CN111789854A

CN111789854A - Medical application of 2-alkene-1-alcohol glycyrrhetinic acid methyl ester in preparation of antiviral hepatitis B

Info

Publication number: CN111789854A
Application number: CN202010728992.5A
Authority: CN
Inventors: 巫秀美; 何尹来; 陈德华; 刘进; 普翠芬; 张石云; 李珍华; 赵昱
Original assignee: Dali University
Current assignee: Dali University
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2020-10-20

Abstract

The invention relates to application of 2-alkene-1-alcohol glycyrrhetinic acid methyl ester in preparing a medicine for treating hepatitis B virus infection diseases, and particularly provides application of 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester in preparing a medicine for resisting hepatitis B virus infection diseases. The compound has obvious activity of inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells, and the strength of inhibiting the secretion of the HBsAg and the HBeAg is higher than that of positive medicaments of alpha-interferon and lamivudine; the inhibition of HBV-DNA replication was also higher than that of interferon-alpha at day 8 at a concentration of 100. mu.g/ml. The results show that the 2-alkene-1-alcohol glycyrrhetinic acid methyl ester can be expected to be used for preparing non-nucleoside medicaments for treating hepatitis B virus infection diseases, and particularly, the compound has the application of preparing HBV-DNA inhibitors, HBsAg inhibitors and HBeAg inhibitors; the preparation method has simple steps, low cost and wide raw material sources, and is easy to carry out industrialized production.

Description

Medical application of 2-alkene-1-alcohol glycyrrhetinic acid methyl ester in preparation of antiviral hepatitis B

Technical Field

The invention relates to the technical field of medicines, in particular to application of 2-alkene-1-alcohol glycyrrhetinic acid methyl ester in preparing a medicine for treating hepatitis B virus infection diseases. The compound has the structure of 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester, is pentacyclic triterpenic acid methyl ester, has the obvious activity of inhibiting HepG2.2.15 cells from secreting HBsAg and HBeAg, can obviously inhibit the replication of HBV-DNA in HepG2.2.15 cells, and can be expected to be used for preparing non-nucleoside-type medicaments for clearing the HBsAg and the HBeAg, inhibiting the replication of the HBV-DNA and treating hepatitis B virus infection diseases.

Background

Hepatitis b is an infectious disease caused by hepatitis b virus (HBV, hepatitis b virus), and is also called viral hepatitis b. HBV is a member of hepadnaviridae, a partial circular DNA virus, and is in the shape of a spherical particle with a diameter of 42 nm, widely present in tissues such as liver, pancreas, lymphocytes, etc., and is continuously replicated. HBV is a peculiar virus, less infectious in other animals and can replicate only in humans or primate chimpanzees. The virus is transmitted through blood, saliva, semen and vaginal secretion of hepatitis B virus carriers and patients, and has chronic carrying state. The disease is widely popularized in China, and is divided into various modes such as vertical transmission, horizontal transmission, in-home transmission, iatrogenic transmission and sexual transmission, so that the infection rate of people is high, and the infection rate in certain areas reaches more than 35%. According to the relevant data, the number of patients who have positive hepatitis detection reaches 1.89 hundred million, and the number of people who should see no treatment (carriers) is nearly 4 hundred million. Is one of the most serious infectious diseases endangering the health of people at present. Hepatitis B is clinically manifested in a variety of forms, and is likely to develop into chronic hepatitis and liver cirrhosis, and a few patients can turn into primary liver cancer. Hepatitis B virus in blood is easier to remove, but hepatitis B virus in tissue cells is difficult to remove.

Hepatitis B surface antigen (HBsAg) is the coat protein of hepatitis B virus, and HBsAg positive is the gold standard for determining HBV infection. HBsAg positive, but without hepatitis symptoms present, becomes HBV virus carrier. The greater the HBsAg titer, the greater the probability that it will bind the hepatitis B core antigen HBeAg, be HBV-DNA positive and have increased DNA polymerase activity, and thus be more infectious. Therefore, inhibiting the secretion and replication of HBsAg is an important target and detection target in the development of anti-hepatitis B virus drugs. Wushuyun, Beijing Di Tan Hospital: there is some correlation between HBsAg clearance and hepatitis b closed-loop covalent dna (cccDNA), clearance of HBsAg is a sign of significant reduction in cccDNA levels. In 2002, the research results published in the journal of new england medicine suggest that: for Chronic Hepatitis B (CHB) patients, if HBsAg is effectively eliminated before cirrhosis, the incidence of cirrhosis and hepatocellular carcinoma will be reduced by 60 times. HBsAg serum clearance was used as one of the therapeutic endpoint criteria in the hepatitis B treatment guidelines of the American Association for liver disease research (AASLD), the Asia-pacific liver research Association (APASL), and the European Association for liver research (EASL). The annual meeting of the european society for liver research in 2008 reports: the PEG-IFN-alpha-2 a treatment of CHB patients is stopped after 48 weeks, and the HBsAg clearance rates of 1, 2,3 and 4 years are respectively 3%, 6%, 8% and 11%, while the clearance rates of the lamivudine alone on the HBsAg patients are only 0%, 0% and 3% in 1, 2,3 and 4 years after the medicine is stopped. At present, although new HBsAg medicines enter clinical tests at home and abroad, specific medicines for removing the HBsAg at a target point are not found in first-line medicines for treating acute and chronic hepatitis B.

The hepatitis B e antigen HBeAg is a structural protein of the core of hepatitis B virus HBV and is produced in large quantity during the propagation of hepatitis B virus. Hepatitis b virus HBV has the smallest genome of all known DNA viruses (only 3.2kb), and its genes mainly encode five proteins (S, C, E, P, X). Protein C is the viral core protein, while protein E is part of protein C, and becomes hepatitis b E antigen (HBeAg), a protein that is already encoded but not assembled into viral particles, and is secreted into the patient's blood when the virus replicates. Clinically, serum HBeAg is often used as an important marker for HBV replication, infectivity, severity of the disease and for the evaluation of its therapeutic response. The antigen is closely related to HBV-DNA and is a very practical serum marker for clinically expressing virus replication. The serum HBeAg positive patient shows that HBV replication exists in the body of the patient, so the patient has higher infectivity; higher expression of HBeAg in a patient indicates that the patient is more contagious. Similarly, the inhibition of the secretion and replication of HBeAg is an important target and detection target in the research and development of anti-hepatitis B virus drugs. HBeAg clearance indicates that the body has continuous HBV inhibition, ALT normal, tissue inflammation and necrosis reduction and the incidence of liver cirrhosis is reduced. Serum HBeAg is therefore thought to reflect a more stable therapeutic effect, and serum clearance of HBeAg marks the onset of action of the patient's immune system. In 2002, the research results published in the journal of new england medicine suggest that: for CHB patients, if HBeAg clearance is achieved before cirrhosis, the incidence of cirrhosis and hepatocellular carcinoma will be reduced by 10-fold. Serum clearance of HBeAg was used as one of the therapeutic endpoint criteria in the guidelines of the american association for liver disease research AASLD, the asia-pacific liver research association APASL and the european association for liver research EASL. Therefore, the medicine capable of inhibiting and reducing the expression or activity of HBeAg belongs to the medicine capable of effectively treating the hepatitis B virus infection diseases.

In recent years, as liver diseases are studied, standardized HBV-DNA analysis has been developed, and the understanding of the conditions of hepatitis B patients has been greatly advanced. Quantitative analysis of HBV-DNA can predict the severity and prognosis of hepatitis B, since persistent positive HBV-DNA (i.e., persistent viremia) is likely to progress and aggravate hepatitis B; high hepatitis B virus (HBV-DNA) content is easy to promote the formation of cirrhosis; the persistence of HBV-DNA is a high risk factor for the development of hepatocellular carcinoma (HCC). In particular, patients with high virus content, long course of disease, aging or other liver diseases, and the high concentration of HBV-DNA in vivo can cause the significant increase of the mortality rate of the compensatory liver cirrhosis and the primary severe liver disease. It must also be recognized that HBV-DNA levels are extremely closely related to liver histology: the literature reports that the improvement and elimination of liver fibrosis are obvious after antiviral treatment; recent international conference on liver disease reports that potent and low-drug resistant antiviral treatments, with the decrease and negative reversal of HBV-DNA, can be observed with varying degrees of reversal of cirrhosis. Thus, it is now claimed that cirrhosis should also be treated with antiviral therapy.

Therefore, the use of HBV-DNA markers in antiviral therapy also plays a significant role: the level of HBV-DNA is an important index for determining whether chronic hepatitis B needs antiviral treatment; in antiviral treatment, whether virology early response exists or not is judged according to the treatment response of HBV-DNA, and then a long-term medication strategy is determined to obtain continuous virology response so as to achieve the aim of continuous virus inhibition; striving for virus continuous negative according to HBV-DNA continuous inhibition condition to achieve antiviral final treatment target; different degrees of improvement and disappearance of cccDNA were also shown according to the continuous complete suppression of HBV-DNA; in antiviral treatment, the change of HBV-DNA is used for evaluating and preventing virus variation caused by antiviral drugs and the risk of drug resistance; once viral variation or resistance occurs, HBV-DNA changes are the only first sign and diagnostic basis and are the guide and basis for therapeutic resistance and changing therapeutic strategies.

Therefore, the inhibition degree of HBV-DNA has a new significance in further diagnosis and treatment of hepatitis B, and has a great guiding effect on observation of curative effect, prognosis of hepatitis B and drug resistance risk assessment. Therefore, HBV-DNA was not detected as one of the therapeutic endpoints of hepatitis B virus patients by both the Asia-Pacific liver institute and the European liver institute. The inhibitory strength of the tested compound on HBV-DNA is also regarded as an important index for evaluating the drug effect of the hepatitis B treatment drug in the new drug development guide of China.

At present, the drugs for patients with hepatitis B are mainly classified into a plurality of categories including liver protection, enzyme reduction, virus resistance, hepatic fibrosis resistance, immunity regulation and the like. The antivirus is the fundamental method, while the liver protection and enzyme reduction are only the adjuvant therapy, and the treatment is mainly temporary and permanent. Although there have been some advances in recent years in the treatment of hepatitis B with antiviral drugs; however, the current clinical treatment scheme for viral hepatitis B can only achieve the aim of inhibiting HBV replication and secondary infection in serum, and the most main drugs are nucleoside drugs such as lamivudine (3-TC), entecavir, Adefovir (ADV), telbivudine and the like, and emtricitabine, tenofovir, clevudine and the like in clinical trials. The nucleoside drugs have the advantages of: has high bioavailability and is safe to take orally. However, they can temporarily control the disease condition, but once they are expensive to sell; drug resistance can be caused after long-term use, and indexes such as HBV-DNA, ALT, liver histology and the like rebound to different degrees after drug withdrawal; thirdly, the obvious well-known adverse effects of long-term use of nucleoside drugs, such as kidney damage, infant teratogenesis and the like. The headaches are: the occurrence of virus resistance greatly reduces the cure rate, and because the nucleoside drugs are reversible to virus replication, the treatment course is more than one year for most patients to achieve the maximum curative effect, so the occurrence of the virus resistance can not achieve the expected effect. And nucleoside drugs also have the defects of difficulty in clearing cccDNA, difficulty in negative conversion of HBsAg after one year treatment and the like.

Biological engineering antiviral drugs derived from human leucocytes, such as interferon (alpha and beta), recombinant interferon and the like, recently become hot drugs for researching and treating CHB, and have double effects of resisting virus and regulating immunity. It can inhibit virus replication through antivirus function, thereby relieving liver cell inflammatory reaction, reducing liver cell damage, delaying disease development, and improving clinical symptoms and liver physiological function of patients; but also can enhance the immunity, and especially can promote the killing of the T cell to kill the cell infected by the virus by enhancing the action of the natural killer cell and the helper T cell in vivo, thereby indirectly playing the role of antivirus. Therefore, the interferon is gradually the first choice medicine for clinically treating the chronic hepatitis B virus every day, but the side effects and adverse reactions of the interferon are more reported; as long as hepatitis B virus deoxyribonucleic acid (HBV-DNA) is positive, the hepatitis B virus in the body of the patient is likely to have variation, the virus is actively replicated and is infectious, the varied virus is not sensitive to antiviral drugs, and the recurrence rate is high, so that the interferon treatment on the hepatitis B is always low in efficiency, expensive in price and high in economic burden of patients, and the clinical application is difficult. And is not suitable for patients with decompensated liver cirrhosis. In order to overcome the defects that the side effect, the adverse reaction and the like of the alpha-interferon restrict the clinical application of the alpha-interferon, the invention also uses the alpha-interferon as a positive control medicament to carry out a control test.

It must be noted that: the antiviral drugs currently used are only inhibitors of viral replication and cannot directly kill viruses and destroy virosomes, otherwise host cells are damaged. These antiviral drugs (mostly nucleoside drugs) also have the disadvantages of great toxic and side effects, easy viral gene mutation, easy rebound after drug withdrawal, and the like, so the development of novel antiviral drugs is a urgent task in the field of current drug development. It has extremely important social and economic significance for treating a large number of hepatitis B patients and virus carriers in China, controlling infection sources and the like. Therefore, the discovery of new non-nucleoside hepatitis B virus inhibitors and lead compounds capable of inhibiting HBV-DNA replication from natural medicines used by ethnic nationality for a long time has great instructive significance and has wide development prospect.

Based on the purpose, the inventor has previously completed the technology and product research of a plurality of anti-hepatitis B virus natural products and the structure improvement derivatives thereof, and discovers a plurality of compounds for eliminating HBsAg or HBeAg and inhibiting HBV-DNA replication, thereby indicating that the screening of innovative medicaments capable of preventing and treating hepatitis B virus infection from the natural products and the synthetic derivatives thereof is feasible. [ see: "medical use of enantiomorphous eudesmol sesquiterpenes for inhibiting hepatitis B virus" (Zhao Yi, Liuguang, Yurongmi, Lihaibo, etc.; ZL 200610053827.4); "medicinal use of 2 β -hydroxyilicic acid for inhibiting hepatitis B virus" (Li school \22531, Zhao Yi, Huangkexin, Li Hai Bo, etc.; ZL 200610053749.8); "medicinal use of 2 α,3 β -dihydroxy-5, 11(13) -dieneudesman-12-oic acid for inhibiting hepatitis B virus" (Zhao Yi, Zhang He, Sun Han Dong, Li Hao, etc.; ZL 200610053601.4); the use of eremophilane lactone for inhibiting hepatitis B virus and its pharmaceutical composition (Zhao Yi, Li Hai Bo, Yangrel, Zhongchang, etc.; ZL 03153691.3); "an eremophilane lactone acid natural product and its application" (Zhao Yi, Zhongchang Xin, Shizuyun, Wang Xiaoyu, etc.; ZL 200610053575.5); "A eudesmane type sesquiterpene acid and its uses" (Zhao Yi, Liu Guang Ming, Li Hai Bo, Wuxiu Mei, etc.; ZL 200610053579.3); the application of six-edge chrysanthemum plant extract in preparing medicine composition for inhibiting herpes simplex virus and hepatitis B virus (Zhao Yi, Zhongchangxi, Yirong Ming, white Ye; ZL 200510132508.8); "medical use of 1 β -oxo-5, 11(13) -diene eudesmane-12-oic acid for inhibiting hepatitis B virus" (Zhao Yi, Li school 22531, Huangkexin, Li Hai Bo, etc.; ZL 200610053610.3); "medical use of 1 β -hydroxyilicic acid for inhibiting hepatitis B virus" (Zhao Yi, Li school \22531, Huangkexin, sago Xiumei, etc.; ZL 200610053625. X); 1-O-substituted benzoyl quinic acid compounds and their use for inhibiting hepatitis B virus (Li school 22531, Huli hong, Wu Xiumei, Zhao Yi, etc.; ZL 200810062451.2); recently, the group of the present inventors invented new anti-HBV active compounds and their use in the preparation of anti-HBV drugs from derivatives synthesized from natural products as starting templates: contains bromine dihydroflavonol lignan (ZL 201010181451.1), ring A coupling flavonolignan (ZL 201010181892.1), benzyloxy flavonolignan (ZL 201010181644.7), B/E bis-methoxy silybin (ZL 201010181499.2), quercetin dimer flavone (ZL 201010181869.2), a benzo phenylpropanoin (ZL 201010181533.6), B-ring ethoxy dihydroflavonol (ZL 201010181512.4), substituted isosilybin (ZL 201010181679.0), ring A substituted silybin ester (ZL 201010181721.9), ring E bromine substituted silybin (ZL 201010181632.4), ring E demethoxy silybin (ZL 201010181731.2), acetamide dehydrosilybin (ZL 201010181523.2), an angle type flavonolignan (ZL 201010181503.5), diallyl flavonolignan (201010181908.9), bis-methyl dehydrosilybin (ZL 201010181775.5), diamine formyl dehydrosilybin (ZL 010181504.X), flavonolignan (+/-) Scutella prostin A (ZL 201010181362.7), aryl carbamoyl dehydrogenated silybin (ZL201010181414.0), E-ring iodine substituted silybin (ZL 201010181661.0), B-ring ethoxy silybin (ZL201010181500.1), A-ring dioxane flavonolignan (ZL 201010181411.7), dehydrogenated silybin diether (ZL201010117317.5), dehydrogenated silybin trialkyl ether (ZL 200910099405.4), isopentenyloxy substituted dehydrogenated silybin ether (ZL 200910099404.X), 7-and 20-position dehydrogenated silybin dialkyl ether (ZL200910099403.5), A-ring substituted silybin ether (200910099042.4) and diallyl propyl substituted silybin ether (ZL 200910099041. X). Needless to say, it is necessary and urgent to continuously search for lead compounds capable of effectively preventing and treating HBV from natural products and structurally modified derivatives thereof, and the lead compounds are listed as one of the major items for new drug development by the national ministry of science and technology.

The traditional Chinese medicine liquorice is a plant belonging to genus glycyrrhiza of leguminosae, and the herbal medicine is listed as the superior product in Shen nong Ben Cao Jing. Tang Dynasty, the book of materia Medica of drug Property: in the medicines, the liquorice is called as the Chinese old because the liquorice is the monarch medicine and is used for treating seventy-two kinds of mammary stone toxin and relieving one thousand two hundred common herb toxin and harmonizing the active ingredients of the medicines. The main effects of apricot forest are clearing away heat and toxic material, harmonizing drug property, etc., so far there is a theory that no herb is in the way. In the prescription database recorded in the TCM120, the usage frequency of licorice in the prescription is listed as the first. The root tuber and rhizome of licorice contains a large amount of glycyrrhizic acid, also known as glycyrrhizin. In 2003, scientists at the university of Frankfurt, Germany, found that glycyrrhizic acid can inhibit the replication of SARS-associated virus in Vero cells, namely SARS virus clinical isolates FFM-1 and FFM-2 (Jindrich Cinatl Jret al, Lancet,2003,361: 2045-. Glycyrrhizic acid can inhibit virus replication and also inhibit virus adsorption and penetration. The glycyrrhizic acid is added in the virus adsorption period and after the adsorption period, so that the effect is more obvious. Therefore, glycyrrhizic acid is a potential effective lead compound against high-risk viruses. Based on this, scientists continuously research the derivatives to screen more efficient antiviral preparations, and find that the sulfate has the anti-HIV effect 4 times of that of glycyrrhizic acid, so that the development of efficient antiviral preparations or immunopotentiators is very hopeful.

Chinese scientists find that: glycyrrhizic acid can also protect the liver and delay and reduce the rise of serum transaminase [ tianqinglai et al, research and development of pharmacological action of effective components of licorice, research and development of natural products, 2006 (18): 343-; clinical tests of 136 Chronic Hepatitis B (CHB) patients also find that the magnesium isoglycyrrhizinate injection can significantly improve clinical symptoms, physical signs and biochemical indicators of liver function of patients [ xu qingjie and the like, clinical research on treatment of chronic hepatitis b by magnesium isoglycyrrhizinate, clinical medicine 2011,31 (7): 74-75 ]; therefore, in recent years, glycyrrhizic acid preparations in China have been used for adjuvant therapy of CHB, and although viral hepatitis B cannot be cured, certain effects on improvement of CHB symptoms and the like are achieved.

Glycyrrhizic acid is diglucuronide of glycyrrhetinic acid, and glycyrrhetinic acid is clinically used as adrenocortical hormone and adrenocorticotropic hormone medicine, and can replace deoxycorticosterone for treating Addison's disease. Although glycyrrhetinic acid has various physiological functions of resisting inflammation, enhancing nonspecific cellular immune function, scavenging oxygen free radicals and the like, glycyrrhetinic acid glycoside pentacyclic triterpene compounds represented by the glycyrrhetinic acid pentacyclic triterpene compounds have not been directly applied to the resistance of HBV infection. Therefore, we choose it as the starting material, reasonably modify its structure, and design a series of 18 β -glycyrrhetinic acid derivatives including the structure shown in formula (1) by means of computer aided design, and one of our purposes is: the lead compound of 18 beta-glycyrrhetinic acid derivatives is expected to be found to effectively inhibit secretion of HBsAg or HBeAg and inhibit replication of HBV-DNA, so that the lead compound is further developed into an innovative medicine for clearing HBsAg or HBeAg, inhibiting replication of HBV-DNA and treating CHB.

The 18 beta-glycyrrhetinic acid derivative has no effective development in treating DNA virus infection, and especially has no effective development in the aspect of resisting hepatitis B virus, so that the method for searching the active compound in the field of resisting hepatitis B virus from the glycyrrhetinic acid pentacyclic triterpene derivative, namely the method for modifying the structure to have the activity of resisting DNA virus is a brand-new field. It is a very desirable challenge to find lead compounds from them that effectively inhibit HBsAg or HBeAg secretion, HBV-DNA replication. To explore this area, we designed and prepared an enol functional structure with the A ring containing 2,3 en-1-ol to increase the SP of the A ring²Hybrid molecules and hybrid regions, and then 30-carboxyl of glycyrrhetinic acid is methylated into carboxymethyl ester, so that not only is the lipophilicity of the molecules changed, but also the space distance of the electronegative group of the A ring of the pentacyclic triterpenic acid is adjusted, and a lead compound with activities of eliminating HBsAg/HBeAg and inhibiting HBV-DNA replication is discovered.

Disclosure of Invention

In view of the above, the present invention provides the use of 1 α -hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester shown in formula (1) for preparing the drugs for preventing and treating hepatitis b virus infection diseases, wherein the compound shown in formula (1) can effectively resist hepatitis b virus HBV.

Wherein Me is methyl.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of the compound shown in the formula (1) in preparing a drug of an HBsAg inhibitor.

The invention provides application of the compound shown in the formula (1) in preparation of a medicament of an HBeAg inhibitor.

The invention provides application of the compound shown in the formula (1) in preparing a medicament of an HBV-DNA inhibitor.

The invention also provides a method for preparing the compound shown in the formula (1), which is characterized by comprising the following steps: firstly, forming methyl ester from 30-site carboxyl of commercially available glycyrrhetinic acid, then conducting mesylation on 3-site hydroxyl of glycyrrhetinic acid, further reacting to remove methanesulfonic acid, forming double bonds on A ring, then oxidizing allylic positions of the double bonds on the A ring into a 2-alkene-1-acetoxyl structure by selenium dioxide under the action of acetic acid, and finally removing acetyl under the action of potassium hydroxide to form 2-alkene-1-alcohol glycyrrhetinic acid methyl ester shown in the formula (1).

The compound of formula (1) with the effects of eliminating HBsAg and HBeAg and inhibiting HBV-DNA replication, or the pharmaceutically acceptable salt and the solvate thereof can be combined with pharmaceutic adjuvants or carriers to form a pharmaceutical composition for treating viral hepatitis B, and is characterized by comprising a mixture of the compound of formula (1) and a therapeutically effective amount of the compound serving as an active ingredient. The dosage form of the pharmaceutical composition can be tablets, capsules, injections, aerosols, suppositories, membranes, dropping pills, patches, subcutaneous implants, external liniments, oral liquids or ointments, and can also adopt controlled-release or sustained-release dosage forms or nano preparations known in the modern pharmaceutical industry.

Compared with natural glycyrrhetinic acid, the compound 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-methyl carboxylate designed by the inventor has the characteristics of various differences in structure and physicochemical properties, including hydrophobicity, aromaticity, Gibbs free energy, hydrogen bond receptors, electrical property, intermolecular van der Waals force, 3D conformation, extension direction, molecular gravity center, conjugation degree, electrical distribution center and the like, of the compound, namely the compound 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester, which is greatly different from the glycyrrhetinic acid; and the molecular weight of the compound of the formula (1) is increased by 12 mass units compared with glycyrrhetinic acid. The above characteristics all determine that the ligand-receptor binding complex forms and binding modes of the three-dimensional conformation of the compound shown in formula (1) and the 3D space structure of HBsAg, HBeAg and HBV-DNA can generate large differences, the binding sites and binding modes, the binding free energy and the like can generate large changes, and therefore, the compound has unexpected effects on inhibiting secretion of HBsAg or HBeAg and replication of HBV-DNA.

HepG2.2.15 cells are derived from the human hepatoma cell line HepG2 cells transfected with HBV genes, the cell line can stably replicate HBV genomes, and HBV-DNA can be detected from cell supernatants. We tested the effect of the compound of formula (1) on the secretion of HBsAg and HBeAg by HepG2.2.15 cells and its inhibitory activity on HBV-DNA replication in HepG2.2.15 cells in order to finally obtain a chemical entity capable of effectively eliminating HBsAg or HBeAg and inhibiting the autonomous intellectual property right of HBV-DNA replication. The test results show that: the pentacyclic triterpenic acid methyl ester has remarkable activity of inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells, and the strength of inhibiting HBsAg secretion of the compound at the concentration of 100 micrograms/ml is 1.39 times that of a positive control medicament 1(100 micrograms/ml lamivudine) and 2.94 times that of a positive control medicament 2(10000 units/ml alpha-interferon) respectively on the 8 th day of co-culture; the strength of inhibiting HBeAg secretion at the concentration of 100 micrograms/ml is 3.31 times that of a positive control medicament 1(100 micrograms/ml lamivudine) and 2.77 times that of a positive control medicament 2(10000 units/ml alpha-interferon) respectively; the inhibitory activity of the compound of formula (1) on HBV-DNA replication at high concentration (100. mu.g/ml) exceeds that of the positive control interferon-alpha, reaching 54.4% of that of the positive control lamivudine. The compounds of formula (1) have unexpected anti-HBV effects, and thus it is expected that they will continue to be developed as active lead compounds for eliminating HBsAg or HBeAg, inhibiting HBV-DNA replication, and treating viral hepatitis B. And can be expected to be further developed into innovative non-nucleoside innovative medicaments for eliminating hepatitis B HBsAg or HBeAg antigen and inhibiting HBV-DNA replication.

In conclusion, the pentacyclic triterpene methyl ester derived from glycyrrhetinic acid has structural uniqueness and novelty of antiviral action, and finds that the pentacyclic triterpene methyl ester has the unusual activity of inhibiting the secretion of HBsAg and HBeAg of hepatitis B surface antigens and inhibiting HBV-DNA replication in an anti-hepatitis B virus activity test, so that the compound is expected to be an active lead compound of non-nucleoside medicaments for treating CHB. Through the detailed reference of the inventor, no report about the compound for treating the hepatitis B virus infectious diseases and preparing anti-hepatitis B virus medicines exists so far. The pentacyclic triterpenic acid compound shown in the formula (1) is an unexpected discovery for strong inhibition of HBsAg, HBeAg and HBV-DNA, and has definite originality, thereby completing the invention.

The invention has the advantages that: the compound 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-methyl carboxylate shown in the formula (1) is found to have the functions of eliminating HBsAg or HBeAg, inhibiting HBV-DNA replication and preventing and treating hepatitis B virus, and provides a new material basis for developing non-nucleoside innovative drugs for resisting HBV and innovative drugs for treating viral hepatitis B. Has potential huge social benefit and economic benefit. The invention has the following further characteristics: the synthetic starting material glycyrrhetinic acid is convenient to obtain and synthesize. The preparation method of the compound shown in the formula (1) is simple and easy to implement, the raw materials are convenient and easy to obtain, the cost is low, the pollution is small, and the large-scale production under the conditions of energy conservation and emission reduction is facilitated. The industrialization prospect is very clear.

Detailed description of the preferred embodiments

The invention obtains the pentacyclic triterpenic acid compound shown in the formula (1) which is derived from glycyrrhetinic acid and can effectively inhibit the secretion of hepatitis B HBsAg and HBeAg and the replication activity of HBV-DNA through chemical synthesis and purification by various chromatographic means, and the chemical structure of the compound is deduced and verified through comprehensive analysis such as mass spectrum, nuclear magnetic resonance spectrum and the like. The inventor finds that the compound shown in the formula (1) has obvious inhibition effect on the secretion of hepatitis B HBsAg and HBeAg secreted by HepG2.2.15 cells and the replication of HBV-DNA, and prompts that the compound has the characteristics of safe administration, strong effect of eliminating the HBsAg or HBeAg and inhibition of the replication of the HBV-DNA. Therefore, according to the research of the inventor, the 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester shown in the formula (1) designed and synthesized by the inventor can be used for preparing non-nucleoside medicaments for treating hepatitis B virus infectious diseases.

In order to better understand the essence of the present invention, the following results of the preparation of the compound of formula (1) and the test of its inhibitory effect on the replication of HBsAg and HBeAg, HBV-DNA secreted from HepG2.2.15 cells, respectively, illustrate its novel use in the pharmaceutical field. The examples present the synthesis, structural identification and activity data for the compounds of formula (1). Unless otherwise specified, the percentages in the present invention refer to weight percentages. It must be noted that the examples of the present invention are for illustrating the present invention and not for limiting the present invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.

Example 1: preparation of compound 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester of formula (1)

1.1 instruments and reagents

Measuring the ultraviolet spectrum by using a Shimadzu UV-240 ultraviolet spectrophotometer; nuclear magnetic resonance spectroscopy was measured by an INOVA superconducting nuclear magnetic resonance spectrometer (VARIAN INOVA-400MHz) (tetramethylsilyl ether TMS as internal standard); electrospray mass spectrometry ESI-MS was determined by Bruker Esquire 3000+ mass spectrometer; silica gel (100-200 meshes, 200-300 meshes and 300-400 meshes) for column chromatography and silica gel GF254 (10-40 meshes) for thin layer chromatography are produced by Qingdao ocean factories; all the used reagents are analytically pure, wherein the boiling range of petroleum ether is 60-90 ℃; high Performance Liquid Chromatography (HPLC) using an agilent 1100 instrument; thin layer preparative chromatography (PTLC) was performed using aluminum foil silica gel plates from Merck; sephadex LH-20 for column chromatography is a product of company AB, Amersham Pharmacia Biotech, Sweden; thin plate (TLC) detection with UV lamps at 254nm and 365 nm; the developer is iodine vapor, 10% sulfuric acid-ethanol and phosphomolybdic acid solution.

Wherein, glycyrrhetic acid refers to glycyrrhetinic acid, CH₂N₂Is diazomethane, Et₂O is diethyl ether solution, MsCl is methanesulfonyl chloride, CH₂Cl₂Refers to methylene chloride, Li₂CO₃Lithium carbonate, DMF dimethylformamide, SeO₂Selenium dioxide, HOAc acetic acid, KOH potassium hydroxide and reflux reaction.

1.2 preparation of intermediate Compound methyl Glycyrrhetinic acid ester of formula (2)

In a dry reaction flask, 4.70 g of glycyrrhetinic acid (obtained from Seisan Chen Biotech Co., Ltd., purity 99% by HPLC) was dissolved in 40 ml of a mixed solution of diethyl ether and tetrahydrofuran (1: 1, V/V). In a fume hood, a newly prepared ethyl ether solution of diazomethane is added dropwise by a separating funnel in a 15 mmol manner under magnetic stirring to generate a large amount of white flocculent solid, and the reaction is continued for 50 minutes after bubbles overflow and disappear. Dropping 1M hydrochloric acid solution until no gas is discharged (eliminating excess diazomethane), filtering with a Buchner funnel, separating white solid from water layer, washing the solid with 30 ml 0.1N sodium hydroxide solution, washing with double distilled water until the filtrate is neutral, and drying under reduced pressure to obtain 4.58 g white solid. Performing silica gel column chromatography, eluting with chloroform-acetone (100: 1-1: 1), detecting by TLC thin layer chromatography, and collecting pure product to obtain intermediate compound methyl glycyrrhetinate (4.34 g) shown in formula (2), melting point 240-241 deg.C (dichloromethane), and R_f(petroleum ether: chloroform: methanol: 4: 0.6: 0.52; the yield thereof was found to be 89.7%.

Hydrogen spectrum of nuclear magnetic resonance¹H-NMR (400MHz, deuterated chloroform): 0.80 (singlet, 6H), 1.00 (singlet, 3H), 1.12 (singlet, 3H), 1.13 (singlet, 3H), 1.14 (singlet, 3H), 1.36 (singlet, 3H), 1.50 to 2.28 (multiplet ), 2.34 (singlet, 1H, H-9), 2.79(1H, broad doublet, J ═ 13.6Hz, H-18), 3.23(1H, doublet, J ═ 10.8,5.6Hz, H-3), 3.66 (singlet, 3H, CO ═ 13.6Hz, H-3)₂ Me) 5.66 (singlet, 1H, H-12); nuclear magnetic resonance carbon spectrum¹³C-NMR (100MHz, deuterated chloroform): 16.6(q),17.1(q),19.4(t),23.2(q),23.1(q),25.5(q),26.2(t),26.9(q),27.1(t),27.2(t),28.4(q),32.2(t),35.6(t),32.1(s),36.6(t),38.7(t),37.8(s),38.8(s),41.8(t),44.0(s),44.6(s),47.4(s),49.2(d),52.6(q),55.5(d),61.1(d),79.8(d),128.3(d),169.7(s),177.6(s), 201.1(s). Electrospray mass spectrometry ESI-MS: m/z 485[ M + H ]]⁺. The product obtained by the preparation is presumed to be 3 beta-hydroxy-11-carbonyl oleanane-12-alkene-30-carboxylic acid methyl ester shown in the formula (2) according to the spectrogram data.

1.3 preparation of Compound 3 β -methylsulfonyl-18 β -Glycyrrhetinic acid methyl ester of formula (3)

2.42 g of methyl glycyrrhetinate of the compound of the formula (2) prepared under the condition of 1.2 is added into a dry reaction bottle, and stirred and dissolved by 30 ml of dichloromethane; under the condition of ice salt bath, 6 ml of methanesulfonyl chloride is dropwise added under stirring, and the temperature is controlled to be not higher than 10 ℃ in the dropwise adding process. Distilling the brown solution under reduced pressure to remove the solvent to obtain a crude product 3 beta-methylsulfonyl-18 beta-glycyrrhetinic acid methyl ester; recrystallization from chloroform gave 2.24 g of an orange-yellow solid. Electrospray mass spectrometry ESI-MS: m/z 565[ M + H]⁺。

1.4 preparation of 2, 3-ene-18 beta-methyl glycyrrhetinate, a compound of formula (4)

1.13 g of methyl 3 β -methanesulfonyl-18 β -glycyrrhetinate represented by the formula (3) prepared by the preparation method under item 1.3 was dissolved in 40 ml of dimethylformamide, and 0.5 g of lithium carbonate was added with stirring and heated under reflux for half an hour. After the completion of the TLC test reaction, the solvent was evaporated under reduced pressure, the product was purified by column chromatography on silica gel (50 g), and the product was eluted with a gradient of dichloromethane/ethyl acetate (10: 1 to 2: 1), followed by TLC test and combination, and finally, the solvent was evaporated under reduced pressure to give 812 mg of methyl 2, 3-ene-18 β -glycyrrhetinate. R_f(petroleum ether: ethyl acetate: 3: 1): 0.69; electrospray mass spectrometry ESI-MS: m/z 467[ M + H ]]⁺. Melting point: 210-212 ℃ (dichloromethane); [ alpha ] to]²⁵ _D+125.2 ° (c 0.36, dichloromethane); hydrogen spectrum of nuclear magnetic resonance¹H-NMR (400MHz, deuterated chloroform): 0.80 (singlet, 3H), 0.90 (singlet, 3H), 0.97 (singlet, 3H), 1.14 (singlet, 3H), 1.16 (singlet, 3H), 1.17 (singlet, 3H), 1.36 (singlet, 3H), 2.42 (singlet, 1H, H-9), 3.05 (doublet, 1H, H-1, J ═ 17.6,5.6Hz), 3.69 (singlet, 3H, CO₂ Me) 5.32(1H, bimodal, J ═ 10.4Hz, H-3), 5.42(1H, multimodal, H-2), 5.69 (monomodal)1H, H-12). The prepared product is presumed to be the compound 2, 3-alkene-18 beta-methyl glycyrrhetinate of the formula (4) according to the spectrogram data.

1.5 preparation of Compound 1 α -acetoxy-11-carbonyl Olean-2, 12-diene-30-Carboxylic acid methyl ester of formula (5)

To a solution of 466 mg of 2, 3-en-18 β -glycyrrhetinic acid methyl ester of the compound of the formula (4) prepared by the method described in 1.4 in 7 ml of acetic acid, selenium dioxide (224 mg, 2.0 mmol) was added. The reaction mixture was stirred under reflux for 1 hour. After cooling, filtration was carried out. The filtrate was added with 10 ml of water and 10 ml of dichloromethane. After separation, the aqueous layer was extracted three times with 10 ml of dichloromethane each time, the organic layers were combined, washed slightly basic with saturated sodium bicarbonate solution and washed neutral with water. The mixture was dried over anhydrous magnesium sulfate overnight, filtered, and the solvent was removed under reduced pressure. And repeatedly performing column chromatography on the crude product by using 30 g of silica gel, performing gradient elution by using petroleum ether and ethyl acetate (20: 1-1: 1), checking and combining by using TLC (thin layer chromatography), and finally performing reduced pressure evaporation to remove the solvent to obtain 325 mg of the product with the HPLC purity of more than 98%. R_f(petroleum ether: ethyl acetate: 8: 1): 0.52; electrospray mass spectrometry ESI-MS: m/z 525[ M + H ]]⁺. Melting point: 229-230 ℃ (dichloromethane); [ alpha ] to]²⁵ _D+132.4 ° (c 0.34, dichloromethane); hydrogen spectrum of nuclear magnetic resonance¹H-NMR (400MHz, deuterated chloroform): 0.82 (singlet, 3H), 0.92 (singlet, 3H), 1.00 (singlet, 3H), 1.15 (singlet, 3H), 1.17 (singlet, 3H), 1.18 (singlet, 3H), 1.36 (singlet, 3H), 1.95 (singlet, 3H, OAc), 3.20 (broad singlet, 1H, H-18),3.36 (singlet, 1H, H-9), 3.69 (singlet, 3H, CO₂ Me) 5.60(1H, bimodal, J ═ 10.0Hz, H-3), 5.69 (monomodal, 1H, H-12), 5.80(2H, multimodal, H-1)&H-2). The product obtained is presumed to be 1 alpha-acetoxyl-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester according to the spectrogram data.

1.6 preparation of Compounds of formula (1)

To a solution of 210 mg of 1 α -acetoxy-11-carbonyl-oleanane-2, 12-diene-30-carboxylic acid methyl ester of the compound of the formula (5) prepared by the method under 1.5 in 7 ml of ethanol was added 1 ml of an aqueous solution of potassium hydroxide (180 mg, 3.2 mmol). The reaction mixture is stirred under refluxStirring for 1 hour. After completion of the TLC detection reaction, most of the solvent was distilled off under reduced pressure, and 10 ml of water and 10 ml of ethyl acetate were added to the cooled residue. After separation, the aqueous layer was extracted three times with 10 ml of ethyl acetate each time, the organic layers were combined, washed with 1 mol/l hydrochloric acid solution to slightly acidic, and then washed with water to neutral. The mixture was dried over anhydrous magnesium sulfate overnight, filtered, and the solvent was removed under reduced pressure. And repeatedly performing column chromatography on the crude product by using 15 g of silica gel, performing gradient elution by using petroleum ether and ethyl acetate (20: 1-1: 1), checking and combining by using TLC (thin layer chromatography), and finally performing reduced pressure evaporation to remove the solvent to obtain 48.3 mg of the product with the HPLC purity of more than 98%. R_f(5: 1) petroleum ether/ethyl acetate 0.22; electrospray mass spectrometry ESI-MS: m/z 483[ M + H]⁺. Melting point: 215-216 ℃ (dichloromethane); [ alpha ] to]²⁵ _D+125.2 ° (c 0.36, dichloromethane); hydrogen spectrum of nuclear magnetic resonance¹H-NMR (400MHz, deuterated chloroform): 0.84 (singlet, 3H), 0.91 (singlet, 3H), 1.00 (singlet, 3H), 1.09 (singlet, 3H), 1.14 (singlet, 3H), 1.19 (singlet, 3H), 1.39 (singlet, 3H), 3.32 (singlet, 1H, H-9), 3.70 (singlet, 3H, CO)₂ Me) 4.69 (bimodal, 1H, J ═ 5.6Hz, H-1), 5.50(1H, bimodal, J ═ 10.4Hz, H-3), 5.68 (bimodal, 1H, J ═ 10.4,5.6Hz, H-2), 5.72 (monomodal, 1H, H-12); nuclear magnetic resonance carbon spectrum¹³C-NMR (100MHz, deuterated chloroform): 14.2(C-25),17.7(C-26),19.5(C-6),23.8(C-27),25.9(C-24),26.1(C-15),26.7(C-16),27.8(C-28),28.5(C-23),28.7(C-29),31.3(C-21),32.3(C-7),33.1(C-17),34.8(C-4),36.7(C-22),45.4(C-10),41.9(C-19),43.9(C-20),44.5(C-8),44.0(C-14),47.8(C-18),48.3(C-5),52.5(OMe),53.3(C-9),70.7(C-1),126.7(C-2),128.8(C-12),141.4(C-3), 170.13 (C-13),177.9(C-30),201.1 (C-11). In the NOE test, H-1 has a remarkable NOE effect with the C-25 methyl group, but no correlation with H-9 is observed, so that the hydrogen at the 1 position and the C-25 methyl group are in the same side beta configuration. The product obtained is presumed to be 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester according to the spectrogram data. The structure can be further verified by combining HMBC and HSQC spectra obtained by other nuclear magnetic resonance two-dimensional spectrum tests.

Example 2: inhibition of hepatitis B surface antigen (HBsAg) by Compounds of formula (1)

2.1 cell culture:

HepG2.2.15 cells were cultured in DMEM medium containing 10% inactivated fetal calf serum, 100U/ml penicillin, 100U/ml streptomycin, 100. mu.g/ml G418 at 37 ℃ with 5% CO₂And culturing in an incubator with 100% relative humidity.

2.2 determination of the inhibitory action of the test samples on HBsAg secreted by HepG2.2.15 cells:

taking HepG2.2.15 cells in logarithmic growth phase, diluting the cells to 1 × 10 with culture medium⁵Perml, seeded in 96-well cell culture plates at 100 ml per well, 5% CO at 37 ℃₂After 24 hours of incubation in an incubator with 100% relative humidity, the test sample diluted with medium was added, each concentration having three multiple wells of 200. mu.l per well, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in an incubator with 100% relative humidity, changing culture medium containing samples with the same concentration every 4 days, and mixing the changed culture medium with the same concentration of the same sample in equal volume to serve as a sample to be detected. The concentration of hepatitis B surface antigen (HBsAg) in the medium was measured on day eight using ELISA kit and expressed as P/N. Wherein the compound of formula (1) is prepared according to example 1 at concentrations of 100. mu.g/ml, 20. mu.g/ml and 4. mu.g/ml; lamivudine (3-TC) was used as a positive control 1, and the test concentrations were 100. mu.g/ml, 20. mu.g/ml and 4. mu.g/ml; alpha-interferon was used as a positive control 2 and tested at 10000 units/ml, 5000 units/ml and 1000 units/ml.

2.3 Experimental results:

the experimental results are shown in table 1, and the compound 2-alkene-1-alcohol glycyrrhetinic acid methyl ester in the formula (1) has a remarkable effect of inhibiting hepatitis B surface antigen (HBsAg). On the eighth day of the experiment, the inhibitory activity of the compound of formula (1) on HBsAg secreted by HepG2.2.15 cells was higher than that of lamivudine and interferon-alpha.

TABLE 1 inhibition ratio (%)% of hepatitis B surface antigen (HBsAg) secreted from HepG2.2.15 by test sample

2.4 the results show that:

the above experimental results show that: the pentacyclic triterpenic acid compound 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-methyl carboxylate shown in the formula (1) has a remarkable inhibiting effect on hepatitis B surface antigen (HBsAg) secreted by HepG2.2.15 cells, and the HBsAg clearing strength of the pentacyclic triterpenic acid compound is 44.5 percent under the concentration of 100 micrograms/ml, which is respectively 2.94 times of the highest test concentration (10000 units/ml) of a positive control drug alpha-interferon and 1.39 times of the first-line drug lamivudine of the positive control drug. Therefore, the pentacyclic triterpenic acid compound has definite activity of inhibiting the surface antigen secreted by the hepatitis B virus.

HBsAg clearance is the clinically closest state to healing and becomes a very valuable therapeutic end point for CHB in patients with hepatitis b. Therefore, the pentacyclic triterpenic acid compound shown in the formula (1) can be expected to be developed into non-nucleoside innovative medicines for reducing hepatitis B surface antigen and controlling viral hepatitis B symptoms.

Example 3: inhibition of hepatitis B e antigen (HBeAg) by Compounds of formula (1)

3.1 cell culture: the procedure is as in example 2.

3.2 determination of the inhibitory action of the test samples on HBeAg secreted by HepG2.2.15 cells:

taking HepG2.2.15 cells in logarithmic growth phase, diluting the cells to 1 × 10 with culture medium⁵Perml, seeded in 96-well cell culture plates at 100 ml per well, 5% CO at 37 ℃₂After 24 hours of incubation in an incubator with 100% relative humidity, the test sample diluted with medium was added, each concentration having three multiple wells of 200. mu.l per well, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in an incubator with 100% relative humidity, changing culture medium containing samples with the same concentration every 4 days, and mixing the changed culture medium with the same concentration of the same sample in equal volume to serve as a sample to be detected. The concentration of hepatitis B e antigen (HBeAg) in the medium was measured on day eight by ELISA kit and expressed as P/N. Wherein the compound of formula (1) is prepared according to example 1 at concentrations of 100. mu.g/ml, 20. mu.g/ml and 4. mu.g/ml; lamivudine (3-TC) is used as a positive control 1, and the test concentration is 100 microgramsPer ml, 20 μ g/ml and 4 μ g/ml; alpha-interferon was used as a positive control 2 and tested at 10000 units/ml, 5000 units/ml and 1000 units/ml.

3.3 Experimental results:

the results of the experiment are shown in table 2. The pentacyclic triterpenic acid compound 2-alkene-1-alcohol glycyrrhetinic acid methyl ester shown in the formula (1) shows a relatively remarkable effect of inhibiting hepatitis B e antigen (HBeAg). On the eighth day of the experiment, the inhibitory activity of the high-dose compound of formula (1) on HBeAg secreted by HepG2.2.15 cells reaches 50.5%, which is higher than that of positive control 1 (lamivudine) and positive control 2 (alpha-interferon).

TABLE 2 inhibition ratio (%)

3.4 the results show that:

the results of this example show that: the pentacyclic triterpenic acid compound 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-methyl carboxylate shown in the formula (1) has a relatively obvious inhibition effect on hepatitis B e antigen (HBeAg) secreted by HepG2.2.15 cells, and the inhibition activity of the pentacyclic triterpenic acid compound exceeds that of positive control drugs alpha-interferon and lamivudine (3-TC). The strength of inhibiting HBeAg secretion at the concentration of 100 micrograms/ml is 3.30 times that of a positive control medicament 1(100 micrograms/ml 3-TC) and 2.77 times that of a positive control medicament 2(10000 units/ml alpha-interferon) respectively; therefore, the pentacyclic triterpenic acid compound can inhibit the activity of hepatitis B virus for secreting HBeAg, and can be expected to be developed into a medicament for reducing hepatitis B e antigen and controlling viral hepatitis B symptoms.

Example 4: inhibition of hepatitis B Virus deoxyribonucleic acid (HBV-DNA) replication by Compounds of formula (1)

4.1 cell culture: the procedure is as in example 2.

4.2 determination of the inhibitory Effect of test samples on the replication of HBV-DNA secreted by HepG2.2.15 cells:

taking HepG2.2.15 cells in logarithmic growth phase, diluting the cells to 1 × 10 with culture medium⁵Perml, seeded in 96-well cell culture plates at 100 ml per well, 5% CO at 37 ℃₂After 24 hours of incubation in an incubator with 100% relative humidity, the test sample diluted with medium was added, each concentration having three multiple wells of 200. mu.l per well, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in an incubator with 100% relative humidity, changing culture medium containing samples with the same concentration every 4 days, and mixing the changed culture medium with the same concentration of the same sample in equal volume to serve as a sample to be detected. And (3) determining the concentration of the HBV-DNA in the sample to be detected by using an HBV-DNA quantitative PCR kit at the 8 th day. Wherein the compound of formula (1) is prepared according to example 1 at concentrations of 100. mu.g/ml, 20. mu.g/ml and 4. mu.g/ml; lamivudine (3-TC) was used as a positive control 1, and the test concentrations were 100. mu.g/ml, 20. mu.g/ml and 4. mu.g/ml; alpha-interferon was used as a positive control 2 and tested at 10000 units/ml, 5000 units/ml and 1000 units/ml.

4.3 Experimental results:

the results of the experiment are shown in table 3. The compound of formula (1) having pentacyclic triterpenic acid skeleton has stronger effect of inhibiting the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA).

TABLE 3 inhibition ratio (%)% of HBV-DNA replication of HepG2.2.15 cells by test sample

4.4 the results show that:

the results of this example reveal: the pentacyclic triterpenic acid compound 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-methyl carboxylate shown in the formula (1) has a strong inhibition effect on the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA), the replication inhibition activity of the pentacyclic triterpenic acid compound on the HBV-DNA is more than 40% at a high dose (100 micrograms/ml), the replication inhibition activity of positive control alpha-interferon on the HBV-DNA is only 31.3% at the highest test concentration (10000 units/ml), and the replication inhibition strength of the compound shown in the formula (1) on the HBV-DNA is 1.31 times that of the alpha-interferon; the inhibitory activity of the compound shown in the formula (1) on the replication of HBV-DNA of hepatitis B virus at high concentration is 54.4 percent of that of lamivudine as a positive control medicament (see table 3), so that the compound shown in the formula (1) belongs to a natural product for obviously and effectively inhibiting the hepatitis B virus by non-nucleoside and reaches the standard of anti-HBV lead compounds. The pentacyclic triterpenic acid compound is worthy of further attention and intensive research, and can be expected to be further optimized and developed into non-nucleoside innovative medicines for inhibiting HBV-DNA replication.

While the foregoing specification illustrates the invention, examples are provided to illustrate the practice and significance of the invention. The actual use of the invention encompasses all of the usual variations, adaptations, or modifications as come within the scope of the claims and their equivalents.

Claims

1. Use of 2-en-1-ol glycyrrhetinic acid methyl ester with a structure shown in formula (1) for preparing a medicament for preventing and treating hepatitis B virus infection diseases;

the name of the compound of formula (1): 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester.

2. Use of 2-en-1-ol glycyrrhetinic acid methyl ester having a structure represented by formula (1) in claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus deoxyribonucleic acid HBV-DNA, the name of the compound of formula (1) being: 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester.

3. Use of methyl 2-en-1-ol glycyrrhetinate having a structure represented by formula (1) in claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus surface antigen HBsAg, the name of the compound of formula (1) being: 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester.

4. Use of methyl 2-en-1-ol glycyrrhetinate having the structure shown in formula (1) in claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus core antigen HBeAg, the name of the compound of formula (1) being: 1 alpha-hydroxy-11-carbonyl oleanane-2, 12-diene-30-carboxylic acid methyl ester.