CN111789854A - Medicinal use of methyl 2-en-1-ol glycyrrhetinate for preparing antiviral hepatitis B - Google Patents

Abstract

The present invention relates to the use of methyl 2-ene-1-alcohol glycyrrhetinate for preparing a medicine for treating hepatitis B virus infection. Specifically, the present invention provides 1α-hydroxy-11-carbonyl oleanane-2 , the application of methyl 12-diene-30-carboxylate in the preparation of medicines against hepatitis B virus infection. The compound has obvious activity of inhibiting the secretion of HBsAg and HBeAg by HepG2.2.15 cells, and the intensity of inhibiting the secretion of HBsAg and HBeAg is higher than that of the positive drugs α-interferon and lamivudine; The inhibition rate of HBV-DNA replication was also higher than that of α-interferon. The above shows that the 2-ene-1-alcohol glycyrrhetinic acid methyl ester can be expected to be used for the preparation of non-nucleoside drugs for the treatment of hepatitis B virus infection. , the application of HBsAg inhibitor and HBeAg inhibitor; and the preparation method has simple steps, low cost, wide source of raw materials, and easy industrial production.

Description

Medicinal use of methyl 2-en-1-ol glycyrrhetinate for preparing antiviral hepatitis B

technical field

The present invention relates to the technical field of medicine, in particular to the use of methyl 2-en-1-olglycyrrhetinate for preparing a medicine for treating hepatitis B virus infection. The structure of this compound is 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate methyl ester, which is a pentacyclic triterpene methyl ester, which has a significant inhibitory effect on HepG2.2.15 cells It can secrete the activity of HBsAg and HBeAg, and can significantly inhibit the replication of HBV-DNA in HepG2.2.15 cells. It can be expected to be used for the preparation of non-nucleoside innovations for clearing HBsAg and HBeAg, inhibiting HBV-DNA replication and treating hepatitis B virus infection. type of drug use.

Background technique

Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV, hepatitis B virus), so it is also called viral hepatitis B. HBV is a member of the hepadnaviridae family of hepadnaviridae. It is a partial circular DNA virus with spherical particles with a diameter of 42 nanometers. HBV is a peculiar virus that is less contagious in other animals and can only replicate in humans or in primate chimpanzees. The virus is transmitted through the blood, saliva, semen, and vaginal secretions of HBV carriers and HBV patients, and has a chronic carrier state. The disease is widely prevalent in my country, because it is divided into vertical transmission, horizontal transmission, family transmission, iatrogenic transmission and sexual transmission. According to relevant data, the number of patients who tested positive for hepatitis has reached 189 million, while the number of people who should seek medical attention (carriers) is nearly 400 million. It is one of the most serious infectious diseases that endanger people's health. The clinical manifestations of hepatitis B are diverse, and it is easy to develop into chronic hepatitis and liver cirrhosis, and a small number of patients can be transformed into primary liver cancer. Hepatitis B virus in the blood is relatively easy to clear, but the hepatitis B virus in tissue cells is difficult to clear.

Hepatitis B surface antigen (HBsAg) is the coat protein of hepatitis B virus, and HBsAg positive is the gold standard for judging HBV infection. Those who were HBsAg positive but had no symptoms of hepatitis became HBV carriers. The greater the HBsAg titer, the greater the probability of combining hepatitis B core antigen HBeAg, HBV-DNA positive and DNA polymerase activity, and thus the stronger the infectivity. Therefore, inhibiting the secretion and replication of HBsAg is an important target and detection target in the development of anti-HBV drugs. Beijing Ditan Hospital Wu Shuyun et al reported that there is a certain correlation between HBsAg clearance and hepatitis B closed-loop covalent DNA (cccDNA), and clearance of HBsAg is a sign of significantly reduced cccDNA levels. In 2002, the results of the study published in the "New England Journal of Medicine" concluded that for patients with chronic hepatitis B (CHB), if HBsAg is effectively cleared before cirrhosis, the incidence of cirrhosis and hepatocellular carcinoma will be reduced by 60 times. The American Association for the Study of Liver Diseases (AASLD), the Asia-Pacific Association for the Study of the Liver (APASL) and the European Association for the Study of the Liver (EASL) all use HBsAg seroclearance as one of the criteria for determining the endpoint of treatment in hepatitis B treatment guidelines. According to the 2008 annual meeting of the European Society for the Study of the Liver, the treatment of CHB patients with peginterferon alfa-2a was discontinued after 48 weeks, and the HBsAg clearance rates were 3% and 6%, respectively, after 1, 2, 3, and 4 years of follow-up. , 8% and 11%, while the HBsAg clearance rate of lamivudine alone was only 0%, 0%, 0% and 3% at 1, 2, 3, and 4 years after drug withdrawal. At present, although new HBsAg drugs have entered clinical trials at home and abroad, in the first-line drugs for the treatment of acute and chronic hepatitis B, there is no specific drug whose target is to clear HBsAg.

The hepatitis B e antigen HBeAg is the structural protein of the HBV core of the hepatitis B virus, which is produced in large quantities during the reproduction of the hepatitis B virus. Hepatitis B virus HBV has the smallest genome (only 3.2kb) among all known DNA viruses, and its genes mainly encode five proteins (S, C, E, P, X). The C protein is the core protein of the virus, and the E protein is a part of the C protein, which becomes the hepatitis B e antigen (HBeAg), which is a protein that has been encoded but not assembled into the virus particle, and is secreted into the patient's blood when the virus replicates. . Clinically, serum HBeAg is usually used as an important marker for evaluating HBV replication, infectivity, disease severity and response to treatment. This antigen is closely related to HBV-DNA and is a very practical serum marker for viral replication in clinical practice. A patient with positive serum HBeAg indicates that there is HBV replication in their body, so they have a higher infectivity; the higher the expression of HBeAg in a patient, the stronger the infectivity of the patient. Similarly, inhibiting the secretion and replication of HBeAg is also an important target and detection target in the development of anti-HBV drugs. The clearance of HBeAg indicates that there is continuous HBV suppression, normal ALT, reduced tissue inflammation and necrosis, and reduced incidence of liver cirrhosis. Therefore, serum HBeAg is considered to reflect a more stable treatment effect, and HBeAg seroclearance marks the beginning of the patient's immune system. In 2002, a study published in the "New England Journal of Medicine" concluded that for CHB patients, if HBeAg was cleared before cirrhosis, the incidence of cirrhosis and hepatocellular carcinoma would be reduced by 10 times. The guidelines of the American Association for the Study of Liver Diseases (AASLD), the Asia-Pacific Association for the Study of the Liver (APASL), and the European Association for the Study of the Liver (EASL) all use HBeAg seroclearance as one of the treatment endpoint criteria. Therefore, drugs that can inhibit or reduce the expression or activity of HBeAg belong to the drugs that can effectively treat hepatitis B virus infection.

In recent years, with the study of liver disease, standardized HBV-DNA analysis has been developed, which has greatly advanced the understanding of the condition of hepatitis B patients. Quantitative analysis of HBV-DNA can predict the severity and prognosis of hepatitis B, because persistent positive HBV-DNA (ie persistent viremia) can easily lead to the progression and aggravation of hepatitis B disease; high hepatitis B virus (HBV-DNA) content can easily promote liver cirrhosis HBV-DNA persistence is a high risk factor for hepatocellular carcinoma (HCC). Especially in patients with higher viral content, longer course of disease, older age or combined with other liver diseases, continuous high concentrations of HBV-DNA in the body can lead to a significant increase in the mortality of compensated liver cirrhosis and primary liver disease. At the same time, it must be recognized that the level of HBV-DNA is closely related to liver histology: the literature reports that after antiviral treatment, the improvement and elimination of liver fibrosis are obvious; the recent International Liver Disease Conference reported that strong and low-drug resistance antiviral After treatment, with the reduction of HBV-DNA and negative conversion, different degrees of reversal of liver cirrhosis can be observed. Therefore, it is now advocated that liver cirrhosis should also be treated with antiviral therapy.

Therefore, the application of HBV-DNA indicators in antiviral therapy also plays a pivotal role: the level of HBV-DNA is an important indicator to determine whether chronic hepatitis B needs antiviral therapy; in antiviral therapy, according to HBV-DNA To determine whether the HBV-DNA has an early response to treatment, and then determine whether it is an early virological response, and then decide on a long-term medication strategy to achieve a sustained virological response and achieve sustained viral suppression. Treatment goals; according to the continuous and complete inhibition of HBV-DNA, it also shows different degrees of improvement and disappearance of cccDNA; in antiviral therapy, changes in HBV-DNA are used to evaluate and prevent virus mutation and resistance caused by antiviral drugs. Once the virus is mutated or resistant, the change in HBV-DNA is the only first signal and diagnostic basis, and it is also the guidance and basis for treatment resistance and changing treatment strategies.

Therefore, the degree of inhibition of HBV-DNA has a new significance in the further diagnosis and treatment of hepatitis B, and has a great guiding role in the observation of efficacy, the evaluation of the prognosis of hepatitis B and the risk of drug resistance. Therefore, both the Asia-Pacific Society for the Study of the Liver and the European Society for the Study of the Liver regard undetectable HBV-DNA as one of the treatment endpoints for HBV patients. In my country's new drug development guidelines, the inhibitory intensity of the tested compounds on HBV-DNA is also regarded as an important indicator for evaluating the efficacy of hepatitis B drugs.

At present, the drugs for hepatitis B patients are mainly divided into several categories such as liver protection and enzyme reduction, antiviral, anti-hepatic fibrosis and immune regulation. Anti-virus is the fundamental method, while protecting liver and reducing enzymes is only adjuvant therapy. Although some progress has been made in the treatment of hepatitis B with antiviral drugs in recent years; however, the current clinical treatment regimen for viral hepatitis B can only achieve the inhibition of HBV replication and secondary infection in serum, and the most important drugs are still nucleoside drugs such as Lamivudine (3-TC), entecavir, adefovir (ADV), telbivudine, etc., as well as emtricitabine, tenofovir, clevuding, etc. in clinical trials. Some of the advantages of nucleoside drugs are: high bioavailability and safer oral administration. However, although they can temporarily control the disease, one is expensive; the other is that drug resistance can occur after long-term use, and indicators such as HBV-DNA, ALT, and liver histology may rebound to varying degrees after drug withdrawal; The third is the relatively obvious and well-known adverse effects of long-term use of nucleoside drugs, such as kidney damage and infant teratogenicity. The most headache is: the emergence of virus resistance greatly reduces the cure rate, because nucleoside drugs are reversible to virus replication, so if most patients want to achieve the maximum effect, the course of treatment must be more than one year, so the drug resistance When sex comes along, it doesn't have the desired effect. In addition, nucleoside drugs are difficult to remove cccDNA, and it is difficult to convert HBsAg to negative after one year of treatment.

Bioengineered antiviral drugs derived from human leukocytes, such as interferon (α, β-interferon) and recombinant interferon, have recently become hotspots for research and treatment of CHB, which have dual effects of antiviral and immune regulation. It can not only inhibit virus replication through antiviral effect, thereby reduce the inflammatory response of liver cells, reduce liver cell damage, delay the development of the disease, and improve the clinical symptoms and liver physiological function of patients; it can also enhance the immune function. The role of T cells, in particular, can promote killer T cells to kill virus-infected cells, thus indirectly playing an antiviral role. Therefore, interferon has gradually become the clinical drug of choice for the treatment of chronic hepatitis B virus, but its side effects and adverse reactions are reported more; as long as the hepatitis B virus deoxyribonucleic acid (HBV-DNA) is positive, it is likely that the hepatitis B virus has occurred in the body. Variation, active viral replication, infectious, and mutated viruses are insensitive to antiviral drugs and have a high recurrence rate. Therefore, the total effective rate of interferon in the treatment of hepatitis B is not high, and it causes high prices and large economic burdens for patients, thus causing clinical trials. Difficult to use widely. And it is not suitable for patients with decompensated cirrhosis. In order to overcome the defects of the above-mentioned α-interferon, such as side effects and adverse reactions, which restrict its clinical application, the present invention also uses it as a positive control drug to carry out a control experiment.

It must be noted that the currently used antiviral drugs are actually only inhibitors of virus replication, and cannot directly kill the virus and destroy the virion, otherwise it will damage the host cell. These antiviral drugs (mostly nucleoside drugs) also have the above-mentioned shortcomings such as large toxic and side effects, easy to cause viral gene mutation, and easy rebound after drug withdrawal. Therefore, the development of new antiviral drugs is a top priority in the field of drug research and development. It has extremely important social and economic significance for treating a large number of hepatitis B patients and virus carriers in my country and controlling the source of infection. Therefore, the discovery of new non-nucleoside hepatitis B virus inhibitors and such leading compounds that can inhibit HBV-DNA replication from natural medicines that have been used for a long time by ethnic folk has great guiding significance and has broad development prospects.

Based on this purpose, the inventor has previously completed a number of technologies and product research and development of anti-HBV natural products and their structurally modified derivatives, and discovered a variety of compounds that clear HBsAg or HBeAg and inhibit HBV-DNA replication, thus demonstrating that natural products are derived from natural products. It is feasible to screen out innovative drugs that can prevent and treat hepatitis B virus infection from the synthetic derivatives thereof. [See: "Medical use of a class of enantiocinol sesquiterpenes to inhibit hepatitis B virus" (Zhao Yu, Liu Guangming, Yu Rongmin, Li Haibo, etc.; ZL200610053827.4); "2β-Hydroxy Ilexic Acid Inhibition of Hepatitis B Virus" "Medical Uses" (Li Xiaokun, Zhao Yu, Huang Kexin, Li Haibo, etc.; ZL 200610053749.8); "Medical Uses of 2α,3β-Dihydroxy-5,11(13)-dienecaine-12-acid for Inhibiting Hepatitis B Virus" (Zhao Yu, Zhang Lihe, Sun Handong, Li Haibo, etc.; ZL 200610053601.4); "Use of Erimofenolide for Inhibiting Hepatitis B Virus and Its Pharmaceutical Composition" (Zhao Yu, Li Haibo, Yang Leixiang, Zhou Changxin, etc.; ZL 03153691.3); "A natural product of erimofene lactone acid and its application" (Zhao Yu, Zhou Changxin, Shi Shuyun, Wang Xiaoyu, etc.; ZL200610053575.5); "A eucalyptane-type sesquiterpene acid and its use" ( Zhao Yu, Liu Guangming, Li Haibo, Wu Xiumei, etc.; ZL200610053579.3); "The use of hexagonal chrysanthemum extracts in the preparation of pharmaceutical compositions for inhibiting herpes simplex virus and hepatitis B virus" (Zhao Yu, Zhou Changxin, Yu Rongmin , Bai Hua; ZL 200510132508.8); "Medical use of 1β-oxo-5,11(13)-diene eucalyptane-12-acid to inhibit hepatitis B virus" (Zhao Yu, Li Xiaokun, Huang Kexin, Li Haibo, etc.; ZL200610053610 .3); "Medical Use of 1β-Hydroxy Ilexic Acid for Inhibiting Hepatitis B Virus" (Zhao Yu, Li Xiaokun, Huang Kexin, Wu Xiumei, etc.; ZL 200610053625.X); "1-Oxygen-substituted benzoylquinic acid compounds and its use in inhibiting hepatitis B virus” (Li Xiaokun, Hu Lihong, Wu Xiumei, Zhao Yu, etc.; ZL 200810062451.2); recently, the inventor’s team has invented new anti-HBV active compounds from derivatives synthesized from natural products as starting templates and its application in the preparation of anti-HBV drugs: bromodihydroflavonol lignans (ZL 201010181451.1), A-ring coupled flavonolignans (ZL 201010181892.1), benzyloxy flavonolignans (ZL 201010181644.7), B/E dimethoxysilibinin (ZL 201010181499.2), quercetin dimer flavonoid (ZL 201010181869.2), a benzophenone (ZL 201010181533.6), B cycloethoxy dihydroflavonol ( ZL 201010181512.4), substituted isosilibinin (ZL 201010181679.0), A ring substituted silibinin ester (ZL 201010181721.9), E ring bromine substituted silibinin (ZL 201010181632.4), E ring demethoxysilibinin (ZL 201010181731.2), acetamide dehydrogenation Silibinin (ZL 201010181523.2), an horn-type flavonoid lignan (ZL 201010181503.5), bisallyl flavonoid lignan (ZL 201010181908.9), dimethyl dehydrosilibinin (ZL 201010181775.5), double Carbamate dehydrosilibinin (ZL 201010181504.X), flavonoid lignans (±) Scutella prostin A (ZL 201010181362.7), arylcarbamyl dehydrosilibinin (ZL201010181414.0), E-ring iodine Substituted silibinin (ZL 201010181661.0), B-ring ethoxysilibinin (ZL201010181500.1), A-ring dioxane flavonolignan (ZL 201010181411.7), dehydrosilibinin diether (ZL201010117317) .5), a class of dehydrosilibinin trialkyl ethers (ZL 200910099405.4), isopentenyloxy substituted dehydrosilibinin ethers (ZL 200910099404.X), 7 and 20 dehydrogenated water silybin dialkyl ether (ZL200910099403.5), silybin ether substituted on A ring (ZL 200910099042.4), bisallyl substituted silybin ether (ZL 200910099041.X). Undoubtedly, it is very necessary and urgent to continue to search for leading compounds that can effectively prevent and cure HBV from natural products and their structurally modified derivatives.

Traditional Chinese medicine licorice is a leguminous licorice plant, and this herb is listed as the top grade in "Shen Nong's Materia Medica". In the Tang Dynasty's "Medicinal Materia Medica", it is stated that licorice is the king among all medicines. It cures 72 kinds of milk stone poisons, resolves 1,200 kinds of herbal poisons, and has merit in reconciling various medicines, so it is called "the old country". There has always been a saying in Xinglin that "no herbs can't make a prescription", and its main functions are to clear away heat and detoxify, and to reconcile medicinal properties. In the prescription database recorded in TCM120, the frequency of use of licorice in the prescription is listed as the first. The roots and rhizomes of licorice contain a lot of glycyrrhizic acid, also known as glycyrrhizin. In 2003, scientists at the University Hospital of Frankfurt, Germany found that glycyrrhizic acid could inhibit the replication of SARS-related viruses in Vero cells, namely, clinical isolates of SARS virus FFM-1 and FFM-2 (Jindrich Cinatl Jret al, Lancet, 2003, 361:2045- 2046), giving this natural product more research space. In addition to inhibiting viral replication, glycyrrhizic acid can also inhibit viral adsorption and penetration. The effect of adding glycyrrhizic acid was more obvious during the virus adsorption period and after the adsorption period. Therefore, glycyrrhizic acid is a potentially potent lead compound against high-risk viruses. Based on this, scientists continue to study its derivatives to screen for more efficient antiviral agents, and found that the anti-HIV effect of its sulfate ester is 4 times that of glycyrrhizic acid itself, which is very promising to develop into efficient antiviral agents or immune enhancers.

Scientists in my country found that glycyrrhizic acid can also protect the liver, and can delay and reduce the elevation of serum transaminase [Tian Qinglai, et al., Research Progress on the Pharmacological Effects of Active Ingredients of Licorice, Natural Product Research and Development, 2006, (18): 343-347] A clinical trial on 136 patients with chronic hepatitis B (CHB) also found that magnesium isoglycyrrhizinate injection can significantly improve the clinical symptoms, signs and biochemical indicators of liver function [Xu Qingjie et al., Clinical Research on Magnesium Isoglycyrrhizinate in the Treatment of Chronic Hepatitis B, Clinical Medicine, 2011, 31(7): 74-75]; therefore, in recent years, glycyrrhizic acid preparations in my country have been used for the adjuvant treatment of CHB, although they have not been able to cure viral hepatitis B, but have achieved certain effects in improving the symptoms of CHB.

Glycyrrhizic acid is the diglucuronide of glycyrrhetic acid. Glycyrrhizic acid is clinically used as an adrenal cortex hormone and adrenocorticotropic hormone drug, which can replace deoxycorticosterone for the treatment of Addison's disease. Although glycyrrhizic acid has various physiological functions such as anti-inflammatory, enhancing non-specific cellular immune function, and scavenging oxygen free radicals, the glycyrrhizin-like pentacyclic triterpenoids represented by it have not been directly applied to anti-HBV infection. Therefore, we selected it as the starting material, and rationally modified its structure. By means of computer-aided design, a series of 18β-glycyrrhetinic acid derivatives including the structure shown in formula (1) were designed, One of our goals is to find 18β-glycyrrhetinic acid derivatives that can effectively inhibit the secretion of HBsAg or HBeAg and inhibit HBV-DNA replication, so as to further develop them into HBsAg or HBeAg-clearing and HBV-DNA-inhibiting lead compounds. Innovative drugs for replication and treatment of CHB.

18β-glycyrrhetinic acid derivatives in the treatment of DNA virus infection, especially their new uses for anti-hepatitis B virus have not been effectively developed, so look for the anti-hepatitis B virus field activity from the glycyrrhetinic acid pentacyclic triterpenoid derivatives Compounds, i.e. the modification of such structures to have anti-DNA viroid activity, is a new field. It is a very promising challenge to find lead compounds that can effectively inhibit HBsAg or HBeAg secretion and HBV-DNA replication. To explore this field, we designed and prepared an enol functional group structure containing 2,3 en-1-ol in the A ring to increase the SP ² hybrid state molecule and hybrid region of the A ring, and then the glycyrrhetic acid 30 position The carboxymethylation into carboxymethyl ester not only changes the lipophilicity of the molecule, but also adjusts the spatial distance of the electronegativity group of the A-ring of the pentacyclic triterpene acid, in order to discover the extraordinary ability to clear HBsAg/HBeAg and inhibit HBV. - Active lead compound for DNA replication.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate methyl ester represented by formula (1) for preparing and preventing hepatitis B The use of a drug for viral infections, the compound of formula (1) can effectively resist hepatitis B virus HBV.

其中Me是指甲基。

where Me means methyl.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides the application of the compound of formula (1) in the preparation of a medicine for HBsAg inhibitor.

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

The present invention provides the application of the compound of formula (1) in the preparation of a medicine for HBV-DNA inhibitor.

The invention also provides a method for preparing the compound represented by the formula (1), which is characterized in that: firstly, the 30-position carboxyl group of commercially available glycyrrhetic acid is formed into a methyl ester, and then its 3-position hydroxy group is mesylated, and then further After the reaction removes methanesulfonic acid, a double bond is formed on the A ring, and then under the action of acetic acid, the double bond on the A ring is oxidized to 2-ene-1-acetoxy structure with selenium dioxide. The acetyl group is removed under the action of potassium to form methyl 2-en-1-ol glycyrrhetinate represented by formula (1).

The compound of formula (1), or its pharmaceutically acceptable salt or solvate, which has the efficacy of removing HBsAg and HBeAg and inhibiting HBV-DNA replication provided by the present invention, can be combined with pharmaceutically acceptable excipients or carriers to form a drug combination for treating viral hepatitis B is characterized in that it contains a therapeutically effective amount of a mixture consisting of a compound of formula (1) as an active ingredient. The dosage form of its pharmaceutical composition can be tablets, capsules, injections, aerosols, suppositories, films, drop pills, patches, subcutaneous implants, external liniments, oral liquids or ointments. Controlled-release or sustained-release dosage forms or nanoformulations are well known in the modern pharmaceutical industry.

Compared with natural glycyrrhetic acid, the compound 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylic acid methyl ester designed by the inventors of formula (1) has many differences in structure and physicochemical properties The characteristics of chemical compounds, including their hydrophobicity, aromaticity, Gibbs free energy, hydrogen bond acceptor, electrical property, intermolecular van der Waals force, as well as 3D conformation, extension direction, molecular gravity center, degree of conjugation, electrical distribution center, etc. The properties are quite different from that of glycyrrhetic acid; and the molecular weight of the compound of formula (1) is 12 mass units higher than that of glycyrrhetic acid. The above characteristics all determine that the three-dimensional conformation of the compound represented by formula (1) is combined with the 3D spatial structure of HBsAg, HBeAg, and HBV-DNA. The ligand-receptor binding complex shape and binding mode may produce large differences. Its binding site and binding mode, its binding free energy, etc. will produce great changes, so it may have unexpected effects in inhibiting HBsAg or HBeAg secretion and HBV-DNA replication.

HepG2.2.15 cells are derived from human hepatoma cell line HepG2 cells transfected with HBV gene. The cell line can replicate HBV genome stably, and HBV-DNA can also be detected in the cell supernatant. 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 the ability to effectively clear HBsAg or HBeAg and inhibit HBV-DNA. Reproduction of chemical entities with independent intellectual property rights. The test results showed that the pentacyclic triterpenoid methyl ester had significant activity of inhibiting the secretion of HBsAg and HBeAg by HepG2.2.15 cells. On the 8th day of co-cultivation, the compound inhibited the secretion of HBsAg at a concentration of 100 μg/ml, respectively. It is 1.39 times that of positive control drug 1 (100 μg/ml lamivudine) and 2.94 times that of positive control drug 2 (10000 units/ml α-interferon); it inhibits HBeAg secretion at a concentration of 100 μg/ml They are 3.31 times that of positive control drug 1 (100 μg/ml lamivudine) and 2.77 times that of positive control drug 2 (10000 units/ml α-interferon); formula (1) of high concentration (100 μg/ml) ) compound's inhibitory activity on HBV-DNA replication exceeded that of the positive control α-interferon, reaching 54.4% of the positive control lamivudine. The compound of formula (1) has an unexpected anti-HBV effect as described above, so it can be expected to continue to be developed as an active lead compound for clearing HBsAg or HBeAg, inhibiting HBV-DNA replication and treating hepatitis B virus. It can be expected to be further optimized and developed into innovative non-nucleoside innovative drugs that clear hepatitis B HBsAg or HBeAg antigen and inhibit HBV-DNA replication.

To sum up, the pentacyclic triterpene methyl ester derived from glycyrrhetic acid has both structural uniqueness and novelty of antiviral effect. The usual activity of inhibiting the secretion of hepatitis B surface antigen HBsAg and HBeAg, and inhibiting the replication of HBV-DNA, so this compound is expected to be the active leading compound of non-nucleoside drugs for the treatment of CHB. According to the detailed literature review of the present inventor, so far, there is no report on the compound for treating hepatitis B virus infectious diseases and preparing anti-hepatitis B virus drugs. The potent inhibition of HBsAg, HBeAg and HBV-DNA by the pentacyclic triterpenoid compounds of formula (1) is an unexpected discovery and has definite originality, and the present invention is completed accordingly.

The present invention is beneficial in that it is found for the first time that the compound 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate represented by formula (1) has the ability to clear HBsAg or HBeAg, inhibit HBV -DNA replication, and the potential of medicines in the prevention and treatment of hepatitis B virus, provides a new material basis for the development of innovative non-nucleoside anti-HBV drugs and innovative drugs for the treatment of viral hepatitis B. It has potential huge social and economic benefits. Another feature of the present invention is that the glycyrrhetic acid, the synthetic starting material of the present invention, is convenient to source and easy to synthesize. The preparation method of the compound of formula (1) is simple and easy to implement, the source of raw materials is convenient and easy to obtain, the cost is low, and the pollution is small, which is favorable for large-scale production under the condition of energy saving and emission reduction. The prospect of industrialization is very clear.

specific implementation

The inventors obtained the formula derived from glycyrrhetinic acid, which can not only effectively inhibit the secretion of HBsAg and HBeAg in hepatitis B, but also inhibit the replication activity of HBV-DNA through chemical synthesis and purification by various chromatographic means ( 1) The pentacyclic triterpenoid compounds shown, and their chemical structures were verified by comprehensive analytical deduction such as mass spectrometry and nuclear magnetic resonance spectroscopy. The inventors found that the compound of formula (1) has a significant inhibitory effect on the secretion of hepatitis B HBsAg and HBeAg secreted by HepG2.2.15 cells and the replication of HBV-DNA, suggesting that the compound has drug safety, potent clearance of HBsAg or HBeAg, inhibition of Characteristics of HBV-DNA replication. Therefore, according to the research of the inventors, the 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate methyl ester represented by the formula (1) designed and synthesized by the inventors can be used For the preparation of non-nucleoside drugs for the treatment of hepatitis B virus infectious diseases.

In order to better understand the essence of the present invention, the preparation of the compound of formula (1) and the results of its inhibition test on HBsAg and HBeAg secreted by HepG2.2.15 cells and HBV-DNA replication are used to illustrate its application in the pharmaceutical field. new uses. The examples provide synthesis, structural identification and activity data for compounds of formula (1). Unless otherwise specified, the percentages in the present invention refer to weight percentages. It must be noted that the embodiments of the present invention are used to illustrate rather than limit the present invention. Simple improvements made to the present invention according to the essence of the present invention all belong to the scope of protection of the present invention.

Example 1 : Preparation of compound 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylic acid methyl ester of formula (1)

1.1 Instruments and Reagents

Ultraviolet spectrum was measured by Shimadzu UV-240 ultraviolet spectrophotometer; nuclear magnetic resonance spectrum analysis was measured by INOVA type 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 for column chromatography (100-200, 200-300 and 300-400 mesh) and silica gel GF254 (10-40 mesh) for thin-layer chromatography were produced by Qingdao Ocean Chemical Factory ; The reagents used are all analytically pure, and the boiling range of petroleum ether is 60-90 ℃; Agilent 1100 instrument is used for high performance liquid phase detection (HPLC); Sephadex LH-20 adopts the products of Amersham Pharmacia Biotech AB, Sweden; 254nm and 365nm ultraviolet lamps are used for thin plate (TLC) detection; iodine vapor, 10% sulfuric acid-ethanol and phosphomolybdic acid solution are used as color developing agents.

Among them, glycyrrhetic acid refers to glycyrrhetic acid, CH ₂ N ₂ refers to diazomethane, Et ₂ O refers to ether solution, MsCl refers to methanesulfonyl chloride, CH ₂ Cl ₂ refers to dichloromethane, and Li ₂ CO ₃ refers to Lithium carbonate, DMF means dimethylformamide, SeO2 means selenium _dioxide , HOAc means acetic acid, KOH means potassium hydroxide, and flux means reflux reaction.

1.2 Preparation of intermediate formula (2) compound methyl glycyrrhetinate

4.70 g of glycyrrhetic acid (purchased from Xi'an Chenyi Biotechnology Co., Ltd., 99% purity detected by HPLC) was added to the dry reaction flask, and dissolved in 40 ml of a mixed solution of ether:tetrahydrofuran (1:1, V/V). In a fume hood, 15 mmol of the freshly prepared diazomethane solution in ether was dripped with a separatory funnel under magnetic stirring to generate a large amount of white flocculent solids. After the bubbles overflowed and disappeared, the reaction was continued for 50 minutes. Drop into 1M hydrochloric acid solution until no gas is released (eliminate excess diazomethane), filter through a Buchner funnel, separate the white solid from the water layer, wash the solid first with 30 ml of 0.1N sodium hydroxide solution, and then wash with double distilled water until The filtrate was neutral and dried under reduced pressure to obtain 4.58 g of a white solid. Silica gel column chromatography, eluted with chloroform-acetone (100:1 ~ 1:1), TLC thin layer detection, and the pure product was collected to obtain the intermediate compound methyl glycyrrhetinate represented by formula (2) 4.34 g, melting point 240～241°C (dichloromethane), R _f (petroleum ether:chloroform:methanol=4:4:0.6)=0.52; yield 89.7%.

¹ H-NMR (400MHz, deuterated chloroform) δ: 0.80 (singlet, 6H), 1.00 (singlet, 3H), 1.12 (singlet, 3H), 1.13 (singlet, 3H), 1.14 (singlet, 3H), 1.36 (singlet, 3H), 1.50-2.28 (multiplet, multiple H), 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 ₂ Me ), 5.66 (singlet, 1H, H-12); ^13C -NMR (100MHz, deuterated chloroform) δ: 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] ⁺ . According to the above spectral data, it can be inferred that the prepared product is methyl 3β-hydroxy-11-carbonyloleanane-12-ene-30-carboxylate represented by formula (2).

1.3 Preparation of compound 3β-methylsulfonyl-18β-glycyrrhetinate of formula (3)

In a dry reaction flask, add 2.42 g of methyl glycyrrhetinate, the compound of formula (2) prepared under item 1.2, and stir and dissolve with 30 ml of dichloromethane; under an ice-salt bath, add 6 ml of methanesulfonyl chloride dropwise with stirring , the control temperature during the dropwise addition is not higher than 10 ℃. The brown solution was evaporated under reduced pressure to remove the solvent to obtain a crude product of methyl 3β-methanesulfonyl-18β-glycyrrhetinate; recrystallized from chloroform to obtain 2.24 g of an orange-yellow solid. Electrospray Mass Spectrometry ESI-MS: m/z 565 [M+H] ⁺ .

1.4 Preparation of compound 2,3-ene-18β-glycyrrhetinic acid methyl ester of formula (4)

Dissolve 1.13 g of methyl 3β-methanesulfonyl-18β-glycyrrhetinate represented by the formula (3) prepared according to the preparation method under item 1.3 in 40 ml of dimethylformamide, and add 0.5 g of lithium carbonate under stirring , heated to reflux for half an hour. After the completion of the reaction by TLC, the solvent was evaporated under reduced pressure, and the product was purified by 50 g silica gel column chromatography, eluted with a gradient of dichloromethane:ethyl acetate (10:1-2:1), combined by TLC, and finally reduced under reduced pressure. The solvent was evaporated to obtain 812 mg of methyl 2,3-ene-18β-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°C (dichloromethane); [α] ²⁵ _D +125.2° (c 0.36, dichloromethane); ¹ H-NMR (400MHz, deuterated chloroform) δ: 0.80 (single peak) , 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, CO ₂ Me ), 5.32 (1H, Doublet, J=10.4 Hz, H-3), 5.42 (1H, multiplet, H-2), 5.69 (singlet, 1H, H-12). According to the above spectral data, it can be inferred that the prepared product is the compound of formula (4) 2,3-ene-18β-glycyrrhetinic acid methyl ester.

1.5 Preparation of compound 1α-acetoxy-11-carbonyl oleanane-2,12-diene-30-carboxylic acid methyl ester of formula (5)

To a solution of 466 mg of methyl 2,3-ene-18β-glycyrrhetinate, a compound of formula (4), prepared under Item 1.4, in 7 mL of acetic acid, was added selenium dioxide (224 mg, 2.0 mmol) . The reaction mixture was stirred at reflux for 1 hour. After cooling, filter. The filtrate was added with 10 mL of water and 10 mL of dichloromethane. After the layers were separated, the aqueous layer was extracted three times with 10 ml of dichloromethane each time, and the organic layers were combined, washed with saturated sodium bicarbonate solution until slightly alkaline, and then washed with water until neutral. Dry over anhydrous magnesium sulfate overnight, filter, and remove the solvent under reduced pressure. The crude product was subjected to repeated column chromatography on 30 g of silica gel, eluted with a gradient of petroleum ether:ethyl acetate (20:1-1:1), and combined by TLC inspection. Finally, the solvent was evaporated under reduced pressure to obtain the product 325 with an HPLC purity of more than 98%. mg. R _f (petroleum ether:ethyl acetate=8:1)=0.52; Electrospray Mass Spectrometry ESI-MS: m/z 525 [M+H] ⁺ . Melting point: 229～230°C (dichloromethane); [α] ²⁵ _D +132.4° (c 0.34, dichloromethane); ¹ H-NMR (400MHz, deuterated chloroform) δ: 0.82 (single peak) , 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, CO ₂ Me ), 5.60 (1H, doublet, J=10.0 Hz, H-3), 5.69 (singlet, 1H, H-12), 5.80 (2H, multiplet, H-1 & H-2). According to the above spectral data, it can be inferred that the prepared product is methyl 1α-acetoxy-11-carbonyloleanane-2,12-diene-30-carboxylate.

1.6 Preparation of compound of formula (1)

To a solution containing 210 mg of methyl 1α-acetoxy-11-carbonyloleanane-2,12-diene-30-carboxylate of the compound of formula (5) prepared by the method under item 1.5 in 7 ml of ethanol To the solution, potassium hydroxide (180 mg, 3.2 mmol) in 1 mL of water was added. The reaction mixture was stirred at reflux for 1 hour. After TLC detected the reaction, most of the solvent was evaporated under reduced pressure, and 10 ml of water and 10 ml of ethyl acetate were added to the cooled residue. After the layers were separated, the aqueous layer was extracted three times with 10 ml of ethyl acetate each time, and the organic layers were combined, washed with a 1 mol/L hydrochloric acid solution until slightly acidic, and then washed with water until neutral. Dry over anhydrous magnesium sulfate overnight, filter, and remove the solvent under reduced pressure. The crude product was subjected to repeated column chromatography on 15 grams of silica gel, eluted with a gradient of petroleum ether: ethyl acetate (20:1-1:1), combined by TLC, and finally the solvent was evaporated under reduced pressure to obtain a product with a purity of more than 98% by HPLC 48.3 mg. R _f (petroleum ether:ethyl acetate=5:1)=0.22; Electrospray Mass Spectrometry ESI-MS: m/z 483 [M+H] ⁺ . Melting point: 215～216°C (dichloromethane); [α] ²⁵ _D +125.2° (c 0.36, dichloromethane); ¹ H-NMR (400MHz, deuterated chloroform) δ: 0.84 (single peak) , 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 (doublet, 1H, J=5.6Hz, H-1), 5.50 (1H, doublet, J=10.4Hz, H-3), 5.68 (doublet, 1H, J=10.4, 5.6Hz, H-2), 5.72 (singlet, 1H, H-12); C NMR ¹³ C-NMR (100MHz, deuterated chloroform) δ: 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), 170.3 (C-13), 177.9 (C-30), 201.1 (C-11). In the NOE test, H-1 and C-25 methyl groups had significant NOE effects, but no correlation was observed with H-9. Therefore, the 1-position hydrogen and C-25 methyl groups were in the same side β configuration. According to the above spectral data, it can be inferred that the prepared product is methyl 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate. Combined with the HMBC and HSQC spectra obtained from other NMR two-dimensional spectral experiments, the above structures can be further verified.

Example 2 : Inhibitory effect of compound of formula (1) on hepatitis B surface antigen (HBsAg)

2.1 Cell culture:

HepG2.2.15 cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum, 100U/ml penicillin and 100U/ml streptomycin, 100 μg/ml G418 at 37°C, 5% CO ₂ , 100% Culture in a relative humidity incubator.

2.2 Determination of the inhibitory effect of the test sample on the HBsAg secreted by HepG2.2.15 cells:

Take HepG2.2.15 cells in logarithmic growth phase, dilute the cells with medium to 1×10 ⁵ /ml, and inoculate them in a 96-well cell culture plate, 100 ml per well, at 37°C, 5% CO ₂ , 100% relative After culturing in a humidified incubator for 24 hours, the test samples diluted with culture medium were added, and three replicate wells were set up for each concentration, with ₂₀₀ microliters per well. Culture in an incubator, change the medium containing the sample with the same concentration every 4 days, and mix the same volume of the medium with the same concentration of the same sample, as the sample to be tested. On the eighth day, the concentration of hepatitis B surface antigen (HBsAg) in the medium was measured by ELISA kit, expressed as P/N. Wherein, the concentration of the compound of formula (1) prepared according to Example 1 is 100 μg/ml, 20 μg/ml and 4 μg/ml; taking lamivudine (3-TC) as the positive control 1, its test concentration were 100 μg/ml, 20 μg/ml and 4 μg/ml; with α-interferon as the positive control 2, the test concentrations were 10,000 units/ml, 5,000 units/ml and 1,000 units/ml.

2.3 Experimental results:

The experimental results are shown in Table 1. The compound of formula (1), 2-en-1-ol glycyrrhetinic acid methyl ester, has a significant inhibitory effect on 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 α-interferon.

Table 1. Inhibition rate (%) of test samples to HepG2.2.15 secreted hepatitis B surface antigen (HBsAg)

2.4 Result description:

The above experimental results show that the pentacyclic triterpenoid acid compound 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate methyl ester represented by formula (1) secretes HepG2.2.15 cells The Hepatitis B Surface Antigen (HBsAg) has a significant inhibitory effect, and its clearance of HBsAg is 44.5% at a concentration of 100 μg/ml, which is the highest tested concentration of the positive control drug alpha-interferon (10000 units/ml) 2.94 times that of the positive control first-line drug lamivudine, and 1.39 times that of the positive control first-line drug lamivudine. It can be seen that the pentacyclic triterpenoid compounds have the exact activity of inhibiting the secretion of surface antigen of hepatitis B virus.

HBsAg clearance is the clinical state closest to cure, and for hepatitis B patients, its HBsAg clearance has become a very valuable CHB treatment endpoint. Therefore, the pentacyclic triterpenoid compounds represented by formula (1) can be expected to be developed into non-nucleoside innovative drugs for reducing hepatitis B surface antigen and controlling the symptoms of viral hepatitis B.

Example 3 : Inhibitory effect of the compound of formula (1) on hepatitis B e antigen (HBeAg)

3.1 Cell culture: the method is the same as that in Example 2.

3.2 Determination of the inhibitory effect of the test sample on HBeAg secreted by HepG2.2.15 cells:

Take HepG2.2.15 cells in logarithmic growth phase, dilute the cells with medium to 1×10 ⁵ /ml, and inoculate them in a 96-well cell culture plate, 100 ml per well, at 37°C, 5% CO ₂ , 100% relative After culturing in a humidified incubator for 24 hours, the test samples diluted with culture medium were added, and three replicate wells were set up for each concentration, with ₂₀₀ microliters per well. Culture in an incubator, change the medium containing the sample with the same concentration every 4 days, and mix the same volume of the medium with the same concentration of the same sample, as the sample to be tested. On the eighth day, the concentration of hepatitis B e antigen (HBeAg) in the culture medium was measured by ELISA kit, expressed as P/N. Wherein, the concentration of the compound of formula (1) prepared according to Example 1 is 100 μg/ml, 20 μg/ml and 4 μg/ml; taking lamivudine (3-TC) as the positive control 1, its test concentration were 100 μg/ml, 20 μg/ml and 4 μg/ml; with α-interferon as the positive control 2, the test concentrations were 10,000 units/ml, 5,000 units/ml and 1,000 units/ml.

3.3 Experimental results:

The experimental results are shown in Table 2. The pentacyclic triterpene acid compound 2-en-1-ol glycyrrhetinic acid methyl ester represented by formula (1) exhibits a relatively significant inhibitory effect on hepatitis B e antigen (HBeAg). On the eighth day of the experiment, the high-dose compound of formula (1) inhibited HBeAg secreted by HepG2.2.15 cells by 50.5%, which was higher than that of positive control 1 (lamivudine) and positive control 2 (α-interferon). Inhibitory activity against HBeAg.

Table 2. Inhibition rate (%) of tested samples to HepG2.2.15 secreted hepatitis B e antigen (HBeAg)

3.4 Result description:

The results of this example show that the pentacyclic triterpene acid compound 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate methyl ester represented by the formula (1) has an effect on HepG2.2.15 cells The secreted hepatitis B e antigen (HBeAg) has a relatively significant inhibitory effect, and its inhibitory activity exceeds that of the positive control drugs α-interferon and lamivudine (3-TC). The intensity of inhibiting HBeAg secretion at a concentration of 100 μg/ml was 3.30 times that of the positive control drug 1 (100 μg/ml 3-TC) and 2.77 times that of the positive control drug 2 (10000 units/ml α-interferon); It can be seen that the pentacyclic triterpenoid compounds can inhibit the activity of HBeAg secreted by hepatitis B virus, so it can be expected to be developed as a drug for reducing hepatitis B e antigen and controlling the symptoms of viral hepatitis B.

Example 4 : Inhibitory effect of the compound of formula (1) on the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA)

4.1 Cell culture: the method is the same as that in Example 2.

4.2 Determination of the inhibitory effect of the test sample on the replication of HBV-DNA secreted by HepG2.2.15 cells:

Take HepG2.2.15 cells in logarithmic growth phase, dilute the cells with medium to 1×10 ⁵ /ml, and inoculate them in a 96-well cell culture plate, 100 ml per well, at 37°C, 5% CO ₂ , 100% relative After culturing in a humidified incubator for 24 hours, the test samples diluted with culture medium were added, and three replicate wells were set up for each concentration, with ₂₀₀ microliters per well. Culture in an incubator, change the medium containing the sample with the same concentration every 4 days, and mix the same volume of the medium with the same concentration of the same sample, as the sample to be tested. On the 8th day, the concentration of HBV-DNA in the samples to be tested was determined by HBV-DNA quantitative PCR kit. Wherein, the concentration of the compound of formula (1) prepared according to Example 1 is 100 μg/ml, 20 μg/ml and 4 μg/ml; taking lamivudine (3-TC) as the positive control 1, its test concentration 100 μg/ml, 20 μg/ml and 4 μg/ml; with α-interferon as the positive control 2, the test concentrations were 10000 units/ml, 5000 units/ml and 1000 units/ml.

4.3 Experimental results:

The experimental results are shown in Table 3. The compound of formula (1) having a pentacyclic triterpenoid skeleton has a relatively potent effect of inhibiting the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA).

Table 3 Inhibition rate (%) of test samples on HBV-DNA replication of HepG2.2.15 cells

4.4 Result description:

The results of this example reveal that the pentacyclic triterpene acid compound 1α-hydroxy-11-carbonyloleanane-2,12-diene-30-carboxylate methyl ester represented by formula (1) deoxygenates hepatitis B virus The replication of ribonucleic acid (HBV-DNA) has a relatively potent inhibitory effect, and its inhibitory activity on the replication of hepatitis B virus HBV-DNA exceeds 40% at high doses (100 μg/ml), while the positive control α-interferon is in The highest test concentration (10000 units/ml) has only 31.3% inhibitory activity on HBV-DNA replication, the compound of formula (1) has 1.31 times the inhibitory strength of HBV-DNA replication than α-interferon; the compound of formula (1) The inhibitory activity to HBV-DNA replication of hepatitis B virus at high concentration is 54.4% of that of the positive control drug lamivudine (see Table 3), so the compound of formula (1) belongs to a significant non-nucleoside inhibitory natural product of hepatitis B virus, Meet the anti-HBV lead compound standard. The pentacyclic triterpenoids deserve further attention and in-depth research, and can be expected to be further optimized and developed into a non-nucleoside innovative drug that inhibits HBV-DNA replication.

In describing the present invention in the foregoing specification, the purpose of providing the embodiments is to illustrate the actual operation of the present invention and the meaning of the present invention. Practical application of this invention includes all general changes, adaptations, or modifications when coming within the scope of the claims of this invention and their equivalents.

Claims (4)

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.