CN101829091B - Use of acetamide dehydrosilibinin as a drug for the preparation of viral hepatitis B - Google Patents

Abstract

The invention relates to the use of acetamide dehydrosilibinin as a drug for treating viral hepatitis B, in particular to a dehydrosilibinin ester flavonoid lignan substituted by carbamoylmethoxy on the A ring or its equivalent Medicinal salts are used to prepare medicines for removing HBsAg and HBeAg, and drugs for inhibiting HBV DNA replication, which have extremely significant activity of inhibiting HBsAg and inhibiting HBeAg, and the strength of clearing HBsAg and HBeAg at a concentration of 20 micrograms/ml is 90.5 respectively % and 63.6%, 5.6 times and 3.8 times more than the positive control drug α-interferon; at the same time, at this concentration, it showed an inhibition rate of 90.4% on HBV DNA, 12% higher than lamivudine, and higher than α - Interferon 2.4 times. The above shows that the flavonoid lignan or its pharmaceutically acceptable salt can be expected to be used in the preparation of non-nucleoside drugs for treating hepatitis B virus infection.

Description

Use of acetamide dehydrosilibinin as a drug for the preparation of viral hepatitis B

technical field

The invention relates to the field of medical technology, in particular, the invention relates to a dehydrosilibinin ester flavonoid lignan or a pharmaceutically acceptable salt thereof which is substituted by dicarbamoylmethoxy on the A ring and is used for the preparation of Uses of hepatitis B virus surface antigen HBsAg and hepatitis B e antigen HBeAg, inhibiting HBV DNA replication, and treating hepatitis B virus infection diseases. This flavonoid lignan has a very significant activity of inhibiting HBsAg and HBeAg, and its intensity of removing HBsAg and HBeAg is respectively 90.5% and 63.6% at a concentration of 20 micrograms per milliliter, surpassing positive control drugs (α of 10000 units/ml) respectively. -interferon) 5.6 times and 3.8 times; more noteworthy is that it shows 90.4% inhibition rate to HBV DNA at this concentration, which is 12% higher than lamivudine and 2.4 times higher than α-interferon . The above pharmacodynamic results show that the flavonoid lignan 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. the

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. the

Hepatitis B surface antigen (HBsAg) is the coat protein of hepatitis B virus, and HBsAg positive is the gold standard for judging HBV infection. People who are HBsAg positive 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. the

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 treating HBeAg-negative CHB patients. It can be seen that the discovery of drugs that can efficiently clear HBsAg has significant social and economic benefits. the

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. the

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. the

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 in CHB patients treated with interferon 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. the

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. the

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; When there is mutation or drug resistance, the change of HBV DNA is the only first 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. the

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. the

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 large toxic 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. the

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 plant extracts from the genus Prismus to inhibit herpes simplex virus and hepatitis B virus" (Zhao Yu, Zhou Changxin, Yu Rongmin, Bai Hua; CN 1989989A); "1β-oxo-5,11(13)-diene eucalyptane -12-Acid Inhibits the Medical Application of Hepatitis B Virus” (Zhao Yu, Li Xiaokun, Huang Kexin, Li Haibo, etc.; CN 1927197A); “1β-Hydroxypectic Acid Inhibits the Medical Application of Hepatitis B Virus” (Zhao Yu, Li Xiaokun, Huang Kexin , Wu Xiumei, etc.; CN 1935131A); "Enanti-erimofantanoic acid and its medicinal application of inhibiting hepatitis B surface antigen" (Huang Kexin, Li Xiaokun, Wang Xiaoyu, Zhao Yu, etc.; CN 101239054); "1-Oxy- Substituted benzoylquinic acid compounds and their use for inhibiting hepatitis B virus" (Li Xiaokun, Hu Lihong, Wu Xiumei, Zhao Yu, etc.; CN101293836); the inventors have published articles on inhibiting the activities of HBsAg, HBeAg and HBV DNA, see: " 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), Y u Zhao*(赵瑜) et al, Antiviral Chemistry & Chemotherapy, 2005, 16, 277-282; "Evaluation of Antiviral Activity of Compounds Isolated from Ranunculus sieboldii Miq. ), Xiumei Wu (吴秀美), Yu Zhao*(赵瑜), 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. Therefore, it is listed as a major project for new drug development by the Ministry of Science and Technology one. the

R.等，Curr.Med.Chem.2007，14，315-338； 

Z.等，Phytother.Res.2003，17，524-530； 

R.等，Bioorg.Med.Chem.2004，12，5677-5687；Varga，Z.等；Phytothe.Res.2001，15，608-612.Singh，R.P.等，Curr.Cancer Drug Tar.2004，4，1-11)。因此，以水飞蓟宾为代表的黄酮木质素类化合物引起了越来越多的关注，如发明人于2006-2009年间制备并报道的多个系列水飞蓟宾类衍生物 也具有显著的抗氧化活性(杨雷香、赵昱等，“Design，synthesis andexamination of neuron protective properties of alkenylated and amidateddehydro-silybin derivatives”，Journal of Medicinal Chemistry，2009，52(23)，7732-7752；汪峰，赵昱等，“Preparation ofC-23 esterified silybinderivatives and evaluation of their lipid peroxidation inhibitory and DNAprotective properties”，Bioorganic & Medicinal Chemistry，2009，17(17)，6380-6389；杨雷香，赵昱等，“Synthesis and antioxidant propertiesevaluation of novel silybin analogues”，Journal of Enzyme Inhibition andMedicinal Chemistry，2006，21(4)，399-404；等等)。在发明人报道的上述文章中，经发明人设计并合成出的多个系列之A环、B环、E环、23位取代的黄酮木脂素类化合物都显示出强效捕获DPPH自由基和超氧阴离子自由基的活性、抗氧化活性、以及保护PC12细胞的活性。但是显而易见：上述研究仅集中于研究水飞蓟宾类黄酮木脂素的抗氧化作用和细胞保护作用。  Among the above-mentioned drugs for treating CHB, there is also a class of hepatoprotective drugs, the typical example of which is widely used clinically is silymarin present in the seeds of the Compositae plant milk thistle. Milk thistle has been widely used clinically, and its trade name is Legalon ^TM or Flavobion ^TM on the market, and its representative compound is the flavonoid lignan silybin. Flavonoid lignin compounds belong to heterolignins, which are a kind of natural products composed of a molecule of phenylpropanoid and a molecule of flavone. Milk thistle contains the most silybin and has the highest activity. The main effects of the drug are as follows: (1) Anti-free radical activity: silymarin has a protective effect on liver damage caused by carbon tetrachloride, galactosamine, alcohols and other liver toxins. In 1990, Lotteron et al. reported that in mouse liver microsomes, silymarin could reduce in vitro lipid peroxidation caused by carbon tetrachloride metabolism and peroxidation caused by reduced coenzyme alone, all of which indicated that silymarin was a chain-interrupting anti-oxidant. Oxidants or free radical scavengers. (2) Protect the liver cell membrane: maintain the fluidity of the cell membrane and protect the liver cell membrane by resisting lipid peroxidation. It can also block the binding of mycotoxins phalloidin and α-amanitin to specific receptors on liver cells, inhibit its attack on liver cells and transmembrane transport, interrupt its enterohepatic circulation, thereby strengthening the liver cell membrane Resistance to multiple damaging factors. (3) Promote the repair and regeneration of liver cells: After entering the cells, silibinin can bind to estradiol receptors and activate them. The activated receptors can enhance the activity of RNA polymerase 1 in the nucleus of liver cells, making RNA Enhance transcription, promote the synthesis of enzymes and proteins, and indirectly promote the synthesis of DNA, which is beneficial to the repair and regeneration of liver cells. (4) Anti-tumor effect: Various reactive oxygen species can oxidize guanine to form 8-hydroxyguanine, causing DNA damage, and then causing tumors. As an effective anti-free radical substance, silibinin also shows the effect of preventing and treating tumors. effect. More than 30 years of clinical trials have proved that the drug has definite curative effect and low toxicity (see Flora, K. et al., Am.J.Gastroenterol, 1998, 93, 139-143; Saller R. et al., Drugs, 2001, 61( 14), 2035-2063;

R. et al., Curr. Med. Chem. 2007, 14, 315-338;

Z. et al., Phytother. Res. 2003, 17, 524-530;

R. et al., Bioorg.Med.Chem.2004, 12, 5677-5687; Varga, Z. et al.; Phytothe.Res.2001, 15, 608-612. , 1-11). Therefore, flavonoid lignin compounds represented by silibinin have attracted more and more attention. For example, several series of silybin derivatives prepared and reported by the inventor during 2006-2009 also have significant Antioxidant activity (Yang Leixiang, Zhao Yu, etc., "Design, synthesis and examination of neuron protective properties of alkylated and amidateddehydro-silybin derivatives", Journal of Medicinal Chemistry, 2009, 52(23), 7732-7752; Wang Feng, Zhao Yu, etc., "Preparation of C-23 esterified silybinderivatives and evaluation of their lipid peroxidation inhibitory and DNAprotective properties", Bioorganic & Medicinal Chemistry, 2009, 17(17), 6380-6389; Yang Leixiang, Zhao Yu et al., "Synthesis and antioxidant properties evaluation bin of novel si ", Journal of Enzyme Inhibition and Medicinal Chemistry, 2006, 21(4), 399-404; etc.). In the above-mentioned articles reported by the inventor, the flavonoid lignans of multiple series of A ring, B ring, E ring, and 23-position substitutions designed and synthesized by the inventor all show strong capture of DPPH free radicals and The activity of superoxide anion free radicals, antioxidant activity, and the activity of protecting PC12 cells. But it is obvious: the above studies have only focused on the antioxidant and cytoprotective effects of silybin flavonoid lignans.

Recently (2006), Xie Jun reported the results of combined application of interferon with antiviral and immunomodulatory dual effects and liver-protecting silibinin phospholipid complex in the treatment of chronic hepatitis B. The reduction of AST value is more obvious than that of interferon alone, indicating that the silibinin phospholipid complex can strengthen the effect of interferon in the treatment of chronic hepatitis B. However, similar treatment schemes still use silybin and other flavonoid lignans as auxiliary drugs, and are mainly used to protect liver cell membranes rather than directly use them to implement antiviral effects. 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. Natural products, in which there is no relevant record that any flavonoid lignans have anti-HBV activity. In particular, silybin-like natural products and their derivatives are hardly involved in China, and only the team of inventors has carried out structural modification and antioxidant activity research on them that have not been paid attention to before. the

Although the flavonoid lignans represented by silibinin have the above-mentioned antioxidant effects, there are relatively few literatures on their antiviral treatment. The flavonoid lignans treat DNA virus infection especially Its new application for anti-hepatitis B virus (including inhibition of HBsAg or HBeAg, inhibition of HBV DNA replication) has not been effectively developed, so the active compound in the field of anti-hepatitis B virus is found from flavonoid lignans, that is, the structure of flavonoid lignans Engineering it to have anti-DNA viroid activity is a new field. It is an unprecedented challenge to find a lead compound that clears HBsAg or HBeAg and inhibits HBV DNA replication. In order to explore this field, we designed and prepared a new flavonoid lignan derivative with a different structure from silibinin, that is, the diethylcarbamoylmethoxy group on the A ring of the original silibinin Substituted dehydrosilibinin ester flavonoid lignans, the parent skeleton is changed to dehydrosilibinin, and then a diethylcarbamoylmethoxy substituent is connected to the A ring, so that the The conjugation range and conjugation strength of the entire molecule are extended, and the substitution length of the original A ring module is extended, and nitrogen atoms and new hydrogen bond acceptors or donors are introduced at the same time. This design can generate a new spatial structure different from that of milk thistle. A new class of flavonoid lignans. In order to discover the extraordinary activity of clearing HBsAg or HBeAg, inhibiting HBV DNA replication, and even lead compounds, so as to further develop them into innovative drugs that can clear HBsAg or HBeAg, inhibit HBV DNA replication, and treat CHB. invention. the

Contents of the invention

The object of the present invention is to provide dehydrosilibinin ester flavonoid lignans or pharmaceutically acceptable salts thereof that are substituted by carbamoylmethoxy on the A ring shown in formula (1) and are used for preparing scavenging HBsAg or HBeAg, Inhibiting HBV DNA replication, a new application in the treatment of hepatitis B virus drugs. the

The name of the compound of formula (1) is: N, N-diethyl-2-{2-[3-(3-methoxyl-4-hydroxyl-phenyl)-2-hydroxymethyl-2,3- Dihydro-benzo[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide. the

The present invention also provides a method for preparing dehydrosilibinin ester flavonoid lignans substituted by carbamoylmethoxy group on the A ring shown in formula (1), characterized in that: commercially available Or self-made silibinin and 2-bromo-N, N-diethyl-acetamide are obtained by condensation reaction under alkaline conditions. the

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. the

Compared with the natural flavonoid lignan compound silybin, the dehydrosilybin ester flavonoid lignan compound (1) substituted by diethylcarbamoylmethoxy on the A ring of the present invention has many structures and physicochemical properties, including its hydrophobicity, aromaticity, Gibbs free energy, hydrogen bond acceptor, electrical properties, intermolecular van der Waals force, and 3D conformation, stretching direction, molecular center of gravity, conjugation degree, The properties such as the electrical distribution center are significantly different from silibinin; and the molecular weight of compound (1) is 112 mass units higher than that of silybin. The above-mentioned characteristics all determine that the three-dimensional conformation of the compound shown in formula (1) is combined with the 3D spatial structure of HBsAg or HBeAg or even HBV DNA, and the ligand-receptor binding complex morphology and binding mode may have large differences. Its binding site and binding mode, its binding free energy, etc. will have a large change, so it may have unexpected effects in clearing HBsAg or HBeAg and inhibiting HBV DNA replication. the

We tested the growth inhibitory effect of the compound on HepG2.2.15 cells, and tested its inhibitory activity on HBsAg, HBeAg secreted by HepG2.2.15 cells and HBV DNA replication. The test results found that the dehydrosilibinin ester flavonoid lignans substituted by bisethylcarbamoylmethoxy on the A ring synthesized in the present invention are N, N-diethyl-2-{2-[ 3-(3-Methoxy-4-hydroxy-phenyl)-2-hydroxymethyl-2,3-dihydro-benzo[1,4]dioxane-6-yl]-3,5 -Dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide has extremely significant inhibitory activity on HBsAg and HBeAg secreted by HepG2.2.15 cells, and its clearance is at a concentration of 20 μg/ml The intensity of HBsAg and HBeAg is 90.5% and 63.6% respectively, surpasses respectively positive control drug (10000 unit/ml alpha-interferon) 5.6 times and 3.8 times; What's more exciting is: at this concentration, its effect on HBV DNA It showed an inhibition rate of 90.4%, 12% higher than that of lamivudine, and 2.4 times higher than that of α-interferon. 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. Through 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 compound of flavonoid lignan formula (1) is an unexpected discovery for the potent inhibition of HBsAg, HBeAg and HBV DNA, and has definite originality. the

In summary, the bisethylcarbamoylmethoxy substituted dehydrosilibinin ester flavonoid lignans we synthesized on the A ring have both structural uniqueness and novel antiviral effects. In the anti-hepatitis B virus activity test, not only the extremely potent activity of inhibiting HBsAg, but also the significant activity of inhibiting HBeAg, and the extremely potent activity of inhibiting HBV DNA replication were found; it is expected to become a powerful It is the lead compound of non-nucleoside drugs for clearing HBsAg or HBeAg, inhibiting HBV DNA replication and treating CHB. the

The present invention is beneficial in that it is found for the first time that the dehydrosilibinin ester flavonoid lignans substituted by carbamoylmethoxy on the A ring shown in formula (1) has the ability to remove HBsAg or HBeAg, inhibit HBV DNA replication, The drug potential in the prevention and treatment of hepatitis B virus infectious diseases 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. the

Detailed ways

The present inventor obtained the diethylcarbamoyl methoxy substitution on the A ring through chemical synthesis and purification by various chromatographic means, which can not only strongly inhibit the secretion of hepatitis B HBsAg and HBeAg, but also effectively inhibit the replication activity of HBV DNA. The dehydrosilibinin ester flavonoid lignan active compound, and 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 flavonoid lignan 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 hepatitis B virus infection disease. the

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 the partial synthesis, structure identification and activity data of the compound 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. the

Embodiment 1: Formula (1) compound N, N-diethyl-2-{2-[3-(3-methoxyl-4-hydroxyl-phenyl)-2-hydroxymethyl-2,3- Preparation of dihydro-benzo[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide

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 compound (1)

Add 100 mg of silibinin (purchased from Liaoning Panjin Tianyuan Pharmaceutical Co., Ltd., or use the person who prepared in this room, HPLC detection purity 98%), 50 mg of potassium carbonate and 10 mg of potassium iodide in the dry reaction flask, and use 5 ml of dried DMF solvent was dissolved, and 40 mg of 2-bromo-N, N-diethyl-acetamide solution in dry THF solvent was gradually added dropwise to the solution under the protection of argon, and stirred at 45°C for 12 Hour. The mixture was poured into 10 ml of ice water and extracted with chloroform (5×8 ml). The organic layer was separated, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Silica gel column chromatography, eluting with chloroform/methanol (40:1). Then it was purified by Sephadex LH-20 column chromatography. 25 mg of yellow solid was obtained, the yield was 20%. R _f (chloroform:methanol=30:1): 0.38; ¹ H NMR (400MHz, deuterated acetone): δ1.18 (triplet, 3H, J=7.2Hz, NCH ₂ CH ₃ ); 1.22 (triplet, 3H, J=7.2Hz, _NCH2CH3 ), 3.38 (quartet _, 2H, J=7.2Hz, _NCH2CH3 ), 3.42 ₍ quartet, 2H, J=7.2Hz, NCH ₂ CH ₃ ), 3.56 (multiplet, 1H, H-23b), 3.76 (singlet, 3H, OCH ₃ ), 3.78 (multiplet, 1H, H-23a), 4.24 (multiplet, 1H, H-10 ), 4.93 (singlet, 2H, OCH ₂ C=O), 5.00 (doublet, 1H, J=8.0Hz, H-11), 6.33 (broad singlet, 1H, H-6), 6.72 (broad singlet peak, 1H, H-8), 7.89-6.78 (multiplet, 6H, Ar-H), 12.58 (singlet, 1H, OH-5); electrospray mass spectrometry ESI-MS: m/z 594 [M+H ] ⁺ .

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 culture medium at a concentration of 1000 μg/ml, 200 μg/ml, 40 μg/ml and 8 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. After culturing for 72 hours, add 10 microliters of MTT reagent to each well, continue culturing for 4 hours, discard Remove the medium, add 200 microliters of DMSO to each well, shake with a shaker for 20 minutes, and measure the OD value with a microplate reader at a wavelength of 570 nm. 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%. Experiments were repeated three times.

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 20 μg/ml, 4 μg/ml and 0.8 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 HBsAg concentration in the culture medium with an ELISA kit, and express it with P/N; take lamivudine (3-TC) as positive control 1 (note: the test concentration is 100 micrograms/ml, 20 micrograms/ml and 4 micrograms/ml milliliter); take α-interferon as positive control 2 (note: the test concentration is 10000 units/ml, 5000 units/ml and 1000 units/ml).

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 the hepatitis B surface antigen HBsAg secreted by HepG2.2.15 cells is higher than that of lamivudine and α-interferon at high doses on the eighth day. the

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

2.4 Result description:

The results of this example illustrate: the flavonoid lignan compound N shown in formula (1), N-diethyl-2-{2-[3-(3-methoxy-4-hydroxyl-phenyl)-2 -Hydroxymethyl-2,3-dihydro-benzo[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yl Oxygen}-acetamide has a significant inhibitory effect on the hepatitis B surface antigen (HBsAg) secreted by HepG2.2.15 cells, and its intensity of removing HBsAg is 90.5% at a concentration of 20 micrograms per milliliter, surpassing the positive control drug 2 (10000 unit/ml of α-interferon) 5.6 times; also more than positive control 1 lamivudine 7.9 times; it can be seen that this flavonoid lignan has a strong activity of inhibiting virus secretion surface antigen. the

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 flavonoid lignan compound represented by the formula (1) can be expected to be developed into a non-nucleoside innovative drug that reduces the hepatitis B surface antigen and controls the symptoms of viral hepatitis B. the

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. the

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. the

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 20 μg/ml, 4 μg/ml and 0.8 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 culture medium with an ELISA kit, and express it with P/N; take lamivudine (3-TC) as positive control 1 (note: the test concentration is 100 μg/ml, 20 μg/ml and 4 μg/ml milliliter); take α-interferon as positive control 2 (note: the 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. Formula (1) compound N, N-diethyl-2-{2-[3-(3-methoxy-4-hydroxyl-phenyl)-2-hydroxymethyl-2,3-dihydro-benzene And[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide showed a very significant inhibition of ethanol The role of hepatitis 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 20 micrograms/ml, its intensity of removing HBeAg is 63.6%, which exceeds the positive control drug 2 (α- Interferon) 3.8 times; while positive control 1 lamivudine is zero to HBeAg inhibitory activity. the

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 flavonoid lignan compound shown in formula (1) has no obvious inhibitory effect on the growth of HepG2.2.15 cells at a concentration of 40 micrograms/ml, but it has a very significant inhibition of HBeAg activity, at 20 micrograms/ml Its strength of removing HBeAg under the milliliter concentration is 63.6%, 3.8 times more than the positive control drug (10000 units/ml alpha-interferon); even more than the positive control lamivudine showing no inhibitory activity. It can be seen that the flavonoid lignans have significant activity of inhibiting the secretion of HBeAg by hepatitis B virus, and thus can be expected to be developed as a medicine for reducing hepatitis B e antigen and controlling viral hepatitis B symptoms. the

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. the

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. the

4.3 Flavonoid lignan compounds shown in the assay formula (1) inhibit 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 dilute the cells to 1 × ¹⁰⁵ cells/ml, inoculated in 96-well cell culture plate, 100 microliters per hole, cultured in an incubator at 37°C, 5% CO ₂ , and 100% relative humidity for 24 hours, and then added the formula ( 1) The flavonoid lignan compounds shown in the concentration are 20 micrograms/ml, 4 micrograms/ml and 0.8 micrograms/ml respectively, 200 microliters per well, three replicate wells for each concentration, placed at 37°C, 5 %CO ₂ , cultured in an incubator with 100% relative humidity, the culture medium containing the same sample concentration was changed every 4 days, and the same volume of the culture medium replaced by the same sample and the same concentration was mixed uniformly as the sample to be tested. 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 flavonoid lignan formula (1) has a strong effect of inhibiting the replication of hepatitis B virus deoxyribonucleic acid. the

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

4.5 Result shows: the flavonoid lignan compound shown in formula (1) has extremely potent inhibitory action to the replication of hepatitis B virus deoxyribonucleic acid (HBV DNA), exciting is: at 20 micrograms/ml concentration It showed 90.4% inhibitory rate to HBV DNA, 12% higher than lamivudine, and 2.4 times higher than α-interferon. Therefore, the dehydrosilybin ester flavonoid lignan compound substituted with diethylcarbamoylmethoxy on the A ring is an extremely significant and effective non-nucleoside natural product for inhibiting hepatitis B virus, and it is worthy of further attention and in-depth research , and can be expected to be further optimized and developed into a non-nucleoside innovative drug that inhibits HBV DNA replication. the

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. the

Claims (4)

1. The carbamoylmethoxy-substituted dehydrosilibinin ester flavonoid lignan or its pharmaceutically acceptable salt on the A ring having the structure shown in formula (1) is used for the preparation of a drug for the treatment of hepatitis B the purpose of; The name of the compound of formula (1) is: N, N-diethyl-2-{2-[3-(3-methoxyl-4-hydroxyl-phenyl)-2-hydroxymethyl-2,3- Dihydro-benzo[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide. 2. The dehydrosilibinin ester flavonoid lignans or their pharmaceutically acceptable salts substituted with carbamoylmethoxy groups on the A ring having the structure shown in formula (1) are used for the preparation of reducing hepatitis B virus surface antigen Use of HBsAg drugs;

The name of the compound of formula (1) is: N, N-diethyl-2-{2-[3-(3-methoxyl-4-hydroxyl-phenyl)-2-hydroxymethyl-2,3- Dihydro-benzo[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide. 3. Dehydrosilibinin ester flavonoid lignans or their pharmaceutically acceptable salts substituted with carbamoylmethoxy groups on the A ring with the structure shown in formula (1) are used for the preparation of drugs for removing hepatitis B e antigen HBeAg use; The name of the compound of formula (1) is: N, N-diethyl-2-{2-[3-(3-methoxyl-4-hydroxyl-phenyl)-2-hydroxymethyl-2,3- Dihydro-benzo[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide. 4. The dehydrosilibinin ester flavonoid lignans or their pharmaceutically acceptable salts substituted by carbamoylmethoxy on the A ring with the structure shown in formula (1) are used to prepare deoxyribose for inhibiting hepatitis B virus The use of nucleic acid HBV DNA replication medicine,

The name of the compound of formula (1) is: N, N-diethyl-2-{2-[3-(3-methoxyl-4-hydroxyl-phenyl)-2-hydroxymethyl-2,3- Dihydro-benzo[1,4]dioxan-6-yl]-3,5-dihydroxy-4-oxo-4H-benzopyran-7-yloxy}-acetamide.