of a class novel wooden that the new spatial structure is different from silibinin; in the hope of finding; flavanolignan's lead compound that can suppress the HBV dna replication dna; have the novelty medicine that can suppress HBV dna replication dna treatment CHB thereby it is developed further into, finish the present invention in view of the above.
Summary of the invention
The purpose of this invention is to provide the purposes that silybin ester that the benzoyl of structure shown in the formula (1) replaces or its officinal salt are used to prepare the medicine for the treatment of hepatitis B;
The name of formula (1) chemical compound is called: (±)-benzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester.
The present invention also provides the method for the flavanolignan's compounds shown in a kind of preparation formula (1), it is characterized in that:, carry out condensation reaction and get in the presence of triphenyl phosphorus and diethyl azodiformate with commercially available or homemade silibinin and benzoic acid.
Another object of the present invention provides a kind of pharmaceutical composition that is used to suppress HBV dna replication dna, treatment hepatitis B, it is characterized by to contain (1) chemical compound of the formula as active component of treat effective dose or the mixture of its officinal salt and pharmaceutically acceptable auxiliaries composition.Its pharmaceutical dosage form can be that tablet, capsule, injection, aerosol, suppository, membrane, drop pill, paster agent, subcutaneous planting bury agent, externally-applied liniment, oral liquid or ointment, can also adopt the known controlled release of modern pharmaceutical circle or slow release formulation or nanometer formulation.
The silybin ester type flavanolignan (1) that benzoyl of the present invention replaces compares with natural flavone Lignanoids compounds silibinin, feature with differentiation on many structures and the physico-chemical property comprises that speciality such as its hydrophobicity, armaticity, Gibbs free energy, hydrogen bond receptor, electrical, intermolecular Van der Waals force and 3D conformation, direction of extension, molecule center of gravity, electrical distribution center all have obviously different with silibinin; And chemical compound (1) molecular weight ratio silibinin has increased 168 mass units.The ligand-receptor that above-mentioned feature has all determined the three-dimensional conformation of chemical compound shown in the formula (1) to combine with the 3d space structure of HBV DNA all may produce bigger difference in conjunction with complex form and combination, its binding site and binding pattern, it all can produce bigger change in conjunction with free energy etc., thereby may suppress aspect the HBV dna replication dna beyond thought effect arranged.
We have tested the growth inhibited effect of this chemical compound to the HepG2.2.15 cell, have tested its inhibition activity to the hepatitis B HBV dna replication dna of HepG2.2.15 emiocytosis simultaneously.Result of the test is found: the flavanolignan's chemical compound shown in the formula (1) has more intense inhibitory action to duplicating of hepatitis B virus DNA (deoxyribonucleic acid) (HBV DNA), it suppresses the inhibition activity of specific activity alpha-interferon when the maximum concentration 10000 units per ml concentration to duplicating of hepatitis B viruses (HBV) DNA and exceeds twice nearly when high dose (100 mcg/ml), belong to potent non-nucleoside and suppress the hepatitis B virus natural product.Above formula (1) chemical compound has beyond thought anti-HBV effect, thereby can expect that it can be used as the active lead compound that suppresses HBV dna replication dna, treatment hepatitis B and continues exploitation.
Through the detailed Literature Consult of the inventor, up to the present, still there is not report about this compounds for treating hepatitis B virus infection disease and preparation anti-hepatic-B virus medicine.Potent inhibition all belongs to beyond thought discovery to flavanolignan's formula (1) chemical compound for HBV DNA, and definite originality is arranged.In sum, uniqueness on this flavanolignan's existing structure that we prepare, the novelty that the research of antivirus action aspect is arranged again, and in anti-hepatitis B activity test, found the activity of uncommon inhibition HBV DNA to be expected to become the lead compound that suppresses HBV dna replication dna and treatment CHB.
Usefulness of the present invention is: the silybin ester type flavanolignan of the replacement of the benzoyl shown in the discoverable type (1) first has inhibition HBV dna replication dna effect and the patent medicine potentiality aspect the control hepatitis B virus, and the original new drug that becomes treatment hepatitis B virus original new drug, exploitation inhibition HBV dna replication dna for exploitation provides new material base.Have potential huge social benefit and economic benefit.The present invention's characteristics again is: the present invention's synthetic starting material convenient sources, and synthetic convenient.Its preparation method is simple, and raw material sources conveniently are easy to get, and cost is low, pollutes for a short time, is beneficial to the large-scale production under the energy-saving and emission-reduction condition.Industrialization prospect is very clear and definite.
The specific embodiment
The inventor is simply synthetic by multistep, and obtains this by the chromatography means and can effectively suppress the active flavanolignan's class reactive compound of HBV dna replication dna, derives its chemical constitution through integration analysis such as mass spectrum and NMR (Nuclear Magnetic Resonance) spectrum again.The inventor finds, formula (1) chemical compound does not have obvious inhibitory action to the growth of HepG2.2.15 cell, and the duplicating of HBV DNA of HepG2.2.15 emiocytosis had significant inhibitory effect, point out this chemical compound to have drug safety, suppress HBV dna replication dna characteristics of high efficiency.Therefore; according to the inventor's research, the silibinin flavonolignan chemical compound that 23 shown in the designed and synthetic formula of inventor (1) go up the benzoyl replacement can be used to prepare the medicine of treatment hepatitis B virus infection disease and be used for the treatment of the hepatitis B virus infection disease.
In order to understand essence of the present invention better, use the preparation of formula (1) chemical compound below respectively and, its new purposes in pharmaceutical field is described to the HepG2.2.15 cell growth inhibition and to the result of the inhibitory action test of the HBV dna replication dna of HepG2.2.15 emiocytosis.Embodiment has provided partial synthesis, the structure of formula (1) chemical compound and has identified and activity data.Mandatory declaration, embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention, and essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 1:The preparation of formula (1) chemical compound
1.1 instrument and reagent:
Ultraviolet spectra is measured with Shimadzu UV-240 ultraviolet spectrophotometer; Proton nmr spectra
1H-NMR measures (tetramethylsilane ether TMS is interior mark) by INOVA type NMR spectrometer with superconducting magnet (VARIAN INOVA-400MHz); Electrospray Mass Spectrometry ESI-MS is measured by BrukerEsquire 3000+ mass spectrograph, and column chromatography is produced by Haiyang Chemical Plant, Qingdao with silica GF254 (10-40 order) with silica gel (100-200,200-300 and 300-400 order) and thin layer chromatography; Agents useful for same is analytical pure, and wherein the petroleum ether boiling range is 60-90 ℃; Thin layer preparative chromatography (PTLC) the aluminium foil silica gel plate of Merck company; Column chromatography adopts Sweden Amersham Pharmacia Biotech AB company product with polydextran gel Sephadex LH-20; Reverse phase silica gel RP-18 adopts the Chromatorex product of Japanese Fuji Silysia Chemical company; MCI is a Mitsubishi chemical company product, and thin plate (TLC) detects the uviol lamp with 254nm and 365nm; Developer iodine vapor, 10% sulphuric acid-ethanol and phosphorus molybdenum acid solution.
1.2 the preparation of chemical compound (1):
Add 1 gram silibinin in exsiccant reaction bulb, 0.49 gram benzoic acid and 1.6 gram triphenyl phosphorus are with 20 milliliters of anhydrous tetrahydro furan dissolvings, add 1 gram diethyl azodiformate, add the back in stirring at room 10 hours, distilling under reduced pressure removes and desolvates, and adds 5 milliliters of chloroforms, remove by filter white solid, mother solution is through 200-300 order silica gel column chromatography repeatedly, and 10: 1 eluting of chloroform/methanol are through the gel filtration chromatography purification, final that buff powder 0.76 restrains yield: 54%.
(±)-benzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester: R
f(chloroform: ethyl acetate: formic acid=50: 1: 0.25): 0.20; Proton nmr spectra
1H NMR (400MHz, deuterochloroform, δ ppm): 3.85 (s, 3H, OCH
3), 4.21 (m, 1H, H-23a), 4.28 (m, 1H, H-10), 4.52 (m, 2H, H-3,23b), 4.96 (d, J=12.0Hz, 1H, H-2), 5.02 (d, J=8.0Hz, 1H, H-11), 6.02 (s, 1H, H-6), 6.06 (s, 1H, H-8), 6.92-8.06 (m, 11H, Ar-H), 11.20 (s, 1H, 5-OH); Electrospray Mass Spectrometry ESI-MS:m/z 585[M-H]
+
Embodiment 2:The inhibitory action that formula (1) chemical compound duplicates the hepatitis B virus DNA (deoxyribonucleic acid) (HBV DNA) of HepG2.2.15 emiocytosis
2.1 cell culture:
In containing 10% inactivated fetal bovine serum, 100U/ ml penicillin and 100U/ milliliter streptomycin in the DMEM culture medium of 100 mcg/ml G418, are put 37 ℃, 5%CO with the HepG2.2.15 cell culture
2, cultivate in the incubator of 100% relative humidity.
Measure the inhibitory action of formula (1) chemical compound 2.2 adopt mtt assay to the growth of HepG2.2.15 cell:
The take the logarithm HepG2.2.15 cell of trophophase becomes 1 * 10 with culture medium with cell dilution
5Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO
2, cultivate the chemical compound (1) that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 1000 mcg/ml, 200 mcg/ml, 40 mcg/ml and 8 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, to cultivate after 72 hours, every hole adds MTT reagent 10 microlitres, continues to cultivate 4 hours, discards culture medium, and every hole adds the DMSO200 microlitre, with agitator vibration 20 minutes, measures the OD value with microplate reader under the 570nm wavelength.With the culture hole that only adds culture medium is control wells.Suppression ratio (%)=(control wells OD value-experimental group OD value)/control wells OD value * 100%.The experiment triplicate.
2.3 the inhibitory action that the flavanolignan's chemical compound shown in the mensuration formula (1) duplicates hepatitis B virus DNA (deoxyribonucleic acid) (HBV DNA): the HepG2.2.15 cell of the trophophase of taking the logarithm becomes 1 * 10 with culture medium with cell dilution
5Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO
2, cultivate the flavanolignan's chemical compound that adds after 24 hours with shown in the formula (1) of culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 100 mcg/ml, 20 mcg/ml and 4 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, place 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.Measure HBV DNA concentration in the culture medium with HBV DNA quantitative PCR kit in the time of the 8th day.(annotate: the lamivudine test concentrations is 100 mcg/ml with the positive contrast 1 of lamivudine (3-TC), 20 mcg/ml and 4 mcg/ml), with the positive contrast 2 of alpha-interferon (annotate: the alpha-interferon test concentrations is 10000 units per ml, 5000 units per ml and 1000 units per ml).
2.4 experimental result:
Experimental result illustrates as shown in table 1, and flavanolignan's formula (1) chemical compound of preparing has the effect of potent inhibition hepatitis B virus DNA replication.
Table 1 sample in the time of the 8th day to the suppression ratio of the HBV dna replication dna of HepG2.2.15 cell
This embodiment presentation of results: the flavanolignan's chemical compound shown in the formula (1) has more intense inhibitory action to duplicating of hepatitis B virus DNA (deoxyribonucleic acid) (HBV DNA), it suppresses the inhibition activity of specific activity alpha-interferon when the maximum concentration 10000 units per ml concentration to duplicating of HBV DNA and exceeds twice nearly when high dose (100 mcg/ml), the inhibition of duplicating to HBV DNA when median dose (20 mcg/ml) just surpasses 50%; Therefore this chemical compound belongs to effective non-nucleoside and suppresses the hepatitis B virus natural product, is worth further paying close attention to and further investigation, and can expects that further optimized development is for suppressing the medicine of HBV dna replication dna.
When above-mentioned description elaboration was of the present invention, the purpose that embodiment is provided simultaneously was to illustrate actual mechanical process of the present invention and meaning of the present invention.In the time of in entering claim of the present invention and its equivalent scope, practical application of the present invention comprises all general variations, cooperates, or improves.
Claims (2)
1. the silybin ester or its officinal salt that have the benzoyl replacement of structure shown in the formula (1) are used for the purposes that the hepatitis B medicine is treated in preparation;
The name of formula (1) chemical compound is called: (±)-benzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester.
2. the silybin ester or its officinal salt that have the benzoyl replacement of structure shown in the formula (1) are used for the purposes that preparation suppresses hepatitis B virus DNA (deoxyribonucleic acid) HBV dna replication dna medicine;
The name of formula (1) chemical compound is called: (±)-benzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010181906 CN101829105A (en) | 2010-05-25 | 2010-05-25 | Application of benzoly-substituted silybin in preparing drugs for treating virus hepatitis |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1418637A (en) * | 2002-12-19 | 2003-05-21 | 王登之 | Silicibinin-N-methylglucamine disperser for treating hepatitis, and its prepn. method |
| CN1732918A (en) * | 2005-08-25 | 2006-02-15 | 江苏大学 | Nanometer preparation of silybin and preparation method thereof |
| CN1990484A (en) * | 2005-12-26 | 2007-07-04 | 浙江海正天华新药研发有限公司 | Silybin esters derivatives and preparation and use thereof |
| CN1990485A (en) * | 2005-12-26 | 2007-07-04 | 浙江海正天华新药研发有限公司 | Silybin flavonolignan and their production method and use |
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- 2010-05-25 CN CN 201010181906 patent/CN101829105A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1418637A (en) * | 2002-12-19 | 2003-05-21 | 王登之 | Silicibinin-N-methylglucamine disperser for treating hepatitis, and its prepn. method |
| CN1732918A (en) * | 2005-08-25 | 2006-02-15 | 江苏大学 | Nanometer preparation of silybin and preparation method thereof |
| CN1990484A (en) * | 2005-12-26 | 2007-07-04 | 浙江海正天华新药研发有限公司 | Silybin esters derivatives and preparation and use thereof |
| CN1990485A (en) * | 2005-12-26 | 2007-07-04 | 浙江海正天华新药研发有限公司 | Silybin flavonolignan and their production method and use |
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