Design, Synthesis And Anti-HBV Research Of Novel 2-Pyridone Derivatives

Hepatitis B virus (HBV) is a cause of chronic liver disease worldwide, affecting more than 5% of the world's population. HBV can cause both acute and chronic infections. Chronic HBV infection leads to liver damage, cirrhosis, and liver cancer with high mortality. There are about 400 million people who are chronic infected, resulting in 0.5 - 1.2 million deaths annually. More seriously, China is one of the countries of high incidence of hepatitis B; there are more than 700 million people who were infected by HBV. According to the latest news, there are 140 million HBV carriers, and nearly 0.3 million of whom are dying for the related diseased per year. HBV infection represents a serious global medical, social, and economic burden. However, the current anti-HBV therapeutic measures are limited and suffer from drug related toxicity, drug resistance and serious drug-drug interactions. Therefore, there is an emergent need to develop novel anti-HBV agents with higher efficiency and lower toxicity.Shegan (Belamcanda Chinensis D.C.), a kind of Chinese traditional medicine used as antivirus therapy, was chosen as the source of natural products. To identify the anti-HBV active constituents, this dissertation employed the bio-assay guided isolation and fractionation for the chemical investigation. Six isoflavonoid compounds have been separated, all of which showed good anti-HBV activity in vitro. Then a small compound library consisted of isoflavonoids was established using the C-C coupling reactions catalyzed by Pd. And on the process of modification of the product of Heck coupling reaction, a kind of novel scaffold of 2-pyridone derivative was discovered. The anti-HBV assays showed most of the small compounds possess moderate anti-HBV activity. Surprisingly, the 2-pyridone derivatives showed much higher activity than isoflavonoids in the library. On the basis of results, it is significant to do more modification and optimization on the 2-pyridone scaffold.In order to improve the anti-HBV activity and study the structure and activity relationships (SARs) of 2-pyridone derivatives, two serials of 2-pyridones have been designed and synthesized: 24 of 5-(2-hydroxy-4-methoxybenzoyl)-1-substituted-pyridin- 2(1H)-one (serial LK041) and 16 of (E)-5-((2-hydroxy-4-methoxyphenyl)(substituted- limino)methyl)-1- substituted-pyridin-2(1H)-one (serial LK042). The anti-HBV assay revealed that all the 2-pyridone derivatives showed good anti-HBV activity and compounds LK041-4, LK041-6, LK041-7, LK041-8, LK041-14 and LK042-3, LK042-13 had excellent potency against the secretion of HBsAg, HBeAg and the replication of HBV DNA. The EC50 of anti-HBV DNA of LK041-4 and LK042-13 were 0.206μM and 0.12μM respectively, which were much better than positive control adefovir.To know more information about the active sites of the target enzyme, a rational pharmacophore model has been constructed. From the model, we knew thatπ-πhydrophobic interaction and hydrogen bonding interaction were the main elements of 2-pyridone derivatives interacting with the target enzymes. The rational pharmacophore model is significant for the prediction of activity, further optimization and the discovery of novel anti-HBV scaffold.Based on the information of pharmacophore model, 13 of 5-methoxy-2-(6-oxo-1-o- tolyl-1, 6– dihydropyridine-3- carbonyl) phenyl substituted ester and 20 of 5-(2-((1-sub- stituted-benzyl-1H-1, 2, 3-triazol-4-yl) methoxy)-4-methoxybenzoyl) -1-o-tolylpyridin-2 (1H) -one ( serial LK043 ), 16 of 5- ( 2- hydroxyl -5- acetamido-benzoyl) -1- substituted- pyridin- 2 (1H) - one (serial LK044), 18 of 5 - ( 2, 4 - dihydroxybenzoyl) -1-substituted- pyridin- 2 (1H) -one (serial LK045), 9 of 5 - ( 2-hydroxyl-3-nitro-benzoyl)-1-substituted- pyridin-2(1H)-one (serial LK046) and 9 of 5-(2-hydroxy-4-(methoxy-methoxy)benzoyl)- 1-substituted-pyridin- 2 (1H) -one (serial LK047) were designed and synthesized. All the target molecules were reported firstly and their structures were determined by 1H NMR, 13C NMR,LC-MS,IR and element analysis. Furthermore, the single crystals of key structures were obtained and the structure analyzed and determined by X-Ray diffraction.(E)-methyl 3-(7-methoxy-4-oxo-4H-chromen-3-yl)acrylate (Ⅵ) is the key synthetic intermediate of 2-pyridone derivatives and its synthetic conditions were optimized in this dissertation. According to literature, the yield was 75%, nevertheless, the byproduct 7-methoxy-4H-chromen-4-one (Ⅲ) would make the next step reaction much more complicated. In the process of optimization, the solvent, catalyst, temperature, base and time of the reaction were considered. The yield of the new process was improved from 75% to more than 90% and pure product was obtained without any byproductⅢ. What the most important was the cycle of the reaction was shortened to 10 min from 18 hours, which made the reaction a more timesaving and energy-saving one.HBV antigen and HBV DNA inhibitory efficacies of the target compounds were evaluated in HepG 2.2.15 cells transfected by HBV. MTT, ELISA and probe PCR methods were used to detect the cytotoxicity, inhibitory of HBsAg and HBeAg, and the replication of HBV DNA, respectively. Most of the compounds showed good ant-HBV activity. Especially, the anti-HBsAg activity of LK041-07, LK041-08 and LK042-03, LK042-05 were in the range of 13 to 18μM;the anti-HBeAg activity of LK041-06, LK041-07, LK041-14 and LK042-03 were in the range of 0.1 to 0.73μM; the anti-HBV DNA activity of LK041-04, LK041-06, LK041-08, LK042-07, 13 and LK045-05, LK045-06 were in the range of 0.12 to 1μM. Among them, compound LK041-06, 0 LK041-7 and LK042-03 showed wonderful potency to all the three elements.From the biological data, preliminary structure-activity relationships of 2-pyridones derivatives of anti-HBV activity were summarized as below: (1) Electron-donating groups like alkyl and alkoxy connected with the 1-N were good to the anti-HBV activity, while the electron-withdrawing groups made the compounds lose activity. (2) Alkyl substitution was better than aryl and benzyl substitutions while oxygen atom of the linker was substituted by the same groups together with 1-N atom. (3) Long hydrophobic side chain and heterocycle binding with 2'-O were not good to the anti-HBV activity; however, the activity was maintained when the aryl substitutions were connected with 2'-O through 3~4 binds. (4) Strong electron-withdrawing groups, such as nitro and acetamido groups, on the benzene ring connected with linker made compounds lose anti-HBV activity. (5) The dissociative hydroxyl group on 4'of the benzene ring enhanced the ant-HBV activity, which revealed that the hydrogen-donating groups were favorable for the anti-HBV activity.In this dissertation, a kind of novel 2-pyridone scaffold possessing anti-HBV activity was discovered firstly on the basis of total synthesis and optimization of natural anti-HBV products. Together with the guidance of pharmacophore model of anti-HBV activity, 125 of new compounds have been designed and synthesized. Based on the results of anti-HBV assay, the affection of multi-site modification was investigated and some valuable structures were obtained. The anti-HBV mechanism will be the main task in the future.