Design, Synthesis, and Bioactive Screen In Vitro of Cyclohexyl (E)-4-(Hydroxyimino)-4-Phenylbutanoates and Their Ethers for Anti-Hepatitis B Virus Agents

A series of oxime Cyclohexyl (E)-4-(hydroxyimino)-4-phenylbutanoates and their ethers were designed, synthesized, and evaluated for anti-hepatitis B virus (HBV) activities with HepG 2.2.15 cell line in vitro. Most of these compounds possessed anti-HBV activities, and among them, compound 4B-2 showed significant inhibiting effects on the secretion of HBsAg (IC50 = 63.85 ± 6.26 μM, SI = 13.41) and HBeAg (IC50 = 49.39 ± 4.17 μM, SI = 17.34) comparing to lamivudine (3TC) in HBsAg (IC50 = 234.2 ± 17.17 μM, SI = 2.2) and HBeAg (IC50 = 249.9 ± 21.51 μM, SI = 2.07). Docking study of these compounds binding to a protein residue (PDB ID: 3OX8) from HLA-A2 that with the immunodominant HBcAg18–27 epitope (HLA-A2.1- restricted CTL epitope) active site was carried out by using molecular operation environment (MOE) software. Docking results showed that behaviors of these compounds binding to the active site in HLA-A protein residue partly coincided with their behaviors in vitro anti-HBV active screening.


Introduction
Hepatitis B virus (HBV) remains a serious health problem for leading risks of liver related diseases for many people in the world. An estimation of 257 million people is chronically infected with HBV in the world, and among them, about 15-40% develop liver cirrhosis, hepatic failure, and hepatocellular carcinoma. More than 887,000 people die every year for complications of HBV [1,2]. Therapies of hepatitis B infection currently include methods of direct-acting antivirals (just as with nucleoside analogs) and host-targeting antivirals (just as with interferon) [3]. The nucleoside analogs in treatment of anti-HBV play a role of Troy horse in synthesis of HBV DNA and suppressed replication of HBV, but they are not effective to eliminate virus completely from patients [4][5][6]. Meanwhile, therapies of HBV with nucleoside analogs in the long term cause side effects and resistance [7][8][9]. Therefore, it is still a tremendous challenge to develop new anti-HBV agents for further improvement of anti-HBV therapy. Many non-nucleoside analog compounds with anti-HBV activities have been found and invented from synthesized compounds [10,11] and natural products [12][13][14]. Researches showed that HLA-A2 with the immunodominant HBcAg18-27 epitope (HLA-A2.1-restricted CTL epitope) binding peptides of vaccine or of HBcAg initiated specific respond of T cell and resolved cute HBV infection [15][16][17][18][19]. Other peptides specifically bound to the HLA-A2 residue activate CTL responds to prevent infection and eliminate HBV [20,21]. Therefore, a proposal can be implied that other non-peptide compounds specifically interact with protein residue in HLA-A2.1 restricted CTL epitope may initiate CTL respond Inhibition of secreting HBsAg and HBeAg of these synthesized compounds were assayed in HepG 2.2.15 cells with lamivudine (3TC) as a positive control. Results of cytotoxicity and anti-HBV activities were showed in Table 1

Molecular Docking Study
The docking software MOE 2008.10 was used for molecular docking of oxime derivatives and a protein residue in HLA-A2.1-restricted CTL epitope (PDB ID: 3OX8) was selected for docking study. Molecular docking results clearly revealed interactions between ligands and protein in the active site. Affinity scoring function ∆G of the protein-ligand complexes and other docking results were shown in Table 2. Significant interactions with protein residue in docking were compound 4A-3 and 4D-3 for −22.8858 kcal/mol and −21.2040 kcal/mol, respectively. Two hydrogen bonds of lengths 2.29 and 2.58 Å for N of O-N in the oxime group with Tyr27 and Tyr63, respectively, was found for compound 4A-3 (Figures 2 and 3).

Structure-Activity Relationship (SAR)
Oximes (4A-1, 4B-1, 4C-1 and 4D-1) which R=H showed weaker anti-HBsAg and anti-HBeAg activities, weaker affinities to the selected target protein in docking than those compounds which R=Me and benzyl. When these oximes were etherified, anti-HBV activities of their ethers increased, meanwhile cytotoxicity of these ethers decreased by comparing to their relative oximes. Methyl ethers 4A-2, 4B-2, 4C-2 and 4D-2 showed more significant anti-HBV active in their relative groups ( Figure 4). Results also revealed that substitute groups in phenyl (groups 4B, 4C and 4D) increased anti-HBV activities by comparing to relative compounds in 4A group. Docking results showed that N or O atoms in ON fragment and O atom in C=O groups in the oxime derivatives interacted with ammonic acid residues by hydrogen bond, and their docking affinity partly coincided with their anti-HBV activities in vitro. Similar structure-activity relationships were found in our previous works [23].
ammonic acid residues by hydrogen bond, and their docking affinity partly coincided with their anti-HBV activities in vitro. Similar structure-activity relationships were found in our previous works [23].  ammonic acid residues by hydrogen bond, and their docking affinity partly coincided with their anti-HBV activities in vitro. Similar structure-activity relationships were found in our previous works [23].

Materials and Methods
Melting points were measured on an uncorrected WRX-4 electrothermal melting point apparatus (Shanghai, China). 1 H NMR and 13 C NMR spectra were recorded on Bruker AVANCE III HD600 ( 1 H/ 13 C, 600MHz/150MHz) spectrometer (Bruker, Bremerhaven, Germany) using TMS as an internal standard. The mass spectra were recorded on a Finnigan LCQ Deca XP MAX mass spectrometer (Thermo Fisher, San Jose, CA, USA) equipped with an ESI source and an ion trap analyzer in the

Materials and Methods
Melting points were measured on an uncorrected WRX-4 electrothermal melting point apparatus (Shanghai, China). 1 H NMR and 13 C NMR spectra were recorded on Bruker AVANCE III HD600 ( 1 H/ 13 C, 600MHz/150MHz) spectrometer (Bruker, Bremerhaven, Germany) using TMS as an internal standard. The mass spectra were recorded on a Finnigan LCQ Deca XP MAX mass spectrometer (Thermo Fisher, San Jose, CA, USA) equipped with an ESI source and an ion trap analyzer in the

Materials and Methods
Melting points were measured on an uncorrected WRX-4 electrothermal melting point apparatus (Shanghai, China). 1 H NMR and 13 C NMR spectra were recorded on Bruker AVANCE III HD600 ( 1 H/ 13 C, 600MHz/150MHz) spectrometer (Bruker, Bremerhaven, Germany) using TMS as an internal standard. The mass spectra were recorded on a Finnigan LCQ Deca XP MAX mass spectrometer (Thermo Fisher, San Jose, CA, USA) equipped with an ESI source and an ion trap analyzer in the positive ion mode/in the negative ion. Silica gel GF-254 was used in thin-layer chromatography, and silica gel H used in flash column chromatography (Qingdao Haiyang Chemical, Qingdao, China).
All solvents and reagents were analytical grade. The purity of target compounds was assessed on the basis of analytical HPLC (Thermo Fisher Ultimate 3000), and the results were > 95%.

Preparation of Oximes and Their Ethers
Compound 3 (3A, 3B, 3C, and 3D, 3.84mmol) and substituted hydroxylamine hydrochloride (7.7 mmol) in CH 2 Cl 2 (20 mL) with pyridine (2 mL) was refluxed for 12 h (Scheme 1). After stopping, the resultant solution was evaporated to give a residue under vacuum. This residue was dissolved in ethyl acetate (20 mL), then washed with saturated sodium chloride solution (20 mL × 3). The organic phase was dried with anhydrous sodium sulfate, and evaporated to give a crude product. This crude product was purified by flash column chromatography with an eluent of ethyl acetate/petroleum ether

Statistical Analysis
SPSS 20.0 and MICROSOFT EXCEL 2010 were used for the statistical analysis. All data were expressed as the mean ± standard error of mean (S.E.M.). The differences among the groups were analyzed by one-way analysis of variance (ANOVA) with a Tukey post hoc test when comparing multiple groups. P values lower than 0.05 were regarded as statistically significant.

Conclusions
A series of cyclohexyl (E)-4-(alkyl imino)-4-(4-substituted-phenyl) butanoates possessed obvious and significant anti-HBV activities with SI HBsAg values from 0.89 to 13.41 and SI HBeAg values from 1.06 to 17.34 compared to control lamivudine in SI HBsAg values 2.20 and SI HBeAg values 2.07. Results also revealed that oximes were weaker in inhibiting HBV, and more cytotoxic than their relative ethers; meanwhile, methyl ethers were the most active in inhibiting HBV among compounds in every group. Compound 4B-2 among these compounds showed the best inhibition of HBsAg and HBeAg secretion.