Discovery of Oxime Ethers as Hepatitis B Virus (HBV) Inhibitors by Docking, Screening and In Vitro Investigation

A series of oxime ethers with C6-C4 fragment was designed and virtually bioactively screened by docking with a target, then provided by a Friedel–Crafts reaction, esterification (or amidation), and oximation from p-substituted phenyl derivatives (Methylbenzene, Methoxybenzene, Chlorobenzene). Anti-hepatitis B virus (HBV) activities of all synthesized compounds were evaluated with HepG2.2.15 cells in vitro. Results showed that most of compounds exhibited low cytotoxicity on HepG2.2.15 cells and significant inhibition on the secretion of HBsAg and HBeAg. Among them, compound 5c-1 showed the most potent activity on inhibiting HBsAg secretion (IC50 = 39.93 μM, SI = 28.51). Results of the bioactive screening showed that stronger the compounds bound to target human leukocyte antigen A protein in docking, the more active they were in anti-HBV activities in vitro.


Introduction
Hepatitis B virus (HBV) infection is a serious worldwide health problem, which causes acute and chronic hepatitis B, cirrhosis, hepatocellular carcinoma, and other hepatic diseases [1]. According to the World Health Organization, 240 million people in the world suffer chronic infection and develop into HBV carriers. About 0.78 million people die per year from HBV-related diseases [2]. The nucleoside analogs used in treatment of anti-HBV play the role of Trojan horse in the synthesis of HBV DNA and suppress replication of HBV [3][4][5][6][7], but they are not effective in eliminating the virus from patients. Meanwhile, HBV therapies with nucleoside analogs in the long term cause serious side effects and resistance [8][9][10][11][12]. For the improvement of HBV therapies, the structural modification of nucleoside analogs has focused and developed many derivatives [13][14][15][16]. Although more and more effective nucleoside analogs agents for treating HBV have been invented and developed, they only inhibit or stop replication of HBV DNA, and do not eliminate HBV cccDNA from patients. The cccDNA in patients will cause HBV DNA replication. Methods to stop and eliminate HBV DNA and cccDNA from HBV patients are still a great challenge for researchers. In searching for more effective anti-HBV agents, many significant anti-HBV non-nucleoside analogs from synthesized compounds [17][18][19][20][21][22][23] and natural products [24][25][26] have been found. Some were designed according to their interactions with receptor by simulating screen [27]. Researchers showed that HLA-A2 with the immunodominant HBcAg18-27 epitope (or HLA-A2.1-restricted CTL epitope) binds peptide of vaccine or of HBcAg to initiate a specific respond of T cell and resolve acute HBV infection [28][29][30]. Some other peptides Molecular docking studies of the oxime ester derivatives were carried out using MOE 2008.10 as docking software in order to rationalize the biological activity results and understand the various interactions between ligand and protein in the active site in detail. HLA-A protein (HLA-A*02:03, PDB ID: 3OX8) [36] was used for docking study. The "Site Finder" tool of this program was used to search for its active site. Three docking procedures for each ligand were performed and the best configuration of each of the ligand-receptor complexes was selected based on energetic grounds. The affinity-scoring function δG was used to assess and rank the ligand-receptor complexes. The docking scores and the hydrogen bonding strength of all the molecules are shown in Table 1. These oxime ester derivatives had a dock score ranging from −12.0185 to −9.1113, and all of them were involved in at least one hydrogen-bonding interaction with the active site of 3OX8 protein. Among them, N in the oxime group (-O-N=C) of 14 compounds interacted with amino acid residues by a hydrogen bond in the active site, which is similar to the reported works [33]. O in the carbonyl group (-C=O) of five compounds interacted with amino acid residues by a hydrogen bond in the active sites. This indicated in theory the roles of the oxime group and carbonyl group in anti-HBV activity. Bioactive results in vitro showed the compound 5c-1 had the most potent anti-HBV activity with IC 50 values of 39.93 µM for HBsAg, the next was 5a-1 (HBsAg IC 50 =74.92 µM), followed by 3c-2 (HBsAg IC 50 = 94.71 µM, HBeAg IC 50 = 93.91 µM). Compounds 5c-1 ( Figure 1) and 3c-2 ( Figure 2) formed two hydrogen bonds in length 2.50 and 2.44 Å, 2.63 and 2.61 Å with O of O=C in amide group of Tyr27 and Tyr63, respectively (Figures 1 and 2), and compound 5a-1 formed only one hydrogen bond in length 2.40 Å with N of C=N in oxime group of Tyr26 ( Figure 3).

Anti-Hepatitis B Virus (HBV) Activity
All synthesized derivatives were assayed for their anti-HBV activities in vitro, which included inhibiting the secretion of HBsAg and HBeAg in HepG2.2.15 cells with lamivudine (3TC, a clinically popular anti-HBV agent) as a positive control. The anti-HBV activities of the compounds were expressed as the concentration of compound that achieved 50% inhibition (IC50) to the secretion of HBsAg and HBeAg. Some compounds in which the inhibition rate of the secretion of HBsAg or HBeAg did not reach 50% after 9d would not have the IC50 value calculated ( Figure 4A,B). The cytotoxicity of compounds was expressed as the concentration of compound required to kill 50% (TC50) of the HepG2.2.15 cells. Parts of compounds were shown to be more active for inhibiting the secretion of HBsAg and had lower cytotoxicity than lamivudine. Eleven among the 18 derivatives displayed higher inhibitory activity against the secretion of HBsAg than lamivudine ( Figure 5), and five among those derivatives demonstrated better inhibitory effect in the secretion of HBeAg than lamivudine ( Figure 6). Among them, compounds 5c-1 and 5a-1 had the most potential as anti-HBV agents.

Anti-Hepatitis B Virus (HBV) Activity
All synthesized derivatives were assayed for their anti-HBV activities in vitro, which included inhibiting the secretion of HBsAg and HBeAg in HepG2.2.15 cells with lamivudine (3TC, a clinically popular anti-HBV agent) as a positive control. The anti-HBV activities of the compounds were expressed as the concentration of compound that achieved 50% inhibition (IC 50 ) to the secretion of HBsAg and HBeAg. Some compounds in which the inhibition rate of the secretion of HBsAg or HBeAg did not reach 50% after 9d would not have the IC 50 value calculated ( Figure 4A,B). The cytotoxicity of compounds was expressed as the concentration of compound required to kill 50% (TC 50 ) of the HepG2.2.15 cells. Parts of compounds were shown to be more active for inhibiting the secretion of HBsAg and had lower cytotoxicity than lamivudine. Eleven among the 18 derivatives displayed higher inhibitory activity against the secretion of HBsAg than lamivudine ( Figure 5), and five among those derivatives demonstrated better inhibitory effect in the secretion of HBeAg than lamivudine ( Figure 6). Among them, compounds 5c-1 and 5a-1 had the most potential as anti-HBV agents.

Structure-Activity Relationship (SAR)
Most of these compounds showed low cytotoxicity to HepG2.2.15 cells lines except the derivatives 3c-2 and 4b-1. The inhibition ratio of part of the derivatives on HBeAg was less than 50% in the test concentration range. The anti-HBV activity of the derivatives was evaluated from their inhibition on the secretion of HBsAg . Compounds 5a-1, 5a-2, 5b-1, 5b-2, 5c-1 and 5c-1 with similar structures but different oxime ether groups showed different anti-HBV activity ( Table 2). Compound 5a-1, with IC50 values of 74.92 μM and 273.87 μM for HBsAg and HBeAg, respectively, was shown to be more effective at inhibiting HBsAg secretion but weaker at HBeAg secretion than that of compound 5a-2 (HBsAg IC50 = 156.27 μM, HBeAg IC50 = 220.09 μM). It was similar to the compounds

Structure-Activity Relationship (SAR)
Most of these compounds showed low cytotoxicity to HepG2.2.15 cells lines except the derivatives 3c-2 and 4b-1. The inhibition ratio of part of the derivatives on HBeAg was less than 50% in the test concentration range. The anti-HBV activity of the derivatives was evaluated from their inhibition on the secretion of HBsAg . Compounds 5a-1, 5a-2, 5b-1, 5b-2, 5c-1 and 5c-1 with similar structures but different oxime ether groups showed different anti-HBV activity ( Table 2). Compound 5a-1, with IC50 values of 74.92 μM and 273.87 μM for HBsAg and HBeAg, respectively, was shown to be more effective at inhibiting HBsAg secretion but weaker at HBeAg secretion than that of compound 5a-2 (HBsAg IC50 = 156.27 μM, HBeAg IC50 = 220.09 μM). It was similar to the compounds
According to the results mentioned above, the SARs were summarized. The N-phenyl amide-substituted methoxy oxime ether derivatives possessed higher anti-HBV activity than other analogs.

Molecular Docking
The docking study was performed using the MOE 2008.10 to understand the ligand-receptor interactions in detail. The crystal structure of human leukocyte antigen (HLA-A) protein (PDB ID: 3OX8) [36] was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). Conformation of all the compounds was constructed in ChemDraw Ultra 7.0 and optimized in HyperChem 8.0.7 Software, and included hydrogen addition, 3D structure conversion, force-field optimization, and geometry optimization. The optimized ligands were built using the builder interface of the MOE program. The crystal structure was imported into MOE and chain A was considered for the docking process as the protein is a dimer consisting of A and B chains. The structure was protonated, unbound waters were deleted, polar hydrogens were added, and energy minimization was carried out. The active site was correlated with the 'Site Finder' module of MOE to define the docking site for the ligands [33][34][35]. The docking procedure was followed using the standard protocol implemented in MOE 2008.10 and the geometry of the resulting complexes was studied using the MOE's Pose Viewer utility.

Materials and Methods
Melting points were determined using a fully automatic melting point apparatus MP420 (Jinan, China) and were uncorrected. Mass spectrometry (MS) spectra ware recorded on Thermo Scientific ITQ 1100 instrument (Thermo Fisher Scientific, Waltham, MA, USA), Varian CP 3800 + Satum 2200 instrument (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were recorded on a Bruker AV III HD 600 MHz ( 1 H/ 13 C, 600 MHz/150 MHz) spectrometer (Brucker, Corp, MA, USA) by using CDCl 3 , CD 3 OH, or DMSO as solvent and TMS as an internal standard. All the chemicals were obtained from local suppliers and were used without further purification.

Chemistry
The general procedure (Scheme 1) for the preparation of compounds (2a, 2b and 2c): succinic anhydride (1 equiv, 10 mmol) was reacted with an appropriate aromatic compound (substituted benzene, 1 equiv, 10 mmol) in DCM (20 mL) in the presence of anhydrous aluminium chloride (1.5 equiv, 15 mmol). The reaction mixture was stirred under anhydrous conditions overnight at room temperature and then ice-cold diluted hydrochloric acid solution was added dropwise. A solid mass separated out which was filtered and purified by recrystallization to give 2a, 2b and 2c [44].
The general procedure for the preparation of compounds (3a, 3b and 3c): a mixture of the appropriate acid 2a-c (1 equiv, 10 mmol) and p-toluenesulfonic acid (0.4 equiv, 4 mmol) in EtOH (20 mL) was refluxed for 6-8 h and evaporated to remove EtOH. The residue was suspended in H 2 O (30 mL) and extracted with EtOAc (2 × 50 mL) which was further purified by column chromatography on silica gel eluting with AcOEt-petroleum ether (1:4, v/v) to get 3a-c as crystals. The general procedure for the preparation of compounds (4a-c-5a-c): an appropriate acid 2a-b (1 equiv, 10 mmol), and 4-dimethylamioprdine (0.02 equiv, 0.2 mmol) was added to a solution of the furfuryl alcohol or aniline (1 equiv, 10 mmol) in THF (20 mL). The mixture was stirred and cooled to 0 • C and then N,N-dicyclohexylcarbodiimide (DCC) (1.1 equiv, 11 mmol) was added over a 5-min period and the reaction was stirred under anhydrous conditions for 6-8 h at room temperature. The mixture was filtered and the filtrate was evaporated to yield a crude product which was finally purified by recrystallization to give 4a, 4b, 4c, 5a, 5b and 5c as crystals [38][39][40][41][42][43].

Cells and Cell Culture
HepG2.2.15 (clonal cells derived from human hepatoma cell line G2) cells were provided by the Chinese Academy of Medical Sciences (P.R. China) and maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum and 380 µg/mL of G418, 50 ug/mL of kanamycin, and 0.03% L-glutamine at 37 • C in a 5% CO 2 atmosphere with 100% humidity [26,33].

Drug Treatment
HepG2.2.15 cells were seeded at a density of 1 × 10 5 cells/ml (200 mL/well) in 96-well plates and maintained at 37 • C for 24 h prior to extract addition, followed by treatment with various concentrations of drugs. Lamivudine (3TC) was served as the positive control. Cells were treated with drug-containing fresh medium every 3 d for up to 9 d in a time-dependent experiment. Medium was taken at the 9th day of the treatment and stored at −20 • C until analysis. The IC 50 and selected index (SI) of each compound were calculated, respectively.

Cell Toxicity
Logarithmically growing cells were seeded in 96-well culture plates at a density of 1 × 10 5 cells/mL (200 mL/well). They were cultured for 24 h and then treated with various concentrations of drugs. Optical density (OD) values were read at 450 nm after 9 days and the percentage of cell death was calculated; the cells were treated with drug-containing fresh medium every 3 d for up to 9 d. After drug treatment, the cytotoxicity was measured using the MTT assay [45,46].

Method for HBsAg and HBeAg Inhibition Assays
The levels of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the supernatant of the HepG2.2.15 cell were simultaneously detected using enzyme-linked immunosorbent assay (ELISA) kits (Rongsheng Biotechnology Co. Ltd., Shanghai, China) according to the manufacturer's instructions.The synthesized derivatives were expressed as the concentration of compound that achieved 50% inhibition (IC 50 ) to the secretion of HBsAg and HBeAg. The selectivity index (SI) was determined as the ratio of CC 50 to IC 50, which is a major pharmaceutical parameter of estimates possible for future clinical application.

Conclusions
In a summary, most of the compounds in the series of oxime ethers with a C 6 -C 4 fragment, which were designed and virtually bioactively screened by docking with a target protein, showed anti-HBV activities in an in vitro assay. Among them, compound 5c-1 showed the most potent activity inhibiting HBsAg secretion (IC 50 = 39.93 µM, SI = 28.51). The results of bioactive screening showed that the stronger the compounds bound to a target protein (human leukocyte antigen A protein) in docking, the more active they were for anti-HBV in vitro.