Design, Synthesis and Bioactive Evaluation of Oxime Derivatives of Dehydrocholic Acid as Anti-Hepatitis B Virus Agents

Oxime derivatives of dehydrocholic acid and its esters were designed for anti-hepatitis B virus (HBV) drugs according to principles of assembling active chemical fragments. Twelve compounds were synthesized from dehydrocholic acid by esterification and oxime formation, and their anti-hepatitis B virus (HBV) activities were evaluated with HepG 2.2.15 cells. Results showed that 5 compounds exhibited more effective inhibition of HBeAg than positive control, among them 2b-3 and 2b-1 showed significant anti-HBV activities on inhibiting secretion of HBeAg (IC50 (2b-3) = 49.39 ± 12.78 μM, SI (2b-3) = 11.03; IC50 (2b-1) = 96.64 ± 28.99 μM, SI (2b-1) = 10.35) compared to the Entecavir (IC50 = 161.24 μM, SI = 3.72). Molecular docking studies showed that most of these compounds interacted with protein residues of heparan sulfate proteoglycan (HSPG) in host hepatocyte and bile acid receptor.


Introduction
Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), and still a major global health problem, which causes chronic infection, and puts people at a high risk of death from cirrhosis and liver cancer [1,2]. According to World Health Organization (WHO) reports, an estimated 257 million people are living with the hepatitis B virus infection in the world (defined as hepatitis B surface antigen positive). In 2015, hepatitis B resulted in 887,000 deaths, mostly from complications (including cirrhosis and hepatocellular carcinoma) [3]. The nucleos(t)ide analogues are recommended for the treatment of chronic hepatitis B in the current consensus guidelines due to their significant suppression of HBV replication [4][5][6][7]. Unfortunately, this treatment is not satisfactory due to the limitations and side effects of nucleos(t)ide drugs. HBV therapy with nucleoside analogs, in long term, has developed resistance and obvious decreased inhibition effects [8][9][10]. The disadvantages of nucleoside analogs prompted us and other researchers to invent and find new structural non-nucleoside analog compounds [11][12][13][14][15][16]. Many anti-HBV bioactive non-nucleoside analog compounds have been designed and developed on the basis of their interactions with receptor using molecular docking [17][18][19][20][21]. When HBV receptor binding domain PreS1 and PreS2 protein (including L protein, M protein and S protein) interact with small molecules, the virus will not allow entry to hepatocyte. A receptor, heparan sulfate proteoglycan (HSPG), which is critical for virus attachment and helps enrich virions on the cell surface (bringing them in close proximity to the receptor) from pre-S2 in hepatocyte interacts with small molecules; the virus will be inhibited to attach the hepatocyte and endocytosis will not be allowed [22]. Cholic acids and their derivatives were reported as liver-targeted vehicles for drug delivery due to their existence in the liver with no side effects and metabolization through enterohepatic circulation [23][24][25][26]. Dehydrocholic acid (DHCA) is a derivative of cholic acid [27] containing one carboxyl group and three carbonyl groups. Drugs targeted and concentrated to the liver organ will increased their anti-HBV effectivity and decreased side effects. Therefore DHCA, with reactive functional groups, was considered as a liver-target vehicle to deliver drugs to the liver in our present work. Based on the bioactivities of DHCA and oximes in our previous works [28][29][30], the introduction of the oxime group to DHCA should be suggested to possess liver-targeted and anti-HBV activities. So, a series of oxime derivatives of DHCA were designed, synthesized, and screened for anti-HBV activity in vitro in this work, and molecular docking studies were carried out to investigate the relationship of structure and bioactivity of these compounds using a molecular operating environment (MOE).

Structure-Activity Relationship (SAR)
Results of the bioactive assay showed that DHCA exhibited no activity against the secretion of HBeAg and HBsAg, while most of the derivatives exhibited more or less activity against the secretion of HBsAg and HBeAg, as shown in Table 1.
(selectivity index) = CC50/IC50; d HBeAg: hepatitis B e antigen; e HBsAg: hepatitis B surface antigen; f The inhibition ratio less than 50% in the test concentration range; g Dehydrocholic acid (DHCA) is the raw material of reaction; h Entecavir (ETV) as the positive control. Data were expressed as mean ± S.D. (n = 3). * Compared with the positive control index: p < 0.05. ** Compared with the positive control index: p < 0.01.

Structure-Activity Relationship (SAR)
Results of the bioactive assay showed that DHCA exhibited no activity against the secretion of HBeAg and HBsAg, while most of the derivatives exhibited more or less activity against the secretion of HBsAg and HBeAg, as shown in Table 1.

Molecular Docking Study
To further investigating relationship of structures of the bioactivity and interactions between the ligand and protein of these oxime derivatives, docking studies were carried out using MOE 2008.10. The "Site Finder" tool in this program was used to reach for the active site. Docking study of these oxime derivatives with bile acid receptor protein residue (PDB: 3bej) and HSPG protein residue (PDB: 3sh5) was achieved. The docking scores (S) and the hydrogen bond strength of all the molecules are shown in Tables S2 and S3.
Docking studies showed that DHCA and cholic acid interacted to bile acid receptor with scores in −11.35 and −12.35 kcal/mol, respectively, and bound to residues Lys321 and Ile468 (Figure 3). These results confirmed DHCA possessed hepatocyte targeting activity, theoretically. These oxime

Molecular Docking Study
To further investigating relationship of structures of the bioactivity and interactions between the ligand and protein of these oxime derivatives, docking studies were carried out using MOE 2008.10. The "Site Finder" tool in this program was used to reach for the active site. Docking study of these oxime derivatives with bile acid receptor protein residue (PDB: 3bej) and HSPG protein residue (PDB: 3sh5) was achieved. The docking scores (S) and the hydrogen bond strength of all the molecules are shown in Tables S2 and S3.
Docking studies showed that DHCA and cholic acid interacted to bile acid receptor with scores in −11.35 and −12.35 kcal/mol, respectively, and bound to residues Lys321 and Ile468 (Figure 3). These results confirmed DHCA possessed hepatocyte targeting activity, theoretically. These oxime derivatives had strong interaction with the dock score ranging from −13.46 to −10.85 kcal/mol with bile acid receptor (Table S2, 2b-1, 2b-3, and 0-2 compounds, 2a-1 and 2c-1 with effective inhibition of HBV showed no interaction to the HSPG protein. These docking results did not coincide with anti-HBV activities. Docking results revealed strong interaction between these oxime derivatives of DHCA and the bile acid receptor, which implied these DHCA derivatives might concentrate to hepatocyte, and possess liver-target activity. Although DHCA and cholic acid docked to the HSPG protein, and showed moderate interactions, they did not exhibit inhibition of HBV in vitro assay (Table S1, Figure S8

Cell Culture and Drug Treatment
HepG2.2.15 cells were kindly provided by the Chinese Academy of Medical Sciences (Beijing, China), derived from human hepatoma cell line G2. These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1.5 g/L of sodium bicarbonate, 10 mL/L of penicillin, and streptomycin, respectively, and 200 mg/L of G418 at 37 • C under 5% carbon dioxide in a 95-98% humidity. The compounds were diluted to the desired concentrations in culture medium. Before treated with various concentrations of compounds, cells were seeded at a density of 1 × 10 5 cells/mL in 96-well plates and incubated for 24 h at 37 • C. Every three days in a nine day period, supernatant of each well was replaced with compound-containing fresh medium [23].

Method for Cell Toxicity and HBsAg and HBeAg Inhibition Assays
The levels of HBeAg and HBsAg in the supernatant of the cells were measured using ELISA assay following the manufacturer's protocol (Shanghai Kehua Bio-engineering Co., Ltd., Shanghai, China). The synthesized compounds were expressed as the concentration that achieved 50% inhibition (IC 50 ) to the secretion of HBeAg and HBsAg [29].
The cytotoxicity activity of the synthesized compounds was determined by MTT assay [35]. After refreshing the supernatant, 20 µL MTT (5 mg/mL) was added to each well, which was further cultured for 4 h at 37 • C. Then the supernatant of each well was carefully removed, formazan crystals were dissolved in 150 mL of DMSO and the absorbance at 450 nm was recorded [36]. Cytotoxicity of these compounds was expressed as the concentration of compound required to kill 50% (CC 50 ) of the HepG2.2.15 cells.

Molecular Docking
The crystal structures of heparan sulfate proteoglycan (HSPG) protein (P98160, PDB: 3sh5) and bile acid receptor protein (Q96RI1, PDB:3bej) were downloaded from The UniProt Knowledgebase (https://www.uniprot.org/). Molecular Docking simulations of the compounds inside the protein, which from HSPG and bile acid receptor, were carried out by using Molecular Operating Environment (MOE) 2008.10. Initially, structures of compounds were protonated with addition of polar hydrogens, and then converted to three-dimensional (3D) structures, followed by energy minimization with force-field using HyperChem 8.0.7 to get stabilized conformers. The stabled conformer of compounds were introduced to MOE, and then proceeded an "energy minimize" process to offer structurally optimized compounds, and were saved as PDB format files, respectively. After crystal structures of the receptor protein were introduced to MOE, unbound water, other small molecules, and ions were removed, then "protonate 3D" was proceeded, to add proteins to the proteins, subsequently followed by an "energy minimize" process to give structurally optimized protein. A structurally optimized compound was then introduced to optimized protein to proceed docking simulation. Docking score