CN111759845A - Application of beta-hydroxyketene methyl oleanolic acid in preparing medicament for treating viral hepatitis B - Google Patents
Application of beta-hydroxyketene methyl oleanolic acid in preparing medicament for treating viral hepatitis B Download PDFInfo
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- CN111759845A CN111759845A CN202010726159.7A CN202010726159A CN111759845A CN 111759845 A CN111759845 A CN 111759845A CN 202010726159 A CN202010726159 A CN 202010726159A CN 111759845 A CN111759845 A CN 111759845A
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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Abstract
The invention relates to application of beta-hydroxyketene methyl oleanolic acid 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-hydroxy-11-carbonyl oleanane-12-alkene-28-carboxylic acid methyl ester, in preparing a medicament for preventing and treating hepatitis B virus infection diseases. The compound of the formula (1) has relatively remarkable activity of inhibiting HBeAg secreted by HepG2.2.15 cells, and the strength of inhibiting HBeAg secretion at the concentration of 100 micrograms/ml is 2.68 times that of alpha-interferon (10000 units/ml) of a positive control medicament and 3.18 times that of lamivudine (100 micrograms/ml); at this concentration it showed an inhibitory rate of more than 88% against HBV-DNA replication, whereas the highest tested concentration (10000 units/ml) of interferon-alpha showed an inhibitory activity of only 31.5% against HBV-DNA replication. The results show that the pentacyclic triterpenic acid compound can be expected to be used for preparing non-nucleoside medicaments for treating hepatitis B virus infection diseases.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of beta-hydroxyketene methyl oleanolic acid in preparing a viral hepatitis B medicine. The compound is a pentacyclic triterpenic acid derivative with a 3 beta-hydroxy-11-carbonyl oleanane-12-alkene-28-carboxylic acid methyl ester structure, 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 medicines 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 (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 (ZL200910099405.4), 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 HBeAg secretion, HBV-DNA replication.
In order to explore the field, the natural pentacyclic triterpenic acid is selected as an initiator, the structure of the natural pentacyclic triterpenic acid is reasonably modified, a methyl carboxylate derivative of oleanolic acid shown in a formula (1) is designed and prepared by a computer aided design means, 28-site carboxyl of the oleanolic acid is methylated into carboxymethyl, so that the lipophilicity of the whole molecule is enhanced, 11-site B of the oleanolic acid is oxidized into carbonyl, so that an enone structure is formed on the B ring, the conjugation degree and the electron cloud density are changed, and the lead compound with the activities of eliminating HBeAg and inhibiting replication of HBV-DNA based on the natural medicine is discovered. One of our objectives is: the oleanolic acid derivative lead compound capable of inhibiting HBeAg secretion and HBV-DNA replication is hoped to be found, so that the oleanolic acid derivative lead compound can be further developed into an innovative medicine capable of clearing HBeAg, inhibiting HBV-DNA replication and treating chronic hepatitis B.
Disclosure of Invention
The invention aims to provide a new application of beta-hydroxyketene oleanolic acid methyl ester shown in a formula (1) in preparing medicines for eliminating HBeAg, inhibiting HBV-DNA replication and treating hepatitis B virus infection diseases;
the name of the compound of formula (1): the IUPAC name of the 3 beta-hydroxy-11-carbonyl oleanane-12-alkene-28-carboxylic acid methyl ester is as follows: (4aS,6aS,6bR,8aR,10S,12aS,12bR,14bS) -methyl-10-hydroxy-2,2,6a,6b,9,9,12 a-heptathienyl-13-oxo-1, 2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14 b-icosahydroprocene-4 a-carboxylate.
The invention also provides a method for preparing the compound shown in the formula (1), which is characterized by comprising the following steps: the methyl ester is formed by the 28-carboxyl group of the commercial oleanolic acid, and then Jone's oxidation is carried out to form the methyl 3 beta-hydroxy-11-carbonyl oleanan-12-ene-28-carboxylate shown in the formula (1).
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 oleanolic acid, the 3 beta-hydroxy-11-carbonyl oleanane-12-ene-28-methyl carboxylate of the compound shown in the formula (1) designed by the inventor has the characteristics of structural and physicochemical property differentiation, 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 other characteristics of the compound are greatly different from that of the oleanolic acid; and the molecular weight of the compound of formula (1) is increased by 28 mass units compared with oleanolic 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.
The American college synthesized the compound of formula (1) and studied its inhibitory effect on mouse macrophage interferon-gamma induced nitric oxide (Honda T, et al, Novel synthetic oligosaccharide and ura trisections with variant olefin functions in the ring A as inhibitors of nitrile oxidase in cancer therapeutics, Journal of Medicinal Chemistry,2000,31(30): 1866-membered 1877). The inhibitory activity of the compound of formula (1) against the replication of hepatitis B core antigen HBeAg and HBV-DNA has not been reported so far.
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 respectively 2.68 times that of a positive control medicament alpha-interferon (10000 units/ml) and 3.18 times that of lamivudine (100 micrograms/ml) on the 8 th day of co-culture; the inhibitory activity of the compound of formula (1) on HBV-DNA replication at this concentration (100. mu.g/ml) exceeded 88%, whereas the inhibitory activity of interferon-alpha at the highest tested concentration (10000 units/ml) was only 31.5%. 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, the pentacyclic triterpenic acid compound derived from oleanolic acid has novelty in anti-HBV effect, and the activity of inhibiting hepatitis B HBeAg and the activity of inhibiting HBV-DNA replication are found to be unusual in an 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 of strong inhibition of HBeAg and HBV-DNA, has definite originality and is completed according to the invention.
The invention has the advantages that: the compound 3 beta-hydroxy-11-carbonyl oleanane-12-alkene-28-methyl carboxylate shown in the formula (1) is found to have the potential 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. Has potential huge social benefit and economic benefit. The invention has the following further characteristics: 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, which can effectively inhibit HBeAg secretion and HBV-DNA replication activity, is obtained by chemical synthesis and purification through various chromatographic means, and the chemical structure of the pentacyclic triterpenic acid compound is verified through comprehensive analysis deduction 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 beta-hydroxyketene oleanolic 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 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 the 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 starting material methyl oleanolic acid:
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, Rf(Petroleum ether: ethyl acetate: 5: 1) 0.31, melting point 189-191 deg.C (dichloro-methane)Methane). 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-2 Me) 5.28 (broad singlet, 1H, H-12); electrospray mass spectrometry ESI-MS: m/z 471[ M + H]+According to the above-mentioned spectral data, said intermediate compound is identified to be 3 β -hydroxy-oleanane-12-ene-28-carboxylic acid methyl ester, i.e. methyl oleanolic acid.
1.3 preparation of Compound 3 β -hydroxy-11-carbonyl oleanane-12-ene-28-carboxylic acid methyl ester of formula (1)
Wherein Me is methyl; oleanolic acid methyl ester refers to methyl Oleanolic acid; CrO3Refers to chromium trioxide; HOAc refers to acetic acid; ac of2O is acetic anhydride.
1.18 g of methyl oleanolic acid, which is an intermediate compound represented by the formula (2) prepared in the above item 1.2, was dissolved in 60 ml of an acetic acid solution containing 5% acetic anhydride, and 0.30 g of analytically pure chromium trioxide powder was added with stirring. After the reaction mixture was stirred at room temperature for 4 hours, the reaction was terminated by TLC detection. 40 ml of water and 40 ml of dichloromethane are added and the separated aqueous layer is extracted twice with 40 ml of dichloromethane each time. And combining organic phases, washing the organic phases to be slightly alkaline by using saturated sodium bicarbonate solution, washing the organic phases to be neutral by using distilled water, drying the organic phases over night by using anhydrous magnesium sulfate, filtering the organic phases, evaporating the filtrate under reduced pressure to remove the solvent to obtain a green jelly, performing gradient elution by using silica gel column chromatography (100: 1-3: 1), detecting by using a TLC (thin layer chromatography), combining and desolventizing the green jelly, and collecting a pure product to obtain 0.73 g of a white non-shaped solid. Rf(petroleum ether: ethyl acetate: 5: 1): 0.10; melting point 188-189 deg.C (dichloromethane). Hydrogen spectrum of nuclear magnetic resonance1H NMR (400MHz, deuterated chloroform): 0.79 (singlet, 6H), 0.91 (singlet, 3H), 0.92 (singlet, 3H), 0.93 (singlet, 3H), 0.99 (singlet, 3H), 1.09 (singlet, 3H), 1.35 (singlet, 3H), 2.31 (singlet, 1H, H-9), 2.82 (1H)Doublet, J ═ 13.2,13.2,3.2Hz, H-1), 2.98 (doublet, 1H, J ═ 10.4,6.4Hz, H-18), 3.21 (multiplet, 1H, H-3), 3.63 (singlet, 3H, CO2 Me) 5.63 (broad singlet, 1H, H-12); nuclear magnetic resonance carbon spectrum13C NMR (100MHz, deuterated chloroform): 15.5(C-24), 16.1(C-26), 17.4(C-6), 18.9(C-25), 22.9(C-30), 23.4(C-16), 23.5(C-27), 27.3(C-16), 27.7(C-23), 28.1(C-2), 29.6(C-20), 30.6(C-29), 31.6(C-7), 32.8(C-8), 32.9(C-22), 33.7(C-21), 37.3(C-10), 39.1(C-1), 41.6(C-4), 43.5(C-14), 44.2(C-18), 45.0(C-19), 46.2(C-17), 51.8 (OCH)3) 55.0(C-5), 61.8(C-9), 78.7(C-3), 127.9(C-12), 168.6(C-13), 177.4(C-28), 200.3 (C-11). Electrospray mass spectrometry ESI-MS: m/z 485[ 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 by using a 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) as a positive control 1, with a test concentration of 100 μmG/ml, 20. mu.g/ml and 4. mu.g/ml, α -interferon as positive control 2, 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-hydroxy-11-carbonyl oleanane-12-ene-28-carboxylic acid methyl ester shown in the formula (1) has extremely strong inhibition effect on the replication of hepatitis B virus deoxyribonucleic acid (HBV-DNA), and is exciting: the replication inhibition activity of the pentacyclic triterpenic acid compound to HBV-DNA is more than 88% at a dose of 100 micrograms/milliliter, while the inhibition activity of positive control alpha-interferon to HBV-DNA is only 31.5% at the highest test concentration (10000 units/milliliter), so the pentacyclic triterpenic acid compound belongs to a natural product for remarkably and effectively inhibiting the hepatitis B virus by non-nucleosides, the replication inhibition activity to the HBV-DNA at high concentration is close to that of a positive control medicament lamivudine, is 2.82 times of that of the alpha-interferon, and reaches the anti-HBV lead compound standard. 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 by using a 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% CO2The method comprises the steps of culturing in an incubator with 100% relative humidity, changing a culture medium containing samples with the same concentration every 4 days, uniformly mixing the changed culture medium with the same concentration of the samples in equal volume to obtain samples to be tested, measuring the concentration of hepatitis B e antigen (HBeAg) in the culture medium by using an ELISA kit on the eighth day by using P/N, wherein the concentration of the compound of the formula (1) prepared according to the embodiment 1 is 100 micrograms/ml, 20 micrograms/ml and 4 micrograms/ml, the test concentration of lamivudine (3-TC) is 100 micrograms/ml, 20 micrograms/ml and 4 micrograms/ml, and the test concentration of α -interferon is 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 beta-hydroxyketene oleanolic acid methyl ester shown in the formula (1) shows a remarkable 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 48.7%, 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 beta-hydroxy-11-carbonyl oleanane-12-alkene-28-methyl carboxylate shown in the formula (1) has a remarkable inhibiting 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 3.18 times that of a positive control medicament 1(100 micrograms/ml 3-TC) and 2.68 times that of a positive control medicament 2(10000 units/ml alpha-interferon); therefore, the pentacyclic triterpenic acid compound can 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 (3)
1. The application of beta-hydroxyketene oleanolic acid methyl ester with the structure shown in the formula (1) in preparing a medicament for treating viral hepatitis B;
the name of the compound of formula (1): 3 beta-hydroxy-11-carbonyl oleanane-12-ene-28-carboxylic acid methyl ester.
2. Use of beta-hydroxyketene oleanolic acid methyl ester having a 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: 3 beta-hydroxy-11-carbonyl oleanane-12-ene-28-carboxylic acid methyl ester.
3. Use of beta-hydroxyketene oleanolic acid methyl ester having a structure represented by formula (1) in claim 1 for preparing a medicament for inhibiting hepatitis b virus core antigen HBeAg, the name of the compound of formula (1) being: 3 beta-hydroxy-11-carbonyl oleanane-12-ene-28-carboxylic acid methyl ester.
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| CN202010726159.7A Pending CN111759845A (en) | 2020-07-27 | 2020-07-27 | Application of beta-hydroxyketene methyl oleanolic acid in preparing medicament for treating viral hepatitis B |
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| Country | Link |
|---|---|
| CN (1) | CN111759845A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105998032A (en) * | 2016-06-02 | 2016-10-12 | 贵州医科大学 | Application of oleanolic acid derivative and pharmaceutical compositions thereof in medicine |
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2020
- 2020-07-27 CN CN202010726159.7A patent/CN111759845A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105998032A (en) * | 2016-06-02 | 2016-10-12 | 贵州医科大学 | Application of oleanolic acid derivative and pharmaceutical compositions thereof in medicine |
Non-Patent Citations (2)
| Title |
|---|
| YAN WENQIANG等: "Series of Oleanolic Acid Derivatives as Anti-Hepatitis B Virus Agents: Design, Synthesis, and in Vitro and in Vivo Biological Evaluation", 《MOLECULES》, vol. 21, pages 48 - 11 * |
| 刘厚佳等: "木瓜中齐墩果酸抗乙型肝炎病毒研究", 《解放军药学学报》, vol. 18, no. 5, pages 272 - 274 * |
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Application publication date: 20201013 |