Design,Synthesis And Biological Evaluation Of Novel Fused Heterocycle Derivatives As Non-nucleoside HBV Inhibitors

Hepatitis B is an infectious inflammatory disease of the liver, which is caused by hepatitis B virus (HBV). Currently, clinically available drugs against HBV infection approved by FDA comprise interferon and nucleos(t)ide analogues. Regrettably, the marketed drugs are not idea for the appearance of severe side effects and the emergence of drug-resistant virus strains, which encourage the development of novel non-nucleoside analogues targeting non-polymerase viral. Non-nucleoside HBV inhibitors, possessing different structure and various mechanism, gradually become the hot spot in the field of HBV study. And the small synthetic heterocyclic molecules, with the characteristics of easy synthesis and drug-likeness, have distinguishing advantages. However, the candidates failed to be approved because of the high cytotoxicity, poor activity and pharmacokinetic properties. Therefore, there is an urgent need for the development of non-nucleoside inhibitors with safety, high efficiency, low toxicity and anti-resistance.Benzimidazole derivatives are of wide interest because of their diverse biological activities and clinical applications. Increasing number of benzimidazoles display effective anti-HBV activity, which can be selected as benchmark compounds for further investigation. To improve the water solubility and the oral bioavailability of the leads, a series of novel 3H-imidazo[4,5-b] pyridine derivatives was designed by introducing the nitrogen atom in benzene ring. The active groups of the lead compounds were retained to guarantee the anti-HBV activity. Linkers with different length and structure between the nuclear core and the right-side ring were introduced to evaluate their influence towards anti-HBV activity. Meanwhile, as the isostere of benzimidazoles, another series of purine analogues was designed for further structure-activity relationships study.Two series of compounds with 15 3H-imidazo[4,5-6]pyridines and 20 purine analogues were synthesized. All of the target compounds were characterized by mass spectrometry,1H nuclear magnetic resonance (1H NMR) spectroscopy and 13C nuclear magnetic resonance (13C NMR).Using 3-TC as the control compound, the target compounds were evaluated for their anti-HBV activity and cytotoxicity on the HepG2.2.15 cell line, which include the activity of inhibit HBsAg and HBeAg secretion and DNA replication by the methods of ELISA and PCR respectively. The results were expressed as IC50 (concentration of compound required for 50% inhibition of HBeAg, HBsAg and DNA replication), CC50 (Concentrations of compounds required for 50% extinction of HepG 2.2.15 cells). And selectivity index (SI) determined as the value of CC50/IC50.Similar to benzimidazoles,3H-imidazo[4,5-6]pyridine derivatives exhibited potent activity against HBV DNA replication activity with low cytotoxicity and moderate activity against HBsAg or HBeAg secretion. Introducing 4-methylbenzoyl to the N-1 position of the core could increase the activity. The alkyl linker was important for potent antiviral activity. Notably, compounds 6f (IC50= 5.06μM, SI>19.76) and 6g (IC50= 2.43μM, SI> 41.15) displayed similar activity with 3-TC (IC50= 1.15μM, SI> 86.96), which can be chosen as the benchmark for the further optimization to develop favourable candidates for the treatment of HBV infection.Consistently, the results further proved that derivatives with relatively good activities should bear a flexible linker and 4-methylbenzoyl at the N-1 position. Unlike the previous compounds, most purine analogues lost activity against DNA replication. However, compound LN-S-10 was found to inhibit the secretion of HBsAg (IC50= 6.63μM, SI=1.17) and HBeAg (IC50= 4.43μM, SI=1.72) potently, which suggested the possible novel mechanism unlike the series one.In summary, based on benzimidazoles and classical bioisosterism strategy, two series of novel compounds were designed, synthesized and evaluated for their anti-HBV activities. Among them, several inhibitors were found with potential anti-HBV activity and novel mechanism, which pave the way for the anti-HBV treatment.