CN111920820A

CN111920820A - Application of acetyl glycyrrhetinic acid methyl ester in preparation of medicine for treating viral hepatitis B

Info

Publication number: CN111920820A
Application number: CN202010728951.6A
Authority: CN
Inventors: 巫秀美; 赵微; 杜雯雯; 吴俊珠; 赵键挺; 孙敏哲; 金凡茂; 赵昱
Original assignee: Dali University
Current assignee: Dali University
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2020-11-13

Abstract

The invention relates to application of acetyl glycyrrhetinic acid methyl ester in preparing a medicament for treating viral hepatitis B. Specifically, the invention provides an application of a compound shown as a formula (1), namely 3 beta-acetoxyl-11-carbonyl oleanane-12-alkene-30-carboxylic acid methyl ester, in preparing a medicament for preventing and treating hepatitis B virus infection diseases. The compound of the formula (1) has remarkable activity of inhibiting HBeAg secreted by HepG2.2.15 cells, and has the strength of inhibiting HBeAg secretion of 51.4% at the concentration of 100 micrograms/ml, which is 3.78 times that of alpha-interferon (10000 units/ml) serving as a positive control medicament and 4.94 times that of lamivudine (100 micrograms/ml); it showed 94.6% inhibition of HBV-DNA replication at this concentration, which was higher than the lamivudine inhibitory strength at the same concentration (88.4% inhibition of HBV-DNA by 3-TC at 100. mu.g/ml), which was 3.1 times higher than that of a high concentration of interferon-alpha (10000 units/ml). The above shows that the acetyl glycyrrhetinic acid methyl ester can be expected to be used for preparing non-nucleoside medicaments for treating hepatitis B virus infection diseases, in particular, the compound has the application for preparing HBV-DNA inhibitors and HBeAg inhibitors, and the preparation method has the advantages of simple steps, low cost, wide raw material sources and easy industrial production.

Description

Application of acetyl glycyrrhetinic acid methyl ester in preparation of medicine for treating viral hepatitis B

Technical Field

The invention relates to the technical field of medicines, in particular to application of a glycyrrhetinic acid derivative, namely 3 beta-acetoxyl-18 beta-methyl glycyrrhetinate, in preparation of a medicine for treating hepatitis B virus infection diseases. The compound is a pentacyclic triterpenic acid derivative, has the obvious activity of inhibiting HBeAg secretion of 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 medicaments for clearing 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.

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 reproduction of HBV. Hepatitis b virus has the smallest genome of all known DNA viruses (only 3.2kb), whose 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, inhibition of secretion and replication of HBeAg is an important target and detection target in the development of anti-HBV 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 drug capable of inhibiting and reducing the expression or activity of HBeAg belongs to the drug capable of effectively treating HBV 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 and development of a plurality of anti-hepatitis B virus natural products and structurally modified derivatives thereof, and discovers a plurality of compounds for eliminating HBeAg and inhibiting HBV-DNA replication, thereby showing that the screening of innovative medicaments capable of preventing and treating hepatitis B virus infection from natural products and 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 (ZL 201010181414.0), E-ring iodine substituted silybin (ZL 201010181661.0), B-ring ethoxy silybin (ZL 201010181500.1), A-ring dioxane flavonolignan (ZL 201010181411.7), dehydrogenated silybin diether (ZL 201010117317.5), dehydrogenated silybin trialkyl ether (ZL200910099405.4), isopentenyloxy substituted dehydrogenated silybin ether (ZL 200910099404.X), 7-and 20-position dehydrogenated silybin dialkyl ether (ZL 200910099403.5), A-ring substituted silybin ether (200910099042.4) and diallyl propyl substituted silybin ether (ZL 200910099041. X). Needless to say, it is necessary and urgent to continuously search for lead compounds capable of effectively preventing and treating HBV from natural products and structurally modified derivatives thereof, and the lead compounds are listed as one of the major items for new drug development by the national ministry of science and technology.

The traditional Chinese medicine liquorice is a plant belonging to genus glycyrrhiza of leguminosae, and the herbal medicine is listed as the superior product in Shen nong Ben Cao Jing. Tang Dynasty, the book of materia Medica of drug Property: in the medicines, the liquorice is called by the old in China because the liquorice is the monarch medicine for treating seventy-two kinds of mammary stone toxin and relieving one thousand two hundred common herb toxin and harmonizing the active effects of the medicines. The main effects of apricot forest are clearing away heat and toxic material, harmonizing drug property, etc., so far there is a theory that no herb is in the way. In the prescription database recorded in the TCM120, the usage frequency of licorice in the prescription is listed as the first. The root tuber and rhizome of licorice contains a large amount of glycyrrhizic acid, also known as glycyrrhizin. In 2003, scientists at the university of Frankfurt, Germany, found that glycyrrhizic acid can inhibit the replication of SARS-associated virus in Vero cells, namely SARS virus clinical isolates FFM-1 and FFM-2 (Jindrich Cinatl Jr et al, Lancet,2003,361:2045-2046), and further give more research space to the natural product. Glycyrrhizic acid can inhibit virus replication and also inhibit virus adsorption and penetration. The glycyrrhizic acid is added in the virus adsorption period and after the adsorption period, so that the effect is more obvious. Therefore, glycyrrhizic acid is a potential effective lead compound against high-risk viruses. Based on this, scientists continuously research the derivatives to screen more efficient antiviral preparations, and find that the sulfate has the anti-HIV effect 4 times of that of glycyrrhizic acid, so that the development of efficient antiviral preparations or immunopotentiators is very hopeful.

Chinese scientists find that: glycyrrhizic acid also has the effect of protecting liver, and can delay and reduce the rise of serum transaminase [ tianqinglai, etc., pharmacological action research progress of effective components of licorice, natural product research and development, 2006, (18): 343-347 ]; clinical tests of 136 Chronic Hepatitis B (CHB) patients also find that the magnesium isoglycyrrhizinate injection can significantly improve clinical symptoms, physical signs and biochemical indicators of liver function of patients [ xu qingjie and the like, clinical research on treatment of chronic hepatitis b by magnesium isoglycyrrhizinate, clinical medicine 2011, 31 (7): 74-75 ]; therefore, in recent years, glycyrrhizic acid preparations in China have been used for adjuvant therapy of CHB, and although viral hepatitis B cannot be cured, certain effects on improvement of CHB symptoms and the like are achieved.

Glycyrrhizic acid is diglucuronide of glycyrrhetinic acid, and glycyrrhetinic acid is clinically used as adrenocortical hormone and adrenocorticotropic hormone medicine, and can replace deoxycorticosterone for treating Addison's disease. Although glycyrrhetinic acid has various physiological functions of resisting inflammation, enhancing nonspecific cellular immune function, scavenging oxygen free radicals and the like, glycyrrhetinic acid has not been directly applied to anti-HBV infection. The new application of the 18 beta-glycyrrhetinic acid derivative in treating DNA virus infection, particularly in the aspect of resisting hepatitis B virus is not effectively developed, so that the method for searching an active compound in the field of resisting hepatitis B virus from the glycyrrhetinic acid pentacyclic triterpene derivative, namely the method for modifying the structure to have the activity of resisting DNA virus is a brand-new field. It is a very desirable challenge to find lead compounds from which HBeAg can be cleared and HBV-DNA replication can be inhibited. In order to explore the field, we select it as a starting material, reasonably modify its structure, and design a series of 18 beta-glycyrrhetinic acid derivatives including the structure shown in formula (1) by means of computer aided design, wherein one of the purposes is: it is hoped to find 18 beta-glycyrrhetinic acid derivative lead compounds capable of inhibiting HBeAg secretion and HBV-DNA replication, so that the lead compounds can be further developed into innovative medicaments capable of clearing HBeAg, inhibiting HBV-DNA replication and treating CHB.

For this reason, we have designed and prepared a methyl ester derivative of 18 β -glycyrrhetinic acid containing an acetoxy group at the 3-position, i.e., a carboxymethylate at the 30-position of glycyrrhetinic acid, and acetylate the β -hydroxyl group at the 3-position of glycyrrhetinic acid, thereby enhancing the lipophilicity of the whole molecule and enhancing the electron withdrawing property of the pentacyclic triterpenic acid A ring, in order to find out the activity of eliminating HBeAg, inhibiting HBV-DNA replication, and even lead compounds, which are far more unusual.

Disclosure of Invention

The invention aims to provide a new application of 3 beta-acetoxyl-18 beta-glycyrrhetinic acid methyl ester shown in a formula (1) in preparing a medicament for eliminating HBeAg, inhibiting HBV-DNA replication and treating hepatitis B virus infection diseases.

The name of the compound of formula (1): 3 beta-acetoxy-11-carbonyl oleanane-12-ene-30-carboxylic acid methyl ester, the IUPAC name of which is: (2S,4aS,6aS,6bR,8aR,10S,12aS,12bR,14bR) -methyl-10-acetoxy-2, 4a, 6a,6b, 9,9,12a-hepta methyl-13-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14 b-icosahydroprocene-2-carboxylate.

The invention also provides a method for preparing the compound shown in the formula (1), which is characterized by comprising the following steps: using diazomethane to generate methyl carboxylate from commercially available glycyrrhetinic acid in one step, and then performing acetylation on 3-hydroxyl of glycyrrhetinic acid by an acetic anhydride/pyridine method to obtain the glycyrrhetinic acid methyl carboxylate.

Another object of the present invention is to provide a composition for the preparation of a medicament for eliminating HBeAg, inhibiting HBV-DNA replication, and treating viral hepatitis B, characterized by containing a therapeutically effective amount of a mixture consisting of the compound of formula (1) as an active ingredient. The medicament can be tablets, capsules, injections, aerosols, suppositories, membranes, dropping pills, sticking tablets, subcutaneous implants, external liniments, oral liquid or ointments, and can also adopt controlled release or sustained release formulations or nano preparations known in the modern pharmaceutical industry.

Compared with natural glycyrrhetinic acid, the 3 beta-acetoxyl-18 beta-glycyrrhetinic acid methyl ester compound designed by the inventor has the characteristics of various differences in structure and physicochemical properties, including hydrophobicity, aromaticity, Gibbs free energy, hydrogen bond receptors, electrical property, intermolecular van der Waals force, 3D conformation, extension direction, molecular gravity center, conjugation degree, electrical distribution center and the like, and has certain difference with the glycyrrhetinic acid; and the molecular weight of the compound of the formula (1) is increased by 56 mass units compared with glycyrrhetinic acid. The above characteristics all determine that the three-dimensional conformation of the compound represented by formula (1) may be different from that of the ligand-receptor binding complex which binds to the 3D space structure of HBeAg or HBV-DNA, and the binding site and binding mode, binding free energy, etc. may be greatly changed, so that the compound may have unexpected effects in inhibiting HBeAg secretion and HBV-DNA replication.

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 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 HBeAg and inhibiting the autonomous intellectual property of HBV-DNA replication. The test results show that: the pentacyclic triterpenic acid methyl ester has remarkable activity of inhibiting HBeAg secreted by HepG2.2.15 cells, and the strength of the compound for inhibiting HBeAg secretion at the concentration of 100 micrograms/ml is 51.4 percent on the 8 th day of co-culture, which is respectively 3.78 times of positive control medicament alpha-interferon (10000 units/ml) and 4.94 times of lamivudine (100 micrograms/ml); the compound of formula (1) showed 94.6% inhibition of HBV-DNA at this concentration (100. mu.g/ml), which was higher than the lamivudine inhibitory strength at the same concentration (88.4% inhibition of HBV-DNA by 3-TC at 100. mu.g/ml), and was 3.1 times higher than that of high concentration interferon-alpha (10000 units/ml). 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 HBeAg, inhibiting HBV-DNA replication, and treating viral hepatitis B. And can be expected to be further developed into innovative non-nucleoside innovative drugs for eliminating hepatitis B HBeAg antigen and inhibiting HBV-DNA replication.

In conclusion, 3 beta-acetoxyl-18 beta-glycyrrhetinic acid methyl ester derived from glycyrrhetinic acid has structural uniqueness and novelty of anti-HBV effect, and finds unusual activity of inhibiting hepatitis B HBeAg and activity of inhibiting HBV-DNA replication in anti-hepatitis B virus 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 (1) have been found to be a potent inhibitor of HBeAg and HBV-DNA, and are of definite originality, and the invention has been completed based on this finding.

The invention has the advantages that: the compound 3 beta-acetoxyl-18 beta-glycyrrhetinic acid methyl ester shown in the formula (1) is found to have the functions of eliminating HBeAg, inhibiting HBV-DNA replication and preventing and treating hepatitis B virus, and provides a new material basis for developing innovative drugs for treating hepatitis B virus and developing non-nucleoside innovative drugs for inhibiting HBeAg secretion and HBV-DNA replication for the first time. Has potential huge social benefit and economic benefit. The invention has the following further characteristics: the synthetic starting material glycyrrhetinic acid and/or glycyrrhizic acid is convenient to obtain, the preparation method of the compound shown in the formula (1) is simple and feasible, the raw materials are convenient and easy to obtain, the cost is low, the pollution is small, and the large-scale production under the conditions of energy conservation and emission reduction is facilitated. The industrialization prospect is very clear.

Detailed description of the preferred embodiments

The invention obtains the pentacyclic triterpenic acid compound shown in the formula (1) which is derived from glycyrrhetinic acid and can effectively inhibit HBeAg secretion and HBV-DNA replication activity by chemical synthesis and purification through a plurality of chromatographic means, and the chemical structure of the compound is verified by comprehensive analysis derivation of mass spectrum, nuclear magnetic resonance spectrum and the like. The inventor finds that the compound shown in the formula (1) has a remarkable inhibiting effect on the replication of HBeAg and HBV-DNA secreted by HepG2.2.15 cells, and the compound is characterized by safe administration, strong effect of eliminating HBeAg and high effect of inhibiting the replication of HBV-DNA. Therefore, according to the research of the inventor, the 3 beta-acetoxyl-18 beta-glycyrrhetinic 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 and treating hepatitis B virus infectious diseases.

In order to better understand the essence of the present invention, the following uses the preparation of the compound of formula (1) and the results of the test of its inhibitory effect on the replication of HBeAg and HBV-DNA secreted from HepG2.2.15 cells, respectively, to illustrate its new use in the pharmaceutical field. The examples present partial 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 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 3 beta-acetoxyl-18 beta-methyl glycyrrhetinate compound of formula (1)

1.1 instruments and reagents

Measuring the ultraviolet spectrum by using a Shimadzu UV-240 ultraviolet spectrophotometer; hydrogen spectrum of nuclear magnetic resonance¹H-NMR was measured by an INOVA type superconducting nuclear magnetic resonance spectrometer (VARIAN INOVA-400MHz) (tetramethylsilyl ether TMS as an 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 Compound methyl Glycyrrhetinic acid (2)

Wherein, glycyrrhetic acid refers to glycyrrhetinic acid, CH₂N₂,Et₂O is diazomethane ether solution.

4.70 g of glycyrrhetinic acid (obtained from Seisan Chen Biotech Co., Ltd., purity 99% by HPLC) was dissolved in 40 ml of a mixed solution of diethyl ether and tetrahydrofuran (1:1, V/V) in a dry reaction flask. In a fume hood, a newly prepared ethyl ether solution of diazomethane is added dropwise by a separating funnel in a 15 mmol manner under magnetic stirring to generate a large amount of white flocculent solid, and the reaction is continued for 50 minutes after bubbles overflow and disappear. Dropping 1M hydrochloric acid solution until no gas is discharged (eliminating excess diazomethane), filtering with a Buchner funnel, separating white solid from water layer, washing the solid with 30 ml 0.1N sodium hydroxide solution, washing with double distilled water until the filtrate is neutral, and drying under reduced pressure to obtain 4.58 g white solid. Performing silica gel column chromatography, eluting with chloroform-acetone (100: 1-1: 1), detecting by TLC thin layer, and collecting pure product to obtain intermediate compound methyl glycyrrhetinate (4.34 g) shown in formula (2), melting point 240-241 deg.C (dichloromethane), R_f(petroleum ether: chloroform: methanol: 4: 0.6: 0.52; the yield thereof was found to be 89.7%. Hydrogen spectrum of nuclear magnetic resonance¹H NMR (400MHz, deuterated chloroform): 0.80 (singlet, 6H), 1.00 (singlet, 3H), 1.12 (singlet, 3H), 1.13 (singlet, 3H), 1.14 (singlet, 3H), 1.36 (singlet, 3H), 1.50 to 2.28 (multiplet ), 2.34 (singlet, 1H, H-9), 2.79(1H, broad doublet, J ═ 13.6Hz, H-18), 3.23(1H, doublet, J ═ 10.8,5.6Hz, H-3), 3.66 (singlet, 3H, CO ═ 13.6Hz, H-3)₂ Me) 5.66 (singlet, 1H, H-12); nuclear magnetic resonance carbon spectrum¹³C NMR (100MHz, deuterated chloroform): 16.6(q),17.1(q),19.4(t),23.2(q),23.1(q),25.5(q),26.2(t),26.9(q),27.1(t),27.2(t),28.4(q),32.2(t),35.6(t),32.1(s),36.6(t),38.7(t),37.8(s),38.8(s),41.8(t),44.0(s),44.6(s),47.4(s),49.2(d),52.6(q),55.5(d),61.1(d),79.8(d),128.3(d),169.7(s),177.6(s), 201.1(s). Electrospray mass spectrometry ESI-MS: m/z 485[ M + H ]]⁺. According to the spectrogram data, the prepared compound is the compound of the formula (2), namely the methyl glycyrrhetinate.

1.3 preparation of Compounds of formula (1)

485 mg of methyl glycyrrhetinate, which is an intermediate compound represented by the formula (2) prepared under 1.2, was dissolved in 15 ml of anhydrous pyridine, and 20 ml of acetic anhydride was added thereto, followed by reaction at 90 ℃ overnight. The remaining solution was distilled off under reduced pressure in a water bath, the solid in the flask was dissolved in 50 ml of ethyl acetate, the separatory funnel was washed with 20 ml of water each time to neutrality, and the organic layer solution was distilled under reduced pressure to give 515 mg of a crude product. Performing silica gel column chromatography, repeatedly eluting with chloroform-acetone (100: 1-1: 1), detecting by TLC thin layer, collecting pure product (with purity of above 98.5%) to obtain pure 3 β -acetoxy-18 β -glycyrrhetinic acid methyl ester represented by formula (1) 488 mg, white solid, yield 92.6%; r_f(petroleum ether: chloroform: methanol: 4: 0.6: 0.74; hydrogen spectrum of nuclear magnetic resonance¹H NMR (400MHz, deuterated chloroform): 0.98 (singlet, 6H), 1.04 (singlet, 3H), 1.05 (singlet, 3H), 1.06 (singlet, 3H), 1.32 (singlet, 3H), 1.36 (singlet, 3H), 1.39 (singlet, 3H), 1.40 (singlet, 1H, H-5), 2.21 (singlet, 3H, OAc), 3.65 (singlet, 3H, CO)₂ Me) 5.71 (singlet, 1H, H-12); nuclear magnetic resonance carbon spectrum¹³C NMR (100MHz, deuterated chloroform): 15.7(q),17.2(q),16.9(q),18.9(t),22.6(q),23.5(q),23.7(q),24.8(t),26.5(q),26.8(t),27.0(t),27.2(q),28.1(q),30.3(t),32.3(s),32.9(t),33.7(s),36.2(t),36.4(t),36.8(s),39.2(s),39.7(s),42.3(t),43.6(s),48.6(d),50.9(d),55.6(q),66.5(s),82.1(d),122.4(d),168.9(s),174.7(s), 201.9(s). Electrospray mass spectrometry ESI-MS: m/z 528[ M + H ]]⁺。

Example 2:inhibition of HBV-DNA replication secreted by HepG2.2.15 cells 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 and 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 Effect of the Compound of formula (1) on the replication of hepatitis B Virus deoxyribonucleic acid (HBV-DNA):

taking HepG2.2.15 cells in logarithmic growth phase, diluting the cells to 1 × 10 with culture medium⁵One/ml, seeded in 96-well cell culture plates, 100. mu.l/well, at 37 ℃ in 5% CO₂Adding pentacyclic triterpenic acid compound shown in formula (1) diluted by a culture medium into an incubator with 100% relative humidity for 24 hours, wherein the concentration is 100 micrograms/ml, 20 micrograms/ml and 4 micrograms/ml respectively, each hole is 200 microliters, each concentration is provided with three multiple holes, and placing at 37 ℃ 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. On day 8, the HBV-DNA concentration in the medium was measured using the HBV-DNA quantitative PCR kit. Lamivudine (3-TC) was used as a positive control 1, and the test concentrations were 100. mu.g/ml, 20. mu.g/ml and 4. mu.g/ml; alpha-interferon was used as a positive control 2 and tested at 10000 units/ml, 5000 units/ml and 1000 units/ml.

2.3 results of the experiment

As shown in Table 1, the compound of formula (1) having a pentacyclic triterpenic acid skeleton has a potent effect of inhibiting the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA).

TABLE 1 inhibition ratio (%)% of HBV-DNA replication of HepG2.2.15 cells by test sample

2.4 the results show

The results of this example illustrate that: the pentacyclic triterpenic acid compound 3 beta-acetoxyl group-18 beta-glycyrrhetinic acid methyl ester shown in the formula (1) has extremely strong inhibiting effect on the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA), and is exciting: the inhibition activity to HBV-DNA replication of 100 microgram/ml exceeds 94%, while positive control alpha-interferon has only 30.5% inhibition activity to HBV-DNA at the highest test concentration (10000 units/ml), so the pentacyclic triterpenic acid compound belongs to a natural product of remarkably effective non-nucleoside inhibition hepatitis B virus, the inhibition activity to HBV-DNA replication of high concentration is higher than that of positive control drug lamivudine, which is 3.1 times of alpha-interferon, and reaches the standard of anti-HBV lead compound. Is worthy of further attention and intensive research, and can be expected to be further developed into a non-nucleoside innovative medicament for inhibiting the replication of the hepatitis B virus.

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

3.1 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.

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 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.

3.3 Experimental results:

the results of the experiment are shown in table 2. The pentacyclic triterpenic acid compound 3 beta-acetoxyl group-18 beta-glycyrrhetinic acid methyl ester shown in the formula (1) shows a definite effect of inhibiting hepatitis B e antigen (HBeAg). On the eighth day of the experiment, the inhibitory activity of the high-dose compound of formula (1) on HBeAg secreted by HepG2.2.15 cells reaches 51.4%, which is higher than that of positive control 1 (lamivudine) and positive control 2 (alpha-interferon) at high concentration.

TABLE 2 inhibition ratio (%)

3.4 the results show that:

the results of this example show that: the pentacyclic triterpenic acid compound 3-acetoxyl group-18-glycyrrhetinic acid methyl ester shown in the formula (1) has obvious inhibition effect on hepatitis B e antigen (HBeAg) secreted by HepG2.2.15 cells. The strength of the compound for inhibiting HBeAg secretion at the concentration of 100 micrograms/ml is 4.94 times that of a positive control medicament 1(100 micrograms/ml 3-TC) and 3.78 times that of a positive control medicament 2(10000 units/ml alpha-interferon); therefore, the pentacyclic triterpenic acid compound can obviously 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.

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

Claims

1. Use of methyl acetyl glycyrrhetinate having a structure represented by formula (1) for preparing a medicament for treating viral hepatitis B;

the name of the compound of formula (1): 3 beta-acetoxyl group-11-carbonyl oleanane-12-alkene-30-carboxylic acid methyl ester.

2. Use of methyl acetylglycyrrhetinate having a structure represented by formula (1) in claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus deoxyribonucleic acid HBV-DNA, the name of the compound of formula (1) being: 3 beta-acetoxyl group-11-carbonyl oleanane-12-alkene-30-carboxylic acid methyl ester.

3. Use of methyl acetyl glycyrrhetinate having a structure represented by formula (1) in claim 1 for the preparation of a medicament for the inhibition of hepatitis b virus core antigen HBeAg, the name of the compound of formula (1) being: 3 beta-acetoxyl group-11-carbonyl oleanane-12-alkene-30-carboxylic acid methyl ester.