| Viral hepatitis type B, referred to as Hepatitis B, is a serious infectious disease caused by the hepatitis B virus (HBV). Long-term development of Hepatitis B can lead to acute or chronic viral hepatitis, severe hepatitis, liver cirrhosis (LC) and hepatocellular carcinoma (HCC). According to the report of World Health Organization(WHO), about 240 million people worldwide had been involved in chronic HBV infection, and more than 686,000 people die every year. Due to the high incidence, long course and difficulty to cure, hepatitis B has become a major disease which seriously affect people's health and social development, therefore research of effective drugs of HBV has become the top priority. China is a high prevalence country of hepatitis B with about 90 million HBV carriers and an average 28 million people have chronic hepatitis B (CHB).HBV is a member of hepadnaviridae, whose genome is partially double-stranded circular. But its replication process possesses the same characteristics as RNA retroviruses replication process, which including: adsorption and fusion to the target cells, DNA repair and transcription, the translation and reverse transcription of progenome RNA, viral particle assembly and budding, etc. Based on the understanding of HBV life cycle and molecular biology, some drugs have been developed for the treatment of CHB. Those currently used drugs mainly include interferon,immunomodulatory drugs, and DNA polymerase inhibitors. Despite the dual role of immunomodulation and anti-virus of a-interferon (IFN-a), it is effective only to 30%-40% of patients, moreover adverse reactions limit its clinical application. Thymosin-al(Tal) and other immunomodulatory drugs can improve the body's specific immunity to HBV but lack of pertinency, so they can be used only as adjuvant drugs or as part of a combined treatment with other HBV drugs. Five nucleoside/nucleotide HBV DNA polymerase inhibitors (lamivudine, adefovir dipivoxil, entecavir, telbivudine and tenofovir) are widely used in clinic. However, because of the genetic heterogeneity of HBV genome, the virus can easily develop resistance to this kind of drugs. Therefore,discovery and development of novel anti-HBV inhibitors with improved potency, low toxicity, or novel modes of action is undoubtedly essential to combat the HBV infection.Targeting the capsid protein of hepatitis B virus (HBV) and thus interrupting normal capsid formation has been an attractive approach to block the replication of HBV viruses. In the second chapter, drug candidate NZ-4 and another capsid protein inhibitor AT-130 were employed as lead compounds, eight small series of heterocycle derivatives (pyrazole, thiazole, pyrazine, pyrimidine and pyridine analogs) were designed as potential HBV non-nucleoside inhibitors through scaffold hopping,bioisosterism and pharmacophore hybrid-based strategies. In Series ?, the thiazole ring was replaced by pyrazole ring through bioisosterism, and optimal group thiazole was emploied at 1-position of pyrazole ring, besides, with continuous exploration of structure-activity relationships (SARs) at 4-position of pyrazole, diverse groups were introduced to improve antiviral activity. In Series ?, the thiazole ring was also replaced by pyrazole ring based on the bioisosterism principle, meanwhile, different substituents were introduced to 1 -position of pyrazole ring to find optimal groups. In addition, while 3-F phenyl group was introduced to 1-position of pyrazole, SARs of 4-position was further discussed. In Series ?, the thiazole ring was also replaced by pyrazole ring and the effects of 3 -position of pyrazole on the activity of the antiviral were discussed. In Series IV, the designed compounds retained the advantaged scafold of 2',2-bis-thiazole,and 4-position of lead compound was further diversely modified to find high efficiency and low toxicity lead compounds. In Series ?-?, the thiazole scaffold of lead compound NZ-4 was replaced by diverse heterocycles (pyrazine, pyridine and pyridine),which was first designed to explore the anti-HBV activity of these derivatives. In Series VIII, the privileged structure thiazole scaffold and pharmacophores of lead compound NZ-4 were also employed, however, N and S of thiazole scaffold as hydrogen bond receptors were exchanged each other, which will lay the foundation for further optimization.The in vitro anti-HBV assay demonstrated that the scaffold of thiazole was better than pyrazine, pyridine and pyridine. The designed compound IV-8c displayed the most potent activity against the replacation of HBV DNA with IC50 of 2.2±1.1 pM,which was better than lead compound NZ-4 (2.3±0.5?M); Besides,compound I-8g also exhibited potent anti-HBV DNA replication activity (ICs50 3.8±0.3 ?M), which was similar with NZ-4; In addition, I-8g also showed better selective index (SI > 26.3) than NZ-4 (SI = 25.7),and is worthy of further investigation as a lead compound.Moreover, surface plasmon resonance (SPR) experiment confirmed that the target of these inhibitors was HBV core protein, and compounds I-8g and IV-8c showed the simmilar affinity constant (KD=60.8 ?M and 60.0 ?M) with lead compound NZ-4 (KD?50.6 ?M), which was consistent with cell activity. These results encourage further optimization of them as novel lead compounds for development of more potent HBV inhibitors.In the third chapter, drug candidate GLS4 was employed as lead compound,based on the interactions between heteroaryldihydropyrimidine analogys (HAPs) and HBV capsid protein investigated by a comprehensive molecular simulation approach including CoMFA and CoMSIA in 3D-QSAR and molecular docking, a series of novel HAPs-triazole derivatives was designed and synthesised. Among them, morpholine substitudent was replaced by triazole group to improve the metabolic stability; and some hydrophilic groups have the potential to form hydrogen bonding with the surrounding amino acid residue Ser121, some hydrophobic groups will form hydrophobic forces with the amino acid residue Pro 138, which will enhance its antiviral activity. The molecular modeling study revealed that the designed compound (R = 2-aminophenyl) generally overlapped the lead compound GLS4, and 2-bromo-4-fluorophenyl group was occupied the same hydrophobic pocket defined by the residues Pro25. Asp29, Leu30, Thr33, Trp102, Ile105 and Ser106; The thiazole group was occupied the hydrophobic pocket formed by residues Trp 102,Phe23, Phe 122 and Tyr118, meanwhile the nitrogen atom of thiazole group form hydrogen bonding interactions with the surrounding amino acid residue Leu 140 through water bridges; In addition, ester group was located at the top of the cavity defined by the amino acid residues Thr109, Phe 110, Thr33 and Leu37.Activity results showed that minor changes in substituents may cause significant changes in activity, and some even disappeared; In R substitudent, polar group in the ortho position was beneficial to anti-HBV DNA replication activity; However, the R group was directed toward the solvent opening region, and the size of the substituent greatly affected the activity, larger volumes of amides and sulfonamide substituents resulted in loss of antiviral activity. Several compounds exhibited potent antiviral activity. Among them, compound XIV-5a displayed the most potent activity against the replacation of HBV DNA with IC50 of 0.35±0.04 ?M, which was better than the positive drug lamivudine (0.54±0.18 ?M); It also showed less cytotoxicity than lead compound. In addition, the selectivity index (SI) of the inhibition of HBV DNA replication is superior to or equivalent to the lead compound GLS4 and the marketed drug lamivudine, which can be further modified as a lead compound.In the fourth chapter of this paper, based on pharmacophore model study,according to bioisosterism and molecular hybridization, taking 2-pyridone (natural products derivatives with potent anti-HBV activity) and thiazolide non-nucleoside HBV inhibitors as lead compounds, we designed and synthesized a series of novel 3-aryl-5-amide substituted 2-pyridone derivertives. The NH group was introduced into the central core to improve the hydrophilic profile; moreover, on the right wing, C-N bond between phenyl and pyridone ring was replaced by widely applicable C-C linkage;at the left wing, amide group was also introduced, which have the potential to improve anti-HBV activity and water solubility.The antiviral activity in the cell-based assay showed that several designed compounds displayed potent anti-HBV DNA replication activity. Among them,compound XII-5u exhibited best activity with IC50 of 4.5±2.3 ?M. which was similar with lead compound and lightly less than the marked drug lamivudine. Besides, it showed potent activity against the secretion of HBeAg with IC50 of 9.8 ±2.8 ?M. In addition, water solubility of compound XII-5u was evaluated by HPLC method. It was shown that the solubility in the pure water and PBS buffer (PH=7.4) was 13.26 ?g/mL and 28.68 ?g/mL, respectively, which improved certain water solubility and verified the experiment design. In a word, XII-5u can be as the lead compound for further modification.In the fifth chapter, based on the mechanism of nucleoside anti-HBV drugs, SARs and anti-resistance drug design strategy, marked drug telbivudine was taken as lead compound, and novel benzothiadiazine was designed as novel bases of nucleoside.Among them, telbivudine base 2-carbonyl group was replaced by sulfone through bioisosterism principle to make stronger hydrogen bonding donor of ortho NH and enhance the hydrogen bonds binding force between the bases and natural nucleoside analogues bases, which can improve the affinity of nucleoside analogues with DNA polymerase and the synthesis rate of its incorporation in DNA; Regarding the part of ribose, the open chain ribose of adefovir dipivoxil-phosphoric acid methyl ethyl oxygen(PME) was employed to enhance the activity aganist HBV resistant strains and reduce severe side effects; Meanwhile, the phosphate group was changed into ester according to the prodrug principles, in order to improve antiviral activity, pharmacokinetic properties and bioavailability. The antiviral activity in the cell-based assay showed that the designed compounds XIV-5, XIV-7a and XIV-7c displayed certain anti-HBV DNA replication activity under 50 ?M concentration with the inhibition rate of 68.3%, 64.1%and 57.6%, respectivity, which was weaker than market drug lamivudine (88.9%).Innovation: Because of the genetic heterogeneity of HBV genome, the virus can easily develop resistance to marked drugs. Therefore, discovery and development of novel anti-HBV inhibitors with improved potency, low toxicity, or novel modes of action is undoubtedly essential to combat the HBV infection. Based on scaffold hopping,bioisosterism and pharmacophore hybrid, prodrug and stucture-based rational drug design principle, eight new class heterocycle HBV inhibitors (pyrazole, thiazole,pyrazine, pyrimidine, pyridine, pyridone, benzothiadiazine and dihydropyrimidine analogs) were designed and synthesised.Based on the structure of the target compound, 14 synthetic routes, 50 intermediates and 112 target compounds were designed and synthesized through literature research and retrosynthetic analysis, among them, more than 130 compounds have not been reported in the literature. The anti-HBV activity of 108 synthsized target compounds were evaluated, their cytotoxicity, anti-HBV antigen secretion activities(HBsAg and HBeAg) and anti-HBV DNA replication activity were assayed with cell counting kit-8 (CCK-8), enzyme linked immunosorbent assay (ELISA) and PCR methods, respectively. Besides, the affinity of target compounds and HBV core protein was evaluated by surface plasma resonance experiment. Among them, compound IV-8c displayed the potent activity against the replacation of HBV DNA with IC50 of 2.2±1. 1 ?M, which was better than lead compound NZ-4 (2.3±0.5 ?M); and compound XIV-5a showed less cytotoxicity than lead compound and displayed the most potent activity against the replacation of HBV DNA with IC50 of 0.35±0.04 ?M, which was better than the positive drug lamivudine (0.54±0.18 ?M); In addition,compound ?-5u also exhibited potent activity against the replacation of HBV DNA with IC50 of 4.5±2.3 ?M,which was similar with lead compound and improved certain water solubility, which provided valuable information for the development of anti-HBV drug research. |
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