Dihydroisoindolo [ 2 , 1-a ] quinazoline-5 , 11-dione derivatives as potent and selective inhibitors targeting hepatitis B virus

The construction of dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives (4a–4m), by  the condensation isatoic anhydride, appropriate amines and 2-formylbenzoic acid by using silica sulfuric acid as catalyst was reported. These dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives (DIQ) were identified as potent inhibitors of HBV capsid assembly. The newly synthesized dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives 4a-4m were characterized by 1 H NMR, 13 C NMR, and Mass spectrum and evaluated for their anti-HBV activity. Majority of the synthesized compounds inhibited the expression of viral antigens at low concentration. But five compounds, 4a, 4b, 4c, 4f, and 4m were shown potent inhibition of HBV DNA replication at submicromolar range. Of these compounds, compound 4a was the most active when compared with lamivudine.


Introduction
Worldwide hepatitis B virus (HBV) becomes serious problem which is causing disease for more than 2 billion people.More than 360 million individuals were chronically infected from liver cirrhosis and hepatocellular carcinoma (Dény and Zoulim, 2010).The current therapies including vaccines, immunomodulators, interferon-, polyethylene glycol interferon- and nucleoside drugs for treating HBV are still unsatisfactory, due to high recurrence, drug resistance and inevitable side effects including influenza-like illness, myalgia, headache, reduction of neutrophilic granulocyte and blood platelet, etc (Sato and Mori, 2010;Locarnini and Mason, 2006;Wong et al., 1993;Fattovich et al., 1998).Therefore, it is important to explore novel classes of drugs with different antiviral targets and mechanisms for anti-HBV purposes.
either from sigma or Merck, and all reagents were of analytical reagent grade.Thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates and visualized under UV light. 1 H NMR spectra were recorded with Varian Mercury Plus 400 MHz instrument. 13C NMR spectra were recorded with a Varian Gemini 100 MHz instrument.All the chemical shifts are reported in δ (ppm) using TMS as an internal standard.Multiplicity is indicated by one or more of the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad); the coupling constants (J) correspond to the order of the multiplicity assignment.Mass spectra were recorded with a PE Sciex model API 3000 instrument.All the reactions were carried out under nitrogen atmosphere.

Biological evaluation
Cell culture: HepG2 2.2.15 cells, derived from HepG2 human hepatocellular carcinoma cells, were stably transfected with a head-to-tail HBV DNA dimer and were maintained in MEM with heat-inactivated 10% fetal bovine serum (FBS) and 1% antibiotics.In parallel experiments, human Huh7 hepatoma cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with heat-inactivated 10% FBS and 1% antibiotics.HepG2 2.2.15 and Huh7 cells were both grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

Cell viability assay:
The cytotoxic effects of synthesized compounds were determined using a Cell Titer 96 cell proliferation assay kit (MTS) (Promega, Madison, WI, USA).In order to pinpoint the toxicity limits in HepG2 2.2.15 and Huh7 cells, they were plated into 96-well plates at a density of 4 x 10 4 cells/mL for 24 hours.Cells were then treated with serial dilutions of compounds ranging 2.5-160 mg/mL, and the mixture was incubated for 3 days.Cell toxicity was calculated according to the maker's protocol.All the results were performed in triplicates, and results are presented as relative percentages over that of the control group.

Determination of HBV expression levels:
After treating HepG2 2.2.15 cells or HBV-transfected Huh7 cells, levels of the HBsAg and HBeAg proteins were measured in culture media using an EIA kit (Johnson and Johnson, Skillman, NJ, USA) according to the manufacturer's instructions.
SEC general procedure: Capsid assembly was initiated by mixing Cp149 with test compounds in 2 buffer incubation 24 hours, respectively.Assembly reactions were examined on a Superose column (Biosep-SEC-S3000) mounted on HPLC system equipped with an auto injection module.The column was equilibrated with 100 mM HEPES pH 7.5, 300 mM NaCl at 0.6 mL/min.

Results and Discussion
In the initial studies we investigated the cylclization reaction of 2-formylbenzoic acid (3) with isatoic anhydride (1) and 2,4-difluoro aniline (2a) using SSA as catalyst in ethanol under reflux (Scheme-1) for 2 hours which yielded the required compound 4a in 89% (Table I, entry 1).Several amines were used for checking the compatibility of the method.All the amines including aliphatic, aromatic and also heterocyclic amines were well tolerated with the current methodology to yield the desired products in good to better yields.Thus, the structures of the synthesized target compounds are listed in Table I.
Inhibition activities for HBV replication of the compounds 4a-4m were determined in the HepG2.2.15 cells, which constitutively produces HBV genomes, and secretes virus-like particles (Korba and Gerin, 1992).Lamivudine (3TC) was used as positive control.To ascertain the cytotoxic effects of the tested compounds, the cell viability was determined after the cells exposed to the compounds for 48 hours (Table I).Hep G2.2.15 cell contained multiple copies of the HBV genome, which were stably integrated into the host cell genome and was widely used as a useful 'in vitro' model for evaluation of novel anti-HBV drugs.So, in the experiment, the Hep G2.2.15 cell line as in vitro cellular model was chosen.
All the synthesized derivatives were tested in vitro in HepG2.2.15 cells for cytotoxicity and anti-HBV activity.The properties of target compounds were summarized in Table II, in which they were compared to the drug lamivudine.Compounds 4a, 4b, 4c, 4f, 4n, and 4m displayed good to better anti HBV activity.
A comparison of the IC50 values of the derivatives 4a-4m clearly indicated that the polarity of substituent and size of the substituent on the substitution had noticeable effect on its antiviral activities (Table II).4a (IC50 = 4.13 µM), bearing a 2,4-difluorophenyl substituent, was comparably potent to the known inhibitor of  Among all the compounds 4a, 4b, and 4m which are showing better anti-HBV activities, compounds containing the electron-withdrawing groups such as fluorine or chlorine and heterocyclic system.
To gain better understand into the mechanisms of our compounds, 4a was investigated to examine the effect on preformed Hepatitis B virus (HBV) capsid and on HBV capsid assembly by size exclusion chromatography (SEC) (Stray et al., 2005).Recovered protein was assigned to the void (aberrant capsid induced by 4a, 8.6 min), capsid (9.6 min) and dimer (12.6 min) based on the HPLC chromatogram.
The results suggested that 4a can break the equilibrium and change the product of HBV core protein selfassembly.By structural biology, we established a screening system for anti-HBV compounds that target on nucleocapsid.SEC would be a much better method to discover the strong antiviral compounds because of its objectivity, convenience and precision.

Figure 1 :
Figure 1: SEC showed the effect of 4a on capsid assembly

Table II Anti-HBV activity and cytotoxicity of target compounds in vitro
a TC50 is 50% cytotoxic concentration in HepG2.2.15 cells; b IC50 is 50% inhibitory concentration; c Selectivity index (SI: TC50/IC50); d No antiviral activity at the concentration lower than its TC50; e 3TC -Lamivudine