CN101829103B - Application of flavonoid quercetin dimmer as medicament for treating viral hepatitis B - Google Patents

Abstract

The present invention relates to the use of quercetin dimer flavone as a drug for treating viral hepatitis B, in particular to a quercetin dimer flavone or a pharmaceutically acceptable salt thereof for preparing a drug for removing HBsAg and HBeAg and inhibiting HBV DNA replication The use of the drug, which has significant inhibition of HBsAg and inhibition of HBeAg activity, at a concentration of 100 micrograms per milliliter, the intensity of clearing HBsAg and HBeAg is 65.7% and 44.8%, respectively, 4.1 times and 2.7 times more than the positive control drug α-interferon ; Simultaneously, it shows 44.8% inhibition rate to HBV DNA at this concentration, and is 117% of the inhibition rate of alpha-interferon at the highest test concentration. The above shows that the quercetin dimer flavone or its pharmaceutically acceptable salt can be expected to be used in the preparation of non-nucleoside drugs for eliminating HBsAg and HBeAg, inhibiting HBV DNA replication, and treating hepatitis B virus infection diseases.

Description

Use of quercetin dimer flavone as a drug for treating viral hepatitis B

technical field

The present invention relates to the technical field of medicine, specifically, the present invention relates to a kind of quercetin dimer flavone or its pharmaceutically acceptable salt, which is used to prepare and reduce hepatitis B virus surface antigen HBsAg and hepatitis B e antigen HBeAg, inhibit HBV DNA replication, treat Use of medicines for hepatitis B virus infection. This dimer flavonoid has quite significant activity of inhibiting HBsAg and HBeAg, and its intensity of removing HBsAg and HBeAg is respectively 65.7% and 44.8% under the concentration of 100 micrograms/milliliter, surpasses positive control drug (10000 units/milliliter α- Interferon) 4.1 times and 2.7 times; Simultaneously, it shows 44.8% inhibitory rate to HBV DNA at this concentration, and is 117% of the inhibitory rate of alpha-interferon when the highest test concentration. The above pharmacodynamic results show that the quercetin dimer flavone or its pharmaceutically acceptable salt can be expected to be used in the preparation of non-nucleoside drugs for eliminating HBsAg and HBeAg, inhibiting HBV DNA replication, and treating hepatitis B virus infection.

Background technique

Hepatitis B (HBV) is an infectious disease caused by the hepatitis B virus, HBV. HBV is a member of the hepadnaviridae family of hepadnaviridae, and its shape is a spherical particle with a diameter of 42 nanometers. HBV is a peculiar virus that is less contagious in other animals and can only replicate in humans or the primate chimpanzee. The virus is transmitted through the blood, saliva, semen, and vaginal secretions of hepatitis B virus carriers and hepatitis B patients, and has a chronic carrier state. Hepatitis B is widely prevalent in my country, because it can be divided into vertical transmission, horizontal transmission, family transmission, iatrogenic transmission and sexual transmission, etc. It has a high infection rate among the population, and the infection rate in some areas reaches more than 35%. According to relevant data, the number of patients who tested positive for hepatitis has reached 189 million, and the number of people who should seek medical treatment but have not (carriers) is nearly 400 million. It is one of the most serious infectious diseases that endanger people's health. The clinical manifestations of hepatitis B are diverse, and it is easy to develop into chronic hepatitis B (CHB) and liver cirrhosis, and a small number of patients can be transformed into primary liver cancer.

Hepatitis B surface antigen (HBsAg) is the coat protein of hepatitis B virus, and HBsAg positive is the gold standard for judging HBV infection. Those who are positive for HBsAg but have no symptoms of hepatitis become carriers of HBV virus. The greater the titer of HBsAg, the greater the probability of combining hepatitis B core antigen HBeAg, HBV DNA positive and elevated DNA polymerase activity, and thus the stronger the infectivity. Similarly, inhibiting the secretion and replication of HBsAg is also an important target and detection target in the development of anti-HBV drugs. Wu Shuyun from Beijing Ditan Hospital reported that there is a certain correlation between the clearance of hepatitis B HBsAg and closed-circle covalent DNA (cccDNA), and the clearance of HBsAg is a sign of a significant decrease in the level of cccDNA. In 2002, researchers who published research results in the "New England Journal of Medicine" believed that for CHB patients, if HBsAg was cleared before liver cirrhosis, the incidence of liver cirrhosis and hepatocellular carcinoma would be reduced by 60 times. The guidelines of the American Association for the Study of Liver Diseases (AASLD), Asia Pacific Association for the Study of the Liver (APASL), and European Association for the Study of the Liver (EASL) all use HBsAg serum clearance as one of the treatment endpoint criteria.

According to the 2008 European Society for the Study of the Liver report: Pegylated interferon α-2a treatment of CHB patients stopped after 48 weeks, followed up for 1, 2, 3, and 4 years, and the HBsAg clearance rates were 3% and 6% respectively. , 8% and 11%, while those who used lamivudine alone had only 0%, 0%, 0% and 3% of the HBsAg clearance rate in 1, 2, 3, and 4 years after drug withdrawal. Therefore, this interferon is considered to be the best treatment option for HBeAg-negative CHB patients. It can be seen that the discovery of drugs that can efficiently remove HBsAg has significant social and economic benefits.

Hepatitis B e antigen HBeAg is the structural protein of the core of hepatitis B virus HBV, which is produced in large quantities during the reproduction of hepatitis B virus. Hepatitis B virus HBV has the smallest genome (only 3.2kb) among all known DNA viruses, and its genes mainly encode five proteins (S, C, E, P, X). Protein C is the core protein of the virus, and protein E is a part of protein C, which becomes hepatitis B e antigen (HBeAg), which is a protein that has been encoded but not assembled into the virus particle, and will be secreted into the blood of the patient when the virus replicates . Clinically, serum HBeAg is usually used as an important marker for the evaluation of HBV replication, infectivity, disease severity and response to treatment. This antigen is closely related to HBV DNA and is a very practical serum marker for clinically expressed virus replication. Serum marker HBeAg-positive patients indicate that there is HBV replication in their bodies, so they have higher infectivity. The higher the expression of HBeAg in a patient, the stronger the infectivity of the patient. Similarly, inhibiting the secretion and replication of HBeAg is also an important target and detection target in the development of anti-HBV drugs. The removal of HBeAg shows that there is continuous HBV suppression in the body, ALT is normal, tissue inflammation and necrosis are reduced, and the probability of liver cirrhosis is reduced. Therefore, serum HBeAg is considered to reflect a more stable therapeutic effect, and HBeAg serum clearance marks the beginning of the patient's immune system. In 2002, the researchers who published their research results in the "New England Journal of Medicine" believed that for CHB patients, if HBeAg is cleared before liver cirrhosis, the incidence of liver cirrhosis and hepatocellular carcinoma will be reduced by 10 times. The guidelines of the American Association for the Study of Liver Diseases (AASLD), Asia Pacific Association for the Study of the Liver (APASL), and European Association for the Study of the Liver (EASL) all use HBeAg serum clearance as one of the criteria for judging treatment endpoints. Therefore, drugs that can inhibit or reduce the expression or activity of HBeAg are also effective drugs for treating hepatitis B virus infection.

At present, the drugs for hepatitis B patients are mainly divided into several categories such as liver protection and enzyme reduction, anti-virus, anti-hepatic fibrosis and immune regulation. Anti-virus is the fundamental method, while protecting the liver and reducing enzymes is only an adjuvant treatment, which mostly treats the symptoms but rarely cures the root cause. What has made progress in recent years is the research on antiviral drug treatment. However, the current clinical treatment options for viral hepatitis B can only achieve inhibition of HBV replication and secondary infection, and the most important drugs are still nucleoside drugs such as lamivudine (3-TC), entecavir, adefovir ( ADV), telbivudine, etc., as well as emtricitabine, tenofovir, clevuding, etc. that are in clinical trials. In my country, lamivudine has become a medical insurance drug and is used in a large number of HBV patients. Some advantages of nucleoside drugs are: high bioavailability and relatively safe oral administration. However, although they can effectively control the disease, they are expensive on the one hand; drug resistance can appear after long-term use, and HBV DNA, ALT and liver histology rebound in varying degrees after drug withdrawal; The relatively obvious and well-known adverse effects of nucleoside drugs, such as kidney damage, infant teratogenicity, etc. The most troublesome thing is: the emergence of viral drug resistance greatly reduces the cure rate, because nucleoside drugs are reversible to virus replication, so if most patients want to achieve the maximum curative effect, the course of treatment must be more than one year, so that drug resistance Sexuality then appears, and the desired effect cannot be achieved. Nucleoside drugs also have the disadvantages that it is difficult to remove cccDNA, and HBsAg is difficult to turn negative after one year of treatment.

Bioengineered antiviral drugs derived from human leukocytes, such as interferon (α, β-interferon) and recombinant interferon, have recently become hot research and treatment drugs for CHB, which have dual functions of antiviral and immune regulation. It can not only inhibit virus replication through antiviral effect, so as to reduce the inflammatory response of liver cells, reduce liver cell damage, delay the progression of the disease, thereby improving the patient's clinical symptoms and liver physiological functions; it can also enhance immune function, by strengthening natural killer cells and The role of helper T cells, in particular, can promote killer T cells to kill virus-infected cells, thus indirectly playing an antiviral role. The HBsAg seroconversion obtained by interferon treatment in CHB patients is more durable than that of lamivudine. A study in the Journal of Gastrointestinal Tract in 2003 showed that the 3-year recurrence rate of HBsAg in the interferon treatment group was significantly lower than that of lamivudine group; and peginterferon can be used once a week, which is more convenient. Therefore, interferon has gradually become one of the first-selected drugs for clinical treatment of chronic hepatitis B virus, but its side effects and adverse reactions are reported more, the total effective rate is not high, the price is expensive, and the patient's economic burden is large, thus causing it to be difficult to be widely used clinically. It is not suitable for use in patients with decompensated cirrhosis.

In recent years, with the study of liver disease, the development of standardized HBV DNA analysis has greatly promoted the understanding of the condition of hepatitis B patients. Quantitative analysis of HBV DNA can predict the severity and prognosis of hepatitis B, because continuous positive HBV DNA (i.e. persistent viremia) is likely to make the progression and aggravation of hepatitis B; high content of hepatitis B virus (HBV DNA) is likely to promote the formation of liver cirrhosis; The persistent presence of HBV DNA is a high-risk factor for the occurrence of hepatocellular carcinoma (HCC), especially in patients with high viral content, long course of disease, older age or other liver diseases; persistent high concentration of HBV DNA can lead to decompensated The mortality rate of liver cirrhosis and primary hepatic severe disease increased significantly. At the same time, it must be recognized that the level of HBV DNA is closely related to liver histology: it has been reported in the literature that liver fibrosis can be significantly improved and eliminated after antiviral treatment; the recent International Liver Disease Conference reported that strong and low drug resistance antiviral treatment , With the decrease and negative of HBV DNA, it was observed that liver cirrhosis can be reversed to varying degrees, so it is now advocated that liver cirrhosis should also be treated with antiviral therapy.

Therefore, the application of HBV DNA indicators in antiviral treatment also plays a pivotal role: the level of HBV DNA is an important indicator to determine whether chronic hepatitis B requires antiviral treatment; Different treatment standards and requirements for HBeAg negative; in antiviral treatment, according to the treatment response of HBVDNA, judge whether there is an early virological response and then determine the strategy of long-term medication to achieve a sustained virological response and achieve the purpose of sustained viral suppression; The virological response of HBV DNA creates the basis and conditions for HBeAg seroconversion so as to achieve its good intermediate treatment goal; according to the continuous suppression of HBV DNA, strive for continuous negative virus, so as to achieve the ultimate goal of antiviral treatment; according to HBV DNA Sustained complete suppression also showed varying degrees of improvement and disappearance of cccDNA; in antiviral treatment, changes in HBV DNA were used to evaluate and prevent the risk of virus mutation and drug resistance caused by antiviral drugs; In the case of mutation or drug resistance, the change of HBV DNA is the only initial signal and diagnostic basis, and it is also the guidance and basis for treating drug resistance and changing treatment strategies. In summary, the degree of inhibition of HBV DNA is of great significance in the further diagnosis and treatment of hepatitis B, and the observation of curative effect has a great guiding role in evaluating the prognosis of hepatitis B and the risk of drug resistance.

From the above-mentioned factors, it can be seen that another fundamental link for inhibiting the proliferation of hepatitis B virus in vivo is to inhibit the replication of HBV DNA. The reduction of HBV DNA level or below the detection level is another golden key to test antiviral drugs. Therefore, both the Asia-Pacific Society for the Study of the Liver and the European Society for the Study of the Liver regard undetectable HBV DNA as one of the treatment endpoints for patients with HBV. In my country's new drug development guidelines, the inhibitory strength of the tested compound to HBV DNA is also regarded as one of the test items that must be completed. The reason why lamivudine can become the preferred nucleoside drug is because of its potent activity of inhibiting HBV DNA replication. Therefore, compounds that can inhibit both HBV DNA replication and HBsAg will be more promising as innovative drugs for treating hepatitis B patients.

It must be explained that the antiviral drugs currently used are actually only inhibitors of virus replication, and cannot directly kill the virus and destroy the virion, otherwise it will damage the host cell. These antiviral drugs (mostly nucleoside drugs) also have the above-mentioned disadvantages such as high toxicity and side effects, easy mutation of viral genes, and easy rebound after drug withdrawal. Therefore, the development of new antiviral drugs is an urgent task in the field of drug research and development today. It has extremely important social and economic significance for treating a large number of hepatitis B patients and virus carriers in my country and controlling the source of infection. Therefore, the discovery of new non-nucleoside hepatitis B virus inhibitors and such lead compounds that can reduce HBsAg, HBeAg or inhibit HBV DNA replication from the natural medicines that have been used for a long time in the folk has great guiding significance and has a vast development prospect.

Based on this purpose, the inventor has previously completed a number of patents and articles on anti-hepatitis B virus natural products and their structurally modified derivatives, and discovered a variety of anti-hepatitis B virus surface antigen HBsAg or hepatitis B virus e-antigen HBeAg activity, inhibiting HBV DNA replication Compounds, so that it is feasible to screen out innovative drugs that can reduce HBsAg or HBeAg and prevent and treat hepatitis B virus infection from natural products and their synthetic derivatives "The medical application of 2β-hydroxypectic acid in inhibiting hepatitis B virus" (Zhao Yu, Liu Guangming, Yu Rongmin, Li Haibo, etc.; ZL 200610053827.4); ); "2α, 3β-dihydroxy-5, 11(13)-diene eucalyptus-12-acid inhibits the medical application of hepatitis B virus" (Zhao Yu, Zhang Lihe, Sun Handong, Li Haibo, etc.; ZL200610053601.4 ); "The use of erimoferne lactone to inhibit hepatitis B virus and its pharmaceutical composition" (Zhao Yu, Li Haibo, Yang Leixiang, Zhou Changxin, etc.; ZL 03153691.3); "A natural product of erimoferne lactone and its Application" (Zhao Yu, Zhou Changxin, Shi Shuyun, Wang Xiaoyu, etc.; ZL 200610053575.5); "A eucalyptane-type sesquiterpene acid and its application" (Zhao Yu, Liu Guangming, Li Haibo, Wu Xiumei, etc.; ZL 200610053579.3); The use of Chrysanthemum plant extracts to inhibit herpes simplex virus and hepatitis B virus” (Zhao Yu, Zhou Changxin, Yu Rongmin, Bai Hua; CN 1989989A); The medical use of 12-acid to inhibit hepatitis B virus” (Zhao Yu, Li Xiaokun, Huang Kexin, Li Haibo, etc.; CN 1927197A); Wu Xiumei et al.; CN 1935131A); "Enanti-erimofantanoic acid and its medicinal application for inhibiting hepatitis B surface antigen" (Huang Kexin, Li Xiaokun, Wang Xiaoyu, Zhao Yu, etc.; CN 101239054); "1-oxo-substituted Benzoylquinic acid compound and its use for inhibiting hepatitis B virus” (Li Xiaokun, Hu Lihong, Wu Xiumei, Zhao Yu, etc.; CN101293836); the inventor’s published articles on inhibiting the activities of HBsAg, HBeAg and HBV DNA can be found in: “In Vitro Antiviral Activity of Three Enantiomeric Sesquiterpene Lactones from Senecio Species Against Hepatitis B Virus", Haibo Li(李海波), Changxin Zhou(周长新), Xiumei Wu(吴秀美), Yu Zhao*(赵玉) et al., Antiviral Chemistry&Chemotherapy, 2005, 16, 277-282; Wu (Wu Xiumei), Yu Zhao* (Zhao Yu), etc., Planta Medica, 2005, 71(12), 1128-1133; "Application of high-speed counter-current chromatography for the isolation of antiviraleremophilenolides from Ligularia atroviolacea", Shi, Shu-Yun (Shi Shuyun), Hai-Bo (Li Haibo), Zhao, Yu (Zhao Yu), etc., Biomedical Chromatography, 2008, 22(9), 985-991; "Purification and identification of antiviral components from Laggera pterodonta by high-speed counter-current chromatography", Shuyun Shi (Shi Shuyun), Yu Zhao (Zhao Yu), etc., Journal of Chromatography B, 2007, 859, 119-124]. Undoubtedly, it is very necessary and urgent to continue to search for lead compounds that can clear HBsAg or HBeAg and inhibit HBV DNA replication from natural products and their structurally modified derivatives. one.

Quercetin is a flavonol, and Hirose et al found that quercetin can form dimers [Hirose et al, Chemistry Letters, 1999, 8, 775-776]. Since it has almost twice the molecular weight of quercetin and contains more reactive groups such as hydroxyl groups, it may have different antiviral abilities than quercetin. Zuo Guoying et al. (Zuo Guoying, Liu Shuling, Xu Guili, World Chinese Journal of Digestion, 2006, Vol. 14, No. 13, pp. 1241-1246) reviewed the research progress of the anti-HBV activity of medicinal plant ingredients in vitro in the past 20 years. For natural products, there is no record that any quercetin dimer flavonoids have anti-HBV activity, and only the inventor team has carried out a new synthesis method and research on anti-HBV activity that have not been noticed before. One of our purposes is: hope to find dimeric flavonoid lead compounds that can reduce HBsAg or HBeAg and inhibit HBV DNA replication, so as to further develop them into innovative drugs that can clear HBsAg or HBeAg, inhibit HBV DNA replication, and treat CHB drug. Based on this, the present invention has been completed.

Contents of the invention

The purpose of the present invention is to provide quercetin dimer flavonoids shown in formula (1) or its pharmaceutically acceptable salts for the preparation of new applications in the preparation of HBsAg or HBeAg, inhibition of HBV DNA replication, and treatment of hepatitis B drugs .

The name of the compound of formula (1) is: 1,3,11a-trihydroxyl-9-(3,5,7-trihydroxyl-4H-1-benzopyran-4-ketone-2)-5a-(3 , 4-dihydroxyphenyl)-5,6,11-hexahydro-5,6,11-trioxo-tetracen-12-one.

The present invention also provides a method for preparing quercetin dimer flavonoids represented by formula (1), which is characterized in that: commercially available or self-made quercetin is used under anhydrous conditions to be catalyzed by silver salts It is obtained by free radical coupling reaction coupling.

Another object of the present invention is to provide a pharmaceutical composition for preparing HBsAg or HBeAg, inhibiting HBV DNA replication, and treating viral hepatitis B, which is characterized by containing a therapeutically effective amount of the formula ( 1) A mixture of a compound or its pharmaceutically acceptable salt and pharmaceutically acceptable excipients. Its dosage form can be tablets, capsules, injections, aerosols, suppositories, films, dripping pills, patches, subcutaneous implants, liniments for external use, oral liquids or ointments. Known controlled-release or sustained-release dosage forms or nano-formulations.

Compared with the natural flavonoid lignan compound quercetin, the quercetin dimer flavonoid compound (1) of the present invention has many different characteristics in structure and physicochemical properties, including its hydrophobicity, aromaticity, Gibb The free energy, hydrogen bond acceptor, electrical property, intermolecular van der Waals force, and 3D conformation, stretching direction, molecular center of gravity, conjugation degree, electrical distribution center and other properties are significantly different from quercetin; and the compound (1 ) molecular weight increased by 300 mass units than quercetin. The above-mentioned characteristics have all determined that the three-dimensional conformation of the compound shown in formula (1) is combined with the 3D space structure of HBsAg or HBeAg or even HBV DNA. The ligand-receptor binding complex morphology and binding mode may have larger differences. The binding site, binding mode, and its binding free energy will all change greatly, so it may have unexpected effects in clearing HBsAg or HBeAg and inhibiting HBV DNA replication.

We tested the growth inhibitory effect of the compound on HepG2.2.15 cells, and tested its inhibitory activity on beta HBsAg, HBeAg secreted by HepG2.2.15 cells and HBV DNA replication. Test result finds: the quercetin dimer flavone synthesized in the present invention has extremely significant inhibitory activity to HBsAg and HBeAg secreted by HepG2. 65.7% and 44.8%, 4.1 times and 2.7 times more than the positive control drug (10000 units/ml of α-interferon) respectively; at the same time, it showed a 44.8% inhibitory rate to HBV DNA at this concentration, and it was α- The inhibition rate of interferon at the highest tested concentration was 117%. All the above shows that the compound of formula (1) has unexpected anti-HBV effect, so it can be expected that it can continue to be developed as an active lead compound for removing HBsAg or HBeAg, inhibiting HBV DNA replication, and treating hepatitis B.

According to the inventor's detailed literature review, so far, there is no report about the compound for treating hepatitis B virus infectious diseases and preparing anti-hepatitis B virus drugs. The discovery that the compound quercetin dimer flavonoids of formula (1) can effectively inhibit HBsAg, HBeAg and HBV DNA is an unexpected discovery, and has exact originality. In summary, the quercetin dimer flavone we synthesized has both structural uniqueness and novelty in the study of antiviral effects, and found extremely potent anti-HBV activity tests. Inhibit the activity of HBsAg, found significant inhibition of HBeAg activity, and found that it has a very strong inhibitory activity of HBV DNA replication; it is expected to become a non-nucleoside drug that can effectively eliminate HBsAg or HBeAg, inhibit HBV DNA replication and treat CHB the lead compound.

The present invention is beneficial in that it is first discovered that the quercetin dimer flavone shown in formula (1) has the effect of removing HBsAg or HBeAg, inhibiting HBV DNA replication, and its potential as a drug in the prevention and treatment of hepatitis B virus infection, It provides a new material basis for the development of innovative drugs for the treatment of hepatitis B virus, the development of non-nucleoside innovative drugs that clear hepatitis B surface antigen HBsAg or hepatitis B e antigen HBeAg, and inhibit HBV DNA replication. It has potentially huge social and economic benefits. Another feature of the present invention is: the source of the synthetic starting material of the present invention is convenient, and the synthesis is convenient. The preparation method is simple and easy, the raw material source is convenient and easy to obtain, the cost is low, the pollution is small, and it is beneficial to large-scale production under the condition of energy saving and emission reduction. The prospect of industrialization is very clear.

Detailed ways

The present inventor obtained a quercetin dimer flavonoid active compound that can effectively inhibit the secretion of hepatitis B HBsAg and HBeAg and effectively inhibit HBV DNA replication activity through chemical synthesis and purification by various chromatographic means. Its chemical structure was deduced by comprehensive analysis of mass spectrometry and nuclear magnetic resonance spectrum. The inventors have found that the compound of formula (1) has no significant inhibitory effect on the growth of HepG2.2.15 cells, but has significant inhibitory effect on the secretion of hepatitis B HBsAg and HBeAg secreted by HepG2.2.15 cells and the replication of HBV DNA, suggesting that the compound It has the characteristics of safe medication, powerful removal of HBsAg or HBeAg, and high efficiency in inhibiting HBV DNA replication. Therefore, according to the inventor's research, the quercetin dimer flavonoid compound shown in the formula (1) designed and synthesized by the inventor can be used for the preparation of non-nucleoside drugs for the treatment of hepatitis B virus infectious diseases and for the treatment of HBV infectious disease.

In order to better understand the essence of the present invention, the preparation of the compound of formula (1) and its inhibitory effect on HepG2.2.15 cell growth and the inhibitory effect test on HBsAg, HBeAg and HBV DNA replication of HepG2.2.15 cell secretion are respectively below As a result, its new use in the pharmaceutical field is illustrated. The examples give partial synthesis, structure identification, and activity data of compounds of formula (1). It must be noted that the embodiments of the present invention are used to illustrate the present invention rather than limit the present invention. The simple improvements made to the present invention according to the essence of the present invention all belong to the protection scope of the present invention.

Embodiment 1: the preparation of formula (1) compound

1.1 Instruments and reagents:

The ultraviolet spectrum was measured with a Shimadzu UV-240 ultraviolet spectrophotometer; the hydrogen nuclear magnetic resonance spectrum ¹ H-NMR was measured by an INOVA superconducting nuclear magnetic resonance spectrometer (VARIAN INOVA-400MHz) (tetramethylsilyl ether TMS was used as internal standard); Spray mass spectrometry ESI-MS was determined by BrukerEsquire 3000+ mass spectrometer, silica gel for column chromatography (100-200, 200-300 and 300-400 mesh) and silica gel GF254 for thin layer chromatography (10-40 mesh) were all provided by Qingdao Marine Produced in a chemical factory; all reagents used were of analytical grade, and thin-layer preparative chromatography (PTLC) used aluminum foil silica gel plates from Merck Company; Sephadex LH-20 used for column chromatography was produced by Amersham Pharmacia Biotech AB in Sweden; reverse phase Silica gel RP-18 adopts the Chromatorex product of Fuji Silysia Chemical Company of Japan; MCI is the product of Mitsubishi Chemical Company of Japan, and thin plate (TLC) detection uses 254nm and 365nm ultraviolet lamps; Color developer uses iodine vapor, 10% sulfuric acid-ethanol and phosphorus molybdenum acid solution.

1.2 Preparation of formula (1) compound:

Add 343 milligrams of quercetin (prepared by ourselves, HPLC detection purity 98%), 550 milligrams of silver carbonates in the dry reaction bottle, add 20 milliliters of dried acetone and 60 milliliters of anhydrous benzene solution under nitrogen protection, in Stir between 55-60°C for 20 hours. The mixture was left standing, and insoluble matter was filtered off. The mother liquor was concentrated under reduced pressure to obtain a yellow solid. It was chromatographed on a 200-300 mesh silica gel column and eluted with chloroform/methanol (20:1). 182 mg of a yellow solid was obtained. R _f (chloroform:methanol=10:1)=0.18; Infrared IR (KBr tablet) cm ^-1 : 3195, 1689, 1647, 1588, 1495; ¹ H NMR (400MHz, deuterated acetone): 6.05 (doublet, J=2.0Hz, 1H, H-6'), 6.10 (doublet, J=2.0Hz, 1H, H-8'), 6.28 (doublet, J=2.0Hz, 1H, H- 6), 6.62 (double peak, J=2.0Hz, 1H, H-8), 6.81 (double peak, J=8.4Hz, 1H, H-13), 7.16 (double double peak, J=2.4, 8.4Hz, 1H, H-14'), 7.29 (doublet, J=8.0Hz, 1H, H-13'), 7.35 (doublet, J=2.4Hz, H-10'), 7.96 (doublet, J=2.0 Hz, 1H, H-10), 8.00 (double doublet, J=2.0, 8.8Hz, 1H, H-14); ¹³ C NMR (100MHz, deuterated acetone, δppm): 91.4 (C- 3'), 94.6(C-8), 97.3(C-8'), 98.0(C-6'), 99.3(C-6), 100.9(C-4'a), 101.6(C-2') , 104.2(C-4a), 115.4(C-9′), 116.5(C-14′), 117.7(C-10), 118.2(C-13), 121.1(C-10′), 123.4(C- 14), 126.2 (C-9′), 126.9 (C-13), 137.6 (C-3), 141.8 (C-11), 143.0 (C-12), 145.4 (C-2, C-11′) , 147.6(C-12'), 157.8(C-8a), 160.7(C-8'a), 162.3(C-4a), 165.1(C-5'), 165.2(C-7), 169.2(C -7'), 176.7 (C-4), 189.0 (C-4'); Electrospray mass spectrometry ESI-MS: m/z 603 [M+H] ⁺ (100).

Embodiment 2: Compound (1) inhibits the hepatitis B surface antigen (HBsAg) secreted by HepG2.2.15 cells

2.1 Cell culture:

Culture HepG2.2.15 cells in DMEM medium containing 10% inactivated fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin, 100 μg/ml G418 at 37°C, 5% CO ₂ , 100% cultured in a relative humidity incubator.

2.2 The inhibitory effect of the compound of formula (1) on HepG2.2.15 cell growth was measured by MTT method:

Take HepG2.2.15 cells in the logarithmic growth phase, dilute the cells to 1×10 ⁵ cells/ml with medium, inoculate in 96-well cell culture plate, 100 microliters per well, at 37°C, 5% CO ₂ , 100 After culturing in an incubator with % relative humidity for 24 hours, add compound (1) diluted with medium, the concentrations are 1000, 200, 40 and 8 micrograms/ml respectively, 200 microliters per hole, and three duplicate holes are set for each concentration , placed in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity. After culturing for 72 hours, add 10 microliters of MTT reagent to each well, continue culturing for 4 hours, discard the medium, and add 200 DMSO to each well. Microliter, shake with a shaker for 20 minutes, and measure the OD value with a microplate reader at a wavelength of 570nm. The wells in which only medium was added were used as control wells. Inhibition rate (%)=OD value of control well-OD value of experimental group)/OD value of control well×100%.

Determination of the inhibitory effect of compound 1 on hepatitis B surface antigen (HBsAg): Take the HepG2.2.15 cells in the logarithmic growth phase, dilute the cells to 1×10 ⁵ /ml with culture medium, and inoculate them in 96-well cell culture plates. Wells of 100 ml were cultured in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity for 24 hours, and then samples diluted with medium were added at concentrations of 100, 20 and 4 μg/ml, 200 μl per well , set three duplicate wells for each concentration, culture in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity, change the medium containing the same concentration of samples every 4 days, and replace the same sample with the same concentration Mix equal volumes of culture medium and use it as the sample to be tested. Measure the HBsAg concentration in the culture medium with an ELISA kit, expressed as P/N; take lamivudine (3-TC) as positive control 1 (the test concentration is 100, 20 and 4 μg/ml); use α-interferon It is positive control 2 (test concentration is 10000, 5000 and 1000 units/ml). Experiments were repeated three times.

2.3 Experimental results:

The experimental results are shown in Table 1, the compound of formula (1) has a significant effect of inhibiting hepatitis B surface antigen (HBsAg). It has no obvious inhibitory effect on the growth of HepG2.2.15 cells, but its inhibitory activity on HBsAg secreted by HepG2.2.15 cells is higher than that of lamivudine and α-interferon at high doses on the eighth day.

Table 1. The inhibition rate of hepatitis B surface antigen secreted by HepG2.2.15 on the eighth day of the sample

2.4 Result description:

The results of this example illustrate: the quercetin dimer flavonoid compound shown in formula (1) has a significant inhibitory effect on the hepatitis B surface antigen (HBsAg) secreted by HepG2.2.15 cells. The intensity of removing HBsAg is 65.7%, 4.1 times more than the positive control drug (10000 units/ml of α-interferon); also 4.1 times more than the positive control 2 lamivudine; it can be seen that the quercetin dimer flavone has a strong Inhibition of viral secreted surface antigen activity.

HBsAg clearance is the closest clinical state to cure. For hepatitis B patients, HBsAg clearance has become a very valuable end point of CHB treatment. Therefore, the quercetin dimer flavonoid compound represented by formula (1) can be expected to be developed into a non-nucleoside innovative drug that reduces hepatitis B surface antigen and controls the symptoms of viral hepatitis B.

Embodiment 3: Compound (1) inhibits the hepatitis B e antigen (HBeAg) secreted by HepG2.2.15 cells

3.1 Cell culture: the method is the same as in Example 2.

3.2 Determination of the inhibitory effect of the compound of formula (1) on the growth of HepG2.2.15 cells by MTT method: the method is the same as in Example 2.

3.3 Determination of the inhibitory effect of the compound on hepatitis B e antigen (HBeAg): Take the HepG2.2.15 cells in the logarithmic growth phase, dilute the cells to 1×10 ⁵ /ml with medium, and inoculate them in a 96-well cell culture plate. Wells of 100 ml were cultured in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity for 24 hours, and then samples diluted with medium were added at concentrations of 100 μg/ml, 20 μg/ml and 4 μg/ml , 200 microliters per well, set three duplicate wells for each concentration, culture in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity, change the medium containing the same concentration of samples every 4 days, and mix the same The exchanged culture medium with the same concentration as the sample was mixed in equal volume and used as the sample to be tested. Measure the HBeAg concentration in the medium with an ELISA kit, and express it with P/N; take lamivudine (3-TC) as positive control 1 (note: the test concentration of lamivudine is 100 micrograms/ml, 20 micrograms/ml and 4 micrograms/ml); take α-interferon as positive control 2 (Note: α-interferon test concentration is 10000 units/ml, 5000 units/ml and 1000 units/ml).

3.4 Experimental results: The experimental results are shown in Table 2, the compound of formula (1) showed a very significant inhibitory effect on hepatitis B e antigen (HBeAg). It has no obvious inhibitory effect on the growth of HepG2.2.15 cells at a concentration of 40 micrograms/ml, but at a concentration of 100 micrograms/ml, the intensity of its removal of HBeAg is 44.8%, exceeding the positive control drug 2 (α- interferon) 2.7 times; and the positive control 1 lamivudine is zero to HBeAg inhibitory activity when the highest concentration (100 micrograms/ml).

Table 2. The inhibition rate of Hepatitis B e antigen secreted by HepG2.2.15 on the eighth day of the sample

3.5 The result shows: the quercetin dimer flavonoid compound shown in formula (1) has no obvious inhibitory effect on the growth of HepG2.2.15 cells at a concentration of 40 micrograms per milliliter, but it has very significant inhibition of HBeAg activity, but At 100 micrograms/ml concentration, its HBeAg removal intensity is 44.8%, 2.7 times higher than the positive control drug 2 (10000 units/ml alpha-interferon); more than the positive control lamivudine that does not show inhibitory activity. It can be seen that the quercetin dimer flavone has significant activity of inhibiting the secretion of HBeAg by hepatitis B virus, and thus can be expected to be developed as a drug for reducing hepatitis B e antigen and controlling the symptoms of viral hepatitis B.

Embodiment 4: formula (1) compound is to HepG2.2.15 cell secreted hepatitis B virus deoxyribonucleic acid (HBV DNA) replication inhibitory effect

4.1 Cell culture: the method is the same as in Example 2.

4.2 MTT method was used to measure the inhibitory effect of the flavonoid lignan compound represented by formula (1) on the growth of HepG2.2.15 cells: the method was the same as in Example 2.

4.3 The inhibitory effect of the quercetin dimer flavonoid compound shown in the assay formula (1) on the replication of hepatitis B virus deoxyribonucleic acid (HBV DNA): get the HepG2.2.15 cells in the logarithmic growth phase, and use the culture medium to divide the cells Diluted to 1× ¹⁰⁵ cells/ml, inoculated in 96-well cell culture plate, 100 microliters per well, cultured in an incubator at 37°C, 5% CO ₂ , 100% relative humidity for 24 hours, then added diluted with medium The quercetin dimer flavonoid compound shown in the formula (1), the concentration is respectively 100 micrograms/ml, 20 micrograms/ml and 4 micrograms/ml, 200 microliters in every hole, each concentration establishes three duplicate holes, Place it in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity. Change the culture medium containing the same sample concentration every 4 days. Test samples. On the 8th day, the HBV DNA concentration in the culture medium was measured with a HBV DNA quantitative PCR kit. With lamivudine (3-TC) as positive control 1 (note: the test concentration of lamivudine is 100 micrograms/ml, 20 micrograms/ml and 4 micrograms/ml), with α-interferon as positive control 2 ( NOTE: α-interferon tested at concentrations of 10,000 units/mL, 5,000 units/mL, and 1,000 units/mL).

4.4 Experimental results: Examples of experimental results As shown in Table 3, the compound of quercetin dimer flavone formula (1) has a strong effect of inhibiting the replication of hepatitis B virus DNA.

The inhibiting rate of the HBV DNA replication of HepG2.2.15 cells in the 8th day of table 3 sample

4.5 Result shows: the quercetin dimer flavonoid compound shown in formula (1) has extremely potent inhibition to the replication of hepatitis B virus deoxyribonucleic acid (HBV DNA), at 100 micrograms/ml concentration its to HBV DNA showed an inhibition rate of 44.8%, which was 117% of that of alpha-interferon at the highest tested concentration. Therefore, this quercetin dimer flavonoid compound is a significant and effective non-nucleoside natural product for inhibiting hepatitis B virus, which is worthy of further attention and in-depth research, and can be expected to be further optimized and developed as an innovative non-nucleoside drug that inhibits HBV DNA replication.

When the above description explains the present invention, the purpose of providing examples is to illustrate the practical operation process of the present invention and the significance of the present invention. The practice of the present invention includes all common changes, adaptations, or improvements when coming within the scope of the claims of the present invention and their equivalents.

Claims (4)

1. the flavonoid quercetin dimmer that has structure shown in the formula (1) is used for the purposes of preparation treatment hepatitis B medicine;

2. the flavonoid quercetin dimmer that has structure shown in the formula (1) is used for the purposes that preparation reduces hepatitis B virus surface antigen HBsAg medicine,

3. the flavonoid quercetin dimmer that has structure shown in the formula (1) is used for the purposes that hepatitis B virus e antigen HBeAg medicine is removed in preparation,

4. the flavonoid quercetin dimmer that has structure shown in the formula (1) is used for the purposes that preparation suppresses hepatitis B virus DNA HBV dna replication dna medicine,