阅读说明：本技术 异环烯酮齐墩果酸甲酯用于制备防治病毒性乙肝的药物用途 (Application of isocycloenone oleanolic acid methyl ester in preparing medicine for preventing and treating viral hepatitis B ) 是由 巫秀美 张云智 张定芝 陈弈彤 杨倩筠 常石伟 段银祥 孙颖 赵昱 于 2020-07-27 设计创作，主要内容包括：本发明涉及异环烯酮齐墩果酸甲酯用于制备防治病毒性乙肝的药物用途,具体而言,本发明提供了3β-羟基-12-羰基齐墩果烷-13(18)-烯-28-羧酸甲酯在制备抗乙型肝炎病毒感染疾病的药物中的应用。该化合物具有显著的抑制HepG2.2.15细胞分泌的HBsAg和HBeAg活性,在第8天时于100微克/毫升浓度下其抑制HBsAg和HBeAg分泌的强度皆超过阳性对照药物α-干扰素和拉米呋啶,该浓度下其对HBV-DNA复制更显示出超过97％的抑制率。以上表明该异环烯酮齐墩果酸甲酯可预期用于制备治疗乙型肝炎病毒感染疾病之非核苷类药物的用途；具体而言,该化合物具有用于制备HBV-DNA抑制剂、HBsAg抑制剂、HBeAg抑制剂的用途,且其制备方法步骤简单、成本低,且原料来源广泛,容易进行产业化生产。(The invention relates to an application of isocycloenone methyl oleanolic acid in preparing a medicament for preventing and treating viral hepatitis B, and particularly provides an application of 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-methyl carboxylate in preparing a medicament for resisting hepatitis B virus infection diseases. The compound has remarkable activity of inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells, the strength of inhibiting the secretion of the HBsAg and the HBeAg exceeds that of a positive control medicament alpha-interferon and lamivudine at the concentration of 100 micrograms/ml on day 8, and the compound shows more than 97% inhibition rate on HBV-DNA replication at the concentration. The results show that the isocycloenone oleanolic acid methyl ester can be expected to be used for preparing non-nucleoside medicaments for treating hepatitis B virus infection diseases; specifically, the compound has the application of preparing HBV-DNA inhibitor, HBsAg inhibitor and HBeAg inhibitor, and the preparation method has the advantages of simple steps, low cost, wide raw material source and easy industrial production.)

1. Use of isocycloenone oleanolic acid methyl ester having a structure represented by formula (1) for preparing a medicament for preventing and treating hepatitis B virus infection diseases;

the name of the compound of formula (1): 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-carboxylic acid methyl ester.

2. Use of the methyl isocycloalkenone oleanolic acid having the structure represented by the formula (1) in claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus deoxyribonucleic acid HBV-DNA, the name of the compound of the formula (1) being: 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-carboxylic acid methyl ester.

3. Use of methyl isocycloalkenone oleanolic acid having a structure represented by the formula (1) of claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus surface antigen HBsAg, the name of the compound of the formula (1) being: 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-carboxylic acid methyl ester.

4. Use of methyl isocycloalkenone oleanolic acid having a structure represented by the formula (1) in claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus core antigen HBeAg, the name of the compound of the formula (1) being: 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-carboxylic acid methyl ester.

5. A process for preparing a compound having the structure of formula (1) as defined in claim 1, characterized in that: using the oleanolic acid sold in the market to form methyl oleanolic acid under the action of diazomethane; in the presence of selenium dioxide, oxidizing and isomerizing a 12-position double bond of methyl oleanolic acid to form C/D cyclic conjugated diene, forming an epoxy intermediate under the action of m-chloroperoxybenzoic acid mCPBA, and oxidizing and opening a ring under the action of concentrated hydrochloric acid to prepare the 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-carboxylic acid methyl ester shown in the formula (1).

Technical Field

The invention relates to the technical field of medicines, in particular to application of isocycloenone methyl oleanolic acid in preparing a medicine for preventing and treating viral hepatitis B. The compound is a pentacyclic triterpenic acid derivative with a 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-carboxylic acid methyl ester structure, has very remarkable activity of inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells, can remarkably inhibit HBV-DNA replication in the HepG2.2.15 cells, and can be expected to be used for preparing non-nucleoside-type medicaments for clearing the HBsAg and the HBeAg, inhibiting the 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 (ZL 201010181451.1), ring A coupling flavonolignan (ZL 201010181892.1), benzyloxy flavonolignan (ZL 201010181644.7), B/E bis-methoxy silybin (ZL 201010181499.2), quercetin dimer flavone (ZL 201010181869.2), a benzo phenylpropanoin (ZL 201010181533.6), B-ring ethoxy dihydroflavonol (ZL 201010181512.4), substituted isosilybin (ZL 201010181679.0), ring A substituted silybin ester (ZL 201010181721.9), ring E bromine substituted silybin (ZL 201010181632.4), ring E demethoxy silybin (ZL 201010181731.2), acetamide dehydrosilybin (ZL 201010181523.2), an angle type flavonolignan (ZL 201010181503.5), diallyl flavonolignan (201010181908.9), bis-methyl dehydrosilybin (ZL 201010181775.5), diamine formyl dehydrosilybin (ZL 010181504.X), flavonolignan (+/-) Scutella prostin A (ZL 201010181362.7), aryl carbamoyl dehydrogenated silybin (ZL201010181414.0), E-ring iodine substituted silybin (ZL 201010181661.0), B-ring ethoxy silybin (ZL201010181500.1), A-ring dioxane flavonolignan (ZL 201010181411.7), dehydrogenated silybin diether (ZL201010117317.5), dehydrogenated silybin trialkyl ether (ZL 200910099405.4), isopentenyloxy substituted dehydrogenated silybin ether (ZL 200910099404.X), 7-and 20-position dehydrogenated silybin dialkyl ether (ZL200910099403.5), A-ring substituted silybin ether (200910099042.4) and diallyl propyl substituted silybin ether (ZL 200910099041. X). Needless to say, it is necessary and urgent to continuously search for lead compounds capable of effectively preventing and treating HBV from natural products and structurally modified derivatives thereof, and the lead compounds are listed as one of the major items for new drug development by the national ministry of science and technology.

The 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 method designs and prepares the isocycloalkenone oleanolic acid methyl ester shown in the formula (1), the double bond originally existing in the C ring is used for preparing the isocycloalkenone structure conjugated by the carbonyl group at the 12-position of the C ring and the double bond of the D ring through the modern synthesis technology, and the SP of the C/D ring is increased²A hybrid region is used for enlarging the conjugation degree, changing the folding condition of C/D ring molecules and carrying out methyl esterification on 28-site carboxylic acid; based on the natural medicine, the lead compound with the activity of eliminating HBsAg and/or HBeAg and inhibiting HBV-DNA replication is found out unusually.

Disclosure of Invention

In view of the above, the present invention provides the use of methyl 3 β -hydroxy-12-carbonyl oleanane-13 (18) -ene-28-carboxylate represented by formula (1) for preparing a medicament for preventing and treating hepatitis b virus infection diseases, wherein the compound represented by 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 the oleanolic acid sold in the market to form methyl oleanolic acid under the action of diazomethane; in the presence of selenium dioxide, oxidizing and isomerizing a 12-position double bond of methyl oleanolic acid to form C/D cyclic conjugated diene, forming an epoxy intermediate under the action of m-chloroperoxybenzoic acid mCPBA, and oxidizing and opening a ring under the action of concentrated hydrochloric acid to prepare the 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-carboxylic acid methyl ester shown in the formula (1).

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

Compared with natural oleanolic acid, the compound 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-methyl carboxylate designed by the inventor has the characteristics of structural and physicochemical properties of differentiation, including hydrophobicity, aromaticity, Gibbs free energy, hydrogen bond receptors, electrical property, intermolecular van der Waals force, 3D conformation, stretching direction, molecular gravity center, conjugation degree, electrical distribution center and the like of the compound, namely the compound 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-methyl carboxylate, which is different from the oleanolic acid; and the molecular weight of the compound of formula (1) is increased by 28 mass units compared with that of oleanolic acid. In addition, the C-ring and D-ring electron cloud configuration is significantly altered compared to oleanolic acid. The above characteristics all determine that the ligand-receptor binding complex forms and binding modes of the three-dimensional conformation of the isocycloalkone oleanolic acid methyl ester shown in the formula (1) and the 3D space structure of HBsAg, HBeAg and HBV-DNA can be different, the binding sites and binding modes, the binding free energy and the like can be greatly changed, and therefore, the ligand-receptor binding complex can have unexpected effects on inhibiting secretion of HBsAg or HBeAg and replication of HBV-DNA.

HepG2.2.15 cells are derived from the human hepatoma cell line HepG2 cells transfected with HBV genes, the cell line can stably replicate HBV genomes, and HBV-DNA can be detected from cell supernatants. We tested the effect of the compound of formula (1) on the secretion of HBsAg and HBeAg by HepG2.2.15 cells and its inhibitory activity on HBV-DNA replication in HepG2.2.15 cells in order to finally obtain a chemical entity capable of effectively eliminating HBsAg or HBeAg and inhibiting the autonomous intellectual property right of HBV-DNA replication. The test results show that: the pentacyclic triterpenic acid compound has the obvious activity of inhibiting HBsAg and HBeAg secreted by HepG2.2.15 cells; on day 8 of co-culture, the compound inhibited HBsAg secretion at a concentration of 100. mu.g/ml by 1.4 times that of positive control 1 (100. mu.g/ml lamivudine) and 2.8 times that of positive control 2(10000 units/ml interferon-. alpha.); the strength of the inhibitor for inhibiting HBeAg secretion at the concentration of 100 micrograms/ml is 5.21 times that of alpha-interferon (10000 units/ml) as a positive control medicament and 3.53 times that of lamivudine (100 micrograms/ml); at a concentration of 100. mu.g/ml, the compound of formula (1) has an inhibitory activity on HBV-DNA replication of more than 97%, while a high concentration of interferon-alpha (10000 units/ml) has an inhibitory activity on HBV-DNA replication of only 33.6%; the inhibitory strength of the compound of formula (1) on HBV-DNA replication at high concentration is 2.89 times that of alpha-interferon, and the inhibitory activity on HBV-DNA replication at high and medium concentration is close to that of lamivudine at corresponding concentration. 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 isocycloalkenone methyl oleanolic acid derived from oleanolic acid has structural uniqueness and novelty of 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 the 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 triterpenic acid compound shown in the formula (1) is an unexpected discovery for strong inhibition of HBsAg, HBeAg and HBV-DNA, and has definite originality, thereby completing the invention.

The invention has the advantages that: the compound 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-28-methyl carboxylate shown in the formula (1) is found to have the functions of eliminating HBsAg or HBeAg, inhibiting HBV-DNA replication and preventing and treating hepatitis B virus for the first time, 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 feasible, has rich and easily obtained raw material sources, low cost and little pollution, and is beneficial to large-scale production under the conditions of energy conservation and emission reduction. 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 3 beta-hydroxy-12-carbonyl oleanane-13 (18) -alkene-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.