| Hepatitis B virus(HBV)is a member of the hepadnaviridae family of viruses,which spread through blood,mother-to-child,sexual contact and skin mucosal damage.Long-term infection leads to chronic hepatitis B(CHB),liver metabolic disorders,liver cirrhosis(LC),and hepatocellular carcinoma(HCC).According to the World Health Organization(WHO),there are about 260 million persons infected with HBV worldwide,mainly distributing in Asia and Africa.Incidence of hepatitis B is very high in China,with about 20 million people suffering from chronic hepatitis B.Hepatitis B,with high incidence and long disease course,cannot be cured by approved drugs such as the nucleoside analogues.Therefore,discovery of safe and efficient non-nucleoside HBV inhibitors is of great significance.With the development of structural biology,multiple proteins in the HBV life cycle have been reported as drug targets in recent years.Among them,HBV capsid protein plays an important role in the life cycle of HBV,including the reverse transcription of pre-genomic RNA,the synthesis of relaxed-circular DNA and the formation of subviral particles.Mechanisms of capsid protein inhibitors include interfering with the normal assembly of nucleocapsids,inducing the formation of irregular polymers,promoting degradation of core proteins and promoting the formation of "envelopes" that do not contain viral genes.HBV capsid protein inhibitors represent a class of the most studied non-nucleoside small molecular inhibitors,not only do they have good inhibitory activity against both wild-type strains and mutant strains,but also they can exert synergistic antiviral effects with nucleoside drugs.GLS4,a heteroaryldihydropyrimidine(HAP)-based inhibitor of HBV capsid protein,showed nanomolar in vitro antiviral activity inhibiting HBV DNA replication(HepG2.2.15 cell line,IC50 = 12 nM).It also demonstrated high activity against adefovir-resistant strains(rtA181V,IC50 = 0.161 μM;rtN236T,IC50 = 0.131μM).GLS4 is currently studied in Phase Ⅱ by Guangdong Dongyangguang Pharmaceutical Company.However,the water solubility and in vivo oral bioavailability(cLogP = 4.7,F = 14%)of GLS4 are poor,which limit its clinical application.In the second chapter of this thesis,the prodrug strategy was employed to solve this problem.The privileged motif "5-methyl-2-oxo-1,3-dioxole" was introduced to the nitrogen atom of the dihydropyrimidine ring.Thus,prodrug of GLS4(1-5)was synthesized and evaluated for its in vitro antiviral activity(cytotoxicity,inhibitory effect on HBV DNA replication,inhibitory effect on HBsAg and HBeAg secretion),water solubility,acute toxicity and pharmacokinetic properties.The results showed that 1-5 demonstrated low cytotoxicity(CC50>50 pM);the IC50 value of 1-5 in anti-HBV DNA replication assay was 0.041 ± 0.045 μM,which was comparable to GLS4(IC50=0.035± 0.025 μM),being 40 times higher than the approved drug lamivudine(IC50= 1.73± 0.28 μM);What’s more,the IC50 values of anti-HBsAg and HBeAg secretion activities of 1-5 were 17.87± 0.8 μM and 0.39 ± 0.1 μM,respectively,which were comparable to the lead compound GLS4(IC50 was 15.82± 0.4 μM and 0.18± 0.03±M,respectively)and much higher than lamivudine(IC50>50 μM).In addition,the solubility of 1-5 in PBS buffer at pH 7.4(15.13 μg/mL)was much higher than the solubility of GLS4 under the same conditions(<0.32 μg/mL).When 1-5 was orally administered to mice at 2 g/kg,it showed weak acute toxicity.However,1-5 demonstrated extreme low in vivo stability and poor pharmacokinetic properties,it may metabolize into other byproducts,and could not smoothly release the active form.In the third chapter,based on the preliminary structure-activity relationships of dihydropyrimidine-based HBV capsid inhibitors,and the crystal structures of capsid protein-ligand complexes,two novel series of dihydropyrimidine derivatives(namely,sulfonamide and phosphoramide-bearing compounds)were designed by targeting the"protein-solevent interface" using knowledge-based pharmacophore hybridization and bioisosterism design strategies.The target compounds were synthesized and evaluated for their in vitro cytotoxicities,inhibitory effects on HBV DNA replication and inhibitory effects on HBsAg and HBeAg secretion.The molecular docking study of the representative compound was also performed.The results showed that the nitro-bearing R1 substituent was favored for the bioactivity in sulfonamide series.Different positions of the substituent make significantly effects on the activity:ortho-and para-substitution contribute to antiviral activity while the meta-substitution impaire the activity.Among the R2 substituents of the phosphoramide series,benzyl was the best,the introduction of fluorine atom further contributed to the activity.However,larger substituents resulted in higher toxicity.Among all synthesized compounds,compound Ⅱ-6m displayed the most potent anti-HBV DNA replication activity(IC50 = 0.76 ± 0.12 μM),which was comparable to the approved drug lamivudine(IC50 = 0.62 ± 0.31 μM).Besides,Ⅱ-6m showed no cytotoxicity under the tested concentration(CC50>50 μM)and moderate anti-HBeAg secretion activity(IC50 = 18.12± 7.9 μM).Molecular docking study showed that the sulfonamide group of Ⅱ-5m forms a hydrogen bond with the main chain of Leu140,which verifies our design idea.Taking Ⅱ-6m as a lead compound,further investigations are warranted and are currently in progress to develop dihydropyrimidine derivatives with improved activity and to better elucidate their antiviral mechanism of action.In summary,on the basis of the structural characteristics of anti-HBV clinical drug candidate GLS4,preliminary structure-activity relationships of dihydropyrimidine-based HBV capsid inhibitors and the crystal structures of capsid protein-ligand complexes,the prodrug,crystallographic overlays-based molecular hybridization and bioisosterism strategies we employed in the drug design process,which led to the discovery of three series of small HAP molecules with high in vitro anti-HBV activity,including "5-methyl-2-oxo-1,3-dioxole"-bearing prodrug of GLS4 and two novel series of dihydropyrimidine derivatives(namely,sulfonamide and phosphoramide-bearing compounds).Furthermore,the structure-activity relationships of the target compounds were discussed and the binding model of the representative compound was analyzed,which laid the foundation of further optimization of HAPs. |
PDF Full Text Request