CN111789853A - Use of methyl methanesulfonyldiene oleanolic acid for preparing medicine for preventing and treating viral hepatitis B - Google Patents

Abstract

The present invention relates to the use of methyl methanesulfonyl diene oleanolic acid for preparing medicines for the prevention and treatment of viral hepatitis B, in particular, the present invention provides 3β-methylsulfonyloxy oleanane-11,13( 18) the application of-diene-28-carboxylate methyl ester in the preparation of the medicine of anti-hepatitis B virus infection disease. The compound has obvious activity of inhibiting the HBsAg and HBeAg secreted by HepG2.2.15 cells, and at a concentration of 100 μg/ml on the 8th day, the intensity of inhibiting the secretion of HBsAg and HBeAg and the replication of HBV-DNA is higher than that of the positive control drug α-interferon and lamivudine. The above shows that the methyl methanesulfonyl diene oleanolic acid can be expected to be used for the preparation of non-nucleoside drugs for the treatment of hepatitis B virus infection. The application of the HBsAg inhibitor and the HBeAg inhibitor; and the preparation method has simple steps, low cost, wide source of raw materials, and easy industrial production.

Description

Methylsulfonyldiene oleanolic acid methyl ester used in the preparation of drugs for the prevention and treatment of viral hepatitis B use

technical field

The present invention relates to the technical field of medicine, in particular to the use of methyl methanesulfonyldiene oleanolic acid for preparing medicines for preventing and treating viral hepatitis B. The compound is a pentacyclic triterpene acid with the structure of methyl 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate, which has the exact 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 (chronic hepatitis B, CHB), if HBsAg is effectively cleared before cirrhosis, the incidence of cirrhosis and hepatocellular carcinoma will be reduced. 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, anti-virus, 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; ZL200510132508.8); "Medical use of 1β-oxo-5,11(13)-dienecine-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 their Uses for Inhibiting Hepatitis B Virus" (Li Xiaokun, Hu Lihong, Wu Xiumei, Zhao Yu, etc.; ZL 200810062451.2); Recently, the inventor's team invented a new type of anti-HBV activity from derivatives synthesized from natural products as starting templates Compound and its application in the preparation of anti-HBV drugs: bromodihydroflavonol lignan (ZL 201010181451.1), A-ring coupled flavonolignan (ZL 201010181892.1), benzyloxyflavonol lignan (ZL 201010181644.7) , B/E dimethoxysilibinin (ZL 201010181499.2), quercetin dimer flavonoid (ZL 201010181869.2), a phenylpropanoid (ZL 201010181533.6), B cycloethoxy dihydroflavonol (ZL 201010181512.4), substituted isosilibinin (ZL 201010181679.0), A ring substituted silybin ester (ZL 201010181721.9), E ring bromine substituted silibinin (ZL 201010181632.4), E ring demethoxysilybin Bin (ZL 201010181731.2), acetamide dehydrogenated water Libribin (ZL 201010181523.2), an angular flavonoid lignan (ZL 201010181503.5), bisallyl flavonoid lignan (ZL 201010181908.9), dimethyl dehydrosilibinin (ZL 201010181775.5), diamine Formyl dehydrosilibinin (ZL 201010181504.X), flavonolignan (±) Scutella prostin A (ZL 201010181362.7), arylcarbamoyl dehydrosilibinin (ZL201010181414.0), E ring iodine substitution 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), prenyloxy-substituted dehydrosilibinin ethers (ZL 200910099404.X), 7 and 20-position dehydrosilibines Cibinin dialkyl ether (ZL200910099403.5), silybin ether substituted on A ring (ZL 200910099042.4), bisallyl substituted silibinin 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.

The natural product oleanolic acid is a natural active ingredient of pentacyclic triterpenoid acids widely distributed in the plant kingdom, mainly found in Oleaceae, Gentianaceae, Umbelliferae, Araliaceae, Cucurbitaceae, etc. , are distributed in stems and leaves. Pharmacological research reports: it can alleviate the acute and chronic liver injury caused by carbon tetrachloride in rats, and can restore the enlarged mitochondria and the expanded rough endoplasmic reticulum; it can also make hepatocytes balloon-like in acute and chronic liver injury. Degeneration, necrosis, reduce inflammation. Oleanolic acid treatment can reduce the accumulation of triglyceride in the liver of injured rats and increase the amount of glycogen. Oleanolic acid also significantly reduced serum alanine aminotransferase and serum γ-globulin in experimental animals with acute and chronic liver injury and liver cirrhosis, which was consistent with the reduction of liver-related inflammation observed in histological observations. Oleanolic acid can significantly increase the number of mitoses in the residual liver of rats, which has the function of promoting liver cell regeneration; taking oleanolic acid can also inhibit the degree of fibrosis in rats with liver fibrosis, and reduce the content of hepatic collagen. , which has the effect of preventing liver cirrhosis. The clinical reports of oleanolic acid include: Shanghai Infectious Disease Hospital and other units used to treat 280 cases of acute jaundice hepatitis, and the cure rate was as high as 64.8%; 298 cases of chronic hepatitis were treated, and the effective rate was 43.7%. The above pharmacological studies or clinical application studies all use the natural product or its derivatives as hepatoprotective drugs, and its structurally modified derivatives are reported in relatively few literatures on antiviral therapy. Oleanolic acid derivatives are used for the treatment of DNA virus-like infections. , especially its new use for anti-hepatitis B virus (including inhibition of hepatitis B HBsAg and/or HBeAg antigen, inhibition of HBV-DNA replication) has not been effectively developed, so from oleanolic acid derivatives to find anti-HBV field Active 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.

Therefore, we selected the natural pentacyclic triterpene acid as the starting material, and rationally modified its structure. By means of computer-aided design, a series of oleanders including the structure shown in formula (1) were designed. Fruit acid derivatives, one of our goals is to find oleanolic acid derivatives that can inhibit the secretion of HBsAg and/or HBeAg and inhibit HBV-DNA replication, so as to further develop them into compounds that can clear HBsAg and / or HBeAg, innovative drugs for inhibiting HBV-DNA replication and treating chronic hepatitis B.

In order to explore this field, we designed and prepared the methanesulfonyldiene methyl ester derivative of oleanolic acid represented by the formula (1). In the heterocyclic conjugated double bonds of the C ring and the D ring, the number of SP ² hybrid molecules is increased, so that the degree of conjugation of the oleanolic acid derivative is increased; at the same time, the 28-position carboxylic acid is methylated, and the A methanesulfonyl group is attached to the 3-hydroxyl group, in order to discover an extraordinary lead compound with the activity of scavenging HBsAg and/or HBeAg and inhibiting HBV-DNA replication based on this natural medicine.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylic acid methyl ester represented by formula (1) for preparing The use of a drug for preventing and treating hepatitis B virus infection, the compound of formula (1) can effectively resist hepatitis B virus HBV; wherein MsO refers to methylsulfonyloxy, and Me refers to 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 present invention also provides a method for preparing the compound represented by formula (1), which is characterized in that: methyl oleanolic acid is formed from commercially available oleanolic acid under the action of diazomethane; and then in the presence of selenium dioxide In the next step, the 12-position double bond is oxidatively isomerized, and then a methanesulfonyl group is linked to the 3-hydroxyl group with methanesulfonyl chloride. Thus, 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylic acid methyl ester represented by formula (1) is prepared.

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 oleanolic acid, the compound 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate designed by the inventors of formula (1) has many structures and physicochemically differentiated features, including its hydrophobicity, aromaticity, Gibbs free energy, hydrogen bond acceptor, electricality, intermolecular van der Waals forces, as well as 3D conformation, extension direction, molecular gravity center, degree of conjugation, The electrical distribution center and other characteristics are different from oleanolic acid; and the molecular weight of the compound of formula (1) is 90 mass units higher than that of oleanolic acid. In addition, the C-ring and D-ring electron cloud configurations are significantly changed compared to oleanolic acid. The above characteristics all determine that the three-dimensional conformation of the compound represented by the formula (1) is combined with the 3D spatial structure of HBsAg, HBeAg, HBV-DNA. The ligand-receptor binding complex shape and binding mode may be different, and its binding site may be different. The point and binding mode, its binding free energy, etc. will all 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 triterpenoids had significant activity of inhibiting the secretion of HBsAg and HBeAg by HepG2.2.15 cells. On the 8th day of co-culture, the compound inhibited the secretion of HBsAg at a concentration of 100 μg/ml. The positive control drug 1 (100 μg/ml lamivudine) was 1.27 times, and the positive control drug 2 (10000 units/ml α-interferon) was 1.61 times; the intensity of its inhibition of HBeAg secretion at a concentration of 100 μg/ml were respectively It is 3.31 times that of the positive control drug α-interferon (10000 units/ml) and 9.13 times that of lamivudine (100 μg/ml). The inhibition rate was 3.60 times higher than that of high concentration alpha-interferon (10000 units/ml), exceeding the inhibitory intensity of lamivudine at the same concentration. The compound of formula (1) has an unexpected anti-HBV effect, so it can be expected to continue to be developed as an active lead compound for clearing HBsAg and/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.

In summary, our methyl methanesulfonyldiene oleanolic acid derived from oleanolic acid has both structural uniqueness and novel anti-HBV effect, and has been tested in anti-HBV activity test. Unusual activities of inhibiting HBsAg and/or HBeAg activity of hepatitis B and HBV-DNA replication were found in this study; it is expected to become an active lead compound of non-nucleoside drugs for the treatment of chronic hepatitis B (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 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate represented by formula (1) has the ability to scavenge HBsAg or The potential of HBeAg, inhibition of HBV-DNA replication, and 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 synthetic starting material of the present invention, oleanolic acid, has wide distribution, convenient source and low price. The preparation method of the compound of formula (1) is simple and feasible, the raw material sources are abundant and readily available, 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 (1) derived from an oleanolic acid which can not only effectively inhibit the secretion of HBsAg and HBeAg in hepatitis B, but also effectively inhibit the replication activity of HBV-DNA through chemical synthesis and purification by chromatography. The chemical structures of the pentacyclic triterpenoids shown were verified by comprehensive analytical deductions such as mass spectrometry and nuclear magnetic resonance spectroscopy. The inventors found that the compound of formula (1) has definite and significant inhibitory effects 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 safe and potent clearance of HBsAg and HBeAg. , Inhibit the characteristics of HBV-DNA replication. Therefore, according to the research of the inventors, the 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylic acid represented by the formula (1) designed and synthesized by the inventors Methyl ester can be used to prepare non-nucleoside drugs for treating 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 3-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylic acid methyl ester of formula (1)

Wherein, Oleanolic acid refers to oleanolic acid; Oleanolic acid methyl ester refers to methyl oleanolic acid; CH ₂ N ₂ refers to diazomethane, Et ₂ O refers to diethyl ether; SeO ₂ refers to selenium dioxide; AcOH means acetic acid; MsCl means methylsulfonyl chloride; _Et3N means triethylamine _{; CH2Cl2} means dichloromethane; Me means methyl; _MeO2SO means methylsulfonyloxy; refers to the reflux reaction; rt refers to room temperature.

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.

1.2 Preparation of intermediate starting material Formula (2) compound 3β-hydroxy-oleanane-12-ene-28-carboxylic acid methyl ester

4.57 g of oleanolic acid (purchased from Sichuan Heyikang 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 4.62 g of a white solid crude product. Silica gel column chromatography, eluted with chloroform-acetone (100:1～1:1), detected by TLC thin layer, and collected the pure product to obtain the compound of formula (2), that is, the intermediate starting material methyl oleanolic acid 3.68 g, R _f (petroleum ether: ethyl acetate = 5: 1) = 0.31, melting point 189-191 °C (dichloromethane). ^1H -NMR (400MHz, deuterated chloroform) δ: 0.72 (singlet, 3H), 0.78 (singlet, 3H), 0.89 (singlet, 3H), 0.90 (singlet, 3H), 0.92 (singlet, 3H), 0.98 (singlet, 3H), 1.13 (singlet, 3H), 2.86 (1H, doublet, J=14.0, 4.0Hz, H-18), 3.20 (1H, doublet , J=10.4, 4.0Hz, H-3), 3.66 (singlet, 3H, CO ₂ Me ), 5.28 (broad singlet, 1H, H-12); ESI-MS: m/z 471[ M+H] ⁺ . The above spectroscopic data are consistent with the compound 3β-hydroxy-oleanane-12-ene-28-carboxylic acid methyl ester of formula (2) reported in the literature, namely methyl oleanolic acid.

1.3 Preparation of intermediate formula (3) compound 3β-hydroxyoleanane-11,13(18)-diene-28-carboxylic acid methyl ester:

Add 470 mg of methyl oleanolic acid, an intermediate starting material prepared under item 1.2, into a dry reaction flask, stir and dissolve with 8 ml of acetic acid; add 280 mg of selenium dioxide under stirring, and the reaction mixture is refluxed for reaction 4 Hour. After the completion of the reaction was detected by TLC, filtered after cooling, and 10 mL of water and 10 mL of dichloromethane were added. After separation, the aqueous layer was extracted three times with 10 mL of dichloromethane; the organic layers were combined, washed with saturated sodium bicarbonate solution until slightly alkaline, and then washed with water until neutral. It was dried with anhydrous magnesium sulfate overnight, filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was repeatedly chromatographed on a 30-gram silica gel column, eluted with petroleum ether-ethyl acetate (100:1-1:1), and detected by TLC. After combining the fractions containing the target product, the solvent was distilled off under reduced pressure to obtain 82.3 mg of a white amorphous solid. Electrospray Mass Spectrometry ESI-MS: m/z 469 [M+H] ⁺ . Melting point: 109～110°C (dichloromethane); ¹ H-NMR (400MHz, deuterated chloroform) δ: 0.79 (singlet, 9H), 0.92 (singlet, 6H), 0.96 (singlet, 6H) 3H), 0.99 (singlet, 3H), 2.52 (broad doublet, 1H, J=14.8Hz, H-19), 3.23 (1H, multiplet, H-3), 3.66 (singlet, 3H, OCH ₃ ), 5.27 (broad doublet, 1H, J=10.4Hz, H-11), 6.42 (broad doublet, 1H, J=10.4Hz, H-12); carbon nuclear magnetic resonance spectroscopy ¹³ C-NMR (100MHz, deuterium Chloroform) δ: 17.3(q), 18.5(q), 18.9(t), 21.1(q), 23.5(q), 26.0(q), 26.3(t), 28.1(t), 28.9(q), 29.0(q), 32.2(t), 32.6(t), 32.9(t), 34.5(s), 36.7(t), 38.3(s), 39.0(t), 40.9(s), 41.2(t), 41.3(s), 41.9(s), 49.1(s), 52.6(q), 55.4(d), 54.4(d), 78.8(d), 126.1(d), 127.2(d), 132.5(s), 136.7(s), 178.0(s). According to the above spectroscopic data, the structure of the compound of formula (3) was identified as methyl 3β-hydroxyoleanane-11,13(18)-diene-28-carboxylate.

1.4 Preparation of compound of formula (1)

In a dry reaction flask, dissolve 47 mg of the compound of formula (3), methyl 3β-hydroxyoleanane-11,13(18)-diene-28-carboxylate, in 10 mL of dichloromethane and 6 mL of triethyl amine. 1.6 ml of methanesulfonyl chloride was added dropwise to the mixture under an ice bath, and the reaction temperature was controlled at about 0°C during the dropwise addition. After the addition was complete, the reaction was continued to stir for 3 hours. After filtration, the filtrate was washed with 1 mol/L hydrochloric acid solution until slightly acidic, and then washed with distilled water until neutral. Dry over anhydrous magnesium sulfate overnight, filter, and evaporate the filtrate to remove the solvent under reduced pressure. The filtrate is subjected to 20 g silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate (100:1-1:1), and TLC detection by thin layer chromatography The pure products containing the same target product were combined, and the solvent was evaporated under reduced pressure to obtain 35.4 mg of a yellow solid. The purity detected by HPLC was greater than 99%. Electrospray Mass Spectrometry ESI-MS: m/z 547 [M+H] ⁺ . Melting point: 105～106℃ (dichloromethane); ¹ H-NMR (400MHz, deuterated chloroform) δ: 0.72 (singlet, 3H), 0.79 (singlet, 3H), 0.86 (singlet, 3H) 3H), 0.90 (singlet, 3H), 0.94 (singlet, 3H), 0.95 (singlet, 3H), 1.12 (singlet, 3H), 2.51 (broad doublet, 1H, J=14.0Hz, H- 19), 3.04 (singlet, 3H, _SO2Me ), 3.67 (singlet, 3H, _OCH3 ), 4.13 (1H, doublet, J=7.2, 14.0Hz, H-3), 5.59 (broad doublet Peak, 1H, J=10.4Hz, H-11), 6.43 (broad doublet, 1H, J=10.4Hz, H-12); C-NMR ^13C -NMR (100MHz, deuterated chloroform) δ: 17.4 (q), 18.6(q), 18.5(t), 20.9(q), 23.7(q), 25.3(q), 26.0(t), 26.8(t), 28.9(q), 29.0(q), 32.3 (t), 32.5(t), 33.0(t), 33.7(s), 36.5(s), 36.7(t), 38.2(q), 38.5(t), 40.8(s), 41.0(s), 42.2 (s), 40.2(t), 49.0(s), 52.7(q), 55.2(d), 54.4(d), 86.8(d), 126.2(d), 127.1(d), 132.5(s), 136.9 (s), 177.8(s). According to the above spectroscopic data, the structure of the compound of formula (1) was identified as methyl 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate.

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), 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate methyl ester, has an exact inhibitory effect on the surface of hepatitis B. The role of 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 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate represented by formula (1) has an effect on HepG2 .2.15 Hepatitis B surface antigen (HBsAg) secreted by cells has a definite inhibitory effect, and its clearance of HBsAg at a concentration of 100 μg/ml is 24.7%, which is the highest test concentration of the positive control drug α-interferon (10,000 units). /ml) 1.61 times; it is 1.27 times the positive control first-line drug lamivudine (100 μg/ml). 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 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate methyl ester represented by formula (1) showed exact inhibition of beta-type The role of hepatitis e antigen (HBeAg). On the eighth day of the experiment, the inhibitory activity of the high dose of the compound of formula (1) on HBeAg secreted by HepG2.2.15 cells reached 36.7%, 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 methyl ester of 3-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate represented by formula (1) can inhibit the secretion of ethyl acetate by HepG2.2.15 cells. Hepatitis e antigen (HBeAg) has a definite inhibitory effect. Its inhibition of HBeAg secretion at a concentration of 100 μg/ml is 9.13 times that of the positive control drug 1 (100 μg/ml 3-TC) and 3.31 times that of the positive control drug 2 (10000 units/ml α-interferon); It can be seen that the pentacyclic triterpenoid compounds can significantly 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 very 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 suggest that the methyl 3-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate represented by the formula (1) is effective against hepatitis B virus deoxyribonucleic acid (HBV-DNA) replication has an extremely potent inhibitory effect; what is exciting is that its inhibitory activity on hepatitis B virus HBV-DNA replication at high doses (100 μg/ml) exceeds 95%, while the positive control α -Interferon has only 26.5% inhibitory activity on HBV-DNA at the highest test concentration (10000 units/ml), the compound of formula (1) has an inhibitory strength on HBV-DNA replication that is 3.60 times that of α-interferon; formula ( 1) The replication inhibitory activity of the compound to hepatitis B virus HBV-DNA at high concentrations is close to the inhibitory activity at the corresponding test concentration of the clinical first-line drug lamivudine (see Table 3), so the pentacyclic triterpenoids are significantly effective. The non-nucleosides inhibit the natural products of hepatitis B virus and meet the standard of anti-HBV lead compounds. The compound of formula (1) deserves 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. the purposes of the methyl methanesulfonyldiene oleanolic acid having the structure shown in the formula (1) for the preparation of a medicine for preventing and treating hepatitis B virus infection;

The name of the compound of formula (1) is: methyl 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate. 2. the application of the methyl methanesulfonyl diene oleanolic acid methyl ester of the structure shown in the formula (1) in claim 1 for the preparation of the hepatitis B virus deoxyribonucleic acid HBV-DNA inhibitor, formula (1) The name of the compound is: methyl 3β-methylsulfonyloxyoleanane-11,13(18)-diene-28-carboxylate. 3. the application of the methyl methanesulfonyl diene oleanolic acid having the structure shown in the formula (1) in claim 1 for the preparation of the medicine of the hepatitis B virus surface antigen HBsAg inhibitor, the name of the compound of the formula (1) It is: 3β-methylsulfonyloxy oleanane-11,13(18)-diene-28-carboxylic acid methyl ester. 4. the application of the methyl methanesulfonyldiene oleanolic acid having the structure shown in the formula (1) in claim 1 for the preparation of the medicine of the hepatitis B virus core antigen HBeAg inhibitor, the name of the compound of the formula (1) It is: 3β-methylsulfonyloxy oleanane-11,13(18)-diene-28-carboxylic acid methyl ester.