Discovery of Novel Small Molecule Anti-HCV Agents via the CypA Inhibitory Mechanism Using O-Acylation-Directed Lead Optimization

In this work, the relationship between cyclophilin A (CypA) and HCV prompted us to screen a series of small molecule CypA inhibitors which were previously reported by our group. Among them, compound 1, discovered as a non-immunosuppressive anti-HCV agent with an EC50 value of 0.67 μM in a virus assay, was selected for further study. Subsequent chemical modification by O-acylation led to a novel class of molecules, among which compound 25 demonstrated the most potent anti-HCV activity in the virus assay (EC50 = 0.19 μM), but low cytotoxicity and hERG cardiac toxicity. The following studies (a solution stability assay and a simple pharmacokinetic test together with a CypA enzyme inhibition assay) preliminarily indicated that 25 was a prodrug of 1. To the best of our knowledge, 25 is probably the most potent currently reported small molecule anti-HCV agent acting via the CypA inhibitory mechanism. Consequently, our study has provided a new potential small molecule for curing HCV infection.


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2-(2-Benzyloxyethoxy)-6-fluorobenzonitrile (36). To a solution of 2-benzyloxyethanol (0.96 g) in dimethyl sulfoxide (DMSO, 5 mL), was added 60% sodium hydride (0.156 g) and the mixture was stirred in an atmosphere of nitrogen at room temperature for 3 h. To the mixture 2,6-difluorobenzonitrile (35, 0.47 g) was added and then the temperature was increased to 110 °C. The reaction mixture was stirred for 10 h and, after cooling, poured into 100 mL water, and extracted with dichloromethane (DCM, 40mL) three times. The organic layers were combined, dried with anhydrous sodium sulfate, and then concentrated to dryness. The residue was purified by flash column chromatography on silica gel, eluted with a mixture of ethyl acetate (EA)/PE (1:5, v/v) 2,6-Bis-(2-benzyloxyethoxy)-benzonitrile (41). To a solution of 2-benzyloxyethanol (1.7 g) in DMSO (5 mL), was added 60% sodium hydride (0.45 g) and the mixture was stirred in an atmosphere of nitrogen at room temperature for 3 h. To the mixture, 2,6-difluorobenzonitrile (35, 0.47 g) was added and then the temperature was increased to 110 °C. The reaction mixture was stirred for 10 h and, after cooling it was poured into water (100 mL), and extracted with DCM (40mL) three times. The organic layers were combined, dried with anhydrous sodium sulfate, then concentrated to dryness. 2-Benzyloxy-6-(2-benzyloxyethoxy)-benzonitrile (37). To a solution of sodium benzyloxide (0.48 g) in DMSO (10 mL), was added 2-(2-benzyloxyethoxy)-6-fluorobenzonitrile (36, 0.45 g) with stirring and then the temperature was rapidly increased to 110 °C for 10 h. The reaction mixture was cooled and poured into water (100 mL), and extracted with DCM (40 mL) three times. The organic layers were combined, dried with anhydrous sodium sulfate, then concentrated to dryness. The residue was purified by flash column chromatography on silica gel, eluted with a mixture of EA/PE (1:5, v/v), to afford 37 (0.53 g, 89%) as a white solid: 1 (40). To a solution of 9H-fluoren-9-yl isocyanate (39, 0.21g) was added 38 (0.32 g) with stirring at room temperature. The mixture was heated to 110 °C for 10 h. The solvent was evaporated under vacuum to give the crude product that was purified by flash column chromatography on silica gel, eluted with a mixture of THF/DCM/PE (1:5:3, v/v/v), to afford 40 (347 mg, 70%) as a white solid: EI-MS m/z 584.2 (M + ).

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HPLC Analysis Data of Compounds 14-33 Table S1. HPLC analysis data of compounds 14-33. The purities of identified compounds were essential to the conclusions drawn in the text and were determined by the same instrumentation under several different conditions given in the following table. The peak purity was checked by UV spectraoscopy.

Establishment of HCV Virus and Subgenomic Replicon Cell Lines
The HCV virus assay was constructed by using the method developed as previously described with a small modification [2,3]. In brief, the pRluc-JFH-1 plasmid was constructed as follows: based on the plasmid of pJFH-1, a gift from Apath, LLC (New York, NY, USA), a humanized Renilla luciferase reporter gene was introduced into the C-terminus of NS5A in the JFH-1 genome. The plasmid pRluc-JFH-1 was made through digestion with XbaI restriction enzyme, and used as a template for RNA transcription. The virus transcripts were prepared in vitro by using the Ambion MEGAscript Kit, and then 10 μg RNA was mixed with 400 mL of Huh7.5.1 cells, which were a kind gift of Jin Zhong (Institute Pasteur of Shanghai, Chinese Academy of Science, Shanghai, China), at a concentration of 1 × 10 7 cells·mL −1 . After electroporation, the Huh7.5.1 cells containing virus transcripts were seeded in a 10 cm dish. After cells were cultured for 4 days, the supernatant was collected and filtered to obtain the stock solution of virus JFH-1. To obtain virus titer, the virus stocks were diluted at a gradient of 1:10, and incubated the Huh7.5.1 cells for 48 h at 37 °C. Then the cells were harvested and the luminescence was detected as manufacturer's protocol of Renilla-Glo™ Luciferase Assay System (Promega, Beijing, China).

Solution Stability Assay
The most potent compound 25 was selected as a candidate for further testing by the HPLC method [5]. To a solution of 25 (1 mg) in DMSO (6 mL) was added PBS buffer (48 mg NaCl, 1.2 mg KCl, 22 mg Na2HPO4·12H2O, 1.4 mg KH2HPO4, pH 7.4) at 0 °C. Then the samples were incubated at 37 °C. At defined times the solution was analyzed and samples were run on a BECKMAN ULTRASPHERE C18 ODS column (4.6 × 250 mm, 5 μm particle size) at 25 °C with a flow rate of 0.5 mL·min −1 of an isocratic eluent composed of methanol/water (70/30, v/v), and the eluted analytes were detected at 254 nm.

Pharmacokinetic Studies
The pharmacokinetic studies of compound 25 were carried out in SD rats (male SD rats (200 ± 20 g) × 3, provided by the Sun Yat-sen University Animals Center). Before administration, to a solution of 25 (4 mg) in N,N-dimethylacetamide (DMA, 100 μL) was added castor oil (900 μL), and then the solution was diluted with normal saline to 10 mL. The rats were administered at a dose of 5 mL·kg −1 intravenously.

Cell Culture and Cell Requirements
A CHO cell line stably transfected with hERG cDNA and expressing hERG channels was used for the study. The cells were cultured in medium containing the following components: Ham's F12, 10% (v/v) heat inactivated FBS, 100 µg·mL −1 hygromycin B and 100 µg·mL −1 geneticin. The cells used in QPatch study must meet following criteria: under microscopy examination, the majority of cells in suspension should be single and isolated; their viability should be greater than 95%, with only a few debris and cell clumps (which may clog the holes in QPlate during whole-cell clamp recording); cell density should be ranged within 3-8 × 10 6 cells·mL −1 in the final suspension before applying to the QPatch stir chamber. After leaving CO2 incubator, cells are maintained in serum-free medium buffered HEPES. Cells in such condition can be used for recording only for four hours after harvesting.

Recording System
Whole-cell recordings were performed using automated QPatch (Sophion, Ballerup, Denmark). The cells were voltage clamped at a holding potential of −80 mV. The hERG current was activated by S7 depolarizing at +20 mV for 5 s, after which the current was taken back to −50 mV for 5 s to remove the inactivation and observe the deactivating tail current. The maximum amount of tail current size was used to determine hERG current amplitude. The composition of the solutions used for the electrophysiological recordings were described in Table S4.

Automated QPatch Procedures
After achieving break-in (whole-cell) configuration, the cells were recorded for 120 s to assess current stability. The voltage protocol described above was then applied to the cells every 20 s throughout the whole procedure. Only stable cells with recording parameters above threshold were allowed to enter the drug addition procedure.
External solution containing 0.2% DMSO (vehicle) was applied to the cells to establish the baseline. After allowing the current to stabilize for 3 min, compound 25 (20 mM stocked in DMSO) was applied. The solution of 25 (40, 8, 1.6, 0.32, 0.064 and 0.0128 µM, first diluted in DMSO, and then serial-diluted in external solution to the final µM ranges before QPatch test. Final DMSO concentration was 0.2%) was added and the cells were kept in the test solution until the compound's effect reached a steady state or for a maximum of 3 min. Washout with external solution might be performed until the recovery of the current reached a steady state. Positive control cisapride (dosing started at 3 µM) is used in the experiments to test the same batch of cells used for test compounds to ensure the normal response and the good quality of the cells. The method described above was performed according to [6][7][8].