CN111789858A - Application of A-ring double-bond oleanolic acid methyl ester in preparation of antiviral hepatitis B drugs - Google Patents

Abstract

The invention relates to application of methyl oleanolic acid with an A-ring double bond in preparation of an antiviral hepatitis B medicament, and particularly provides application of methyl oleanane 2, 12-diene-28-carboxylate in preparation of a medicament for preventing and treating hepatitis B virus infection diseases. The compound has obvious activity of inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells, the strength of inhibiting secretion of the HBsAg and the HBeAg and HBV-DNA replication is higher than that of positive drug alpha-interferon, and is stronger than or equal to lamivudine. The results show that the A-ring double-bond oleanolic acid methyl ester can be expected to be used for preparing non-nucleoside medicaments for treating hepatitis B virus infection diseases, and particularly, the compound has the application of preparing HBV-DNA inhibitors, HBsAg inhibitors and HBeAg inhibitors; the preparation method has simple steps, low cost and wide raw material sources, and is easy to carry out industrialized production.

Description

Application of A-ring double-bond oleanolic acid methyl ester in preparation of antiviral hepatitis B drugs

Technical Field

The invention relates to the technical field of medicines, in particular to application of A-ring double-bond methyl oleanolic acid in preparation of antiviral hepatitis B medicines. The compound is a pentacyclic triterpenic acid derivative which is synthesized by taking oleanolic acid as an initiator and has an oleanane 2, 12-diene-28-carboxylic acid methyl ester structure, has the activity of exactly inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells, can obviously inhibit HBV-DNA replication in the HepG2.2.15 cells, and can be expected to be used for preparing non-nucleoside innovative medicines for clearing the HBsAg and the HBeAg, inhibiting the HBV-DNA replication and treating hepatitis B virus infection diseases.

Background

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

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

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

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

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

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

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

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

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

Based on the purpose, the inventor has previously completed the technology and product research of a plurality of anti-hepatitis B virus natural products and the structure improvement derivatives thereof, and discovers a plurality of compounds for eliminating HBsAg or HBeAg and inhibiting HBV-DNA replication, thereby indicating that the screening of innovative medicaments capable of preventing and treating hepatitis B virus infection from the natural products and the synthetic derivatives thereof is feasible. [ see: "medical use of enantiomorphous eudesmol sesquiterpenes for inhibiting hepatitis B virus" (Zhao Yi, Liuguang, Yurongmi, Lihaibo, etc.; ZL 200610053827.4); "medicinal use of 2 β -hydroxyilicic acid for inhibiting hepatitis B virus" (Li school \22531, Zhao Yi, Huangkexin, Li Hai Bo, etc.; ZL 200610053749.8); "medicinal use of 2 α,3 β -dihydroxy-5, 11(13) -dieneudesman-12-oic acid for inhibiting hepatitis B virus" (Zhao Yi, Zhang He, Sun Han Dong, Li Hao, etc.; ZL 200610053601.4); the use of eremophilane lactone for inhibiting hepatitis B virus and its pharmaceutical composition (Zhao Yi, Li Hai Bo, Yangrel, Zhongchang, etc.; ZL 03153691.3); "an eremophilane lactone acid natural product and its application" (Zhao Yi, Zhongchang Xin, Shizuyun, Wang Xiaoyu, etc.; ZL 200610053575.5); "A eudesmane type sesquiterpene acid and its uses" (Zhao Yi, Liu Guang Ming, Li Hai Bo, Wuxiu Mei, etc.; ZL 200610053579.3); the application of six-edge chrysanthemum plant extract in preparing medicine composition for inhibiting herpes simplex virus and hepatitis B virus (Zhao Yi, Zhongchangxi, Yirong Ming, white Ye; ZL 200510132508.8); "medical use of 1 β -oxo-5, 11(13) -diene eudesmane-12-oic acid for inhibiting hepatitis B virus" (Zhao Yi, Li school 22531, Huangkexin, Li Hai Bo, etc.; ZL 200610053610.3); "medical use of 1 β -hydroxyilicic acid for inhibiting hepatitis B virus" (Zhao Yi, Li school \22531, Huangkexin, sago Xiumei, etc.; ZL 200610053625. X); 1-O-substituted benzoyl quinic acid compounds and their use for inhibiting hepatitis B virus (Li school 22531, Huli hong, Wu Xiumei, Zhao Yi, etc.; ZL 200810062451.2); recently, the group of the present inventors invented new anti-HBV active compounds and their use in the preparation of anti-HBV drugs from derivatives synthesized from natural products as starting templates: contains bromine dihydroflavonol lignan (ZL201010181451.1), ring A coupling flavonolignan (ZL 201010181892.1), benzyloxy flavonolignan (ZL201010181644.7), B/E bis-methoxy silybin (ZL 201010181499.2), quercetin dimer flavone (ZL201010181869.2), a benzo phenylpropanoin (ZL 201010181533.6), B-ring ethoxy dihydroflavonol (ZL201010181512.4), substituted isosilybin (ZL 201010181679.0), ring A substituted silybin ester (ZL201010181721.9), ring E bromine substituted silybin (ZL 201010181632.4), ring E demethoxy silybin (ZL201010181731.2), acetamide dehydrosilybin (ZL 201010181523.2), an angle type flavonolignan (ZL201010181503.5), diallyl flavonolignan (201010181908.9), bis-methyl dehydrosilybin (ZL201010181775.5), bis-carbamoyl dehydrosilybin (ZL 010181504.X), flavonolignan (Juutella) propestin A (ZL 201010181362.7), arylcarbamoyl silybin (ZL201010181414.0), e ring iodine substituted silybin (ZL 201010181661.0), B ring ethoxy silybin (ZL201010181500.1), a ring dioxane flavonolignan (ZL 201010181411.7), dehydrogenated silybin diether (ZL201010117317.5), a class of dehydrogenated silybin trialkyl ether (ZL 200910099405.4), isopentenyl oxygen substituted dehydrogenated silybin ether (ZL200910099404.X), 7 and 20 position dehydrogenated silybin dialkyl ether (ZL200910099403.5), a ring a substituted silybin ether (ZL 200910099042.4), and bis allyl substituted silybin ether (ZL 200009199041. X). Needless to say, it is necessary and urgent to continuously search for lead compounds capable of effectively preventing and treating HBV from natural products and structurally modified derivatives thereof, and the lead compounds are listed as one of the major items for new drug development by the national ministry of science and technology.

The natural product oleanolic acid is a pentacyclic triterpenic acid natural active ingredient widely distributed in plant kingdom, is mainly found in Oleaceae, Gentianaceae, Umbelliferae, Araliaceae, Cucurbitaceae and the like, and is distributed in root tuber, stem leaf and other parts of various plants. The pharmacological research reports that: it has effects of relieving acute and chronic liver injury of rat caused by carbon tetrachloride, and recovering enlarged mitochondria and expanded rough endoplasmic reticulum; can also induce ballooning degeneration and necrosis of liver cells with acute and chronic liver injury, and relieve inflammation reaction. Oleanolic acid treatment can reduce triglyceride accumulation in liver of injured rat and increase glycogen amount. Oleanolic acid also significantly reduces serum glutamic pyruvic transaminase of acute and chronic liver injury and liver cirrhosis experimental animals, reduces serum gamma-globulin, and is consistent with the reduction of related inflammation reaction of liver observed by histology. The oleanolic acid can promote the number of the nuclear division images of the residual liver of the rat to be obviously increased, thereby having the function of promoting the regeneration of liver cells; the oleanolic acid can also inhibit fibrosis degree of hepatic fibrosis rats, and reduce liver collagen content, thereby preventing liver cirrhosis. The reports of the clinical application of oleanolic acid include: 280 cases of acute icteric hepatitis are treated by units such as Shanghai infectious disease hospitals, and the cure rate is as high as 64.8 percent; 298 cases of chronic hepatitis have the obvious efficiency of 43.7 percent. The natural product or the derivative thereof is used as a liver protection drug in the pharmacological research or clinical application research, the structural modification derivative of the natural product or the derivative thereof has relatively few documents reported in the aspect of antiviral treatment, and the new application of the oleanolic acid derivative in the aspect of treating DNA virus infection, particularly in the aspect of resisting hepatitis B virus (including inhibiting hepatitis B HBsAg and/or HBeAg antigen and inhibiting HBV-DNA replication) is not effectively developed, so that the method for searching an active compound in the field of resisting hepatitis B virus from the oleanolic acid derivative, namely modifying the structure to have the activity of resisting DNA virus is a brand-new field. It is a very desirable challenge to find lead compounds from them that effectively inhibit HBsAg or HBeAg secretion, HBV-DNA replication.

Therefore, we select the natural pentacyclic triterpenic acid as a starting material, reasonably modify the structure of the natural pentacyclic triterpenic acid, and design a series of oleanolic acid derivatives including the structure shown in the formula (1) by means of computer-aided design, wherein one of the purposes is as follows: the oleanolic acid derivative lead compound capable of inhibiting secretion of HBsAg and/or HBeAg and inhibiting replication of HBV-DNA is hoped to be found, so that the oleanolic acid derivative lead compound can be further developed into an innovative medicine capable of clearing HBsAg and/or HBeAg, inhibiting replication of HBV-DNA and treating chronic hepatitis B.

In order to explore the field, the oleanolic acid derivative shown in the formula (1) is designed and prepared, the hydroxyl originally existing in the ring A is dehydrated to prepare the ring A double bond, and SP is increased²The number of hybrid molecules, which methylate the 28 th carboxyl of oleanolic acid, enhances the hydrophobicity of the molecules in the region, so that the lead compound with the activities of removing HBsAg and/or HBeAg and inhibiting HBV-DNA replication is discovered based on the natural drug.

Disclosure of Invention

In view of the above, the present invention provides the use of methyl oleanane 2, 12-ene-28-carboxylate shown in formula (1) for preparing the drugs for preventing and treating hepatitis b virus infection diseases, and the compound shown in formula (1) can effectively resist hepatitis b virus HBV.

Wherein Me is methyl.

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

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

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

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

The invention also provides a method for preparing the compound shown in the formula (1), which is characterized by comprising the following steps: using commercial oleanolic acid to generate 28-carboxylic acid methyl ester by diazomethane, then using methanesulfonyl chloride to link an upper methanesulfonyl group on a 3-position hydroxyl group, and reacting the formed methyl sulfonate compound in the presence of N, N-Dimethylacetamide (DMAC) and lithium carbonate to remove a methanesulfonyl group to form an A ring double bond; thereby obtaining the oleanane 2, 12-diene-28-carboxylic acid methyl ester shown as the formula (1).

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

Compared with natural oleanolic acid, the compound oleanane 2, 12-diene-28-methyl carboxylate designed by the inventor has a plurality of characteristics of different structures and physicochemical properties, including hydrophobicity, aromaticity, Gibbs free energy, hydrogen bond receptors, electrical property, intermolecular van der Waals force, 3D conformation, extension direction, molecular gravity center, conjugation degree, electrical distribution center and the like, which are different from the oleanolic acid; and the molecular weight of the compound of formula (1) is reduced by 4 mass units compared with oleanolic acid. In addition, the A-ring electron cloud configuration is significantly changed compared to oleanolic acid. The above characteristics all determine that the three-dimensional conformation of the compound shown in formula (1) and the 3D space structure of HBsAg, HBeAg and HBV-DNA are combined, the form and the combination mode of the ligand-receptor binding complex are possibly different, the combination site, the combination mode, the combination free energy and the like are greatly changed, and therefore, the compound has unexpected effects on inhibiting the secretion of HBsAg or HBeAg and the replication of HBV-DNA.

HepG2.2.15 cells are derived from the human hepatoma cell line HepG2 cells transfected with HBV genes, the cell line can stably replicate HBV genomes, and HBV-DNA can be detected from cell supernatants. We tested the effect of the compound of formula (1) on the secretion of HBsAg and HBeAg by HepG2.2.15 cells and its inhibitory activity on HBV-DNA replication in HepG2.2.15 cells in order to finally obtain a chemical entity capable of effectively eliminating HBsAg or HBeAg and inhibiting the autonomous intellectual property right of HBV-DNA replication. The test results show that: the pentacyclic triterpenic acid compound has remarkable activity of inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells, and the strength of inhibiting the HBsAg secretion of the compound at the concentration of 100 micrograms/ml is similar to the inhibition activity of a positive control medicament (lamivudine at 100 micrograms/ml) on the 8 th day of co-culture; the strength of the inhibitor for inhibiting HBeAg secretion at high, medium and low concentrations exceeds the inhibitory activity of lamivudine at the highest test concentration (100 micrograms/ml); at a concentration of 100. mu.g/ml, the inhibitory activity of the compound of formula (1) on HBV-DNA replication reaches 57.8% of the inhibitory intensity of lamivudine at a corresponding concentration, and exceeds the inhibitory activity of alpha-interferon (10000 units/ml) at a high concentration on HBV-DNA replication. The compounds of formula (1) have unexpected anti-HBV effects, and thus it is expected that they will continue to be developed as active lead compounds for eliminating HBsAg and/or HBeAg, inhibiting HBV-DNA replication, and treating viral hepatitis B. And can be expected to be further developed into innovative non-nucleoside innovative medicaments for eliminating hepatitis B HBsAg or HBeAg antigen and inhibiting HBV-DNA replication.

In conclusion, the A-ring double bond methyl oleanolic acid derived from oleanolic acid has structural uniqueness and novelty in anti-HBV effect, and finds that the activity of inhibiting hepatitis B HBsAg and/or HBeAg and the activity of inhibiting HBV-DNA replication are unusual in anti-HBV activity test; is expected to be an active lead compound of non-nucleoside medicaments for treating Chronic Hepatitis B (CHB). Through the detailed reference of the inventor, no report about the compound for treating the hepatitis B virus infectious diseases and preparing anti-hepatitis B virus medicines exists so far. The pentacyclic triterpenes of formula (1) are unexpected findings of strong inhibition of HBsAg, HBeAg and HBV-DNA, and have definite originality, thus completing the invention.

The invention has the advantages that: the oleanane 2, 12-diene-28-carboxylic acid methyl ester shown in the formula (1) is found to have the functions of eliminating HBsAg or HBeAg, inhibiting HBV-DNA replication and preventing and treating hepatitis B virus, and provides a new material basis for developing non-nucleoside innovative drugs for resisting HBV and developing innovative drugs for treating viral hepatitis B. Has potential huge social benefit and economic benefit. The invention has the following further characteristics: the oleanolic acid as the synthetic starting material of the present invention has the advantages of wide distribution, convenient source and low cost. The preparation method of the compound shown in the formula (1) is simple and easy to implement, the raw materials are convenient and easy to obtain, the cost is low, the pollution is small, and the large-scale production under the conditions of energy conservation and emission reduction is facilitated. The industrialization prospect is very clear.

Detailed description of the preferred embodiments

The pentacyclic triterpenic acid compound shown in the formula (1) and derived from oleanolic acid can be obtained by chemical synthesis and purification through a chromatography means, can effectively inhibit the secretion of hepatitis B HBsAg and HBeAg, and can effectively inhibit the replication activity of HBV-DNA, and the chemical structure of the pentacyclic triterpenic acid compound is deduced and verified through comprehensive analysis such as mass spectrum, nuclear magnetic resonance spectrum and the like. The inventor finds that the compound shown in the formula (1) has obvious inhibition effect on the secretion of hepatitis B HBsAg and HBeAg secreted by HepG2.2.15 cells and the replication of HBV-DNA, and prompts that the compound has the characteristics of safe administration, strong effect of eliminating HBsAg and HBeAg and inhibiting the replication of HBV-DNA. Therefore, according to the research of the inventor, the oleanane 2, 12-diene-28-carboxylic acid methyl ester shown in the formula (1) designed and synthesized by the inventor can be used for preparing non-nucleoside medicaments for treating hepatitis B virus infectious diseases.

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

Example 1: preparation of oleanane 2, 12-diene-28-carboxylic acid methyl ester compound of formula (1)

1.1 instruments and reagents

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

1.2 preparation of intermediate starting material methyl oleanolic acid and intermediate compound methyl 3 beta-methylsulfonyloxy oleanane-12-ene-28-carboxylate of formula (2)

4.57 g of oleanolic acid (obtained from Sichuan Heiykang Biotech Co., Ltd., purity 99% by HPLC) was dissolved in 40 ml of a mixed solution of diethyl ether and tetrahydrofuran (1: 1, V/V) in a dry reaction vessel. In a fume hood, a newly prepared ethyl ether solution of diazomethane is added dropwise by a separating funnel in a 15 mmol manner under magnetic stirring to generate a large amount of white flocculent solid, and the reaction is continued for 50 minutes after bubbles overflow and disappear. Dropping 1M hydrochloric acid solution until no gas is discharged (eliminating excess diazomethane), filtering by a Buchner funnel, separating a white solid from a water layer, washing the solid by 30 ml of 0.1N sodium hydroxide solution, washing by double-distilled water until the filtered liquid is neutral, and drying under reduced pressure to obtain 4.62 g of a white solid crude product. Performing silica gel column chromatography, eluting with chloroform-acetone (100: 1-1: 1), detecting by TLC thin layer chromatography, and collecting pure product to obtain intermediate starting material methyl oleanolic acid 3.68 g, R_f(Petroleum ether: ethyl acetate: 5: 1) 0.31, mp 189-191 deg.C (dichloromethane). Hydrogen spectrum of nuclear magnetic resonance¹H-NMR (400MHz, deuterated chloroform): 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), c₂ Me) 5.28 (broad singlet, 1H, H-12); electrospray mass spectrometry ESI-MS: m/z 471[ M + H]⁺. The above-mentioned spectroscopic data are in agreement with the literature reports of methyl 3 β -hydroxy-oleanan-12-ene-28-carboxylate (i.e., methyl oleanolic acid).

Wherein Oleanolic acid refers to Oleanolic acid; oleanolic acid methyl ester refers to methyl Oleanolic acid; CH (CH)₂N₂Refers to diazomethane; et (Et)₂O is diethyl ether; MsCl refers to methylsulfonyl chloride; et (Et)₃N is triethylamine; CH (CH)₂Cl₂Refers to dichloromethane; me is methyl; MeO₂SO means methylsulfonyloxy.

Adding 3.52 g of intermediate initiator methyl oleanolic acid into a dry reaction bottle, and stirring and dissolving the intermediate initiator methyl oleanolic acid with 45 ml of dichloromethane; and (3) dropwise adding 9 ml of methanesulfonyl chloride under stirring in a salt bath with ice, wherein the temperature is controlled to be not higher than 10 ℃ in the dropwise adding process. The brown solution was distilled under reduced pressure to remove the solvent to obtain a crude product, which was repeatedly recrystallized from chloroform to obtain 2.12 g of a white amorphous solid. Electrospray mass spectrometry ESI-MS: m/z 549[ M + H]⁺. Melting point: 107-109 ℃ (dichloromethane); [ alpha ] to]²⁵ _D117.6 ° (c 0.34, chloroform); hydrogen spectrum of nuclear magnetic resonance¹H-NMR (400MHz, deuterated chloroform): 0.72 (singlet, 3H), 0.86 (singlet, 3H), 0.89 (singlet, 3H), 0.92 (singlet, 3H), 0.94 (singlet, 3H), 1.02 (singlet, 3H), 1.12 (singlet, 3H), 2.86 (doublet, 1H, J ═ 14.0Hz, H-18), 3.01 (singlet, 3H, SO)_{2 3}CH) 3.62 (singlet, 3H, CO)_{2 3}CH) 4.35 (multiplet, 1H, H-3), 5.28 (broad singlet, 1H, H-12). From the above-mentioned spectroscopic data, it was found that the compound obtained was 3 β -methylsulfonyloxy oleanane-12-ene-28-carboxylic acid methyl ester of formula (2).

1.3 preparation of Compounds of formula (1)

Wherein, MeO₂SO means methylsulfonyloxy; DMAC means N, N-dimethylacetamide; li₂CO₃Refers to lithium carbonate; reflux refers to a reflux reaction.

1.12 g of intermediate 3 β -methylsulfonyloxyoleanan-12-ene-28-carboxylic acid methyl ester of formula (2) prepared under item 1.2 was dissolved in 40 ml of N, N-Dimethylacetamide (DMAC), and 310 mg of lithium carbonate was added to the solution. The mixed solution is stirred and refluxed for half an hour, and the reaction solution is filtered after cooling. 20 ml of water and 20 ml of ether were added to the filtrate, and after separation of the layers, the aqueous layer was extracted 3 times with 20 ml of ether. The organic phases are combined, washed to slightly acidic with 1 mol/l hydrochloric acid solution and then washed to neutral with water. The residue was subjected to silica gel column chromatography (50 g), gradient elution with petroleum ether and ethyl acetate (100: 1 to 1: 1), Thin Layer Chromatography (TLC) and chromatography, and the combined purified products containing the same target product were combined and evaporated under reduced pressure to give 780 mg of a white amorphous solid. Purity greater than 99% by HPLC. Electrospray mass spectrometry ESI-MS: m/z 453[ M + H ]]⁺. Melting point: 66-67 ℃ (dichloromethane); [ alpha ] to]²⁵ _D+62.5 ° (c 0.64, dichloromethane); hydrogen spectrum of nuclear magnetic resonance¹H-NMR (400MHz, deuterated chloroform): 0.70 (singlet, 3H), 0.82 (singlet, 3H), 0.83 (singlet, 3H), 0.86 (singlet, 3H), 0.88 (singlet, 3H), 1.04 (singlet, 3H), 2.80 (broad doublet, 1H, J ═ 13.4Hz, H-18), 3.61 (singlet, 3H, CO ═ 13.4Hz, H-18)_{2 3}CH) 5.23 (broad singlet, 1H, H-12), 5.31-5.37 (multiplet, 2H, H-2, H-3); nuclear magnetic resonance carbon spectrum¹³C-NMR (100MHz, deuterated chloroform): 14.7(C-25),15.8(C-26),19.9(C-6),23.8(C-23),23.9(C-24),23.4(C-16),23.9(C-11),25.9(C-27),26.7(C-24),28.5(C-15),29.6(C-30),30.8(C-20),32.8(C-7),32.9(C-22),33.9(C-21),36.7(C-1),37.9(C-4),38.6(C-10),39.7(C-8),41.3(C-14),41.6(C-18),46.0(C-19),46.6(C-17),46.2(C-9),52.1(C-5),52.6(OMe),121.3(C-2),123.2(C-12), 2(C-12), 143.6(C-13),177.6 (C-28). Based on the above-mentioned spectral data, the formula (A)1) The structure of the compound is oleanane 2, 12-diene-28-carboxylic acid methyl ester.

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

2.1 cell culture:

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

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

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

2.3 Experimental results:

as shown in Table 1, the compound of formula (1), methyl oleanane 2, 12-diene-28-carboxylate, has a definite effect of inhibiting hepatitis B surface antigen (HBsAg). On the eighth day of the experiment, the inhibitory activity of the compound of formula (1) on HBsAg secreted by HepG2.2.15 cells was comparable to that of lamivudine.

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

2.4 the results show that:

the above experimental results show that: the pentacyclic triterpenic acid compound oleanane 2, 12-diene-28-carboxylic acid methyl ester shown in the formula (1) has a definite inhibitory effect on hepatitis B surface antigen (HBsAg) secreted by HepG2.2.15 cells, and the intensity of removing the HBsAg is 34.9% at the concentration of 100 micrograms/ml, and is similar to the inhibitory intensity of a positive control first-line drug lamivudine (100 micrograms/ml).

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

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

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

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

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

3.3 Experimental results:

the results of the experiment are shown in table 2. The pentacyclic triterpenic acid compound oleanane 2, 12-diene-28-methyl carboxylate shown in the formula (1) has certain effect of inhibiting hepatitis B e antigen (HBeAg). On the eighth day of the experiment, the inhibitory activity of the compound of formula (1) at a high dose on HBeAg secreted from HepG2.2.15 cells was higher than that of the positive control.

TABLE 2 inhibition ratio (%)

3.4 the results show that:

the results of this example show that: the oleanane 2, 12-diene-28-carboxylic acid methyl ester shown in the formula (1) has certain inhibition effect on hepatitis B e antigen (HBeAg) secreted by HepG2.2.15 cells. The strength of inhibiting HBeAg secretion at 100. mu.g/ml concentration is 28.4%, while the positive control drug (3-TC at 100. mu.g/ml) showed only 15.3% of inhibitory activity; and the inhibitory concentration of the compound of formula (1) at medium and low concentrations still exceeds the inhibitory concentration of the positive control drug lamivudine at the highest concentration (100. mu.g/ml) (see Table 2). Therefore, the pentacyclic triterpenic acid compound can exactly inhibit the activity of hepatitis B virus for secreting HBeAg, and can be expected to be developed into a medicament for reducing hepatitis B e antigen and controlling viral hepatitis B symptoms.

Example 4: inhibition of hepatitis B Virus deoxyribonucleic acid (HBV-DNA) replication by Compounds of formula (1) 4.1 cell culture: the procedure is as in example 2.

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

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

4.3 Experimental results:

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

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

4.4 the results show that:

the results of this example suggest: the oleanane 2, 12-diene-28-carboxylic acid methyl ester shown in the formula (1) has stronger inhibiting effect on the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA): the compound has the inhibitory activity of more than 43 percent on HBV-DNA replication of hepatitis B virus at higher dose (100 micrograms/ml), achieves 57.8 percent of the inhibitory activity of the corresponding concentration of clinical first-line drug lamivudine (positive control 1) on HBV-DNA replication, and has only 31.3 percent of the inhibitory activity of the other positive control alpha-interferon on HBV-DNA at the highest test concentration (10000 units/ml). Therefore, the pentacyclic triterpenic acid compound belongs to an effective non-nucleoside hepatitis B virus inhibition natural product, is worthy of further attention and intensive research, and can be expected to be further optimized and developed into a non-nucleoside innovative medicine for inhibiting HBV-DNA replication.

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

Claims (4)

1. Use of A-ring double bond methyl oleanolic acid having a structure represented by formula (1) in preparation of a medicament for preventing and treating hepatitis B virus infection diseases;

the name of the compound of formula (1): oleanane 2, 12-diene-28-carboxylic acid methyl ester.

2. Use of methyl oleanolic acid having a double bond of ring a of the structure represented by formula (1) in claim 1 for preparing a medicament for hepatitis b virus deoxyribonucleic acid HBV-DNA inhibitor, the name of the compound of formula (1) being: oleanane 2, 12-diene-28-carboxylic acid methyl ester.

3. Use of methyl oleanolic acid having an a-ring double bond represented by the formula (1) in claim 1 for preparing a medicament of an inhibitor of hepatitis b virus surface antigen HBsAg, the name of the compound of the formula (1) being: oleanane 2, 12-diene-28-carboxylic acid methyl ester.

4. Use of methyl oleanolic acid having an a-ring double bond represented by the formula (1) in claim 1 for preparing a medicament of an inhibitor of hepatitis b virus core antigen HBeAg, the name of the compound of the formula (1) being: oleanane 2, 12-diene-28-carboxylic acid methyl ester.